The
gonad is an outstanding experimental model where a primary fate decision in
bipotential precursor cells has the dramatic consequence of controlling the phenotypic
sex of the organism. We have focused on how this fate decision is executed and
maintained at the level of the transcriptional network, as well as how
divergent organogenesis of a testis or ovary results from this primary fate
decision. Transcriptome analysis of the three principle precursor populations
in the bipotential XX and XY gonad (supporting cell precursors, steroidogenic
cell precursors, and germ cells) identified distinct signatures specific to
each of these lineages, that are shared in XX and XY gonads. At the bipotential
stage, more genes associated with the female pathway are expressed in both XX
and XY supporting cell precursors, suggesting a female bias in lineage
priming. In mammals, divergence along
the male pathway is initiated by transcriptional activation of the Y-linked
gene, Sry, at E10.5 in supporting
cell precursors. To define the dynamic
establishment of sexually dimorphic expression patterns downstream of Sry, we conducted transcriptome analyses
comparing XX and XY gonads at six 4-hour intervals between E11.0-E12.0. Expression
of Sry initiates sequential upregulation
of male pathway genes, as well as sequential downregulation of female pathway
genes in XY gonads. Deletion of Fgf9, which acts as a repressor of Wnt4, a signal that drives the female
pathway, leads to male to female sex reversal and ovary development in XY
offspring. This and other experiments suggest that repression of female genes
is critical to establish the male pathway. To investigate how this
fate commitment is reflected in the chromatin landscape in pre-Sertoli cells,
we identified DNAse hypersensitivity sites (DHS) in chromatin of E13.5 and
E15.5 Sertoli progenitors. DHS identified regions of open chromatin both in genes
associated with the male pathway and actively transcribed in Sertoli
progenitors, and those that were silent and associated with the female
pathway. Chromatin immunoprecipitation
(ChIP) analysis using an antibody against the active chromatin mark, H3K27ac,
distinguished those DHS peaks that are associated with active transcription
from those that are likely associated with factors that repress expression, identifying
enhancers across the genome, and defining the chromatin landscape that
regulates commitment to Sertoli fate.
This study was funded by grants from NICHD
and NIDDK to BC.