**2. The early development of endodermal epithelial organs**

#### **2.1. Specification and primordium development**

The process of development followed by these epithelial tissues is well exemplified by the early development of salivary glands. The primordium of submandibular glands raises from an evaginated thickening of ectoderm-derived oral epithelium into the neural crest-derived mesenchyme at the base of tongue [1]. The evaginated epithelium proliferates forming an epithelial "stalk" and a terminal bud. The stalk will evolve into excretory duct cells, and the buds will establish the named "pseudoglandular" area by repeated elongation and branching morphogenesis, which will finally differentiate into functional acini (**Figure 1**) [2].

In mice, mammary placodes are visible at E11-E12 and become buds at E13 when surrounded by several layers of mesenchyme. Signals from the mesenchyme of cardia and septum transversum determine the hepatic fate in the foregut endoderm, [3] inducing expression of the transcription factor Hhex, but not Pdx1, whereas the Hhex-Pdx1+ foregut endoderm will differentiate into the extrahepatic bile ducts and the ventral pancreas [4]. Apart from this ventral area, the embryonic pancreas in vertebrates forms from a dorsal protrusion of the primitive gut epithelium, which express Mnx1 [5]. These two pancreatic buds grow, branch, and fuse to form a multilayered epithelium (E9.0 to E11.5), which forms the definitive pancreas. This stratified epithelium consists of two domains: an outer layer of semipolarized "cap" cells, which express only basal markers, and an inner "body" of nonpolarized cells [6, 7] (**Figure 1**).

At E9.0-E9.5, Nkx2–1, a transcription factor specific of the lung, is determined on the ventral side of the anterior foregut by Wnt ligands expressed in the surrounding ventral mesoderm that activates the canonical Wnt pathway in the epithelium. One day later, Nkx2–1+ cells extend ventrally forming a primitive trachea and two lung buds, whereas Sox 2 expression restricted on the distal foregut endoderm will determine the esophagus. Next, the trachea and the esophagus become fully separated [8] (**Figure 1**). Thus, absence of Wnt signaling

**Figure 1.** Different models of branching morphogenesis occurring during development of lung (branching of tubes), salivary gland (branching of an unpolarized primordium and later de novo lumen formation), and pancreas (polarization and remodeling of an unpolarized mass resulting in more synchronous branching, lumen formation, and

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differentiation). Modified from [5, 6, 140].

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In the present chapter, we will examine prior studies supporting the claim that the development of other branching organs as lung, salivary gland, pancreas, or kidney, despite their morphological and functional differences, follows common patterning programs under the control of epithelium-underlying mesenchyme interactions governed by a few families of molecules (FGF/FGFR, Wnt, BMP/TGFβ, Shh), and that the thymus, an epithelial primary lymphoid organ derived from the ventral endoderm of the third pharyngeal pouch, despite following the same pattern, constitutes a special case. Remarkably, its functions are not related to those of other epithelia of similar origin but rather to the establishment of a 3D epithelial network necessary for the functional maturation of thymocytes. Before acquiring their specific features, distinct epithelial organs, therefore, follow a common complex pattern of development which includes different processes. After a first step of specification from the original embryonic layer, they undergo a process of **tubulogenesis** consisting of outgrowth and extension of the epithelial primordium forming a tubular structure. A complex program of **branching morphogenesis helps to** increase the functional area of the organ. Finally, **terminal epithelial differentiation** prepares the primordium to become a functional organ.

**2. The early development of endodermal epithelial organs**

The process of development followed by these epithelial tissues is well exemplified by the early development of salivary glands. The primordium of submandibular glands raises from an evaginated thickening of ectoderm-derived oral epithelium into the neural crest-derived mesenchyme at the base of tongue [1]. The evaginated epithelium proliferates forming an epithelial "stalk" and a terminal bud. The stalk will evolve into excretory duct cells, and the buds will establish the named "pseudoglandular" area by repeated elongation and branching

In mice, mammary placodes are visible at E11-E12 and become buds at E13 when surrounded by several layers of mesenchyme. Signals from the mesenchyme of cardia and septum transversum determine the hepatic fate in the foregut endoderm, [3] inducing expression of the transcription factor Hhex, but not Pdx1, whereas the Hhex-Pdx1+ foregut endoderm will differentiate into the extrahepatic bile ducts and the ventral pancreas [4]. Apart from this ventral area, the embryonic pancreas in vertebrates forms from a dorsal protrusion of the primitive gut epithelium, which express Mnx1 [5]. These two pancreatic buds grow, branch, and fuse to form a multilayered epithelium (E9.0 to E11.5), which forms the definitive pancreas. This stratified epithelium consists of two domains: an outer layer of semipolarized "cap" cells, which express only basal markers, and an inner "body" of nonpolarized cells [6, 7] (**Figure 1**). At E9.0-E9.5, Nkx2–1, a transcription factor specific of the lung, is determined on the ventral side of the anterior foregut by Wnt ligands expressed in the surrounding ventral mesoderm that activates the canonical Wnt pathway in the epithelium. One day later, Nkx2–1+ cells extend ventrally forming a primitive trachea and two lung buds, whereas Sox 2 expression restricted on the distal foregut endoderm will determine the esophagus. Next, the trachea and the esophagus become fully separated [8] (**Figure 1**). Thus, absence of Wnt signaling

morphogenesis, which will finally differentiate into functional acini (**Figure 1**) [2].

**2.1. Specification and primordium development**

20 Histology

**Figure 1.** Different models of branching morphogenesis occurring during development of lung (branching of tubes), salivary gland (branching of an unpolarized primordium and later de novo lumen formation), and pancreas (polarization and remodeling of an unpolarized mass resulting in more synchronous branching, lumen formation, and differentiation). Modified from [5, 6, 140].

courses with lack of Nkx2–1 expression, absence of tracheal morphogenesis, and lung agenesis [9]. BMP expression in the ventral mesoderm is necessary to establish a proper location of the lung along the proximal distal axis of the foregut [8]. Also, FGF molecules expressed in the ventral mesoderm reinforce early lung specification of the foregut endoderm [8]. Murine embryos deficient in either FGF10 or FGFR2b exhibit a stopped salivary gland development at the initial bud stage [10], and conditioned deletion of FGF8 in the ectoderm results in arrest of salivary gland development [11]. The process could be more complex because specific overexpression of FGF7 in the salivary gland epithelium produces small glands that exhibit delayed differentiation [12], and elimination of FGF signaling antagonists, Sprouty 1 and 2, impairs salivary epithelium development [13]. Indeed, multiple branching organs undergo agenesis after deletion of either FGF10 or its receptor FGFR2b [10].

As above indicated, the establishment of cell polarity, in which cues provided by neighboring cells and ECM play major roles, has particular relevance for epithelial tubulogenesis. These cues activate signaling pathways, particularly those mediated by Wnt ligands and their receptors [15], which modify the cytoskeleton, cell contractibility, and trafficking, as well as the transcription program. Wnt ligands and receptors arrange in the epithelial cells in a polarized manner. Wnt5a and 3a are released specifically throughout the basolateral cell surface, where their specific receptors, Fz2, LRP6, and Rer2 are expressed [21]. In the embryonic midgut, Wnt5a is produced by mesenchyme cells under the basement membrane and it activates Wnt5a receptor (Fz2, Rer2) on the basolateral domains of epithelial cells, resulting in Racdependent adhesion, establishment of apical/basal polarization, formation of cell junctions, and organization of intracellular molecule trafficking necessary to establish different apical

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The developing submandibular gland expresses numerous Wnt ligands and receptors, as well as antagonists in both epithelium and mesenchyme and are accurately regulated spatially and temporally [13]. Wnt signaling promotes duct development by coordinating canonical and noncanonical pathways. Canonical activation through Wnt/β-catenin signaling inhibits end bud formation, whereas Wnt 5b activates the noncanonical Wnt pathway to determine duct formation with the concourse of the transcription factor, TFCP2L1. Inhibition of end bud formation is a consequence of the absence of Wnt distally, regulated by FGF signaling that represses Wnt5b expression and upregulates the Wnt inhibitor, SFRP1, (secreted related

Likewise, retinoid acid produced by foregut mesenchyme before lung specification [23] signals through retinoid acid receptor B in the mesoderm to regulate FGF expression [24]. Shh

Distinct members of the EGF family (EGF, TGFα) and their receptors (EGFR 1, 2, 3) as well as heparin-binding EGF (HBEGF) and neuregulin are differentially expressed in the salivary glandular epithelium and mesenchyme, and the activation of EGF receptors modulates ductal

Hedgehog proteins, mainly Shh and their receptors Patched1 and Smoothed, also participate in the organization of a salivary duct and a preacinar end bud (prostate, sebaceous glands, mammary glands, lung) [11], but its effects on these organs are indirect because Hh signals in the mesenchyme, whereas in the salivary gland, the action is directly exerted on duct epithelium [26]. On the other hand, overexpression of Gli-1, one effector of Hh pathway in keratin+ epithelial cells, results in large lumens, duct expansion, and loss of acini [27]. Again, Hh and FGF8 appear to cooperate in these processes. FGF is a Gli3-mediated target of Hh signaling pathway. Both FGF8 and Shh positively upregulate each other [28], and the former rescues defects in salivary gland development produced by cyclopamine, a blocker of Hh signaling [11]. Shh could collaborate with other molecules, such as ectodysplasin [29] or TGFβ [30] in

Branching morphogenesis constitutes a developmental program that induces the building of an arborized network, in which new tubules arise from the pre-existing ones by repeated

signaling in the mesoderm is also a regulator of the initial lung bud outgrowth [25].

morphogenesis by governing progenitor cell differentiation and expansion [26].

the formation of the salivary gland duct, but results are contradictory.

**2.3. Branching morphogenesis**

and basal domains.

frizzled protein 1), which sequesters Wnt proteins [22].

#### **2.2. Tubulogenesis**

Both mono- and pluristratified epithelia have the capacity to fold and form tubes [14]. Distinct mechanisms of cellular binding have been reported, including the orientation of cells via cellto-cell and cell-ECM interactions, the establishment of apical-basal polarity, changes in the cellular shape and migration capacities, and formation and expansion of the luminal spaces, which eventually fuse establishing a unique cavity [14, 15].

Although some tubules show lumens surrounded by a single cell, they normally consist of multicellular lumens sealed by cell junctions. In addition, tubules may branch (see later) and/ or differentiate into end buds or cap-like structures, as acini (pancreas, mammary, and salivary glands) or alveoli (lung) [16] (**Figure 1**). Further variability in the process of tubulogenesis is provided by the distinct mechanisms used for the formation of lumens. Budding, wrapping, entrapment, cavitation, and hollowing have been described in organs, which undergo tubulogenesis during their development. Budding or wrapping occur in polarized epithelia, as is the case of the lung, whereas the formation of tubules by entrapment, cavitation, or hollowing is performed by nonpolarized cells [16]. In the entrapment, migrating cells trap an extracellular space and form a lumen [17]. By contrast, the formation of lumens by cavitation, reported in mammary and salivary glands, implies programmed cell death to create a cell-free space [18], whereas in the hollowing, the luminal space is organized de novo via exocytosis of intracellular vesicles [19]. The salivary glands, the liver, or the pancreas undergo polarization from unpolarized primordia (**Figure 1**). In the pancreas, E10.5–11.5 individual cells within the inner body of pancreatic buds acquire apico-basal polarity and rearrange to form microlumina by fusion of apical membrane-containing vesicles with the cell membrane. During this process, the asynchronous apical constriction of individual polarized cells generates rosettes with a central lumen that later expand and eventually fuse to generate an immature, highly interconnected tubular plexus, consisting of stratified epithelial cells surrounded by an epithelial periphery [7]. Their reorganization will form the ductal system and primordial endocrine islets and the acinar exocrine cells, respectively (**Figure 1**) [5, 6].

In the salivary gland, lumen formation takes place and evolves along the forming branched structure, following branching progression. Initially, epithelial cell polarization results in multiple microlumens that fuse to form a contiguous lumen [20] (**Figure 1**). The signaling events controlling microlumen fusion to establish a common single lumen are just beginning to emerge [15].

As above indicated, the establishment of cell polarity, in which cues provided by neighboring cells and ECM play major roles, has particular relevance for epithelial tubulogenesis. These cues activate signaling pathways, particularly those mediated by Wnt ligands and their receptors [15], which modify the cytoskeleton, cell contractibility, and trafficking, as well as the transcription program. Wnt ligands and receptors arrange in the epithelial cells in a polarized manner. Wnt5a and 3a are released specifically throughout the basolateral cell surface, where their specific receptors, Fz2, LRP6, and Rer2 are expressed [21]. In the embryonic midgut, Wnt5a is produced by mesenchyme cells under the basement membrane and it activates Wnt5a receptor (Fz2, Rer2) on the basolateral domains of epithelial cells, resulting in Racdependent adhesion, establishment of apical/basal polarization, formation of cell junctions, and organization of intracellular molecule trafficking necessary to establish different apical and basal domains.

The developing submandibular gland expresses numerous Wnt ligands and receptors, as well as antagonists in both epithelium and mesenchyme and are accurately regulated spatially and temporally [13]. Wnt signaling promotes duct development by coordinating canonical and noncanonical pathways. Canonical activation through Wnt/β-catenin signaling inhibits end bud formation, whereas Wnt 5b activates the noncanonical Wnt pathway to determine duct formation with the concourse of the transcription factor, TFCP2L1. Inhibition of end bud formation is a consequence of the absence of Wnt distally, regulated by FGF signaling that represses Wnt5b expression and upregulates the Wnt inhibitor, SFRP1, (secreted related frizzled protein 1), which sequesters Wnt proteins [22].

Likewise, retinoid acid produced by foregut mesenchyme before lung specification [23] signals through retinoid acid receptor B in the mesoderm to regulate FGF expression [24]. Shh signaling in the mesoderm is also a regulator of the initial lung bud outgrowth [25].

Distinct members of the EGF family (EGF, TGFα) and their receptors (EGFR 1, 2, 3) as well as heparin-binding EGF (HBEGF) and neuregulin are differentially expressed in the salivary glandular epithelium and mesenchyme, and the activation of EGF receptors modulates ductal morphogenesis by governing progenitor cell differentiation and expansion [26].

Hedgehog proteins, mainly Shh and their receptors Patched1 and Smoothed, also participate in the organization of a salivary duct and a preacinar end bud (prostate, sebaceous glands, mammary glands, lung) [11], but its effects on these organs are indirect because Hh signals in the mesenchyme, whereas in the salivary gland, the action is directly exerted on duct epithelium [26]. On the other hand, overexpression of Gli-1, one effector of Hh pathway in keratin+ epithelial cells, results in large lumens, duct expansion, and loss of acini [27]. Again, Hh and FGF8 appear to cooperate in these processes. FGF is a Gli3-mediated target of Hh signaling pathway. Both FGF8 and Shh positively upregulate each other [28], and the former rescues defects in salivary gland development produced by cyclopamine, a blocker of Hh signaling [11]. Shh could collaborate with other molecules, such as ectodysplasin [29] or TGFβ [30] in the formation of the salivary gland duct, but results are contradictory.

#### **2.3. Branching morphogenesis**

courses with lack of Nkx2–1 expression, absence of tracheal morphogenesis, and lung agenesis [9]. BMP expression in the ventral mesoderm is necessary to establish a proper location of the lung along the proximal distal axis of the foregut [8]. Also, FGF molecules expressed in the ventral mesoderm reinforce early lung specification of the foregut endoderm [8]. Murine embryos deficient in either FGF10 or FGFR2b exhibit a stopped salivary gland development at the initial bud stage [10], and conditioned deletion of FGF8 in the ectoderm results in arrest of salivary gland development [11]. The process could be more complex because specific overexpression of FGF7 in the salivary gland epithelium produces small glands that exhibit delayed differentiation [12], and elimination of FGF signaling antagonists, Sprouty 1 and 2, impairs salivary epithelium development [13]. Indeed, multiple branching organs undergo agenesis

Both mono- and pluristratified epithelia have the capacity to fold and form tubes [14]. Distinct mechanisms of cellular binding have been reported, including the orientation of cells via cellto-cell and cell-ECM interactions, the establishment of apical-basal polarity, changes in the cellular shape and migration capacities, and formation and expansion of the luminal spaces,

Although some tubules show lumens surrounded by a single cell, they normally consist of multicellular lumens sealed by cell junctions. In addition, tubules may branch (see later) and/ or differentiate into end buds or cap-like structures, as acini (pancreas, mammary, and salivary glands) or alveoli (lung) [16] (**Figure 1**). Further variability in the process of tubulogenesis is provided by the distinct mechanisms used for the formation of lumens. Budding, wrapping, entrapment, cavitation, and hollowing have been described in organs, which undergo tubulogenesis during their development. Budding or wrapping occur in polarized epithelia, as is the case of the lung, whereas the formation of tubules by entrapment, cavitation, or hollowing is performed by nonpolarized cells [16]. In the entrapment, migrating cells trap an extracellular space and form a lumen [17]. By contrast, the formation of lumens by cavitation, reported in mammary and salivary glands, implies programmed cell death to create a cell-free space [18], whereas in the hollowing, the luminal space is organized de novo via exocytosis of intracellular vesicles [19]. The salivary glands, the liver, or the pancreas undergo polarization from unpolarized primordia (**Figure 1**). In the pancreas, E10.5–11.5 individual cells within the inner body of pancreatic buds acquire apico-basal polarity and rearrange to form microlumina by fusion of apical membrane-containing vesicles with the cell membrane. During this process, the asynchronous apical constriction of individual polarized cells generates rosettes with a central lumen that later expand and eventually fuse to generate an immature, highly interconnected tubular plexus, consisting of stratified epithelial cells surrounded by an epithelial periphery [7]. Their reorganization will form the ductal system and primordial endocrine

In the salivary gland, lumen formation takes place and evolves along the forming branched structure, following branching progression. Initially, epithelial cell polarization results in multiple microlumens that fuse to form a contiguous lumen [20] (**Figure 1**). The signaling events controlling microlumen fusion to establish a common single lumen are just beginning

after deletion of either FGF10 or its receptor FGFR2b [10].

which eventually fuse establishing a unique cavity [14, 15].

islets and the acinar exocrine cells, respectively (**Figure 1**) [5, 6].

**2.2. Tubulogenesis**

22 Histology

to emerge [15].

Branching morphogenesis constitutes a developmental program that induces the building of an arborized network, in which new tubules arise from the pre-existing ones by repeated rounds of sprouting [15]. Two morphological models can be distinguished: de novo branching from the surface of a primordial epithelium or the lateral side of a pre-existing branch (budding) and the splitting of a pre-existing branch tip into several tips (clefting) [31]. Moreover, branching morphogenesis can be stereotypic as occurs in the kidney branches [32] or stochastic, without a defined pattern, as reported in mammary gland or salivary gland [31]. At the cellular level, new branch formation can be driven by collective cell migration, patterned cell proliferation and differential growth, coordinated cell deformation or epithelial folding, and/or cell arrangement and matrix-driven branching [31]. Budding in blood vessels and Drosophila trachea follows an invasive form of collective cell migration, whereas in mammalian epithelial organs (i.e., mammary gland) budding appears to be powered by a noninvasive form of collective cell migration along with cell proliferation [33].

are strongly expressed but downregulated in pregnancy [47] and overexpression of Wnt 4

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Both canonical and noncanonical Wnt signaling pathways are necessary for lung branching morphogenesis. Reduced canonical Wnt/β-catenin signaling in the pulmonary epithelium causes enlarged bronchioles and reduced epithelial branches and alveoli [49], and conditional deletion of β-catenin or overexpression of Wnt inhibitor Dickkopf 1 severely impairs branching morphogenesis [49, 50] by regulating FGFR2 and BMP4 effects on lung epithelium [50]. BMP4 seems to limit FGF10-mediated lung epithelial outgrowth [51]. Wnt5a appears to be a key for determining the effects of noncanonical Wnt pathway in lung branching morphogenesis. Wnt5a−/− mice exhibit increased formation of peripheral airways [52]. In this case, effects of Wnt5a are mediated through Shh: Wnt5a regulates Shh expression in the lung epithelium

Several members of the FGF family and their receptors are expressed in the renal stroma whereas FGF stimulation induces the appearance of branched tubular structures [53], and the

FGFR1 and FGFR2 are expressed in pubertal and adult mammary glands, and the specific deletion of FGFR1 in keratin 14+ cells produces a transitional delay of the gland development with reduced ductal outgrowth and branch points [55], whereas the deletion of FGFR2 produces an incomplete branching [56]. In addition, FGF10 directs the early stages of epithelial migration and branching, whereas FGF2 is responsible for epithelial expansion and duct

FGF/FGFR signaling is also a key for generating new branches in the developing lung [58]. FGFR2b, which binds four ligands (FGF1, FGF3, FGF7, and FGF10) detected on mesenchymal cells [58] is largely expressed in the airway epithelium, and FGF signaling in lung is associated with Shh pathway [59]. Activation of FGFR2b on epithelial cells by FGF10 secreted by mesenchyme cells induces Shh expression that creates a negative feedback loop by regulating

Recently, results on the effects of TGFβ1 have been contradictory. TCFβ1 that accumulates in mesenchyme inhibits the branching by inducing components of ECM [61], but its in vivo elimination does not result in altered branching, perhaps due to the existence of other similar

At the end of the development, specialized epithelial cell types appear and gradually mature. In the case of salivary glands, final differentiation of end buds into secretory acini is followed

Complex signaling established between lung epithelium, mesenchyme, ECM and vasculature is essential for normal alveolar space organization. Between E16.5 and 3-5PN, lungs develop at the distal end of branches saccules that finally form alveoli (3-14PN in mice) establishing a proximal-distal polarity in the just formed branches (**Figure 1**). Thus, whereas Sox-2

by further growth and functional differentiation [26] (**Figure 1**).

lack of FGFR2b generates significantly smaller kidneys with reduced branches [54].

increases branching while lacking results in delayed ductal branching [48].

and, in turn, Shh regulates FGF10 signaling in the mesenchyme [52].

elongation [57].

FGF10 levels [60].

factors as TGFβ2 and TGFβ3 [62].

**2.4. Cell differentiation**

Cell proliferation is related to organ growth, and differential cell proliferation may be related to branched budding [31]. Blocking cell proliferation abolishes budding in cultured mouse lung [34] and mammary gland [33], whereas clefting in salivary gland still proceeds [35] mediated by cytoskeleton and ECM remodeling [36]. Clefting at the branch tip in lung and kidney requires proliferation to enlarge the tip, which deforms and splits [37]. In kidney, other factors contribute because less mesenchyme cells correlate with less branching [38], and studies on the 3D morphology of fetal organs demonstrate that the local geometry of the epithelial buds determine the pattern of branching [32].

In lung, branching involve cytoskeleton-mediated constriction of the apical surface of cells [39], with the concourse of Rho GTPases and the involvement of Wnt-dependent planar cell polarity pathway [15].

ECM elements also play an important role in branching morphogenesis. Thus, fibronectin accumulates at branch point constriction and its block inhibits cleft formation [40]. In addition, the loss of β1 integrin that interacts with fibronectin blocks the branching morphogenesis inducing a multilayered epithelium [41]. Degradation of collagen 1 and collagen 3 reduces cleft formation and, therefore, branching [42], and the blockade of laminin α1 or γ1 inhibits branching in culture [43], whereas laminin α5−/− embryos show reduced branching [44].

In many organs, branching occurs through repetitive clefting and elongation of epithelial end buds at distal ends, but whereas in some of them, such as the pancreas, lumen formation occurs concomitantly with branching [7]; in others (i.e., salivary glands), there is a substantive delay between the two processes [45] (**Figure 1**). In pancreas, lumen formation gives rise to a plexus and, at the same time, the epithelial bud is progressively transformed into a lobulated surface of multiple minor protruding tips interrupted by epithelial ridges. Progressive remodeling of the pancreatic plexus in an outside-in continuous manner, eventually leading to a single-layered epithelial network surrounding a single lumen [6, 46] (**Figure 1**).

In the same manner as previous stages of epithelium development, branching morphogenesis is controlled through epithelial-mesenchyme interactions mediated by a network of signaling pathways that includes largely Wnt, FGF, Shh, and TGFβ/BMP. Mammary glands undergo several processes of branching morphogenesis, associated with their physiological cycle, under control of Wnt signaling [15]. In virgin glands, Wnt2, Wnt5a, and Wnt7b are strongly expressed but downregulated in pregnancy [47] and overexpression of Wnt 4 increases branching while lacking results in delayed ductal branching [48].

Both canonical and noncanonical Wnt signaling pathways are necessary for lung branching morphogenesis. Reduced canonical Wnt/β-catenin signaling in the pulmonary epithelium causes enlarged bronchioles and reduced epithelial branches and alveoli [49], and conditional deletion of β-catenin or overexpression of Wnt inhibitor Dickkopf 1 severely impairs branching morphogenesis [49, 50] by regulating FGFR2 and BMP4 effects on lung epithelium [50]. BMP4 seems to limit FGF10-mediated lung epithelial outgrowth [51]. Wnt5a appears to be a key for determining the effects of noncanonical Wnt pathway in lung branching morphogenesis. Wnt5a−/− mice exhibit increased formation of peripheral airways [52]. In this case, effects of Wnt5a are mediated through Shh: Wnt5a regulates Shh expression in the lung epithelium and, in turn, Shh regulates FGF10 signaling in the mesenchyme [52].

Several members of the FGF family and their receptors are expressed in the renal stroma whereas FGF stimulation induces the appearance of branched tubular structures [53], and the lack of FGFR2b generates significantly smaller kidneys with reduced branches [54].

FGFR1 and FGFR2 are expressed in pubertal and adult mammary glands, and the specific deletion of FGFR1 in keratin 14+ cells produces a transitional delay of the gland development with reduced ductal outgrowth and branch points [55], whereas the deletion of FGFR2 produces an incomplete branching [56]. In addition, FGF10 directs the early stages of epithelial migration and branching, whereas FGF2 is responsible for epithelial expansion and duct elongation [57].

FGF/FGFR signaling is also a key for generating new branches in the developing lung [58]. FGFR2b, which binds four ligands (FGF1, FGF3, FGF7, and FGF10) detected on mesenchymal cells [58] is largely expressed in the airway epithelium, and FGF signaling in lung is associated with Shh pathway [59]. Activation of FGFR2b on epithelial cells by FGF10 secreted by mesenchyme cells induces Shh expression that creates a negative feedback loop by regulating FGF10 levels [60].

Recently, results on the effects of TGFβ1 have been contradictory. TCFβ1 that accumulates in mesenchyme inhibits the branching by inducing components of ECM [61], but its in vivo elimination does not result in altered branching, perhaps due to the existence of other similar factors as TGFβ2 and TGFβ3 [62].

#### **2.4. Cell differentiation**

rounds of sprouting [15]. Two morphological models can be distinguished: de novo branching from the surface of a primordial epithelium or the lateral side of a pre-existing branch (budding) and the splitting of a pre-existing branch tip into several tips (clefting) [31]. Moreover, branching morphogenesis can be stereotypic as occurs in the kidney branches [32] or stochastic, without a defined pattern, as reported in mammary gland or salivary gland [31]. At the cellular level, new branch formation can be driven by collective cell migration, patterned cell proliferation and differential growth, coordinated cell deformation or epithelial folding, and/or cell arrangement and matrix-driven branching [31]. Budding in blood vessels and Drosophila trachea follows an invasive form of collective cell migration, whereas in mammalian epithelial organs (i.e., mammary gland) budding appears to be powered by a noninvasive

Cell proliferation is related to organ growth, and differential cell proliferation may be related to branched budding [31]. Blocking cell proliferation abolishes budding in cultured mouse lung [34] and mammary gland [33], whereas clefting in salivary gland still proceeds [35] mediated by cytoskeleton and ECM remodeling [36]. Clefting at the branch tip in lung and kidney requires proliferation to enlarge the tip, which deforms and splits [37]. In kidney, other factors contribute because less mesenchyme cells correlate with less branching [38], and studies on the 3D morphology of fetal organs demonstrate that the local geometry of the

In lung, branching involve cytoskeleton-mediated constriction of the apical surface of cells [39], with the concourse of Rho GTPases and the involvement of Wnt-dependent planar cell

ECM elements also play an important role in branching morphogenesis. Thus, fibronectin accumulates at branch point constriction and its block inhibits cleft formation [40]. In addition, the loss of β1 integrin that interacts with fibronectin blocks the branching morphogenesis inducing a multilayered epithelium [41]. Degradation of collagen 1 and collagen 3 reduces cleft formation and, therefore, branching [42], and the blockade of laminin α1 or γ1 inhibits branching in culture [43], whereas laminin α5−/− embryos show reduced branching [44].

In many organs, branching occurs through repetitive clefting and elongation of epithelial end buds at distal ends, but whereas in some of them, such as the pancreas, lumen formation occurs concomitantly with branching [7]; in others (i.e., salivary glands), there is a substantive delay between the two processes [45] (**Figure 1**). In pancreas, lumen formation gives rise to a plexus and, at the same time, the epithelial bud is progressively transformed into a lobulated surface of multiple minor protruding tips interrupted by epithelial ridges. Progressive remodeling of the pancreatic plexus in an outside-in continuous manner, eventually leading

In the same manner as previous stages of epithelium development, branching morphogenesis is controlled through epithelial-mesenchyme interactions mediated by a network of signaling pathways that includes largely Wnt, FGF, Shh, and TGFβ/BMP. Mammary glands undergo several processes of branching morphogenesis, associated with their physiological cycle, under control of Wnt signaling [15]. In virgin glands, Wnt2, Wnt5a, and Wnt7b

to a single-layered epithelial network surrounding a single lumen [6, 46] (**Figure 1**).

form of collective cell migration along with cell proliferation [33].

epithelial buds determine the pattern of branching [32].

polarity pathway [15].

24 Histology

At the end of the development, specialized epithelial cell types appear and gradually mature. In the case of salivary glands, final differentiation of end buds into secretory acini is followed by further growth and functional differentiation [26] (**Figure 1**).

Complex signaling established between lung epithelium, mesenchyme, ECM and vasculature is essential for normal alveolar space organization. Between E16.5 and 3-5PN, lungs develop at the distal end of branches saccules that finally form alveoli (3-14PN in mice) establishing a proximal-distal polarity in the just formed branches (**Figure 1**). Thus, whereas Sox-2 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 types 1 and 2 alveolar cells do so in the distal zone [63].

Afterward, at a prefolicular growth stage, the thyroid grows by branching morphogenesis of epithelial cords radiating from the UBB remnant, reminding the pseudoglandular stage of salivary gland before duct generation [72]. Finally, cells polarize locally forming cystic lumens leading to cords of back-to-back connected follicles. This folliculogenesis occurs synchronously, not in a proximal/distal direction and is related to Sox9 expression, which is firstly expressed in some cells in the placode and finally accumulates in the distal portions as

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Therefore, thyroid development is equivalent to that of an exocrine gland (i.e., salivary gland) in which the ductal system is not fully differentiated but is regressed, and the endocrine portion detached. An initially forming continuous branching structure polarizes locally leading

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].

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

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

in other branching organs, remaining in mature follicular cells [72].

to isolated cystic lumens and to the generation of isolated follicles [72].

**molecules**

**4.1. Early thymus development**

or even not contribute to the thymus rudiment [80].

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

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 and endothelial cells favors trunk development [46].

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

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].

## **3. The development of the thyroid**

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

Afterward, at a prefolicular growth stage, the thyroid grows by branching morphogenesis of epithelial cords radiating from the UBB remnant, reminding the pseudoglandular stage of salivary gland before duct generation [72]. Finally, cells polarize locally forming cystic lumens leading to cords of back-to-back connected follicles. This folliculogenesis occurs synchronously, not in a proximal/distal direction and is related to Sox9 expression, which is firstly expressed in some cells in the placode and finally accumulates in the distal portions as in other branching organs, remaining in mature follicular cells [72].

Therefore, thyroid development is equivalent to that of an exocrine gland (i.e., salivary gland) in which the ductal system is not fully differentiated but is regressed, and the endocrine portion detached. An initially forming continuous branching structure polarizes locally leading to isolated cystic lumens and to the generation of isolated follicles [72].
