Gonadal sex reversal at single-cell resolution in Znrf3-deficient mice

Abstract

The role of anti-WNT ZNRF3 is central to determining gonadal fate: XY mice lacking functional ZNRF3 exhibit a highly variable gonadal sex reversal phenotype in the fetal period, characterised by appearance of ovarian tissue. To investigate this sex reversal further, we used single-cell RNA-seq to examine the transcriptomes of XY Znrf3-deficient gonads during the mouse sex-determining period. Analyses of cell trajectories in mutant gonads reveal the failure of pre-supporting cells to commit to the Sertoli cell fate, XY granulosa cell development, unstable commitment in those cells that reach the Sertoli path and enhanced contribution to a supporting-like cell fate. By developing a machine learning-based score for transcriptomic similarity to Sertoli and granulosa, we show pervasive disruption to acquisition of testicular cell fate in the mutant supporting cell lineage, with large numbers of cells co-expressing pro-Sertoli and pro-granulosa markers. These data reveal that loss of Znrf3 results in transcriptomic and cellular heterogeneity, with shifts in cellular sex identity that undermine a simple binary model in which mutant supporting cell precursors achieve either Sertoli or granulosa cell differentiation.

Keywords: Genetics of sex; Mouse; Ovotestis; Sex determination; Sex reversal; Testis; Znrf3.

Fig. 1.

scRNA-seq analyses of gonadal sex determination in C57BL/6J (B6J) wild-type and Z-del mouse embryos. (A) UMAP showing ∼68,000 gonadal cells from three genotypes (XY WT, XX WT and XY Z-del) at three stages (11.5, 12.5 and 14.5 dpc). All major gonadal lineages are present. (B) UMAP of all cells coloured by stage (blue, 11.5 dpc; coral, 12.5 dpc; green,14.5 dpc). (C) Dot plots detailing expression specific to each cell type. Main supporting lineages express Kctd14; gonad progenitors express Upk3b; Leydig cells express Star; Sertoli cells express Sox9 and Amh; SLCs express Pax8, although this is also found in other cell-types. CycPS, cycling pre-supporting cells; G. Prog, gonad progenitor; PS, pre-supporting; PST, pre-steroidogenic; SLC, supporting-like cells; Meso-like, Mesonephros-like.

Fig. 2.

Transcriptomic comparison of sex determination in the CD1 and B6J mouse strains. (A) Combined CD1/B6J UMAP showing constituent cell types (left), strain of origin (middle) and stage of origin (right). The dotted lines group multiple sub-cell-types into one larger cell type. (B) Proportions of 13 different cell-types in B6J and CD1 datasets at 11.5 (upper) and 12.5 dpc (lower). (C) Violin plots showing expression of Wnt4 and canonical WNT marker Lef1 in the supporting cell lineage of the B6J and CD1 datasets at 11.5 dpc. (D) Plots of GCI (y-axis) and SCI (x-axis) scores found in cells from the B6J and CD1 supporting cell lineage datasets. Each dot represents an individual cell and its position on the SCI/GCI plot reflects its cellular identity regarding attainment of supporting cell (Sertoli or granulosa cell) fate. CycPS, cycling pre-supporting cells; GCI, granulosa cell identity; G.Prog, gonad progenitor; PS, pre-supporting cells; PST, pre-steroidogenic; SCI, Sertoli cell identity; SLC, supporting-like cells.

Fig. 3.

The impact of loss of Znrf3 function (Z-del) on gonadal cell transcriptomes in B6J. (A) The supporting cell precursor and supporting cell regions of the all-cell UMAP, indicating the location of XX WT, XY WT and XY Z-del cells. (B) Proportions of supporting cell-types in the same three genotypes as A, at 11.5 dpc (left) 12.5 dpc (middle) and 14.5 dpc (right); cell-types are indicated by colour. (C) Pie-charts showing cell-cycle stage of Sertoli cells in XY WT and XY Z-del cells. (D) Principal component analysis of all samples analysed. (E) UMAPs derived from individual gonad samples at 14.5 dpc: XX WT (GX47), XY WT (GX37) and two XY Z-del samples; GX28 (an ovotestis) and GX04 (an ovoTESTIS). Cell-types are indicated by colour (key shown in panel B, right). CycPS, cycling pre-supporting; PS1, pre-supporting 1; PS2, pre-supporting 2; SLC, supporting-like cells.

Fig. 4.

Predicted alteration to cellular differentiation trajectories in B6J XY Z-del gonads. (A-C) RNA velocity trajectories (arrowheads on black lines) are shown on UMAPs from XY WT, which combines samples from all stages (A), XY Z-del samples from all stages (B) and individual XY Z-del samples at 11.5 and 12.5 dpc (C). The red arrows further indicate the general direction of the individual trajectories. For XY Z-del, in the absence of a Sertoli cluster, the predicted trajectory is towards pre-supporting and SLC cell-types. PS1, pre-supporting 1; PS2, pre-supporting 2; SLC, supporting-like cells.

Fig. 5.

Pervasive disruption to attainment of cellular sex identity in B6J XY Z-del gonads. (A) Density scatter-plots of GCI and SCI scores for all supporting cells in XX WT, XY Z-del and XY WT gonads. Density scatter plots chart the variation in SCI (x-axis) and GCI (y-axis) scores in these three genotypes across three stages – 11.5 dpc (top), 12.5 dpc (middle) and 14.5 dpc (lower) – in a 3×3 grid. Each dot represents an individual cell and its identity regarding attainment of supporting cell (Sertoli and granulosa cell) fate. For XY Z-del at 14.5 dpc, ‘A’ refers to a low SCI score group, corresponding to supporting cells that are not pre-Sertoli/Sertoli; ‘B’ refers to a high SCI score group comprising pre-Sertoli/Sertoli cells. (B) GCI/SCI plot for all supporting cells, showing cells with similar GCI and SCI scores (‘double-positive’, DPs) indicated in red. At 12.5 dpc and 14.5 dpc, the proportion of DPs in Z-dels has increased compared with XY WT. (C) GCI/SCI plot for supporting-like cells (SLC), with Foxl2 expression levels indicated by colour. The average SCI and GCI score for SLCs is indicated by a red and blue trend line, respectively. SLCs have increased Foxl2 expression and higher than average GCI in Z-dels when compared with XY WTs. (D) GCI/SCI plot for SLCs, with Pax8 expression levels indicated by colour. Trend lines shown are as in C. SLCs have decreased Pax8 expression in cells with high GCI. (E) GCI/SCI density scatter plot for Sertoli cells, with Amh expression levels indicated by colour. In XY Z-del gonads, Sertoli cells have a reduced SCI score, with significantly lower Amh expression when compared with XY WT, at 12.5 dpc. (F) GCI/SCI plot for Sertoli cells, with Foxl2 expression levels indicated by colour. In XY Z-del gonads, Sertoli cells have a reduced SCI score, with significantly higher Foxl2 expression compared with XY WT, at 12.5 dpc. GCI, granulosa cell identity; SCI, Sertoli cell identity.