Loss of NR5A1 in mouse Sertoli cells after sex determination changes cellular identity and induces cell death by anoikis

ABSTRACT To investigate the role of the nuclear receptor NR5A1 in the testis after sex determination, we analyzed mice lacking NR5A1 in Sertoli cells (SCs) from embryonic day (E) 13.5 onwards. Ablation of Nr5a1 impaired the expression of genes characteristic of SC identity (e.g. Sox9 and Amh), caused SC death from E14.5 onwards through a Trp53-independent mechanism related to anoikis, and induced disorganization of the testis cords. Together, these effects caused germ cells to enter meiosis and die. Single-cell RNA-sequencing experiments revealed that NR5A1-deficient SCs changed their molecular identity: some acquired a ‘pre-granulosa-like’ cell identity, whereas other reverted to a ‘supporting progenitor-like’ cell identity, most of them being ‘intersex’ because they expressed both testicular and ovarian genes. Fetal Leydig cells (LCs) did not display significant changes, indicating that SCs are not required beyond E14.5 for their emergence or maintenance. In contrast, adult LCs were absent from postnatal testes. In addition, adult mutant males displayed persistence of Müllerian duct derivatives, decreased anogenital distance and reduced penis length, which could be explained by the loss of AMH and testosterone synthesis due to SC failure.

The sizes of the expected fragments are indicated on the right: tg, Cre transgene; L2 and +, loxP-flanked and wild-type alleles, L-, excised, null allele.Note that the mutant testis contained only traces of the Nr5a1 L-allele (asterisk), indicating that SC bearing the excised allele were no longer present at birth.(B) PCR analysis of genomic DNA extracted from FACS-purified YFP-positive (YFP+) and negative (YFP-) cells contained in Nr5a1 SC-/-;Trp53 SC-/-mutants fetuses at E14.5.Upper panel shows genotyping of Trp53 alleles; lower panel shows genotyping of Nr5a1 alleles.The YFP-negative cells (i.e., all cells except SC) contained only the unexcised (L2) alleles, while the YFP-positive cells (i.e., SC) contained only the cre-recombined (L-), null alleles.Note however that traces of the Nr5a1 L2 alleles were detected in YFP-positive cells.

Fig. S8. Selected ligand-receptor interactions between SC and GC.
Diagram of the ligand-receptor interactions between SC and GC, as revealed from the single cell transcriptomes.Each node of the network corresponds to a given gene encoding a ligand (oval shape) or a receptor (rectangle shape).Intercellular communication network among SC and GC were predicted using the CellTalkDB database, which contains literature-supported ligand-receptor pairs.Downregulated and upregulated genes are blue and yellow coloured, respectively.Grey boxes indicated receptors whose expression in not changed.The arrows point from the ligand to the receptors.

Mice
To construct the targeting vector a 1.9 kb-long DNA fragment encompassing exon 7 (ENSMUSE00000 693512) was amplified by PCR using 129/SvPass genomic DNA and cloned into an iCS proprietary vector containing a loxP site, as well as a loxP-and FRTflanked neomycin resistance cassette (step1 plasmid).Then, 3 kb-and 3.7 kb-long fragments corresponding to 5' and 3' homology arms were amplified by PCR and introduced into step1 plasmid to generate the targeting construct.This linearized construct was electroporated into 129/SvPass mouse embryonic stem (ES) cells.After selection, targeted clones were identified by PCR using external primers and confirmed by Southern blots (5' and 3' digests) hybridized with neomycin, 5' and 3' external probes.One positive ES clone was injected into C57BL/6J blastocysts.To remove the selection cassette from the Nr5a1 locus, chimeric males were crossed with Gt(ROSA)26Sor tm1(FLP1)Dym females (Farley et al., 2000).Germline transmission was obtained, and a further breeding step was needed to segregate animals bearing the Nr5a1 L2 allele (also called Nr5a1 tm1.1Ics ) from animals bearing the transgene.To inactivate Nr5a1 in SC, female mice bearing Plekha5 Tg(AMH-cre)1Flor (Lécureuil et al., 2002) and Gt(ROSA)26Sor tm1(EYFP)Cos (Srinivas et al., 2001) transgenes, and heterozygous for the L2 allele of Nr5a1 were mated with males heterozygous or homozygous for L2 alleles of Nr5a1.The resulting Plekha5 Tg(AMH-cre)1Flor ;Nr5a1 +/+ ; Gt(ROSA)26Sor tm1(EYFP)Cos and Plekha5 Tg(AMH-cre)1Flor ;Nr5a1 L2/L2 ; Gt(ROSA)26Sor tm1(EYFP)Cos males are referred to as control and Nr5a1 SC-/-mutant fetuses, respectively.To test for the role of TRP53, the L2 allele of Trp53 gene (Jonkers et al., 2001) was further introduced in the mice described above.

Fig. S4 .
Fig. S4.Identification of cell clusters generated from the single-cell transcriptomes.(A-B) UMAP projection of the 8,998 cells coloured by cell clusters (panel A) or by phases of the cellcycle, as indicated (panel B).Associated cell annotation is indicated close to the corresponding cell clusters (named C1-23).(C) Dot plot with the expression of selected markers (x axis) for each cell cluster (y axis).The dot size represents the percentage of cells expressing a given gene within a given cell cluster.The colour intensity (from light to dark red) indicates the average expression (log normalized counts) of a given gene within a given cell cluster.Legend: coel.ep., coelomic epithelium; ctrl, control; mut, mutant; PTM, peritubular myoid.

Fig. S5 .
Fig. S5.Number of differentially expressed genes and associated GO terms.Bar plot representing the number of significantly downregulated (left side, in blue) and upregulated (right side, in yellow) genes between control and Nr5a1 SC-/-cells (x axis), in each cell-type (y axis).Enriched GO terms and their associated p values are given for each cell-type.Legend: coel.ep., coelomic epithelium; PTM, peritubular myoid.

Fig. S7 .
Fig. S7.Cluster C20 corresponds to rete testis cells, in which gene excision by cre recombinase is not operational.(A) Diagram illustrating the location and organization of rete testis in the mouse.Seminiferous cords are in green, while rete testis is in brown.(B) Magnification of UMAP plot (rotated by 90° with respect to Fig. 6) for clusters C19, C20 and C21.Cells belonging to C19 (from control testes, YFP-negative cells) and to C21 (from Nr5a1 SC-/-testes, YFP-positive) are in grey.Cells belonging to C20 and expressing distinct combination between Nr5a1, Yfp, Pax8 and Aldh1a3 are depicted by a colour code: pink represents cells expressing Nr5a1 but not Yfp, Pax8 or Aldh1a3 (SC); blue stands for cells expressing Nr5a1, Pax8 and/or Aldh1a3, but not Yfp (rete testis cells); green represents cells expressing Yfp but not Nr5a1, Pax8 or Aldh1a3 (mutant SC).Circles and triangles represent control and Nr5a1 SC-/-cells, respectively.Note that the YFP-reporter transgene was absent in the control gonads but present in the Nr5a1-deficient gonads used for the scRNA-seq experiments.(C-J) Detection of PAX8 and NR5A1 (magenta signals) together with YFP,

Fig. S9 .
Fig. S9.Identification of cell clusters generated from the atlas.(A-B) UMAP projection of the 94,705 cells from the single-cell transcriptomic atlas of gonad development recently published (Mayère et al., 2022) coloured by cell clusters (in panel A) or

Fig. S10 .
Fig. S10.Identification of cell clusters generated from the single-cell transcriptomes of gonad somatic cells only.(A-B) UMAP projection of the 8,998 cells coloured by cell clusters (panel A) or by genotype, as indicated (panel B).Associated cell annotation is indicated close to the corresponding cell clusters (named C1-C19).(C) Proportions of control and mutant cells in each cluster is indicated as coloured bars.Legend: Legend: coel.ep., coelomic epithelium; ctrl, control; mut, mutant; PTM, peritubular myoid cells.(D) Dot plot with the expression of selected markers (x axis) for each cell cluster (y axis).The dot size represents the percentage of cells expressing a given gene within a given cell cluster.The colour intensity (from light to dark red) indicates the average expression (log normalized counts) of a given gene within a given cell cluster.

Fig. S11 .
Fig. S11.Identification of somatic male cell clusters generated from the atlas.(A-B) UMAP projection of the male somatic cells from the single-cell transcriptomic atlas of gonad development (Mayère et al., 2022) coloured by cell clusters (in panel A) or by developmental stage, from E10.5 to E16.5 as indicated (in panel B).Cell clusters (C1-C28) and their associated cell annotation are indicated.(C) Stacked bar plot showing the proportion of the developmental stages (from light at E10.5 to dark blue at E16.5) amongst the different male cell clusters.(D) Dot plot with the expression of selected markers (x axis) for each cell cluster (y axis).The dot size represents the percentage of cells expressing a given marker within a given cell cluster.The colour intensity (from light to dark red) indicates the average expression (log normalized counts) of a given marker within a given cell cluster.Legend: AGP, adrenal-gonadal primordium; coel.ep., coelomic epithelium; E, embryonic day; prog., progenitor; PTM, peritubular myoid cells; supp.prog., supporting progenitor cells.

Fig. S12 .
Fig. S12.NR5A1-deficient SC express Nr2f2 and display a transcriptomic signature corresponding to an earlier stage of development for SC.(A) Expression of Nr2f2 in control (blue) and Nr5a1 SC-/-(pink) somatic cells projected on the reference single-cell transcriptomic atlas of male somatic cell development (Mayère et al., 2022).The color intensity (from white to dark blue or dark pink) indicates the level of expression.(B) Boxplot showing the pseudo time prediction for each of the major somatic cell-types of the control (blue boxes) and Nr5a1 SC-/-(pink boxes) testes.The NR5A1-deficient SC population appears younger than the control SC population.Legend: coel.ep., coelomic epithelium; PTM, peritubular myoid cells; supp.prog., supporting progenitor cells.

Fig. S13 .
Fig.S13.ATP synthesis is not dramatically altered in NR5A1-deficient SC. (A) Tukey box plots illustrating medians, ranges and variabilities of log normalized expression of the indicated genes belonging to NADH dehydrogenase (complex I of respiratory chain), succinate dehydrogenase (complex II), cytochrome c reductase (complex III), cytochrome c oxidase (complex IV) and ATPase (complex V) in SC of control (blue boxes) and Nr5a1 SC-/- (pink boxes) testes.A scheme of the respiratory chain is also depicted.(B) ATP quantities measured in of FACS-purified control (blue dots, n =6 batches) and NR5A1-deficient (pink dots, n =6 batches) SC.RLU, relative luminescence units.The difference is not statistically significant.(C) Intermediate metabolites and genes involved in the glycolytic pathway.Genes that were up-regulated and down-regulated in SC from Nr5a1 SC-/-testes are indicated by red and green boxes, respectively.(D) Tukey box plots illustrating medians,

Fig. S14 .
Fig. S14.Ablation of Nr5a1 in SC impairs ACTA2 expression in peritubular myoid cells.(A-F) Detection of ACTA2 (magenta signal) and AMH (green signal) on histological sections of control (A) and Nr5a1 SC-/-(B) testes at E15.5.Arrows point to ACTA2-positive peritubular myoid (PTM) cells, whereas arrowheads point to ACTA2-negative PTM cells.Note that ACTA2 expression is lost in PTM cells adjacent to SC where AMH expression is reduced or lost in Nr5a1 SC-/-testes.Conversely, ACTA2 expression is retained in PTM cells adjacent to AMH-positive SC, which were preferentially located on the mesonephric side of the in Nr5a1 SC-/-testes.Scale bar (in B): 10 µm.

Table S5 .
Comparison of the outcome of Nr5a1 or SC ablation during fetal development according to four distinct studies.

Table S6 .
Primary antibodies used for immunohistochemistry experiments.

Table S7 .
Secondary antibodies used for immunohistochemistry experiments.