The distinction between sexes is one of the most obvious example of morphological dimorphism in the animal kingdom, that highlights one of the most crucial fate decisions made in utero; to become a male or female. In order for sexual development to occur, the formation of gonad anlagen is first required. Mammalian gonads are unique among the animal kingdom as they 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, but it’s regulatory network, and that of the UGR is poorly understood. 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 was preformed for LHX9 to target regions in the chromatin where LHX9 regulates. In addition ChIP-seq was also performed on two histone modification marks, H3K4me3 and H3K27me3, which highlight regions of active and repressive transcriptional states respectively. Regions in the genome where LHX9 or the histone marks were found were validated using ChIP-qPCR and in situ hybridization.
Targets of LHX9 validated by ChIP-qPCR were found to be required for processes such as sex determination, sexual differentiation, cell proliferation, angiogenesis and cell migration. In addition, several other targets whose expression patterns in the UGR were not previously characterized was determined 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. Several genes of the Wnt family were validated via ChIP-qPCR. 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.