The Wilms tumor protein WT1 is an essential factor for kidney development. In humans, mutations in WT1 lead to Wilms tumor, a pediatric kidney cancer as well as to developmental anomalies concerning the urogenital tract. Inactivation of Wt1 in mice causes multiple organ defects most notably agenesis of the kidneys. In zebrafish, two paralogous wt1 genes exist, wt1a and wt1b. The wt1 genes are expressed in a similar and overlapping but not identical pattern. Here, we have examined the role of both wt1 genes in early kidney development employing a transgenic line with pronephros specific GFP expression and morpholino knockdown experiments. Inactivation of wt1a led to failure of glomerular differentiation and morphogenesis resulting in a rapidly expanding general body edema. In contrast, knockdown of wt1b was compatible with early glomerular development. After 48 h, however, wt1b morphant embryos developed cysts in the region of the glomeruli and tubules and subsequent pericardial edema at 4 days post-fertilization. Thus, our data suggest different functions for wt1a and wt1b in zebrafish nephrogenesis. While wt1a has a more fundamental and early role in pronephros development and is essential for the formation of glomerular structures, wt1b functions at later stages of nephrogenesis.

The Wilms' tumor suppressor gene Wt1 encodes a zinc-finger transcription factor that plays an essential role in organ development, most notably of the kidney. Despite its importance for organogenesis, knowledge of the regulation of Wt1 expression is scarce. Here, we have used transgenesis in zebrafish harboring two wt1 genes, wt1a and wt1b, in order to define regulatory elements that drive wt1 expression in the kidney. Stable transgenic lines with approximately 30 kb of the upstream genomic regions of wt1a or wt1b almost exactly recapitulated endogenous expression of the wt1 paralogs. In the case of wt1b, we have identified an enhancer that is located in the far upstream region that is necessary and sufficient for reporter gene expression in the pronephric glomeruli. Regarding wt1a, we could also identify an enhancer that is located approximately 4 kb upstream of the transcriptional start site that is required for expression in the intermediate mesoderm. Interestingly, this intermediate mesoderm enhancer is highly conserved between fish and mammals, is bound by members of the retinoic acid receptor family of transcription factors in gel shift experiments and mediates responsiveness to retinoic acid both in vivo and in cell culture. To our knowledge, this is the first functional demonstration of defined regulatory elements controlling Wt1 expression in vivo. The identification of kidney-specific enhancer elements will help us to better understand the integration of extracellular signals into intracellular networks in nephrogenesis.

The Wilms' tumor suppressor gene wt1 encodes a zinc-finger transcription factor that plays an important role in the development of the mammalian genitourinary system. Mutations in WT1 in humans lead to anomalies of kidney and gonad development and cause Wilms' tumor, a pediatric kidney cancer. The inactivation of both wt1 alleles in mice gives rise to multiple organ defects, among them agenesis of kidney, spleen, and gonads. In zebrafish, an ortholog of wt1 has been described that is expressed in the pronephric field and is later restricted to the podocytes. Here, we report the existence of a second wt1 gene in zebrafish, which we have named wt1b (we named the initial gene wt1a). The overall sequence identity of the two Wt1 proteins is 70% and 92% between the zinc-finger regions, respectively. In contrast to wt1a, wt1b is expressed from the earliest stages of development onward, albeit at low levels. Both wt1a and wt1b are expressed in the intermediate mesoderm, with wt1b being restricted to a smaller area lying at the caudal end of the wt1a expression domain. In adult fish, high expression levels for both genes can be found in gonads, kidney, heart, spleen, and muscle. Developmental Dynamics 235:554 -561, 2006.

The Wilms' tumor suppressor gene 1 (WT1) encodes a zinc finger transcription factor. Mutation of WT1 in humans leads to Wilms' tumor, a pediatric kidney tumor, or other kidney diseases, such as Denys-Drash and Frasier syndromes. We showed previously that inactivation of WT1 in podocytes of adult mice results in proteinuria, foot process effacement, and glomerulosclerosis. However, the WT1-dependent transcriptional network regulating podocyte development and maintenance in vivo remains unknown. Here, we performed chromatin immunoprecipitation followed by high-throughput sequencing with glomeruli from wild-type mice. Additionally, we performed a cDNA microarray screen on an inducible podocyte-specific WT1 knockout mouse model. By integration of cistromic and transcriptomic analyses, we identified the WT1 targetome in mature podocytes. To further analyze the function and targets of WT1 in podocyte maturation, we used an Nphs2-Cre model, in which WT1 is deleted during podocyte differentiation. These mice display anuria and kidney hemorrhage and die within 24 hours after birth. To address the evolutionary conservation of WT1 targets, we performed functional assays using zebrafish as a model and identified Nphs2, Mafb, and Magi2 as novel WT1 target genes required for podocyte development. Our data also show that both Mafb and Magi2 are required for normal development of the embryonic zebrafish kidney. Collectively, our work provides insights into the transcriptional networks controlled by WT1 and identifies novel WT1 target genes that mediate the function of WT1 in podocyte differentiation and maintenance.

There was an error published in J. Cell Sci. 126,[196][197][198][199][200][201][202][203][204][205][206][207][208] The sequence of MO sdpI_e3i3 was reported incorrectly in the Materials and Methods section. The correct sequence of the MO sdpI_e3i3 used for this study is gtcaaaatccttcctcacCTCTCTC.We apologise for this mistake. Summary During development, general body plan information must be translated into distinct morphologies of individual cells. Shaping cells is thought to involve cortical cytoskeletal components and Bin-Amphiphysin-Rvs167 (BAR) superfamily proteins. We therefore conducted comprehensive side-by-side loss-of-function studies of zebrafish orthologs of the F-BAR protein syndapin I and the actin nucleator Cobl. Zebrafish syndapin I associates with Cobl. The loss-of-function phenotypes of these proteins were remarkably similar and suggested a common function. Both cobl-and syndapin I-morphant fish showed severe swimming and balance-keeping defects, reflecting an impaired organization and function of the lateral line organ. Their lateral line organs lacked several neuromasts and showed an impaired functionality of the sensory hair cells within the neuromasts. Scanning electron microscopy revealed that sensory hair cells of both cobl-and syndapin I-morphant animals showed defects in the formation of both microtubule-dependent kinocilia and F-actinrich stereocilia. Consistent with the kinocilia defects in sensory hair cells, body length was shortened and the development of body laterality, a process depending on motile cilia, was also impaired. Interestingly, Cobl and syndapin I both localized to the base of forming cilia. Rescue experiments demonstrated that proper formation of ciliated sensory hair cell rosettes relied on Cobl's syndapin Ibinding Cobl homology domain, the actin-nucleating C-terminus of Cobl and the membrane curvature-inducing F-BAR domain of syndapin I. Our data thus suggest that the formation of distinct types of ciliary structures relies on membrane topology-modulating mechanisms that are based on F-BAR domain functions and on complex formation of syndapin I with the actin nucleator Cobl.