Abstract

WT1 encodes a transcription factor involved in kidney development and tumorigenesis. Using representational difference analysis, we identified a new set of WT1 targets, including a homologue of the Drosophila receptor tyrosine kinase regulator, sprouty. Sprouty1 was up-regulated in cell lines expressing wild-type but not mutant WT1. WT1 bound to the endogenous sprouty1 promoter in vivo and directly regulated sprouty1 through an early growth response gene-1 binding site. Expression of Sprouty1 and WT1 overlapped in the developing metanephric mesenchyme, and Sprouty1, like WT1, plays a key role in the early steps of glomerulus formation. Disruption of Sprouty1 expression in embryonic kidney explants by antisense oligonucleotides reduced condensation of the metanephric mesenchyme, leading to a decreased number of glomeruli. In addition, sprouty1 was expressed in the ureteric tree and antisense-treated ureteric trees had cystic lumens. Therefore, sprouty1 represents a physiologically relevant target gene of WT1 during kidney development.

Highlights

  • The development of the mammalian metanephric kidney is a model for the study of cellular and molecular mechanisms of organogenesis [1]

  • Disruption of spry1 expression in kidney explants altered normal glomeruli formation and ureteric tree morphology. These results demonstrate that spry1 is a Wilms Tumor suppressor gene 1 (WT1) target gene that may mediate some of the regulatory effects of WT1 during kidney organogenesis

  • RT-PCR analysis (Fig. 1A) showed that spry1 expression was low in NIH3T3 cells and increased in WT1-expressing cell lines (WR16, WR35, WR14)

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Summary

Introduction

The development of the mammalian metanephric kidney is a model for the study of cellular and molecular mechanisms of organogenesis [1]. We previously showed that NIH3T3 cells engineered to express the WT1A isoform were growth-inhibited and displayed partial epithelial differentiation [9]. These morphological changes correlated with significant alterations in gene expression consistent with a role for WT1 in the MET. Precise spatial and temporal control of the Ras/Raf/mitogen-activated protein kinase cascade, a major pathway for growth factors to mediate cell proliferation or differentiation, is required for normal development. Disruption of spry expression in kidney explants altered normal glomeruli formation and ureteric tree morphology Taken together, these results demonstrate that spry is a WT1 target gene that may mediate some of the regulatory effects of WT1 during kidney organogenesis

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