Transcriptional Control of Epithelial Differentiation during Kidney Development

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1 J Am Soc Nephrol 14: S9 S15, 2003 Transcriptional Control of Epithelial Differentiation during Kidney Development DAVID RIBES,* EVELYNE FISCHER, AMÉLIE CALMONT,* and JEROME ROSSERT* *INSERM U489 and The University Pierre and Marie Curie, Paris, France; and Pasteur Institute, Paris, France. In Mammals, kidney development proceeds in three stages (reviewed in 1). The first two stages lead to the formation of transient structures, the pronephros and the mesonephros, and the third stage gives rise to the metanephros, which is the permanent kidney. However, similar pathways seem to be involved in the development of all three structures. First, the pronephric tubules and the pronephric duct form at the cervical end of the intermediate mesoderm and fuse to form the pronephros. These two components of the pronephros are epithelial structures that arise from an epithelial transformation of nephrogenic mesoderm. Second, the Wolffian duct, which is an extension of the pronephric duct, grows caudally, reaches the mesonephric mesenchyme, and induces the formation of the mesonephros. It induces the mesonephric mesenchyme to condense, form mesonephric tubules, and give rise to nephrons that form along the Wolffian duct. These nephrons consist of glomerulus-like structures and of proximal and distal tubules. Third, starting at 10.5 to 11 d post coitum (pc) in mouse and at 35 to 37 d pc in Human, reciprocal inductive interactions between a mesenchymal structure of the intermediate mesoderm, the metanephric blastema, and an outgrowth of the Wolffian duct, the ureteric bud, lead to the formation of the metanephros. The metanephric mesenchyme induces the ureteric bud to grow, branch, and give rise to the collecting duct system. At the same time, the ureteric bud induces the metanephric mesenchymal cells that surround it to condense around its tips, forming pretubular aggregates, and then to differentiate into epithelial structures that will ultimately form the epithelial components of the nephrons through a multistep process. The condensed mesenchyme successively differentiates into vesicles, comma-shaped bodies, S-shaped bodies, and then nephrons. At the same time, mesenchymal cells that are located in between the developing nephrons differentiate into stromal cells. Parallel to this differentiation process, the distal parts of the S-shaped bodies fuse with collecting ducts, and the proximal parts of these structures become highly vascularized and form glomeruli. Correspondence to Dr. Jerome A. Rossert, INSERM U489 and Department of Nephrology, Tenon Hospital, 4 rue de la Chine, Paris, France; Phone: ; Fax: ; jerome.rossert@tnn.ap-hop-paris.fr / Journal of the American Society of Nephrology Copyright 2003 by the American Society of Nephrology DOI: /01.ASN F As for many other structures, the combinatorial action of different cell-specific transcription factors is very likely to play a critical role in the development of the ureteric bud and of the metanephric mesenchyme. In this review, we focus on transcription factors that have been shown to play a role in vivo in the differentiation of metanephric mesenchymal cells into epithelial or stromal cells and in the differentiation of ureteric bud cells into epithelial cells of the excretory system. Early Differentiation of Metanephric Mesenchymal Cells In Drosophila melanogaster, the genes eyeless, sine oculis, eyes absent, and dachshund form a regulatory network that directs formation of the eye (reviewed in 2). A mutation in one of these genes leads to abnormal development of the eye, whereas ectopic expression of eyeless, eyes absent, or dachshund leads to ectopic eye formation. Analysis of this network has shown that eyes absent and sine oculis act downstream of eyeless and that eyes absent is upstream of sine oculis. In Mammals, the genes homologous to eyeless (Pax6), sine oculis (Six1, 4), and eyes absent (Eya1, 2, 4), as well as other members of the Pax, Eya, and Six families, also form a network that is involved in the formation of different organs, including muscle, eye lens, placode, inner ear, and possibly kidney (reviewed in 3). During kidney development, Pax2, Eya1, and possibly Six2 seem to be involved in early stages of metanephric blastema differentiation. Furthermore, other genes, such as Hox11 genes and Foxc1, also seem to interact with this network and modulate early differentiation of metanephric blastema (Figure 1). Pax2 belongs to a family of homeobox genes that contain a paired domain, which mediates specific DNA binding. In Human, heterozygous mutations in the PAX2 gene can be responsible for renal hypoplasia (4). During kidney development, Pax2 is expressed in the Wolffian duct, the ureteric bud, and the collecting ducts but also in the metanephric blastema at early stages of metanephrogenesis (5). It is expressed in the metanephric mesenchyme before induction by the ureteric bud, in mesenchymal condensates, and in comma-shaped bodies, whereas its expression decreases in S-shaped bodies, where it is expressed only in regions adjacent to the branching ureteric buds, and it is absent in mature nephrons. Generation of mice homozygous mutant for null alleles of Pax2 has shown that Pax2 is indispensable for ureteric bud development (6). These mice lack kidneys, ureters, and genital tracts, and analysis of

2 S10 Journal of the American Society of Nephrology J Am Soc Nephrol 14: S9 S15, 2003 Figure 1. Model of interactions between transcription factors involved in early kidney development. This model is mostly derived from analysis of knockout mice. At least three different pathways contribute to early differentiation of metanephric mesenchymal cells: the Wt1 pathway; the Pax2-Eya1-Six2-Hox11 pathway, which leads to production of glial cell line derived neurotrophic factor (Gdnf) and is inhibited by Foxc1; and the Sall1 pathway, which is downstream of the two previous ones. Foxd1 and the genes encoding the RAR and 2 are necessary for differentiation of stromal cells. Emx2 and possibly Pax2 and Lim1 induce the differentiation of ureteric bud cells. Reciprocal inductive interactions are necessary for differentiation of metanephric mesenchymal cells and ureteric bud cells. Part of these interactions is mediated by the binding of Gdnf to its receptor Ret and coreceptor Gdnfr1. Differentiation of metanephric mesenchymal cells and ureteric bud cells also requires interactions with stromal cells. developing embryos has shown that the Wolffian duct develops only partially and that the ureteric bud does not form. The absence of ureteric bud formation is associated with a loss of glial cell line derived neurotrophic factor (Gdnf) gene expression in the metanephric blastema of Pax2 / mice (7). Gdnf is a member of the TGF- superfamily that is produced by the metanephric mesenchyme and that is necessary for ureteric budding (8). A possible role for Pax2 thus would be the induction of mesenchymal competence and of Gdnf expression, which is in agreement with the ability of Pax2 to activate the transcription of Gdnf in vitro. However, analysis of Eya1 / mice and of Hox11 null mutant mice has shown that Pax2 is not sufficient to activate Gdnf expression (cf. infra) and that induction of the metanephric blastema probably requires a combinatorial action of different transcription factors. The Eya genes encode proteins that possess transactivation activity but do not seem to bind DNA and probably act as coactivators. They contain a so-called Eya domain that is indispensable for coactivation activity. In Human, haploinsufficiency for Eya1 results in the branchio-oto-renal and branchio-otic syndromes that associate craniofacial abnormalities, hearing loss, and, for the branchio-oto-renal syndrome, kidney defects (9). During kidney development, Eya1 is expressed in the metanephric mesenchyme but not in the ureteric bud or its derivatives (10). At birth, Eya1 knockout mice lack kidneys and ureters because of a failure of the ureteric bud to form, and analysis of kidney development in Eya1 / embryos has shown that Pax2 is expressed but that expression of Gdnf and Six2 cannot be detected (11). Thus, Eya1 seems to act downstream of Pax2 and to be indispensable for acquisition of mesenchymal competence and for expression of Gdnf by metanephric mesenchymal cells.

3 J Am Soc Nephrol 14: S9 S15, 2003 Transcriptional Control of Epithelial Differentiation during Kidney Development S11 The Hoxa11, Hoxc11, and Hoxd11 genes are expressed in the metanephric mesenchyme but not in the Wolffian duct or the ureteric bud or its derivatives (12 14). At birth, mice harboring null mutant alleles for all three of these genes have no kidney, and analysis of kidney development has shown that the ureteric bud never forms (15). In situ hybridization experiments have shown that Wt1, Pax2, and Eya1 are normally expressed, whereas Six2 and Gdnf are absent. Furthermore, analysis of embryos with only five mutant alleles support the hypothesis that the levels of expression of Six2 are dependent on the numbers of functional Hox11 alleles, because these mutants, which have one functional allele, show weak expression of Six2 (15). These data are in agreement with the hypothesis that Pax2, Eya1, and Six2 form a functional network and that induction of Six2 by Eya1 and Hox11 gene products is necessary for Gdnf induction (Figure 1). Six2 is a transcription factor that is characterized by the presence of a Six-type homeodomain and of a Six domain located N terminal to the homeodomain. Six and Eya have been shown to interact directly and to induce synergistic activation of promoters (16). Unfortunately, targeted disruption of Six2 has not yet been reported, and the role of this gene in metanephric blastema differentiation and induction of Gdnf expression is purely hypothetical. Foxc1/Mf1/Fkh1 is a transcription factor that belongs to the forkhead/winged-helix family and that seems to interact with the Pax2-Eya1-Six2 pathway. The forkhead family contains transcription factors that share an evolutionarily conserved DNA-binding domain named forkhead domain. Studies of knockout mice have shown that members of this family play essential roles during embryonic development and in particular that they regulate cell proliferation, cell fate determination, and differentiation. During embryonic development, Foxc1 is expressed in various tissues, including the metanephric mesenchyme, but not in the Wolffian duct or the ureteric bud (17). A proportion of mice that lack functional Foxc1 genes display duplex kidneys associated with double ureters (17). The additional ureter arises from the Wolffian duct more anteriorly to the normal ureter and is not connected to the bladder. It probably results from anterior persistence of Gdnf expression domain, which suggests that Foxc1 may negatively regulate the expression of Gdnf. Because the expression of Eya1 also extends anteriorly in mice that do not have functional Foxc1 genes (17), Foxc1 may inhibit the expression of Eya1, which, in turn, inhibits the expression of Gdnf (Figure 1). The Pax2- Eya1-Six2-Hox11-Foxc1 pathway is not the only one to be involved in early induction of metanephric mesenchyme, and other genes that do not seem to interact directly with this pathway, such as Wt1 or Sall1, are also indispensable for early kidney development. The Wilms tumor-suppressor gene Wt1 encodes a DNAbinding protein that contains four zinc fingers of the C 2 H 2 type. It exists as different isoforms that are generated by alternative splicing and by usage of alternative translation start sites and that have roles as transcription factors but also in RNA processing (18). In Human, mutations in WT1 are responsible for Wilms tumors but also for the WAGR (Wilms tumor Aniridia genitourinary abnormalities mental retardation), Denys-Drash, and Frasier syndromes, which all are characterized by the presence of kidney abnormalities (18). During kidney development, Wt1 is expressed at relatively low levels in the metanephric mesenchyme before induction by the ureteric bud and at higher levels in condensing mesenchymal cells, in vesicles, in comma-shaped bodies, and at the proximal part of S-shaped bodies in cells that will become podocytes (19,20). In Wt1-null mice, the metanephric blastema forms, but the ureteric bud does not sprout from the Wolffian duct, and thus the metanephric mesenchyme undergoes apoptosis and kidney does not form (21). Ex vivo coculture experiments using metanephric blastema from Wt1 / mice have shown that it is unable to condense and that Wt1 is indispensable for induction of the metanephric mesenchyme by normal ureteric buds (21). Wt1-null mice complemented with a YAC transgene spanning the Wt1 locus survive until birth, which allows a more precise analysis of the role of Wt1 during kidney development (22). Analysis of these mice has shown that Wt1 is required not only for early induction of the metanephric mesenchyme but also at later stages of kidney development. Although the majority of mice show a total absence of ureteric bud development, similar to what is seen in uncomplemented null mice, some embryos have hypodysplastic kidneys where metanephric mesenchyme condenses around the tips of the ureteric bud branches but forms no or very few epithelial structures. Furthermore, whereas Pax2 expression is present but reduced in kidney mesenchyme of Wt1 / mice, it is normal in complemented mice. This suggests that Wt1 modulates the early differentiation of metanephric mesenchyme but does not directly upregulate Pax2. Thus, Wt1 would act in combination with Pax2, Eya1, Six2, and Hox genes to induce early metanephric mesenchyme differentiation (Figure 1). Sall1 (Sal-like 1) is the mammalian homolog of the Drosophila region-specific homeotic gene Spalt. It is a homeotic gene that contains 10 zinc finger motifs and that is expressed mostly in the metanephric mesenchyme surrounding the ureteric bud during metanephros development (23). Heterozygous mutations of SALL1 in Human lead to the Townes-Brocks syndrome, which is characterized by dysplastic ears, polydactyly, imperforate anus, and kidney and heart abnormalities (24). Sall1-null mice have either complete kidney agenesis or severe renal dysplasia (23). Analysis of metanephros development has shown that the ureteric bud forms but fails either to invade the metanephric mesenchyme or to grow and branch within the metanephric mesenchyme, which leads to its apoptosis. Coculture experiments have shown that Sall1 is required for the metanephric mesenchyme to attract the ureteric bud, and analysis of genes expression has shown that it does not have direct effects on Wt1, Pax2, Eya1, orgdnf expression (23). Thus, still another pathway seems to be needed for proper development of the metanephric mesenchyme, besides the Pax2-Eya1-Six2 pathway and besides Wt1 (Figure 1). It is of note that analysis of knockout mice has shown that Sall2, which is closely related to Sall1 and which is also expressed in the metanephric mesenchyme surrounding the ureteric bud, is dispensable for kidney development (25).

4 S12 Journal of the American Society of Nephrology J Am Soc Nephrol 14: S9 S15, 2003 Differentiation of Tubular Epithelial Cells and of Podocytes Mature tubules are formed by a variety of highly specialized epithelial cells, and it is likely that many different transcription factors are necessary for normal tubulogenesis. However, so far, only a few of them have been identified (Figure 2). Pod1 is a basic helix-loop-helix transcription factor that is expressed in condensing metanephric mesenchyme surrounding the ureteric bud branches, in podocytes at all stages of differentiation, and in interstitial cells (26). In contrast, epithelial cells other than podocytes do not express this gene. Pod1- null mice display severely hypoplastic kidneys, and analysis of kidney development shows that the conversion of condensed mesenchyme into epithelial tubular cells is not only delayed but also severely impaired, with formation of a reduced number of nephrons and blockage in differentiation of tubular epithelial cells (26). Podocytes also start to differentiate but then fail to undergo terminal differentiation, and glomerular formulation is arrested at the capillary loop stage (26). HNF1 /LF-B1 is a dimeric homeodomain transcription factor that is expressed in different organs, including liver and kidney. It can bind DNA as homo- or heterodimers, and HNF1 /vhnf1/lf-b3 and DcoH (dimerization cofactor for HNF1) have been shown to heterodimerize with HNF1. During kidney development, HNF1 is expressed selectively in proximal tubular cells, and HNF1 / mice display defects in terminal differentiation of proximal tubular cells responsible Figure 2. Transcription factors involved in late stages of differentiation of metanephric mesenchymal cells into epithelial cells. This list is derived from analysis of knockout mice. Wt1, Pod1, and Lmx1b are required for normal differentiation of podocytes. Pod1 is necessary for the conversion of condensed mesenchymal cells into epithelial tubular cells, and HNF1 is necessary for terminal differentiation of proximal tubular cells. Pax2 is very likely to be required for tubulogenesis, and Lim1 may also be required for differentiation of epithelial cells. for Fanconi syndrome (27,28). In particular, they have glucosuria, aminoaciduria, and phosphaturia. In Human, heterozygous mutations of HNF1 also lead to impaired renal glucose reabsorption (29). As described above, Pax2 / mice lack ureteric bud (6). Thus, the metanephric mesenchyme undergoes apoptosis, and the role of Pax2 during late stages of metanephrogenesis cannot be analyzed. However, in organotypic cultures, inhibition of Pax2 expression prevents the condensation and epithelial conversion of metanephric mesenchymal cells (30). Furthermore, overexpression of Pax2 in transgenic mice is responsible for abnormal differentiation of tubular cells (31). Thus, Pax2 may play a role in epithelial conversion of mesenchymal cells and in differentiation of tubular cells. Lim1 is a transcription factor that contains a LIM class homeodomain and two LIM domains. During kidney development, Lim1 is expressed in ureteric bud branches and in collecting ducts but also in vesicles, comma-shaped bodies, S- shaped bodies, and developing tubules (32). Analysis of the role of Lim1 during kidney development in Mammals is difficult because null mutant mice lack head structures and die around day 10 pc, before development of the metanephros. However, the four mice that survived until birth completely lacked kidneys and gonads (33). Study of the role of Xlim1 during development of Xenopus embryos has shown that this gene synergizes with Pax8 to induce pronephric tubule differentiation and growth (34). In Mammals, Lim1 thus could also be involved in inducing epithelial cell differentiation. Although podocytes are highly differentiated cells that express many specific markers, only a few transcription factors that are involved in podocyte differentiation have been identified by using knockout mice (Figure 2). Because early kidney development is grossly abnormal in mice that completely or partially lack functional Wt1 alleles, the role of Wt1 in podocyte differentiation cannot be assessed using these mice (21,22). However, the presence of glomerular defects in subjects with Denys-Drash syndrome or Frasier syndrome suggested that this gene was necessary for normal differentiation of podocytes (18). This hypothesis has been recently confirmed by generation of mice specifically lacking the KTS or the KTS isoform of Wt1 (35). These two isoforms result from the existence of two alternative splice donor sites at the end of exon 9, and they differ by the presence ( KTS) or absence ( KTS) of three amino acids between zinc fingers 3 and 4. In mice that lack the KTS isoform of Wt1, podocytes remain cuboidal and formation of foot processes is severely impaired (35). Mice that lack the KTS isoform have very small glomeruli and synaptopodin, and 3 integrin are almost completely absent, which suggests that early steps of podocyte differentiation are impaired (35). Furthermore, in vitro experiments suggest that Wt1 directly activates the transcription of podocalyxin, a gene that encodes a podocyte-specific protein (36). As stated above, Pod1 is expressed in podocytes at all stages of differentiation, and Pod1 null mice show a clear defect in podocyte differentiation (26). In particular, they remain cuboidal with few foot processes. Furthermore, the density of the

5 J Am Soc Nephrol 14: S9 S15, 2003 Transcriptional Control of Epithelial Differentiation during Kidney Development S13 capillary network is markedly reduced, suggesting that podocyte differentiation is necessary for normal development of the capillary tuft. Lmx1b is a DNA-binding protein that contains two LIM domains separated by one homeodomain. In Human, heterozygous mutations of LMX1B are responsible for the nail-patella syndrome (37). During embryonic development, Lmx1b is expressed in limbs but also in kidney, where its expression is restricted to podocytes (38). Lmx1b / mice display abnormal glomeruli that are not fully differentiated, contain dysplastic podocytes, and lack a capillary network (38 41). The podocytes remain cuboidal, and no foot processes or slit diaphragms can be detected. Furthermore, they do not express podocin, and they synthesize decreased amounts of pro- 3(IV) and pro- 4(IV) collagen chains (39 41). Differentiation of Stromal Cells During embryonic development, differentiation of most if not all epithelial tissues requires interactions with an adjacent mesenchyme. Similarly, during kidney development, differentiation of tubular cells requires interactions not only with the ureteric bud branches but also with adjacent stromal cells. The role of stromal cells has been mostly highlighted by analysis of Foxd1/Bf2 null mice and of retinoic acid receptors (RAR) Rara and Rarb2 double mutant mice. Foxd1 is a member of the forkhead/winged helix family of transcription factors, which is expressed in different organs, including kidney, during embryonic development. In kidney, it is selectively expressed in stromal cells that surround condensed mesenchymal cells and differentiating nephrons and at lower levels in stromal cells located in the medulla around ureteric bud branches (42). Foxd1 / mice die at birth, while they have small kidneys that are abnormally positioned. Analysis of kidney development in these mice shows a dramatic reduction in the number of S-shaped and comma-shaped bodies, and the presence of large amounts of condensed mesenchymal cells, which suggests first that stromal cells do not differentiate normally and second that they are necessary for epithelial differentiation of condensed mesenchymal cells (42). Furthermore, because expression of Ret (a receptor that is expressed by ureteric bud cells and that binds Gdnf) is abnormal in ureteric bud cells of Foxd1 / mice, stromal cells may also be necessary for normal differentiation of ureteric bud cells (42). This hypothesis is in agreement with results obtained by analyzing null-mutant mice for RAR (cf. infra). RAR are transcription factors that belong to the nuclear receptor superfamily and transduce the retinoid signal. In the presence of retinoic acid, they bind to enhancer elements and activate transcription. Analysis of double mutant mice that lack Rara (retinoic acid receptor ) and Rarb2 (retinoic acid receptor 2) shows that retinoic acid is indispensable for normal development of stromal cells. During kidney development, Rara is expressed at low levels throughout the embryonic kidney, whereas Rarb2 is expressed selectively in stromal cells (43). Unlike Foxd1, Rarb2 is expressed at similar levels in all stromal cells (43). Rara / Rarb2 / double mutant mice have severely dysplastic kidneys with reduced branching of the ureteric bud and accumulation of stromal cells beneath the renal capsule (43). This inhibition of ureteric bud branching is associated with a reduced expression of Ret in ureteric bud cells, and overexpression of Ret in these cells can almost completely rescue renal development (44). Thus, binding of retinoic acid to its receptor is indispensable for normal development of stromal cells, and interactions between stromal cells and ureteric bud cells are necessary for normal development of the excretory system. However, these interactions may be either direct or indirect, mediated by interactions between stromal cells and epithelium-forming mesenchymal cells. Differentiation of Ureteric Bud Cells Surprising is that only one transcription factor expressed by ureteric bud cells has been clearly shown to be necessary for their differentiation, even if Pax2 and Lim1 are also likely to play a direct role in ureteric bud development. Emx2 is a homeobox gene that is expressed in the Wolffian duct, in the ureteric bud and its derivatives, and in early epithelial structures forming vesicles and comma-shaped bodies (45). In nullmutant mice, the ureteric bud invades the metanephric mesenchyme, but it does not branch, does not induce the metanephric mesenchyme to condense, and rapidly degenerates (46). This is associated with a large reduction of the expression of Lim1, Pax2, and Ret in the ureteric bud cells. Furthermore, expression of Gdnf is normal before invasion of the metanephric mesenchyme by the ureteric bud, but later on it is greatly reduced around the invading ureteric bud, and the gene encoding Wnt4 (a soluble molecule that is normally produced by condensed mesenchymal cells) is not expressed in pretubular aggregates. Coculture experiments show that the ureteric bud is unable to branch or induce an epithelial transformation of wild-type mesenchyme but that the development of the mutant metanephric mesenchyme is normal (46). Taken together, these experiments suggest that Emx2 regulates the differentiation of ureteric bud cells and the production by these cells of a signal that induces epithelial differentiation of metanephric mesenchymal cells. A role of Pax2 in differentiation of ureteric bud cells is mostly suggested by analysis of nephric duct formation. Pax8 is another member of the Pax family of transcription factors that is expressed during pro-, meso-, and metanephros development, but kidney development is normal in Pax8 / mice (47,48). Whereas initial development of the nephric duct is normal in Pax2 / mice, in Pax2 / Pax8 / doublemutant embryos, the intermediate mesenchyme does not undergo mesenchymal-epithelial transition and the nephric duct does not form (47 49). In zebrafish, lack of functional Pax2.1 gene leads to abnormal epithelial differentiation of the pronephric duct (50). Conversely, in chick embryos, ectopic expression of Pax2 in the intermediate mesoderm and genital ridge can induce the formation of ectopic nephric ducts (50). Similarly, a role for Lim1 in ureteric development is suggested by the fact that ectopic expression of XPax8 and Xlim1 can induce the formation of additional pronephric tubules in Xenopus embryos (34). In conclusion, although different transcription factors that

6 S14 Journal of the American Society of Nephrology J Am Soc Nephrol 14: S9 S15, 2003 are necessary for early differentiation of metanephric mesenchymal cells have been identified, little is known about the genes that regulate terminal differentiation of tubular epithelial cells or of ureteric bud cells. However, understanding the development of the renal excretory system is of critical importance because, in children, congenital abnormalities of kidney and urinary tract are the main cause of end-stage kidney disease. One approach could be to identify the enhancers that drive gene expression in a temporally and spatially controlled manner during differentiation of ureteric bud cells. References 1. Ekblom P, Miettinen A, Virtanen I, Wahlstrom T, Dawnay A, Saxen L: In vitro segregation of the metanephric nephron. Dev Biol 84: 88 95, Kumar JP, Moses K: Eye specification in Drosophila: Perspectives and implications. 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