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