**5. After acquisition of thymus fate, thymic primordium undergoes tubulogenesis and branching morphogenesis**

E11.5 thymic primordium consists of a bi/pluristratified epithelium polarized with respect to a ramified central lumen resulting from the evagination of pharyngeal epithelium where K5+ Cld 3/4+ cells line the lumen [138, 139]. In the following days, the thymus grows and the K5+ Cld 3/4 + cell cords increase their total length and branching degree. At the same time, external clefts determine an incipient lobulation that became clearly evident by day E14.5. Beyond E12.5, the initial lumen is almost totally closed although a central lumen is still visible at E12,5 and to some extent at E13,5 [139]. Secondary forming lumens can be observed in the K5 + Cld 3/4+ branching cell cords up to E13.5 (**Figure 2**) [139]. Thus, in these initial steps of thymus development, its histological organization is quite similar to that of organs undergoing branching morphogenesis in which the lumen formation and elongation take place within a proliferating bud (**Figure 2**) [20, 140]. These results indicate that between E11.5 and E13.5, a primary lumen connects with secondary and growing order lumens through branched microlumens or polarized canals, giving rise to a continuous formed or forming luminal structure that grows hierarchically (**Figure 2**). Therefore, in the thymus, clefting/branching and lumen formation seems to be more synchronic and not as regionally separated as in the salivary gland, similar to the pancreas condition (**Figure 2**).

However, in the thymus, a definitive duct is not developed, nor is terminal end buds, acini or other differentiated distal secreting structures, but instead the thymus remains as a concentric structure in which the central Cld + K5+ cells will differentiate into the thymic medulla [138, 139]. This central medulla does not apparently present lumen and is surrounded by the thymic cortex differentiated from Cld 3/4- cells (**Figure 2**) [139]. On the other hand, the fact that the branching pattern of the K5 + Cld 3/4+ cell cords appears to be similar between different mice [139] and that rat adult thymic medulla has also been described as a 4–5 order ramified structure in which the ratio of branches sizes is mathematically constant [141], suggesting that thymus development follows a programmed branching pattern.

On the other hand, in the absence of Foxn1, the existence of a tubular-branched structure in which both ductal and acinar components can be distinguished [144] and that cannot be colonized by lymphoid progenitors [76] is clearly evident. This is the situation of Nude (Foxn1−/−) thymi, in which the transcription factor Foxn1 central for thymic epithelial differentiation, lacks. The earliest stages of Foxn1−/− thymus development appear to occur in the same way as those of wt thymus, and the expression of claudin 3/4 and wt thymus takes place in similar ways [145], suggesting that wt thymus organogenesis might be considered as a modification of the tubulogenesis and epithelial branching morphogenesis, which occur in the nude thymus (**Figure 2**). Thus, FoxN1 expression would preclude lumen formation and generate concentric layers of distinct TEC subsets (Muñoz et al., 2018 submitted). Moreover, the conditioned removal of FoxN1 in K14+ epithelial cells results in the progressive polarization of medullary cells, Cld3/4 expression, and lumen formation [146]. Other defects affect mainly thymic branching morphogenesis without importantly altering thymic-specific differentiation. Transgenic expression of Noggin under the control of a FoxN1 promoter leads to a hypoplastic spheric thymus always containing big cystic structures [125]. These structural alterations seem to be the result of a branching defect in consonance with the known role of BMP signaling in regulating branching morphogenesis of different organs [26] and to affect Foxn1 expression [147].

**Figure 2.** Thymus development follows a branching morphogenesis process similar to those of salivary gland and

Epithelial Development Based on a Branching Morphogenesis Program: The Special Condition…

http://dx.doi.org/10.5772/intechopen.81193

33

pancreas (see **Figure 1**).

If, as above indicated, the branching morphogenesis of the developing thymus has some particular features, as the lack of a definitive duct or terminal end buds or acini, the early lymphoid seeding introduces in the thymus development other important differences with respect to other epithelial organs inducing the specific three-dimensional network formed by dendritic-shaped TECs. Thymocyte precursors enter the thymus at around E11.5 through the surrounding mesenchyme [142], and at E12.5 CD45+ , lymphoid progenitors appear associated with nonpolarized TECs that express little or no K5 [139]. This 3D arrangement of thymic epithelium is, to some extent, precluded in the absence of thymocytes [143] in which the presence of medullary luminal or cystic structures becomes more evident, presumably representing a default pathway of epithelial differentiation when thymocytes are missing [143].

Epithelial Development Based on a Branching Morphogenesis Program: The Special Condition… http://dx.doi.org/10.5772/intechopen.81193 33

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,

E11.5 thymic primordium consists of a bi/pluristratified epithelium polarized with respect to a ramified central lumen resulting from the evagination of pharyngeal epithelium where K5+ Cld 3/4+ cells line the lumen [138, 139]. In the following days, the thymus grows and the K5+ Cld 3/4 + cell cords increase their total length and branching degree. At the same time, external clefts determine an incipient lobulation that became clearly evident by day E14.5. Beyond E12.5, the initial lumen is almost totally closed although a central lumen is still visible at E12,5 and to some extent at E13,5 [139]. Secondary forming lumens can be observed in the K5 + Cld 3/4+ branching cell cords up to E13.5 (**Figure 2**) [139]. Thus, in these initial steps of thymus development, its histological organization is quite similar to that of organs undergoing branching morphogenesis in which the lumen formation and elongation take place within a proliferating bud (**Figure 2**) [20, 140]. These results indicate that between E11.5 and E13.5, a primary lumen connects with secondary and growing order lumens through branched microlumens or polarized canals, giving rise to a continuous formed or forming luminal structure that grows hierarchically (**Figure 2**). Therefore, in the thymus, clefting/branching and lumen formation seems to be more synchronic and not as regionally separated as in the salivary

However, in the thymus, a definitive duct is not developed, nor is terminal end buds, acini or other differentiated distal secreting structures, but instead the thymus remains as a concentric structure in which the central Cld + K5+ cells will differentiate into the thymic medulla [138, 139]. This central medulla does not apparently present lumen and is surrounded by the thymic cortex differentiated from Cld 3/4- cells (**Figure 2**) [139]. On the other hand, the fact that the branching pattern of the K5 + Cld 3/4+ cell cords appears to be similar between different mice [139] and that rat adult thymic medulla has also been described as a 4–5 order ramified structure in which the ratio of branches sizes is mathematically constant [141], sug-

If, as above indicated, the branching morphogenesis of the developing thymus has some particular features, as the lack of a definitive duct or terminal end buds or acini, the early lymphoid seeding introduces in the thymus development other important differences with respect to other epithelial organs inducing the specific three-dimensional network formed by dendritic-shaped TECs. Thymocyte precursors enter the thymus at around E11.5 through the

with nonpolarized TECs that express little or no K5 [139]. This 3D arrangement of thymic epithelium is, to some extent, precluded in the absence of thymocytes [143] in which the presence of medullary luminal or cystic structures becomes more evident, presumably representing a

default pathway of epithelial differentiation when thymocytes are missing [143].

, lymphoid progenitors appear associated

gesting that thymus development follows a programmed branching pattern.

**5. After acquisition of thymus fate, thymic primordium undergoes** 

modulate the effects produced by Wnt4 overexpression [133].

32 Histology

**tubulogenesis and branching morphogenesis**

gland, similar to the pancreas condition (**Figure 2**).

surrounding mesenchyme [142], and at E12.5 CD45+

**Figure 2.** Thymus development follows a branching morphogenesis process similar to those of salivary gland and pancreas (see **Figure 1**).

On the other hand, in the absence of Foxn1, the existence of a tubular-branched structure in which both ductal and acinar components can be distinguished [144] and that cannot be colonized by lymphoid progenitors [76] is clearly evident. This is the situation of Nude (Foxn1−/−) thymi, in which the transcription factor Foxn1 central for thymic epithelial differentiation, lacks. The earliest stages of Foxn1−/− thymus development appear to occur in the same way as those of wt thymus, and the expression of claudin 3/4 and wt thymus takes place in similar ways [145], suggesting that wt thymus organogenesis might be considered as a modification of the tubulogenesis and epithelial branching morphogenesis, which occur in the nude thymus (**Figure 2**). Thus, FoxN1 expression would preclude lumen formation and generate concentric layers of distinct TEC subsets (Muñoz et al., 2018 submitted). Moreover, the conditioned removal of FoxN1 in K14+ epithelial cells results in the progressive polarization of medullary cells, Cld3/4 expression, and lumen formation [146]. Other defects affect mainly thymic branching morphogenesis without importantly altering thymic-specific differentiation. Transgenic expression of Noggin under the control of a FoxN1 promoter leads to a hypoplastic spheric thymus always containing big cystic structures [125]. These structural alterations seem to be the result of a branching defect in consonance with the known role of BMP signaling in regulating branching morphogenesis of different organs [26] and to affect Foxn1 expression [147].
