RESUMO
Development of the metanephric kidney depends on tightly regulated interplay between self-renewal and differentiation of a nephron progenitor cell (NPC) pool. Several key factors required for the survival of NPCs have been identified, including fibroblast growth factor (FGF) signaling and the transcription factor Wilms' tumor suppressor 1 (WT1). Here, we present evidence that WT1 modulates FGF signaling by activating the expression of growth arrest-specific 1 (Gas1), a novel WT1 target gene and novel modulator of FGF signaling. We show that WT1 directly binds to a conserved DNA binding motif within the Gas1 promoter and activates Gas1 mRNA transcription in NPCs. We confirm that WT1 is required for Gas1 expression in kidneys in vivo. Loss of function of GAS1 in vivo results in hypoplastic kidneys with reduced nephron mass due to premature depletion of NPCs. Although kidney development in Gas1 knockout mice progresses normally until E15.5, NPCs show decreased rates of proliferation at this stage and are depleted as of E17.5. Lastly, we show that Gas1 is selectively required for FGF-stimulated AKT signaling in vitro. In summary, our data suggest a model in which WT1 modulates receptor tyrosine kinase signaling in NPCs by directing the expression of Gas1.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Fatores de Crescimento de Fibroblastos/metabolismo , Néfrons/metabolismo , Transdução de Sinais , Células-Tronco/metabolismo , Proteínas WT1/metabolismo , Animais , Proteínas de Ciclo Celular/genética , Proliferação de Células , DNA/genética , Ativação Enzimática/efeitos dos fármacos , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Silenciamento de Genes , Camundongos Knockout , Modelos Animais , Néfrons/anormalidades , Néfrons/embriologia , Néfrons/patologia , Técnicas de Cultura de Órgãos , Regiões Promotoras Genéticas/genética , Ligação Proteica , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-ret/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
The transcription factor Wilms' tumor suppressor 1 (WT1) is key to podocyte development and viability; however, WT1 transcriptional networks in podocytes remain elusive. We provide a comprehensive analysis of the genome-wide WT1 transcriptional network in podocytes in vivo using chromatin immunoprecipitation followed by sequencing (ChIPseq) and RNA sequencing techniques. Our data show a specific role for WT1 in regulating the podocyte-specific transcriptome through binding to both promoters and enhancers of target genes. Furthermore, we inferred a podocyte transcription factor network consisting of WT1, LMX1B, TCF21, Fox-class and TEAD family transcription factors, and MAFB that uses tissue-specific enhancers to control podocyte gene expression. In addition to previously described WT1-dependent target genes, ChIPseq identified novel WT1-dependent signaling systems. These targets included components of the Hippo signaling system, underscoring the power of genome-wide transcriptional-network analyses. Together, our data elucidate a comprehensive gene regulatory network in podocytes suggesting that WT1 gene regulatory function and podocyte cell-type specification can best be understood in the context of transcription factor-regulatory element network interplay.
