Dimorphic expression of multiple male- and female-enriched genes in B6 is delayed compared to 129S1 mice.

<p>(A) Expression of male- (top panel) and female-enriched (bottom panel) genes. Dimorphic expression for these genes is delayed by ∼5 hours in B6 compared to 129S1. (B) Heatmap showing dimorphic expression at E11.6 in 129S1 and comparison of same genes in B6. While a few genes show earlier dimorphic expression in B6 mice compared to 129S1, the dominant pattern shows a ∼5 hr delay between B6 and 129S1 mice. (C) Matrix showing the time of onset of dimorphism in 129S1 and B6 mice for male-enriched (top panel) and female-enriched (bottom panel) genes. For example, out of the 32 male-enriched probes that became dimorphically expressed at E11.4 in 129S1, 9 probes were also dimorphically expressed starting at E11.4 in B6 while 18 showed dimorphic expression starting at E11.6 in B6 mice. However, 4 genes are dimorphic in B6 XY gonads at E11.4, but not in 129S1 until E11.6. * indicates significant overlap with p<0.001 evaluated by a hypergeometric test. The highlighted diagonals show the number of genes showing similar onset of dimorphism in 129S1 and B6 mice. Note that some genes that are male- or female-enriched in one strain do not show dimorphism in the other strain.</p

Cascades of dimorphic expression involving both activation and repression in XY gonads.

<p>(A) Examples of genes showing higher expression in XY (male-enriched genes, top panel) and XX gonads (female-enriched genes, bottom panel) from 129S1 mice. Blue and red vertical lines show the time point when dimorphic expression is significant. (B) Cascades of dimorphic gene expression identified by the HMM in XY (top panel) and XX gonads (bottom panel). Colors indicate the log fold change between XY and XX gonads at a specific time point for the 129S1 strain. The genes are arranged in order of time of onset of dimorphic expression. (C) Contribution to changes in expression between E12.0 and the time point before the onset of dimorphism are shown for each gene in (B) in XY (column 1) and XX (column 2) gonads. Top panel: male-enriched genes. Bottom panel: female-enriched genes. This analysis shows that male-enriched genes are mostly up-regulated in XY gonads while female-enriched genes are mostly down-regulated in XY gonads.</p

Changes in XX and XY gonads contribute to expression fold change between E11.0 and E12.0.

<p>Gene expression in XY and XX gonads was compared at the beginning and end of the 24-hour developmental window. For probes that exhibited a 1.5-fold or greater change in expression in either sex between E11.0 and E12.0, log of the Fold Change in the XY gonad is plotted on the Y-axis, and log of the Fold Change in the XX gonad is plotted on the X-axis. Probes that are similarly up-regulated or down-regulated in both sexes appear in gray in the upper right and lower left quadrants, respectively. Probes that become enriched in XY gonads relative to XX are shown in blue, while genes that become enriched in XX gonads relative to XX are shown in red. Examples from each category are highlighted, and their expression patterns in XY (blue line) and XX (red line) gonads are displayed. From this perspective, it is clear that enrichment in one sex is achieved by activation, repression, or both regulatory mechanisms.</p

Validation of <i>Lmo4</i> as a novel regulator of gene expression in the fetal gonad.

<p>(A) <i>Lmo4</i> exhibits an expression pattern indicative of a role in sex determination and consistent with expectations for a gene underlying a strain eQTL regulating early sex determination and male pathway genes. It is expressed at similar levels in XY and XX gonads before E11.6, becomes enriched in XY gonads as early as E11.6 in both strains, and shows reduced expression levels in B6 (dashed lines), consistent with the observed allelic effects of the Chromosome 3 eQTL. (B) E12.5 XY gonads were dissected free of the mesonephroi, pooled by sex, dissociated into single cell suspensions, plated on tissue culture plates at t = 0 with lentiviral particles containing shRNA targeted to the candidate gene of interest, and cultured for 72 hours. Quantitative RT-PCR was conducted to assay expression of predicted targets. (C) Lentiviral shRNA-mediated knockdown of Lmo4 in cultured XY primary gonadal cells resulted in the consistent down-regulation of multiple Chromosome 3 eQTL target genes relative to the nontargeting control (gray bar) using two different shRNA hairpins targeting Lmo4 (light/medium blue bars in graph). Expression was normalized to the housekeeping gene <i>Gapdh</i>. Both male pathway genes, <i>Fgf9</i> and <i>Col9a3</i>, were significantly down-regulated following Lmo4 knockdown with both clones. Similarly, one of the putative targets with a role in early gonadogenesis, <i>SF1/Nr5a1</i>, was significantly reduced, however expression of the other gene involved in early gonadogenesis, <i>Wt1</i>, was not significantly affected by <i>Lmo4</i> knockdown. The important male pathway regulator <i>Sox9</i> was found to be significantly down-regulated as a result of <i>Lmo4</i> knockdown. <i>Canx</i> (a second normalization gene not predicted to be a target of LMO4) showed no difference in expression compared to the control. Error bars show minimum and maximum expression. Significance was calculated by comparing control and data across all independent runs.</p

Identification of candidate genes in prominent trans-band eQTLs based on dynamic expression patterns.

<p>Strain differences in temporal gene expression are informative for predicting regulatory genes underlying trans-band eQTLs. Multiple criteria were established to identify the best candidate genes in the trans-band eQTLs mapped in Munger et al. <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003630#pgen.1003630-Munger1" target="_blank">[1]</a>. First, the putative regulatory gene must be expressed above background at or before E11.5 to exert any effects on downstream target genes. Genes implicated in the list of target genes that exhibited a sexually-dimorphic expression pattern consistent with the sexual differentiation pathway (male or female) were prioritized. Genes that met both of the above criteria, and exhibited strain expression differences (either in overall levels of transcript abundance or in timing of onset of sexual dimorphism) in a pattern consistent with the observed allelic effects for that eQTL, were prioritized as the highest candidates. A few eQTLs harbor genes in which abnormal sex determination phenotypes have been noted in the null mutant mouse, and these genes were given similar high priority. Using these criteria, confidence intervals containing 60–526 protein coding genes were narrowed down to eight or fewer high priority candidates.</p

A Hidden Markov Model (HMM) to identify patterns of dimorphic expression in the gonad transcriptome.

<p>Fold differences between XY and XX gonads at each time point in both strains were calculated for all probes passing the ANOVA filtering step. This data was then used to initialize and train the Hidden Markov Model (HMM) (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003630#s4" target="_blank">Materials and Methods</a>). The most probable (Viterbi) state path reflects possible dimorphic expression patterns between XX and XY gonads and was used to cluster genes. Heatmaps illustrate 3 clusters with state paths indicated by circles at the top of each heatmap.</p

Models of differentiation for the different gonadal lineages.

<p>The interstitial/stromal cells differentiate asymmetrically over the time period examined, as we detected few genes specific to the XX stroma by E13.5, whereas, the XY interstitial population acquired a larger set of lineage-specific genes. Supporting cells are primed with a female bias. The natural progression of the primed state may be to adopt the female differentiated state, but in the presence of <i>Sry</i> the cells repress the female program and adopt the male fate. Conversely, germ cells are primed with a male bias. An extrinsic signal may be required from the mesonephros to induce the adoption of the female fate; otherwise, germ cells adopt the male fate.</p

Data from sorted <i>Sf1-EGFP</i> cells also supported female-biased priming for supporting cells.

<p>(A–B) Graphical illustrations of the genes included in our analysis of priming in the <i>Sf1-EGFP</i> data. Because the <i>Sf1</i>-positive population is a mixture of lineages, we used two methods to identify the primed genes associated with supporting cells. XY cells are illustrated in this example, but the same operations were also performed for XX cells. (A) “<i>Sf1</i> primed and supporting cell enriched” genes were both male-primed in the <i>Sf1-EGFP</i> data (comparing E11.0 and E12.5) and lineage-specifically enriched in our XY <i>Sry-EGFP/Sox9-ECFP</i> purified supporting cells at E12.5. Red indicates genes being removed from the analysis, and green indicates genes being retained. (B) For the “<i>Sf1</i> primed, removing interstitial/stromal genes”, we removed genes associated with the interstitial/stromal cells at E12.5 (i.e., sexually dimorphic in the interstitium/stroma) from the <i>Sf1-EGFP</i> primed genes. Genes that were expressed sexually dimorphically in both the interstitial/stromal cells and the supporting cells were removed only if expression was higher in the interstitial/stromal cells than in the <i>Sry-EGFP/Sox9-ECFP</i> supporting cells. The <i>Sf1-EGFP</i> primed genes that were enriched in the <i>Sry-EGFP/Sox9-ECFP</i> supporting cells (C, D, and G) and those that were identified by removing interstitial/stromal genes (E, F, and H) were analyzed separately. (C and E) The percentages of primed genes that were male-primed and female-primed. Both methods showed a female bias. The boxes contain the p-values from the binomial test with the expected percentages of the extreme models, and all extreme models could be rejected as having a p-value<0.05. (D and F) The percentage of male or female genes that were primed showed a significant (*) bias toward the female pathway, as determined by the hypergeometric test (p-value<0.05). (G and H) However, primed genes in both sexes were predominantly expressed at similar levels in progenitors and E12.5 supporting cells of one sex. While supporting cell progenitors have a female bias, they also express some markers of the male pathway at levels similar to male supporting cells at E12.5. Gene lists and permutation tests are provided in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002575#pgen.1002575.s005" target="_blank">Dataset S5</a>.</p

Supporting cells showed lineage priming with a female bias.

<p>(A and H) Graphs of the log-transformed, normalized intensity values of genes. The error bars are standard error. Only the values for supporting cells are shown, except in the depleted and primed example where all cell types are shown. (A) <i>Mdk</i> and <i>Rasgrp1</i> are examples of male- and female-primed genes and <i>Cenpa</i> is an example of a female-primed depleted gene. As in the germ cell analysis, we examined all primed genes (B, C, and I), primed and lineage-specifically enriched genes (D, E, and J), and primed and lineage-specifically depleted genes (F, G, and K). (B, D, and F) The percentages of primed genes that were male-primed and female-primed. The boxes contain the p-values from the binomial test with the expected percentages of the extreme models. (B) Using the first method, all of the extreme models could be excluded because they had a p-value<0.05. (D and F) However, using the second and third methods, the balanced and female models could not be excluded, respectively. (C, E, and G) Nevertheless, examining the percentage of male or female genes that were primed, all methods showed a significant (*) bias toward the female pathway, as determined by the hypergeometric test (p-value<0.05). Taken together, the data supported female-biased priming. (H) Graphs illustrating two primed genes, whose expression in the progenitor is “similar” to the differentiated cell of one sex, or “intermediate” between the two sexes. (I–K) The female-primed genes were predominantly similarly expressed, but the male-primed genes showed more variability. Gene lists and permutation tests are provided in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002575#pgen.1002575.s004" target="_blank">Dataset S4</a>.</p

Sorted cell lineages and microarray validation.

<p>(A) Illustration of the developing XX and XY gonad with supporting cells (blue), interstitial/stromal cells (purple), germ cells (green), and endothelial cells (red). (B, C, and F) Graphs of the log-transformed, normalized intensity values from the microarrays for control genes known to be specific to each lineage. The color for each lineage is conserved in all figures and matches the illustration (A), with XX (♀) values shown as dashed lines, and XY (♂) values shown as solid lines. The error bars are standard error of the mean (“standard error”) of the log transformed values. The Y-axis scale differs for each graph because each transcript cluster has its own intensity range. (B) The control genes were found in the expected lineage, except for (C) genes characteristic of Leydig cells. Leydig cell genes were highly expressed in both the interstitium (as expected) and the endothelial cell fraction. (D) Immunofluorescence of E13.5 XY gonads with <i>Flk1-mCherry</i> (red), PECAM1 (germ and endothelial cells, blue), and 3β-HSD (Leydig cells, green). Arrowheads indicate <i>Flk1-mCherry</i> and PECAM1 double positive endothelial cells. Arrows indicate <i>Flk1-mCherry</i> positive, PECAM1 negative cells that were positive for 3β-HSD, confirming aberrant reporter expression in some Leydig cells. Asterisks indicate germ cells positive for PECAM1 alone. Scale bar = 25 µm. (E) The XY interstitial cells have very low expression of the endogenous <i>Flk1</i> (<i>Kdr</i>) transcript at E13.5, supporting our conclusion that the <i>Flk1-mCherry</i> transgene is aberrantly expressed in Leydig cells.</p