Identification of novel markers of mouse fetal ovary development

In contrast to the developing testis, molecular pathways driving fetal ovarian development have been difficult to characterise. To date no single master regulator of ovarian development has been identified that would be considered the female equivalent of Sry. Using a genomic approach we identified a number of novel protein-coding as well as non-coding genes that were detectable at higher levels in the ovary compared to testis during early mouse gonad development. We were able to cluster these ovarian genes into different temporal expression categories. Of note, Lrrc34 and AK015184 were detected in XX but not XY germ cells before the onset of sex-specific germ cell differentiation marked by entry into meiosis in an ovary and mitotic arrest in a testis. We also defined distinct spatial expression domains of somatic cell genes in the developing ovary. Our data expands the set of markers of early mouse ovary differentiation and identifies a classification of early ovarian genes, thus providing additional avenues with which to dissect this process

Expression analysis of somatic cell genes.

<p>ISH with sagittal section of XX and XY mouse embryos from 11.5 to 13.5 dpc, 5 weeks (wk) postnatal mouse ovaries and testes showed that <i>Slitrk1</i> (<b>A</b>) and <i>oncRNA3</i> (<b>B</b>) are expressed in ovarian somatic cells at 11.5 and 12.5 dpc. <i>Slitrk1</i> is also expressed in the granulosa cells of the mature ovary. Scale bars, 100 µm.</p

Molecular compartmentalization of the early embryonic ovary.

<p>(<b>A</b>) ISH with sagittal section of 13.5 dpc XX embryos for <i>Fst</i>, <i>Wnt4</i>, <i>Rspo1</i> and <i>Irx3</i> followed by IHC for FOXL2 (first and second panel) and section ISH for <i>Lypd6</i> (third panel) identified three different expression domains of somatic cell genes as represented in the schematic on the left of each panel. Scale bar, 100 µm. (<b>B</b>) ISH with transverse sections of 12.5 dpc XX embryos for <i>Foxl2</i>, <i>Wnt4</i>, <i>oncRNA3</i> and <i>Lypd6</i> showed the extent to which these genes were expressed in the mesonephric and coelomic domains respectively. (<b>C</b>) High magnification of ISH with sagittal sections of 13.5 dpc XX embryos for <i>Fst</i>, <i>Wnt4</i>, <i>Rspo1</i> and <i>Irx3</i> followed by IHC for FOXL2 suggested that <i>Fst</i> and <i>Wnt4</i>, but not <i>Rspo1</i> and <i>Irx3</i>, are co-expressed to a large extent with FOXL2. Scale bar, 100 µm (<b>A</b> and <b>B</b>), 20 µm (<b>C</b>).</p

Temporal and spatial expression analysis of <i>D6Mm5e</i>.

<p>Whole mount ISH of mouse embryonic XX and XY gonads from 11.5 to 15.5 dpc (<b>A</b>) and ISH with sagittal section of mouse embryonic XX and XY gonads from 11.5 to 13.5 dpc, (<b>B</b>), showing wave-like upregulation of <i>D6Mm5e</i> reminiscent of PGC (arrowhead) entry into meiosis. Scale bar, 100 µm. (<b>C</b>) <i>D6Mm5e</i> section ISH hybridization (purple staining) followed by IHC for the germ cell marker E-cadherin (ECAD, brown staining) of 13.5 dpc mouse ovaries confirmed <i>D6Mm5e</i> expression in XX PGCs (arrowheads). Scale bars, 100 µm (low magnification, top panel) and 20 µm (high magnification, bottom panel).</p

qRT-PCR validation of genes expressed in the developing ovary identified by microarray.

<p>qRT-PCR analysis of mRNA from isolated XX and XY gonads from 11.5, 12.5 and 13.5 dpc mouse embryos using gene-specific primers for <i>Slitrk1</i> (<b>A</b>), <i>Fam196b</i> (<b>B</b>), <i>D630039A03Rik</i> (<b>C</b>), <i>Tmem174</i> (<b>D</b>), <i>Lrrc34</i> (<b>E</b>), <i>Lypd6</i> (<b>F</b>), <i>Egfl6</i> (<b>G</b>), <i>Magi2</i> (<b>H</b>), <i>Dmrtc1c1</i> (<b>I</b>), <i>Smc1b</i> (<b>J</b>), <i>Spdya</i> (<b>K</b>), <i>D6Mm5e</i> (<b>L</b>), <i>Ccdc41</i> (<b>M</b>), <i>Foxl2</i> (<b>N</b>) and <i>Amh</i> (<b>O</b>) relative to <i>Sdha</i> (mean +SEM of at least three independent experiments; two-tailed, unpaired t-test; *p≤0.05, **p≤0.01, ***p≤0.001). Individual experiments were performed in triplicate on RNA obtained from pooled gonads from 3–4 littermates. All candidate genes were confirmed to be higher expressed in the ovary compared to testis at least at one of the developmental stages investigated. <i>Foxl2</i> served as control gene for ovary, <i>Amh</i> as control gene for testis samples.</p

Sexually dimorphic expression of long ncRNAs during early mouse gonad development.

<p>(<b>A</b>) Microarray analysis identified a number of lncRNAs (red numbers) and mRNAs (blue numbers) to be differentially expressed during gonad development from 11.5 to 14.5 dpc. (<b>B</b>) Cluster analysis of microarray data identified four different classes (A, B1, B2 and C) of lncRNAs expressed specifically or preferentially in the developing ovary (annotations for lncRNA see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041683#pone.0041683.s008" target="_blank">Table S2</a>).</p

<i>Lrrc34</i> expression during gonad development.

<p>Whole-mount ISH (<b>A</b>) of XX and XY mouse embryonic gonads from 11.5 to 15.5 dpc and ISH (<b>B</b>) with sagittal section of XX and XY mouse embryos from 11.5 to 13.5 dpc, 5 weeks (wk) postnatal mouse ovaries and testes as well as section ISH (purple staining) followed by IHC (brown staining) for the germ cell marker E-cadherin of 13.5 dpc ovaries (<b>B,</b> last panel) demonstrated that <i>Lrrc34</i> is XX germ cell-specifically expressed before 13.5 dpc. <i>Lrrc34</i> expression is down-regulated in an anterior-to-posterior wave from 13.5 dpc onwards (<b>A</b>, <b>B</b>). Scale bars, 100 µm.</p

oncRNA6 expression during gonad development.

<p>Whole-mount ISH (<b>A</b>) of XX and XY mouse embryonic gonads from 11.5 to 15.5 dpc and ISH with sagittal section (<b>B</b>) of XX and XY mouse embryos from 11.5 to 13.5 dpc, as well as section ISH (purple staining) followed by IHC (brown staining) for the germ cell marker E-cadherin of 13.5 dpc ovaries (<b>B,</b> last panel) demonstrated that <i>oncRNA6</i> is XX germ cell-specifically expressed before 13.5 dpc. Scale bars, 100 µm (<b>B</b>, first three panels), 50 µm (<b>B</b>, ISH/IHC low magnification), 20 mm (<b>D</b>, ISH/IHC high magnification).</p