The basic-helix-loop-helix Transcription Factor TCF4 Regulates Supporting Cell Differentiation in the Embryonic Gonad

Eleanore V. O’Neil¹, Shannon M. Dupont¹, and Blanche Capel¹

Abstract Text:

The bipotential gonad is the precursor organ to both the ovary and testis and begins to develop as part of the embryonic urogenital system at embryonic day (E) 9.5 in mice. Coelomic epithelial (CE) cells lining the mesonephros begin to proliferate and differentiate into the somatic cell types of the gonad: the pre-supporting and interstitial cell lineages. While these cell populations are essential for adult gonadal function, the mechanisms that initiate their differentiation remain largely unclear. Previous studies have identified that NUMB promotes the differentiation of CE cells into somatic gonadal cell types. In this study we sought to understand whether helix-loop-helix (HLH) transcription factors, which commonly act downstream of NUMB, promote somatic cell differentiation in the gonad. According to transcriptomic data, transcription factor 4 (TCF4) is the highest expressed HLH factor in the bipotential gonad. Immunostaining across developmental timepoints showed that TCF4 is expressed in the CE and is higher expressed in GATA4+ somatic gonadal cells prior to sex determination. When sex determination initiates at ~E11.0, TCF4 is lost in the SOX9+ supporting cells of the testis (pre-Sertoli cells) and is maintained in the testis interstitium. In the ovary, TCF4 is expressed at low levels in the RUNX1+ supporting cells (pre-granulosa cells), and at high levels in the ovary interstitium. Thus, TCF4 is broadly expressed across somatic cell types in the gonad and shows cell type specificity across gonad differentiation. We calculated the frequency of TCF4 binding motifs upstream of gonadal differentiation genes and observed that motifs are present in approximately half of all genes that increase as somatic cells differentiate. ChIP-sequencing for TCF4 in XX and XY gonadal cells at E12.5 identified TCF4 target genes. Surprisingly, we observed that the most common targets of TCF4 were genes that broadly promote transcription and chromatin remodeling rather than genes known to be involved in gonadogenesis. Mass spectrometry on immunoprecipitated TCF4 in sorted somatic gonadal cells revealed that TCF4 binds with the Mediator complex, a multiprotein complex that coordinates transcription factor binding and epigenetic regulators with RNA Polymerase, and core members of the RNA polymerase holoenzyme. Collectively, we believe these data support the hypothesis that TCF4 promotes differentiation of the gonadal lineage by shifting the transcriptional and epigenetic landscape of gonadal somatic cells. To understand how TCF4 affects gonad differentiation in vivo, we obtained mice which contain a premature STOP codon upstream of the TCF4 DNA-binding domain, Tcf4^MUT/+, and bred them to assess gonad differentiation in Tcf4^MUT/MUT offspring. XY E15.5 Tcf4^MUT/MUT testes showed similar histoarchitecture and Sertoli to interstitial cell ratios as wildtype littermates, with elevated expression of SOX9 in Sertoli cells. In contrast, Tcf4^MUT/MUT ovaries had a higher ratio of granulosa to interstitial cells compared to wildtype littermates, as well as lower levels of the transcription factors FOXL2 and NR2F2 (COUPTFII) in granulosa and interstitial cells, respectively. Collectively, these results support the hypothesis that TCF4 promotes differentiation of the gonadal lineages by shifting the transcriptional and epigenetic landscape of somatic cells and regulates differentiation and balance of the ovarian cell types. These findings improve our understanding of the underlying mechanisms of gonad development.