Notch signaling, wt1 and foxc2 are key regulators of the podocyte gene regulatory network in Xenopus.

Podocytes are highly specialized cells in the vertebrate kidney. They participate in the formation of the size-exclusion barrier of the glomerulus/glomus and recruit mesangial and endothelial cells to form a mature glomerulus. At least six transcription factors (wt1, foxc2, hey1, tcf21, lmx1b and mafb) are known to be involved in podocyte specification, but how they interact to drive the differentiation program is unknown. The Xenopus pronephros was used as a paradigm to address this question. All six podocyte transcription factors were systematically eliminated by antisense morpholino oligomers. Changes in the expression of the podocyte transcription factors and of four selected markers of terminal differentiation (nphs1, kirrel, ptpru and nphs2) were analyzed by in situ hybridization. The data were assembled into a transcriptional regulatory network for podocyte development. Although eliminating the six transcription factors individually interfered with aspects of podocyte development, no single gene regulated the entire differentiation program. Only the combined knockdown of wt1 and foxc2 resulted in a loss of all podocyte marker gene expression. Gain-of-function studies showed that wt1 and foxc2 were sufficient to increase podocyte gene expression within the glomus proper. However, the combination of wt1, foxc2 and Notch signaling was required for ectopic expression in ventral marginal zone explants. Together, this approach demonstrates how complex interactions are required for the correct spatiotemporal execution of the podocyte gene expression program.