**4. The early development of the thymus: phases and involved molecules**

Similarly, the thymus follows a pattern of development whose stages resemble the specification, tubulogenesis, and branching morphogenesis previously described. Remarkably, they appear to be regulated by many molecular families reported to be involved in the early development of other branching epithelial organs. Although the thymus development has been profusely studied [73–75], few studies have highlighted its resemblance with a process of tubulogenesis and branching morphogenesis the way we do in this review. As known, thymus development occurs in two steps: an early organogenesis, independent of the transcription factor Foxn1, in which the pharyngeal endoderm is specified to thymus fate and a later organogenesis in which thymic epithelium differentiates and is organized under the control of Foxn1 and the lymphoid progenitor cells that seed the thymic epithelial primordium [76].

#### **4.1. Early thymus development**

expressing endoderm progenitors that differentiate into ciliated cells, secretory cells and basal cells concentrate in the proximal zone, pluripotent Sox9/Id2+ progenitor cells that will form

Pancreatic progenitors simultaneously proliferate and differentiate into the endocrine, ductal and acinar cell lineages. In the E9.5, early primordium, multipotent, unipotent endocrine, and duct-endocrine bipotent precursor cells are present, while a wave of acinar precursor differentiation takes place at the peripheral portion around E11.5–12 as branching morphogenesis initiates and tip differentiation is induced [46] (**Figure 1**). Mesenchymal factors and ECM components increase acinar/tip formation, whereas the interconnection between epithelium

In both organs, lung and pancreas, notch signaling plays an essential role in the differentiation of distinct cell types. Its chemical inhibition in lung causes expansion of distal progenitor cells and decreased numbers of proximal precursors [64]. On the other hand, during development, increased notch signaling correlates with preferential production of secretory cells versus ciliated and neuroendocrine cells [65]. In addition, activation of Notch in keratin 5+ basal cells promotes secretory cell fate whereas its inhibition favors the differentiation toward ciliated cells [66]. In pancreas, Notch activity regulates tip-trunk patterning. Inducing trunk formation via Nkx6.1 activation and blocking tip fate through Ptf1a repression [6] and regulates the differentiation of Ngn3/Pdx1-positive endocrine progenitors versus Sox9/ Hnf1b-expressing ductal cells from trunk bipotent precursors. The specification, differentiation, and mainte-

After branching morphogenesis, there are changes in the epithelial cells and cap mesenchyme cells of the developing kidney [32]. Remarkably, Wnt ligands are asymmetrically distributed in the epithelial branches. Wnt 9d is extensively expressed in the ureteric epithelium but downregulated in the tips where Wnt 11 is expressed. Also, Six 2-expressing cells show zonation in the cap mesenchyme: a slow dividing Six 2hi cell population occurs in the periphery of cap, whereas fast cycling Six 2lo cells are intimately associated with the pretubular aggregate that will govern the nephron formation [32]. Moreover, at the beginning of branching morphogenesis, four Six 2+ cap cells exist for every one of the epithelial tip cells, but during branching,

the ratio falls to 2:1 and continues to decrease until the end of nephron formation [32].

The condition of endocrine tissues is special because they do not show a ductal system, and the secretion is closely associated with the vascular system. Thyroid fate is induced in the anterior endoderm by the concerted action of FGF2 and BMP4 [68], probably derived from cardiogenic mesoderm [69]. A thyroid initial bud is generated in the midline of the pharyngeal floor under control of Tbx1/FGF8 dependent signals [70]; later it detaches from endoderm, cells proliferate and the primordium bifurcates and grows laterally to generate a bilobulated organ with two lateral thyroid bodies formed by fusion with the paired ultimobranchial bodies (UBB),

types 1 and 2 alveolar cells do so in the distal zone [63].

26 Histology

and endothelial cells favors trunk development [46].

nance of acini from tips are regulated mainly by Ptf1a [6, 67].

which provide C cell precursors to the embryonic thyroid [71].

**3. The development of the thyroid**

The first step for the thymic rudiment formation is the segmentation of the posterior pharynx that culminates with the specification of endodermal cells into thymic epithelial cells (TECs) [75]. At these early stages, an inner sheet of endodermal tissue of the third pharyngeal pouch and an outer layer of ectodermal cells of the third branchial cleft contact and fuse [77]. Although pioneer morphological studies pointed out that the thymic epithelium derived from these two embrionary layers [78, 79], further experiments in birds and mice demonstrated that all TECs have an endodermal origin [80, 81]. Moreover, clonal analysis determined the existence of a bipotent common thymic epithelial progenitor cell capable of giving rise to both cortical (c) and medullary (m) TECs [82]. In fact, many of ectodermal cells die in the contact limits with the endoderm and they could just be inductors of thymus tissue or even not contribute to the thymus rudiment [80].

Thymic rudiment appears at E10–11 in mice constituting a simple epithelial structure surrounded by mesenchyme largely derived from the neural crests (NC). Earlier (E9.5), the endoderm evagination has formed a common primordium that expresses Glial cells missing homolog 2 (Gcm2), the earliest marker of parathyroid, in the anterodorsal domain. In the ventral domain, Foxn1 expression will be detected at E11 [80, 83]. From E 11.5, the common primordium initiates the detachment from the lateral surface of the pharynx through apoptosis [80]. Presumably, NC-derived mesenchyme cells are actively involved in this process because Splotch mutants that lack NC cells show delayed or no pharyngeal detachment of parathyroid/thymus rudiment [84, 85]. Nevertheless, other molecules are also concerned because mutants deficient in either Shh, Pax 9, or Frs 2a also maintain the pharynx connection [86–88]. At E12, the rudiment is totally separated from the pharynx and begins to individualize into two different organs. Then, the lateral thymic lobes descend caudally and medially until the midline, above the heart and behind the sternum. NC-derived mesenchyme as well as BMP4, Ephrin B2, and Hoxa3 are involved in the migration of thymic lobes [84, 85, 89].

pharyngeal pouches [97]. Apart from retinoid acid, Pbx1, which acts in cooperation with several Hox proteins, regulates Tbx1 expression [98]. Also, BMPs appear to affect Tbx1 indirectly. Mice deficient in Chordin, a BMP antagonist, shows reduced Tbx1 expression in both pharyngeal endoderm and head mesenchyme [99]. In addition, FGF8 expression disappears in the pharyngeal endoderm of these mice, suggesting a relationship between Tbx1 and FGF8. Indeed, the FGF family is a target of Tbx1. The expression of Tbx1and FGF8 overlap within the secondary heart field [100] and Tbx1-deficient mice exhibit reduced FGF8 expression in the pharyngeal endoderm but not in tissue where Tbx1 is not expressed [101]. The lack of FGF8 courses with failure of mesoderm to migrate at the primitive steak and then absence of embryonic endoderm tissue [102]). Therefore, Tbx1 acts downstream of Chordin/BMPs but upstream of FGF8. Thus, specific deletion of FGF8 in Tbx1-expressing cells phenocopies the DG and VCF syndromes [103]. Presumably, FGF8 models pharyngeal arches and pouch-derived structures,

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additionally affecting survival of pouch endoderm and NC cell migration [104].

a requisite for proper thymic organization.

*4.2.2. The Eya/Hoxa/Pax complexes*

not detach from the pharynx [85].

[107, 108].

Consequently, Tbx1 homozygous mutants show thymus aplasia [94] but indeed, the lack of Tbx1 results in absence of pharyngeal pouches. As a result, the thymus absence seems to be rather a consequence of this defective pouch formation. More recent studies demonstrate that ectopic Tbx1 expression in the ventral third pharyngeal pouch, the domain in which thymic primordium will be formed, suppresses Foxn1 expression and inhibits TEC proliferation and differentiation but does not reverse thymus fate [105]. Moreover, Tbx1 is downregulated in the ventral domain of wt third pharyngeal pouch [98, 101] and ectopic activation of Shh signaling in the third pharyngeal pouch endoderm (see later) induces Tbx1 expression that results in Foxn1 blockade [105]. All these results suggest that actually Tbx1 negatively regulates TEC growth and differentiation and its disappearance from third pharyngeal pouch endoderm is

There are nine Pax (Paired box) proteins in mammals, subdivided in four groups. Pax 1 and Pax 9, belonging to the same group, and Pax 3 are necessary for early thymus development [88, 106]. In addition, Pax function is closely related to that of Hoxa3, Eya1, and Tbx1, suggesting that they share common signaling pathways or follow parallel, complementary routes

Pax 3 specifies third pharyngeal pouch endoderm to TEC fate [109]. Pax3−/− mice (Splotch mutants), that have severe deficiency of NC cells, organize the thymus and the parathyroid normally but from E11.5 onward a change in the limits of parathyroid/thymus domains produces an enlarged thymus and a small parathyroid. In addition, the common rudiment does

Pax1 appears firstly in the foregut endoderm (E 8.5) and 2 days later in the endoderm of the third pharyngeal pouch remaining in the developing thymus. In the adult thymus, Pax1+ cells are restricted to a small group of cTECs [106]. Pax9 expression follows the same pattern but is also detected in NC-derived mesenchyme [110]. Pax1 mutants exhibit smaller thymic than those of wt mice and contain large cysts accumulating DP thymocytes [106], whereas Pax 9−/− embryos do not fold away from foregut and the thymus rudiment does not move

In the branchial arches, the mesenchyme derives from both mesoderm and neural crests [90], although presumably the role of NC-derived cells is more important [91]. NC-derived mesenchyme contributes to organize the outer connective tissue capsule and interlobular septae of developing thymus [92], but their relevance decreases in the adult thymus where mesenchyme could derive from mesoderm [91]. Accordingly, NC-derived mesenchyme is not required for the initial specification of endoderm but, as in other branching suffering epithelial organ, it is important for thymus development participating in the determination of the third pharyngeal pouches, the establishment of the limits between thymus and parathyroid domains, and the signaling necessary for the separation from common rudiment of the pharyngeal endoderm and later of the parathyroid and thymic lobes [85]. Finally, NC-derived cells are involved in the migration of thymic lobes into the thoracic cavity [93].

#### **4.2. Molecules involved in early thymic development**

It is difficult to establish the temporal sequence of functioning of distinct molecules, particularly because any defect in the formation and/or organization of pharyngeal pouches or arches will finally affect the thymus development, even though this development is not regulated directly by it. Furthermore, several molecules act at different stages of the thymus development even exerting opposite effects. Two main systems seem to govern the early thymus development: the one constituted by Hoxa3 and Pax1/9, together with other related molecules, Eya 1 and Six1/4 [73], and Tbx1. Tbx1 is related to human defects in chromosome 22q11.2, responsible of three phenotypes: Di George syndrome (DGS), velocardiofacial syndrome, (SCFS) and conotruncal anomaly face syndrome [94]. Both systems target morphogens of the families FGF, BMP/ TGFβ, Shh, and Wnt, which in turn regulate transcription factor activity making it difficult to establish a conclusive picture. As in other branching organs, many of these molecules are regulated by retinoid acid that would diffuse from adjacent NC-derived mesenchyme specifying pharyngeal endoderm [95]. In support of this, treatment with retinoid acid antagonists or mutant deficient in retinoid acid signaling courses with thymus agenesis [95, 96].

#### *4.2.1. The Tbx1 complex*

Tbx1 is expressed in the third pharyngeal pouch endoderm and surrounding mesenchyme, and its lack produces thymic hypoplasia and defects in other derivatives of third and fourth pharyngeal pouches [97]. Apart from retinoid acid, Pbx1, which acts in cooperation with several Hox proteins, regulates Tbx1 expression [98]. Also, BMPs appear to affect Tbx1 indirectly. Mice deficient in Chordin, a BMP antagonist, shows reduced Tbx1 expression in both pharyngeal endoderm and head mesenchyme [99]. In addition, FGF8 expression disappears in the pharyngeal endoderm of these mice, suggesting a relationship between Tbx1 and FGF8. Indeed, the FGF family is a target of Tbx1. The expression of Tbx1and FGF8 overlap within the secondary heart field [100] and Tbx1-deficient mice exhibit reduced FGF8 expression in the pharyngeal endoderm but not in tissue where Tbx1 is not expressed [101]. The lack of FGF8 courses with failure of mesoderm to migrate at the primitive steak and then absence of embryonic endoderm tissue [102]). Therefore, Tbx1 acts downstream of Chordin/BMPs but upstream of FGF8. Thus, specific deletion of FGF8 in Tbx1-expressing cells phenocopies the DG and VCF syndromes [103]. Presumably, FGF8 models pharyngeal arches and pouch-derived structures, additionally affecting survival of pouch endoderm and NC cell migration [104].

Consequently, Tbx1 homozygous mutants show thymus aplasia [94] but indeed, the lack of Tbx1 results in absence of pharyngeal pouches. As a result, the thymus absence seems to be rather a consequence of this defective pouch formation. More recent studies demonstrate that ectopic Tbx1 expression in the ventral third pharyngeal pouch, the domain in which thymic primordium will be formed, suppresses Foxn1 expression and inhibits TEC proliferation and differentiation but does not reverse thymus fate [105]. Moreover, Tbx1 is downregulated in the ventral domain of wt third pharyngeal pouch [98, 101] and ectopic activation of Shh signaling in the third pharyngeal pouch endoderm (see later) induces Tbx1 expression that results in Foxn1 blockade [105]. All these results suggest that actually Tbx1 negatively regulates TEC growth and differentiation and its disappearance from third pharyngeal pouch endoderm is a requisite for proper thymic organization.

#### *4.2.2. The Eya/Hoxa/Pax complexes*

homolog 2 (Gcm2), the earliest marker of parathyroid, in the anterodorsal domain. In the ventral domain, Foxn1 expression will be detected at E11 [80, 83]. From E 11.5, the common primordium initiates the detachment from the lateral surface of the pharynx through apoptosis [80]. Presumably, NC-derived mesenchyme cells are actively involved in this process because Splotch mutants that lack NC cells show delayed or no pharyngeal detachment of parathyroid/thymus rudiment [84, 85]. Nevertheless, other molecules are also concerned because mutants deficient in either Shh, Pax 9, or Frs 2a also maintain the pharynx connection [86–88]. At E12, the rudiment is totally separated from the pharynx and begins to individualize into two different organs. Then, the lateral thymic lobes descend caudally and medially until the midline, above the heart and behind the sternum. NC-derived mesenchyme as well as BMP4, Ephrin B2, and Hoxa3 are involved in the migration of thymic lobes [84, 85, 89].

In the branchial arches, the mesenchyme derives from both mesoderm and neural crests [90], although presumably the role of NC-derived cells is more important [91]. NC-derived mesenchyme contributes to organize the outer connective tissue capsule and interlobular septae of developing thymus [92], but their relevance decreases in the adult thymus where mesenchyme could derive from mesoderm [91]. Accordingly, NC-derived mesenchyme is not required for the initial specification of endoderm but, as in other branching suffering epithelial organ, it is important for thymus development participating in the determination of the third pharyngeal pouches, the establishment of the limits between thymus and parathyroid domains, and the signaling necessary for the separation from common rudiment of the pharyngeal endoderm and later of the parathyroid and thymic lobes [85]. Finally, NC-derived cells are involved in

It is difficult to establish the temporal sequence of functioning of distinct molecules, particularly because any defect in the formation and/or organization of pharyngeal pouches or arches will finally affect the thymus development, even though this development is not regulated directly by it. Furthermore, several molecules act at different stages of the thymus development even exerting opposite effects. Two main systems seem to govern the early thymus development: the one constituted by Hoxa3 and Pax1/9, together with other related molecules, Eya 1 and Six1/4 [73], and Tbx1. Tbx1 is related to human defects in chromosome 22q11.2, responsible of three phenotypes: Di George syndrome (DGS), velocardiofacial syndrome, (SCFS) and conotruncal anomaly face syndrome [94]. Both systems target morphogens of the families FGF, BMP/ TGFβ, Shh, and Wnt, which in turn regulate transcription factor activity making it difficult to establish a conclusive picture. As in other branching organs, many of these molecules are regulated by retinoid acid that would diffuse from adjacent NC-derived mesenchyme specifying pharyngeal endoderm [95]. In support of this, treatment with retinoid acid antagonists or

mutant deficient in retinoid acid signaling courses with thymus agenesis [95, 96].

Tbx1 is expressed in the third pharyngeal pouch endoderm and surrounding mesenchyme, and its lack produces thymic hypoplasia and defects in other derivatives of third and fourth

the migration of thymic lobes into the thoracic cavity [93].

**4.2. Molecules involved in early thymic development**

*4.2.1. The Tbx1 complex*

28 Histology

There are nine Pax (Paired box) proteins in mammals, subdivided in four groups. Pax 1 and Pax 9, belonging to the same group, and Pax 3 are necessary for early thymus development [88, 106]. In addition, Pax function is closely related to that of Hoxa3, Eya1, and Tbx1, suggesting that they share common signaling pathways or follow parallel, complementary routes [107, 108].

Pax 3 specifies third pharyngeal pouch endoderm to TEC fate [109]. Pax3−/− mice (Splotch mutants), that have severe deficiency of NC cells, organize the thymus and the parathyroid normally but from E11.5 onward a change in the limits of parathyroid/thymus domains produces an enlarged thymus and a small parathyroid. In addition, the common rudiment does not detach from the pharynx [85].

Pax1 appears firstly in the foregut endoderm (E 8.5) and 2 days later in the endoderm of the third pharyngeal pouch remaining in the developing thymus. In the adult thymus, Pax1+ cells are restricted to a small group of cTECs [106]. Pax9 expression follows the same pattern but is also detected in NC-derived mesenchyme [110]. Pax1 mutants exhibit smaller thymic than those of wt mice and contain large cysts accumulating DP thymocytes [106], whereas Pax 9−/− embryos do not fold away from foregut and the thymus rudiment does not move vetrocaudally remaining in the larynx. Although the primordium is colonized by lymphoid progenitors, it shows decreased proportions of proliferating cells and increased apoptosis finally resulting, as Pax1-deficient thymi, in small thymi [111].

*4.2.5. BMP family*

*4.2.6. Wnt family*

Particularly relevant is the role played by BMPs and Wnt molecules in the early thymic development, as direct controllers of Foxn1 expression, the key transcription factor mandatory for the late embryogenesis of thymus [124]. In addition, both signaling pathways constitute the major means for NC-derived mesenchyme to signal thymic epithelial rudiment [93, 124, 125]. Possibly, FGF8 produced by the primordial endoderm signals to the adjacent mesenchyme inducing BMP4 expression [126]. BMP4 and its antagonist Noggin govern the parathyroid/ thymus individualization and the Foxn1-dependent TEC maturation. In general, BMP4 is essential for the early stages of thymus development prior to the onset of Foxn1 expression [93]. BMP4 is expressed in the ventral domain of the pouch and Noggin in the dorsal area colocalizing with Gcm2 in the parathyroid domain [83]. Furthermore, BMP4 seems to be also involved in the full parathyroid/thymus separation, as BMP4 deletion delays the process [93]. Inhibition of BMP signaling provokes decline of Foxn1 expression in the zebrafish thymic primordium [83, 125], and BMP4 signaling promotes Foxn1 expression in early chicken thymus [126], as well as in mouse FTOCs [127]. Loss of BMP4 from pharyngeal endoderm and underlying mesenchyme prior to the onset of Foxn1 expression does not affect patterning, separation from the pharynx, or initial organ formation, although it alters some important morphogenetic processes such as lumen closure, organ separation and migration, initial lymphoid seeding, and formation of mesenchyme thymic capsule [93]. The sequence established between BMP and Foxn1 is the following: FGF8-mediated mesenchymal BMP signaling initiates the expression of both Foxn1 and BMP4 in the endodermal cells [126]. Then, endodermal BMP4 expression targets a regulatory feedback loop [128] for maintaining BMP4 and Foxn1 expression in the future thymic epithelium rather to directly affect Foxn1 [129]. In these conditions, if BMP signaling is blocked, the expression of both molecules ceases and nonfunctional Foxn1-TECs would remain in the thymus. If this occurs during concrete periods of midgestation, thymopoiesis will irreversibly fail [129]. Therefore, the balance between BMP4 and its inhibitors (i.e., Noggin) becomes critical for a proper maturation of thymic epithelium.

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Wnt family members are extensively expressed in developing and adult thymi in both TECs and fibroblasts [130], whereas their receptors are only detected on TECs [124]. Particularly, the noncanonical Wnt4 and Wnt5b, but also the canonical Wnt10b, coexpress with Foxn1 in third pharyngeal pouch and later in E13 and adult thymus [131] and are involved in its control [124]. Thus, overexpression of Dkk1, a Wnt4 inhibitor, in TECs induces thymic atrophy with reduced epithelial progenitors and TEC proliferation and appearance of TEC proliferation [132]. However, recent results indicate that a proper thymus development can only occur when β-catenin-dependent Wnt signaling is low or lacking [133]. Thus, β-catenin-deficient thymi exhibit Foxn1 expression, and stabilized β-catenin overexpression shows decreased rather than increased Foxn1 transcripts [133, 134]. Therefore, these results suggest that β-catenin is dispensable for Foxn1 expression in fetal TECs. Remarkably, during branching morphogenesis of lung and lacrimal glands, Wnt overexpression, stimulated Wnt signaling and conditional overexpression of β-catenin all result in decreased branching morphogenesis [135]. However, it is important to remark that sustained Wnt signaling promotes the production of secreted

The control exerted by Pax1, perhaps also by Pax 9, and Hoxa3 on early thymus development presumably follows a common pathway [107, 108]. Hoxa 3 is expressed in both endoderm of third pharyngeal pouch and NC-derived mesenchyme [107]. When this expression is downregulated in E10.5–11 Hoxa3−/− mice, the formation of parathyroid/thymus rudiment is blocked, increases the proportion of apoptotic endodermal cells and there is reduced proliferation of mesenchyme cells [112]. More importantly, the expression of both Pax1 and Pax9 decreases in the third pharyngeal pouch of E10,5 Hoxa3-deficient embryos [107], suggesting that Pax 1/9 act downstream of Hoxa3 but all three molecules have synergistic and dosedependent effects on early thymus maturation [88, 113, 114]. Thus, Hoxa3+/− Pax1+/− double heterozygous mice have a similar phenotype as Pax1−/− mutants, but Hoxa3+/− Pax1−/− hypoplastic thymi exhibit a more severe phenotype than Pax1−/− [114].

Eya1 is involved in the regulation of genes controlling cell growth, activating the repressor Six (Sine oculis). The expression of Eya1, Six, and Pax genes colocalizes in the NC cells and the pharyngeal endoderm [115]. In the absence of Eya1, the third pharyngeal pouch does not detach from the pharyngeal endoderm, and consequently, the thymic primordium is not formed. Foxn1, Pax1, and Pax3 are not expressed in the thymic area, but Hoxa3, Pax1, and Pax3 appear in the E10.5 pouch endoderm [115]. On the other hand, endodermal Six expression is Eya1 dependent, and loss of Six1 in Eya1−/− embryos contributes to the induced thymic defects [116]. Accordingly, Six1 acts downstream of Eya1, whereas Hoxa3, Pax1, and Pax3 do it upstream or independently of Eya 1 [117].

#### *4.2.3. FGF family*

FGF is an extensive family of molecules that influences cell survival, proliferation, and differentiation of many epithelial organs, as repeatedly mentioned in this review. FGF8 is expressed in the pouch epithelium, whereas FGF10 is produced by underlying NC-derived mesenchyme both being involved in the maturation of endoderm [104]. After pouch formation, FGF7 and again FGF10, activate FGFR2iiib receptor on fetal TECs for inducing their proliferation. Accordingly, deficient mice either in the receptor or FGF10 show severe thymic hypoplasia and reduced TEC proliferation [118–120]. Likewise, removal of surrounding mesenchyme from E12 fetal thymus inhibits the growth but not the differentiation of epithelial cells [119, 121]. Other studies demonstrate that FGF7 produced by thymic blood vessels also promotes expansion but not differentiation of TECs [118].

#### *4.2.4. Shh*

Shh is a promoting factor for parathyroid development via Tbx1 [122], whereas negatively regulating the growth of thymus domain. Consequently, Shh functions as an antagonist of BMP4 signaling [87]. Shh is expressed early in the posterior endoderm of second pouch and then in the third arch endoderm, acting upstream of Tbx1 [123] and affecting the patterning of pharyngeal pouches [77].

#### *4.2.5. BMP family*

vetrocaudally remaining in the larynx. Although the primordium is colonized by lymphoid progenitors, it shows decreased proportions of proliferating cells and increased apoptosis

The control exerted by Pax1, perhaps also by Pax 9, and Hoxa3 on early thymus development presumably follows a common pathway [107, 108]. Hoxa 3 is expressed in both endoderm of third pharyngeal pouch and NC-derived mesenchyme [107]. When this expression is downregulated in E10.5–11 Hoxa3−/− mice, the formation of parathyroid/thymus rudiment is blocked, increases the proportion of apoptotic endodermal cells and there is reduced proliferation of mesenchyme cells [112]. More importantly, the expression of both Pax1 and Pax9 decreases in the third pharyngeal pouch of E10,5 Hoxa3-deficient embryos [107], suggesting that Pax 1/9 act downstream of Hoxa3 but all three molecules have synergistic and dosedependent effects on early thymus maturation [88, 113, 114]. Thus, Hoxa3+/− Pax1+/− double heterozygous mice have a similar phenotype as Pax1−/− mutants, but Hoxa3+/− Pax1−/−

Eya1 is involved in the regulation of genes controlling cell growth, activating the repressor Six (Sine oculis). The expression of Eya1, Six, and Pax genes colocalizes in the NC cells and the pharyngeal endoderm [115]. In the absence of Eya1, the third pharyngeal pouch does not detach from the pharyngeal endoderm, and consequently, the thymic primordium is not formed. Foxn1, Pax1, and Pax3 are not expressed in the thymic area, but Hoxa3, Pax1, and Pax3 appear in the E10.5 pouch endoderm [115]. On the other hand, endodermal Six expression is Eya1 dependent, and loss of Six1 in Eya1−/− embryos contributes to the induced thymic defects [116]. Accordingly, Six1 acts downstream of Eya1, whereas Hoxa3, Pax1, and Pax3

FGF is an extensive family of molecules that influences cell survival, proliferation, and differentiation of many epithelial organs, as repeatedly mentioned in this review. FGF8 is expressed in the pouch epithelium, whereas FGF10 is produced by underlying NC-derived mesenchyme both being involved in the maturation of endoderm [104]. After pouch formation, FGF7 and again FGF10, activate FGFR2iiib receptor on fetal TECs for inducing their proliferation. Accordingly, deficient mice either in the receptor or FGF10 show severe thymic hypoplasia and reduced TEC proliferation [118–120]. Likewise, removal of surrounding mesenchyme from E12 fetal thymus inhibits the growth but not the differentiation of epithelial cells [119, 121]. Other studies demonstrate that FGF7 produced by thymic blood vessels also

Shh is a promoting factor for parathyroid development via Tbx1 [122], whereas negatively regulating the growth of thymus domain. Consequently, Shh functions as an antagonist of BMP4 signaling [87]. Shh is expressed early in the posterior endoderm of second pouch and then in the third arch endoderm, acting upstream of Tbx1 [123] and affecting the patterning

finally resulting, as Pax1-deficient thymi, in small thymi [111].

hypoplastic thymi exhibit a more severe phenotype than Pax1−/− [114].

do it upstream or independently of Eya 1 [117].

promotes expansion but not differentiation of TECs [118].

*4.2.3. FGF family*

30 Histology

*4.2.4. Shh*

of pharyngeal pouches [77].

Particularly relevant is the role played by BMPs and Wnt molecules in the early thymic development, as direct controllers of Foxn1 expression, the key transcription factor mandatory for the late embryogenesis of thymus [124]. In addition, both signaling pathways constitute the major means for NC-derived mesenchyme to signal thymic epithelial rudiment [93, 124, 125]. Possibly, FGF8 produced by the primordial endoderm signals to the adjacent mesenchyme inducing BMP4 expression [126]. BMP4 and its antagonist Noggin govern the parathyroid/ thymus individualization and the Foxn1-dependent TEC maturation. In general, BMP4 is essential for the early stages of thymus development prior to the onset of Foxn1 expression [93]. BMP4 is expressed in the ventral domain of the pouch and Noggin in the dorsal area colocalizing with Gcm2 in the parathyroid domain [83]. Furthermore, BMP4 seems to be also involved in the full parathyroid/thymus separation, as BMP4 deletion delays the process [93]. Inhibition of BMP signaling provokes decline of Foxn1 expression in the zebrafish thymic primordium [83, 125], and BMP4 signaling promotes Foxn1 expression in early chicken thymus [126], as well as in mouse FTOCs [127]. Loss of BMP4 from pharyngeal endoderm and underlying mesenchyme prior to the onset of Foxn1 expression does not affect patterning, separation from the pharynx, or initial organ formation, although it alters some important morphogenetic processes such as lumen closure, organ separation and migration, initial lymphoid seeding, and formation of mesenchyme thymic capsule [93]. The sequence established between BMP and Foxn1 is the following: FGF8-mediated mesenchymal BMP signaling initiates the expression of both Foxn1 and BMP4 in the endodermal cells [126]. Then, endodermal BMP4 expression targets a regulatory feedback loop [128] for maintaining BMP4 and Foxn1 expression in the future thymic epithelium rather to directly affect Foxn1 [129]. In these conditions, if BMP signaling is blocked, the expression of both molecules ceases and nonfunctional Foxn1-TECs would remain in the thymus. If this occurs during concrete periods of midgestation, thymopoiesis will irreversibly fail [129]. Therefore, the balance between BMP4 and its inhibitors (i.e., Noggin) becomes critical for a proper maturation of thymic epithelium.

#### *4.2.6. Wnt family*

Wnt family members are extensively expressed in developing and adult thymi in both TECs and fibroblasts [130], whereas their receptors are only detected on TECs [124]. Particularly, the noncanonical Wnt4 and Wnt5b, but also the canonical Wnt10b, coexpress with Foxn1 in third pharyngeal pouch and later in E13 and adult thymus [131] and are involved in its control [124]. Thus, overexpression of Dkk1, a Wnt4 inhibitor, in TECs induces thymic atrophy with reduced epithelial progenitors and TEC proliferation and appearance of TEC proliferation [132]. However, recent results indicate that a proper thymus development can only occur when β-catenin-dependent Wnt signaling is low or lacking [133]. Thus, β-catenin-deficient thymi exhibit Foxn1 expression, and stabilized β-catenin overexpression shows decreased rather than increased Foxn1 transcripts [133, 134]. Therefore, these results suggest that β-catenin is dispensable for Foxn1 expression in fetal TECs. Remarkably, during branching morphogenesis of lung and lacrimal glands, Wnt overexpression, stimulated Wnt signaling and conditional overexpression of β-catenin all result in decreased branching morphogenesis [135]. However, it is important to remark that sustained Wnt signaling promotes the production of secreted Wnt antagonisms [136] that block thymocyte development in FTOCs [137]. On the other hand, other signaling pathways involved in TEC differentiation, such as those mediated by BMPs, modulate the effects produced by Wnt4 overexpression [133].
