In order for development of the reproductive system to occur, the formation of gonad anlagen is first required. Mammalian gonads arise from a bipotential progenitor gonadal tissue called the urogenital ridge (UGR). However, very little is known about how the molecular networks that shape its formation and the molecular preparations made to allow for two developmental trajectories. The LIM-homeobox gene, Lhx9 is among only a handful of genes known to be required for UGR formation. In order to investigate the molecular underpinnings involved in UGR formation, we took a large-scale approach using chromatin immunoprecipitation followed by next generation sequencing (ChIP-seq) to identify genes involved in this process.
ChIP-seq on mouse UGRs (E11.5) was performed for Lhx9 and for two histone modification marks, H3K4me3 and H3K27me3, which highlight regions of active and repressive transcriptional states respectively. Lhx9 target genes were confirmed by RT-qPCR using a Lhx9 knockout line.Genes required for processes such as sex determination, sexual differentiation, cell proliferation, angiogenesis and cell migration were identified as regulated by Lhx9. Several Lhx9 target genes, whose expression patterns in the UGR were not previously characterized, were analysed further by in situ hybridization.
Furthermore, looking at both histone mark ChIP-seq datasets, many genes were found to possess a ‘bivalent’ histone modification dynamic, whereby both H3K4me3 and H3K27me3 were found in the promoter or enhancer regions. This histone dynamic has been characterized as a feature that highlights certain lineage regulatory genes, holding them in a ‘poised’ transcriptional state. In particular, many genes involved in the Wnt signaling pathway were identified to possess bivalent histone marks. Bringing both ChIP-seq datasets together, we provide a wider scope of the transcriptional and epigenetic regulatory network that is necessary for UGR formation, but also the preparation for sexual development.