Canonical Wnt/β-catenin activity and differential epigenetic marks direct sexually dimorphic regulation of Irx3 and Irx5 in developing gonads

Members of the Iroquois B (IrxB) homeodomain cluster genes, specifically Irx3 and Irx5, are critical for heart, limb, and bone development. Recently, we reported their importance for oocyte and follicle survival within the developing ovary. Irx3 and Irx5 expression begins after sex determination in the ovary but remains absent in the fetal testis. Mutually antagonistic molecular signals ensure ovary vs testis differentiation with canonical Wnt/β-catenin signals paramount for promoting the ovary pathway. Notably, few direct downstream targets have been identified. We report that Wnt/β-catenin signaling directly stimulates Irx3 and Irx5 transcription in the developing ovary. Using in silico analysis of ATAC- and ChIP-Seq databases in conjunction with gonad explant transfection assays, we identified TCF/LEF binding sequences within two distal enhancers of the IrxB locus that promote β-catenin-responsive ovary expression. Meanwhile, Irx3 and Irx5 transcription is suppressed within the developing testis by the presence of H3K27me3 on these same sites. Thus, we resolved sexually dimorphic regulation of Irx3 and Irx5 via epigenetic and β-catenin transcriptional control where their ovarian presence promotes oocyte and follicle survival vital for future ovarian health.