J Am Soc Nephrol 14: S9–S15, 2003 Transcriptional Control of Epithelial Differentiation during Kidney Development

DAVID RIBES,* EVELYNE FISCHER,† AME´ LIE 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 As for many other structures, the combinatorial action of (reviewed in 1). The first two stages lead to the formation of different cell-specific transcription factors is very likely to play transient structures, the pronephros and the mesonephros, and a critical role in the development of the ureteric bud and of the the third stage gives rise to the metanephros, which is the metanephric mesenchyme. In this review, we focus on tran- permanent kidney. However, similar pathways seem to be scription factors that have been shown to play a role in vivo in involved in the development of all three structures. First, the the differentiation of metanephric mesenchymal cells into ep- pronephric tubules and the pronephric duct form at the cervical ithelial or stromal cells and in the differentiation of ureteric bud end of the intermediate mesoderm and fuse to form the prone- cells into epithelial cells of the excretory system. phros. These two components of the pronephros are epithelial structures that arise from an epithelial transformation of neph- Early Differentiation of Metanephric rogenic mesoderm. Second, the Wolffian duct, which is an Mesenchymal Cells extension of the pronephric duct, grows caudally, reaches the In Drosophila melanogaster, the genes eyeless, sine oculis, mesonephric mesenchyme, and induces the formation of the eyes absent, and dachshund form a regulatory network that mesonephros. It induces the mesonephric mesenchyme to con- directs formation of the eye (reviewed in 2). A mutation in one dense, form mesonephric tubules, and give rise to nephrons of these genes leads to abnormal development of the eye, that form along the Wolffian duct. These nephrons consist of whereas ectopic expression of eyeless, eyes absent,ordachs- glomerulus-like structures and of proximal and distal tubules. hund leads to ectopic eye formation. Analysis of this network Third, starting at 10.5 to 11 d post coitum (pc) in mouse and at has shown that eyes absent and sine oculis act downstream of 35 to 37 d pc in Human, reciprocal inductive interactions eyeless and that eyes absent is upstream of sine oculis.In between a mesenchymal structure of the intermediate meso- Mammals, the genes homologous to eyeless (Pax6), sine oculis derm, the metanephric blastema, and an outgrowth of the (Six1, 4), and eyes absent (Eya1, 2, 4), as well as other Wolffian duct, the ureteric bud, lead to the formation of the members of the Pax, Eya, and Six families, also form a network metanephros. The metanephric mesenchyme induces the ure- that is involved in the formation of different organs, including teric bud to grow, branch, and give rise to the collecting duct muscle, eye lens, placode, inner ear, and possibly kidney system. At the same time, the ureteric bud induces the meta- (reviewed in 3). During kidney development, Pax2, Eya1, and nephric mesenchymal cells that surround it to condense around possibly Six2 seem to be involved in early stages of metaneph- its tips, forming pretubular aggregates, and then to differentiate ric blastema differentiation. Furthermore, other genes, such as into epithelial structures that will ultimately form the epithelial Hox11 genes and Foxc1, also seem to interact with this net- components of the nephrons through a multistep process. The work and modulate early differentiation of metanephric blas- condensed mesenchyme successively differentiates into vesi- tema (Figure 1). cles, comma-shaped bodies, S-shaped bodies, and then Pax2 belongs to a family of homeobox genes that contain a nephrons. At the same time, mesenchymal cells that are located paired domain, which mediates specific DNA binding. In Hu- in between the developing nephrons differentiate into stromal man, heterozygous mutations in the PAX2 gene can be respon- cells. Parallel to this differentiation process, the distal parts of sible for renal hypoplasia (4). During kidney development, the S-shaped bodies fuse with collecting ducts, and the prox- Pax2 is expressed in the Wolffian duct, the ureteric bud, and imal parts of these structures become highly vascularized and the collecting ducts but also in the metanephric blastema at form glomeruli. 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, Correspondence to Dr. Jerome A. Rossert, INSERM U489 and Department of Ne- phrology, Tenon Hospital, 4 rue de la Chine, 75020 Paris, France; Phone: ϩ33-1-56- whereas its expression decreases in S-shaped bodies, where it 01-60-29; Fax: ϩ33-1-56-01-69-99; E-mail: [email protected] is expressed only in regions adjacent to the branching ureteric 1046-6673/1400-0009 buds, and it is absent in mature nephrons. Generation of mice Journal of the American Society of Nephrology homozygous mutant for null alleles of Pax2 has shown that Copyright © 2003 by the American Society of Nephrology Pax2 is indispensable for ureteric bud development (6). These DOI: 10.1097/01.ASN.0000067647.05964.9F mice lack kidneys, ureters, and genital tracts, and analysis of 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 activity but do not seem to bind DNA and probably act as only partially and that the ureteric bud does not form. The coactivators. They contain a so-called Eya domain that is absence of ureteric bud formation is associated with a loss of indispensable for coactivation activity. In Human, haploinsuf- glial cell line–derived neurotrophic factor (Gdnf) gene expres- ficiency for Eya1 results in the branchio-oto-renal and bran- sion in the metanephric blastema of Pax2Ϫ/Ϫ mice (7). Gdnf chio-otic syndromes that associate craniofacial abnormalities, is a member of the TGF-␤ superfamily that is produced by the hearing loss, and, for the branchio-oto-renal syndrome, kidney metanephric mesenchyme and that is necessary for ureteric defects (9). During kidney development, Eya1 is expressed in budding (8). A possible role for Pax2 thus would be the the metanephric mesenchyme but not in the ureteric bud or its induction of mesenchymal competence and of Gdnf expres- derivatives (10). At birth, Eya1 knockout mice lack kidneys sion, which is in agreement with the ability of Pax2 to activate and ureters because of a failure of the ureteric bud to form, and the transcription of Gdnf in vitro. However, analysis of analysis of kidney development in Eya1Ϫ/Ϫ embryos has Eya1Ϫ/Ϫ mice and of Hox11 null mutant mice has shown that shown that Pax2 is expressed but that expression of Gdnf and Pax2 is not sufficient to activate Gdnf expression (cf. infra) and Six2 cannot be detected (11). Thus, Eya1 seems to act down- that induction of the metanephric blastema probably requires a stream of Pax2 and to be indispensable for acquisition of combinatorial action of different transcription factors. mesenchymal competence and for expression of Gdnf by met- The Eya genes encode proteins that possess transactivation anephric mesenchymal cells. 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 Aniridia–genitourinary abnormalities–mental retardation), De- the metanephric mesenchyme but not in the Wolffian duct or nys-Drash, and Frasier syndromes, which all are characterized the ureteric bud or its derivatives (12–14). At birth, mice by the presence of kidney abnormalities (18). During kidney harboring null mutant alleles for all three of these genes have development, Wt1 is expressed at relatively low levels in the no kidney, and analysis of kidney development has shown that metanephric mesenchyme before induction by the ureteric bud the ureteric bud never forms (15). In situ hybridization exper- and at higher levels in condensing mesenchymal cells, in iments have shown that Wt1, Pax2, and Eya1 are normally vesicles, in comma-shaped bodies, and at the proximal part of expressed, whereas Six2 and Gdnf are absent. Furthermore, S-shaped bodies in cells that will become podocytes (19,20). In analysis of embryos with only five mutant alleles support the Wt1-null mice, the metanephric blastema forms, but the ure- hypothesis that the levels of expression of Six2 are dependent teric bud does not sprout from the Wolffian duct, and thus the on the numbers of functional Hox11 alleles, because these metanephric mesenchyme undergoes apoptosis and kidney mutants, which have one functional allele, show weak expres- does not form (21). Ex vivo coculture experiments using met- sion of Six2 (15). These data are in agreement with the hy- anephric blastema from Wt1Ϫ/Ϫ mice have shown that it is pothesis that Pax2, Eya1, and Six2 form a functional network unable to condense and that Wt1 is indispensable for induction and that induction of Six2 by Eya1 and Hox11 gene products is of the metanephric mesenchyme by normal ureteric buds (21). necessary for Gdnf induction (Figure 1). Wt1-null mice complemented with a YAC transgene spanning Six2 is a transcription factor that is characterized by the the Wt1 locus survive until birth, which allows a more precise presence of a Six-type homeodomain and of a Six domain analysis of the role of Wt1 during kidney development (22). located N terminal to the homeodomain. Six and Eya have Analysis of these mice has shown that Wt1 is required not only been shown to interact directly and to induce synergistic acti- for early induction of the metanephric mesenchyme but also at vation of promoters (16). Unfortunately, targeted disruption of later stages of kidney development. Although the majority of Six2 has not yet been reported, and the role of this gene in mice show a total absence of ureteric bud development, similar metanephric blastema differentiation and induction of Gdnf to what is seen in uncomplemented null mice, some embryos expression is purely hypothetical. have hypodysplastic kidneys where metanephric mesenchyme Foxc1/Mf1/Fkh1 is a transcription factor that belongs to the condenses around the tips of the ureteric bud branches but forkhead/winged-helix family and that seems to interact with forms no or very few epithelial structures. Furthermore, the Pax2-Eya1-Six2 pathway. The forkhead family contains whereas Pax2 expression is present but reduced in kidney transcription factors that share an evolutionarily conserved mesenchyme of Wt1Ϫ/Ϫ mice, it is normal in complemented DNA-binding domain named forkhead domain. Studies of mice. This suggests that Wt1 modulates the early differentia- knockout mice have shown that members of this family play tion of metanephric mesenchyme but does not directly upregu- essential roles during embryonic development and in particular late Pax2. Thus, Wt1 would act in combination with Pax2, that they regulate cell proliferation, cell fate determination, and Eya1, Six2, and Hox genes to induce early metanephric mes- differentiation. During embryonic development, Foxc1 is ex- enchyme differentiation (Figure 1). pressed in various tissues, including the metanephric mesen- Sall1 (Sal-like 1) is the mammalian homolog of the Dro- chyme, but not in the Wolffian duct or the ureteric bud (17). A sophila region-specific homeotic gene Spalt. It is a homeotic proportion of mice that lack functional Foxc1 genes display gene that contains 10 zinc finger motifs and that is expressed duplex kidneys associated with double ureters (17). The addi- mostly in the metanephric mesenchyme surrounding the ure- tional ureter arises from the Wolffian duct more anteriorly to teric bud during metanephros development (23). Heterozygous the normal ureter and is not connected to the bladder. It mutations of SALL1 in Human lead to the Townes-Brocks probably results from anterior persistence of Gdnf expression syndrome, which is characterized by dysplastic ears, polydac- domain, which suggests that Foxc1 may negatively regulate the tyly, imperforate anus, and kidney and heart abnormalities expression of Gdnf. Because the expression of Eya1 also (24). Sall1-null mice have either complete kidney agenesis or extends anteriorly in mice that do not have functional Foxc1 severe renal dysplasia (23). Analysis of metanephros develop- genes (17), Foxc1 may inhibit the expression of Eya1, which, ment has shown that the ureteric bud forms but fails either to in turn, inhibits the expression of Gdnf (Figure 1). The Pax2- invade the metanephric mesenchyme or to grow and branch Eya1-Six2-Hox11-Foxc1 pathway is not the only one to be within the metanephric mesenchyme, which leads to its apo- involved in early induction of metanephric mesenchyme, and ptosis. Coculture experiments have shown that Sall1 is re- other genes that do not seem to interact directly with this quired for the metanephric mesenchyme to attract the ureteric pathway, such as Wt1 or Sall1, are also indispensable for early bud, and analysis of genes expression has shown that it does kidney development. not have direct effects on Wt1, Pax2, Eya1,orGdnf expression The Wilms’ tumor-suppressor gene Wt1 encodes a DNA- (23). Thus, still another pathway seems to be needed for proper binding protein that contains four zinc fingers of the C2H2 type. development of the metanephric mesenchyme, besides the It exists as different isoforms that are generated by alternative Pax2-Eya1-Six2 pathway and besides Wt1 (Figure 1). It is of splicing and by usage of alternative translation start sites and note that analysis of knockout mice has shown that Sall2, that have roles as transcription factors but also in RNA pro- which is closely related to Sall1 and which is also expressed in cessing (18). In Human, mutations in WT1 are responsible for the metanephric mesenchyme surrounding the ureteric bud, is Wilms’ tumors but also for the WAGR (Wilms’ tumor– dispensable for kidney development (25). S12 Journal of the American Society of Nephrology J Am Soc Nephrol 14: S9–S15, 2003

Differentiation of Tubular Epithelial Cells and for Fanconi syndrome (27,28). In particular, they have glucos- of Podocytes uria, aminoaciduria, and phosphaturia. In Human, heterozy- Mature tubules are formed by a variety of highly specialized gous mutations of HNF1␣ also lead to impaired renal glucose epithelial cells, and it is likely that many different transcription reabsorption (29). factors are necessary for normal tubulogenesis. However, so As described above, Pax2Ϫ/Ϫ mice lack ureteric bud (6). far, only a few of them have been identified (Figure 2). Thus, the metanephric mesenchyme undergoes apoptosis, and Pod1 is a basic helix-loop-helix transcription factor that is the role of Pax2 during late stages of metanephrogenesis can- expressed in condensing metanephric mesenchyme surround- not be analyzed. However, in organotypic cultures, inhibition ing the ureteric bud branches, in podocytes at all stages of of Pax2 expression prevents the condensation and epithelial differentiation, and in interstitial cells (26). In contrast, epithe- conversion of metanephric mesenchymal cells (30). Further- lial cells other than podocytes do not express this gene. Pod1- more, overexpression of Pax2 in transgenic mice is responsible null mice display severely hypoplastic kidneys, and analysis of for abnormal differentiation of tubular cells (31). Thus, Pax2 kidney development shows that the conversion of condensed may play a role in epithelial conversion of mesenchymal cells mesenchyme into epithelial tubular cells is not only delayed and in differentiation of tubular cells. but also severely impaired, with formation of a reduced num- Lim1 is a transcription factor that contains a LIM class ber of nephrons and blockage in differentiation of tubular homeodomain and two LIM domains. During kidney develop- epithelial cells (26). Podocytes also start to differentiate but ment, Lim1 is expressed in ureteric bud branches and in col- then fail to undergo terminal differentiation, and glomerular lecting ducts but also in vesicles, comma-shaped bodies, S- formulation is arrested at the capillary loop stage (26). shaped bodies, and developing tubules (32). Analysis of the HNF1␣/LF-B1 is a dimeric homeodomain transcription fac- role of Lim1 during kidney development in Mammals is diffi- tor that is expressed in different organs, including liver and cult because null mutant mice lack head structures and die kidney. It can bind DNA as homo- or heterodimers, and around day 10 pc, before development of the metanephros. HNF1␤/vHNF1/LF-B3 and DcoH (dimerization cofactor for However, the four mice that survived until birth completely HNF1) have been shown to heterodimerize with HNF1␣. Dur- lacked kidneys and gonads (33). Study of the role of Xlim1 ing kidney development, HNF1␣ is expressed selectively in during development of Xenopus embryos has shown that this proximal tubular cells, and HNF1␣Ϫ/Ϫ mice display defects gene synergizes with Pax8 to induce pronephric tubule differ- in terminal differentiation of proximal tubular cells responsible entiation and growth (34). In Mammals, Lim1 thus could also be involved in inducing epithelial cell differentiation. Although podocytes are highly differentiated cells that ex- press many specific markers, only a few transcription factors that are involved in podocyte differentiation have been identi- fied 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 podo- cyte differentiation cannot be assessed using these mice (21,22). However, the presence of glomerular defects in sub- jects with Denys-Drash syndrome or Frasier syndrome sug- gested that this gene was necessary for normal differentiation of podocytes (18). This hypothesis has been recently confirmed by generation of mice specifically lacking the ϩKTS or the ϪKTS 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 (ϩKTS) or absence (ϪKTS) of three amino acids between zinc fingers 3 and 4. In mice that lack the ϩKTS isoform of Wt1, podocytes remain cuboidal and formation of foot processes is severely impaired (35). Mice that lack the ϪKTS isoform have very small glo- meruli and synaptopodin, and ␣3 integrin are almost com- pletely absent, which suggests that early steps of podocyte differentiation are impaired (35). Furthermore, in vitro exper- Figure 2. Transcription factors involved in late stages of differentia- iments suggest that Wt1 directly activates the transcription of tion of metanephric mesenchymal cells into epithelial cells. This list podocalyxin, a gene that encodes a podocyte-specific protein is derived from analysis of knockout mice. Wt1, Pod1, and Lmx1b are required for normal differentiation of podocytes. Pod1 is necessary for (36). the conversion of condensed mesenchymal cells into epithelial tubular As stated above, Pod1 is expressed in podocytes at all stages cells, and HNF1␣ is necessary for terminal differentiation of proximal of differentiation, and Pod1 null mice show a clear defect in tubular cells. Pax2 is very likely to be required for tubulogenesis, and podocyte differentiation (26). In particular, they remain cuboi- Lim1 may also be required for differentiation of epithelial cells. dal with few foot processes. Furthermore, the density of the J Am Soc Nephrol 14: S9–S15, 2003 Transcriptional Control of Epithelial Differentiation during Kidney Development S13 capillary network is markedly reduced, suggesting that podo- ureteric bud and accumulation of stromal cells beneath the cyte differentiation is necessary for normal development of the renal capsule (43). This inhibition of ureteric bud branching is capillary tuft. associated with a reduced expression of Ret in ureteric bud Lmx1b is a DNA-binding protein that contains two LIM cells, and overexpression of Ret in these cells can almost domains separated by one homeodomain. In Human, heterozy- completely rescue renal development (44). Thus, binding of gous mutations of LMX1B are responsible for the nail-patella retinoic acid to its receptor is indispensable for normal devel- syndrome (37). During embryonic development, Lmx1b is ex- opment of stromal cells, and interactions between stromal cells pressed in limbs but also in kidney, where its expression is and ureteric bud cells are necessary for normal development of restricted to podocytes (38). Lmx1bϪ/Ϫ mice display abnor- the excretory system. However, these interactions may be mal glomeruli that are not fully differentiated, contain dysplas- either direct or indirect, mediated by interactions between tic podocytes, and lack a capillary network (38–41). The stromal cells and epithelium-forming mesenchymal cells. podocytes remain cuboidal, and no foot processes or slit dia- phragms can be detected. Furthermore, they do not express Differentiation of Ureteric Bud Cells podocin, and they synthesize decreased amounts of pro-␣3(IV) Surprising is that only one transcription factor expressed by and pro-␣4(IV) collagen chains (39–41). ureteric bud cells has been clearly shown to be necessary for their differentiation, even if Pax2 and Lim1 are also likely to Differentiation of Stromal Cells play a direct role in ureteric bud development. Emx2 is a During embryonic development, differentiation of most if homeobox gene that is expressed in the Wolffian duct, in the not all epithelial tissues requires interactions with an adjacent ureteric bud and its derivatives, and in early epithelial struc- mesenchyme. Similarly, during kidney development, differen- tures forming vesicles and comma-shaped bodies (45). In null- tiation of tubular cells requires interactions not only with the mutant mice, the ureteric bud invades the metanephric mesen- ureteric bud branches but also with adjacent stromal cells. The chyme, but it does not branch, does not induce the metanephric role of stromal cells has been mostly highlighted by analysis of mesenchyme to condense, and rapidly degenerates (46). This is Foxd1/Bf2 null mice and of retinoic acid receptors (RAR) Rara associated with a large reduction of the expression of Lim1, and Rarb2 double mutant mice. Pax2, and Ret in the ureteric bud cells. Furthermore, expres- Foxd1 is a member of the forkhead/winged helix family of sion of Gdnf is normal before invasion of the metanephric transcription factors, which is expressed in different organs, mesenchyme by the ureteric bud, but later on it is greatly including kidney, during embryonic development. In kidney, it reduced around the invading ureteric bud, and the gene encod- is selectively expressed in stromal cells that surround con- ing Wnt4 (a soluble molecule that is normally produced by densed mesenchymal cells and differentiating nephrons and at condensed mesenchymal cells) is not expressed in pretubular lower levels in stromal cells located in the medulla around aggregates. Coculture experiments show that the ureteric bud is ureteric bud branches (42). Foxd1Ϫ/Ϫ mice die at birth, while unable to branch or induce an epithelial transformation of they have small kidneys that are abnormally positioned. Anal- wild-type mesenchyme but that the development of the mutant ysis of kidney development in these mice shows a dramatic metanephric mesenchyme is normal (46). Taken together, these reduction in the number of S-shaped and comma-shaped bod- experiments suggest that Emx2 regulates the differentiation of ies, and the presence of large amounts of condensed mesen- ureteric bud cells and the production by these cells of a signal chymal cells, which suggests first that stromal cells do not that induces epithelial differentiation of metanephric mesen- differentiate normally and second that they are necessary for chymal cells. epithelial differentiation of condensed mesenchymal cells (42). A role of Pax2 in differentiation of ureteric bud cells is Furthermore, because expression of Ret (a receptor that is mostly suggested by analysis of nephric duct formation. Pax8 expressed by ureteric bud cells and that binds Gdnf) is abnor- is another member of the Pax family of transcription factors mal in ureteric bud cells of Foxd1Ϫ/Ϫ mice, stromal cells may that is expressed during pro-, meso-, and metanephros devel- also be necessary for normal differentiation of ureteric bud opment, but kidney development is normal in Pax8Ϫ/Ϫ mice cells (42). This hypothesis is in agreement with results ob- (47,48). Whereas initial development of the nephric duct is tained by analyzing null-mutant mice for RAR (cf. infra). normal in Pax2Ϫ/Ϫ mice, in Pax2Ϫ/Ϫ Pax8Ϫ/Ϫ double- RAR are transcription factors that belong to the nuclear mutant embryos, the intermediate mesenchyme does not un- receptor superfamily and transduce the retinoid signal. In the dergo mesenchymal-epithelial transition and the nephric duct presence of retinoic acid, they bind to enhancer elements and does not form (47–49). In zebrafish, lack of functional Pax2.1 activate transcription. Analysis of double mutant mice that lack gene leads to abnormal epithelial differentiation of the pro- Rara (retinoic acid receptor ␣) and Rarb2 (retinoic acid recep- nephric duct (50). Conversely, in chick embryos, ectopic ex- tor ␤2) shows that retinoic acid is indispensable for normal pression of Pax2 in the intermediate mesoderm and genital development of stromal cells. During kidney development, ridge can induce the formation of ectopic nephric ducts (50). Rara is expressed at low levels throughout the embryonic Similarly, a role for Lim1 in ureteric development is suggested kidney, whereas Rarb2 is expressed selectively in stromal cells by the fact that ectopic expression of XPax8 and Xlim1 can (43). Unlike Foxd1, Rarb2 is expressed at similar levels in all induce the formation of additional pronephric tubules in Xe- stromal cells (43). 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