**Novel Cellular and Molecular Interactions During Limb Development, Revealed from Studies on the Split Hand Foot Congenital Malformation**

Daniele Conte, Luisa Guerrini and Giorgio R. Merlo

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/60402

### **Abstract**

[66] Shiraishi N, Yamada S, Takakuwa T. Three-dimensional models once again: for re‐ search and teaching of early human development. Congenit Anom. 2013;53(1):58-9.

124 New Discoveries in Embryology

The embryonic development of the limbs is widely used as a paradigm for the comprehension of the cellular processes and molecular mechanisms underlying organogenesis and pattern formation. The chick, mouse and (recently), zebrafish embryos are excellent models, for the ease of experimental manipulation and the availability of several mutant strains with limb malformation defects.

Knowledge on the molecular circuits that control cell expansion and positiondependent cell differentiation in the developing limb bud is rapidly expanding. Recently, a set of human congenital malformations known as split hand foot malformations (SHFM) together with the corresponding animal models have revealed novel molecular players and regulations, important for the function and maintenance of the apical ectodermal ridge, the structure that coordinates limb outgrowth with digit pattern.

In this chapter we illustrate the pathways centred on the master transcription factor p63, and discuss the mechanisms by which these pathways impact on the regulation of signalling molecule controlling growth and shape of the normal limb. Finally we indicate how the signalling networks are misregulated in SHFM, and point to emerging functions of the FGF8 and Wnt5a signalling molecules.

**Keywords:** Limb, Embryonic ectoderm, AER, SHFM, EEC, p63, Dlx5, Wnt5a, FGF8

© 2015 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

### **1. Introduction**

The limbs are projecting paired appendages of an animal body used especially for movement and grasping, for example, wings, arms, and legs. The development of the limb bud is often taken as a paradigm for a cellular and molecular comprehension of the common principles of organogenesis and pattern formation. Embryonic patterning implies that cells acquire positional information, usually by interpreting concentration gradient of signalling molecules. Accordingly, limb pattern is specified along three principal axes: anterior-posterior (A-P) (e.g., thumb to little finger), dorsal-ventral (D-V) (e.g., back of hand to palm) and proximal-distal (P-D) (e.g., shoulder to nails). Digit pattern across the A-P axis is a classic example of a signalling gradient that specifies positional values, linked to a gradient of Sonic-Hedgehog (SHH). D-V patterning is less studied and involves signals from dorsal and ventral ectoderm. The specification of P-D positional values has long been considered to involve a timing mechanism, under the control of ligands of the fibroblast growth factor (FGF) family. A concentration gradient of molecules can also give cells polarity information, recently shown to be critical for patterning and morphogenesis.

The limbs are not essential for life, thus a large number of mutant strains are available for studies on the genetic determinants of limb development, in normal and pathological condi‐ tions. Manipulation of chicken limb buds has been widely used in the past, mainly because of the ease of examination and manipulation, to postulate the first models of limb bud develop‐ ment leading to the identification of important regulatory genes and interactions. In addition to the chicken model, functional genetics has made great advances thanks to spontaneous and engineered loss- and gain-of-function mutant mouse strains, and recently with the advent of the zebrafish embryos as animal models.

In this chapter, we illustrate the pathways centred on the master transcription factor p63, and discuss the mechanisms by which these pathways impact on the regulation of signalling molecules controlling growth and patterning of the normal limb bud. Based on available knowledge, we propose how signalling networks are misregulated in the split hand foot malformation (SHFM) and related developmental conditions, and indicate emerging functions of the FGF8 and Wnt5a diffusible molecules.

### **1.1. Limb initiation**

Around the embryonic age E8.0 in the mouse, limb buds are initiated as four lumps of mesenchymal cells covered by ectoderm, protruding from the main body axis at approximately the position of somites 6–11 (the forelimbs, FL) and somites 24–27 (the hindlimbs, HL). The limb buds are paired along the cephalo-caudal axis and develop at the same fixed locations on this body axis (Figure 1A). How are their positions defined?

It has been proposed that the position of several dorsal organs along the cephalo-caudal axis, their identity and timing of appearance depend on the expression of specific sets of *Hox* genes. The 39 vertebrate *Hox* genes code for homeodomain transcription factors, homologous with the genes of the Drosophila *HOM-C* complex, and are combinatorially expressed along the Novel Cellular and Molecular Interactions During Limb Development, Revealed from Studies on the Split Hand… http://dx.doi.org/10.5772/60402 127

#### Conte et al.

**1. Introduction**

126 New Discoveries in Embryology

to be critical for patterning and morphogenesis.

the zebrafish embryos as animal models.

of the FGF8 and Wnt5a diffusible molecules.

this body axis (Figure 1A). How are their positions defined?

**1.1. Limb initiation**

The limbs are projecting paired appendages of an animal body used especially for movement and grasping, for example, wings, arms, and legs. The development of the limb bud is often taken as a paradigm for a cellular and molecular comprehension of the common principles of organogenesis and pattern formation. Embryonic patterning implies that cells acquire positional information, usually by interpreting concentration gradient of signalling molecules. Accordingly, limb pattern is specified along three principal axes: anterior-posterior (A-P) (e.g., thumb to little finger), dorsal-ventral (D-V) (e.g., back of hand to palm) and proximal-distal (P-D) (e.g., shoulder to nails). Digit pattern across the A-P axis is a classic example of a signalling gradient that specifies positional values, linked to a gradient of Sonic-Hedgehog (SHH). D-V patterning is less studied and involves signals from dorsal and ventral ectoderm. The specification of P-D positional values has long been considered to involve a timing mechanism, under the control of ligands of the fibroblast growth factor (FGF) family. A concentration gradient of molecules can also give cells polarity information, recently shown

The limbs are not essential for life, thus a large number of mutant strains are available for studies on the genetic determinants of limb development, in normal and pathological condi‐ tions. Manipulation of chicken limb buds has been widely used in the past, mainly because of the ease of examination and manipulation, to postulate the first models of limb bud develop‐ ment leading to the identification of important regulatory genes and interactions. In addition to the chicken model, functional genetics has made great advances thanks to spontaneous and engineered loss- and gain-of-function mutant mouse strains, and recently with the advent of

In this chapter, we illustrate the pathways centred on the master transcription factor p63, and discuss the mechanisms by which these pathways impact on the regulation of signalling molecules controlling growth and patterning of the normal limb bud. Based on available knowledge, we propose how signalling networks are misregulated in the split hand foot malformation (SHFM) and related developmental conditions, and indicate emerging functions

Around the embryonic age E8.0 in the mouse, limb buds are initiated as four lumps of mesenchymal cells covered by ectoderm, protruding from the main body axis at approximately the position of somites 6–11 (the forelimbs, FL) and somites 24–27 (the hindlimbs, HL). The limb buds are paired along the cephalo-caudal axis and develop at the same fixed locations on

It has been proposed that the position of several dorsal organs along the cephalo-caudal axis, their identity and timing of appearance depend on the expression of specific sets of *Hox* genes. The 39 vertebrate *Hox* genes code for homeodomain transcription factors, homologous with the genes of the Drosophila *HOM-C* complex, and are combinatorially expressed along the **Figure 1.** Schematic representation of limb development with embryonic timeline for chick wing and mouse forelimb. A) Representation of the prospective limb territories in a stage 14/8 chick (Hamburger-Hamilton stages, HH))/mouse embryo. The forelimbs (FL) and hindlimbs (HL) derive from discrete regions of the lateral plate mesoderm (LPM). *Tbx5* (red) is expressed in the prospective FL, whereas *Tbx4* (red) is expressed in the prospective HL. In this stage, *Pitx1* (yellow) is expressed in a caudal domain that overlaps with *Tbx4*. The somites (blue) are numbered and serve as refer‐ ence for the axial position of the FL and HL fields. B) Model of initiation of FL bud. Hox protein gradients establish the condition for the synthesis of retinoic acid (RA) in the LPM. RA causes induction of the transcription factors Tbx5 (or Tbx4 for the HL). *Tbx5*-expressing mesechymal cells express *FGF10* and induce the ectodermal cells of the surface (yel‐ low square) to activate epithelial-mesenchymal transition (EMT). C) Newly generated mesenchymal cells express *FGF10* that induces the overlaying ectodermal cells to express *FGF8* giving rise to the apical ectodermal ridge (AER). Expression of *FGF8* by the AER induces the mesenchymal cells to express *FGF10*, thus establishing a positive feedback loop for the initial phases of limb outgrowth. D) Schematic representation of the progress zone (PZ) model. The AER maintains cells of the PZ in an unspecified state. For a detailed description of the proposed models of P-D patterning see the text. E) AER-derived FGF8 maintain the expression of *SHH* in the ZPA cells, which in turn gives feedback on the AER cells to maintain *FGFs* expression, via Grem1 and BMP inhibition. This signalling between the AER and ZPA contributes to co ordinate the patterning along the P-D and A-P axes.

main body axis. *Hox* genes are serially organized in four clusters (a, b, c, and d), each located on a different chromosome. Within each cluster, *Hox* genes are organized in a physical order collinear with the cephalo-caudal axis of the growing organism so that the genes lying at the 3' end are expressed earlier and are localized in the most anterior domains. Moving toward the 5' direction, each next gene is expressed in a progressively more posterior territory. Thus, each *Hox* gene has a specific anterior limit of expression, and each A-P embryonic territory expresses a specific combination of *Hox* genes, utilized as positional information.

A key signalling molecule for limb initiation is FGF10, a member of the FGF family of diffusible peptides. The *FGF10* mRNA is detected quite early in the presumptive limb mesenchyme and promotes AER induction (a key organizer and regulator of the P-D limb extension; see below) and initiation of ectopic limb development when applied exogenously [1]. Conversely, in *FGF10*-null mice, limb buds are initiated but the AER does not form, resulting in complete truncation of all four limbs [2 - 4].

The expression of *FGF10* coincides with the time when the trunk is only competent to form an ectopic limb, for example, the time at which the trunk mesenchyme becomes determined and can no longer be redirected to a limb fate (the HH stage 16–17 of the chick embryo) [5, 6]. The initial assumption was that limbs originate from a pre-existing mesenchymal population undergoing a localized regulation of proliferation. In fact, at the HH stage 17–18, there is a substantial decrease in proliferation of the flank mesoderm, while instead higher rates are maintained within the emerging limb buds.

The current model considers that, shortly after gastrulation, a re-epithelization of mesodermal cells occurs so that the entire embryo is essentially epithelial, including also the notochord, the somites, the intermediate mesoderm and the lateral plate mesoderm (LPM). At stage HH 13 in the chick, before limb initiation, the somatopleure displays epithelial rather than mesen‐ chymal features. The LPM of the limb field starts out as an epithelium and ultimately generates limb-bud mesenchyme through a process termed epithelial-mesenchymal transition (EMT) [7] (Figure 1B). In embryos null for *FGF10* and *Tbx5* the proportion of mesenchymal cells com‐ pared to the proportion of epithelial cells was significantly lower than that of wild-type (WT). These mutants show hyperplasia of the somatopleure epithelium, in support of failure of these cells to undergo EMT. These new data show the time in which the trunk is competent to form an ectopic limb, is precisely the time at which the trunk mesenchyme is initially generated. The old experiments of ectopic application of FGF10 need to be re-interpreted, as induction of limb-bud formation from epithelial trunk somatopleure cells and not from mesenchymal cells of the same A-P level.

### **1.2. T-box genes and limb-type specification**

The FL and HL of all vertebrate species are evidently different (e.g., wing vs. leg in the chick embryo, pectoral vs. pelvic fins in fish embryos, arms vs. legs in primates, etc.). The specifi‐ cation of limb-type identity and morphology is established before overt limb initiation. A large body of evidence indicates that two transcription factors of the T-box family participate in the early definition of limb-type identity: *Tbx5* for the presumptive territories of the mouse FL (wing of the chick, pectoral fins of fishes) and of *Tbx4* in the presumptive territory of the mouse HL (leg of the chick, pelvic fins of fishes) (Figure 1A). These genes are expressed very early in the prospective limb mesoderm and, in addition to define limb-type identity, also appear to be necessary and sufficient for early limb induction [8 - 15].

However, although only expressed in FL and HL, respectively, *Tbx5* and *Tbx4* appear not to be the master directors of limb-bud identity/morphology design. Instead, the *Pitx1* gene, which codes for a paired-type homeodomain transcription factor and which is expressed in the HL bud, is the upstream regulator of *Tbx4*, and is directly implicated in HL specification. Multiple independent *cis*-regulatory elements of *Tbx4* expression have been identified, including the HL-specific enhancers [16]. Both may be targets of Pitx1 and other unknown upstream factors. Structural changes of these regulators might be some of the multiple factors responsible for the hind/lower limb morphology specification. However, limb-bud identity/morphology determination remains to a large extent unexplained.

### **1.3. Role of retinoic acid**

main body axis. *Hox* genes are serially organized in four clusters (a, b, c, and d), each located on a different chromosome. Within each cluster, *Hox* genes are organized in a physical order collinear with the cephalo-caudal axis of the growing organism so that the genes lying at the 3' end are expressed earlier and are localized in the most anterior domains. Moving toward the 5' direction, each next gene is expressed in a progressively more posterior territory. Thus, each *Hox* gene has a specific anterior limit of expression, and each A-P embryonic territory

A key signalling molecule for limb initiation is FGF10, a member of the FGF family of diffusible peptides. The *FGF10* mRNA is detected quite early in the presumptive limb mesenchyme and promotes AER induction (a key organizer and regulator of the P-D limb extension; see below) and initiation of ectopic limb development when applied exogenously [1]. Conversely, in *FGF10*-null mice, limb buds are initiated but the AER does not form, resulting in complete

The expression of *FGF10* coincides with the time when the trunk is only competent to form an ectopic limb, for example, the time at which the trunk mesenchyme becomes determined and can no longer be redirected to a limb fate (the HH stage 16–17 of the chick embryo) [5, 6]. The initial assumption was that limbs originate from a pre-existing mesenchymal population undergoing a localized regulation of proliferation. In fact, at the HH stage 17–18, there is a substantial decrease in proliferation of the flank mesoderm, while instead higher rates are

The current model considers that, shortly after gastrulation, a re-epithelization of mesodermal cells occurs so that the entire embryo is essentially epithelial, including also the notochord, the somites, the intermediate mesoderm and the lateral plate mesoderm (LPM). At stage HH 13 in the chick, before limb initiation, the somatopleure displays epithelial rather than mesen‐ chymal features. The LPM of the limb field starts out as an epithelium and ultimately generates limb-bud mesenchyme through a process termed epithelial-mesenchymal transition (EMT) [7] (Figure 1B). In embryos null for *FGF10* and *Tbx5* the proportion of mesenchymal cells com‐ pared to the proportion of epithelial cells was significantly lower than that of wild-type (WT). These mutants show hyperplasia of the somatopleure epithelium, in support of failure of these cells to undergo EMT. These new data show the time in which the trunk is competent to form an ectopic limb, is precisely the time at which the trunk mesenchyme is initially generated. The old experiments of ectopic application of FGF10 need to be re-interpreted, as induction of limb-bud formation from epithelial trunk somatopleure cells and not from mesenchymal cells

The FL and HL of all vertebrate species are evidently different (e.g., wing vs. leg in the chick embryo, pectoral vs. pelvic fins in fish embryos, arms vs. legs in primates, etc.). The specifi‐ cation of limb-type identity and morphology is established before overt limb initiation. A large body of evidence indicates that two transcription factors of the T-box family participate in the early definition of limb-type identity: *Tbx5* for the presumptive territories of the mouse FL (wing of the chick, pectoral fins of fishes) and of *Tbx4* in the presumptive territory of the mouse

expresses a specific combination of *Hox* genes, utilized as positional information.

truncation of all four limbs [2 - 4].

128 New Discoveries in Embryology

of the same A-P level.

maintained within the emerging limb buds.

**1.2. T-box genes and limb-type specification**

A signalling molecule known to act upstream of *Tbx5* during forelimb/pectoral fin develop‐ ment is retinoic acid (RA) [17 - 21]. According to old observations, patterning along the proximal-distal axis of the vertebrate limb is controlled by opposing diffusible signals, in which RA functions as the proximal signal and FGF as the distal one [22, 23].

The mechanism through which RA controls limb development has been widely debated [21], but clear results have only been produced in recent years [17, 19, 22, 24 - 27]. Mouse and zebrafish embryos null for the gene *aldehyde dehydrogenase-1a2 (aldh1a2*) fail to synthesize RA and do not develop limbs/fins. These mutants fail to express *Tbx5*, and the exogenous appli‐ cation of RA can rescue both limb/fin development and *Tbx5* expression [18, 19, 25, 27, 28]. However, a recent paper shows that RA signalling is not required for P-D limb patterning, and instead provides genetic evidence that RA-FGF antagonism occur only along the trunk lateral plate mesoderm, prior to FL budding, to permit induction of *Tbx5* [29]. This study shows that RA controls limb development in a manner much different than that originally envisioned (see below).

### **1.4. Proximal-distal axis**

The limb skeleton is laid down as five cartilage skeletal elements, not just the three referred to as stylopod (humerus/femur), zeugopod (radius-ulna/tibia-fibula), and autopod (digits); in fact two carpal regions between zeugopod and autopod are present, that initially have the same size as the other segments but then grow substantially less.

P-D extension and patterning is strictly linked to the signalling activity of the apical ectodermal ridge (AER), a morphologically distinct ectodermal thickening, extending along the entire A-P length, and lining the D-V border. The AER is present between E9 and E11 in the mouse embryo, consists of a pseudo-stratified epithelium in the chick and pluristratified epithelium in the mouse, and is a dynamic structure constantly undergoing morphogenetic changes [30 - 32].

The AER plays a fundamental role in promoting and regulating the outgrowth and patterning of the P-D limb axis. Experimental removal of the AER in chicken limb buds, causes a devel‐ opmental arrest, and truncation of wing skeleton [33], meanwhile grafts of an AER to a recipient limb bud induces ectopic P-D outgrowth [34]. In 1993, Niswander identified FGFs as the relevant signals produced by the AER to induce P-D limb axis formation and extension. P-D extension and outgrowth is rescued by exogenous application of FGFs on AER removal [35]. This study provided the first molecular insights into how AER-FGF signalling controls in P– D extension and patterning. Four FGF ligands (4, -8, -9 and -17), are expressed by the AER cells with redundant functions during P–D patterning of mouse limb buds. Inactivation of the three FGFs expressed predominantly by the posterior AER (FGF4, -9, -17) does not alter limb-bud development [36]. In contrast, loss of *FGF8*, which is the first and only FGF ligand expressed by the entire AER from early stages onward, disrupts formation of the proximal-most limb skeletal element [37 - 40]. This early and transient disruption of P-D extension is rescued by the activation of FGF4 in the *FGF8*-deficient AER, which results in almost normal development of the more distal limb skeleton [41, 42]. Combined inactivation of both *FGF8* and *FGF4* causes a complete arrest of limb-bud development and limb agenesis [39, 42]. In addition, transient expression of *FGF8* and *FGF4* during initiation of limb-bud outgrowth is sufficient for specification of the entire PD axis, but the progressive proliferative expansion of such specified limb segments is disrupted [42].

Other AER-expressed FGFs, in particular *FGF9*, contribute to the proliferative expansion of the limb mesenchymal progenitors in a P-D sequential order, so that higher AER-expressed FGF levels are required for formation of more distal limb structures [36]. Taken together, this genetic analysis reveals an instructive role of AER-FGF signalling in the specification and proliferative expansion along the P*–*D axis.

The AER is first induced by the expression of *FGF10* in the prospective limb-bud mesenchyme. *FGF10* is expressed in the same territories as *Tbx4 and Tbx5* and interestingly, *FGF10* is a direct transcriptional target of these transcription factors. The expression of *FGF10* is essential to establish AER-expressed FGF signalling, which in turn is required to maintain *Ffg10* expression [1, 42]. The reciprocal induction of *FGF10-FGF8* requires the expression of *Wnt3a*, coding for a ligand of the Wnt family acting through the β-catenin pathway (described in the following sections). The activation of *Wnt3* expression couples *FGF8* and *FGF4* expression from cells of the AER with *FGF10* expression [43, 44]. Thus, in these early phases, a positive feedback loop between *FGF10* and *FGF8* is established in adjacent territories, and is required for reciprocal maintenance (Figure 1A-C).

### **1.5. Limb extension: The progress zone**

Old experiments showed that removing the AER at progressively earlier stages resulted in truncations of the limb skeleton at progressively more proximal levels [33]. Thus, the acquis‐ ition of a P-D positional identity seemed to depend on the time that proliferating/unspecified cells spend near the AER (the progress zone, PZ) under the influence of AER signals. According to the model proposed by Summerbell and Wolpert [45] the mesenchymal progenitor cells leaving the PZ early would acquire proximal identities, whereas the same cells leaving the PZ later would acquire progressively more distal identities (Figure 1D).

The great merit of this model has been to introduce the notion of time as an important factor in morphogenetic signalling; however, as a result of extensive molecular and cellular analyses, the original PZ model has been largely abandoned. First, the loss of proximal but not distal skeletal elements in *FGF8*-deficient mouse limb buds [40] are difficult to reconcile. Second, fate mapping studies in chicken embryos provide good evidence for the presence of pools of progenitor cells with distinct P-D identities, specified very early and then expanded sequen‐ tially by proliferation [46]. Removal of the AER at progressively later stages simply eliminates the distal mesenchyme containing the specified but not yet expanded progenitor pools. These studies provide a straightforward alternative explanation for the loss of distal skeletal elements following AER removal [46]. These and other results led to the proposal of the early specifi‐ cation/expansion model as an alternative to the PZ model [42, 46]. This model proposes that AER-expressed FGF signalling controls survival and sequential proliferative expansion of a pool of progenitor/stem cells in P-D territories, in a dose- and time-dependent highly regulated fashion.

A third model has been proposed, in which P-D patterning is controlled by opposing diffusible signals, with RA functioning as a proximal signal and FGF acting as a distal signal [26]. Chick FL or HL ectopically exposed to RA or FGF8, or to antagonists of RAR or FGF receptor, display P-D fate changes that either expand or contract expression of proximal limb markers [23]. Further evidence has been recently provided, indicating that RA is needed for P–D patterning of both FL and HL [47, 48]. Using recombinant heterotopic chick limb transplantations they propose that the exposure to the activities of Wnt3a, FGF8 (distal molecule), and RA (proximal molecule) maintains the potential to form both proximal and distal structures. While these studies report the ability of RA treatment to reprogram distal limb mesenchyme to a proximal fate and to maintain early limb mesenchyme in a *Meis1*-expressing proximal fate alongside Wnt and FGF treatment [47], they do not address a requirement for endogenous RA in proximal limb mesenchyme. The ability of RA to increase *Meis1/2* (a proximal marker) could be a consequence of loss of FGF signalling, known to repress *Meis1/2* distally [36].

Recently, Cunningham and colleagues [29] provide convincing evidence that RA is not required for limb patterning and that RA-FGF antagonism does not occur along the limb P-D axis, as originally proposed [26]. They suggest that both the initial expression of *Meis1/2* in the LPM and later in the proximal limb bud do not require RA signalling, while the downregula‐ tion of *Meis1/2* expression in the distal limb requires AER-derived FGF8. They suggest that since *FGF8* expression in the AER appears after limb-bud formation [37], the proximal most limb domain is out of range of early AER-derived FGF signals, leading to maintenance of proximal *Meis1/2* expression and restricted distal *Meis1/2* expression (Figure 1D).

### **1.6. Anterior-posterior pattern**

The AER plays a fundamental role in promoting and regulating the outgrowth and patterning of the P-D limb axis. Experimental removal of the AER in chicken limb buds, causes a devel‐ opmental arrest, and truncation of wing skeleton [33], meanwhile grafts of an AER to a recipient limb bud induces ectopic P-D outgrowth [34]. In 1993, Niswander identified FGFs as the relevant signals produced by the AER to induce P-D limb axis formation and extension. P-D extension and outgrowth is rescued by exogenous application of FGFs on AER removal [35]. This study provided the first molecular insights into how AER-FGF signalling controls in P– D extension and patterning. Four FGF ligands (4, -8, -9 and -17), are expressed by the AER cells with redundant functions during P–D patterning of mouse limb buds. Inactivation of the three FGFs expressed predominantly by the posterior AER (FGF4, -9, -17) does not alter limb-bud development [36]. In contrast, loss of *FGF8*, which is the first and only FGF ligand expressed by the entire AER from early stages onward, disrupts formation of the proximal-most limb skeletal element [37 - 40]. This early and transient disruption of P-D extension is rescued by the activation of FGF4 in the *FGF8*-deficient AER, which results in almost normal development of the more distal limb skeleton [41, 42]. Combined inactivation of both *FGF8* and *FGF4* causes a complete arrest of limb-bud development and limb agenesis [39, 42]. In addition, transient expression of *FGF8* and *FGF4* during initiation of limb-bud outgrowth is sufficient for specification of the entire PD axis, but the progressive proliferative expansion of such specified

Other AER-expressed FGFs, in particular *FGF9*, contribute to the proliferative expansion of the limb mesenchymal progenitors in a P-D sequential order, so that higher AER-expressed FGF levels are required for formation of more distal limb structures [36]. Taken together, this genetic analysis reveals an instructive role of AER-FGF signalling in the specification and

The AER is first induced by the expression of *FGF10* in the prospective limb-bud mesenchyme. *FGF10* is expressed in the same territories as *Tbx4 and Tbx5* and interestingly, *FGF10* is a direct transcriptional target of these transcription factors. The expression of *FGF10* is essential to establish AER-expressed FGF signalling, which in turn is required to maintain *Ffg10* expression [1, 42]. The reciprocal induction of *FGF10-FGF8* requires the expression of *Wnt3a*, coding for a ligand of the Wnt family acting through the β-catenin pathway (described in the following sections). The activation of *Wnt3* expression couples *FGF8* and *FGF4* expression from cells of the AER with *FGF10* expression [43, 44]. Thus, in these early phases, a positive feedback loop between *FGF10* and *FGF8* is established in adjacent territories, and is required for reciprocal

Old experiments showed that removing the AER at progressively earlier stages resulted in truncations of the limb skeleton at progressively more proximal levels [33]. Thus, the acquis‐ ition of a P-D positional identity seemed to depend on the time that proliferating/unspecified cells spend near the AER (the progress zone, PZ) under the influence of AER signals. According to the model proposed by Summerbell and Wolpert [45] the mesenchymal progenitor cells

limb segments is disrupted [42].

130 New Discoveries in Embryology

maintenance (Figure 1A-C).

**1.5. Limb extension: The progress zone**

proliferative expansion along the P*–*D axis.

The mammalian limb bud is typically pentadactylous, for example, the autopodium gives rise to five skeletal elements. The digit organization, from anterior (pre-axial, the thumbs) to posterior (post-axial, the little finger) is referred to as the A-P pattern. It has long been recognized that the embryonic tissue mainly implicated in the regulation of the A-P pattern is the zone of polarizing activity (ZPA) (Figure 1E). In 1956, a region within the posteriorproximal limb mesenchyme was identified, that when grafted in the anterior margin of host chicken wing buds results in mirror image duplications of all digits [49, 50]. The ZPA acts as a signalling centre and specifies positional information in the limb-bud mesenchyme by secreting the diffusible molecule Sonic Hedgehog (SHH). Within the limb bud mesenchyme, SHH is present in a posterior (high) to anterior (low) gradient [51, 52]. Genetic studies indicate that the time spent expressing *SHH* provides cells with a kinetic memory relevant to specifi‐ cation of their A-P identities [53 - 56].

SHH signalling is translated into an intracellular, anterior (high) to posterior (low), gradient of the transcriptional repressor Gli3R within the limb mesenchyme [67]. Upon binding to the receptor Patched, SHH counteracts the conversion of Gli3 full-length into its cleaved repressor form. The Gli3R gradient is then required to establish the polarized expression of other genes involved in A-P patterning, and ultimately is translated into digit pattern, in ways not fully clarified [24, 57 - 61].

Further genetic studies in mouse and zebrafish embryos have implicated also HAND2 in the activation of *SHH* expression in both limb and fin buds [62]. Moreover, in the mouse embryo a mutual antagonistic interaction between Hand2 and Gli3, prior to *SHH* expression, estab‐ lishes a A-P pre-pattern [60, 61, 63]. Finally, at later stages of development, the expression of the 5' most members of the *Hoxd* gene cluster is activated within the posterior limb-bud mesenchyme. Cell biochemical studies have revealed a direct interaction of Hoxd proteins with the *cis*-regulatory limb-bud enhancer region of the *SHH* gene [64].

### **1.7. Hox genes and digit identity**

An exhaustive illustration of this topic is beyond the scope of this chapter. Digit patterning has commonly been interpreted in the context of a gradient of expression of *SHH* preventing the processing of Gli3 to its repressor form (Gli3R) [65, 66].

Thus, a SHH gradient is translated into an inverse Gli3R gradient [24, 67]. However, between *Gli3* and *SHH;Gli3* null mutant mice display identical polydactylous limb phenotypes, indicating that an iterative series of (non-patterned) digits can form in the absence of SHH [24, 60], suggesting the existence of a SHH-independent prepatterning.

This observation, rather than supporting the SHH gradient model, is consistent with a Turingtype model of digit patterning [68 - 70]. According to this model, dynamic interactions between activator and inhibitor molecules produce periodic patterns of spots or stripes, serving as a molecular pre-pattern for chondrogenesis. Although the core molecules of a self-organizing mechanism remain poorly known, potential candidates for molecular modulators of the system include the *Hox* genes [70]. Distal *Hoxa* and *Hoxd* genes have a well-known impact on digit number, though their specific role remains unclear. *Hoxd* genes interact with the SHH-Gli3 pathway; these include the mutual transcriptional regulation between Hox genes and SHH and the binding of Hoxd12 to Gli3R, resulting in a blockage of Gli3R repressor activity [71 - 73]. In general, gain- and loss-of-function experiments suggest a positive relation between *Hox* genes and digit number [72, 74] that is also indicated by the ectopic anterior up-regulation of distal *Hoxd* genes in the Gli3-dependent polydactyly [24, 61]. Interestingly the disruption of various *Hox* genes combined with loss of *Gli3* results in polydactyly; more *Hox* genes are removed – more digits are formed [75]. Thus, losing *Hox* genes seemed to shorten the spacing between digits – the wavelength in Turing's mathematical language.

The Turing's model implies the activity of two diffusing and interacting molecules; however, *Hox* genes code for non-diffusible transcription factors, for example, they cannot directly participate. However, evidence that distal *Hox* genes are necessary for correct limb develop‐ ment is overwhelming. Indeed, in addition to a correct digit formation via a Turing-type regulation of SHH signalling, a second role of *Hox* genes in limb P-D patterning has been studied in depth. Caudal, late-expressed paralogs of the *Hox* gene clusters display a P-D as well as A-P gradient of expression within the limb mesenchyme [76, 77] suggesting a combi‐ natorial role of these genes in patterning the limb skeletal elements. Experimental evidence leads to the conclusion that the paralogs 9–13 of the *Hox* gene clusters -*a* and -*d* specify individual limb segments [78]. Indeed *Hoxa11 -/-;Hoxd11 -/-* double mutant embryos lack radius and ulna [79] while *Hoxa13 -/-;Hoxd13 -/-* double mutants lack digits [80].

Finally, in spite of the major role played by posterior *Hox* genes, little is known about the cellular and/or molecular bases for the observed developmental defects. Attempts in this directions [81] show that malformation of the FL zeugopod in *Hoxa11/Hoxd11* double mutant mice results from multiple defects during the formation of the zeugopod, including reduced *FGF8* and *FGF10* expression, formation of smaller mesenchymal condensations, and failure to form normal growth plates at the proximal and distal ends of the zeugopod bones. As a consequence, growth and maturation of these bones is highly disorganized.

### **1.8. AER and ZPA interaction**

recognized that the embryonic tissue mainly implicated in the regulation of the A-P pattern is the zone of polarizing activity (ZPA) (Figure 1E). In 1956, a region within the posteriorproximal limb mesenchyme was identified, that when grafted in the anterior margin of host chicken wing buds results in mirror image duplications of all digits [49, 50]. The ZPA acts as a signalling centre and specifies positional information in the limb-bud mesenchyme by secreting the diffusible molecule Sonic Hedgehog (SHH). Within the limb bud mesenchyme, SHH is present in a posterior (high) to anterior (low) gradient [51, 52]. Genetic studies indicate that the time spent expressing *SHH* provides cells with a kinetic memory relevant to specifi‐

SHH signalling is translated into an intracellular, anterior (high) to posterior (low), gradient of the transcriptional repressor Gli3R within the limb mesenchyme [67]. Upon binding to the receptor Patched, SHH counteracts the conversion of Gli3 full-length into its cleaved repressor form. The Gli3R gradient is then required to establish the polarized expression of other genes involved in A-P patterning, and ultimately is translated into digit pattern, in ways not fully

Further genetic studies in mouse and zebrafish embryos have implicated also HAND2 in the activation of *SHH* expression in both limb and fin buds [62]. Moreover, in the mouse embryo a mutual antagonistic interaction between Hand2 and Gli3, prior to *SHH* expression, estab‐ lishes a A-P pre-pattern [60, 61, 63]. Finally, at later stages of development, the expression of the 5' most members of the *Hoxd* gene cluster is activated within the posterior limb-bud mesenchyme. Cell biochemical studies have revealed a direct interaction of Hoxd proteins with

An exhaustive illustration of this topic is beyond the scope of this chapter. Digit patterning has commonly been interpreted in the context of a gradient of expression of *SHH* preventing

Thus, a SHH gradient is translated into an inverse Gli3R gradient [24, 67]. However, between *Gli3* and *SHH;Gli3* null mutant mice display identical polydactylous limb phenotypes, indicating that an iterative series of (non-patterned) digits can form in the absence of SHH [24,

This observation, rather than supporting the SHH gradient model, is consistent with a Turingtype model of digit patterning [68 - 70]. According to this model, dynamic interactions between activator and inhibitor molecules produce periodic patterns of spots or stripes, serving as a molecular pre-pattern for chondrogenesis. Although the core molecules of a self-organizing mechanism remain poorly known, potential candidates for molecular modulators of the system include the *Hox* genes [70]. Distal *Hoxa* and *Hoxd* genes have a well-known impact on digit number, though their specific role remains unclear. *Hoxd* genes interact with the SHH-Gli3 pathway; these include the mutual transcriptional regulation between Hox genes and SHH and the binding of Hoxd12 to Gli3R, resulting in a blockage of Gli3R repressor activity [71 - 73]. In general, gain- and loss-of-function experiments suggest a positive relation between *Hox* genes and digit number [72, 74] that is also indicated by the ectopic anterior up-regulation

the *cis*-regulatory limb-bud enhancer region of the *SHH* gene [64].

60], suggesting the existence of a SHH-independent prepatterning.

the processing of Gli3 to its repressor form (Gli3R) [65, 66].

cation of their A-P identities [53 - 56].

**1.7. Hox genes and digit identity**

clarified [24, 57 - 61].

132 New Discoveries in Embryology

The maintenance and propagation of *SHH* expression requires AER-derived FGF signalling as part of a positive epithelial-mesenchymal (E-M) feedback loop operating between the ZPA and the AER [82, 83] (Figure 1E). The BMP antagonist Gremlin1 (Grem1) was identified as a crucial mesenchymal component in this E-M feedback signalling system [59, 66, 84]. Grem1 is required to up-regulate AER-FGF signalling and to establish SHH/Grem1/FGF E-M feedback signalling. In *Grem1*-null limb buds, the establishment of this E-M feedback signalling loop is interrupted, and this in turn interferes with specification and expansion of the distal compart‐ ment (zeugopod and autopod) [59, 84].

### **1.9. Dorsal-ventral axis**

Dorsal-ventral (D-V) patterning is mainly organized via signalling by Wnt7a, a diffusible molecule of the Wnt-family expressed in the dorsal ectoderm. Wnt7a is both necessary and sufficient to dorsalize the limb, indeed the loss of *Wnt7a* causes the dorsal side of limbs to acquire a ventral side identity, accompanied by missing posterior digits. Wnt7a is required to maintain expression of *SHH,* explaining the digit loss. Restoring the Wnt7a signal rescues both of these defects [85, 86].

Wnt7a induces the expression of *Lmx1*, coding for a Lim-family homeodomain-containing transcription factor. Lmx-1 is involved in dorsalization of the limb, which was shown by deleting *Lmx1* in mice: *Lmx1* null embryos produced ventral skin on both sides of their paws [87, 88]. There are other factors known to control the D-V patterning; on the ventral side the transcription factor-coding *Engrailed-1* gene, exclusively expressed in the ventral ectoderm, has been shown to repress the dorsalizing effect of Wnt7a in this territory [89] (Figure 1E).

### **2. Distal Limb Malformations in Human**

Congenital limb malformations are relatively common, and are genetically and clinically heterogeneous, with a diverse spectrum in their epidemiology, aetiology and anatomy. They are often difficult to diagnose and categorize, because of their complex phenotypes and their association with other malformations and clinical symptoms. Many etiological factors have been suggested for limb anomalies, including inheritance of mutated genes, teratogenic drugs, environmental chemicals, ionizing radiation (atomic weapons, radioiodine and radiation therapy), infections, metabolic imbalance (e.g., maternal diabetes), or mechanical factors like amniotic band syndrome. With the advent of functional genetics, molecular pathways centred on disease genes are being unravelled.

A wide set of human congenital limb malformations can be attributed to defects in P-D development. In this chapter, we will attempt to link known disease-causing genes with their known or presumed function in the maintenance of the AER. We will focus on the genes for which more functional data are available: namely *Dlx5*, *FGF8*, *p63* and *Wnts*. These genes are co-expressed in the AER cells of the mouse limb [90] as well as in the fins of the zebrafish embryos [91, 92] and are known or proposed diseases-genes for the SHFM and EEC congenital limb malformations.

P-D defect refer to absence or hypoplasia of distal structure of the limb with more or less normal proximal structures. The spectrum of P-D limb reduction anomalies ranges from very mild disorders, such as syndactyly, to very severe forms, such as phocomelia or amelia. The most frequent congenital limb malformations are syndactylies, characterized by the fusion of the soft tissues of fingers and toes with or without bone fusion. Syndactylies are due to the lack of apoptosis in the interdigital mesenchyme and may also occur isolated or with other symptoms in a syndrome [93].

Polydactylies are distinguished by the appearance of supernumerary digits or parts of them, which may be present as a complete duplication of a whole limb or as a duplication of single digits [94]. Pre-axial polydactyly with extra digits located on the side of the hand or of the thumb or postaxial polydactyly where the extra digit is found on the side of the hand or foot of the fifth digit are common isolated limb malformation traits. On molecular level, many forms of polydactyly have been shown to be more or less directly linked to the SHH signal trans‐ duction pathway, which play a major role in A-P patterning of the limb [95, 96].

Brachydactylies are defined by shortened digits and are classified on an anatomic and genetic background into five groups from A to E [93]. Isolated brachydactylies are often inherited in an autosomal dominant manner and are characterized by a high degree of phenotypic variability. Type-B brachydactylies are associated to mutation in the *Ror2* gene, and *Ror2* mutations are also associated with the Robinow syndrome in which brachydactyly is a common feature [97 - 101].

A severe P-D arrest of the developing limb bud gives rise to phocomelia, characterized by undeveloped limbs [102]. Usually the upper limbs are not fully formed and sections of the "hands and arms may be missing". Short arm bones, fused fingers and missing thumbs will often occur. Legs and feet are also affected. Individuals with phocomelia will often lack thigh bones, and the hands or feet may be abnormally small or appear as stumps due to their close attachment to the body. Phocomelia is a known negative effect of the administration of thalidomide to pregnant women, in use in the late 1950s/early 1960s, to treat morning sickness, although the mechanism of action of this teratogen remains controversial [103, 104].

Failure of formation of limb buds gives rise to amelia, the complete absence of one or more limbs. The most severe form of amelia is the tetra-amelia, characterized by the absence of all four limbs, associated with craniofacial, pulmonary and urogenital defects. This autosomal recessive disorder has been linked to mutations of the *WNT3* gene (see the following section).

### **2.1. SHFM and EEC**

deleting *Lmx1* in mice: *Lmx1* null embryos produced ventral skin on both sides of their paws [87, 88]. There are other factors known to control the D-V patterning; on the ventral side the transcription factor-coding *Engrailed-1* gene, exclusively expressed in the ventral ectoderm, has been shown to repress the dorsalizing effect of Wnt7a in this territory [89] (Figure 1E).

Congenital limb malformations are relatively common, and are genetically and clinically heterogeneous, with a diverse spectrum in their epidemiology, aetiology and anatomy. They are often difficult to diagnose and categorize, because of their complex phenotypes and their association with other malformations and clinical symptoms. Many etiological factors have been suggested for limb anomalies, including inheritance of mutated genes, teratogenic drugs, environmental chemicals, ionizing radiation (atomic weapons, radioiodine and radiation therapy), infections, metabolic imbalance (e.g., maternal diabetes), or mechanical factors like amniotic band syndrome. With the advent of functional genetics, molecular pathways centred

A wide set of human congenital limb malformations can be attributed to defects in P-D development. In this chapter, we will attempt to link known disease-causing genes with their known or presumed function in the maintenance of the AER. We will focus on the genes for which more functional data are available: namely *Dlx5*, *FGF8*, *p63* and *Wnts*. These genes are co-expressed in the AER cells of the mouse limb [90] as well as in the fins of the zebrafish embryos [91, 92] and are known or proposed diseases-genes for the SHFM and EEC congenital

P-D defect refer to absence or hypoplasia of distal structure of the limb with more or less normal proximal structures. The spectrum of P-D limb reduction anomalies ranges from very mild disorders, such as syndactyly, to very severe forms, such as phocomelia or amelia. The most frequent congenital limb malformations are syndactylies, characterized by the fusion of the soft tissues of fingers and toes with or without bone fusion. Syndactylies are due to the lack of apoptosis in the interdigital mesenchyme and may also occur isolated or with other symptoms

Polydactylies are distinguished by the appearance of supernumerary digits or parts of them, which may be present as a complete duplication of a whole limb or as a duplication of single digits [94]. Pre-axial polydactyly with extra digits located on the side of the hand or of the thumb or postaxial polydactyly where the extra digit is found on the side of the hand or foot of the fifth digit are common isolated limb malformation traits. On molecular level, many forms of polydactyly have been shown to be more or less directly linked to the SHH signal trans‐

Brachydactylies are defined by shortened digits and are classified on an anatomic and genetic background into five groups from A to E [93]. Isolated brachydactylies are often inherited in an autosomal dominant manner and are characterized by a high degree of phenotypic

duction pathway, which play a major role in A-P patterning of the limb [95, 96].

**2. Distal Limb Malformations in Human**

on disease genes are being unravelled.

limb malformations.

134 New Discoveries in Embryology

in a syndrome [93].

SHFM, also known as ectrodactyly or lobster-claw malformation, is a congenital defect affecting predominantly the central rays of hands and/or feet. It may manifest either as an isolated trait or as part of syndromic conditions comprising other developmental disorders [105]. SHFM occurs with the incidence of about 1 in 18,000 live born infants and accounts for 8–17% of all limb malformations [106, 107]. SHFM is clinically heterogeneous, ranging from a relatively mild defect, such as hypoplasia of a single phalanx or syndactyly, to aplasia of one or more central digits (i.e., classical cleft, also known as lobster-claw anomaly).

Inter-individual and intra-familial variability of the SHFM is very high. Furthermore, variable expressivity of this feature can be so significant, that a different pattern of anomaly is seen in each limb of the same individual patient [93]. SHFM is mostly sporadic, although familial forms are known: in these cases an autosomal dominant transmission with reduced penetrance is the most common mode, but autosomal recessive and X-linked forms have been reported.

SHFM has been linked to (at least) six distinct loci [106] (Table 1). SHFM-I (MIM #183600) is the most frequent type and is linked to mutations and/or deletions/rearrangements of the *DLX5;DLX6* bigenic locus. Deletions, inversions and rearrangements affecting chromosome 7q21 have long been reported [108 - 112]. The smallest region of overlapping deletions encompasses severalothergenes inadditionto*DLX5*, and*DLX6*: as*DYNC1l1,SLC25A13,DSS1,* butonly*Dlx5*and*Dlx6*havebeenshowntobespecificallyexpressedintheAERofthedeveloping mouselimbs[113-115].Recently,apointmutationintheDNA-bindingdomainofDLX5(Q178P) has been reported in a SHFM-I family with a recessive transmission, co-segregating with the limb malformations [116]. In the mouse, the combined disruption of *Dlx5;Dlx6* leads to an ectrodactyly phenotype affecting the HLs [114, 115], fully confirming that the human ortho‐ logs *DLX5* (and presumably *DLX6)* are the disease genes for this malformation. Interestingly, SHFM type-V (MIM #606708) is linked to deletions of a region on chromosome 2 encompass‐

ing the *HOXD* gene cluster, near *DLX1* and *DLX2* [117 - 119]. Although no clear evidence for the involvement of *DLX1* and *DLX2* in this malformation is available, it is tempting to imag‐ ine that similarto *DLX5*, misregulated expression of *DLX1/2* in the human embryonic limb bud could be the molecular mechanism leading to SHFM type-V.

SHFM type-III (MIM# 600095) is associated with duplications/rearrangements around the *DACTYLIN* (*FBXW4*) locus on chromosome 10q and the synthetic one in mice [120, 121]. The genomic lesion involves the *DACTYLIN, LBX1*, *βTRCP* and other more distant genes, but none of these is directly disrupted by the rearrangement and no point mutation has been reported. Interestingly, the *FGF8* locus is located in the proximity of the rearrangement breakpoints [122], and considering its importance for P-D limb development, it represents a valid candidate SHFM type-III disease gene.

Mutations of p63 are associated to SHFM type-IV (MIM #605289), a condition in which ectrodactyly appears as an isolated non-syndromic disorder linked to mutations or chromo‐ somal anomalies in the DBD or in the C-terminal domain of p63*α* [123 - 125]. The *α* tail of p63 contains a sumoylation site, inactivated by *p63* mutations found in SHFM-IV (E639X). Sumoy‐ lation can modulate p63 half-life [126] and naturally occurring mutated p63 proteins often display altered stability, suggesting that the final effect of the mutations could be the persis‐ tence of the mutated protein and consequent misexpression of p63 targets [125, 127, 128]. p63 mutations also cause the ectodermal dysplasia-ectrodactyly-cleft lip/palate syndrome type-III (EEC-3) syndrome (MIM #604292) [129] in which ectrodactyly is a common feature.

SHFM type-VI (MIM #225300) is the only autosomal recessive form of this malformation, and is due to homozygous point mutations of the *WNT10B* gene [130 - 133]. Finally, the X-linked SHFM type-II form (MIM #313350) has been mapped to chromosome Xq26.3 [134] but no disease gene has yet been identified.


Novel Cellular and Molecular Interactions During Limb Development, Revealed from Studies on the Split Hand… http://dx.doi.org/10.5772/60402 137


SHFM – Split Hand/Foot Malformation, EEC – Ectrodactyly-Ectodermal dysplasia-Cleft lip/palate, ADULT – Acro-Dermato-Ungual-Lacrimal-Tooth syndrome, LADD – Lacrimo-Auriculo-Dento-Digital syndrome,

CHARGE syndrome (Coloboma of the eye, Heart defects, atresia of the nasal choanae, Retardation of growth and/or development, Genital and/or urinary abnormalities, Ear abnormalities and deafness), VATER association - vertebral anomalies, anal atresia, cardiovascular anomalies, tracheoesophageal fistula, renal and/or radial anomalies, limb defects.

**Table 1.** Genetic alterations and SHFM-related phenotypes

### **2.2. p63-Dlx5;Dlx6 Regulation**

ing the *HOXD* gene cluster, near *DLX1* and *DLX2* [117 - 119]. Although no clear evidence for the involvement of *DLX1* and *DLX2* in this malformation is available, it is tempting to imag‐ ine that similarto *DLX5*, misregulated expression of *DLX1/2* in the human embryonic limb bud

SHFM type-III (MIM# 600095) is associated with duplications/rearrangements around the *DACTYLIN* (*FBXW4*) locus on chromosome 10q and the synthetic one in mice [120, 121]. The genomic lesion involves the *DACTYLIN, LBX1*, *βTRCP* and other more distant genes, but none of these is directly disrupted by the rearrangement and no point mutation has been reported. Interestingly, the *FGF8* locus is located in the proximity of the rearrangement breakpoints [122], and considering its importance for P-D limb development, it represents a valid candidate

Mutations of p63 are associated to SHFM type-IV (MIM #605289), a condition in which ectrodactyly appears as an isolated non-syndromic disorder linked to mutations or chromo‐ somal anomalies in the DBD or in the C-terminal domain of p63*α* [123 - 125]. The *α* tail of p63 contains a sumoylation site, inactivated by *p63* mutations found in SHFM-IV (E639X). Sumoy‐ lation can modulate p63 half-life [126] and naturally occurring mutated p63 proteins often display altered stability, suggesting that the final effect of the mutations could be the persis‐ tence of the mutated protein and consequent misexpression of p63 targets [125, 127, 128]. p63 mutations also cause the ectodermal dysplasia-ectrodactyly-cleft lip/palate syndrome type-III

(EEC-3) syndrome (MIM #604292) [129] in which ectrodactyly is a common feature.

**Case reported Inheritancepattern Limb**

dominant Autosomal recessive

1 family Autosomal

**SHFM-II** Xq26 1 family X-linked recessive SHFM,

**phenotypes**

syndactyly, metacarpalhypo plasia, phalangeal hypoplasia

**Additional phenotypes**

retardation, sensorineural deafness

SHFM EEC, mental

**References**

Crackower et al. (1996) Marinoni et al. (1995) Shamseldin et al. (2012)

> Faiyaz ul Haque et al. (2005)

SHFM type-VI (MIM #225300) is the only autosomal recessive form of this malformation, and is due to homozygous point mutations of the *WNT10B* gene [130 - 133]. Finally, the X-linked SHFM type-II form (MIM #313350) has been mapped to chromosome Xq26.3 [134] but no

could be the molecular mechanism leading to SHFM type-V.

SHFM type-III disease gene.

136 New Discoveries in Embryology

disease gene has yet been identified.

**Chromosome/gene affected**

7q21.3-q22.1*DLX5* mutation

**SHFM-I** Rearrangements

**SHFM locus**

> SHFM type-IV and EEC are caused by mutations in the *p63* gene, which codes for a highly conserved transcription factor related to the *p53* and *p73* tumour-suppressor genes [129, 135 - 137]. A common feature of these disorders is ectodermal dysplasia, consisting in abnormal maturation and stratification of the skin and abnormal development of hairs, teeth, nails, exocrine glands and cornea. The other two consistent features of *p63*-linked disorders are cleft lip/palate and ectrodactyly.

> p63 is expressed in the basal or progenitor layers of many epithelial tissues [138, 139], and is able to promote the epithelial stratification program typical of the mammalian skin, as well as to control proliferation and exit from the cell cycle of epidermal stem cells. For these activities

p63 has been proposed as a master regulator of epidermal stem cell maintenance, proliferation and stratification [140]. The *TP63* gene is translated into ten protein isoforms [141]: the transactivating (TA) isoforms, closely resembling p53, and the delta-N (ΔN) isoforms, devoid of the TA-domain-1 (TA1). Although the TA isoforms were initially thought to be the ones to possess transcriptional regulatory functions, it has well been established that the ΔN isoforms can activate transcription of a distinct set of target genes via a second TA-domain-2 (TA2) [142]. Five TA and ΔN isoforms are generated by two transcripts which are subjected to alternative splicing, thus the final protein products differ at the carboxyl termini (*α*, *β*, *γ, δ* and *ε*). In addition to TA1 or TA2 domain, the p63 proteins contain a DNA-binding domain (DBD) and an oligomerization domain (OD). The α-isoforms (either TA or ∆N) also contain a sterile alpha motif (SAM) domain, a protein-protein interaction module found in developmentally relevant proteins [143, 144]. Recent studies have identified a transcriptional inhibitory (TI) domain located between the SAM domain and the C-terminus of p63*α*; this domain is believed to be responsible for the lower transcriptional activity of TAp63*α* compared to the -*β* and the -*γ* isoforms [145]. ∆Np63α is the most expressed isoform in the embryonic ectoderm.

Attempts to establish genotype–phenotype correlations are hampered by the variable clinical expressivity observed within families: SHFM type-IV and the EEC syndromes are due to mutations in the DNA-binding domain of p63 [129]. In these cases, all p63 isoforms are affected by the mutations. DBD mutants usually act as dominant-negative effectors and render the WT protein unable to bind DNA [129], explaining the dominant transmission of EEC. In contrast, the Hay Wells or ankyloblepharon-ectodermal dysplasia-cleft palate syndrome (AEC, MIM #106260) manifests with normal limbs but severe skin defects, and is typically associated with heterozygous missense mutations in the SAM domain of p63. The acro-dermato-ungual lacrimal tooth (ADULT, MIM #103285) syndrome is associated with a specific gain-of-function mutation R298Q/G in exon 8, affecting the DNA-binding domain of p63. Finally, both limbmammary syndrome (LMS, MIM #603543), very similar to ADULT and EEC syndromes, and Rapp-Hodgkin syndrome (RHS, MIM #129400), resembling AEC, are due to p63 mutations. *p63* mutations causing EEC are usually not found in AEC, LMS and SHFM [146 - 148].

Mice null for *p63* have been generated by two groups independently [136, 137]; at birth these mice show severe defects affecting their skin, limb and craniofacial skeleton, teeth, hair, and mammary glands. Specifically, the skin appears thin, mostly single layered and translucent, unable to prevent water loss. The HLs fail to form altogether, while the FLs are severely truncated and lack most of their distal skeletal elements. The altered phenotypes observed in these mutant mice are a direct consequence of altered cellular properties affecting the same tissues and organs as in EEC patients [90, 136, 137, 149, 150]. While in the null embryos the *p63* protein is missing altogether (i.e., both the TA and ΔN isoforms), in EEC, AEC, LMS, and SHFM-IV patients the mutated p63 protein coexists with half of the normal dose of the wildtype protein. To better model the disease, the group of Dr. A. Mills (CSHL, USA) has generated mice bearing the *R279H* mutation (found in EEC patients). Homozygous embryos and newborn animals show a global phenotype similar, but not identical, to that of *p63-/-* [90], consisting in the absence of the HL, severely truncated FL, a thin translucent skin and cranio‐ facial and palatal defects. The HL defects in both the *p63* null and in the *p63-R279H* homozy‐ gous embryos are evident as early as E9.5, and are accompanied by loss of AER stratification and *FGF8* expression [90, 136, 137]. Interestingly, mild limb defects are observed in heterozy‐ gous p63-R279H mice, the mouse model closer to EEC.

p63 has been proposed as a master regulator of epidermal stem cell maintenance, proliferation and stratification [140]. The *TP63* gene is translated into ten protein isoforms [141]: the transactivating (TA) isoforms, closely resembling p53, and the delta-N (ΔN) isoforms, devoid of the TA-domain-1 (TA1). Although the TA isoforms were initially thought to be the ones to possess transcriptional regulatory functions, it has well been established that the ΔN isoforms can activate transcription of a distinct set of target genes via a second TA-domain-2 (TA2) [142]. Five TA and ΔN isoforms are generated by two transcripts which are subjected to alternative splicing, thus the final protein products differ at the carboxyl termini (*α*, *β*, *γ, δ* and *ε*). In addition to TA1 or TA2 domain, the p63 proteins contain a DNA-binding domain (DBD) and an oligomerization domain (OD). The α-isoforms (either TA or ∆N) also contain a sterile alpha motif (SAM) domain, a protein-protein interaction module found in developmentally relevant proteins [143, 144]. Recent studies have identified a transcriptional inhibitory (TI) domain located between the SAM domain and the C-terminus of p63*α*; this domain is believed to be responsible for the lower transcriptional activity of TAp63*α* compared to the -*β* and the -*γ*

138 New Discoveries in Embryology

isoforms [145]. ∆Np63α is the most expressed isoform in the embryonic ectoderm.

Attempts to establish genotype–phenotype correlations are hampered by the variable clinical expressivity observed within families: SHFM type-IV and the EEC syndromes are due to mutations in the DNA-binding domain of p63 [129]. In these cases, all p63 isoforms are affected by the mutations. DBD mutants usually act as dominant-negative effectors and render the WT protein unable to bind DNA [129], explaining the dominant transmission of EEC. In contrast, the Hay Wells or ankyloblepharon-ectodermal dysplasia-cleft palate syndrome (AEC, MIM #106260) manifests with normal limbs but severe skin defects, and is typically associated with heterozygous missense mutations in the SAM domain of p63. The acro-dermato-ungual lacrimal tooth (ADULT, MIM #103285) syndrome is associated with a specific gain-of-function mutation R298Q/G in exon 8, affecting the DNA-binding domain of p63. Finally, both limbmammary syndrome (LMS, MIM #603543), very similar to ADULT and EEC syndromes, and Rapp-Hodgkin syndrome (RHS, MIM #129400), resembling AEC, are due to p63 mutations. *p63* mutations causing EEC are usually not found in AEC, LMS and SHFM [146 - 148].

Mice null for *p63* have been generated by two groups independently [136, 137]; at birth these mice show severe defects affecting their skin, limb and craniofacial skeleton, teeth, hair, and mammary glands. Specifically, the skin appears thin, mostly single layered and translucent, unable to prevent water loss. The HLs fail to form altogether, while the FLs are severely truncated and lack most of their distal skeletal elements. The altered phenotypes observed in these mutant mice are a direct consequence of altered cellular properties affecting the same tissues and organs as in EEC patients [90, 136, 137, 149, 150]. While in the null embryos the *p63* protein is missing altogether (i.e., both the TA and ΔN isoforms), in EEC, AEC, LMS, and SHFM-IV patients the mutated p63 protein coexists with half of the normal dose of the wildtype protein. To better model the disease, the group of Dr. A. Mills (CSHL, USA) has generated mice bearing the *R279H* mutation (found in EEC patients). Homozygous embryos and newborn animals show a global phenotype similar, but not identical, to that of *p63-/-* [90], consisting in the absence of the HL, severely truncated FL, a thin translucent skin and cranio‐ facial and palatal defects. The HL defects in both the *p63* null and in the *p63-R279H* homozy‐

Mouse models of the AEC syndrome have also been generated. Compared to EEC patients, AEC patients suffer of extreme skin fragility but have normal limbs. The AEC-mutant p63 proteins appear to act in a dominant-negative fashion. Mice were generated in which either ∆Np63a is down regulated in the skin, as a way to mimic the dominant negative action of mutant p63 in the AEC patients, or an AEC-mutant p63 was introduced [151 - 153]. These mice show severe skin erosion resembling the AEC phenotype, characterized by suprabas‐ al epidermal proliferation, delayed terminal differentiation and altered basement mem‐ brane.

p63 mutations cause limb congenital phenotypes due to their impact on the AER Animal models show p63 is essential for epidermal stratification [90, 139, 154 - 156]. Considering that the AER is one of the earliest attempt of the embryonic (non-neural) ectoderm to organize into a multilayered epithelial tissue [157], it is not surprising that in *p63* null or *p63 R279H* homozygous mice the AER is thinner and poorly stratified. Failure to main‐ tain AER stratification and FGF8 expression is a common feature of various ectrodactyly phenotypes [90, 157 - 159].

p63 is expected to control AER functions via transcriptional regulation of AER-restricted target genes [122, 154 - 156], indeed failure of AER stratification has also been associated with loss of expression of key morphogens for limb development, such as *FGF8* and *Dlx5;Dlx6* [122]. *Dlx* genes are the vertebrate homologs of Drosophila *Dll*, a homeodomain transcription factors required for the specification of distal limb elements in the fly embryo. In *Dll* hypomorphic mutant flies, a variable set of phenotypes is observed depending on the mutation, ranging from fusion of the distal segments (weak mutants) to complete loss of distal and medial leg segments (severe mutants) [160, 161]. In mice *Dlx* genes have a prominent role in specifying the mandible and maxillary skeletal structures [162, 163], as well as controlling normal limb development [114, 115]. Point mutations of *DLX5* have been found to co-segregate in familiar cases of SHFM [116] and the combined deletion of *Dlx5* and *Dlx6* leads to ectrodactyly of the HLs, that is, a true mouse model of SHFM type-I. There is evidence that until E11.5 the AER appears and functions normally, including a normal morphology and normal expression of AER markers (*FGF4, FGF8, Msx2*). On the contrary, at E11.5-E12 the expression of AER markers indicate that the central wedge of the AER fails to function. At about the same time the first signs of dysmorphology are visible. The expression of *FGF8* and other markers declines in the central sector of the limb bud, accompanied by loss of stratification in the same territory [158], while the expression of *SHH, Hand2* and *Tbx4* in the mutant limbs is unchanged. Considering the expression pattern of *Dlx* genes in the limb, the *Dlx5;Dlx6* null defect can be summarized as a cell-autonomous failure of the central AER to maintain and express morphogenetic molecules.

*p63* and Dlx proteins are co-expressed in the AER cells [90] as well as in the fins of the zebrafish embryos [91, 92]. In homozygous *p63* null and *p63EEC* (R279H) mutant limbs, the expression of four *Dlx* genes is strongly reduced. Functionally, when the *p63+/EEC* (heterozygous) mutation is combined with an incomplete loss of *Dlx5* and *Dlx6* alleles, severe limb phenotypes are observed, not present in mice with either mutation alone [90]. Together, there is a clear evidence for p63-Dlx regulatory cascade that is functional for distal limb development.

In vitro, ∆Np63α induces transcription from the *Dlx5* and *Dlx6* promoters, an activity abolished by EEC and SHFM-IV mutations, but not by AEC-associated mutations. ChIP analysis shows that p63 occupies the *Dlx5* and *Dlx6* promoters. This regulation takes place both at the proximal promoter level [90] and via a conserved *cis*-acting genomic element, located 250 kb centromeric to DLX5, an element that is specifically deleted in few SHFM patients [164]. Recent studies have identified a tissue-specific enhancer located within the coding exons 15 and 17 of the *Dync1/1* gene (near the *Dlx5;Dlx6* locus). This genomic element is characterized by an enhanc‐ er-type chromatin signature and physically interacts with a *DLX5/6* promoter region 900 kb distal to *DYNC1/1*, specifically in the limb [165, 166]. Using copy number variation (CNV) analyses in SHFM patients, combined with whole genome sequencing to map deletion and translocation breakpoints, a recent study shows that the *DYNC1I1* enhancers are also critical for limb development in humans [167]. An additional enhancer was identified in an intron of the *Slc25a13* locus, close to *Dlx5;Dlx6*, and was shown to drive *Dlx* gene expression in the otic vesicle, forebrain, branchial arch and limbs of the developing embryo [165, 166]. It is plausible that the SHFM phenotype linked with mutations in these enhancers is caused by an altered regulation of Dlx5/6 transcription.

### **2.3. Downstream of Dlx5;Dlx6**

Sp8 is a transcription factor of the Sp1 zinc-finger family [168, 169], homologous to the Drosophila *D-Sp1* gene that has been implicated in appendage development [170]. In the developing limbs *Sp8* shows restricted expression in the ectoderm, including the AER cells [168]. Mouse embryos null for *Sp8* show severe developmental defects affecting the distal portion of the limbs, associated with a strongly reduced expression of *FGF8* [168, 169, 171], *Sp8* is co-expressed with *Dlx* genes in the murine AER and forebrain [172] and appears in the top 1% of a list of conserved/co-expressed genes in microarray data [173]. Furthermore, conserved Dlx5 DNA-binding sites are predicted near the *Sp8* locus, thus *Sp8* is a likely direct *Dlx5* transcriptional target. A Dlx5-Sp8 transcriptional cascade could be upstream of *FGF8* expres‐ sion, which in turn maintains p63 protein stability.

A number of observations suggest that p63 and Dlx proteins may regulate *FGF8* expression by acting directly on the genomic region corresponding to the SHFM type-III critical region [120, 121]: indeed p63-binding sites are present within the region, as demonstrated by ChIPseq screening [164], and several predicted *Dlx5* binding sites cluster around the *FGF8* locus, in genomic regions conserved across mammalian species [158] (unpublished data).

Considering that the AER of *Dac* heterozygous embryos shows reduced *FGF8* expression and defective cell layering [159], and considering that rearrangements/duplications around *Dactylin* do not disrupt or interrupt the gene, and since *Dactylin* is ubiquitously expressed in mouse tissues, the role of *Dactylin* as disease-gene is doubtful [122]. In alternative, *FGF8* and components of the NFkB pathway might be the disease-genes. It is tempting to speculate that the complex duplication rearrangement modifies the position/organization of *cis-*acting control elements, which in turn affect expression of *FGF8* and components of the NFkB pathway. Thus SHFM type-III could be a genome-misorganization type of genetic disease.

In further support of this, genome-wide CNV analyses on a Chinese family with SHFM type-III revealed a micro-duplication on chromosome 10q24 co-segregating with the SHFM phenotype [174]. This novel duplication contains two discontinuous DNA fragments: the minimal centromeric duplicated segment involves *LBX1*, *POLL* and a disrupted *BTRC*; the telomeric duplication encompasses *DPCD* and part of *FBXW4*. No coding and splice-site mutations of candidate genes in the region were found. Interestingly, the second duplicated fragment comprises Dlx5 and p63 DNA binding sites [164].

Another pathway that links p63 and Dlx5 in the regulation of the *FGF8* locus implicates the gene *IKKα*, a direct transcriptional target of p63 relevant for ectoderm development and limb morphogenesis [175 - 177]. Interestingly, while mutations of *p63* and loss of *Dlx5;6* lead to a reduced *FGF8* expression in the AER, in *IKKα* mutant embryos the AER shows an increased *FGF8* expression [178], which nevertheless results in distal limb truncations and severe malformations.

From the above considerations, it appears that numerous players in the p63-Dlx5 cascade may contribute to regulate *FGF8* expression in the AER. The possibility that the *FGF8* locus is a common target of the p63 and the Dlx5 networks during limb development is in agreement with the well-known functions of FGF8 to sustain epithelial mesenchymal signalling and assure the timely generation of mesenchymal progenitors [36].

### **2.4. Post-translational p63 protein regulations**

are observed, not present in mice with either mutation alone [90]. Together, there is a clear evidence for p63-Dlx regulatory cascade that is functional for distal limb development.

In vitro, ∆Np63α induces transcription from the *Dlx5* and *Dlx6* promoters, an activity abolished by EEC and SHFM-IV mutations, but not by AEC-associated mutations. ChIP analysis shows that p63 occupies the *Dlx5* and *Dlx6* promoters. This regulation takes place both at the proximal promoter level [90] and via a conserved *cis*-acting genomic element, located 250 kb centromeric to DLX5, an element that is specifically deleted in few SHFM patients [164]. Recent studies have identified a tissue-specific enhancer located within the coding exons 15 and 17 of the *Dync1/1* gene (near the *Dlx5;Dlx6* locus). This genomic element is characterized by an enhanc‐ er-type chromatin signature and physically interacts with a *DLX5/6* promoter region 900 kb distal to *DYNC1/1*, specifically in the limb [165, 166]. Using copy number variation (CNV) analyses in SHFM patients, combined with whole genome sequencing to map deletion and translocation breakpoints, a recent study shows that the *DYNC1I1* enhancers are also critical for limb development in humans [167]. An additional enhancer was identified in an intron of the *Slc25a13* locus, close to *Dlx5;Dlx6*, and was shown to drive *Dlx* gene expression in the otic vesicle, forebrain, branchial arch and limbs of the developing embryo [165, 166]. It is plausible that the SHFM phenotype linked with mutations in these enhancers is caused by an altered

Sp8 is a transcription factor of the Sp1 zinc-finger family [168, 169], homologous to the Drosophila *D-Sp1* gene that has been implicated in appendage development [170]. In the developing limbs *Sp8* shows restricted expression in the ectoderm, including the AER cells [168]. Mouse embryos null for *Sp8* show severe developmental defects affecting the distal portion of the limbs, associated with a strongly reduced expression of *FGF8* [168, 169, 171], *Sp8* is co-expressed with *Dlx* genes in the murine AER and forebrain [172] and appears in the top 1% of a list of conserved/co-expressed genes in microarray data [173]. Furthermore, conserved Dlx5 DNA-binding sites are predicted near the *Sp8* locus, thus *Sp8* is a likely direct *Dlx5* transcriptional target. A Dlx5-Sp8 transcriptional cascade could be upstream of *FGF8* expres‐

A number of observations suggest that p63 and Dlx proteins may regulate *FGF8* expression by acting directly on the genomic region corresponding to the SHFM type-III critical region [120, 121]: indeed p63-binding sites are present within the region, as demonstrated by ChIPseq screening [164], and several predicted *Dlx5* binding sites cluster around the *FGF8* locus, in

Considering that the AER of *Dac* heterozygous embryos shows reduced *FGF8* expression and defective cell layering [159], and considering that rearrangements/duplications around *Dactylin* do not disrupt or interrupt the gene, and since *Dactylin* is ubiquitously expressed in mouse tissues, the role of *Dactylin* as disease-gene is doubtful [122]. In alternative, *FGF8* and components of the NFkB pathway might be the disease-genes. It is tempting to speculate that the complex duplication rearrangement modifies the position/organization of *cis-*acting

genomic regions conserved across mammalian species [158] (unpublished data).

regulation of Dlx5/6 transcription.

sion, which in turn maintains p63 protein stability.

**2.3. Downstream of Dlx5;Dlx6**

140 New Discoveries in Embryology

Several biochemical observations indicate that the ∆N- and TAp63 proteins are tightly regulated at post-translational level, via protein modification (phosphorylation, sumoylation and ubiquitination) and protein-protein interactions [126, 158, 179, 180]. Such modifications modulate the stability of the p63 protein, regulate its transcriptional activity and ultimately modulate its ability to orchestrate the timing of exit from the cell cycle and the dynamic of stratification of mammalian ectoderm [156, 181, 182].

Among the interacting or modifying proteins, MDM2 and p53 have been previously recog‐ nized [179, 180]. Recently we have shown that the peptidyl-prolyl *cis/trans* isomerase NIMAinteracting-1, Pin1, is a regulator of ∆Np63α protein stability, inducing its proteasomemediated degradation [158] resulting in diminished transcription of two p63 targets [183] (Figure 2).

Another modification is acetylation, catalyzed by histone acetyl-transferase on lysine residues, and known to finely regulate p53 and p73 stability and transcriptional activity [184 - 189]. p73 is acetylated by p300 on lysine residues in the DBD and Oligomerization Domain [190] enhancing p73 ability to bind and activate proapototic target genes [191]. The p73-p300 interaction requires the prolyl-isomerase Pin1, which induces conformational changes following phosphorylation by the tyrosine kinase c-Abl [192]. Acetylation of p53 correlates with its stabilization and activation by antagonizing the activity of the MDM2 ubiquitin-ligase. It is interesting to note that a naturally occurring p63 mutation found in SHFM type-IV patients

#### Conte et al.

**Figure 2.** Schematic representation of the molecules and their interactions that regulate the stability of ΔNp63 during the AER stratification. p63 regulates its own stability via to the expression of *FGF8*; this pathway includes the *Dlx5/ Dlx6* disease genes. Fgf8 stabilizes p63 by counteracting the activity of Pin1 to induce proteasome-mediated degrada‐ tion of ΔNp63α. Novel results indicate that FGF8 activates a signalling cascade leading to activation of c-Abl that pro‐ motes phosphorylation of ΔNp63α on tyrosine residues. This phosphorylation event is required for the interaction of ΔNp63α with the p300 acetyl-transferase, which modulated ΔNp63α stabilization and transcriptional activity. Al‐ though only shown in vitro, we speculate that regulation may also occur in the AER cells (dotted line).

changes lysine 193 into glutamic acid (K193E) [125, 146, 147, 193]. Our unpublished data show that ∆Np63α is acetylated by p300 on the K193 residue, and that the K193E mutation prevents this modification (Guerrini and Restelli, unpublished) (Figure 2).

### **2.5. Emerging roles of FGF8**

Expression of *FGF8* is strongly reduced in the AER of the *p63* null, *R279H p63* mutant, and *Dlx5;Dlx6* mutant embryos [90, 115] as well as several other mouse strains with distal limb defects. The AER of these mutants appears poorly stratified. Thus, loss of AER stratification and reduced *FGF8* expression are a common theme during the onset of this specific class of malformations. The link between *FGF8* expression and AER stratification is not totally clear. When *FGFR2* gene is deleted in the AER cells, via conditional genetics, the AER loses stratifi‐ cation as well as Fgf*8* expression. In this case, the AER cells cannot respond to (AER-derived?) FGFs [194] and it can be concluded that AER-expressed FGFs are needed for AER maintenance, apparently in an autocrine fashion.

An emerging role of FGF8 is the control of p63 stability in the AER cells. The AER of *Dlx5/ Dlx6* null mice shows poor stratification as well as reduced *FGF8* expression, similar to what is seen in *p63* mutant mice. We have documented that *Dlx5;Dlx6* are transcriptional targets of p63, and that in turn *FGF8* is a target of Dlx5. As already said, ∆Np63α protein stability is negatively regulated by the interaction with Pin1, via proteasome-mediated degradation. Recently we have shown that FGF8 counteracts Pin1-∆Np63α interaction, thus indicating that FGF8 participates in a feedback loop which involves the p63-Dlx5 cascade [158] (Figure 2).

p63 stability might also be regulated by another post-translational modification, namely acetylation by the p300 histone acetylase. c-Abl is a key regulator of the p53 family members and is known to be activated by treatment with FGF2 [192, 195 - 198]. Recently we have collected new data showing that FGF8 is able to stabilize ∆Np63α also via a novel pathway that requires the c-Abl tyrosine kinase and the protein acetylation by p300 (Guerrini and Restelli, unpublished). Thus, Dlx5, p63, Pin1, p300 and FGF8 participate in a time- and locationrestricted regulatory loop that seems to be able to self-maintain and whose normal functioning is necessary for AER stratification, hence for normal extension and patterning of the limb buds. These results shed new light on the general molecular mechanisms at the basis of the SHFM and EEC limb malformations (Figure 2).

In an interesting set of experiments using cultured embryonic limbs, it was recently shown that the FGF/MAPK pathway establishes a high-distal to low-proximal gradient that controls the migration velocity of mesenchymal cells [199]. These cell movements enable continuous rearrangement of the cells at the distal tip of the limb bud. The effect of FGF/MAPK signalling emanating from the AER is different than the effect induced by Wnt5a in the limb bud. While Wnt5a induces directional movement of cells, FGF8 acts to induce rapid, yet disorganized, movements. Ultimately, the activity of both Wnt5a and FGF results in distal elongation (Figure 3). These observations suggest that FGF8 acts by inducing random movements, but with a higher velocity as cells move close to the source. A study proposes that the FGF pathway drives tail-bud elongation in the chick embryo by promoting random cell movements [200]. Accord‐ ing to these authors FGF creates a gradient of cell motility and that the tail bud elongates by mass action of random cell movement at the posterior end of the embryo. Although this data indicate a similar mode of FGF action, cells in the limb bud additionally undergo oriented processes of cell division and directional movements under the influence of Wnt5a. This study indicates that it is the combined action of non-canonical WNT and FGF that integrates orientation and movement, consequently driving limb-bud elongation and thereby establish‐ ing a progenitor field of the proper dimensions for the subsequent patterning and morpho‐ genesis of limb anatomy.

### **2.6. Wnt signalling and limb development**

changes lysine 193 into glutamic acid (K193E) [125, 146, 147, 193]. Our unpublished data show that ∆Np63α is acetylated by p300 on the K193 residue, and that the K193E mutation prevents

though only shown in vitro, we speculate that regulation may also occur in the AER cells (dotted line).

**Figure 2.** Schematic representation of the molecules and their interactions that regulate the stability of ΔNp63 during the AER stratification. p63 regulates its own stability via to the expression of *FGF8*; this pathway includes the *Dlx5/ Dlx6* disease genes. Fgf8 stabilizes p63 by counteracting the activity of Pin1 to induce proteasome-mediated degrada‐ tion of ΔNp63α. Novel results indicate that FGF8 activates a signalling cascade leading to activation of c-Abl that pro‐ motes phosphorylation of ΔNp63α on tyrosine residues. This phosphorylation event is required for the interaction of ΔNp63α with the p300 acetyl-transferase, which modulated ΔNp63α stabilization and transcriptional activity. Al‐

Expression of *FGF8* is strongly reduced in the AER of the *p63* null, *R279H p63* mutant, and *Dlx5;Dlx6* mutant embryos [90, 115] as well as several other mouse strains with distal limb defects. The AER of these mutants appears poorly stratified. Thus, loss of AER stratification and reduced *FGF8* expression are a common theme during the onset of this specific class of malformations. The link between *FGF8* expression and AER stratification is not totally clear. When *FGFR2* gene is deleted in the AER cells, via conditional genetics, the AER loses stratifi‐ cation as well as Fgf*8* expression. In this case, the AER cells cannot respond to (AER-derived?) FGFs [194] and it can be concluded that AER-expressed FGFs are needed for AER maintenance,

this modification (Guerrini and Restelli, unpublished) (Figure 2).

**2.5. Emerging roles of FGF8**

Conte et al.

142 New Discoveries in Embryology

apparently in an autocrine fashion.

Wnt molecules are the vertebrate homologs of the Drosophila *wingless* gene, required for wing development. Wnt molecules are involved in all aspects of embryonic development, from patterning to morphogenesis and cell-tissue interactions [201 - 203].

Several members of the Wnt family of ligands are expressed in the ectoderm and mesenchyme of the developing limbs. At early stages, *Wnt8c* and *Wnt2b* are transiently expressed in the

**Figure 3.** Schematic representation of the mesenchymal cells orientation and organization in the early limb bud. These cellular events are regulated by the combined activities of the WNT and FGF pathways. Wnt5A/Jnk/PCP pathway is necessary for the proper orientation of cell movements and cell division. In contrast, the FGF/MAPK signaling path‐ way, emanating from the AER establishes a gradient of cell velocity. The combination of oriented cell divisions and movements drives the P-D extension of the limb bud necessary for subsequent morphogenesis.

LPM and participate in the initiation of HL and FL outgrowth, respectively [43]. At later stages, *Wnt3/Wnt3a* and *Wnt5a* are expressed by the AER cells while *Wnt7a* is expressed in the dorsal ectoderm.

Wnt ligands signal through the Frizzled (Fz) seven-pass trans-membrane receptors. In the "canonical" pathway, binding of Wnt ligands to Fz receptors represses the axin/glycogen synthase kinase-3β (GSK3β) complex, which in the absence of the ligand promotes the degradation of β-catenin via the ubiquitin pathway (reviewed in reference [204]). In Wntactivated cells, cytoplasmic β-catenin accumulates and translocates to the nucleus where, in conjunction with T cell-specific factor/lymphoid enhancer binding factor-1 (Tcf/Lef1) tran‐ scription factors, activates transcription of target genes.

A role of "canonical" Wnt signalling in limb development has long been recognized [205]. In the chick limb bud, the Wnt/β-catenin pathway is essential for the induction and maintenance of the AER. Indeed, ectopic expression of Wnts in the interflank region prior to limb outgrowth induces ectopic *FGF10* expression and limb formation. FGF10 subsequently induces *Wnt3a* expression in the AER, which in turn switches on the expression of Fgf8, again via the β-catenin pathway, and promotes AER formation [43, 44, 206].

In the chick embryo *Wnt3a* mediates the Wnt/β-catenin signalling required for establishment of the AER. In the mouse, old data indicate that mouse embryos lacking the Wnt/β-catenin pathway component *LRP6,* or simultaneously lacking *Lef1* and *Tcf1*, exhibit defective AER formation and limb defects, indicating that his pathway is indeed essential for AER formation [207, 208]. However, *Wnt3a* is not expressed in the limb ectoderm of the mouse embryo, and *Wnt3a* null embryos do not show limb defects [209, 210]. Instead, the closely related *Wnt3* gene is expressed in the limb ectoderm [210] and the conditional removal of *Wnt3* in the limb ectoderm leads to severe distal limb truncations and AER malfunction. Similar results were obtained by the conditional removal of β-catenin in the limb ectoderm [211], strongly sug‐ gesting that the murine *Wnt3* is functionally homologous to chick *Wnt3a*, and that a pre-AER active Wnt3/β-catenin pathway in the embryonic ectoderm is essential for AER formation and maintenance. Notably, homozygous mutations of *WNT3* in human are associated with a rare autosomal recessive congenital disorder known as tetra-amelia [212] characterized by the absence of all four limbs.

Wnt signalling has been implicated in removing "excess" tissue by programmed cell death and sculpting the limb shape. Indeed, the ability of BMP4 to induce cell death in the developing limb appears to be mediated by Dkk1 [213]. Loss of function of Dkk1 in mice results in the downregulation of *Msx1*, a component of the cell death pathway, in the anterior and posterior necrotic zones and the interdigital mesenchyme, whilst gain of Dkk1 function in chicks causes excessive cell death via activation of the c-jun pathway [213, 214]. The decrease in cell death in the mouse mutants contributes to the polydactyly and fusion of digits that occur in *Dkk1* mutant mice [214]. In addition, Fz2, –3 and –4, and Dkk2, and –3, are expressed in the interdigit mesenchyme, suggesting that a fine balance of Wnt signalling controls cell death/survival in this region [215, 216].

### **2.7. Emerging role of Wnt5a and non-canonical signalling**

LPM and participate in the initiation of HL and FL outgrowth, respectively [43]. At later stages, *Wnt3/Wnt3a* and *Wnt5a* are expressed by the AER cells while *Wnt7a* is expressed in the dorsal

movements drives the P-D extension of the limb bud necessary for subsequent morphogenesis.

**Figure 3.** Schematic representation of the mesenchymal cells orientation and organization in the early limb bud. These cellular events are regulated by the combined activities of the WNT and FGF pathways. Wnt5A/Jnk/PCP pathway is necessary for the proper orientation of cell movements and cell division. In contrast, the FGF/MAPK signaling path‐ way, emanating from the AER establishes a gradient of cell velocity. The combination of oriented cell divisions and

Wnt ligands signal through the Frizzled (Fz) seven-pass trans-membrane receptors. In the "canonical" pathway, binding of Wnt ligands to Fz receptors represses the axin/glycogen synthase kinase-3β (GSK3β) complex, which in the absence of the ligand promotes the degradation of β-catenin via the ubiquitin pathway (reviewed in reference [204]). In Wntactivated cells, cytoplasmic β-catenin accumulates and translocates to the nucleus where, in conjunction with T cell-specific factor/lymphoid enhancer binding factor-1 (Tcf/Lef1) tran‐

A role of "canonical" Wnt signalling in limb development has long been recognized [205]. In the chick limb bud, the Wnt/β-catenin pathway is essential for the induction and maintenance of the AER. Indeed, ectopic expression of Wnts in the interflank region prior to limb outgrowth induces ectopic *FGF10* expression and limb formation. FGF10 subsequently induces *Wnt3a* expression in the AER, which in turn switches on the expression of Fgf8, again via the β-catenin

In the chick embryo *Wnt3a* mediates the Wnt/β-catenin signalling required for establishment of the AER. In the mouse, old data indicate that mouse embryos lacking the Wnt/β-catenin pathway component *LRP6,* or simultaneously lacking *Lef1* and *Tcf1*, exhibit defective AER formation and limb defects, indicating that his pathway is indeed essential for AER formation [207, 208]. However, *Wnt3a* is not expressed in the limb ectoderm of the mouse embryo, and *Wnt3a* null embryos do not show limb defects [209, 210]. Instead, the closely related *Wnt3* gene

scription factors, activates transcription of target genes.

pathway, and promotes AER formation [43, 44, 206].

ectoderm.

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144 New Discoveries in Embryology

Wnt ligands can also activate two other branches of "non-canonical" pathways; one of these is known as the planar cell polarity (PCP) pathway, involves Fz receptors and dishevelled (Dvl), which interact with a distinct set of "PCP proteins" such as Van Gogh (Vang) and Prickle [217]. The PCP pathway recruits the small GTPases Rho and Cdc42 and the c-Jun N-terminal kinase (JNK) [218 - 220]. Initially identified in Drosophila, PCP establishes cellular polarity in the plane of an epithelium, perpendicular to the apical-basal orientation [217]. Studies in vertebrate model systems, including Xenopus and zebrafish, indicate that the PCP pathway also regulates a morphogenetic process known as convergent extension (CE). CE was first demonstrated in gastrulating *Xenopus* embryos in which mesodermal cells underwent mediolaterally oriented intercalation, leading to concomitant tissue lengthening and narrowing [221]. Imaging experiments in zebrafish indicate that, in addition to polarized cell intercalation, the PCP pathway also regulates directional cell migration and oriented cell division underlying CE [222 - 225]. A second branch of "non-canonical" Wnt transduction pathways leads to the release of intracellular Ca2+ and the activation of protein kinase C (PKC) and Ca2+/Calmodu‐ lin-dependent Kinase-II (CamKII) [226 - 229]. The choice of the pathway being activated by a Wnt ligand appears to depend mostly on the receptor profile and on the intracellular signalling molecules available in a given cell type, and little on the Wnt ligand itself.

A role of "non-canonical" Wnt signalling during limb development has been recognized, although the cellular and molecular mechanisms are not fully clarified. The vertebrate *Wnt5a* gene, the homolog to Drosophila *Dwnt-5* gene essential for limb and appendage development, is considered the typical non-canonical Wnt, involved in the establishment of PCP [230 - 232]. Wnt5a together with Wnt11 mediates the activation of PCP during the CE in frogs and zebrafish [223, 233, 234], and during mouse limb development *Wnt5a* is expressed in a gradient from the AER to the proximal mesechymal cells, is regulated by FGF signalling from the AER and has been shown to inhibit β-catenin degradation [235, 236].

In addition to the PCP pathway, Wnt5a has been shown to activate at least two other noncanonical pathways. The first is known as the Wnt–Ca2+ pathway, in which Wnt5a stimulation induces Ca2+ release and subsequent activation of the Ca2+-sensitive kinases protein kinase C and Ca2+/calmodulin-dependent kinase [226, 227, 237, 238]. Over-expression of the core PCP proteins, Dvl and Pk, can also activate the Wnt–Ca2+ cascade in zebrafish and *Xenopus*, suggesting that the Wnt–Ca2+ and PCP pathways either overlap substantially or are compo‐ nents of the same signalling network [229, 239, 240]. Second, in mammalian cells Wnt5a has been shown to antagonize the canonical Wnt pathway by either promoting GSK3β-independ‐ ent β-catenin degradation [236] or by inhibiting β-catenin-dependent transcription [241].

Wnt5a can signal through different Fz receptors and co-receptors, but also via nonconventional tyrosine-kinase like receptors (Ror2 and Ryk) and can activate both the canonical and the non-canonical Wnt pathways [241, 242]. Activation of the canonical pathway entails the Lrp5 and Lrp6 co-receptors, which through cytoplasmic Dvl promote stabilization of β-catenin, its nuclear translocation and the activation of gene transcription [243, 244]. However, the distinct phenotypes observed between *Wnt3, β-catenin*, *Lrp5/Lrp6* and *Wnt5a* mutant mice [245] argues that during limb development Wnt5a does not signal through the β-catenin pathway [246].

In human, missense mutations in *WNT5A* have been documented in an autosomal dominant form of RRS (MIM #180700) [247 - 249] implying that a disruption of Wnt5a signalling may underlie both RRS and BDB1. Homozygous *ROR2* mutations have been linked to the autoso‐ mal recessive form of Robinow syndrome (or COVESDEM syndrome) (MIM #268310), while heterozygous *ROR2* mutations lead to type brachydactyly (MIM #113000) [250] and autosomal Dominant Brachydactyly type-B (BDB1, MIM #113000). BDB1 is the most severe form of brachydactyly and is characterized by loss of nails and varying number of phalanges [100, 251]. In contrast, RRS patients display broader skeletal dysplasia including mesomelic limb shortening and dwarfism, and may or may not display brachydactyly [97, 98, 101].

In mice, the disruption of *Wnt5a* results in short metacarpal elements, absence of phalanges and truncations of proximal elements [236, 252, 253]. The remaining limb skeletal elements are significantly shortened and the severity of the phenotype follows a gradient, with distal bones more affected than proximal ones, reminiscent of mesomelic limb shortening in RRS patients. Interestingly, the AER appears normally stratified and expresses *FGF8* [252]. Strong evidence of the involvement of the Wnt5-dependent pathways in limb development is derived from phenotypes of mice with loss of Wnt5a receptors. In addition to Fz receptors, Wnt5a binds to both Ryk and Ror2 receptors and regulates PCP by promoting Vangl2 stability during limb extension [242, 254]. Ryk and Ror2 are single-pass tyrosine-kinase type of receptors [241, 255]. Ror2 (an orphan tyrosine kinase receptor) activates JNK [256] and in Xenopus has been shown to interact with Wnt11 and Fz7 to regulate CE, suggesting that it may be part of the PCP pathway [257]. Upon binding with Wnt5a, Ror2 inhibits the canonical Wnt signalling. Fur‐ thermore, Ror2 also plays an important role in chondrogenesis. *Ror2* is selectively expressed in chondrocytes of cartilage anlagen, and is thus probably important in their initial growth and patterning. Mice mutant for *Ror2* and double mutants for *Ror1;Ror2* exhibit phenotypes that correspond to human RRS malformation, and bear similarities with the *Wnt5a* mutant mice [258, 259]. *Ryk* is another unconventional Wnt5a receptor, consisting in a single trans‐ membrane pass, catalytically-inactive, tyrosine kinase molecule. Ryk mutant mice show limb truncation similar to those of *Wnt5a* null embryos [260]. Finally, disruption of PCP signalling as Vangl2 in mice causes limb morphogenesis and skeletal defects and may underlie the Robinow syndrome and brachydactyly type B [261]. Together, these observations indicate that Wnt5a, Ryk and Ror2 molecules produce similar phenotypes when lost, for example, the disruption of components of the Wnt non-canonical pathway causes similar limb develop‐ mental defects.

### **2.8. Wnt5a controls aspects of PCP and CE in limb development**

zebrafish [223, 233, 234], and during mouse limb development *Wnt5a* is expressed in a gradient from the AER to the proximal mesechymal cells, is regulated by FGF signalling from the AER

In addition to the PCP pathway, Wnt5a has been shown to activate at least two other noncanonical pathways. The first is known as the Wnt–Ca2+ pathway, in which Wnt5a stimulation induces Ca2+ release and subsequent activation of the Ca2+-sensitive kinases protein kinase C and Ca2+/calmodulin-dependent kinase [226, 227, 237, 238]. Over-expression of the core PCP proteins, Dvl and Pk, can also activate the Wnt–Ca2+ cascade in zebrafish and *Xenopus*, suggesting that the Wnt–Ca2+ and PCP pathways either overlap substantially or are compo‐ nents of the same signalling network [229, 239, 240]. Second, in mammalian cells Wnt5a has been shown to antagonize the canonical Wnt pathway by either promoting GSK3β-independ‐ ent β-catenin degradation [236] or by inhibiting β-catenin-dependent transcription [241].

Wnt5a can signal through different Fz receptors and co-receptors, but also via nonconventional tyrosine-kinase like receptors (Ror2 and Ryk) and can activate both the canonical and the non-canonical Wnt pathways [241, 242]. Activation of the canonical pathway entails the Lrp5 and Lrp6 co-receptors, which through cytoplasmic Dvl promote stabilization of β-catenin, its nuclear translocation and the activation of gene transcription [243, 244]. However, the distinct phenotypes observed between *Wnt3, β-catenin*, *Lrp5/Lrp6* and *Wnt5a* mutant mice [245] argues that during limb development Wnt5a does not signal

In human, missense mutations in *WNT5A* have been documented in an autosomal dominant form of RRS (MIM #180700) [247 - 249] implying that a disruption of Wnt5a signalling may underlie both RRS and BDB1. Homozygous *ROR2* mutations have been linked to the autoso‐ mal recessive form of Robinow syndrome (or COVESDEM syndrome) (MIM #268310), while heterozygous *ROR2* mutations lead to type brachydactyly (MIM #113000) [250] and autosomal Dominant Brachydactyly type-B (BDB1, MIM #113000). BDB1 is the most severe form of brachydactyly and is characterized by loss of nails and varying number of phalanges [100, 251]. In contrast, RRS patients display broader skeletal dysplasia including mesomelic limb

In mice, the disruption of *Wnt5a* results in short metacarpal elements, absence of phalanges and truncations of proximal elements [236, 252, 253]. The remaining limb skeletal elements are significantly shortened and the severity of the phenotype follows a gradient, with distal bones more affected than proximal ones, reminiscent of mesomelic limb shortening in RRS patients. Interestingly, the AER appears normally stratified and expresses *FGF8* [252]. Strong evidence of the involvement of the Wnt5-dependent pathways in limb development is derived from phenotypes of mice with loss of Wnt5a receptors. In addition to Fz receptors, Wnt5a binds to both Ryk and Ror2 receptors and regulates PCP by promoting Vangl2 stability during limb extension [242, 254]. Ryk and Ror2 are single-pass tyrosine-kinase type of receptors [241, 255]. Ror2 (an orphan tyrosine kinase receptor) activates JNK [256] and in Xenopus has been shown to interact with Wnt11 and Fz7 to regulate CE, suggesting that it may be part of the PCP pathway [257]. Upon binding with Wnt5a, Ror2 inhibits the canonical Wnt signalling. Fur‐ thermore, Ror2 also plays an important role in chondrogenesis. *Ror2* is selectively expressed

shortening and dwarfism, and may or may not display brachydactyly [97, 98, 101].

and has been shown to inhibit β-catenin degradation [235, 236].

through the β-catenin pathway [246].

146 New Discoveries in Embryology

Recent data [199] shed light on the cellular functions of Wnt5a during limb development. Inspired by the CE process and the PCP pathway, first described in lower organisms, the authors examined the proliferative expansion and migration of mesenchymal cells of the mouse limb bud; in particular, they examined the orientation of cell division and movements in response to Wnt5a. The combination of oriented cell divisions and movements drives the P-D elongation of the limb bud necessary to set the stage for subsequent morphogenesis. They show that Wnt5a via the JNK PCP pathway is needed for the proper orientation of mesenchy‐ mal cell movements and cell division reminiscent of CE in *Xenopus* and zebrafish [222 - 225] (Figure 3).

Although these recent studies implicate Wnt5a in the oriented migration and cell division of the mesenchymal cells, little is known about the ectoderm cells, and in particular the AER cells, in which *Wnt5a* is expressed. It is conceivable that the AER cells might be the prime (autocrine) cellular target of Wnt5a, and that the acquisition of a correct planar orientation is a requisite for correct AER formation. Wnt5a and Dlx5 have an overlapping expression pattern, and the phenotype of *Wnt5a* null mice, although not identical, is quite similar to that of *Dlx5/Dlx6* mutant. One possibility is that a deregulation of *Wnt5a* expression, secondary to the disruption of *Dlx5;Dlx6* may underlie ectrodactyly of the *Dlx5;Dlx6* mutant embryos (Figure 4). In support of this, we have evidence that *Dlx* genes promote neuronal differentiation via *Wnt5a*, and that Dlx2 and Dlx5 physically occupy conserved genomic elements near the *Wnt5a* locus and activate its transcription [262]. This interaction and regulation is likely to occur also in the AER cells, a possibility that remains to be investigated.

### **2.9. Quantitative and dynamic gene expression in limb development**

An emerging theme in developmental biology is the importance of gene dosage and dynamic gene expression for correct morphogenesis [56]. Several *Dlx* (*1, 2, 3, 5* and *6*) and *FGF* (*4, 8, 9* and *17*) genes are co-expressed in the AER, and their expression is dynamically regulated, both with respect to time (embryonic age) and location (territory of expression). In addition, there is evidence that *Dlx* and *FGF* genes are functionally redundant, at least in part. For example, no limb phenotype is observed in mice null for only one *Dlx* gene, while ectrodactyly is

#### Conte et al.

**Figure 4.** Proposed model of regulation of the AER cell orientation. Dlx5 is known to regulate the transcription of both *FGF8* and *Wnt5a*. In turn, FGF8 is required for AER maintenance and stratification, via p63, while the function of Wnt5a for AER maintenance is still poorly known. Recent data of the regulation of orientation and velocity of mesen‐ chymal cells by, respectively, the Wnt5a/PCP and the FGF8-MAP-Erk pathways open the possibility that Wnt5a may regulate the orientation/motility of the AER cell and assure a correct stratification (dotted line).

observed in *Dlx2*;*Dlx5* null mice [161] and the ectrodactyly of *Dlx5;Dlx6* null mice is fully rescued by the re-expression of *Dlx5* alone [115]. Likewise, an increased severity of craniofacial phenotypes correlates with progressive loss of *Dlx* gene [263, 264]. All these are indications of gene-dosage effects between functionally redundant genes.

We propose that the portion of the p63 network that (direct or indirect) regulates *FGF8* expression is exerted in a quantitative and dynamic mode. To support this, we should consider that although *p63* null and *p63EEC* homozygous mice show severe limb trunca‐ tion or absence, the heterozygous mice appear to be normal. When heterozygous EEC mice are bred with heterozygous *Dlx5;Dlx6* ones (the latter have normal limbs), anomalies are clearly observed [90].

A gene-dosage effect combined with the co-expression of functionally redundant genes implies the existence of a threshold level to be maintained to assure AER stratification and signalling functions. Indeed, we have noted that the expression of *Dlx2* and *Dlx5* is lower in the central portion of the AER, compared to the anterior or posterior segments [122]. Thus, the central AER might be more sensitive to reduced *Dlx* expression due to intrinsic lower expression. On the same line, there is evidence that certain amount of AER-derived pan-FGF is required to induce and maintain the underlying mesenchymal progenitors [36, 56, 157]. In fact, in the *Dlx5*; *Dlx6* mutant limbs, the reduction of *FGF8* expression is restricted to the central AER, the region where epithelial-mesenchymal signalling is primarily defective and the region where mor‐ phogenesis fails [114, 115]. Thus, the entire p63-Dlx-FGF cascade is sensitive to gene dosage and position of expression.

### **3. Concluding remarks**

p63 is a master regulator of ectodermal cell proliferation, differentiation and stratification, and has a key role in the establishment of a positive loop that maintains *FGF8* expression. In turn, our recent data reveal a novel role of FGF8 to (directly and indirectly) stabilize the p63 proteins and modulate their transcriptional activity. Thus, in the biology and development of the ectoderm, p63 post-translational modifications are as important as *p63* gene expression and may reveal novel targets to be used in p63 modulation.

We illustrate that the p63-Dlx5 transcriptional regulation is at the centre of a pathway relevant for the SHFM malformation. The stability of p63 and the activation of the pathway appear to be under the regulation of FGF8, which in turn is regulated by the pathway. In addition to decipher this positive regulatory loop, these data support a model to attempt to explain the SHFM-III pathogenesis in terms of genome positional effects on the *FGF8* locus.

FGF8 and Wnt5a provide instructions for mesoderm cells as to which direction and orientation to take, at the basis of AER formation and proper migration of mesenchymal cells. This instruction adopts molecules of the PCP pathway, most likely inducing convergent extension. While this has been recently demonstrated for the mesenchymal cells, the possibility that a Wnt5a-dependent PCP pathway is also functional for the organization and stratification of the AER cells remains to be addressed. Notably, data from the human malformation diseases and the corresponding animal models clearly suggest so.

The study of animal models of EEC and SHFM diseases has provided much of this knowledge, and will continue to do so. The big hope is that, once the pathways will be elucidated, we might be able to exploit diffusible molecules and attempt to correct the limb malformation defects. Preliminary attempts are being conducted on whole-organ cultured limbs.

### **Nomenclature**

observed in *Dlx2*;*Dlx5* null mice [161] and the ectrodactyly of *Dlx5;Dlx6* null mice is fully rescued by the re-expression of *Dlx5* alone [115]. Likewise, an increased severity of craniofacial phenotypes correlates with progressive loss of *Dlx* gene [263, 264]. All these are indications of

**Figure 4.** Proposed model of regulation of the AER cell orientation. Dlx5 is known to regulate the transcription of both *FGF8* and *Wnt5a*. In turn, FGF8 is required for AER maintenance and stratification, via p63, while the function of Wnt5a for AER maintenance is still poorly known. Recent data of the regulation of orientation and velocity of mesen‐ chymal cells by, respectively, the Wnt5a/PCP and the FGF8-MAP-Erk pathways open the possibility that Wnt5a may

We propose that the portion of the p63 network that (direct or indirect) regulates *FGF8* expression is exerted in a quantitative and dynamic mode. To support this, we should consider that although *p63* null and *p63EEC* homozygous mice show severe limb trunca‐ tion or absence, the heterozygous mice appear to be normal. When heterozygous EEC mice are bred with heterozygous *Dlx5;Dlx6* ones (the latter have normal limbs), anomalies are

A gene-dosage effect combined with the co-expression of functionally redundant genes implies the existence of a threshold level to be maintained to assure AER stratification and signalling functions. Indeed, we have noted that the expression of *Dlx2* and *Dlx5* is lower in the central portion of the AER, compared to the anterior or posterior segments [122]. Thus, the central AER might be more sensitive to reduced *Dlx* expression due to intrinsic lower expression. On the same line, there is evidence that certain amount of AER-derived pan-FGF is required to

gene-dosage effects between functionally redundant genes.

regulate the orientation/motility of the AER cell and assure a correct stratification (dotted line).

clearly observed [90].

Conte et al.

148 New Discoveries in Embryology

A-P, anterior-posterior D-V, dorsal-ventral P-D, proximal-distal SHH, sonic hedgehog FGF, fibroblast growth factor FL, forelimb HL, hindlimb ZPA, zone of polarizing activity AER, apical ectodermal ridge PZ, progress zone KO, knock-out PCP, planar cell polarity CE, convergent extension LPM, lateral plate mesoderm

### **Author details**

Daniele Conte1 , Luisa Guerrini2 and Giorgio R. Merlo1\*

\*Address all correspondence to: giorgioroberto.merlo@unito.it

1 Department of Molecular Biotechnology and Health Sciences, Università degli Studi di Torino, Italy

2 Department of Biosciences, Università degli Studi di Milano, Italy

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150 New Discoveries in Embryology

PZ, progress zone

**Author details**

Daniele Conte1

Torino, Italy

**References**

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## **Protein Kinase A and Protein Kinase C Connections: What Could Angiogenesis Tell Us?**

Beatriz Veleirinho, Daniela Sousa Coelho, Viviane Polli, Simone Kobe Oliveira, Rosa Maria Ribeiro-Do-Valle, Marcelo Maraschin and Paulo Fernando Dias

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/60401

### **Abstract**

The formation of embryonic blood vessels, defined as vasculogenesis, is a complex morphogenetic process ultimately related to tubulogenesis, carried out from *in situ* differentiation of mesoderm-recruited or proliferated progenitor endothelial cells (angioblasts) to endothelial cells for structuring a primary vascular plexus. Subse‐ quent events involving apoptosis versus cell survival (remodeling) in the vessel network stabilizes the primordial microvasculature, which through the angiogenesis process yields new capillaries by sprouting from the preexisting ones. Methylxan‐ thinic alkaloids such as caffeine (compounds present in a number of beverages consumed worldwide) exert some well-known effects upon heart and other cardio‐ vascular structures, in part, by negatively interplaying with phosphodiesterase (PDEs) enzymes. Once caffeine as well as *Ilex paraguariensis* (yerba mate) infusion extract have shown to enhance the vessel formation (vasculogenesis and angiogene‐ sis), we discuss the impact afforded by *I. paraguariensis* constituents on the (PDEsrelated) quantities and stability of Protein kinase A (PKA) and Protein kinase C (PKC) enzymes. Besides, the text reflects on a suggested dual roles displayed by PKA and PKC enzymatic pathways in the developmental angiogenic events.

**Keywords:** Protein kinase A (PKA) and protein kinase C (PKC), Cyclases and phosphodiesterases, Methylxanthinic alkaloids, Vessels remodeling, Angiogenesis and vasculogenesis

© 2015 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

### **1. Introduction**

Angiogenesis and vasculogenesis are the better studied processes of vessel formation [1]. Angiogenesis starts from preexistent vasculature, these last structures being either the primitive vascular plexuses primordially formed by vasculogenesis in the embryo or the postcapillary venous compartment of the mature vascular systems [2, 3].

Vasculogenesis is defined as the formation of early embryonic blood vessels from *in situ* differentiation of mesoderm-recruited/proliferated progenitor endothelial cells (angioblasts) to endothelial cells [4, 5]. This process involves endothelial precursor cell clusters organization (blood islands), in the yolk sac membrane (YSM), laying down a primary vascular plexus [6– 8]. A subsequent remodeling of this vascular network – a process that combines events of cell death or regression in some vessels and survival or enhancement in others – gives rise to a more refined and effective microvasculature [9–11].

Further proliferation of capillaries sprouting from preexisting vessels is referred to as angio‐ genesis [12], a process involving coordinated endothelial cell proliferation and migration as well as recovering of extracellular matrix (ECM), tubule formation (tubulogenesis), and expansion of the surrounding vascular tissues [13–15]. Despite angiogenesis in adults being a rare event, it plays a fundamental role in physiological processes, such as the reproductive cycle of fertile women and the wound healing process [16, 17].

There are evidences that the vasculogenesis process that works in the early embryo forming primary vessels at high rates to keep pace with the growth of the body has been adapt‐ ed, under certain situations, in the adult [4, 18, 19], since bone marrow–derived endothe‐ lial progenitor cells in the peripheral blood of adult animals and humans have been shown to be incorporated into neovascularization [3, 20]. Under such conditions, cytokines can be produced to induce the formation of vascular networks alluding to vasculogenic mimicry [13, 21]. Thus, in accordance with this concept, the embryonic cellular mechanisms (proliferation and differentiation) underlying vasculogenesis process would be, in some level, recapitulated in adult life [21–23].

The cardiovascular system is susceptible to positive chronotropic and inotropic actions afforded by a class of compounds like xanthines which cause dilatation in a number of blood vessels (on lung and kidney, e.g.) and constriction in some others, such as the one occurring in brain vessels, revealing their controversial pharmacological features and biological targets diversity [24]. Methylxanthinic alkaloids, such as caffeine and theophylline are majoritarian compounds present in the coffee and cola beverages as well as in various tea extracts [25, 26]. Thus, in particular, caffeine may possibly be one of the most consumed substances all over the world. Its tropism on the cardiovascular structures and other organ systems is already reasonably known [27], as the specific-tissue mechanisms of action in some processes waits for further elucidation. Otherwise, methylxanthinic alkaloid interaction with protein kinase A (PKA) pathway has a remarkable effect on several vessel-related events. For example, Shafer et al. has verified that the treatment with caffeine and other methylxanthines increases cAMP level by inhibiting cAMP phosphodiesterase (PDE) [28]. As cAMP activates PKA, glycolysis is elevated which increases the amount of ATP available for muscle contraction and relaxation. Caffeine, as well as *Ilex paraguariensis* St. Hill., Aquifoliaceae (e.g., mate) infusion extract (1.03–4.12 µM), have been shown to increase the microvessels number, due to the enhance‐ ment on vasculogenesis and angiogenesis rates, in the model of yolk sac and chorioallanto‐ ic membranes of chick embryos [29]. Moreover, an additional stimulant property on embryonic metabolism was evidenced by the increase in the body growth (defined by the body length). The pharmacological effects of caffeine and theophylline present in the mate drinks on the cardiovascular system are mainly addressed to PDEs inhibition, which directly impacts the quantity, stability, and cell activities of PKA and Protein kinase C (PKC) [30]. In fact, the relaxant effect in the smooth muscle is attributed to PDE inhibition, with the consequent increase in cyclic adenosine monophosphate (cAMP) concentration [27, 31]. Moreover, the heart muscle stimulation and the bronchial muscle relaxation are mediated by beta-adrenoceptors stimulation and adenylate cyclase (AC) stimulation. It is also suggested that the competitive antagonism exhibited by methylxanthines on the adeno‐ sine receptors (A1 and A2) determines some of its complex effects [32, 33].

The action mechanism of caffeine and mate extract/tea upon the processes of vessel formation remains unclear despite the important evidences of xanthine involvement-related biological targets (PDE–AC) on the cardiovascular physiology. Thus, it seems important to pay attention to the suggested dual roles of PKA and PKC enzymatic pathways in the angiogenesis.

### **2. Distinct roles of PKC and PKA in angiogenesis**

**1. Introduction**

170 New Discoveries in Embryology

Angiogenesis and vasculogenesis are the better studied processes of vessel formation [1]. Angiogenesis starts from preexistent vasculature, these last structures being either the primitive vascular plexuses primordially formed by vasculogenesis in the embryo or the

Vasculogenesis is defined as the formation of early embryonic blood vessels from *in situ* differentiation of mesoderm-recruited/proliferated progenitor endothelial cells (angioblasts) to endothelial cells [4, 5]. This process involves endothelial precursor cell clusters organization (blood islands), in the yolk sac membrane (YSM), laying down a primary vascular plexus [6– 8]. A subsequent remodeling of this vascular network – a process that combines events of cell death or regression in some vessels and survival or enhancement in others – gives rise to a

Further proliferation of capillaries sprouting from preexisting vessels is referred to as angio‐ genesis [12], a process involving coordinated endothelial cell proliferation and migration as well as recovering of extracellular matrix (ECM), tubule formation (tubulogenesis), and expansion of the surrounding vascular tissues [13–15]. Despite angiogenesis in adults being a rare event, it plays a fundamental role in physiological processes, such as the reproductive

There are evidences that the vasculogenesis process that works in the early embryo forming primary vessels at high rates to keep pace with the growth of the body has been adapt‐ ed, under certain situations, in the adult [4, 18, 19], since bone marrow–derived endothe‐ lial progenitor cells in the peripheral blood of adult animals and humans have been shown to be incorporated into neovascularization [3, 20]. Under such conditions, cytokines can be produced to induce the formation of vascular networks alluding to vasculogenic mimicry [13, 21]. Thus, in accordance with this concept, the embryonic cellular mechanisms (proliferation and differentiation) underlying vasculogenesis process would be, in some

The cardiovascular system is susceptible to positive chronotropic and inotropic actions afforded by a class of compounds like xanthines which cause dilatation in a number of blood vessels (on lung and kidney, e.g.) and constriction in some others, such as the one occurring in brain vessels, revealing their controversial pharmacological features and biological targets diversity [24]. Methylxanthinic alkaloids, such as caffeine and theophylline are majoritarian compounds present in the coffee and cola beverages as well as in various tea extracts [25, 26]. Thus, in particular, caffeine may possibly be one of the most consumed substances all over the world. Its tropism on the cardiovascular structures and other organ systems is already reasonably known [27], as the specific-tissue mechanisms of action in some processes waits for further elucidation. Otherwise, methylxanthinic alkaloid interaction with protein kinase A (PKA) pathway has a remarkable effect on several vessel-related events. For example, Shafer et al. has verified that the treatment with caffeine and other methylxanthines increases cAMP level by inhibiting cAMP phosphodiesterase (PDE) [28]. As cAMP activates PKA, glycolysis is elevated which increases the amount of ATP available for muscle contraction and relaxation.

postcapillary venous compartment of the mature vascular systems [2, 3].

more refined and effective microvasculature [9–11].

level, recapitulated in adult life [21–23].

cycle of fertile women and the wound healing process [16, 17].

PKC isoforms are key mediators in hormone, growth factor, and neurotransmitter-triggered pathways of cell activation [34]. Proteomic technologies (gel-based and gel-free analyses methods) and metabolomics have been successfully used in the study of protein kinases. The application of these novel tools and strategies in the field of kinase signaling has been focused on the role of PKC in the heart (for review, see [35]). Another recent review provides, with particular attention, information on the role of PKC isoforms in the cardiovascular complica‐ tions [36]. A scheme of endothelial signaling pathways is displayed in Figure 1. As reported by Wright and co-workers, the DAG–PKC pathway activated by vascular endothelial growth factors (VEGFs) contributes to the vascular function in many ways, such as the regulation of endothelial permeability, vasoconstriction, extracellular matrix (ECM) synthesis/turnover, leukocyte adhesion, cytokine activation, cell growth, and ultimately, angiogenesis (Figure 1-1) [37]. In fact, such role of PKC on the angiogenesis activation was confirmed by *in vitro* and *in ovo* experiments.

An interesting study related with the PKA *versus* PKC actions on angiogenesis was performed by DeFouw and DeFouw [38]. These researchers showed that whereas the exogenous activa‐ tion of cAMP by PKA pathway signaling acts decreases the macromolecules extravasation in the chick chorioallantoic membrane (CAM) during early angiogenesis (4.5-day CAM, i.e., 4.5 days of embryonic development; stage 24-HH) [39], the PKC activity contributes, at least in part, to CAM endothelial hyper permeability (a crucial pro-angiogenic event) at the 4.5-day chick embryo. Nevertheless, it was already reported [40] that the cyclooxygenase (COX-2) **Figure 1** 

**Figure 1. Schemas of endothelial signaling pathways.** Basic fibroblast growth factor (bFGF) has been shown to acti‐ vate a number of intracellular signaling pathways. Some well characterized processes that have been reported in endo‐ thelial cells and other cell types are shown. Many details in the steps of the processes were omitted for the sake of clearness and the numbers are included to enable the signals/effectors identification (then numbers not necessarily rep‐ resent a sequence on transduction pathways, which are often non-exclusive). The autophosphorylation is activated by several tyrosine residues of FGFR and VEGFR. Some of the phosphotyrosine residues are binding sites for proteins with phosphotyrosine-binding domains such as FGF receptor substrate 2 (FRS2) that functions as docking protein and binds to the GRB2 which then can activate RAS. RAS may recruit RAF-1, a kinase whose action results in activation of a mitogen-activated protein kinases (MAPK) cascade. MAPK translocation to the nucleus proceeds activating tran‐ scription factors. PLC activation also plays a relevant role by causing the hydrolysis of phosphatidylinositol (PIP2) to inositol-3-phosphate and diacylglycerol (DAG) leading to calcium release and activation of protein kinase C (PKC). These kinase/eicosanoid-mediated signal transduction pathways can lead to a number of biological responses on the cell housekeeping that involve cell proliferation, migration, and the other mechanisms related to the endothelial cell phenotype **(1**–**4)**. Guanylyl cyclase (GC) mediated survival promotion by means of AKT-NOS activation **(5**–**8)** and gua‐ niline triphosphate/cyclic guaniline monophosphate (GC-cGMP)-PKC, as well as PKG activation pathways (**5**–**9)**. Phos‐ phodiesterase (PDE) inactivation, as attained by xanthines (caffeine, for example), with the consequent up-regulation of cAMP-PKA signaling and the down-regulation of cGMP-PKG (6-7). A PDE compensatory role on the cAMP/PKA probable anti-proliferative (and/or anti-EC migration) effects afforded, as suggested, by a potent stimulus (from PIP2, for example) on the PKC mitogenic pathway, with subsequent COX-2 activation **(10)**, or also by pro-vascular signals transmission contributions (11).

pathway, as well the AC–PKA signaling, enhances angiogenesis *in vivo* through induction by VEGF. Other studies have also indicated PKA as a positive angiogenesis regulator [41–45]. In this sense, PKA inhibition with H89 (PKA inhibitor) blocks vasoactive intestinal peptideinduced VEGF production and inhibits brain vascular endothelial cells proliferation [41], while PKA stimulation via Forskolin increases angiogenesis through PKA-dependent VEGF expression [42]. Also, Zhang et al. have demonstrated that the proinflammatory prostaglandin E2 (PGE2) promotes angiogenesis through activation of endothelial cell-expressed EP4 and PKA catalytic γ subunits. Furthermore, suppressing the expression of PKA activated sub‐ strates (i.e., Rap1A, HSPB6, or endothelial NO synthase) inhibits the tube formation, while the knockdown of RhoA or glycogen synthase kinase 3β, that are inactivated after PKA phos‐ phorylation, increases the tube formation of human microvascular endothelial cells [43].

**Figure 1** 

172 New Discoveries in Embryology

**5**

GTP

**8 9**

AKT

P70S6K

c-*fos*  c-*jun*  c-*myc* 

GSK3

*Mitosis Apoptosis* 

transmission contributions (11).

(P53)

GC

IGF Insulin Progesterone -

cGMP

**11**

NOS

NO PKC

**<sup>2</sup> <sup>3</sup> 4**

bFGFR

Tyrk

**P**

*HSPG* 

FRS2

GRB-2

**P**

GTP

GDP+Pi

**Figure 1. Schemas of endothelial signaling pathways.** Basic fibroblast growth factor (bFGF) has been shown to acti‐ vate a number of intracellular signaling pathways. Some well characterized processes that have been reported in endo‐ thelial cells and other cell types are shown. Many details in the steps of the processes were omitted for the sake of clearness and the numbers are included to enable the signals/effectors identification (then numbers not necessarily rep‐ resent a sequence on transduction pathways, which are often non-exclusive). The autophosphorylation is activated by several tyrosine residues of FGFR and VEGFR. Some of the phosphotyrosine residues are binding sites for proteins with phosphotyrosine-binding domains such as FGF receptor substrate 2 (FRS2) that functions as docking protein and binds to the GRB2 which then can activate RAS. RAS may recruit RAF-1, a kinase whose action results in activation of a mitogen-activated protein kinases (MAPK) cascade. MAPK translocation to the nucleus proceeds activating tran‐ scription factors. PLC activation also plays a relevant role by causing the hydrolysis of phosphatidylinositol (PIP2) to inositol-3-phosphate and diacylglycerol (DAG) leading to calcium release and activation of protein kinase C (PKC). These kinase/eicosanoid-mediated signal transduction pathways can lead to a number of biological responses on the cell housekeeping that involve cell proliferation, migration, and the other mechanisms related to the endothelial cell phenotype **(1**–**4)**. Guanylyl cyclase (GC) mediated survival promotion by means of AKT-NOS activation **(5**–**8)** and gua‐ niline triphosphate/cyclic guaniline monophosphate (GC-cGMP)-PKC, as well as PKG activation pathways (**5**–**9)**. Phos‐ phodiesterase (PDE) inactivation, as attained by xanthines (caffeine, for example), with the consequent up-regulation of cAMP-PKA signaling and the down-regulation of cGMP-PKG (6-7). A PDE compensatory role on the cAMP/PKA probable anti-proliferative (and/or anti-EC migration) effects afforded, as suggested, by a potent stimulus (from PIP2, for example) on the PKC mitogenic pathway, with subsequent COX-2 activation **(10)**, or also by pro-vascular signals

DAG

caffeine

G-prot

PDE

Ras

Raf-1

nucleus

MAPK

**7 6**

**P**

c-AMP

PKA

ATP AC

IRec

PKA, PKC

PKG

HIFs

caspase-9

**1**

VEGFR2

Tyrk

PKC

Raf

MEK

*ANGIOGENESIS* 

**P**

**10**

PC

COX-2

PGs TXs

endothelial permeability regulation vasoconstriction extracellular matrix synthesis / turnover cytokine activation cell proliferation, cell growth / migration

(PGE1,2)

VEGF *expression* 

AC

AA

LPs

cam

LCs

*αVβ3 / 5* 

PI3 Ca+2

PLC<sup>γ</sup> DAG

ER

CELL

PI

In opposition to the concept of PKA-activated angiogenic events, some evidences have established a profile of angiogenesis inhibition and an endothelial cell survival decrease mediated by PKA [46]. However, these authors have also demonstrated that basic fibroblast growth factor (bFGF)-stimulated blood vessel branch points were non-abolished by concom‐ itant treatments with cAMP or PKAcat. A subsequent study [47] demonstrated, in human granulosa cells, the PKA-mediated negative regulation of vessel formation (as well as the modulation of endothelial cell survival) related to the increase on mRNA levels of angiopoie‐ tin-2 (ANG-2; a pro-apoptotic agent) by both PKA and PKC activators (8-Cl-cAMP and ADMB), whereas the respective inhibitors (GÖ 6983 and Rp-cAMP) markedly decreased the levels of ANG-2 mRNA. Concurrently, VEGF-induced human umbilical vein endothelial cells (HUVECs) migration and proliferation were decreased by PDE2 and PDE4 inhibitors [48]. Additionally, Jin et. al. have shown that PKA activation blocks pp60Src-dependent vascular endothelial–cadherin phosphorylation which stimulates cell–cell adhesion and inhibits endothelial cell polarization and migration, which consequently blocks sprouting in newly forming embryonic blood vessels [49]. In prostate tumor epithelial cells, the cAMP derivative 8-pCPT-2'-O-Me-cAMP, a weak agonist of PKA, acts via stimulation of that kinase that, in its turn, antagonizes Rap1 and hypoxic induction of 1α protein expression, VEGF production and, ultimately, angiogenesis [50]. More recently, Liu et al. have proposed that the major PKA function in physiological condition may be to inhibit angiogenesis through REGγ–proteasome mediated regulation. It has been shown that REGγ interacts with protein kinase A catalytic subunit-α (PKAca reducing its intracellular stability) in HUVECs and mouse embryonic fibroblast cells (MEFs). The study has evidenced that REGγ antagonizes PKA pathway and facilitates VEGF-induced expression of pro-angiogenic genes (e.g., vascular cell adhesion molecule-1 gene [*VCAM-1]* and endothelial-Selectin gene [*E-Selectin*]) through PKA-FoxO1 pathway. Nevertheless, authors empathize that the role of PKA on angiogenesis can vary depending upon different cell context and various signal cascades in physiological or patho‐ logical environments [51]. The anti-angiogenic role of PKA through different mechanisms represents useful tools to inhibit pathologic angiogenesis. Taken in the whole, the above cited results show contrasting actions upon angiogenesis, not only between PKA and PKC actions, but also involving each enzymatic pathway, *per se*.

### **3. How can xanthines interplay with vascular mediators?**

As referred earlier [29], the treatments performed by 1.03–4.12 µM caffeine and mate extract, besides increasing vasculogenesis and angiogenesis concomitantly, have promoted embryonic growth as featured by increase in body total length of treated 4-day chick embryos. These findings may be better understood taking into consideration the findings previously reported by Shibley and Pennington [52]. These researchers have demonstrated that non-acute *in vivo* treatment of cultured 5-day-old chick embryo cells with 1 µM phorbol ester leads to downregulation (instead of up-regulation as afforded by acute treatments) of PKC activity, signifi‐ cantly increasing the insulin-dependent amino acid intake/uptake and transport that are crucial processes for embryonic growth.

On the other hand, PKC has also been shown to be involved in the regulation of glucose (a well-known angiogenic activator and fetal weight and length-increasing factor) transport in adipocytes [53] and that this transportation activity was blocked by PKC inhibition. Indeed, hyperglycemia (15 mM glucose), as well as VEGF, are able, via VEGFR-2, to up-regulate PlGF (placental growth factor; a member of the VEGF family), which also acts as a survival factor for microvascular endothelial cells by preventing apoptosis [54, 55]. These evidences are concurrent with a time-dependent diacylglycerol (DAG)-mediated PKC activation event (Figure 1-2) in response to insulin and insulin-like growth factors activation [56].

Even though the impairment on nutrient transport related to PKC inhibition has been already demonstrated by Christensen et al. [53], possible remarkable compensatory responses exerted, for example, by insulin-like growth factor interaction with AC on the body length of the caffeine-treated embryos should be considered (Figure 1-3) [27].

### **4. What about phosphodiesterases?**

Bearing in mind that the evidences of vasculogenesis and angiogenesis inhibition are related to PKC/PKA pathways, one could yet ponder that those effects not necessarily point to PDErelated action or additional AC-cAMP inhibitors, as the progesterone hormone, for example, It is plausible to assume that caffeine and mate effects might, at least in part, involve other angiogenic pathways than AC-cAMP-PKA inhibition, such as those related to phosphatidyl inositol-2-kinase (PI2K) and calcium/DAG-PKC activation, or its collateral induction by bFGF [57], which is a crucial angiogenic growth factor (Figure 1-4). Besides, the tumor necrosis factoralpha (TNF-α) and/or the guanylyl cyclase-cyclic guaniline monophosphate (GC-cGMP-PKC/ PKG), pro-angiogenic activating pathways are also worth mentioning (Figure 1-5). Notwith‐ standing, the relevance of PDE involvement in vasculature development is evidenced by the concept which the differentiation of a restrictive angiogenic–endothelial barrier function *in vivo* would include inactivation of PDE III and PDE IV. This implies in up-regulation of cAMP-PKA signaling (Figure 1-6) and down-regulation on cGMP-PKG pathway [38]. Moreover, (1) PDE2, PDE3, PDE4, and PDE5 are expressed in HUVEC; (2) both EHNA (20 µM), a PDE2 selective inhibitor, and RP73401 (10 µM), a PDE4 selective inhibitor, are able to enhance the cAMP intracellular levels in HUVECs; (3) EHNA and RP73401 are able to inhibit cell prolif‐ eration, mitotic cycle progression and migration on HUVECs stimulated by VEGF; (4) HUVEC treatments with the cAMP analogue 8-Br-cAMP (600 µM) mimicry the cAMP i*n vitro* inhibitory effects; and (5) only the association of EHNA and RP73401 (co-treatment by PDE2 and PDE4 selective inhibitors) blocks angiogenesis *in vivo*, indicating that to start antiangiogenic activity both migration and cell proliferation must be conjointly abolished [48].

**3. How can xanthines interplay with vascular mediators?**

crucial processes for embryonic growth.

174 New Discoveries in Embryology

As referred earlier [29], the treatments performed by 1.03–4.12 µM caffeine and mate extract, besides increasing vasculogenesis and angiogenesis concomitantly, have promoted embryonic growth as featured by increase in body total length of treated 4-day chick embryos. These findings may be better understood taking into consideration the findings previously reported by Shibley and Pennington [52]. These researchers have demonstrated that non-acute *in vivo* treatment of cultured 5-day-old chick embryo cells with 1 µM phorbol ester leads to downregulation (instead of up-regulation as afforded by acute treatments) of PKC activity, signifi‐ cantly increasing the insulin-dependent amino acid intake/uptake and transport that are

On the other hand, PKC has also been shown to be involved in the regulation of glucose (a well-known angiogenic activator and fetal weight and length-increasing factor) transport in adipocytes [53] and that this transportation activity was blocked by PKC inhibition. Indeed, hyperglycemia (15 mM glucose), as well as VEGF, are able, via VEGFR-2, to up-regulate PlGF (placental growth factor; a member of the VEGF family), which also acts as a survival factor for microvascular endothelial cells by preventing apoptosis [54, 55]. These evidences are concurrent with a time-dependent diacylglycerol (DAG)-mediated PKC activation event

Even though the impairment on nutrient transport related to PKC inhibition has been already demonstrated by Christensen et al. [53], possible remarkable compensatory responses exerted, for example, by insulin-like growth factor interaction with AC on the body length of the

Bearing in mind that the evidences of vasculogenesis and angiogenesis inhibition are related to PKC/PKA pathways, one could yet ponder that those effects not necessarily point to PDErelated action or additional AC-cAMP inhibitors, as the progesterone hormone, for example, It is plausible to assume that caffeine and mate effects might, at least in part, involve other angiogenic pathways than AC-cAMP-PKA inhibition, such as those related to phosphatidyl inositol-2-kinase (PI2K) and calcium/DAG-PKC activation, or its collateral induction by bFGF [57], which is a crucial angiogenic growth factor (Figure 1-4). Besides, the tumor necrosis factoralpha (TNF-α) and/or the guanylyl cyclase-cyclic guaniline monophosphate (GC-cGMP-PKC/ PKG), pro-angiogenic activating pathways are also worth mentioning (Figure 1-5). Notwith‐ standing, the relevance of PDE involvement in vasculature development is evidenced by the concept which the differentiation of a restrictive angiogenic–endothelial barrier function *in vivo* would include inactivation of PDE III and PDE IV. This implies in up-regulation of cAMP-PKA signaling (Figure 1-6) and down-regulation on cGMP-PKG pathway [38]. Moreover, (1) PDE2, PDE3, PDE4, and PDE5 are expressed in HUVEC; (2) both EHNA (20 µM), a PDE2 selective inhibitor, and RP73401 (10 µM), a PDE4 selective inhibitor, are able to enhance the

(Figure 1-2) in response to insulin and insulin-like growth factors activation [56].

caffeine-treated embryos should be considered (Figure 1-3) [27].

**4. What about phosphodiesterases?**

In addition, the relevant study published by Netherton and Maurice [58] punctuates that human vascular endothelial cells (VECs) express variants of PDE2, PDE3, PDE4, and PDE5 families and demonstrate that the levels of these enzymes differ among VECs derived from aorta, umbilical vein, and micro vascular structures as those present in the yolk sac/chorioal‐ lantoic membrane (YSM/CAM) of chick embryos. As stated by those investigators, it is noteworthy that the selective inhibition of PDE2 does not only fail to increase cAMP in any VECs lineage, but also it did not inhibit migration in the VECs studied.

Otherwise, the inhibition of PDE4 activity decreased cell migration but, in association with forskolin (an AC/GC activator), increased cAMP in all VECs studied [58]. PDE3 inhibition potentiated forskolin-induced increases in cAMP and also inhibited migration in VECs derived from aorta and umbilical vein, but not on microvascular VECs. From these data, one should expect that methylxanthines had reduced vessel number in the early extra-embryonic mem‐ branes (YSM and CAM) in response to PDE inhibition (Figure 1-7), by antagonizing adenosine, or indeed by protecting cAMP from degradation. However, there are some evidences con‐ cerning the process of microvessels development where the opposite has just been found. The cAMP pathway truly "rivals" with the angiogenic microenvironment in complexity (for inhibiting inflammatory cytokines) and constitutes a kind of cross-junction to which converges a significant number of cell signaling ways. Then, during vessel formation, cAMP (and its distinct cellular roles) is surely under influence of factors as diverse as different time–space conditions, distinct main regulative pathways, and a number of second messengers/effectors in various signaling routs/cascades. Moreover, these events are dependent on each vascular endothelial cell lineage and the biological system or study model considered.

### **5. Focusing on the environment of developmental microvessels**

Embryonic microvessels (such as those growing in the 4-day chick YSMs/CAMs) are structures physiologically under one primordial choice: that is potentially "life or death" [10]. Therefore, despite the proinflammatory cytokines blockade due to cAMP increase mediated by PDE inhibition in response to methylxanthines action, and also the presence of eventual apoptotic stimulus (such as insulin/IGFs-PKA interaction-mediated cell death), the embryonic endothe‐ lial cells may be concomitantly exposed to powerful survival stimuli, for example, vascular growth factors; survival factors (i.e., ANG-1), guanylyl cyclase (GC)-Akt (i.e., GC-PKB) [59] (Figure 1-8), pericyte-support; blood flow; and others. Besides, specific pro-angiogenic signals/ conditions (NO-synthase/NO-GC, intermittent hypoxia, and GC-PKC, e.g.) would be prepon‐ derant to protect the ECs (Figure 1-9) [60, 61]. In the light of these evidences, it is still plausible to suggest that both caffeine and the *I. paraguariensis* extract may exert a compensatory role on the cAMP/PKA probable anti-EC proliferative effect and/or anti-EC migration effect, by means of potent stimuli (from PIP2, Ca2+, e.g.) to the PKC mitogenic pathway, with supplementary COX-2 prostaglandin-E (PGE1, 2) activation (Figure 1-10). Additionally, pro-vascular integrins/ cytokines contributions and GC-Akt-P70SK-related c-*fos* and c-*jun* activation (Figure 1-11) should be considered. In the context of the dual effect between the AC-cAMP and GC-cGMP functions in the ECs (concerning the up-regulation of cAMP-PKA signaling against the downregulation on cGMP-PKG pathway), it is possible to ponder on a non-improbable straightfor‐ ward antagonist action of PKC on the PKA pathway. In fact, this idea is in part supported by evidences that PKC is able to phosphorylate also PKA-specific consensus sites of Tnl (troponin 1), a cardiac myofilament [62].

As an alternative hypothesis concerning a compensatory mechanism on angiogenesis, negative modulation by cAMP, we suggest the improvement of glucose (an angiogenic activator) uptake by ECs, possibly mediated by insulin/IGF-AC activation in response to methylxanthine administration. As support for this idea, data provided by Hashimoto et al. [63] have shown that inhibitors of PKA and PI3K completely attenuated the NO-induced *in vitro* endothelial tube formation (from human aortic endothelial cells). These findings strongly suggest that PKA (Figure 1-12) and PI3K might both be mediating the angiogenesis process.

### **6. Conclusion**

In conclusion, we should not rescind from the importance of considering some apoptotic level *per se* on the endothelial cells lineages (*anoikis*) during the transition events from immature vasculature, yielded by vasculogenesis, to a more stable and sophisticated one attained by angiogenesis. In the context of angiogenic remodeling [64], some microvessels "have to die for others to survive" becoming stable/quiescent vascular structures [9]. Many "puzzle pieces" of kinases pathways appear to be, up to date, lacking. For example, how to begin solving the metabolome matter related to PKA *versus* PKC pathways in the angiogenesis? In accordance with Agnetti et al. [36], the "one protein at a time" approach is unlikely to provide a compre‐ hensive picture of the cellular signaling due to the concerted action of "several molecular players at the same time." Thus, the activities of both PKC and PKA should not be considered so mutually exclusive characters in the scenery of developmental microvessel formation. However, the remarkable evidences on phosphodiesterases as possible pivotal target mole‐ cules for the angiogenic effects of caffeine and *Ilex paraguariensis* extract strongly suggest an antagonistic role of the protein kinases A and C in the same events.

### **Acknowledgements**

The authors are grateful to the Research and Extension Pro-Rectory of Federal University of Santa Catarina (PRPE/UFSC, Brazil) for their support.

### **Author details**

to suggest that both caffeine and the *I. paraguariensis* extract may exert a compensatory role on the cAMP/PKA probable anti-EC proliferative effect and/or anti-EC migration effect, by means of potent stimuli (from PIP2, Ca2+, e.g.) to the PKC mitogenic pathway, with supplementary COX-2 prostaglandin-E (PGE1, 2) activation (Figure 1-10). Additionally, pro-vascular integrins/ cytokines contributions and GC-Akt-P70SK-related c-*fos* and c-*jun* activation (Figure 1-11) should be considered. In the context of the dual effect between the AC-cAMP and GC-cGMP functions in the ECs (concerning the up-regulation of cAMP-PKA signaling against the downregulation on cGMP-PKG pathway), it is possible to ponder on a non-improbable straightfor‐ ward antagonist action of PKC on the PKA pathway. In fact, this idea is in part supported by evidences that PKC is able to phosphorylate also PKA-specific consensus sites of Tnl (troponin

As an alternative hypothesis concerning a compensatory mechanism on angiogenesis, negative modulation by cAMP, we suggest the improvement of glucose (an angiogenic activator) uptake by ECs, possibly mediated by insulin/IGF-AC activation in response to methylxanthine administration. As support for this idea, data provided by Hashimoto et al. [63] have shown that inhibitors of PKA and PI3K completely attenuated the NO-induced *in vitro* endothelial tube formation (from human aortic endothelial cells). These findings strongly suggest that PKA (Figure 1-12) and PI3K might both be mediating the angiogenesis process.

In conclusion, we should not rescind from the importance of considering some apoptotic level *per se* on the endothelial cells lineages (*anoikis*) during the transition events from immature vasculature, yielded by vasculogenesis, to a more stable and sophisticated one attained by angiogenesis. In the context of angiogenic remodeling [64], some microvessels "have to die for others to survive" becoming stable/quiescent vascular structures [9]. Many "puzzle pieces" of kinases pathways appear to be, up to date, lacking. For example, how to begin solving the metabolome matter related to PKA *versus* PKC pathways in the angiogenesis? In accordance with Agnetti et al. [36], the "one protein at a time" approach is unlikely to provide a compre‐ hensive picture of the cellular signaling due to the concerted action of "several molecular players at the same time." Thus, the activities of both PKC and PKA should not be considered so mutually exclusive characters in the scenery of developmental microvessel formation. However, the remarkable evidences on phosphodiesterases as possible pivotal target mole‐ cules for the angiogenic effects of caffeine and *Ilex paraguariensis* extract strongly suggest an

The authors are grateful to the Research and Extension Pro-Rectory of Federal University of

antagonistic role of the protein kinases A and C in the same events.

Santa Catarina (PRPE/UFSC, Brazil) for their support.

1), a cardiac myofilament [62].

176 New Discoveries in Embryology

**6. Conclusion**

**Acknowledgements**

Beatriz Veleirinho1 , Daniela Sousa Coelho2 , Viviane Polli2 , Simone Kobe Oliveira2 , Rosa Maria Ribeiro-Do-Valle1 , Marcelo Maraschin1 and Paulo Fernando Dias1,2\*

\*Address all correspondence to: paulo.fernando.dias@ufsc.br

1 NANOBIOMAT, Federal University of Santa Catarina, Florianópolis, Brazil

2 Department of Cell Biology, Embryology, and Genetics, Federal University of Santa Cata‐ rina, Florianópolis, Brazil

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**Emrbyo Technology**

### **Chapter 8**

## **A Novel Discipline in Embryology — Animal Embryo Breeding**

Bin Wu, Linsen Zan, Fusheng Quan and Hai Wang

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/61299

### **Abstract**

The modern animal biotechnologies, such as animal cloning, transgenesis, sex determina‐ tion, stem cells, designing new livestock, must be performed on animal gametes includ‐ ing sperm and oocytes, and embryos based on embryology theory. Currently, some key biotechnologies in embryology have become the most powerful tool for animal scientists and breeders to improve genetic construction of animal herds. Here, authors put forward a new concept of **Animal Embryo Breeding** Science to describe this discipline formation, development, and application in animal genetic improvement and breeding. The relation‐ ship of embryo breeding with other disciplines has been profiled. Thus, animal scientists and breeders can easily understand and apply embryo breeding theory and related key techniques to accelerate animal improvement speed, to modify genetic construction of animal population, and to design and create new animal individual or breed.

**Keywords:** Discipline, embryo breeding, biotechnology, livestock

### **1. Introduction**

Animal breeding sciences concern the management and care of farm animals by humans for profit. Not only does it refer to the practice of selectively breeding and raising livestock to promote desirable traits in animals for utility, sport, pleasure, or research [1], but also it refers to the efficient exploitation of a species in agriculture advantageous to humans. The genetic improvement of livestock depends on defining breeding objectives and accurately identifying the right animals to be used for future breeding. Traditional breeding programs involve 1) the design of animal breeding goals including improvement traits, such as milk, wool, growth, carcass and fertility, females vs. males, progeny test and nucleus vs. commercial animal population; 2) application techniques, such as artificial insemination and embryo transfer, are

© 2015 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

used as methods not only to guarantee that females breed regularly but also to help improve herd genetics; 3) based on quantitative genetics theory, estimation of breeding value by phenotype, pedigree, BLUP (best linear unbiased prediction) method, and genetic markers; 4) selection and culling of individuals based on genetic evaluation, balancing rate of change, and inbreeding; and 5) determining mating system. This is a long-term process for livestock genetic improvement.

As modern biotechnology develops, some new techniques can be applied to animal breeding programs 1) to accelerate genetic progression by shortening generation interval and increasing female reproduction; 2) to add new genetic trait to animal body by transgenic technology or to remove bad traits from animal body by gene knockout method [2]; and 3) to create new animal individual or breed by modern biotechnologies including nuclear transfer, cloning, and genetic modification. These new technologies will make it easier to manipulate animal genomes, but food products from genetically engineered animals face a long road to market. Examples of biotechnology applications of particular interest to the department include cell culture, genomics, molecular-marker-assisted breeding, cloning, bioprocessing, and diagnos‐ tic testing, as well as gene technology (genetic modification). Genetic modification deliberates change of an organism's genetic material by moving, introducing, or eliminating specific genes, such as taking a single gene from an animal cell and inserting it into another animal cell to give the second cell a desired characteristic. The terms "gene technology," "genetic engi‐ neering" and "genetic manipulation," "genetic enhancement," "gene splicing," "transgenics," or the use of "recombinant DNA" are used to describe genetic modification processes. Genes can be found in and moved between different plants, animals or microorganisms such as viruses or bacteria, for example, transferring worm fat-1 gene to pig to produce more omega-3 fat acid in pork meat [3]. Genes can also be changed within a specific plant or animal individual. For instance, "knocking out" an undesirable characteristic gene such as susceptibility to a particular disease can be beneficial to the plant or animal life.

In mammals, the realization of these goals must depend upon *in vitro* manipulation of animal oocytes and embryos. Thus, embryology has become a core of these biotechnologies (Figure 1). Currently, embryo biotechnology, which most people call **embryo bioengineering,** has gradually become the most powerful tool for animal scientists and breeders to improve genetic construction of their animal herds or populations. Embryo transfer in cattle has recently gained considerable popularity with seedstock dairy and beef producers. Many kinds of species have been cloned and some transgenic animals have been produced. Thus, embryology has become a core of modern biotechnologies in animal genetic modification and breeding. Combining the new advances in modern biotechnology with future application, authors put forward the new concept of **Animal Embryo Breeding Science** to describe embryology development and application in animal genetic improvement and breeding.

### **2. Concept of animal embryo breeding**

Breeding is the reproductive process, which is producing of elite offspring in animals or plants. Animal breeding programs involve the selection or culling of parents (such as bull and cow)

used as methods not only to guarantee that females breed regularly but also to help improve herd genetics; 3) based on quantitative genetics theory, estimation of breeding value by phenotype, pedigree, BLUP (best linear unbiased prediction) method, and genetic markers; 4) selection and culling of individuals based on genetic evaluation, balancing rate of change, and inbreeding; and 5) determining mating system. This is a long-term process for livestock genetic

As modern biotechnology develops, some new techniques can be applied to animal breeding programs 1) to accelerate genetic progression by shortening generation interval and increasing female reproduction; 2) to add new genetic trait to animal body by transgenic technology or to remove bad traits from animal body by gene knockout method [2]; and 3) to create new animal individual or breed by modern biotechnologies including nuclear transfer, cloning, and genetic modification. These new technologies will make it easier to manipulate animal genomes, but food products from genetically engineered animals face a long road to market. Examples of biotechnology applications of particular interest to the department include cell culture, genomics, molecular-marker-assisted breeding, cloning, bioprocessing, and diagnos‐ tic testing, as well as gene technology (genetic modification). Genetic modification deliberates change of an organism's genetic material by moving, introducing, or eliminating specific genes, such as taking a single gene from an animal cell and inserting it into another animal cell to give the second cell a desired characteristic. The terms "gene technology," "genetic engi‐ neering" and "genetic manipulation," "genetic enhancement," "gene splicing," "transgenics," or the use of "recombinant DNA" are used to describe genetic modification processes. Genes can be found in and moved between different plants, animals or microorganisms such as viruses or bacteria, for example, transferring worm fat-1 gene to pig to produce more omega-3 fat acid in pork meat [3]. Genes can also be changed within a specific plant or animal individual. For instance, "knocking out" an undesirable characteristic gene such as susceptibility to a

In mammals, the realization of these goals must depend upon *in vitro* manipulation of animal oocytes and embryos. Thus, embryology has become a core of these biotechnologies (Figure 1). Currently, embryo biotechnology, which most people call **embryo bioengineering,** has gradually become the most powerful tool for animal scientists and breeders to improve genetic construction of their animal herds or populations. Embryo transfer in cattle has recently gained considerable popularity with seedstock dairy and beef producers. Many kinds of species have been cloned and some transgenic animals have been produced. Thus, embryology has become a core of modern biotechnologies in animal genetic modification and breeding. Combining the new advances in modern biotechnology with future application, authors put forward the new concept of **Animal Embryo Breeding Science** to describe embryology development and

Breeding is the reproductive process, which is producing of elite offspring in animals or plants. Animal breeding programs involve the selection or culling of parents (such as bull and cow)

particular disease can be beneficial to the plant or animal life.

application in animal genetic improvement and breeding.

**2. Concept of animal embryo breeding**

improvement.

186 New Discoveries in Embryology

Figure 1. Embryology has become a core of modern biotechnologies in animal genetic modification and breeding. Any new developed biotechniques such as nuclear **Figure 1.** Embryology has become a core of modern biotechnologies in animal genetic modification and breeding. Any new developed biotechniques such as nuclear transplantation, cloning, and transgenesis, finally must be performed on animal oocytes or embryos. MOET represents multiple ovulation and embryo transfer.

and then determination of mating system. They must be female and male sex combination. However, **Animal Embryo Breeding** is an asexual reproduction of specific oocytes or embryos artificially by current developed biotechnology. The Science of Animal Embryo Breeding is to study how to use the embryo manipulation technologies to improve, create, and clone new animal individual or breed. Current developed techniques include nuclear transfer, cytoplas‐ mic transfer or replacement, in vitro fertilization (IVF), sperm cytoplasmic injection (ICSI), parthenogenesis and androgenesis, embryo cloning, sex selection, transgenesis, gene knock out, stem cells and somatic cell cloning, etc. Although embryo breeding is a branch of tradi‐ tional animal breeding discipline, the science of animal breeding is concerned with the application of the principles of population genetics and qualitative genetics to the improve‐ ment of domestic animals. However, Animal Embryo Breeding is concerned with application of the developed embryo biotechnologies to new animal individual creation, genetic cloning and preservation of animal breeds. The research main body of this discipline focuses on sperm, oocyte and embryo. After the desired animal type (genetic improved goal) has been designed, by means of a serial micromanipulation on oocyte or embryo, such as nuclear transfer, foreign DNA microinjection to egg pronucleus and stem cell technique, a modified improved embryo may be produced in vitro and then transferred into animal uterine cavity so that a new animal individual could be created. In the last couple of decades, many kinds of animals including transgenic pigs, cattle, sheep, and goat, have been produced [4]. transplantation, cloning, and transgenesis, finally must be performed on animal oocytes or embryos. MOET represents multiple ovulation and embryo transfer. **<H1>Concept of Animal Embryo Breeding** Breeding is the reproductive process, which is producing of elite offspring in animals or plants. Animal breeding programs involve the selection or culling of parents (such as bull and cow) and then determination of mating system. They must be female and male sex combination. However, **Animal Embryo Breeding** is an asexual reproduction of specific oocytes or embryos artificially by current developed biotechnology. The Science of Animal Embryo Breeding is to study how to use the embryo manipulation technologies to improve, create, and clone new animal individual or breed. Current developed techniques include nuclear transfer, cytoplasmic transfer or replacement, in vitro fertilization (IVF), sperm cytoplasmic injection (ICSI), parthenogenesis and androgenesis, embryo cloning, sex selection, transgenesis, gene knock out, stem cells and somatic cell cloning, etc. Although embryo breeding is a branch of traditional animal breeding discipline, the science of animal breeding is concerned with the application of the principles of population genetics and qualitative genetics to the improvement of domestic animals. However, Animal Embryo Breeding is concerned with application of the developed embryo biotechnologies to new animal individual creation, genetic cloning and preservation of animal breeds. The research main body of this discipline focuses on sperm, oocyte and embryo. After the desired animal type (genetic improved goal) has

#### **3. The relationship of animal embryo breeding science with other disciplines** a modified improved embryo may be produced in vitro and then transferred into animal uterine cavity so that a new animal individual could be created. In the last couple of

As a new developing subject, Animal Embryo Breeding Science mainly depends upon modern biotechnology development, especially molecular biology, genetics, and reproductive biology

been designed, by means of a serial micromanipulation on oocyte or embryo, such as nuclear transfer, foreign DNA microinjection to egg pronucleus and stem cell technique, with embryology. However, it also has a close association with other subjects such as repro‐ ductive biology and embryology, animal genetics and breeding (Figure 2).

Figure 2. The designed relationship of Animal Embryo Breeding with other disciplines. The Embryo breeding is a core subject which combines molecular biology/genetics with animal genetics and breeding as well as reproductive biology and embryology. **Figure 2.** The designed relationship of Animal Embryo Breeding with other disciplines. The Embryo breeding is a core subject which combines molecular biology/genetics with animal genetics and breeding as well as reproductive biology and embryology.

The goal of animal breeding program can be realized by the current embryo breeding technology. Using molecular biological technique, a specific gene type for the desired animal may be designed. The new developed biotechnologies to attempt to modify animal genetic traits must be conducted on animal oocyte and embryo. The embryo in vitro production and animal individual birth must depend upon animal reproductive technology. Embryology may supply a good condition to produce many high-quality embryos. Thus, the Embryo Breeding is a core subject which combines molecular biology/genetics with animal genetics and breeding as well as reproductive biology and embryology. **<H1>Major research scope and content of Animal Embryo Breeding** The goal of animal breeding program can be realized by the current embryo breeding tech‐ nology. Using molecular biological technique, a specific gene type for the desired animal may be designed. The new developed biotechnologies to attempt to modify animal genetic traits must be conducted on animal oocyte and embryo. The embryo in vitro production and animal individual birth must depend upon animal reproductive technology. Embryology may supply a good condition to produce many high-quality embryos. Thus, the Embryo Breeding is a core subject which combines molecular biology/genetics with animal genetics and breeding as well as reproductive biology and embryology.

Animal Embryo Breeding Science is based on the current developed embryo

#### biotechnology. The core of current embryo biotechnology is oocyte in vitro fertilization (IVF). As human IVF technique rapidly develops in infertility treatment, not only animal IVF has offered a very valuable tool to study mammalian fertilization and early embryo **4. Major research scope and content of animal embryo breeding**

development, but also its commercial applications have being increased. Based on IVF research, some new developed embryo technologies consisting of nuclear transfer, transgenesis, cloning, and stem cells, etc., can be used to create new animal individual or population, and accelerate genetic progression of animal population during the period from early oocyte stage (oogenesis) to preimplantation embryo stage (Figure 3). Animal Embryo Breeding Science is based on the current developed embryo biotechnology. The core of current embryo biotechnology is oocyte in vitro fertilization (IVF). As human IVF technique rapidly develops in infertility treatment, not only animal IVF has offered a very valuable tool to study mammalian fertilization and early embryo development, but also its commercial applications have being increased. Based on IVF research, some new developed embryo technologies consisting of nuclear transfer, transgenesis, cloning, and stem cells, etc., can be used to create new animal individual or population, and accelerate genetic progression of animal population during the period from early oocyte stage (oogenesis) to preimplantation embryo stage (Figure 3).

with embryology. However, it also has a close association with other subjects such as repro‐

Embryo Breeding

Molecular Genetics Animal Breeding

Genetics

Molecular

Figure 2. The designed relationship of Animal Embryo Breeding with other disciplines. The Embryo breeding is a core subject which combines molecular biology/genetics with

**Figure 2.** The designed relationship of Animal Embryo Breeding with other disciplines. The Embryo breeding is a core subject which combines molecular biology/genetics with animal genetics and breeding as well as reproductive biology

& Development

Molecular Reproduction

The goal of animal breeding program can be realized by the current embryo breeding technology. Using molecular biological technique, a specific gene type for the desired animal may be designed. The new developed biotechnologies to attempt to modify animal genetic traits must be conducted on animal oocyte and embryo. The embryo in vitro production and animal individual birth must depend upon animal reproductive technology. Embryology may supply a good condition to produce many high-quality embryos. Thus, the Embryo Breeding is a core subject which combines molecular biology/genetics with animal genetics and breeding as well as reproductive biology and

The goal of animal breeding program can be realized by the current embryo breeding tech‐ nology. Using molecular biological technique, a specific gene type for the desired animal may be designed. The new developed biotechnologies to attempt to modify animal genetic traits must be conducted on animal oocyte and embryo. The embryo in vitro production and animal individual birth must depend upon animal reproductive technology. Embryology may supply a good condition to produce many high-quality embryos. Thus, the Embryo Breeding is a core subject which combines molecular biology/genetics with animal genetics and breeding as well

> Animal Embryo Breeding Science is based on the current developed embryo biotechnology. The core of current embryo biotechnology is oocyte in vitro fertilization (IVF). As human IVF technique rapidly develops in infertility treatment, not only animal IVF has offered a very valuable tool to study mammalian fertilization and early embryo development, but also its commercial applications have being increased. Based on IVF research, some new developed embryo technologies consisting of nuclear transfer, transgenesis, cloning, and stem cells, etc., can be used to create new animal individual or population, and accelerate genetic progression of animal population during the period

Animal Embryo Breeding Science is based on the current developed embryo biotechnology. The core of current embryo biotechnology is oocyte in vitro fertilization (IVF). As human IVF technique rapidly develops in infertility treatment, not only animal IVF has offered a very valuable tool to study mammalian fertilization and early embryo development, but also its commercial applications have being increased. Based on IVF research, some new developed embryo technologies consisting of nuclear transfer, transgenesis, cloning, and stem cells, etc., can be used to create new animal individual or population, and accelerate genetic progression

animal genetics and breeding as well as reproductive biology and embryology.

**<H1>Major research scope and content of Animal Embryo Breeding**

**4. Major research scope and content of animal embryo breeding**

from early oocyte stage (oogenesis) to preimplantation embryo stage (Figure 3).

embryology.

as reproductive biology and embryology.

and embryology.

188 New Discoveries in Embryology

ductive biology and embryology, animal genetics and breeding (Figure 2).

Biology Reproductive

Biology and Embryology

**Sperm, egg, embryo and somatic cell cryopreservation** 

**Figure 3.** Schematic representation of main embryo biotechnologies which can impact on the genetic improvement programs on animal embryo breeding.

Based on this schematic picture, we may focus on several fields for Animal Embryo Breeding research. In the early stage of oogenesis and oocyte maturation, some key techniques such as genomic reconstruction, nuclear transfer, androgenesis and parthenogenesis, cytoplasm replacement, etc., may be used to change animal genetic construction [5]. At the fertilization stage, the sexing sperm may be used to produce specific-sex (female or male) animal popula‐ tion to achieve better economic results [6]. Using intracytoplasmic sperm injection (ICSI) technique may make an elite performance bull with a very few sperm produce a lot of offspring. At the pronuclear stage, the foreign DNA may be injected to zygote to produce transgenic animals. In the preimplantation cleavage and blastocyst stage, preimplantation genetic diagnosis (PGD) or preimplantation genetic screening (PGS), embryo cloning, mosaic animal and embryo stem cell techniques may be used to produce various different types of animals. Also, at any stage, sperm, egg and embryo, as well as somatic cells may be cryopreserved for future use [7]. Thus, we may profile the outline of Animal Embryo Breeding study as shown in Table 1 (Table 1).



**Table 1.** Outline of Animal Embryo Breeding discipline

**Early Gamete Manipulation**

190 New Discoveries in Embryology

**Embryo Transfer**

**Embryo Cloning**

**Transgenic animals**

*Artificial Insemination*

Sperm sexing Ovulation control Superovulation

Embryo splitting Embryo sexing

*In vitro embryo production (IVP) technology*

Embryo transfer technique

Embryo blastomere cloning

**Transfer gene construct**

**Strategies for gene transfer**

b) Sperm-mediated gene transfer

a) Directly inject a gene into egg pronucleus

c) Stem-cell-mediated gene transfer (transfection) d) Retrovirus and viruses vector for gene transfer

Inserting genes Knockout genes

Somatic cell nuclear Transfer (Dolly) Embryonic stem cell nuclear transfer

Induced pluripotent stem cells (iPS) nuclear transfer

Semen collection and its storage

*Oocyte and egg cryopreservation*

Multiple ovulation (superovulation)

*In vitro* maturation (IVM) of oocytes *In vitro* fertilization (IVF) of oocytes Intracytoplasm sperm injection (ICSI) Culture of *in vitro* fertilized embryos Preimplantation embryo diagnosis

Multiple ovulation with embryo transfer (MOET)

Ultrasound-guided oocyte retrieval (TVOR) or nonsurgical ovum pick up (OPU)

### **5. Research category of animal embryo breeding**

As a new discipline, animal scientists and breeders can apply Animal Embryo Breeding Science theory to animal population to improve genetic traits, to add new benefit traits to animal body and to remove some harmful traits from animal body. Major research categories involve the following several aspects:


### **6. Application of Embryo Breeding in animal improvement program**

**1.** Genomic reconstruction by somatic cloning and parthenogenesis to produce specific animal population

When a bull or cow with elite production performance in beef cattle population is discovered, the breeding aim will be to accelerate this cow or bull reproduction to propagate a new breed of cattle. By normal breeding mating, this cow may lose half its inherent genes in its offspring. However, by the means of cloning techniques, many individuals of the same genotype can be theoretically produced. Thus, the accuracy of evaluation may be greatly increased. In spite of low cloning efficiency, many scientists are still interested in animal cloning techniques, which will eventually be used to clone very valuable animals, such as breeding stock, transgenic animals, and endangered species.

By the means of cell nucleus transfer technology, a new animal can be produced using androgenesis method [7]. Androgenesis is a male parthenogenesis in which only paternal chromosomes are kept in the embryo with the removal of the egg nucleus at the fertilization [8]. This is a reproductive pattern from two male parents. After an oocyte nucleus has been removed, a male diploid cell is transferred into this egg in which the oocyte cytoplasm will induce this diploid cell going through meiosis to become a haploid MII oocyte. After induce‐ ment, a male sperm is injected into this oocyte to produce a paternal embryo. Finally, this modified embryo will be transferred into receipt cow to produce a new individual bull with two male parents.

**5. Research category of animal embryo breeding**

transfer technique to clone this animal somatic cell.

expend its reproduction as traditional breeding program.

following several aspects:

192 New Discoveries in Embryology

animal population

animals, and endangered species.

As a new discipline, animal scientists and breeders can apply Animal Embryo Breeding Science theory to animal population to improve genetic traits, to add new benefit traits to animal body and to remove some harmful traits from animal body. Major research categories involve the

**1.** The objective of embryo breeding study is to create new animal individual or improve animal population. Based on the objective of the animal breeding program – what kind of animal traits you need in the breeding program – you may adopt an appropriate method of embryo biotechnique. For instance, if you want to add new genetic trait into animal body, you may use transgenic method to insert this gene into embryo. If you need to produce a complete same animal, clone method may be used as embryo cloning or nuclear

**2.** The technique selection of embryo breeding: based on your breeding objective, a specific technique should be selected; for instance, in transgenic program, what gene and which method should be used to produce transgenic animals. In the animal cloning program to increase animal population homogeneity, various cloning methods should be evaluated for the best cloning technique, such as embryo cloning, stem cell, or somatic cell cloning.

**3.** Inserting embryo breeding into animal breeding program. In practice, embryo breeding is a trick to produce a specific animal. By the means of transgenic tactics, a given target gene vector may be constructed and transformed to chromosome in cell. Then, a given aim-gene embryo may be formed by nucleus transfer technique. By means of the genetic screening and diagnosis on cell levels, an expected embryo with a specific genotype embryo may be determined on embryonic level. Then, this expected embryo with a specific modified gene may be transferred into animal uterus to produce a specific animal. After individual level diagnosis, the ideal animal may be placed in animal population to

**6. Application of Embryo Breeding in animal improvement program**

**1.** Genomic reconstruction by somatic cloning and parthenogenesis to produce specific

When a bull or cow with elite production performance in beef cattle population is discovered, the breeding aim will be to accelerate this cow or bull reproduction to propagate a new breed of cattle. By normal breeding mating, this cow may lose half its inherent genes in its offspring. However, by the means of cloning techniques, many individuals of the same genotype can be theoretically produced. Thus, the accuracy of evaluation may be greatly increased. In spite of low cloning efficiency, many scientists are still interested in animal cloning techniques, which will eventually be used to clone very valuable animals, such as breeding stock, transgenic

**2.** Create new genetic variation in population by genomic modification during embryogen‐ esis

The current animal breeding strategies are mainly based on the principle of selective breeding including the morphology of animal body, the application of quantitative genetics theory, the estimation of breeding value by phenotype, pedigree, BLUP (best linear unbiased prediction) method, and genetic markers. These methods mainly add genetic improvement by increasing the frequency of advantageous alleles of many loci, but actually very few of gene loci are identified. These techniques do not change gene movement from different species or genera due to reproductive barrier, while the new developed transgenic technique can remove the breeding barriers between different species or genera.

The most efficient method of transgenesis in mammals is the genetic manipulation of the pronuclear stage embryo [9]. By injecting foreign DNA into one of the two pronuclei of the zygote, the birth offspring may contain a functional foreign gene in the genome. In the last 20 years, many kinds of transgenic species have been produced for agriculture and medicine application [10]. For example, the transgenic technology in beef cattle industry may improve animals for faster growth, higher quality beef products, or disease resistance [11-13].

The transgenesis first starts with identification of the genes of interest. Current molecular biotechnology may help us to search for some interesting markers used as reference points for mapping relevant genes. These molecular markers can also be used for identification of the animals carrying the transgenes. Most of the quantitative genetic loci (QTL) are polygenic in nature but the manipulation of transgenesis is a single gene trait [14,15]. The technology holds promises in the future in moving polygenic QTL across the breeding barriers of animals. However, it is expected that molecular markers will serve as a potential tool to geneticists and breeders to evaluate the existing germplasm, and to manipulate it to create animals of desired traits [16].

**3.** Shorten generation interval by embryo in vitro production

As the oocyte in vitro maturation (IVM) and in vitro fertilization (IVF) techniques rapidly develop, the ultrasound-guided oocyte retrieval (TVOR) or nonsurgical ovum pick up (OPU) technique can retrieve many oocytes repeatedly from a cow or a heifer. As many as 1000 oocytes have been collected from one female cattle in a year [17-19]. Thus, the embryo in vitro pro‐ duction (IVP) technology has been able to promote a cow to produce more than one hundred offspring in a year and greatly accelerate herd genetic improvement speed [20]. In order to improve ordinary cattle herd, slaughterhouse ovaries also may be used as in vitro embryo production. A lot of oocytes could be obtained from slaughter house cow ovaries. After maturation, these oocytes may be inseminated with elite bull semen for in vitro fertilization [21]. Although the detail genetic backgrounds of these slaughterhouse animals are not known, these embryos have a very high genetic merit from elite bulls. Using these embryos, an ordinary cow herd could obtain at least 50% genetic improvement.

The multiple ovulation and embryo transfer (MOET) was used initially to produce more embryos from genetic elite cows in shorter time periods. Currently, the MOET breeding schemes have widely established in many countries and their use accounts for about 80% of cattle embryos transferred commercially [22]. Currently, the application of transvaginal ultrasonically guided OPU technique may significantly improve MOET scheme efficiency because about 1000 oocytes may be collected and 300 embryos may be produced *in vitro* from a cow in a year at frequent intervals using IVF technology [19]. Also, oocytes may be collected from prepubertal heifers and cattle generation interval may be shorted for 2-3 years. The combination of MOET program with OPU/IVF technique is providing a more efficient way to produce more embryos from an individual donor donor than superovulation stimulation program [23]. Thus, OPU/IVF technique greatly increases MOET breeding scheme efficiency in milk and beef industry.

**4.** Increased economy from animal population by sex selection

Animal sex selection may increase animal economical value for humans. Embryo breeding theory may provide several ways for animal sex selection, including sperm sex selection and preimplantation embryo sex selection. Sperm sex selection is to try to separate semen into Xor Y-bearing chromosome sperm by flow cytometry [24, 25]. Current sorted sperm has been successfully used in IVF for in vitro embryo production and artificial insemination in cattle [6, 26].

Another sexing pathway is to determine the sex of an embryo prior to transfer. Preimplantation genetic diagnosis (PGD) technique has become an efficient method for sex selection. Y-specific chromosome probe for polymerase chain reaction (PCR) and Fluorescent *In Situ* Hybridization (FISH) are two common methods in animal sex determination. On the ordinary farm, cattle embryos may be sexed by complete cell biopsy and PCR technique. Our clinic farm practice [7] showed that a few of trophoectoderm cells could be microbiopsied from blastocyst embryos by transzonal incision using a microsurgical blade. The mini-tube PCR was carried out for 30 minutes and the gel electrophoresis was run for 20 minutes. The sexing result could be obtained in 2 hours. These results clearly demonstrate that the microsurgical technique and subsequent PCR sex analysis allow the rapid commercial exchange of genetic resources on the basis of fresh or frozen sex-desired embryos in embryo transfer programs.

Fluorescent *in situ* hybridization (FISH) technique has also been used as embryo chromosome set (karyotype) diagnosis. A blastomere is removed from an embryo by micromanipulation, and then used to examine the embryo X/Y chromosomes by FISH. Recently, new developed technologies in PGD allow examining of all chromosomes and identifying certain genes or genetic mutations, such as the competitive genomic hybridization (CGH) and microarray analysis. More recently, novel developed Next Generation Sequencing (NGS) for preimplan‐ tation genetic screen (PGS) is now being offered clinically to provide comprehensive, accurate screening of all 24 chromosomes for selections of euploid embryos. PGS results generated are comparable to those achieved with the CGH technology, with improved accuracy, sensitivity, and resolution for more accurate detection of euploid embryos, aneuploidies, chromosome imbalances (translocations), and embryo mosaicism. NGS is a superior technology because it looks at close to 1.1 million data points on the genome compared to around 3,000 with CGH.

**5.** Preservation breeding

offspring in a year and greatly accelerate herd genetic improvement speed [20]. In order to improve ordinary cattle herd, slaughterhouse ovaries also may be used as in vitro embryo production. A lot of oocytes could be obtained from slaughter house cow ovaries. After maturation, these oocytes may be inseminated with elite bull semen for in vitro fertilization [21]. Although the detail genetic backgrounds of these slaughterhouse animals are not known, these embryos have a very high genetic merit from elite bulls. Using these embryos, an ordinary

The multiple ovulation and embryo transfer (MOET) was used initially to produce more embryos from genetic elite cows in shorter time periods. Currently, the MOET breeding schemes have widely established in many countries and their use accounts for about 80% of cattle embryos transferred commercially [22]. Currently, the application of transvaginal ultrasonically guided OPU technique may significantly improve MOET scheme efficiency because about 1000 oocytes may be collected and 300 embryos may be produced *in vitro* from a cow in a year at frequent intervals using IVF technology [19]. Also, oocytes may be collected from prepubertal heifers and cattle generation interval may be shorted for 2-3 years. The combination of MOET program with OPU/IVF technique is providing a more efficient way to produce more embryos from an individual donor donor than superovulation stimulation program [23]. Thus, OPU/IVF technique greatly increases MOET breeding scheme efficiency

Animal sex selection may increase animal economical value for humans. Embryo breeding theory may provide several ways for animal sex selection, including sperm sex selection and preimplantation embryo sex selection. Sperm sex selection is to try to separate semen into Xor Y-bearing chromosome sperm by flow cytometry [24, 25]. Current sorted sperm has been successfully used in IVF for in vitro embryo production and artificial insemination in cattle [6,

Another sexing pathway is to determine the sex of an embryo prior to transfer. Preimplantation genetic diagnosis (PGD) technique has become an efficient method for sex selection. Y-specific chromosome probe for polymerase chain reaction (PCR) and Fluorescent *In Situ* Hybridization (FISH) are two common methods in animal sex determination. On the ordinary farm, cattle embryos may be sexed by complete cell biopsy and PCR technique. Our clinic farm practice [7] showed that a few of trophoectoderm cells could be microbiopsied from blastocyst embryos by transzonal incision using a microsurgical blade. The mini-tube PCR was carried out for 30 minutes and the gel electrophoresis was run for 20 minutes. The sexing result could be obtained in 2 hours. These results clearly demonstrate that the microsurgical technique and subsequent PCR sex analysis allow the rapid commercial exchange of genetic resources on the basis of

Fluorescent *in situ* hybridization (FISH) technique has also been used as embryo chromosome set (karyotype) diagnosis. A blastomere is removed from an embryo by micromanipulation, and then used to examine the embryo X/Y chromosomes by FISH. Recently, new developed technologies in PGD allow examining of all chromosomes and identifying certain genes or

cow herd could obtain at least 50% genetic improvement.

**4.** Increased economy from animal population by sex selection

fresh or frozen sex-desired embryos in embryo transfer programs.

in milk and beef industry.

194 New Discoveries in Embryology

26].

Many animal breeders are interested in preserving bloodlines of animals, either of a rare breed, or of rare pedigrees within a breed. Therefore, Rahbek [27] put forward a preservation breeding concept to describe the purpose of preservation breeding, which is to protect genetic diversity within a species, and to preserve valuable genetic traits that may not be popular or in fashion in the present, but may be of great value in the future. In the animal embryo breeding program, two kinds of cells including reproductive cells and somatic cells may be cryopreserved in liquid nitrogen for future use. Reproductive cell cryopreservation is an important branch of embryo breeding science because it involves the preservation of gametes (sperm and oocytes), embryos, and reproductive tissues (ovarian and testicular tissues) for future use in the assisted reproductive technology. Practically, animal embryo breeding program may provide a sperm and embryo bank with the objective of avoiding genetic dilution and irreplaceable gene losses of the valuable "naturalized breeds" germplasm. It is much lower in cost than normal animal breeding, preserving rare native animal breed plan. At present, many countries have set up gene banks to store frozen embryos and semen of various animal species including native cattle, pig, and some endangered animals.

The development of embryo freezing technologies has revolutionized cattle breeding. Since then, advancements in cryobiology, cell biology, and domestic animal embryology have enabled the development of embryo preservation methodologies for our other domestic animal species, including sheep and goats. Currently, use of preserved embryos has become a routine breeding alternative for all domestic animal species. This freezing and storage methodology may provide for maternal germplasm, global genetic transport, increased selection pressure of herd genetics, and genetic resource rescue.

In the conventional breeding program, an outstanding bull may maintain normal mating for 5 years. However, if this bull semen is cryopreserved, it will extend the bull's breeding time. In embryo breeding program, when some elite bulls leave very few sperm, we may use intracytoplasmic sperm injection (ICSI) technique to inject a single sperm to an oocyte so that genetic merit embryos are obtained [28]. Also, sperm cell genome cloning technique may be used to produce many copies of a specific sperm [8]. The application of this technique to beef and dairy cattle industry has greatly increased merit bull spread in animal herd [29].

Like normal reproduction, somatic cell nuclear transfer (SCNT) starts with an egg or oocyte, but here the nucleus of the egg needs to be removed. Then the nucleus from a somatic (skin) cell is transferred into the enucleated egg which would be analogous to the sperm entering the oocyte. As this develops into a blastocyst, cells from the inner cell mass can be isolated and purified to serve as a source for pluripotent stem cells. In animal embryo breeding, somatic cell is also an important genetic resource. Therefore, the somatic cells, such as skin, hair, and other cells from rare and endangered animals may be collected and cryopreserved so that they can be used in the future.

### **7. Conclusions**

Currently, the following biotechnologies in embryology have been applied or will be applied in animal genetic improvement [9]: 1) Genomic reconstruction by somatic cloning and parthenogenesis can produce specific animal population; 2) new genetic variation in popula‐ tion can be created by genomic modification during embryogenesis, such as transgenic breeding strategies; 3) animal generation interval may be shortened by embryo in *in vitro* production; 4) economy efficiency from animal population may be significantly increased by embryo sex selection; and 5) a rare breed, or of rare pedigrees within a breed, may be efficiently preserved at low cost in liquid nitrogen. Thus, the development of modern biotechnology has brought into being the concept and theory of **Animal Embryo Breeding Science.** Understand‐ ing and applying its theory and technology will be helpful to animal scientists and students as well as animal breeders to accelerate animal improvement speed, to modify genetic construction of animal population, and to create new animal breeds.

### **Author details**

Bin Wu1,2, Linsen Zan3 , Fusheng Quan4 and Hai Wang1,2

1 Arizona Center for Reproductive Endocrinology and Infertility, Tucson, Arizona, USA

2 Yunnan Jiuzhou Hospital, Kunming, Yunnan, China

3 College of Animal Science and Technology, National Beef Cattle Improvement Center, China

4 College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, China

### **References**

[1] Jarman, MR, Clark G, Grigson C, Uerpmann HP, Ryder ML, 1976: Early animal hus‐ bandry. *The Royal Soc*. 275 (936): 85-97.

[2] Wolfer DP, Crusio WE, Lipp HP, 2002: Knockout mice: simple solutions to the prob‐ lems of genetic background and flanking genes. *Trends Neurosci* 25(7):336-40.

oocyte. As this develops into a blastocyst, cells from the inner cell mass can be isolated and purified to serve as a source for pluripotent stem cells. In animal embryo breeding, somatic cell is also an important genetic resource. Therefore, the somatic cells, such as skin, hair, and other cells from rare and endangered animals may be collected and cryopreserved so that they

Currently, the following biotechnologies in embryology have been applied or will be applied in animal genetic improvement [9]: 1) Genomic reconstruction by somatic cloning and parthenogenesis can produce specific animal population; 2) new genetic variation in popula‐ tion can be created by genomic modification during embryogenesis, such as transgenic breeding strategies; 3) animal generation interval may be shortened by embryo in *in vitro* production; 4) economy efficiency from animal population may be significantly increased by embryo sex selection; and 5) a rare breed, or of rare pedigrees within a breed, may be efficiently preserved at low cost in liquid nitrogen. Thus, the development of modern biotechnology has brought into being the concept and theory of **Animal Embryo Breeding Science.** Understand‐ ing and applying its theory and technology will be helpful to animal scientists and students as well as animal breeders to accelerate animal improvement speed, to modify genetic

and Hai Wang1,2

3 College of Animal Science and Technology, National Beef Cattle Improvement Center,

[1] Jarman, MR, Clark G, Grigson C, Uerpmann HP, Ryder ML, 1976: Early animal hus‐

4 College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, China

1 Arizona Center for Reproductive Endocrinology and Infertility, Tucson, Arizona, USA

construction of animal population, and to create new animal breeds.

, Fusheng Quan4

2 Yunnan Jiuzhou Hospital, Kunming, Yunnan, China

bandry. *The Royal Soc*. 275 (936): 85-97.

can be used in the future.

**7. Conclusions**

196 New Discoveries in Embryology

**Author details**

China

**References**

Bin Wu1,2, Linsen Zan3


### **Assisted Reproductive Technologies in Safeguard of Feline Endangered Species**

[17] Taneja M, Yang X, 1998: Promises and problems of in vitro production of embryos by

[18] Machado SA, Reichenbach HD, Weppert M, Wolf E, Gonçalves PB, 2006: The varia‐ bility of ovum pick-up response and in vitro embryo production from monozygotic

[19] Presicce GA, Xu J, Gong GC, Moreno JF, Chaubal S, Xue F, Bella A, Senatore EM, Yang XZ, Tian XC, Du FL, 2011: Oocyte source and hormonal stimulation for *In vitro* fertilization using sexed spermatozoa in cattle. *Vet Med Int*. Published online 2010

[20] Martinez HR, 2012: Assisted reproductive techniques for cattle breeding in develop‐ ing countries: A critical appraisal of their value and limitations. *Reprod Dom Anim*

[21] Wu B, Ignotz G, Currie WB, Yang X, 1997: Dynamics of maturation-promoting factor and its constituent proteins during in vitro maturation of bovine oocytes. *Bio Reprod*

[22] Thibier M, 2005: The zootechnical applications of biotechnology in animal reproduc‐

[23] Betteridge KJ, 2006: Farm animal embryo technologies: Achievements and perspec‐

[24] Blondin P, Beaulieu M, Fournier V, Morin N, Crawford L, Madan P, King WA, 2009: Analysis of bovine sexed sperm for IVF from sorting to the embryo. *Theriogenology.*

[25] Underwood SL, Bathgate R, Ebsworth M, Maxwell WMC, Evans G, 2010: Pregnancy loss in heifers after artificial insemination with frozen-thawed, sex-sorted, re-frozen-

[26] Pontes JHF, Silva KCF, Basso AC, Rigo AG, Ferreira CR, Santos GMG, Sanches BV, Porcionato JPF, Vieira PHS, Faifer FS, Sterza FAM, Schenk JL, Seneda MM, 2010: Large-scale *in vitro* embryo production and pregnancy rates from *Bos taurus*, *Bos indi‐ cus*, and *indicus-taurus* dairy cows using sexed sperm. *Theriogenology*. 74:1349-1355.

[27] Rahbek C, 1993: Captive breeding-a useful tool in the preservation of biodiversity.

[28] Hara H, Abdalla H, Morita H, Kuwayama M, Hirabayashi M, Hochi S, 2011: Proce‐ dure for bovine ICSI, not sperm freeze-drying, impairs the function of the microtu‐

[29] Abu NMAR, 2010: Intracytoplasmic sperm injection-revolution in human and animal

tion: current methods and perspectives. *Reprod Nutr Dev*. 45:235-42.

thawed dairy bull sperm. *Anim Reprod Sci* 118(1):7-12

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198 New Discoveries in Embryology

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*Biodivers Conserv*. 2, 426-437.

Natascia Cocchia, Simona Tafuri, Lucia Abbondante, Leonardo Meomartino, Luigi Esposito and Francesca Ciani

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/61004

### **Abstract**

The growth of the human population and the escalating consumption of natural resources have reduced wild habitats, modifying the existing balance of biological cycles. Therefore, *ex situ* conservation efforts have received renewed attention as a potential safeguard for species with an uncertain future in the wild. Most wild felid species are classified as rare, vulnerable, or endangered due to poaching and habitat loss. Any directed action taken by humans to enhance animal reproduction results in assisted reproductive technologies (ART) development. These technologies have been included in programs for the conservation of endangered species. Therefore, ART provide a new approach in the safeguard programs of felid biodiversity. Currently, ART mainly include Artificial Insemination (AI); *In Vitro* Embryo Production (IVEP) consisting of *In Vitro* Maturation (IVM), *In Vitro* Fertilization (IVF), *In Vitro* Culture (IVC), Embryo Transfer (ET), and Intra Cytoplasmic Sperm Injection (ICSI); gamete/ embryo cryopreservation; gamete/embryo sexing; gamete/embryo micromanipula‐ tion; Somatic Cell Nuclear Transfer (SCNT); and genome resource banking.

The domestic cat is used as a model for the ART development in *Felid* species and as a successful recipient of embryos from closely related, small, nondomestic cats. The Indian desert cat and African wildcat kittens have been born after IVF-derived embryo transfers.

The creation of the biological resource bank represents a complementary support tool for the application of ART in the *in situ* and *ex situ* conservation of endangered felids. Its chief purpose in the protection of endangered species is to preserve the maximum

© 2015 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

current genetic and biological diversity of the population by the processing and cryopreservation of germinal cells and tissues from dead animals so that these genetic recourses may be used in future reproductive projects. In humans and domestic species, it is usually possible to plan the place and time for gonad explants to recover germplasm, thereby enabling a reduction in the gonad storage time in the transport medium. In wild species, it is impossible to predict when and where the gonads can be collected. The gonads can be recovered postmortem, which entails the possibility that the collection place could be distant from a laboratory for IVEP.

In the present chapter, we will make an overview of the data from detectable literatures and focus our attention on analysis of methods utilized in ART for maximizing their efficiency in feline species.

**Keywords:** Assisted Reproductive Technologies (ART), In Vitro Embryo Production (IVEP), endangered feline species

### **1. Introduction**

The growth of the human population and the escalating consumption of natural resources have reduced wild spaces, modifying the existing balance of the biological cycles. Therefore, *ex situ* conservation efforts have received renewed attention as a potential safeguard for species with an uncertain future in the wild. Assisted Reproductive Technologies (ART), which consist of various techniques such as Artificial Insemination (AI), In Vitro Fertilization (IVF), Embryo Transfer (ET), and cryopreservation of embryos, have greatly promoted animal reproductive efficiency and have become a potential means for the conservation and management of wildlife populations threatened with extinction. [1]. Several species, such as domestic cats, dogs, and ferrets are the most popular pets, while other carnivores, like minks and foxes, have an economic value to the industry of fur farming. Tigers, bears, and other large predators have a major effect on the health of natural ecosystems. Any directed action taken by humans to enhance animal reproduction has resulted in assisted reproductive technologies (ART) development. These technologies have been included in programs for the conservation of species threatened with extinction. ART, therefore, affords investigators a new approach that they can include in the safeguard programs of felid biodiversity. Although this technique has greatly improved animal reproduction, it has not advanced beyond the rudimentary stages for use in the conservation of felines threatened with extinction [1]. As several other more widely studied species, the earliest descriptions of successful production of embryos using in vitro fertilization (IVF) in the cat occurred in the 1970s [2, 3]. Meanwhile, as IVF studies in the most popular laboratory animals, economically important domestic animals, and humans literally exploded during the last two decades of the twentieth century, the field of cat IVF experienced no comparative proliferation in publications. Publications in human IVF area represent approximately 50% of the reports from 1970 through 2000 and approximately 50% from 2001 through 2012. The majority of the total citations listed in the search on "IVF in cats", were published in the current century [4]. Most wild felid species are classified as rare, vulnerable, or endangered due to poaching and habitat loss. A great deal of progress has been made in recent years toward the development of assisted reproductive techniques (ART) for species conservation [5, 6] In fact, ART have been included in the programs for the conservation of species threatened with extinction, but the effectiveness of this application to semi domestic, not domestic, and particularly endangered species such as felines, remains consistently low [4]. These ART tools are potentially important for the captive breeding programs of selected felid species. The domestic cat is often used as a model for developing these techniques in the felid species [5, 6]. In the last few years, the ART application in the domestic cat has allowed researchers to obtain 70% metaphase II oocytes after *in vitro* maturation (IVM) [7], and 80% cleaved embryos after IVF and 70–80% after Intra Cytoplasmic Sperm Injection (ICSI) [8]. However, only 10% cleaved embryos could develop to blastocyst [8-11]. Kittens have been born after embryo transfer [5]. *In vivo* embryo collection is the most popular technique for embryo production, in spite of the fairly rapid development and adoption of *in vitro* embryo production. The domestic cat could also be used as a successful recipient of embryos from closely related small nondomestic cats. In fact, some evidences have confirmed the birth of African wildcat and Indian desert cat kittens after the transfer of IVF-derived embryos in female domestic cats [12]. Recently, African wildcat kittens were produced after the transfer of embryos derived by fusion of adult somatic cells from one species with enucleated oocytes of a closely related species (domestic cat) [13]. However, the application of ART, which tries to produce a single viable offspring, unfortunately, cannot justify the expense, labor, and the handling of animals which is associated with stress. Thus, "ART" must be applied within the programs of population management established to have a real impact on conservation. The immediate value of ART is to assist those responsible for the maintenance of viable populations of felines in captivity. Its wider application will require the creation of a global network of qualified scientists and veterinarians willing to perform these procedures as a reproduction service for keeping cats themselves [1].

### **2. Reproductive cycles of wild felids**

current genetic and biological diversity of the population by the processing and cryopreservation of germinal cells and tissues from dead animals so that these genetic recourses may be used in future reproductive projects. In humans and domestic species, it is usually possible to plan the place and time for gonad explants to recover germplasm, thereby enabling a reduction in the gonad storage time in the transport medium. In wild species, it is impossible to predict when and where the gonads can be collected. The gonads can be recovered postmortem, which entails the possibility

In the present chapter, we will make an overview of the data from detectable literatures and focus our attention on analysis of methods utilized in ART for

**Keywords:** Assisted Reproductive Technologies (ART), In Vitro Embryo Production

The growth of the human population and the escalating consumption of natural resources have reduced wild spaces, modifying the existing balance of the biological cycles. Therefore, *ex situ* conservation efforts have received renewed attention as a potential safeguard for species with an uncertain future in the wild. Assisted Reproductive Technologies (ART), which consist of various techniques such as Artificial Insemination (AI), In Vitro Fertilization (IVF), Embryo Transfer (ET), and cryopreservation of embryos, have greatly promoted animal reproductive efficiency and have become a potential means for the conservation and management of wildlife populations threatened with extinction. [1]. Several species, such as domestic cats, dogs, and ferrets are the most popular pets, while other carnivores, like minks and foxes, have an economic value to the industry of fur farming. Tigers, bears, and other large predators have a major effect on the health of natural ecosystems. Any directed action taken by humans to enhance animal reproduction has resulted in assisted reproductive technologies (ART) development. These technologies have been included in programs for the conservation of species threatened with extinction. ART, therefore, affords investigators a new approach that they can include in the safeguard programs of felid biodiversity. Although this technique has greatly improved animal reproduction, it has not advanced beyond the rudimentary stages for use in the conservation of felines threatened with extinction [1]. As several other more widely studied species, the earliest descriptions of successful production of embryos using in vitro fertilization (IVF) in the cat occurred in the 1970s [2, 3]. Meanwhile, as IVF studies in the most popular laboratory animals, economically important domestic animals, and humans literally exploded during the last two decades of the twentieth century, the field of cat IVF experienced no comparative proliferation in publications. Publications in human IVF area represent approximately 50% of the reports from 1970 through 2000 and approximately 50% from 2001 through 2012. The majority of the total citations listed in the search on "IVF in cats",

that the collection place could be distant from a laboratory for IVEP.

maximizing their efficiency in feline species.

(IVEP), endangered feline species

**1. Introduction**

200 New Discoveries in Embryology

Knowledge of anatomical features and hormones and the cycles of wild reproductive feline is the ability to track feline reproductive activity. The hormone measure is a key technique to develop successful ex situ breeding programs to determine the reproductive activity of domestic feline. The ovaries in the wild felid and the domestic cat are caudal to the respective kidneys and connected proximally by the suspensor ligament and dorsally by the mesovaria. The oviducts are covered by the mesosalphinx that forms, laterally to the ovaries – an ovarian bursa. Each oviduct cranially is localized in the medial aspect of the ovarian bursa; caudally it is located in the lateral aspect before terminating at the uterotubal junction. The mesome‐ trium suspends dorsally the horns of the uterus bicornuate. The uterine body is divided internally by an incomplete septum. The cervix is short, but it opens at an angle close to the vaginal orifice. The lips of the vulva are located just below the anus. After fertilization, the blastocysts are distributed evenly along the uterine horns, with an efficient result of transu‐

terine migration. Cats have a zonary endotheliochorial type placenta. Regarding the hormone profile of wild felines, reasonable results were obtained by noninvasive monitoring of steroid [14]. Reproductive cycle models of ovarian steroids have now been published about half of nondomestic felid species, by analysis of fecal steroid metabolites. There are four phases of the oestrous cycle in the cat: proestrus, oestrus, diestrus, and anestrus (or interestrus) [15]. Proestrus usually lasts less than a day, and is associated with the presence of ovarian follicles, increased circulating estrogens, no sexual interest but occasionally there could be copulation with the male. Oestrous has maximum concentrations of follicular estradiol. It is characterized by coitus and, depending on the species, by special and typical behaviors such as vocalization, rubbing, rolling, lordosis, and foot stamping. The release of gonadotrophins-releasing hormone (GnRH) from the basal medial hypothalamus and successive waves of luteinizing hormone (LH) from the anterior pituitary gland, are considered necessary in most felines [16, 17]. This cascade of events will result in ovulation after mating. Surges of estrogens distinguish oestrous from interestrus periods, with cycles ranging from 2 to 4 weeks and oestrus lasting 3–10 days. Cats have historically been categorized as having "induced ovulation," that is, requiring mating to stimulate ovulation. Nevertheless, we now know that felids exhibit a range of ovulatory patterns, from almost exclusively induced to manifold combinations of induced and spontaneous ovulation. There are differences not only across species, but also between individuals within a species [14]. In fact, spontaneous increase in progestogens after oestrogen surges is rare or inexistent in the tiger (*Panthera tigris*), snow leopard (*Panthera uncia*), ocelot (*Leopardus pardalis*) puma (*Felis concolor*), tigrina (*Leopardus tigrinus*), cheetah (*Acinonyx jubatus*), and lynx (*Lynx pardinus, Lynx canadensi, Lynx lynx*). It happens, at least occasionally, in the lion (*Panthera leo*), Pallas'cat (*Otocolobus manul*), leopard (*Panther pardus*), fishing cat (*Prionailurus viverrinus*), and regularly in the margay (*Leopardus wiedii*), clouded leopard (*Neofelis nebulosa*), and domestic cat. In some species of certain taxonomy, the spontaneous ovulation occurs in a more prevalent way when the females are kept together, while in others the provoked ovulation occurs if they are kept in individual housings. Thus, within the same taxonomy, ovulatory mechanisms are regulated to different degrees depending on species and individual-specific responses to psychosocial and/or physical stimuli.

Several studies report the domestic cat be seasonally poly oestrous animal with positive photoperiod under natural [16]. In general, ovarian cyclic activity and reproductive functions are reduced under decreasing photoperiod and starts again after exposure to increasing light. In the cat, melatonin seems to regulate photoperiod-induced seasonality. The highest concen‐ trations happen during the dark phase [17]. Reproduction is in someway seasonal in many nondomestic felids like the tiger, pallas'cat, clouded leopard, snow leopard, and lynx (Table 1). The follicular activity, conversely, is not influenced by season in lions, bobcats, pumas, leopards, margays, tigrinas, ocelots, jaguars, and fishing cats [14]. Progestogen concentrations during pregnant and nonpregnant luteal phases are quantitatively similar in nondomestic felids and domestic cats [15].

Felids express marked variations in reproductive mechanisms among species. Two character‐ istics impact both natural and assisted breeding efforts: effect of seasonality on reproduction and identifying the type of ovulation (induced *vs* spontaneous). Developing ovulation


**Table 1.** Particularity of reproductive cycles of *domestic cat* and some *wild felids.*

induction protocol, with consistent responses, is high priority. Furthermore, it is important to ensure an optimal maternal environment for fertilization and embryo development. Downregulating endogenous ovarian activity and synchronizing time of oestrus are steps of reproductive cycle that need to be controlled and reinforced [1, 18]. We also need a quick and reliable test for diagnosing pregnancy, preferably a noninvasive method.

### **3. Oestrus induction in felids**

terine migration. Cats have a zonary endotheliochorial type placenta. Regarding the hormone profile of wild felines, reasonable results were obtained by noninvasive monitoring of steroid [14]. Reproductive cycle models of ovarian steroids have now been published about half of nondomestic felid species, by analysis of fecal steroid metabolites. There are four phases of the oestrous cycle in the cat: proestrus, oestrus, diestrus, and anestrus (or interestrus) [15]. Proestrus usually lasts less than a day, and is associated with the presence of ovarian follicles, increased circulating estrogens, no sexual interest but occasionally there could be copulation with the male. Oestrous has maximum concentrations of follicular estradiol. It is characterized by coitus and, depending on the species, by special and typical behaviors such as vocalization, rubbing, rolling, lordosis, and foot stamping. The release of gonadotrophins-releasing hormone (GnRH) from the basal medial hypothalamus and successive waves of luteinizing hormone (LH) from the anterior pituitary gland, are considered necessary in most felines [16, 17]. This cascade of events will result in ovulation after mating. Surges of estrogens distinguish oestrous from interestrus periods, with cycles ranging from 2 to 4 weeks and oestrus lasting 3–10 days. Cats have historically been categorized as having "induced ovulation," that is, requiring mating to stimulate ovulation. Nevertheless, we now know that felids exhibit a range of ovulatory patterns, from almost exclusively induced to manifold combinations of induced and spontaneous ovulation. There are differences not only across species, but also between individuals within a species [14]. In fact, spontaneous increase in progestogens after oestrogen surges is rare or inexistent in the tiger (*Panthera tigris*), snow leopard (*Panthera uncia*), ocelot (*Leopardus pardalis*) puma (*Felis concolor*), tigrina (*Leopardus tigrinus*), cheetah (*Acinonyx jubatus*), and lynx (*Lynx pardinus, Lynx canadensi, Lynx lynx*). It happens, at least occasionally, in the lion (*Panthera leo*), Pallas'cat (*Otocolobus manul*), leopard (*Panther pardus*), fishing cat (*Prionailurus viverrinus*), and regularly in the margay (*Leopardus wiedii*), clouded leopard (*Neofelis nebulosa*), and domestic cat. In some species of certain taxonomy, the spontaneous ovulation occurs in a more prevalent way when the females are kept together, while in others the provoked ovulation occurs if they are kept in individual housings. Thus, within the same taxonomy, ovulatory mechanisms are regulated to different degrees depending on species and

individual-specific responses to psychosocial and/or physical stimuli.

felids and domestic cats [15].

202 New Discoveries in Embryology

Several studies report the domestic cat be seasonally poly oestrous animal with positive photoperiod under natural [16]. In general, ovarian cyclic activity and reproductive functions are reduced under decreasing photoperiod and starts again after exposure to increasing light. In the cat, melatonin seems to regulate photoperiod-induced seasonality. The highest concen‐ trations happen during the dark phase [17]. Reproduction is in someway seasonal in many nondomestic felids like the tiger, pallas'cat, clouded leopard, snow leopard, and lynx (Table 1). The follicular activity, conversely, is not influenced by season in lions, bobcats, pumas, leopards, margays, tigrinas, ocelots, jaguars, and fishing cats [14]. Progestogen concentrations during pregnant and nonpregnant luteal phases are quantitatively similar in nondomestic

Felids express marked variations in reproductive mechanisms among species. Two character‐ istics impact both natural and assisted breeding efforts: effect of seasonality on reproduction and identifying the type of ovulation (induced *vs* spontaneous). Developing ovulation In the late of 1970s, various doses and single versus multiple treatments with either a pituitary extract of porcine FSH (approximately 10–20 mg) or eCG for stimulation of follicular devel‐ opment and induction of oestrus was evaluated [15]; 2.0 mg FSH per day until oestrus was observed to be the optimal dose of FSH despite the elevated average ovulation rate and the presence of residual follicles observed after treatment. Cats in the latter group were given the optimal dose of FSH as determined previously [15]: 2.0 mg/ day for 5 days ¼ 10 mg FSH. In the mid-1980s, the Center for Reproduction of Endangered Wildlife of the Cincinnati Zoo established a domestic cat colony model for developing assisted reproductive technologies to apply in conservation efforts for endangered species. The domestic cat, in addition to its prototypical role, was envisioned as a potential recipient of embryos from other species of similarly sized nondomestic cats, of which most are classified as threatened or vulnerable to extinction. In view of previous results in exogenous gonadotropins for oestrous induction, initial emphasis was directed at determining optimal FSH treatment regimes for ovarian follicular stimulation. In a 1987 published article [19] on ovarian response and embryo recovery after treatment with various doses of FSH (2.75–8.0 mg total) and hCG (0–1500 IU) and natural mating, the greatest average number of viable embryos (15.8 morulae and blastocysts) was recovered from the group receiving 4.0 mg FSH/750 IU hCG. Unexpectedly, there was no difference in the average number of viable embryos recovered from donors given the least amount of FSH (2.75 mg total) versus the greatest amount (8.0 mg total): 6.9 versus 7.9. Also, in 1988, we made our one and only attempt to apply the same methods to a species of nondo‐ mestic cat, the serval (*Leptailurus serval*). After daily FSH treatment, at the time of ovulation induction (with hCG), the female was paired with a male. Seven days later, both uterine horns were flushed, but only degenerating ova (>30) were recovered. The ova were examined microscopically, but no sperm were seen, either attached to or penetrating into the zona pellucida. The mating failure was persuasive evidence that, to achieve our goal of applying assisted breeding technology to nondomestic cats, a program to develop methods for in vitro fertilization/embryo culture in cats would be essential. Coincidently, the first report on the birth of kittens after transfer of IVF-derived embryos to recipient females was published at this time [20]. Moreover, repeated treatment of domestic cats with eCG and hCG may cause an immune-mediated refractoriness to ovarian stimulation, dictating that the suitability of these hormonal combinations should be further investigated [1]. Similarly, protocols using porcine FSH and LH resulted in reduced numbers of follicles at the second treatment as compared with the first, possibly due to a humoral immune response [4]. By considering the feasibility of fecal steroid analyses with radioimmunoassay [14] combined with sexual behavior and ultrasonographic images, it is possible to determine the more ideal time for oocyte recovery by laparoscopy, without the use of exogenous gonadotropins.

### **4. Gamete recovery from nondomestic felids**

The first step for ART development is the gamete recovery. Several methods have been reported for semen collection in animals, such as the use of an artificial vagina [21], digital masturbation of the penile bulb and electroejaculation [22], but only electroejaculation method may be used for gamete recovery from wild felids. In any case, the application on nondomestic cats is based on learning how to use these methods in the domestic cats. Electroejaculation is to obtain both epidydimal spermatozoon and spermatogonial germ cells. In female, oocytes are retrieved and recovered from both antral and preantral follicles in ovarian tissue trans‐ plantation [23].

### **5. Male gamete recovery in felids**

With wild carnivores, electroejaculation is the method of choice due to the difficulty and risks involved in handling these animals. Electroejaculation occurs after introducing of a transrectal probe with three electrodes, connected to an electric stimulator that provokes a controlled electric stimulation to allow the ejaculatory reflex to work. The nerves that supply the reproductive organs are stimulated by a weak electric current. The probe is inserted 7–9 cm into the rectum and the electrodes are directed ventrally. It is necessary to take care to evacuate any feces from the rectum for this kind of manipulation. [24]. Different protocols of electroe‐ jaculation have been used by many researchers [15]. The authors reported three series for a total of 80 electric stimulations. The three series were divided in: 30 stimuli (10 stimuli at 2–4 V series 01), 30 stimuli (10 stimuli at 3–5 V series 02), and 20 stimuli (10 stimuli at 5 and 6 V series 03) for the collection of semen from South African cheetahs (*Acinonyx jubatus*), with 5 min intervals between the series. The animal responds to the stimuli with a rigid extension of the hind legs. If this reaction is not seen in series 01 or if stronger stimulation is observed, the electrode may not be in the proper position in the rectum, or there may be interference in the current transmission due to the presence of feces. To collect semen, a gentle pressure applied at the penile base should allow for penile extrusion, and the ejaculate is collected into a prewarmed test tube that has been placed over the glans penis. Using electroejaculation has collected the semen from more than 28 cat species [25]. Moreover, some researchers have reported successful semen collection from wild felids by using electroejaculation, such as tigers (*Panthera tigris*), snow leopards (*Panthera uncia*), Indian leopards (*Panthera pardus*), caracals (*Caracal caracal*), jaguars (*Panthera onca*), ocelots (*Leopardus pardalis*), margays (*L. wiedii*), and tigrinas (*L. tigrinus*). The electroejaculation has been used to collect semen from nondomestic felids [4] and the semen has been cryopreserved. After thawing, in lions (*Panthera leo*), jaguars (*P. onca*), leopards (*Neofelis nebulosa*), cheetahs (*A. jubatus*), and leopard cats (*Felis bengalensis*) 25–50% sperm motility was preserved, and in the latter, a 70% sperm motility was maintained. Furthermore, they reported finding lesser values of sperm motility, ranging between 1 and 20% post-thaw, for Geoffroy's cats (*Felis geoffroy*), Indian tigers (*P. tigris*), and ocelots (*Felis pardalis*), but unfortunately, the spermatozoa from gold cats (*Felis aurata*) did not survive cryopreservation. In addition, 40% sperm motility post-thaw in Siberian tigers (*P. tigris*) was obtained, in semen collected with electroejaculation by [4]. The epididymis is an anatomical component of the male reproductive tract and is connected to the testicle. One of its main functions is the storage of spermatozoa for ejaculation [25]. Current technologies allow semen to be collected directly from the epididymis and this seems to be a viable alternative method for obtaining gametes from animals that have recently died or from animals unable to ejaculate (Figs. 1, 2, 3). It has been suggested that viable epididymal spermatozoa from Iberian deer (*Cervus elaphus*) could be collected in the 10–20 h postmortem period. However, it must be noted that this could vary depending on the temperature conditions and the weather where the procedure is being executed [26]. Comparing epididymal spermatozoa from domestic cats and ejaculated spermatozoa, it was verified that epididymal spermatozoa require less capacitating time as compared with those ejaculated and are able to penetrate feline oocytes 20 min after in vitro insemination [27, 28]. Fresh feline epididymal spermatozoa were able to fertilize oocytes in vitro, promoting 40.7% cleavage rate. After freezing, a 26% cleavage rate was obtained. After intracytoplasmic sperm injection (ICSI) using feline frozen epididymal spermatozoa, 34.9% of embryos have developed to the morale stage, indicating that sperma‐ tozoa with minimal motility could be used in assisted reproductive techniques [29]. Also, the unilateral intrauterine artificial insemination with frozen–thawed epididymal semen from cats may obtain 23% conception rate [30]. For nondomestic cats, [31] were able to collect sperma‐ tozoa from the finely minced cauda epididymus of leopards (*P. pardus*), tigers (*P. tigris*), lions (*P. leo*), pumas (*F. concolor*) and jaguars (*P. onca*). The samples were treated as described by [32], washing the spermatozoa in Hank's balanced salt solution and extended in medium M199 supplemented with 2.5 mmol/l sodium lactate and 0.4% bovine serum albumin. Progressively motile spermatozoa were 60–85% depending on the various felids. In the same study, the epididymal semen was frozen, and thawing motility is between 25 and 65% for the different

in 1988, we made our one and only attempt to apply the same methods to a species of nondo‐ mestic cat, the serval (*Leptailurus serval*). After daily FSH treatment, at the time of ovulation induction (with hCG), the female was paired with a male. Seven days later, both uterine horns were flushed, but only degenerating ova (>30) were recovered. The ova were examined microscopically, but no sperm were seen, either attached to or penetrating into the zona pellucida. The mating failure was persuasive evidence that, to achieve our goal of applying assisted breeding technology to nondomestic cats, a program to develop methods for in vitro fertilization/embryo culture in cats would be essential. Coincidently, the first report on the birth of kittens after transfer of IVF-derived embryos to recipient females was published at this time [20]. Moreover, repeated treatment of domestic cats with eCG and hCG may cause an immune-mediated refractoriness to ovarian stimulation, dictating that the suitability of these hormonal combinations should be further investigated [1]. Similarly, protocols using porcine FSH and LH resulted in reduced numbers of follicles at the second treatment as compared with the first, possibly due to a humoral immune response [4]. By considering the feasibility of fecal steroid analyses with radioimmunoassay [14] combined with sexual behavior and ultrasonographic images, it is possible to determine the more ideal time for oocyte recovery

The first step for ART development is the gamete recovery. Several methods have been reported for semen collection in animals, such as the use of an artificial vagina [21], digital masturbation of the penile bulb and electroejaculation [22], but only electroejaculation method may be used for gamete recovery from wild felids. In any case, the application on nondomestic cats is based on learning how to use these methods in the domestic cats. Electroejaculation is to obtain both epidydimal spermatozoon and spermatogonial germ cells. In female, oocytes are retrieved and recovered from both antral and preantral follicles in ovarian tissue trans‐

With wild carnivores, electroejaculation is the method of choice due to the difficulty and risks involved in handling these animals. Electroejaculation occurs after introducing of a transrectal probe with three electrodes, connected to an electric stimulator that provokes a controlled electric stimulation to allow the ejaculatory reflex to work. The nerves that supply the reproductive organs are stimulated by a weak electric current. The probe is inserted 7–9 cm into the rectum and the electrodes are directed ventrally. It is necessary to take care to evacuate any feces from the rectum for this kind of manipulation. [24]. Different protocols of electroe‐ jaculation have been used by many researchers [15]. The authors reported three series for a total of 80 electric stimulations. The three series were divided in: 30 stimuli (10 stimuli at 2–4 V series 01), 30 stimuli (10 stimuli at 3–5 V series 02), and 20 stimuli (10 stimuli at 5 and 6 V

by laparoscopy, without the use of exogenous gonadotropins.

**4. Gamete recovery from nondomestic felids**

**5. Male gamete recovery in felids**

plantation [23].

204 New Discoveries in Embryology

species. The frozen semen was then submitted to in vitro fertilization and 18.5% developed to 8-cell embryo. Similarly, some evidences showed that frozen epididymal spermatozoa from jaguars were able to penetrate heterologous zona-free oocytes.

**Figure 1.** Epididymal sperms from dead *Panthera pardus.*

Spermatogenesis is a complex and very efficient process with the mitosis and the differentia‐ tion of spermatogonial stem cells in the basal membrane of seminiferous tubules where they are supported by Sertoli cells [33]. The spermatogonial stem cells in mammals are unique, and thus they can maintain their proliferation in adults: the genetic material can be passed from a generation to the subsequent one. Therefore, these cells are a valuable source for medical research, biological experimentation, agricultural biotechnology, and genetic modification of the species [34]. Recent studies on their recovery and cryopreservation showed the perspective of application in the conservation of genetic material from endangered animal species. Present methods described for spermatogonial isolation from fragments of collected testis consists of elutriation or sedimentation rate in a gradient of bovine serum albumin under gravity force action [35]. Some other isolation techniques have been proposed as immunological markers for posterior magnetic cellular separation [36]. After collection, germ cells can remain for several months in tissue culture media, only resuming spermatogenesis afterward in an environment that provides favorable conditions for their expansion and differentiation [37]. The favorable conditions are generally provided by transplant to other organisms [34]. The first success in the spermatogonial transplant was described by [38]. They showed that the microinjection of a cell heterogeneous suspension of mouse testis into the seminiferous tubules of a recipient sterile mouse resulted in spermatogenesis in the injected animal. After this study, several other researchers showed real possibilities such as the spermatogonial culture among different species: the xenograft [39]. It seems that cryopreservation of testis cell suspensions could be the greatest promise for the storage of germ cells to be used later in transplants. Indeed, after cryopreservation, spermatogenesis can continue [39]. In spite of the progress in this field, some elements remain to be controlled, such as the quantity of germ cells to be Assisted Reproductive Technologies in Safeguard of Feline Endangered Species http://dx.doi.org/10.5772/61004 207

species. The frozen semen was then submitted to in vitro fertilization and 18.5% developed to 8-cell embryo. Similarly, some evidences showed that frozen epididymal spermatozoa from

Spermatogenesis is a complex and very efficient process with the mitosis and the differentia‐ tion of spermatogonial stem cells in the basal membrane of seminiferous tubules where they are supported by Sertoli cells [33]. The spermatogonial stem cells in mammals are unique, and thus they can maintain their proliferation in adults: the genetic material can be passed from a generation to the subsequent one. Therefore, these cells are a valuable source for medical research, biological experimentation, agricultural biotechnology, and genetic modification of the species [34]. Recent studies on their recovery and cryopreservation showed the perspective of application in the conservation of genetic material from endangered animal species. Present methods described for spermatogonial isolation from fragments of collected testis consists of elutriation or sedimentation rate in a gradient of bovine serum albumin under gravity force action [35]. Some other isolation techniques have been proposed as immunological markers for posterior magnetic cellular separation [36]. After collection, germ cells can remain for several months in tissue culture media, only resuming spermatogenesis afterward in an environment that provides favorable conditions for their expansion and differentiation [37]. The favorable conditions are generally provided by transplant to other organisms [34]. The first success in the spermatogonial transplant was described by [38]. They showed that the microinjection of a cell heterogeneous suspension of mouse testis into the seminiferous tubules of a recipient sterile mouse resulted in spermatogenesis in the injected animal. After this study, several other researchers showed real possibilities such as the spermatogonial culture among different species: the xenograft [39]. It seems that cryopreservation of testis cell suspensions could be the greatest promise for the storage of germ cells to be used later in transplants. Indeed, after cryopreservation, spermatogenesis can continue [39]. In spite of the progress in this field, some elements remain to be controlled, such as the quantity of germ cells to be

jaguars were able to penetrate heterologous zona-free oocytes.

206 New Discoveries in Embryology

**Figure 1.** Epididymal sperms from dead *Panthera pardus.*

**Figure 2.** Testis and epididymus (a) and excised epididymus (b) of *Panthera pardus* collected 6 h postmortem; testis and epididymus of domestic cat and *Panthera onca* (c).

transplanted, formation of antibodies against spermatogonial cells by the recipient [40], and poor quality of cells that have developed using these procedures [41]. There is also a problem concerning xenograft related to the different time of spermatogenesis in each species [26, 42]. However, the complete spermatogenesis was observed after transplantation of testicular tissue fragments from species that are phylogenetically more distant, such as pigs and goats, into castrated immunodeficient mice. The new reproductive technologies on stem cells offer several potential advantages for carnivorous species. For example, the development of lines of embryonic stem cells in cats and dogs would allow the creation of a generation of transgenic animal models, which could be used to improve the health of both animals and humans. Techniques such as testis xenografting spermatogonial and stem cell transplantation offer new approaches to diffuse genetically valuable individual males, even if they should die before producing sperm. Therefore, these techniques could be applied to biomedical research, as well as to the programs for the conservation of endangered carnivore species. Recently, spermato‐ gonial stem cell transplantation has been performed in a recipient able to produce sperm of donor genetic origin [26]. Sperm production, from prepubertal testis tissue from both ferrets and cats, was obtained from testis xenografting. These first steps reinforce the need for research on stem cell technologies and for complementary technologies of carnivore assisted repro‐ duction, so clinical benefits and the largest array of research can be achieved [26].

**Figure 3.** Ultrasound guided epididymal sperms collection in *Panthera pardus*.

### **6. Female gamete collection in felids**

transplanted, formation of antibodies against spermatogonial cells by the recipient [40], and poor quality of cells that have developed using these procedures [41]. There is also a problem concerning xenograft related to the different time of spermatogenesis in each species [26, 42]. However, the complete spermatogenesis was observed after transplantation of testicular tissue fragments from species that are phylogenetically more distant, such as pigs and goats, into castrated immunodeficient mice. The new reproductive technologies on stem cells offer several potential advantages for carnivorous species. For example, the development of lines of embryonic stem cells in cats and dogs would allow the creation of a generation of transgenic animal models, which could be used to improve the health of both animals and humans. Techniques such as testis xenografting spermatogonial and stem cell transplantation offer new approaches to diffuse genetically valuable individual males, even if they should die before producing sperm. Therefore, these techniques could be applied to biomedical research, as well as to the programs for the conservation of endangered carnivore species. Recently, spermato‐ gonial stem cell transplantation has been performed in a recipient able to produce sperm of donor genetic origin [26]. Sperm production, from prepubertal testis tissue from both ferrets and cats, was obtained from testis xenografting. These first steps reinforce the need for research on stem cell technologies and for complementary technologies of carnivore assisted repro‐

208 New Discoveries in Embryology

duction, so clinical benefits and the largest array of research can be achieved [26].

**Figure 3.** Ultrasound guided epididymal sperms collection in *Panthera pardus*.

The ovarian follicular population seems to be made up of thousands of follicles in different mammalian females. Therefore, oocyte retrieval represents a rich source of genetic material to be used for genetic bank and assisted reproductive techniques in endangered species preser‐ vation, mainly in relation to the possibility of collecting material originated from postmortem or convalescent animals. The development of efficient methods for in vitro maturation (IVM) or fertilization (IVF) of oocytes collected postmortem or through ovariectomy is an important tool to prevent the species extinction [43-45]. Thus, IVM and IVF techniques are adjusted for several nondomestic animals [5] based on systematic studies in domestic animals [46] includ‐ ing wild carnivores. Moreover, application of oocyte and ovary tissue cryopreservation will help in the conservation of several animal species, with the objective of maintaining biodiver‐ sity [47]. Further, ultrasonographic images of the reproductive tract offer new opportunities for induction of sexual cycles and ovulation, adoption of superovulating regimens, as well as the ovum pickup application. Ovarian follicles are then visualized on a monitor, allowing oocyte collection by puncturing the follicles with a fine needle connected to a tube collector. The collected oocytes could be used in IVM and IVF [48, 49]. This technique is extensively used for oocyte collection in cattle and the findings indicate the possibility of repeated collections in both pregnant and nonpregnant females [50]. Concerning carnivorous species, ovum pickup using ultrasonography has yet to be reported. This may be due to the difficulty of ovarian visualization, because in bitches the ovary is surrounded by a pouch rich in conjunctive tissue [23]. Furthermore, there are no commercial probes developed for intravaginal use in either canids or felids. However, in spite of this difficulty, the presence of antral ovarian follicles can be detected by the fluid accumulation in the antral cavity [51]. A success in the follicular and corpora luteal visualization in ovaries of female African wild dogs (*L. pictus*) was reported by transrectal ultrasonography, suggesting the possibility of oocyte puncture in carnivores too [52]. The adaptation of this technique would be an important alternative, because it is a noninvasive procedure and it could allow oocyte collection without the risks involved with surgical procedures. The potential of ultrasonography is underestimated by researchers for assisted reproduction in endangered canid and felid species. Another possibility for oocytes retrieval is laparoscopy. It is the lowest invasive procedure commonly used for intrauterine deposition of frozen–thawed semen in domestic dogs [23] and cats [54]. For domestic cats, [27] reported the laparoscopic collection of oocytes, which were subject to fertilization in vitro with ejaculated semen. In this study, when the developing embryos reached the 4-cell stage, they were transferred to the oviduct of oocyte donors. Thus, five of the six cats receiving embryos became pregnant. According to [53], laparoscopy is effective in the evaluation of reproductive status, particularly the ovarian anatomy and function, direct visual biopsy of internal organs, and as a surgical means of fertility control. In wild felids, [55] reported the laparoscopic visualization of changes in the reproductive tract during ovarian stimulation by gonadotropins in the ocelot (*F. pardalis*). Moreover, the multiple laparoscopic oocyte retrievals was success‐ fully performed in caracal (*C. caracal*) after repeated ovarian stimulation with equine (eCG) and human (hCG) chorionic gonadotropin [4]. Embryos could also be reliably produced in vitro using cryopreserved spermatozoa and live offspring could be produced after embryo transfer. It was suggested that the collection of ovaries from tigers (*P. tigris*), lions (*P. leo*), pumas (*F. concolor*), cheetahs (*A. jubatus*), leopards (*P. pardus*), and jaguars (*P. onca*) could be accomplished by ovary dissection up to 8 h after the death of these animals, by mechanical follicle isolation [56]. The best results were obtained with lion oocytes, fertilized by lion sperm, with a 31.6% (18/44) conception rate. It was demonstrated that leopard oocytes can be fertilized by domestic cat sperm and used in IVF procedures to produce 22% (2/9) 8-cell embryos. Otherwise, domestic cat oocytes can be fertilized by leopard spermatozoa, producing 19.5% (8/41) 8-cell embryos. Also oocyte collection from domestic and nondomestic cats by laparot‐ omy and posterior ovary dissection was successfully performed. These oocytes were submitted to IVF and then transferred to recipient females [57]. The main result obtained in this study was the interspecies embryo transfer from an Indian desert cat (*Felis silvestris ornata*) embryo to a domestic cat (*F. catus*), which resulted in the birth of two kittens. Afterward, the oocyte collected from domestic cat ovaries after ovariectomy were used to demonstrate that mor‐ phology of the oocyte ooplasm can affect in vitro maturation, as well as the gonadotropin supplementation [12]. According to the morphological aspect by stereomicroscopic exam, cumulus–oocyte complexes were classified as mature, immature, or degenerated. Besides the successful embryo production by IVF using this approach, light and electron microscopic evaluations revealed that ovarian stimulation followed by follicular aspiration resulted in a heterogenous oocyte population with respect to meiotic maturation. The correct assessment of the oocyte maturation status is difficult to perform through stereomicroscopical exam [58]. Oocytes can be preserved if they are not immediately submitted to IVF. However, [59] observed that cooling could cause chromosomal anomalies in mature oocytes, as a conse‐ quence of the temperature decrease on meiotic fusion. [60] performed the IVM of domestic feline oocytes, previously kept under refrigeration at 4 °C for 24 h, and they did not observe deleterious effects of storage on oocyte meiotic progression. Moreover, [61] demonstrated that even brief (2–3 weeks) salt storage significantly affects cat oocyte penetration rate, and the penetration continues to decline as storage duration increases to 2–3 months. However, the authors hypothesized that the composition of the solution may have contributed to reduce sperm penetration. For canine species, [62] found that oocyte storage in hypertonic salt solution damages the zona pellucida, reducing the sperm penetration rates. In mice [63], rabbits [64], and bovines [65], it was possible to obtain the birth of normal offspring following IVF after thaw. In domestic felines, [66] demonstrated that the mature oocyte could be cryopreserved and, soon after, fertilized in vitro with success. The maturation of oocyte recovered from antral follicles is an efficient method for the use of haploid female material and the oocyte activation in initial phases of development is a possible tool that also increases the efficiency of the oocyte utilization [56]. The preantral ovarian follicles (PAF) represent 90% of the follicular population in mammals [67]. Small PAF recovered from the ovaries collected from postmortem animals or through ovariectomy, therefore, are a rich oocyte source, because they can mature in vitro (Fig. 4). [68] reported that feline PAF are capable of developing in vitro to the antral phase. Moreover, [69] demonstrated the isolation of PAF from domestic cats by mechanical ovary dissection. By adapting the methods described for domestic cats to nondomestic felid species, [70] accomplished the isolation and the ultrastructural characterization of PAF from cheetahs, jaguars, lions, and Sumatran, Siberian, and Bengal tigers that had died at local zoos. The similarity among domestic and nondomestic felid PAF was verified. The PAF collection was performed from ovary of several species of nondomestic felids [56] with a recovering of 1867±1144 PAF from each ovary, observing that the follicle growth is possible in the culture media for up to 14 days, with a 20% increase (40–50 mm) on the diameter of preantral follicles of the puma. These promising results suggest the possibility of future use of preantral follicles as a source of oocytes to be used in other biotechniques, and the foundation for germplasm banks. [70] reported that it is possible to maintain the viability of PAF from domestic cats after cryopreservation procedures.

transfer. It was suggested that the collection of ovaries from tigers (*P. tigris*), lions (*P. leo*), pumas (*F. concolor*), cheetahs (*A. jubatus*), leopards (*P. pardus*), and jaguars (*P. onca*) could be accomplished by ovary dissection up to 8 h after the death of these animals, by mechanical follicle isolation [56]. The best results were obtained with lion oocytes, fertilized by lion sperm, with a 31.6% (18/44) conception rate. It was demonstrated that leopard oocytes can be fertilized by domestic cat sperm and used in IVF procedures to produce 22% (2/9) 8-cell embryos. Otherwise, domestic cat oocytes can be fertilized by leopard spermatozoa, producing 19.5% (8/41) 8-cell embryos. Also oocyte collection from domestic and nondomestic cats by laparot‐ omy and posterior ovary dissection was successfully performed. These oocytes were submitted to IVF and then transferred to recipient females [57]. The main result obtained in this study was the interspecies embryo transfer from an Indian desert cat (*Felis silvestris ornata*) embryo to a domestic cat (*F. catus*), which resulted in the birth of two kittens. Afterward, the oocyte collected from domestic cat ovaries after ovariectomy were used to demonstrate that mor‐ phology of the oocyte ooplasm can affect in vitro maturation, as well as the gonadotropin supplementation [12]. According to the morphological aspect by stereomicroscopic exam, cumulus–oocyte complexes were classified as mature, immature, or degenerated. Besides the successful embryo production by IVF using this approach, light and electron microscopic evaluations revealed that ovarian stimulation followed by follicular aspiration resulted in a heterogenous oocyte population with respect to meiotic maturation. The correct assessment of the oocyte maturation status is difficult to perform through stereomicroscopical exam [58]. Oocytes can be preserved if they are not immediately submitted to IVF. However, [59] observed that cooling could cause chromosomal anomalies in mature oocytes, as a conse‐ quence of the temperature decrease on meiotic fusion. [60] performed the IVM of domestic feline oocytes, previously kept under refrigeration at 4 °C for 24 h, and they did not observe deleterious effects of storage on oocyte meiotic progression. Moreover, [61] demonstrated that even brief (2–3 weeks) salt storage significantly affects cat oocyte penetration rate, and the penetration continues to decline as storage duration increases to 2–3 months. However, the authors hypothesized that the composition of the solution may have contributed to reduce sperm penetration. For canine species, [62] found that oocyte storage in hypertonic salt solution damages the zona pellucida, reducing the sperm penetration rates. In mice [63], rabbits [64], and bovines [65], it was possible to obtain the birth of normal offspring following IVF after thaw. In domestic felines, [66] demonstrated that the mature oocyte could be cryopreserved and, soon after, fertilized in vitro with success. The maturation of oocyte recovered from antral follicles is an efficient method for the use of haploid female material and the oocyte activation in initial phases of development is a possible tool that also increases the efficiency of the oocyte utilization [56]. The preantral ovarian follicles (PAF) represent 90% of the follicular population in mammals [67]. Small PAF recovered from the ovaries collected from postmortem animals or through ovariectomy, therefore, are a rich oocyte source, because they can mature in vitro (Fig. 4). [68] reported that feline PAF are capable of developing in vitro to the antral phase. Moreover, [69] demonstrated the isolation of PAF from domestic cats by mechanical ovary dissection. By adapting the methods described for domestic cats to nondomestic felid species, [70] accomplished the isolation and the ultrastructural characterization of PAF from cheetahs, jaguars, lions, and Sumatran, Siberian, and Bengal tigers that had died at local zoos. The

210 New Discoveries in Embryology

**Figure 4.** Cat Cumulus Oocyte Complex (COC) collected from Preantral Follicle (PAF); Cat COC grade: a) grade I: compact and integral and multistratified cumulus and dark ooplasm; b) grade II: compact but not integral and paucis‐ tratified cumulus and not homogeneous dark pigmentation of ooplasm; c) grade III: interrupted and incompact cumu‐ lus and clear ooplasm; d) expanded cumulus.

A further alternative is represented by the ovarian tissue transplantation. [71] was the first to report an ovarian transplantation. Only in the twentieth century was a significant improve‐ ment of the vascular anastomosis techniques of several transplanted organs including the ovary achieved [72]. According to [73], both whole ovary and ovarian fragment transplanta‐ tions could be used for ovarian follicle cultures. Moreover, [74] was reported that a great advantage for the preservation and culture of ovarian tissue is due to the possibility of material collection not dependent upon the age or reproductive status of the donor. Moreover, [75] suggested that the term allotransplantation refers to the transplantation of an organ originating from one individual to another that is genetically different, but belonging to the same species. Ovarian cortex fragments transplantation was successfully performed from domestic cats to the renal capsules of severely immunedeficient infertile mice [76]. After 9 months, the necropsy of the recipient mice was accomplished, when the presence of follicles was verified in the grafts. These ovarian follicles reached a 3 mm diameter, had a normal antral cavity, and appeared to be cytologically normal as follicle in integer cat ovary (Fig. 5). However, ovulation was not observed in any of the grafts. Furthermore, [77] reported that xenotransplanting into the kidney capsule from severe combined immunedeficient mice freeze–thawed of cat ovarian cortex did not allow its surviving, but the follicles containing gonadotropin responsive granulosa cells were able to grow to antral stages. Conversely, [78] declared that oocyte and ovarian tissue cryopreservation is not yet fully established. There are still several obstacles to overcome for this technology to be routinely used. Even so, improvement in the cryopreser‐ vation techniques is seen as an important tool for the formation of ovarian tissue banks, with the purpose of conserving precious genetic material of endangered species [79].

**Figure 5.** Histological section (O.M. 200X) of explanted cat ovarian tissue with follicle at different developmental stage: a) and a1) antral follicle; b) preantral follicle; b1) primary follicle.

### **7. Gamete cryopreservation in felids**

suggested that the term allotransplantation refers to the transplantation of an organ originating from one individual to another that is genetically different, but belonging to the same species. Ovarian cortex fragments transplantation was successfully performed from domestic cats to the renal capsules of severely immunedeficient infertile mice [76]. After 9 months, the necropsy of the recipient mice was accomplished, when the presence of follicles was verified in the grafts. These ovarian follicles reached a 3 mm diameter, had a normal antral cavity, and appeared to be cytologically normal as follicle in integer cat ovary (Fig. 5). However, ovulation was not observed in any of the grafts. Furthermore, [77] reported that xenotransplanting into the kidney capsule from severe combined immunedeficient mice freeze–thawed of cat ovarian cortex did not allow its surviving, but the follicles containing gonadotropin responsive granulosa cells were able to grow to antral stages. Conversely, [78] declared that oocyte and ovarian tissue cryopreservation is not yet fully established. There are still several obstacles to overcome for this technology to be routinely used. Even so, improvement in the cryopreser‐ vation techniques is seen as an important tool for the formation of ovarian tissue banks, with

212 New Discoveries in Embryology

the purpose of conserving precious genetic material of endangered species [79].

**Figure 5.** Histological section (O.M. 200X) of explanted cat ovarian tissue with follicle at different developmental stage:

a) and a1) antral follicle; b) preantral follicle; b1) primary follicle.

Cryopreservation of gametes is an important tool in assisted reproduction programs. In fact, long-term storage of oocytes or spermatozoa is necessary for in vitro fertilization (IVF) or artificial insemination (AI) in the future. When geographical or temporal distance between donors and recipient results in nonsimultaneous availability of male and female gametes, cryopreservation is the only option. Maintenance of biodiversity has intrinsic value for the genetic preservation of valuable domestic cat breeds and an extrinsic value for conserva‐ tion management of taxonomically related nondomestic feline species. New knowledge about felid reproductive physiology will enhance the development of techniques that are potential‐ ly applicable to nondomestic cats. Domestic cat spermatozoa and oocytes have peculiar physical characteristics that increase the difficulty of developing successful cryopreserva‐ tion methods as compared to gametes of some other species. Therefore, even though a variety of procedures have been investigated, optimal cryopreservation techniques, either for spermatozoa or oocytes, are yet to be realized [80]. Cat semen was successfully cryopre‐ served, and kittens were born after AI with frozen–thawed semen [81]. Achievements in cryopreservation of felid semen and different protocols of freezing–thawing ejaculated and epididymal cat semen have been reviewed [80]. In our laboratory we cryopreserved epididymal sperm felines with the following protocol: epididymides were collected from 20 domestic cats during routine neutering procedure and from two wild felines at autopsy. The sperm samples, diluted with 4% glycerol/Tris/egg yolk, were loaded into 0.25 ml ministraws, exposed to nitrogen vapor and stored in liquid nitrogen. After 4 weeks, samples were thawed and reevaluated. The quality of each fresh and frozen–thawed sperm sample was tested by determining the motility (54.7±11.3% and 32±13.1%, respectively, for cat spermato‐ zoa; 38.3±18.7% and 21.5±16.8%, respectively, for tiger spermatozoa), viability (74.3±8.6% and 45.2±9.4%, respectively, for cat spermatozoa; 42.4±14.5% and 33.5±12.9%, respectively, for wild felid spermatozoa), morphology, and acrosomal status. The present study showed that feline epididymal spermatozoa can be frozen in egg-yolk extender with 4.0% glycerol in 0.25 ml straws. The procedure used in the present study for epididymal cat sperm cryopreserva‐ tion may be applied to bank for genetic resources of wild felid species. [82] Protocols for freezing/cryopreservation of cat oocytes [80] are established; nevertheless, this technology is still considered "experimental" because the survival rates of cat oocytes after freezing procedures are still low, but to date, there is evidence that some preantral follicles extract‐ ed from cat ovaries remain structurally intact and physiologically active after freezing/ cryopreservation and subsequent thawing [80]. However, there is evidence that some preantral follicles from cat ovaries remain structurally intact and physiologically active after freezing/cryopreservation and subsequent thawing [70]. Domestic cat oocytes have high lipid droplet content in the ooplasm [83]; thus, oocyte permeability to cryoprotectant solutions may be lower than in oocytes of other species [84-87]. Only a few studies have investigated cat oocyte cryopreservation, and the few successes were only obtained for mature oocyte cryopreservation [80]. In the first study [80], mature and immature oocytes were cryopre‐ served by slow cooling, but no blastocysts were obtained after in vitro fertilization (IVF). In

the second study [88], matured cat oocytes were vitrified in straws and, after IVF with frozen– thawed epididymal spermatozoa, the first two blastocysts were obtained [80]. In a recent study, the first attached cat blastocysts were obtained from matured cat oocytes that were vitrified using a cryo-loop system [89]. Another very recent study reported blastocyst production from vitrified germinal vescicle (GV) cat oocytes exposed to resveratrol (Res) in order to compact the decondensed chromatin contained in the large GV of cat oocytes [90]. Despite the importance of cryoprotectant penetration to avoid intracellular ice crystal formation, the greater cryoprotectant concentrations in vitrification solutions are toxic and may cause osmotic injury [91]. Suggestions for minimizing the toxicity of vitrification solutions include the use of less toxic substances, association with different cryoprotec‐ tants, previous exposure to lesser concentrations of cryoprotectants, and reduction of exposure time to vitrification solutions [92, 93]. The major penetrating cryoprotectants for oocyte cryopreservation are ethylene glycol (EG), glycerol (GLY), dimethylsulfoxide (Me2SO), propylene glycol (PrOH), and acetamide [94]. Another common permeating CPA, 1,2 ethanediol (EG) [94], is also suitable for less permeable immature oocytes, as demonstrated in cattle [95]. A recent study investigating bovine oocyte vitrification demonstrated that a solution of EG + Me2SO is a favorable cryoprotectant combination, as the Me2SO (MW = 78.13) molecule is smaller and consequently more permeable than the glycerol molecule (MW = 92.1) [96]. In our laboratory for the first time we obtained blastocysts from egg vitrified at GV stage from cat [97]. The vitrification was performed in OPS into sucrose medium (1 M sucrose in HSOF + 6% BSA) containing dimethyl sulfoxide (DMSO) (16.5% final concentra‐ tion) and ethylene glycol (EG) (16.5% final concentration) as cryoprotectants. This vitrifica‐ tion protocol ensured a development to blastocyst stage and it is the first report of development of vitrified GV COC and confirmed that the selection of an appropriate cryoprotectant mixture and sample volume reduction are two simple but important param‐ eters in the study of a successful vitrification method for feline species. Ovarian tissue cryopreservation combined with the subsequent transplantation into immunocompromised recipients, in order to resuming follicular development, is considered to be a promising approach for cryobanking female gametes in nondomestic felid species [81].

### **8.** *In vitro* **embryo production in felids**

Several laboratories have independently assessed the potential of maturing and fertilizing domestic cat oocytes, mainly using IVM /IVF. The biological competency of IVM/IVF domestic cat embryos has been demonstrated after embryo transfer. Nonetheless, it has become apparent that IVM/IVF success in the cat is generally less than that reported for other com‐ monly studied species like the cow [4]. For example, it is not unusual for 60–80% of cow antral follicular oocytes to be fertilized and to cleave in vitro [9]. Techniques in the mouse have progressed even further to allow the growth, maturation, and successful fertilization of oocytes from primordial ovarian follicles. In contrast, only about 50–60% of cultured cat oocytes achieve nuclear maturation in culture and, after insemination, usually <40% oocytes are fertilized on the basis of embryo cleavage [9]. Even under optimal culture conditions, <20% of these cleaved embryos grow into blastocysts *in vitro* [9]. Nevertheless, in vitro embryo production has also been successful in felids. In vitro-derived embryos of the domestic cat were successfully frozen and developed to term kittens after cryopreservation and transfer. Various aspects of in vitro maturation of felid oocytes and in vitro culture of felid embryos have been comprehensively reviewed [81]. Cat oocytes can be collected from ovaries from sexually mature queens recovered following routine ovariohysterectomy. Feline oocytes can be collected from ovary explanted postmortem within 6 h from death. Within 3–6 h of excision, each ovary was sliced longitudinally with a scalpel blade followed by lateral mincing of the ovarian cortex in a 35 x 0.7 mm Petri dish, flushed by different media (e.g., HEPES synthetic oviductal fluid HSOF or HMEM or TCM199). Collected oocytes were graded (only oocytes exhibiting uniform, darkly pigmented ooplasm and an intact cumulus cell investment were chosen for culture), gently rinsed in a fresh dish of culture medium and immediately placed in 50 µl drops of culture medium under mineral oil or in 500 µl 4WD. In vitro maturation may be performed (25–50 oocytes/ml) in different media, for example, SOF synthetic oviductal fluid added with amino acids and 6 mg/ml BSA containing 0.1 IU of porcine follicle-stimulating hormone and porcine luteinizing hormone supplemented with 25 ng/ml EGF, 25 ml/ml insulin–transferrin–sodium selenite (ITS) and 1.2 mmol/l L-cysteine or Eagle's minimum essential medium containing 0.026 g pyruvate, 0.292 g L-glutamine, 0.4% (w/v) BSA, 100 IU penicillin, 100 IU streptomycin, 1 pg LH ml, 1 pg FSH and 1 pg oestradiol or MEM minimal essential medium containing Earle's salts and bicarbonate buffer supplemented with 0.4% (w/ v) BSA, 2.0 mmol glutamine, 1.0 mmol pyruvate, 1 µg FSH, 1 µg LH ml, and 1 µg oestradiol (EMEM)] in a 5% CO2 incubator at 38.5° C for 24 h. In vitro matured COC can be in vitro fertilized with fresh/cryopreserved epididymal or ejaculated spermatozoa. Briefly, in order to perform IVF, semen was diluted 1:1 in different media (SOF or Ham's FIO medium (HF10) supplemented with 1.0 mmol pyruvate, 2.0 mmol glutamine, and 5% (v/v) fetal calf serum or Tris extender (3.025% Tris(hydroxymethyl)aminomethane, 1.7% citric acid, 1.25% fructose, 0.06% Sodium Benzyl penicillin, 0.1% streptomycin sulphatee) and centrifuged at 300 g for 8 min. Supernatant was discarded and the remaining pellet overlaid with 100 µl HFlO and the sample maintained at room temperature undisturbed for 1 h. Next, 50 µl was removed from the top layer and evaluated for sperm motility, forward progression, and concentration. Cultured oocytes were washed twice in 90 µ fertilization drops of IVF media or in 500 µl FWD under oil in 5% C02 in air at 38°C. Processed sperm samples were diluted in IVF media in order to obtain a final concentration of 1 x 106 spz/ml. After coincubation for 18 h, oocytes were washed to remove cumulus cells and loosely attached spermatozoa and returned to fresh 50 µ drops of IVC media (SOFaaBSA or F10). At 30 h after insemination, oocytes were evaluated at a stereomicroscope for survival, and those showing cytoplasmic degeneration were discarded. The cleavage to the two-cell stage was assessed as an index of fertilization. Subse‐ quent embryonic development was assessed at intervals of 24 to 48 h. As explained, despite some past difficulties achieving in vitro development beyond the morale stage after IVF, more recently, several reports have shown blastocyst rates from approximately 50% to 80% on Day 6, Day 7, or Day 8 [98-100]. The frequency of blastocyst development is universally considered to be a useful measure of embryo developmental potential. In favorable in vitro culture

the second study [88], matured cat oocytes were vitrified in straws and, after IVF with frozen– thawed epididymal spermatozoa, the first two blastocysts were obtained [80]. In a recent study, the first attached cat blastocysts were obtained from matured cat oocytes that were vitrified using a cryo-loop system [89]. Another very recent study reported blastocyst production from vitrified germinal vescicle (GV) cat oocytes exposed to resveratrol (Res) in order to compact the decondensed chromatin contained in the large GV of cat oocytes [90]. Despite the importance of cryoprotectant penetration to avoid intracellular ice crystal formation, the greater cryoprotectant concentrations in vitrification solutions are toxic and may cause osmotic injury [91]. Suggestions for minimizing the toxicity of vitrification solutions include the use of less toxic substances, association with different cryoprotec‐ tants, previous exposure to lesser concentrations of cryoprotectants, and reduction of exposure time to vitrification solutions [92, 93]. The major penetrating cryoprotectants for oocyte cryopreservation are ethylene glycol (EG), glycerol (GLY), dimethylsulfoxide (Me2SO), propylene glycol (PrOH), and acetamide [94]. Another common permeating CPA, 1,2 ethanediol (EG) [94], is also suitable for less permeable immature oocytes, as demonstrated in cattle [95]. A recent study investigating bovine oocyte vitrification demonstrated that a solution of EG + Me2SO is a favorable cryoprotectant combination, as the Me2SO (MW = 78.13) molecule is smaller and consequently more permeable than the glycerol molecule (MW = 92.1) [96]. In our laboratory for the first time we obtained blastocysts from egg vitrified at GV stage from cat [97]. The vitrification was performed in OPS into sucrose medium (1 M sucrose in HSOF + 6% BSA) containing dimethyl sulfoxide (DMSO) (16.5% final concentra‐ tion) and ethylene glycol (EG) (16.5% final concentration) as cryoprotectants. This vitrifica‐ tion protocol ensured a development to blastocyst stage and it is the first report of development of vitrified GV COC and confirmed that the selection of an appropriate cryoprotectant mixture and sample volume reduction are two simple but important param‐ eters in the study of a successful vitrification method for feline species. Ovarian tissue cryopreservation combined with the subsequent transplantation into immunocompromised recipients, in order to resuming follicular development, is considered to be a promising

approach for cryobanking female gametes in nondomestic felid species [81].

Several laboratories have independently assessed the potential of maturing and fertilizing domestic cat oocytes, mainly using IVM /IVF. The biological competency of IVM/IVF domestic cat embryos has been demonstrated after embryo transfer. Nonetheless, it has become apparent that IVM/IVF success in the cat is generally less than that reported for other com‐ monly studied species like the cow [4]. For example, it is not unusual for 60–80% of cow antral follicular oocytes to be fertilized and to cleave in vitro [9]. Techniques in the mouse have progressed even further to allow the growth, maturation, and successful fertilization of oocytes from primordial ovarian follicles. In contrast, only about 50–60% of cultured cat oocytes achieve nuclear maturation in culture and, after insemination, usually <40% oocytes are

**8.** *In vitro* **embryo production in felids**

214 New Discoveries in Embryology

conditions, cat blastocysts, like those of more widely studied species, undergo further growth, in cell numbers and in overall size. Thus, another widely accepted indication of blastocyst developmental potential is their ability to undergo expansion and "hatch" from the zona pellucida in vitro. Most of Day 8 blastocysts show noticeable expansion of the blastocyst cavity such that the zonal diameter is larger and its thickness is much thinner than at earlier stages. In addition, some of them are "hatching" because the embryonic cells are gradually extruded through one or more small apertures in the zona, rather than popping out through a large crack [4].

### **9. Artificial Insemination (AI) and Embryo Transfer (ET) in felids**

conditions, cat blastocysts, like those of more widely studied species, undergo further growth, in cell numbers and in overall size. Thus, another widely accepted indication of blastocyst developmental potential is their ability to undergo expansion and "hatch" from the zona pellucida in vitro. Most of Day 8 blastocysts show noticeable expansion of the blastocyst cavity such that the zonal diameter is larger and its thickness is much thinner than at earlier stages. In addition, some of them are "hatching" because the embryonic cells are gradually extruded through one or more small apertures in the zona, rather than popping out through a large

crack [4].

216 New Discoveries in Embryology

Embryo transfer (ET) and artificial insemination (AI) are potentially important techniques for the propagation and management of genetically valuable domestic cat and endangered nondomestic cat populations. There are different AI techniques for cats [101]. In early studies, intravaginal insemination was exploited, but the success rate has not exceeded 43%. Later, this technique has been used with more effectiveness and a better success rate [102]. Another approach is intrauterine insemination, either surgical [103] or nonsurgical with the use of specially designed catheters [104]. They performed the first transcervical insemination with fresh or frozen semen in cats. This can be considered the method of choice in almost all cases; it is less invasive than the surgical approach, and a much smaller amount of semen is needed with respect to the intravaginal insemination. Recently, AI with semen cryopreservation has been applied in a number of wild felid species. Unfortunately, the teratospermia problem aggravates freezing/cryopreservation in many felid species [4]. In vitro embryo production has also been successful in felids. In vitro-derived embryos of the domestic cat were successfully frozen and developed to term kittens after cryopreservation and transfer. Various aspects of in vitro maturation of felid oocytes and in vitro culture of felid embryos have been comprehensively reviewed [4]. In 1979, the first successful embryo transfer (ET) in cats was reported [105]. The embryos, recovered from donors after mating during a natural cycle, were transferred into like recipients. Three litters of kittens were born from four pregnancies established in seven recipients. Nonetheless, in the following decade, in most ensuing reports on ET of in vitro-produced cat embryos, mixed morale and early blastocysts were deposit‐ ed into the uterus of Day 4, 5, or 6 recipients [106-108]. This interval was used because it is the approximate length of time required for cat embryos to be transported through the oviduct and enter the uterus [109, 110]. To examine the effect of developmental stage, morula versus blastocyst, on pregnancy rate after ET, [111] recovered 1–4-cell embryos from gonadotropintreated donors and cultured them in vitro (in 20% fetal calf serum) for 3–7 days before transfer into synchronous recipients. All four recipients of morulae (6–12 each) established pregnan‐ cies; two aborted before term, and two delivered a total of 10 kittens. Three of five recipi‐ ents of blastocysts cultured for 4–6 days delivered a total of nine kittens, but none of the three recipients of blastocysts cultured for 7 days became pregnant. Although the zonal status of the blastocysts transferred after 7 days in vitro was not provided, most morulae had developed to the blastocyst stage by Day 7 of culture, with hatching starting to occur on Day 6 in blastocysts that were not fully expanded, which would suggest that most were either hatching or hatched when transferred. In their comments on failure of later-stage blasto‐ cysts (Day 8, 1 day in vivo, and 7 days in vitro) to establish pregnancies, the authors noted that further studies were needed "on in vivo development and hatching of transplanted embryos." [112] found that separate transfer of in vitro-produced Day 5 late morulae and Day 5 early blastocysts into synchronous gonadotropin-treated recipients resulted in equally high pregnancy rates of 71% (5/7) and 80% (8/10), respectively. Each recipient received six morulae or six blastocysts and the average litter size was 2.0 (1–3) and 3.0 (1–3), respective‐ ly. Possibly, the only pregnancy/birth after ET of in vitro-derived (IVM/IVF/IVC) Day 7 blastocysts is the single kitten born from 21 embryos transferred into two synchronous recipients [113]. All of the blastocysts were completely zona-intact when transferred. The transfer of fresh or frozen in vitro-derived embryos has proved to be successful in some wildlife felids. In the lion (*Pantera leo*), in vivo-derived oocytes were inseminated in vitro with fresh semen, and some of these embryos developed up to the blastocyst stage. In the tiger (*Panthera tigris*), term kittens developed after the transfer of in vitro-derived embryos [43], and one live kitten was born after the transfer of African wildcat (*Felis silvestris lybica*) frozen–thawed in vitro-derived embryos into three recipients [5]. Although the rate of success was low (4.5%), this result is important as this was the first kitten born after embryo cryopreservation in a wildlife felid species. Two term kittens born after transferring frozen– thawed embryos of ocelot (*Leopardus pardalis*) have also been reported [110]. Cleavage stage ocelot embryos were conventionally frozen with ethylene glycol and kept in liquid nitro‐ gen [110]. More recently, three live ocelot kittens were born in Cincinnati Zoo after the thawing of ocelot embryos from a cryobank and the transfer of nine frozen–thawed em‐ bryos into eight synchronized recipients. The ocelot is an endangered species at least in some countries, and a cryobank is needed to secure its biodiversity [110]. The birth of live kittens produced by intracytoplasmic sperm injection of domestic cat oocytes matured in vitro has been reported [4]. Also noteworthy, there are experiments on "interspecies in vitro hybridi‐ zation," when oocytes of nondomestic felid species were successfully fertilized in vitro by heterologous (domestic cat) spermatozoa. Cleavage stage "hybrid" embryos have resulted from in vitro fertilization of leopard (*Panthera pardus*) and puma (*Felis concolor*) oocytes with frozen–thawed domestic cat semen [4].

### **10. Cloning of domestic and wild cats and interspecies of felide**

Along with these achievements with cryobanking, the domestic cat has been cloned by two independent groups [114,115]. Recently, a domestic cat recipient female has been reported to have given birth to African wildcat (*F. silvestris lybica*) cloned kittens and sand cat (Felis margarita) kittens [13]. Fibroblast nuclei of African wildcat were fused to domestic cat oocytes (interspecies nuclear transfer) and the cloned embryos were transferred into recipient domestic cat females; 17 kittens were born, but only 8 survived, after birth, up to 1 month. These African wildcat kittens represent the first wild Carnivora species to be produced by nuclear transfer [13]. This study showed the possibility of cloning other felid species beside the domestic cat, but it is also a big success in interspecies nuclear transfer/embryo transfer in felids. Earlier, an interspecies ET was performed between the Indian desert cat (*Felis silvestris ornata*) and the domestic cat [4]. Recently, transgenic clones have also been produced in the cat. Genetically modified adult or fetal fibroblasts have been used as donors of nuclei. These nuclei were moved into cat oocytes and then embryos were developed in vitro. After these embryos were transferred into appropriate recipients, three alive transgenic kittens were obtained [81].

### **11. Laparoscopic oviductal embryo transfer and artificial insemination in felids**

The application of laparoscopy to reproductive studies in felids has been invaluable for helping to alleviate some concerns of animal welfare: sowing reproductive organs through the intraabdominal access through a minimally invasive and traumatic approach [116]. Likely, the extrapolation of ART to the genetic management of wild cats would be unattainable in the future without laparoscopy. The latter, for oocyte collection and intrauterine insemination, has been used largely with numerous cat species over the past 20 years. Recently, laparoscopic approaches have been developed and applied in cats for accessing the oviduct precisely to perform laparoscopic oviductal embryo transfer (LO-ET) and artificial insemination (LO-AI) procedures [117,118]. To our knowledge, just in eight cat hereditary disease models and two nondomestic cat species, the ocelot and sand cat, it has been possible to get viable offspring following LO-ET of nonfrozen and frozen–thawed IVF-derived embryos. LO-AI with low sperm numbers and LO-ET have been demonstrated to be similar in efficacy, resulting in high pregnancy percentages (50–70%) following insemination of domestic cats treated with gonadotrophins. Following LO-AI, multiple kittens have been produced in some hereditary disease models with frozen semen, and both Pallas' cat and ocelot kittens were born after LO-AI with freshly collected semen. The application of LO-ET and LO-AI to felids has brought important and effective improvement in the efficiency of ART for genetic management of these invaluable wild and domestic cat populations [119].

### **12. Conclusions**

blastocysts is the single kitten born from 21 embryos transferred into two synchronous recipients [113]. All of the blastocysts were completely zona-intact when transferred. The transfer of fresh or frozen in vitro-derived embryos has proved to be successful in some wildlife felids. In the lion (*Pantera leo*), in vivo-derived oocytes were inseminated in vitro with fresh semen, and some of these embryos developed up to the blastocyst stage. In the tiger (*Panthera tigris*), term kittens developed after the transfer of in vitro-derived embryos [43], and one live kitten was born after the transfer of African wildcat (*Felis silvestris lybica*) frozen–thawed in vitro-derived embryos into three recipients [5]. Although the rate of success was low (4.5%), this result is important as this was the first kitten born after embryo cryopreservation in a wildlife felid species. Two term kittens born after transferring frozen– thawed embryos of ocelot (*Leopardus pardalis*) have also been reported [110]. Cleavage stage ocelot embryos were conventionally frozen with ethylene glycol and kept in liquid nitro‐ gen [110]. More recently, three live ocelot kittens were born in Cincinnati Zoo after the thawing of ocelot embryos from a cryobank and the transfer of nine frozen–thawed em‐ bryos into eight synchronized recipients. The ocelot is an endangered species at least in some countries, and a cryobank is needed to secure its biodiversity [110]. The birth of live kittens produced by intracytoplasmic sperm injection of domestic cat oocytes matured in vitro has been reported [4]. Also noteworthy, there are experiments on "interspecies in vitro hybridi‐ zation," when oocytes of nondomestic felid species were successfully fertilized in vitro by heterologous (domestic cat) spermatozoa. Cleavage stage "hybrid" embryos have resulted from in vitro fertilization of leopard (*Panthera pardus*) and puma (*Felis concolor*) oocytes with

frozen–thawed domestic cat semen [4].

218 New Discoveries in Embryology

**10. Cloning of domestic and wild cats and interspecies of felide**

Along with these achievements with cryobanking, the domestic cat has been cloned by two independent groups [114,115]. Recently, a domestic cat recipient female has been reported to have given birth to African wildcat (*F. silvestris lybica*) cloned kittens and sand cat (Felis margarita) kittens [13]. Fibroblast nuclei of African wildcat were fused to domestic cat oocytes (interspecies nuclear transfer) and the cloned embryos were transferred into recipient domestic cat females; 17 kittens were born, but only 8 survived, after birth, up to 1 month. These African wildcat kittens represent the first wild Carnivora species to be produced by nuclear transfer [13]. This study showed the possibility of cloning other felid species beside the domestic cat, but it is also a big success in interspecies nuclear transfer/embryo transfer in felids. Earlier, an interspecies ET was performed between the Indian desert cat (*Felis silvestris ornata*) and the domestic cat [4]. Recently, transgenic clones have also been produced in the cat. Genetically modified adult or fetal fibroblasts have been used as donors of nuclei. These nuclei were moved into cat oocytes and then embryos were developed in vitro. After these embryos were transferred into appropriate recipients, three alive transgenic kittens were obtained [81].

In the present chapter, we made an overview of the data and methods detectable in literature and focused our attention on analysis of methods utilized in ART for maximizing their efficiency in feline species. ART include mainly Artificial Insemination (AI); *In Vitro* Embryo Production (IVEP) consisting of IVM (*In Vitro* Maturation), IVF (*In Vitro* Fertilization), IVC (*In Vitro* Culture), ET (Embryo Transfer), and ICSI (Intra Cytoplasmic Sperm Injection); gamete/ embryo cryopreservation; gamete/embryo sexing; gamete/embryo micromanipulation; Somatic Cell Nuclear Transfer (SCNT), and genome resource banking, which has been widely used in genetic improvement and industry in livestock animals. The domestic cat is used as a model for the development of ART in Felidae species and can serve as a successful recipient of embryos from closely related, small, nondomestic cats, as shown by the birth of the Indian desert cat and African wildcat kittens after in vitro fertilization (IVF)-derived embryo transfer. The creation of the Biological Resource Bank represents a complementary support tool for the application of ART in the in situ and ex situ conservation of endangered felids. The chief purpose of ART in the protection of endangered species is to preserve the maximum current genetic and biological diversity of the population by the processing and cryopreservation of germinal cells and tissues from dead animals, which can later be used in future reproductive projects. In humans and domestic species, it is usually possible to plan the place and time for gonadal explants to recover germplasm, thereby enabling a reduction in the gonadal storage time in the transport medium. In wild species, it is impossible to predict when and where the gonads can be collected. The gonads can be recovered postmortem, which entails the possi‐ bility that the collection place could be distant from a laboratory for IVEP.

### **Author details**

Natascia Cocchia, Simona Tafuri, Lucia Abbondante, Leonardo Meomartino, Luigi Esposito and Francesca Ciani\*

\*Address all correspondence to: ciani@unina.it

Department of Veterinary Medicine and Animal Productions, University of Naples Federico II, Naples, Italy

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Department of Veterinary Medicine and Animal Productions, University of Naples Federico

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