# Opposing Pathways in Mammalian Sex Determination

> **NIH NIH R01** · DUKE UNIVERSITY · 2022 · $407,089

## Abstract

ABSTRACT
Sex determination in mammals is a complex biological process leading to the differentiation of the bipotential
gonad into a testis or ovary. It is initiated by a cell fate decision in a subset of somatic cells called the
supporting cell lineage. These cells carry bivalent histone marks at many genes associated with sex
determination pathways and are poised for direction into either the Sertoli cell lineage (leading to testis
development) or the granulosa cell lineage (leading to ovary development). Commitment to Sertoli or
granulosa fate is characterized by the loss of repressive histone marks at genes associated with the active
pathway, and expansion of repressive marks at genes associated with the alternative pathway. Repression,
which we hypothesize is mediated in part by chromatin conformational changes, is important to stabilize fate
commitment. However, how epigenetic modifications and transcription factor cascades are integrated to drive
sex determination is not understood. It is well established that the key step in activating Sertoli cell
differentiation is the transient activation of the Y-encoded transcription factor (TF), SRY, and its direct
downstream target, SOX9. However, since our discovery that supporting cells are derived from the coelomic
epithelium (CE), it has been unclear why Sry and female pathway genes are only activated in cells once they
leave the CE and enter the gonad. We recently showed that NUMB, an inhibitor of the Notch signaling
pathway, is asymmetrically distributed to cells that enter the gonad from the CE and is required for gonadal
cell differentiation. These findings imply that a factor(s) activated in Numb+/Notchlo cells confers competence
for differentiation. Exploration of our transcriptome, ATAC-seq, and histone modification datasets, revealed
that 2 E-box family HLH transcription factors, Tcf4 and Tcf12, are expressed at high levels in the bipotential
gonad, remain highly expressed in the granulosa lineage, but are downregulated in the Sertoli lineage where
they map as binding targets of SOX9 and could be targets of feedback repression to block the granulosa
pathway in XY gonads. E-box binding motifs are enriched in the nucleosome-depleted regions near granulosa
genes but are absent from Sertoli genes, with the exception of Sry itself. Our findings suggest that TCF4 and
TCF12, activated in Numb+/Notchlo cells, license differentiation of the supporting cell lineage, serving as
activators of the granulosa pathway and of Sry in the XY gonad; whereas, SOX9, activated by SRY, acts in a
feedback loop to down-regulate Tcf4 and Tcf12 transcription. In Aim 1, we will test the hypothesis that bHLH
TFs are downstream of Numb, comprise an HLH “code” for differentiation of the supporting cell lineage, and
are repressed by SOX9. In Aim 2, we will test the hypothesis that epigenetic and chromatin conformation
changes occur during Sertoli fate commitment, mediated in part by competition between SOX9 and HLH
pro...

## Key facts

- **NIH application ID:** 10404067
- **Project number:** 5R01HD039963-25
- **Recipient organization:** DUKE UNIVERSITY
- **Principal Investigator:** Blanche Capel
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $407,089
- **Award type:** 5
- **Project period:** 1998-01-01 → 2026-03-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10404067

## Citation

> US National Institutes of Health, RePORTER application 10404067, Opposing Pathways in Mammalian Sex Determination (5R01HD039963-25). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10404067. Licensed CC0.

---

*[NIH grants dataset](/datasets/nih-grants) · CC0 1.0*