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

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.

The PDF file includes: Supplementary Materials and Methods Figs.S1 to S8 Tables S1 and S2 Legends for data S1 to S5 References Other Supplementary Material for this manuscript includes the following:

Whole-mount in situ hybridization
Whole-mount in situ hybridization (WISH) analysis of embryonic gonads and probes for Sox9 and Wnt4 have been previously described (60).At least three independent biological samples from XX and XY embryos were analyzed for each gene.

Table S1
Supplementary Table 1  Legends for data S1-S4 Data S1: Differentially expressed genes between XY SLCs and supporting cells at early, late and all stages -associated GO biological process terms.Data S2: Differentially expressed genes between XX SLCs and supporting cells at early, late and all stages.-associated GO biological process terms.Data S3: Differentially expressed genes between cells from subclusters 5,1 and 5,0.Data S4: Genes selected by lasso modeling for granulosa and Sertoli cells and associated weights Data S5: Differentially expressed genes between XX and XY SLCs at early, late and all stagesassociated GO biological process terms.

Fig. S1 .
Fig. S1.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 average level of expression (log normalized counts).Clusters and genes are ordered by hierarchical clustering based on Spearman correlation.To facilitate the reading, cluster number are associated with inferred cell type annotation.

Fig. S2 .
Fig. S2.EdU labeling of PAX8 + cells reveals that these cell cycle arrest in early population and a mixed population of cells in the rete testis at E15.5.(A-B) Wholemount immunofluorescence of gonads pulsed with EdU (green).Overlap of PAX8 (magenta) and GATA4 (cyan) was used to identify SLCs and the presumptive rete testis (dotted line).Boxes in merged images indicate regions shown as isolated channels on right.(A) XY gonads pulsed with EdU an hour before collection at E11.5 or E13.5.(B) XY gonads pulsed with EdU either 1 hour or 5 days before collection at E15.5.Scale bar = 50 µm.

Fig. S3 .
Fig. S3.Sox9 and Wnt4 are both express in SLC of the rete testis and rete ovarii.(A) Point plot representation of the expression of Wnt4 and Sox9 at the different developmental time points in both 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) Whole-mount in-situ hybridization analysis of Sox9 and Wnt4 in XX and XY gonads at E12.5 and E13.5.Wnt4 and Sox9 transcripts were both detected in the rete testis and rete ovarii region (arrowheads).

Fig. S4 .
Fig. S4.Expression profiles of selected genes in SLC and supporting cells.Point plot representation of the expression of selected marker genes at the different developmental time points in both SLCs and supporting cells.Genes were classified according to their expression profiles or the relevance for a signaling pathway: (A) SLC, (B) Sertoli cells, (C) granulosa cells, (D) retinoic acid signaling and Nr0b1 and Bmp4 (E) miscellaneous.

Fig. S6 .
Fig. S6.SLCs rarely contribute to the population of Leydig cells, interstitial progenitors and peritubular myoid cells in XY gonads.Co-immunofluorescence of XY gonads of Pax8:Cre;Rosa26:tdTomato;Nr5a1:GFP mice at P0 for RFP and 3βHSD, a marker of the Leydig cells, RFP and ARX or COUP-TFII, two markers of interstitial progenitors, and RFP and αSMA, a marker of the peritubular myoid cells.Boxes indicate regions shown on the right.White arrowheads indicate co-labeled cells.Scale bars 200 µm and 50 µm.

Fig. S7 :
Fig. S7: Wnt4 is required for normal rete testis development.Representative double immunofluorescence against PAX8 and AMH in control (Wnt4KO +/+ ) or Wnt4KO (Wnt4:KO -/-) XY embryos at P0. Boxes indicate regions shown on the right magnifying the rete testis and adjacent testis cords.The rete testis appears to be hypoplastic and connects fewer testis cords than in control testes.Scale bars 200 µm and 100 µm in insets.