GZF1, a new GDNF-inducible gene, is required for renal branching morphogenesis

Abstract

The glial cell line-derived neurotrophic factor (GDNF)/ rearrangement during transfection, tyrosine kinase (RET) signaling is recognized as a critical pathway of renal branching morphogenesis, and much attention has been paid to the molecules that function upstream of this pathway. It was suggested that transcription factors, Pax2 and Eya-1 , are responsible for Gdnf expression, and Foxc1 prevents ectopic ureteric budding from Wolffian duct by repressing Gdnf and Eya1 . In addition, vitamin A signal from stromal mesenchyme is necessary for Ret expression. Recently it was reported that Wnt11 and Sprouty proteins are associated with the GDNF/RET signaling, but the detailed process downstream of the GDNF/RET signaling in renal development is still poorly understood. We performed an exhaustive analysis of GDNF-inducible genes by differential display to identify an important molecule which function downstream of the GDNF/RET signaling pathway. As a consequence, we found the increased expression of 14 genes by GDNF. Among them, we focused on a new gene with a BTB/POZ domain and C2H2-type zinc finger motifs that was named GZF1 (GDNF-inducible zinc finger gene 1). To elucidate the importance of GZF1 on kidney development, we stained the mouse embryonic kidney with anti-GZF1 antibody. Interestingly, the ureteric bud epithelia of embryonic metanephroi were strongly stained, whereas S-shaped or comma-shaped bodies were not stained. Using serial sections, we detected the expression of GZF1 and RET in the same ureteric buds. Furthermore, antisense oligonucleotides (ODNs) of the Gzf1 markedly impaired the ureteric bud branching in the metanephric organ culture, suggesting that the induction of GZF1 via the GDNF/RET signaling system is required for renal branching morphogenesis. We investigated the transcriptional activity of GZF1 by luciferase reporter gene assay. In this assay, it was revealed that the GZF1 possesses transcriptional repressive activity. To examine the binding sequence for GZF1, we employed cyclic amplification and selection of targets (CASTing). Finally, a consensus sequence was derived from the sequence alignment of individual clones. We named it GZF1 recognition element (GRE). Next we searched for potential target genes that might be regulated by GZF1 and found that a variety of genes possess the GRE in their 5' upstream regulatory region. We are now investigating the effect of the GRE on the promoter activity of one gene among them. To examine whether the GZF1 binds the promoter region of this gene, we performed modified chromatin immunoprecipitation assay. It was shown that GZF1 binds the promoter region of this gene. Further analysis about the transcriptional effect of GZF1 on this gene is now moving. As described above, this novel gene GZF1 may play a crucial role in renal development regulated by the GDNF/RET signaling. We are now generating Gzf1 -/- mouse. We expect this novel molecule will promote our understanding of the mechanisms of the renal organogenesis. Furthermore, its clinical application to the treatment of renal disease, especially to the regeneration of the kidney, should be explored in the future.