Proceedings of The Physiological Society

Europhysiology 2018 (London, UK) (2018) Proc Physiol Soc 41, C070

Oral Communications

A novel tool for identifying transcriptional targets in kidney development using the example of Wilms tumor protein (WT1)

V. Schmidt1, Q. Gao2, W. Chen2, H. Scholz1, K. M. Kirschner1

1. Inst. of Vegetative Physiology, Charité - Universitätsmedizin Berlin, Berlin, Germany. 2. Max Delbrück Center for Molecular Medicine, Berlin, Germany.

The Wilms tumor transcription factor WT1 is necessary for normal kidney formation. Identification of WT1 downstream target genes may thus allow one to analyze genetic pathways relevant to renal development. Since mice with germline knockout of Wt1 are embryonic lethal and lack kidneys, alternative tools are needed to study WT1-dependent gene networks. By combining vivo-morpholino induced gene knockdown in metanephric explants with next generation sequencing, we established a novel instrument for identifying WT1 downstream targets in kidney development. Kidneys were excised from mouse embryos (E12.5) and grown in hanging droplets of nutrient. The explants were treated for 48 h with either Wt1 antisense or mismatch vivo-morpholino. WT1 knockdown was assessed by immunoblotting and mRNA from pooled kidney samples was subjected to deep sequencing. The DESeq package was used to identify the differentially expressed genes. Deep sequencing data were validated by quantitative PCR of more than a dozen of arbitrarily picked transcripts. Silencing of Wt1 by vivo-morpholino treatment significantly reduced 308 and increased 258 mRNAs in metanephric organ cultures. Comparison with previously published ChIP-sequencing data (Motamedi et al., 2014) showed that WT1 binds to 217 of the down-regulated and 194 of the up-regulated genes in murine embryonic kidneys. Among these differentially expressed genes, 184 could be retrieved from the atlas of gene expression in the developing kidney (Brunskill et al, 2008). According to this atlas, 32 of the 184 genes are predominantly expressed in the metanephric mesenchyme, i.e. the site of WT1 expression. The top candidate WT1 target genes were further analyzed. To spread these results from the complex organic system to a cellular system, the endogenous mRNA levels of these top candidates were measured by quantitative PCR in cells treated with WT1 siRNA or non-targeting siRNA. We used the functionally related M15 cell line (murine mesonephros-derived cells), the U2OS cell line (human osteosarcoma cells) as well as the 786-0 cell line (human kidney cancer cells) to analyze gene expression patterns in human cellular systems. Subsequent co-transfection experiments using various reporter constructs indicated that the promoters of the homeobox b9 (Hoxb9), natriuretic peptide receptor 3 (Npr3) and dishevelled associated activator of morphogenesis 2 (Daam2), among other genes, are significantly stimulated by WT1. With these findings, we identified sets of genes in murine embryonic kidneys that are regulated either directly or indirectly by WT1. In conclusion, combination of vivo-morpholino induced gene knockdown in embryonic kidney explants with deep RNA-sequencing is a useful approach for analyzing transcriptional networks in kidney development.

Where applicable, experiments conform with Society ethical requirements