Origin, specification and differentiation of a rare supporting-like lineage in the developing mouse gonad

Abstract

Gonadal sex determination represents a unique model for studying cell fate decisions. However, a complete understanding of the different cell lineages forming the developing testis and ovary remains elusive. Here, we investigated the origin, specification, and subsequent sex-specific differentiation of a previously uncharacterized population of supporting-like cells (SLCs) in the developing mouse gonads. The SLC lineage is closely related to the coelomic epithelium and specified as early as E10.5, making it the first somatic lineage to be specified in the bipotential gonad. SLC progenitors are localized within the genital ridge at the interface with the mesonephros and initially coexpress Wnt4 and Sox9. SLCs become sexually dimorphic around E12.5, progressively acquire a more Sertoli- or pregranulosa-like identity and contribute to the formation of the rete testis and rete ovarii. Last, we found that WNT4 is a crucial regulator of the SLC lineage and is required for normal development of the rete testis.

Fig. 1.. A single-cell atlas of gonadal development and sex differentiation.

(A) Schematic overview of the experimental procedure and dataset. A representative embryo at E11.5 is shown with (in inset) the region containing the gonads and mesonephros. (B) Barplots show the number of single cells RNA-sequenced per stage and sex. (C to F) UMAP representation of the 94,705 cells colored by sex (C), stage (D), Leiden clustering (E), and annotation of the different cell clusters (F). (G) Proportion of cell types across sex and developmental stages. (H) UMAP representation of the expression level of Pax8 gene. (I) Heatmap showing correlation between transcriptomes of the different cell populations. Populations were ordered using hierarchical clustering based on correlation (Spearman) distance of expression levels between cell populations. (J) Stacked violin plots showing expression (log-normalized counts) of major somatic cell markers of the gonad in annotated cell populations. Populations were ordered as in (I). CE, coelomic epithelial cells; SE, surface epithelial cells; Pre-Sup., presupporting cells; Sertoli, Sertoli cells; Granulosa, pregranulosa cells; EIP, early interstitial progenitors; LIP, late interstitial progenitors; FLC, fetal Leydig cells; IM, invading mesonephric cells; PV, perivascular cells; Imm., immune cells; Meso., mesonephric mesenchymal cells; MT, mesonephric tubules; End., endothelial cells; AS, adrenosympathic cells; GC, germ cells; EC, erythrocytes.

Fig. 2.. Characterization of XY and XX SLC populations.

The analysis was performed with both XY (A to K) and XX (L to V) gonadal cells. PAGA representation of the clusters of XY (A) and XX (L) cells expressing Nr5a1, Gata4, and Wt1. Each node is a Leiden cluster, and the links between clusters represent confidence of relation between two clusters. (B, C, M, and N) UMAP projection of gonadal cell lineage at the origin of the supporting cells and SLCs, colored by cell annotation (B and M) and by developmental stage (C and N). Simplified model of the cell lineage reconstruction in the developing testis (D) and ovary (O). UMAP representations of XY (E to I) and XX (P to T) cells colored by expression levels (log-normalized counts) of selected genes. UMAP and barplot representation of the cell cycle phase of XY (J) and XX (U) cells. Barplot illustrates the proportion of cells in the different cell cycle phase for each population. (K and V) Barplots representing the number of DEGs between SLCs and the supporting lineage either in XY (K) or XX (V) developing gonads at early (E11.5) and late (E12.5, E13.5, and E16.5) stages. e-SLC, early SLCs; l-SLC, late SLCs.

Fig. 3.. Sex-specific differentiation of XX and XY SLCs.

(A) UMAP projection of SLC cell lineage colored by developmental stage, genetic sex, and annotation. Leiden clustering was applied to all cells, and a subclustering was applied to cluster #5, dividing it into two (#5.0 and #5.1). The fourth panel shows the PAGA graph representation where each node is a Leiden cluster, and the links between clusters represent confidence of relation between two clusters. (B) UMAP representation colored by expression levels (log-normalized counts) of CE-derived markers Nr5a1 and Gata4, and SLC markers Pax8 and Enpp2. (C) Heatmap showing correlation between transcriptomes of the different clusters. Populations were ordered using hierarchical clustering based on correlation (Spearman) distance of expression levels between cell populations. Dotplot representation of the expression of marker genes in each Leiden cluster. The size of the node represents the percentage of cells in the cluster expressing the gene, and the color indicates the level of expression (log-normalized counts). (D) UMAP representation colored by expression levels (log-normalized counts) of Sertoli cell markers Ptgds and Dhh, and granulosa cell markers Fst and Foxl2. (E) Scatterplots representing pregranulosa and Sertoli scores of individual cells using a LASSO model. A plot was generated for each cell type, and individual cells were colored by developmental stage. (F) Point plots showing pregranulosa and Sertoli cell scores in XX and XY SLC and supporting cells across time. (G) Schematic model of SLC and supporting lineage specification.

Fig. 4.. A population of gonadal cells expressing both GATA4 and PAX8 is present from E10.5 to E11.0 along the genital ridge next to the mesonephros.

Whole-mount IF of XY (A, C, and E) and XX (B, D, and F) genital ridges at E10.5 (A), E11.0 (B), E11.5 (C and D), and E12.5 (E and F). Boxes in merged images indicate regions shown as isolated channels on right. The dotted line corresponds to the border that separates the GATA4⁺ cells of the genital ridge (G) from the mesonephric cells (M). LAMB1 is expressed in the basement membrane throughout the CE and mesonephric tubules. SOX9 overlaps with a subset of PAX8⁺/GATA4⁺ cells in XY and XX tissues at E11.5. NR2F2 is a marker of gonad interstitial cells and mesonephric mesenchyme. Arrowhead indicates SOX9⁺/PAX8⁺ cell within the XY gonad at E11.0. Arrow indicates PAX8⁺/GATA4⁻ cells adjacent in the region of the presumptive rete that are not within mesonephric tubules. Scale bars, 50 μm.

Fig. 5.. Pax8+ progenitors contribute to both rete testis/ovarii and the pool of Sertoli and pregranulosa cells.

(A) 3D reconstructions of Pax8:Cre;Rosa26:tdTomato;Nr5a1:GFP testis and ovary at E13.5, E16.5, and P0. Pax8⁺ cells are lineage-traced with RFP. Green fluorescent protein (GFP), expressed under the control of the Nr5a1 promoter, is used to delineate gonadal cells. Note the presence of RFP⁺ cells close to the rete testis/ovarii but also throughout the gonads. White arrowhead indicates the Wolffian duct, and black arrowhead the Müllerian duct. (B) Double IF for RFP/AMH and RFP/FOXL2 respectively in XY and XX Pax8:Cre;Rosa26:tdTomato;Nr5a1:GFP mice at P0. Note the presence of RFP⁺ cells increases near the rete testis and rete ovarii. Cells in the rete testis are exclusively RFP⁺ and do not express AMH (inset 1). At the junction between the rete testis and the testis cords, low AMH expression is observed in some RFP⁺ cells (inset 2). RFP⁺/AMH⁺ cells are also present in testis cords (inset 3). Similarly, RFP⁺/FOXL2⁺ cells are present in pregranulosa cells of the developing ovary (inset 4), including in some primordial follicles (inset 5). Magenta arrows indicate RFP⁺ cells, and white arrows indicate RFP⁺/AMH⁺ or RFP⁺/FOXL2⁺ cells. MT, mesonephric tubules; RT, rete testis. Scale bars, 200 μm in (A) and 100 μm in (B).

Fig. 6.. Wnt4 is required for rete testis formation.

(A) Expression profiles of genes involved in the WNT/β-catenin pathway in SLCs and supporting cells. Data were extracted from scRNA-seq analysis of XX and XY embryos at E11.5, E12.5, E13.5, and E16.5. (B) Representative double IF against PAX8 and AMH in control (Sf1:Cre;Sox9^F/+;Wnt4KO^+/+) or Wnt4^KO (Sf1:Cre;Sox9^F/+;Wnt4:KO^−/−) XY embryos at E12.5 and E14.5. Boxes indicate regions shown on the right magnifying the rete testis and adjacent testis cords. Note that the numbers of PAX8⁺ cells, the rete testis, and the testis cords near the rete testis are reduced and disorganized in Wnt4 mutant embryos both at E12.5 and E14.5. White and yellow arrowheads indicate PAX8⁺ and PAX8⁺/AMH⁺ cells, respectively. DAPI was used as a nuclear counterstain. Scale bars, 100 μm. (C) Quantification of PAX8⁺ and PAX8⁺/AMH⁺ cells per 10,000 μm² of testis section of control and Wnt4^KO embryos at E14.5. Data are presented as a box-and-whisker plot to illustrate the heterogeneity of PAX8-positive cells according to the different sections. Each point represents the quantification of one testicular section. Three embryos per genotype were used. Student’s t test, two-sided unpaired (*P < 0.05).