# The –KTS splice variant of WT1 is essential for ovarian determination in mice

Elodie Gregoire, Marie-Cécile de Cian, Roberta Migale, Aitana Perea-Gomez, Sébastien Schaub, Natividad Bellido-Carreras, Isabelle Stévant, Chloé Mayère, Yasmine Neirijnck, Agnès Loubat, et al.

► To cite this version:

Elodie Gregoire, Marie-Cécile de Cian, Roberta Migale, Aitana Perea-Gomez, Sébastien Schaub, et al. The –KTS splice variant of WT1 is essential for ovarian determination in mice. Science, 2023, 382 (6670), pp.600-606. 10.1126/science.add8831. hal-04285584

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# The -KTS splice variant of WT1 is essential for ovarian determination in mice

Elodie P Gregoire<fnref n="1" />, Marie-Cécile De Cian#<fnref n="1" />, Roberta Migale#<fnref n="2" />, Aitana Perea-Gomez<fnref n="1" />, Sébastien Schaub<fnref n="3" />, Natividad Bellido-Carreras<fnref n="1" />, Isabelle Stévant<fnref n="4" />,<fnref n="5" />, Chloé Mayère<fnref n="4" />,<fnref n="5" />, Yasmine Neirijnck<fnref n="1" />, Agnès Loubat<fnref n="1" />, Paul Rivaud<fnref n="6" />, Miriam Llorian Sopena<fnref n="2" />, Simon Lachambre<fnref n="7" />, Margot M. Linssen<fnref n="8" />, Peter Hohenstein<fnref n="8" />, Robin Lovell-Badge<fnref n="2" />, Serge Nef<fnref n="4" />,<fnref n="5" />, Frédéric Chalmel<fnref n="6" />, Andreas Schedl<fnref n="1" />, Marie-Christine Chaboissier<fnref n="1" />,*

## Abstract

Sex determination in mammals depends on the differentiation of the supporting lineage of the gonads into Sertoli or pre-granulosa cells that govern testis and ovary development, respectively.

While the Y-linked testis-determining gene *Sry* has been identified, the ovarian-determining factor remains unknown. Here we identify -KTS, a major alternatively spliced isoform of the Wilms'

<footnote n="1">¹Université Côte d'Azur, Inserm, CNRS, Institut de Biologie Valrose (iBV), 06108 Nice, France</footnote>

<footnote n="2">²The Francis Crick Institute, 1 Midland Road, London NW1 1AT, UK</footnote>

<footnote n="3">³Sorbonne Université, CNRS, Development Biology Laboratory (LBDV), 06234 Villefranche sur Mer, France</footnote>

<footnote n="4">⁴Department of Genetic Medicine and Development, University of Geneva, 1211 Geneva, Switzerland</footnote>

<footnote n="5">⁵iGE3, Institute of Genetics and Genomics of Geneva, University of Geneva, 1211 Geneva Switzerland</footnote>

<footnote n="6">⁶Univ Rennes, Inserm, EHESP, IRSET (Institut de Recherche en Santé, Environnement et Travail)-UMR_S 1085, 35000 Rennes, France</footnote>

<footnote n="7">⁷Infinity, Inserm, CNRS, University Toulouse III, 31024 Toulouse, France</footnote>

<footnote n="8">⁸Central Animal and Transgenic Facility and Dept. Human Genetics, Leiden University Medical Center, 2333ZA Leiden, the Netherlands</footnote>

<footnote># These authors contributed equally to this work.</footnote>

<footnote>This work is licensed under a BY 4.0 International license.</footnote>

<footnote>*Corresponding author. Marie-Christine.CHABOISSIER@univ-cotedazur.fr.</footnote>

<footnote>**Author contributions:**</footnote>

<footnote>EPG, RLB, AS and MCC contributed to conception and design of the study. EPG, MCDC, RM, APG, NBC and AL carried out mouse work, in situ hybridization (ISH), immunofluorescence, microscopy, scRNA-seq and other experimental analysis. SS and SL performed image posttreatments for quantitative analysis of Basescope ISH and immunostainings. IS, YN, CM, PR, MLS and FC performed the bioinformatics analysis. EPG, MML, PH, AS generated transgenesis. EPG, AS and MCC wrote the first draft of the manuscript. All authors contributed to manuscript revision, read, and approved the submitted version. Funding and Resources, SN, RLB and MCC; Supervision, MCC.</footnote>

<footnote>**Competing interests:**</footnote>

<footnote>Authors declare that they have no competing interests.</footnote>
# The -KTS splice variant of WT1 is essential for ovarian determination in mice

Elodie P Gregoire¹, Marie-Cécile De Cian#¹, Roberta Migale#², Aitana Perea-Gomez¹, Sébastien Schaub³, Natividad Bellido-Carreras¹, Isabelle Stévant⁴,⁵, Chloé Mayère⁴,⁵, Yasmine Neirijnck¹, Agnès Loubat¹, Paul Rivaud⁶, Miriam Llorian Sopena², Simon Lachambre⁷, Margot M. Linssen⁸, Peter Hohenstein⁸, Robin Lovell-Badge², Serge Nef⁴,⁵, Frédéric Chalmel⁶, Andreas Schedl¹, Marie-Christine Chaboissier¹,*

¹Université Côte d'Azur, Inserm, CNRS, Institut de Biologie Valrose (iBV), 06108 Nice, France

²The Francis Crick Institute, 1 Midland Road, London NW1 1AT, UK

³Sorbonne Université, CNRS, Development Biology Laboratory (LBDV), 06234 Villefranche sur Mer, France

⁴Department of Genetic Medicine and Development, University of Geneva, 1211 Geneva, Switzerland

⁵iGE3, Institute of Genetics and Genomics of Geneva, University of Geneva, 1211 Geneva Switzerland

⁶Univ Rennes, Inserm, EHESP, IRSET (Institut de Recherche en Santé, Environnement et Travail)-UMR_S 1085, 35000 Rennes, France

⁷Infinity, Inserm, CNRS, University Toulouse III, 31024 Toulouse, France

⁸Central Animal and Transgenic Facility and Dept. Human Genetics, Leiden University Medical Center, 2333ZA Leiden, the Netherlands

# These authors contributed equally to this work.

## Abstract

Sex determination in mammals depends on the differentiation of the supporting lineage of the gonads into Sertoli or pre-granulosa cells that govern testis and ovary development, respectively.

While the Y-linked testis-determining gene *Sry* has been identified, the ovarian-determining factor remains unknown. Here we identify -KTS, a major alternatively spliced isoform of the Wilms'

<footnote>This work is licensed under a BY 4.0 International license.</footnote>

<footnote>*Corresponding author. Marie-Christine.CHABOISSIER@univ-cotedazur.fr.</footnote>

<footnote>**Author contributions:**</footnote>

<footnote>EPG, RLB, AS and MCC contributed to conception and design of the study. EPG, MCDC, RM, APG, NBC and AL carried out mouse work, in situ hybridization (ISH), immunofluorescence, microscopy, scRNA-seq and other experimental analysis. SS and SL performed image posttreatments for quantitative analysis of Basescope ISH and immunostainings. IS, YN, CM, PR, MLS and FC performed the bioinformatics analysis. EPG, MML, PH, AS generated transgenesis. EPG, AS and MCC wrote the first draft of the manuscript. All authors contributed to manuscript revision, read, and approved the submitted version. Funding and Resources, SN, RLB and MCC; Supervision, MCC.</footnote>

<footnote>**Competing interests:**</footnote>

<footnote>Authors declare that they have no competing interests.</footnote>
tumor suppressor WT1, as a key determinant of female sex determination. Loss of -KTS variants
blocks gonadal differentiation in mice, whilst increased expression, as found in Frasier syndrome,
induces precocious differentiation of ovaries independently of their genetic sex. In XY embryos,
this antagonizes *Sry* expression, resulting in male-to-female sex reversal. Our results identify -
KTS as an ovarian-determining factor and demonstrate that its time of activation is critical in
gonadal sex differentiation.

In mice, sex is genetically determined by the constitution of the sex chromosomes. This
leads to testis or ovary development in XY and XX embryos respectively, which in turn
influences the sexual development of the whole individual. Prior to sex determination,
WNT/β-catenin signaling mediated by R-spondin1 (RSPO1) contributes to the proliferation
of the gonadal progenitors in both sexes (1). In XY gonads, at around E(Embryonic
day)11.5, RSPO1/WNT/β-catenin is downregulated and *Sry* and its direct target *Sox9* are
up-regulated in a subset of progenitors derived from the overlying coelomic epithelium
(2–4). These transcription factors induce Sertoli cell differentiation. Once differentiated,
they no longer express *Sry* but express other genes including *Amh* (5–7) and establish the
testis fate. In XX gonads, pre-granulosa cell differentiation occurs slightly later, around
E12.0-12.5, as shown by their loss of bipotentiality (8–10), the de novo expression of the
transcription factor FOXL2 (11), and the stabilization of RSPO1/WNT/β-catenin signaling
(12, 13). However, the gene(s) initiating ovarian differentiation remained unknown (14).

One of the key factors in the early development of the gonad is the Wilms' tumor suppressor WT1, a zinc finger transcriptional regulator (15). *WTI*(human)/*WtI*(mouse) encodes two major alternative spliced isoforms that do or do not include the 3 amino acids KTS between the two last zinc fingers. These isoforms are named +KTS and -KTS respectively. Whilst -KTS acts as a transcriptional activator or repressor depending on the cellular context, the insertion of +KTS abrogates DNA binding and promotes the subnuclear localization of WT1 in nuclear speckles (16, 17). A simple imbalance of the ratio of both isoforms in favor of *-KTS* is the molecular basis of Frasier syndrome, characterized by male-to-female sex reversal (18, 19) associated with the downregulation of *Sry* as evidenced in the mouse model (20).

Results

Distribution of *-KTS* transcripts during gonadal development

To determine the distribution of WT1 splice variants in E11.5 XY mouse gonads, we
carried out BaseScope in situ hybridizations. Scoring revealed cellular heterogeneity, with
cells containing variable amounts of +KTS or -KTS transcripts (Fig. 1A, Fig. S1A). This
observation was confirmed by single-cell RNA sequencing analysis of the splice junction
reads obtained from sorted cells dissected from E11.5 mouse gonads (Fig. 1B). Next, we
examined single-cell transcriptomic data of the supporting cell lineage in both sexes from
E10.5 to E13.5 (8) (Fig. 1C, Fig. S1B). Although +KTS exhibits similar mRNA levels
between XY and XX gonads at E10.5 and E11.5, -KTS transcripts are detected in greater
amounts in XY gonads at E11.5 before increasing in XX gonads at E12.5, timepoints that
coincide respectively with Sertoli and pre-granulosa cell differentiation.
tumor suppressor WT1, as a key determinant of female sex determination. Loss of -KTS variants
blocks gonadal differentiation in mice, whilst increased expression, as found in Frasier syndrome,
induces precocious differentiation of ovaries independently of their genetic sex. In XY embryos,
this antagonizes *Sry* expression, resulting in male-to-female sex reversal. Our results identify -
KTS as an ovarian-determining factor and demonstrate that its time of activation is critical in
gonadal sex differentiation.

In mice, sex is genetically determined by the constitution of the sex chromosomes. This
leads to testis or ovary development in XY and XX embryos respectively, which in turn
influences the sexual development of the whole individual. Prior to sex determination,
WNT/β-catenin signaling mediated by R-spondin1 (RSPO1) contributes to the proliferation
of the gonadal progenitors in both sexes (1). In XY gonads, at around E(Embryonic
day)11.5, RSPO1/WNT/β-catenin is downregulated and *Sry* and its direct target *Sox9* are
up-regulated in a subset of progenitors derived from the overlying coelomic epithelium
(2-4). These transcription factors induce Sertoli cell differentiation. Once differentiated,
they no longer express *Sry* but express other genes including *Amh* (5-7) and establish the
testis fate. In XX gonads, pre-granulosa cell differentiation occurs slightly later, around
E12.0-12.5, as shown by their loss of bipotentiality (8-10), the de novo expression of the
transcription factor FOXL2 (11), and the stabilization of RSPO1/WNT/β-catenin signaling
(12, 13). However, the gene(s) initiating ovarian differentiation remained unknown (14).

One of the key factors in the early development of the gonad is the Wilms' tumor suppressor WT1, a zinc finger transcriptional regulator (15). *WTI*(human)/*WtI*(mouse) encodes two major alternative spliced isoforms that do or do not include the 3 amino acids KTS between the two last zinc fingers. These isoforms are named +KTS and -KTS respectively. Whilst -KTS acts as a transcriptional activator or repressor depending on the cellular context, the insertion of +KTS abrogates DNA binding and promotes the subnuclear localization of WT1 in nuclear speckles (16, 17). A simple imbalance of the ratio of both isoforms in favor of *-KTS* is the molecular basis of Frasier syndrome, characterized by male-to-female sex reversal (18, 19) associated with the downregulation of *Sry* as evidenced in the mouse model (20).

Results

Distribution of *-KTS* transcripts during gonadal development

To determine the distribution of WT1 splice variants in E11.5 XY mouse gonads, we
carried out BaseScope in situ hybridizations. Scoring revealed cellular heterogeneity, with
cells containing variable amounts of +KTS or -KTS transcripts (Fig. 1A, Fig. S1A). This
observation was confirmed by single-cell RNA sequencing analysis of the splice junction
reads obtained from sorted cells dissected from E11.5 mouse gonads (Fig. 1B). Next, we
examined single-cell transcriptomic data of the supporting cell lineage in both sexes from
E10.5 to E13.5 (8) (Fig. 1C, Fig. S1B). Although +KTS exhibits similar mRNA levels
between XY and XX gonads at E10.5 and E11.5, -KTS transcripts are detected in greater
amounts in XY gonads at E11.5 before increasing in XX gonads at E12.5, timepoints that
coincide respectively with Sertoli and pre-granulosa cell differentiation.
## -KTS is required for the differentiation of the supporting cells

To address the contribution of *-KTS* to sex determination, we revisited the mouse model of *-*KTS* ablation (-*KTS*/-*KTS* denoted *-KTS KO*) that results in gonadal dysgenesis (20) (Fig. S2A-B). We performed single-cell transcriptome profiling of wildtype and mutant gonadal cells collected around E12.0 (Fig. 1D-I, Table S1). Cells were projected in a 2D-space using UMAP and partitioned into 39 clusters (Fig. 1D). Cluster annotation identified Sertoli and pre-granulosa cells in the controls based on the expression of known markers (Fig. 1E-G, Fig. S3A-B), however these clusters were not present in *-KTS KO* gonads (Fig. 1E-H, Fig. S3B). Nevertheless, pre-supporting cells were observed in *-KTS* mutants of both sexes, as revealed by the expression of *Runx1*(mRNA)/RUNX1(protein) at E12.0 (Fig. S3B-cluster3) and at E12.5 (Fig. 2A, Fig. S4). XY-*KTS*-deficient gonads exhibited a few scattered cells expressing SOX9, contrasting with the widespread SOX9-positive Sertoli cells forming nascent testis cords in XY controls (Fig. 2B). Furthermore, XY-*KTS*-deficient gonads were devoid of *Amh/AMH* expression (Fig. 2C-D, Fig. S5A-B) and instead abnormally maintained *Rspo1* and SRY at E12.5 and until birth (Fig. S2C-E, Fig. S5C-E). Together, our results indicate that the pre-supporting cells did not differentiate as bona fide Sertoli cells in absence of *-KTS*. Despite the significantly reduced expression of *Sox9* (*p*-value = 0.0035), the XY-*KTS KO* pre-supporting cells failed to differentiate into pre-granulosa cells, as evidenced by the almost complete absence of *Foxl2/FOXL2* expression at E12.5 (Fig. 2B, 2D, Fig. S3). Similarly, *Foxl2/FOXL2* expression was strongly reduced in XX-*KTS KO* gonads, further supporting the importance of *-KTS* for the differentiation of the pre-granulosa lineage and the activation of the female program (Fig. 2B, 2D, Fig. S3). Around birth, the expression of *Sox9/SOX9* and *Foxl2/FOXL2* remained low in *-KTS*-deficient gonads of both sexes, and SOX9/FOXL2 double-positive cells were detected (Fig. S5F-H), indicating poor differentiation of the supporting lineage. Together these data demonstrate that *-KTS* is dispensable for the specification of pre-supporting cells but is necessary to stabilize Sertoli cell differentiation and essential to initiate pre-granulosa cell differentiation.

## Absence of +KTS triggers an increase of -KTS amounts

In patients with Frasier syndrome, heterozygous mutations in WT1 prevent the production of *+KTS*, resulting in higher amounts of *-KTS* variants (18, 19). Given the role of *-KTS* in ovarian determination, we investigated the contribution of this increase to sex reversal in the *+KTS KO* (*+KTS*/-*KTS*-) mouse model (20). At E12.5, XY *+KTS* mutant gonads were enriched for RUNX1 and FOXL2-positive pre-granulosa cells and contained rare Sertoli cells, as expected for male-to- female sex reversal (Fig. 3A, Fig. S6A). Next, we verified that *-KTS* transcripts were twice as abundant in *+KTS KO* gonads compared to controls (Fig. S6B). In addition, total WT1 protein levels were similar in XY *+KTS KO* and control gonads, confirming that absence of +KTS is compensated with an increase of -KTS isoforms (Fig. S6C).

## Precocious pre-granulosa cell differentiation prevents *Sry* activation in the mouse Frasier model

Sex reversal in *+KTS KO* embryos is caused by a failure of *Sry* activation (20, 21). To determine if this was due to the lack of *+KTS* or an increase in *-KTS* variants, we compared
**-KTS is required for the differentiation of the supporting cells**

To address the contribution of *-KTS* to sex determination, we revisited the mouse model of *-*KTS* ablation (-*KTS*/-*KTS* denoted *-*KTS KO*) that results in gonadal dysgenesis (20) (Fig. S2A-B). We performed single-cell transcriptome profiling of wildtype and mutant gonadal cells collected around E12.0 (Fig. 1D-I, Table S1). Cells were projected in a 2D-space using UMAP and partitioned into 39 clusters (Fig. 1D). Cluster annotation identified Sertoli and pre-granulosa cells in the controls based on the expression of known markers (Fig. 1E-G, Fig. S3A-B), however these clusters were not present in *-*KTS KO* gonads (Fig. 1E-H, Fig. S3B). Nevertheless, pre-supporting cells were observed in *-*KTS* mutants of both sexes, as revealed by the expression of *Runx1*(mRNA)/*RUNX1*(protein) at E12.0 (Fig. S3B-cluster3) and at E12.5 (Fig. 2A, Fig. S4). XY *-*KTS*-deficient gonads exhibited a few scattered cells expressing SOX9, contrasting with the widespread SOX9-positive Sertoli cells forming nascent testis cords in XY controls (Fig. 2B). Furthermore, XY *-*KTS*-deficient gonads were devoid of *Amh*/AMH expression (Fig. 2C-D, Fig. S5A-B) and instead abnormally maintained *Rspo1* and SRY at E12.5 and until birth (Fig. S2C-E, Fig. S5C-E). Together, our results indicate that the pre-supporting cells did not differentiate as bona fide Sertoli cells in absence of *-KTS*. Despite the significantly reduced expression of *Sox9* (*p*-value = 0.0035), the XY *-*KTS KO* pre-supporting cells failed to differentiate into pre-granulosa cells, as evidenced by the almost complete absence of *Foxl2/FOXL2* expression at E12.5 (Fig. 2B, 2D, Fig. S3). Similarly, *Foxl2/FOXL2* expression was strongly reduced in XX *-*KTS KO* gonads, further supporting the importance of *-KTS* for the differentiation of the pre-granulosa lineage and the activation of the female program (Fig. 2B, 2D, Fig. S3). Around birth, the expression of *Sox9/SOX9* and *Foxl2/FOXL2* remained low in *-*KTS*-deficient gonads of both sexes, and SOX9/FOXL2 double-positive cells were detected (Fig. S5F-H), indicating poor differentiation of the supporting lineage. Together these data demonstrate that *-*KTS* is dispensable for the specification of pre-supporting cells but is necessary to stabilize Sertoli cell differentiation and essential to initiate pre-granulosa cell differentiation.

**Absence of +KTS triggers an increase of -KTS amounts**

In patients with Frasier syndrome, heterozygous mutations in WT1 prevent the production of *+KTS*, resulting in higher amounts of *-*KTS* variants (18, 19). Given the role of *-*KTS* in ovarian determination, we investigated the contribution of this increase to sex reversal in the *+KTS KO* (*+KTS*/-*KTS*-) mouse model (20). At E12.5, XY *+KTS* mutant gonads were enriched for *RUNX1* and *FOXL2*-positive pre-granulosa cells and contained rare Sertoli cells, as expected for male-to- female sex reversal (Fig. 3A, Fig. S6A). Next, we verified that *-*KTS* transcripts were twice as abundant in *+KTS KO* gonads compared to controls (Fig. S6B). In addition, total WT1 protein levels were similar in XY *+KTS KO* and control gonads, confirming that absence of *+KTS* is compensated with an increase of *-KTS* isoforms (Fig. S6C).

**Precocious pre-granulosa cell differentiation prevents *Sry* activation in the mouse Frasier model**

Sex reversal in *+KTS KO* embryos is caused by a failure of *Sry* activation (20, 21). To determine if this was due to the lack of *+KTS* or an increase in *-*KTS* variants, we compared
the number of SRY-positive cells in +KTS KO and +KTS KO/D compound embryos, both of which lack alleles encoding +KTS and contain two and one allele encoding -KTS, respectively. The number of SRY expressing cells was higher in +KTS KO/D compared to XY +KTS KO gonads, indicating that *Sry* expression does not require +KTS but is antagonized by the higher level of -KTS (Fig. 3B). Furthermore, *Rspo1* was abundant in XY +KTS KO gonads at E11.5, a stage when it is downregulated in XY gonads, and *Foxl2/FOXL2* expression was markedly elevated in XY and XX +KTS KO gonads (Fig. 3C-D, Fig. S6E). This suggests precocious pre-granulosa cell differentiation irrespective of the genetic sex.

**-KTS is sufficient to induce ovarian development**

Single-cell transcriptome profiling of XY and XX +KTS KO gonads at E12.0 (Fig. 1D-F, II, Fig. S3B) identified two pre-granulosa cell clusters (c10, c33) distinct from those found in XX controls (c5, c25) and from XY Sertoli cells (c12). Further comparison of transcriptomes of these clusters confirmed that E12.0 +KTS KO cells are transcriptionally related to pre-granulosa cells (Fig. S7). Next, we used comparative analysis to identify genes that are activated or repressed by -KTS in the context of female sex determination (Fig. S8). In XX -KTS KO pre-supporting cells, the expression of 319 genes was significantly deregulated (FDR adjusted p-value ≤0.05, Data S4). *Pdgfa* and *Tcf21*, reported to be targets of WT1 in other organs (22, 23), were down-regulated, whereas *Igf2*, a direct target of WT1 (24), and genes highly expressed in bipotent pre-supporting cells including *Sprr2d* (25), *Wnt6* (26), *Nr0b1* (27), were up-regulated in XX -KTS KO pre-supporting cells and down-regulated in XX +KTS KO pre-granulosa cells, suggesting that they are repressed by -KTS during sex determination. Altogether our data suggested that increased -KTS rather than loss of +KTS was responsible for XY sex reversal in +KTS KO model. To test whether -KTS was sufficient to induce ovarian differentiation in a XY wildtype gonad, we performed transient additive transgenesis using a BAC construct covering the *Wt1* locus, in which we introduced the classical Frasier mutation in intron 9 (interference with +KTS production). Strikingly, 3 out of 4 XY transgenic animals showed the presence of FOXL2 positive cells indicating that -KTS promotes differentiation of pre-granulosa cells in XY gonads (Fig. 4A-B, Fig. S9A-B). Moreover, RT-qPCR analysis of genotypes producing different levels of -KTS suggested that -KTS must reach a threshold to robustly activate *Foxl2* expression (Fig. S9C).

In summary, we can conclude that the altered expression of +KTS caused by mutations in the donor splice site in intron 9 of *Wt1* promotes an increase of the amount of -KTS, which in turn prematurely activates ovarian differentiation, prevents *Sry* up-regulation, and impairs testis development (Fig. 4C). -KTS thus represents a key actor in gonad development that is required to initiate ovarian development.

**Discussion**

Here, we provide evidence that sex determination does not only depend on the up-regulation of the sex-determining factors, *Sry* and *-KTS* for male and female fates respectively, but also on their timing (28-30). This is an important concept as *-KTS* is an autosomal factor
the number of SRY-positive cells in +KTS KO and +KTS KO/D compound embryos, both of which lack alleles encoding +KTS and contain two and one allele encoding -KTS, respectively. The number of SRY expressing cells was higher in +KTS KO/D compared to XY +KTS KO gonads, indicating that *Sry* expression does not require +KTS but is antagonized by the higher level of -KTS (Fig. 3B). Furthermore, *Rspo1* was abundant in XY +KTS KO gonads at E11.5, a stage when it is downregulated in XY gonads, and *Foxl2/FOXL2* expression was markedly elevated in XY and XX +KTS KO gonads (Fig. 3C-D, Fig. S6E). This suggests precocious pre-granulosa cell differentiation irrespective of the genetic sex.

## -KTS is sufficient to induce ovarian development

Single-cell transcriptome profiling of XY and XX +KTS KO gonads at E12.0 (Fig. 1D-F, II, Fig. S3B) identified two pre-granulosa cell clusters (c10, c33) distinct from those found in XX controls (c5, c25) and from XY Sertoli cells (c12). Further comparison of transcriptomes of these clusters confirmed that E12.0 +KTS KO cells are transcriptionally related to pre-granulosa cells (Fig. S7). Next, we used comparative analysis to identify genes that are activated or repressed by -KTS in the context of female sex determination (Fig. S8). In XX -KTS KO pre-supporting cells, the expression of 319 genes was significantly deregulated (FDR adjusted p-value ≤0.05, Data S4). *Pdgfa* and *Tcf21*, reported to be targets of WT1 in other organs (22, 23), were down-regulated, whereas *Igf2*, a direct target of WT1 (24), and genes highly expressed in bipotent pre-supporting cells including *Sprr2d* (25), *Wnt6* (26), *Nr0b1* (27), were up-regulated in XX -KTS KO pre-supporting cells and down-regulated in XX +KTS KO pre-granulosa cells, suggesting that they are repressed by -KTS during sex determination. Altogether our data suggested that increased -KTS rather than loss of +KTS was responsible for XY sex reversal in +KTS KO model. To test whether -KTS was sufficient to induce ovarian differentiation in a XY wildtype gonad, we performed transient additive transgenesis using a BAC construct covering the *Wt1* locus, in which we introduced the classical Frasier mutation in intron 9 (interference with +KTS production). Strikingly, 3 out of 4 XY transgenic animals showed the presence of FOXL2 positive cells indicating that -KTS promotes differentiation of pre-granulosa cells in XY gonads (Fig. 4A-B, Fig. S9A-B). Moreover, RT-qPCR analysis of genotypes producing different levels of -KTS suggested that *-KTS* must reach a threshold to robustly activate *Foxl2* expression (Fig. S9C).

In summary, we can conclude that the altered expression of +KTS caused by mutations in the donor splice site in intron 9 of *Wt1* promotes an increase of the amount of -KTS, which in turn prematurely activates ovarian differentiation, prevents *Sry* up-regulation, and impairs testis development (Fig. 4C). -KTS thus represents a key actor in gonad development that is required to initiate ovarian development.

## Discussion

Here, we provide evidence that sex determination does not only depend on the up-regulation of the sex-determining factors, *Sry* and *-KTS* for male and female fates respectively, but also on their timing (28-30). This is an important concept as *-KTS* is an autosomal factor
expressed in both XY and XX gonads. In wildtype mice, *Sry* acts before *-KTS*, thus securing testis development in XY gonads. If *Sry* expression is impaired or delayed, or if *-KTS* is up-regulated prematurely, such as in the Frasier syndrome model (*+KTS KO*), the pre-granulosa cell differentiation is accelerated leading to male-to-female sex reversal. After the peak of SRY action (30), -KTS becomes necessary to maintain Sertoli cell differentiation in XY embryos and to initiate pre-granulosa cell differentiation in XX embryos (Fig. 4C). While differences in timing and dynamics of sex determination make a direct comparison between mouse and human data difficult, the sex reversal phenotype in mice carrying intron 9 mutations suggests this is a good mouse model for the human Frasier syndrome. Our data thus indicate that increased expression of -KTS rather than loss (or reduction) of +KTS are the primary cause of sex reversal in Frasier syndrome. Interestingly, a change of +KTS/-KTS ratio in favor of *-KTS* operates when the eggs of *Chelydra serpentina*, a turtle with temperature-dependent sex determination, are shifted from a male to a female-producing temperature (31). This suggests that the -KTS isoform of WT1 is also involved in ovarian determination outside of the mammalian class.

## Supplementary Material

Refer to Web version on PubMed Central for supplementary material.

## Acknowledgements

We thank Marie Cerciat for her help in the preparation of 10X Genomics libraries. Sequencing was performed by the GenomEast platform, a member of the 'France Génomique' consortium (ANR-10-INBS-0009). We are grateful to Fariba Jian Motamedi for her advice on transgenesis. The microscopy was performed at the MICA facility, at the iBV (Institut de Biologie Valrose, Université Côte d'Azur, CNRS, Inserm, iBV, France). We acknowledge the help from members of the PRISM Imaging Platform, the Animal house at iBV and Cécile Passot and Mireille Cutajar-Bossert for the management of funding. We are grateful to FLOW, BABS, ASF, and BRF facilities of the Francis Crick Institute. We are grateful to members of the A. Schedl and M.C. Chaboissier groups for helpful discussions.

## Funding

Agence Nationale de la Recherche grant ANR-19-CE14-0022 SexDiff (MCC)

Fondation Maladies Rares grant FONDATION-GenOmics 2018-1 181003 RNAseq (MCC)

Agence Nationale de la Recherche grant ANR-20-CE14-0022 Ardigerm (MCC)

Swiss National Science Foundation grant 31003A_173070 (SN)

Swiss National Science Foundation grant 310030_200316 (SN)

The Francis Crick Institute receives its core funding from Cancer Research UK (FC001107), the UK Medical Research Council (FC001107), the Wellcome (FC001107), and by the UK Medical Research Council (U117512772) (RM, MLS and RLB).

## Data and materials availability

All data are available in the manuscript or the supplementary materials. The scRNA-seq datasets are available at NCBI Gene Expression Omnibus GEO: GSE207097 and in articles cited in the paper. Programs developed are available on Zenodo (32–34).
expressed in both XY and XX gonads. In wildtype mice, *Sry* acts before *-KTS*, thus securing testis development in XY gonads. If *Sry* expression is impaired or delayed, or if *-KTS* is up-regulated prematurely, such as in the Frasier syndrome model (*+KTS KO*), the pre-granulosa cell differentiation is accelerated leading to male-to-female sex reversal. After the peak of SRY action (30), -KTS becomes necessary to maintain Sertoli cell differentiation in XY embryos and to initiate pre-granulosa cell differentiation in XX embryos (Fig. 4C). While differences in timing and dynamics of sex determination make a direct comparison between mouse and human data difficult, the sex reversal phenotype in mice carrying intron 9 mutations suggests this is a good mouse model for the human Frasier syndrome. Our data thus indicate that increased expression of -KTS rather than loss (or reduction) of +KTS are the primary cause of sex reversal in Frasier syndrome. Interestingly, a change of +KTS/-KTS ratio in favor of *-KTS* operates when the eggs of *Chelydra serpentina*, a turtle with temperature-dependent sex determination, are shifted from a male to a female-producing temperature (31). This suggests that the -KTS isoform of WT1 is also involved in ovarian determination outside of the mammalian class.

## Supplementary Material

Refer to Web version on PubMed Central for supplementary material.

## Acknowledgements

We thank Marie Cerciat for her help in the preparation of 10X Genomics libraries. Sequencing was performed by the GenomEast platform, a member of the 'France Génomique' consortium (ANR-10-INBS-0009). We are grateful to Fariba Jian Motamedi for her advice on transgenesis. The microscopy was performed at the MICA facility, at the iBV (Institut de Biologie Valrose, Université Côte d'Azur, CNRS, Inserm, iBV, France). We acknowledge the help from members of the PRISM Imaging Platform, the Animal house at iBV and Cécile Passot and Mireille Cutajar-Bossert for the management of funding. We are grateful to FLOW, BABS, ASF, and BRF facilities of the Francis Crick Institute. We are grateful to members of the A. Schedl and M.C. Chaboissier groups for helpful discussions.

## Funding

Agence Nationale de la Recherche grant ANR-19-CE14-0022 SexDiff (MCC)

Fondation Maladies Rares grant FONDATION-GenOmics 2018-1 181003 RNAseq (MCC)

Agence Nationale de la Recherche grant ANR-20-CE14-0022 Ardigerm (MCC)

Swiss National Science Foundation grant 31003A_173070 (SN)

Swiss National Science Foundation grant 310030_200316 (SN)

The Francis Crick Institute receives its core funding from Cancer Research UK (FC001107), the UK Medical Research Council (FC001107), the Wellcome (FC001107), and by the UK Medical Research Council (U117512772) (RM, MLS and RLB).

## Data and materials availability

All data are available in the manuscript or the supplementary materials. The scRNA-seq datasets are available at NCBI Gene Expression Omnibus GEO: GSE207097 and in articles cited in the paper. Programs developed are available on Zenodo (32–34).
References

1. Chassot A-A, Bradford ST, Auguste A, Gregoire EP, Pailhoux E, de Rooij DG, Schedi A, Chaboissier M-C. WNT4 and RSPO1 together are required for cell proliferation in the early mouse gonad. Development. 2012; 139: 4461–4472. [PubMed: 23095882]

2. Sekido R, Bar I, Narváez V, Penny G, Lovell-Badge R. SOX9 is up-regulated by the transient expression of SRY specifically in Sertoli cell precursors. Dev Biol. 2004; 274: 271–279. [PubMed: 15385158]

3. Gonen N, Futtner CR, Wood S, Garcia-Moreno SA, Salamone IM, Samson SC, Sekido R, Poulat F, Maatouk DM, Lovell-Badge R. Sex reversal following deletion of a single distal enhancer of Sox9. Science. 2018; 360: 1469–1473. [PubMed: 29903884]

4. Karl J, Capel B. Sertoli cells of the mouse testis originate from the coelomic epithelium. Dev Biol. 1998; 203: 323–333. [PubMed: 9808783]

5. Chaboissier M-C, Kobayashi A, Vidal VIP, Lützkendorf S, van de Kant HJG, Wegner M, de Rooij DG, Behringer RR, Schedl A. Functional analysis of Sox8 and Sox9 during sex determination in the mouse. Development. 2004; 131: 1891–1901. [PubMed: 15056615]

6. De Santa Barbara P, Bonneaud N, Boizet B, Desclozeaux M, Moniot B, Sudbeck P, Scherer G, Poulat F, Berta P. Direct interaction of SRY-related protein SOX9 and steroidogenic factor 1 regulates transcription of the human anti-Müllerian hormone gene. Mol Cell Biol. 1998; 18: 6653–6665. [PubMed: 9774680]

7. Bullejos M, Koopman P. Spatially dynamic expression of Sry in mouse genital ridges. Dev Dyn. 2001; 221: 201–205. [PubMed: 11376487]

8. Stévant I, Kühne F, Greenfield A, Chaboissier M-C, Dermitzakis ET, Nef S. Dissecting Cell Lineage Specification and Sex Fate Determination in Gonadal Somatic Cells Using Single-Cell Transcriptomics. Cell Rep. 2019; 26: 3272–3283. e3 [PubMed: 30893600]

9. Harikae K, Miura K, Shinomura M, Matoba S, Hiramatsu R, Tsunekawa N, Kanai-Azuma M, Kurohmaru M, Morohashi K-I, Kanai Y. Heterogeneity in sexual bipotentiality and plasticity of granulosa cells in developing mouse ovaries. J Cell Sci. 2013; 126: 2834–2844. [PubMed: 23613466]

10. Niu W, Spradling AC. Two distinct pathways of pregranulosa cell differentiation support follicle formation in the mouse ovary. Proc Natl Acad Sci U S A. 2020; 117: 20015–20026. [PubMed: 32759216]

11. Schmidt D, Ovitt CE, Anlag K, Fehsenfeld S, Gredsted L, Treier A-C, Treier M. The murine winged-helix transcription factor Foxl2 is required for granulosa cell differentiation and ovary maintenance. Development. 2004; 131: 933–942. [PubMed: 14736745]

12. Parma P, Radi O, Vidal V, Chaboissier MC, Dellambra E, Valentini S, Guerra L, Schedi A, Camerino G. R-spondin1 is essential in sex determination, skin differentiation and malignancy. Nat Genet. 2006; 38: 1304–1309. [PubMed: 17041600]

13. Maatouk DM, Mork L, Chassot A-A, Chaboissier M-C, Capel B. Disruption of mitotic arrest precedes precocious differentiation and transdifferentiation of pregranulosa cells in the perinatal Wnt4 mutant ovary. Dev Biol. 2013; 383: 295–306. [PubMed: 24036309]

14. Elzaiat M, Todeschini A-L, Caburet S, Veitia RA. The genetic make-up of ovarian development and function: the focus on the transcription factor FOXL2. Clin Genet. 2017; 91: 173–182. [PubMed: 27604691]

15. Kreidberg JA, Sariola H, Loring JM, Maeda M, Pelletier J, Housman D, Jaenisch R. WT-1 is required for early kidney development. Cell. 1993; 74: 679–691. [PubMed: 8395349]

16. Englert C, Vidal M, Maheswaran S, Ge Y, Ezzell RM, Isselbacher KJ, Haber DA. Truncated WT1 mutants alter the subnuclear localization of the wild-type protein. Proc Natl Acad Sci U S A. 1995; 92: 11960–11964. [PubMed: 8618823]

17. Larsson SH, Charlie JP, Miyagawa K, Engelkamp D, Rassoulzadegan M, Ross A, Cuzin F, van Heyningen V, Hastie ND. Subnuclear localization of WT1 in splicing or transcription factor domains is regulated by alternative splicing. Cell. 1995; 81: 391–401. [PubMed: 7736591]
References

1. Chassot A-A, Bradford ST, Auguste A, Gregoire EP, Pailhoux E, de Rooij DG, Schedi A, Chaboissier M-C. WNT4 and RSPO1 together are required for cell proliferation in the early mouse gonad. Development. 2012; 139: 4461-4472. [PubMed: 23095882]

2. Sekido R, Bar I, Narváez V, Penny G, Lovell-Badge R. SOX9 is up-regulated by the transient expression of SRY specifically in Sertoli cell precursors. Dev Biol. 2004; 274: 271-279. [PubMed: 15385158]

3. Gonen N, Futtner CR, Wood S, Garcia-Moreno SA, Salamone IM, Samson SC, Sekido R, Poulat F, Maatouk DM, Lovell-Badge R. Sex reversal following deletion of a single distal enhancer of Sox9. Science. 2018; 360: 1469-1473. [PubMed: 29903884]

4. Karl J, Capel B. Sertoli cells of the mouse testis originate from the coelomic epithelium. Dev Biol. 1998; 203: 323-333. [PubMed: 9808783]

5. Chaboissier M-C, Kobayashi A, Vidal VIP, Lützkendorf S, van de Kant HJG, Wegner M, de Rooij DG, Behringer RR, Schedl A. Functional analysis of Sox8 and Sox9 during sex determination in the mouse. Development. 2004; 131: 1891-1901. [PubMed: 15056615]

6. De Santa Barbara P, Bonneaud N, Boizet B, Desclozeaux M, Moniot B, Sudbeck P, Scherer G, Poulat F, Berta P. Direct interaction of SRY-related protein SOX9 and steroidogenic factor 1 regulates transcription of the human anti-Müllerian hormone gene. Mol Cell Biol. 1998; 18: 6653-6665. [PubMed: 9774680]

7. Bullejos M, Koopman P. Spatially dynamic expression of Sry in mouse genital ridges. Dev Dyn. 2001; 221: 201-205. [PubMed: 11376487]

8. Stévant I, Kühne F, Greenfield A, Chaboissier M-C, Dermitzakis ET, Nef S. Dissecting Cell Lineage Specification and Sex Fate Determination in Gonadal Somatic Cells Using Single-Cell Transcriptomics. Cell Rep. 2019; 26: 3272-3283. e3 [PubMed: 30893600]

9. Harikae K, Miura K, Shinomura M, Matoba S, Hiramatsu R, Tsunekawa N, Kanai-Azuma M, Kurohmaru M, Morohashi K-I, Kanai Y. Heterogeneity in sexual bipotentiality and plasticity of granulosa cells in developing mouse ovaries. J Cell Sci. 2013; 126: 2834-2844. [PubMed: 23613466]

10. Niu W, Spradling AC. Two distinct pathways of pregranulosa cell differentiation support follicle formation in the mouse ovary. Proc Natl Acad Sci U S A. 2020; 117: 20015-20026. [PubMed: 32759216]

11. Schmidt D, Ovitt CE, Anlag K, Fehsenfeld S, Gredsted L, Treier A-C, Treier M. The murine winged-helix transcription factor Foxl2 is required for granulosa cell differentiation and ovary maintenance. Development. 2004; 131: 933-942. [PubMed: 14736745]

12. Parma P, Radi O, Vidal V, Chaboissier MC, Dellambra E, Valentini S, Guerra L, Schedi A, Camerino G. R-spondin1 is essential in sex determination, skin differentiation and malignancy. Nat Genet. 2006; 38: 1304-1309. [PubMed: 17041600]

13. Maatouk DM, Mork L, Chassot A-A, Chaboissier M-C, Capel B. Disruption of mitotic arrest precedes precocious differentiation and transdifferentiation of pregranulosa cells in the perinatal Wnt4 mutant ovary. Dev Biol. 2013; 383: 295-306. [PubMed: 24036309]

14. Elzaiat M, Todeschini A-L, Caburet S, Veitia RA. The genetic make-up of ovarian development and function: the focus on the transcription factor FOXL2. Clin Genet. 2017; 91: 173-182. [PubMed: 27604691]

15. Kreidberg JA, Sariola H, Loring JM, Maeda M, Pelletier J, Housman D, Jaenisch R. WT-1 is required for early kidney development. Cell. 1993; 74: 679-691. [PubMed: 8395349]

16. Englert C, Vidal M, Maheswaran S, Ge Y, Ezzell RM, Isselbacher KJ, Haber DA. Truncated WT1 mutants alter the subnuclear localization of the wild-type protein. Proc Natl Acad Sci U S A. 1995; 92: 11960-11964. [PubMed: 8618823]

17. Larsson SH, Charlieu JP, Miyagawa K, Engelkamp D, Rassoulzadegan M, Ross A, Cuzin F, van Heyningen V, Hastie ND. Subnuclear localization of WT1 in splicing or transcription factor domains is regulated by alternative splicing. Cell. 1995; 81: 391-401. [PubMed: 7736591]
18. Barbaux S, Niaudet P, Gubler MC, Grünfeld JP, Jaubert F, Kutten F, Fékété CN, Souleyreau-Therville N, Thibaud E, Fellous M, McElreavey K. Donor splice-site mutations in WT1 are responsible for Frasier syndrome. Nat Genet. 1997; 17: 467-470. [PubMed: 9398852]

19. Klamt B, Koziell A, Poulat F, Wieacker P, Scambler P, Berta P, Gessler M. Frasier syndrome is caused by defective alternative splicing of WT1 leading to an altered ratio of WT1 +/-KTS splice isoforms. Hum Mol Genet. 1998; 7: 709-714. [PubMed: 9499425]

20. Hammes A, Guo JK, Lutsch G, Leheste JR, Landrock D, Ziegler U, Gubler MC, Schedl A. Two splice variants of the Wilms' tumor 1 gene have distinct functions during sex determination and nephron formation. Cell. 2001; 106: 319-329. [PubMed: 11509181]

21. Bradford ST, Wilhelm D, Bandiera R, Vidal V, Schedl A, Koopman P. A cell-autonomous role for WT1 in regulating Sry in vivo. Hum Mol Genet. 2009; 18: 3429-3438. [PubMed: 19549635]

22. Wang ZY, Madden SL, Deuel TF, Rauscher FJ. The Wilms' tumor gene product, WT1, represses transcription of the platelet-derived growth factor A-chain gene. J Biol Chem. 1992; 267: 21999-22002. [PubMed: 1429549]

23. Bandiera R, Vidal VPI, Motamedi FJ, Clarkson M, Sahut-Barnola I, von Gise A, Pu WT, Hohenstein P, Martinez A, Schedl A. WT1 Maintains Adrenal-Gonadal Primordium Identity and Marks a Population of AGP-like Progenitors within the Adrenal Gland. Developmental Cell. 2013; 27: 5-18. [PubMed: 24135228]

24. Ward A, Pooler JA, Miyagawa K, Duarte A, Hastie ND, Caricasole A. Repression of promoters for the mouse insulin-like growth factor II-encoding gene (Igf-2) by products of the Wilms' tumour suppressor gene wt1. Gene. 1995; 167: 239-243. [PubMed: 8566784]

25. Bouma GJ, Hudson QJ, Washburn LL, Eicher EM. New candidate genes identified for controlling mouse gonadal sex determination and the early stages of granulosa and Sertoli cell differentiation. Biol Reprod. 2010; 82: 380-389. [PubMed: 19864314]

26. Cory AT, Boyer A, Pilon N, Lussier JG, Silversides DW. Presumptive pre-sertoli cells express genes involved in cell proliferation and cell signalling during a critical window in early testis differentiation. Mol Reprod Dev. 2007; 74: 1491-1504. [PubMed: 17410545]

27. Swain A, Zanaria E, Hacker A, Lovell-Badge R, Camerino G. Mouse Dax1 expression is consistent with a role in sex determination as well as in adrenal and hypothalamus function. Nat Genet. 1996; 12: 404-409. [PubMed: 8630494]

28. Eicher EM, Washburn LL. Inherited sex reversal in mice: identification of a new primary sex-determining gene. J Exp Zool. 1983; 228: 297-304. [PubMed: 6663259]

29. Bullejos M, Koopman P. Delayed Sry and Sox9 expression in developing mouse gonads underlies B6-Y(DOM) sex reversal. Dev Biol. 2005; 278: 473-481. [PubMed: 15680364]

30. Hiramatsu R, Matoba S, Kanai-Azuma M, Tsunekawa N, Katoh-Fukui Y, Kurohmaru M, Morohashi K-I, Wilhelm D, Koopman P, Kanai Y. A critical time window of Sry action in gonadal sex determination in mice. Development. 2009; 136: 129-138. [PubMed: 19036799]

31. Rhen T, Fagerlie R, Schroeder A, Crossley DA, Lang JW. Molecular and morphological differentiation of testes and ovaries in relation to the thermosensitive period of gonad development in the snapping turtle, Chelydra serpentina. Differentiation. 2015; 89: 31-41. [PubMed: 25662229]

32. Schaub S, SebastienSchaub/DICHisto: Gregoireetal2023. 2023; doi: 10.5281/ZENODO.8329139

33. Lachambre, Nuclei count in gonad. 2023; doi: 10.5281/ZENODO.8345079

34. Lachambre, Intensity measurement in gonad. 2023; doi: 10.5281/ZENODO.8345091

35. Hacker A, Capel B, Goodfellow P, Lovell-Badge R. Expression of Sry, the mouse sex determining gene. Development. 1995; 121: 1603-1614. [PubMed: 7600978]

36. Wilhelm D, Englert C. The Wilms tumor suppressor WT1 regulates early gonad development by activation of Sf1. Genes Dev. 2002; 16: 1839-1851. [PubMed: 12130543]

37. Polanco JC, Wilhelm D, Davidson T-L, Knight D, Koopman P. Sox10 gain-of-function causes XX sex reversal in mice: implications for human 22q-linked disorders of sex development. Hum Mol Genet. 2010; 19: 506-516. [PubMed: 19933217]

38. Gregoire EP, Stevant I, Chassot A-A, Martin L, Lachambre S, Mondin M, de Rooij DG, Nef S, Chaboissier M-C. NRG1 signalling regulates the establishment of Sertoli cell stock in the mouse testis. Mol Cell Endocrinol. 2018; 478: 17-31. [PubMed: 30040984]
18. Barbaux S, Niaudet P, Gubler MC, Grünfeld JP, Jaubert F, Kutten F, Fékété CN, Souleyreau-Therville N, Thibaud E, Fellous M, McElreavey K. Donor splice-site mutations in WT1 are responsible for Frasier syndrome. Nat Genet. 1997; 17: 467-470. [PubMed: 9398852]

19. Klamt B, Koziell A, Poulat F, Wieacker P, Scambler P, Berta P, Gessler M. Frasier syndrome is caused by defective alternative splicing of WT1 leading to an altered ratio of WT1 +/-KTS splice isoforms. Hum Mol Genet. 1998; 7: 709-714. [PubMed: 9499425]

20. Hammes A, Guo JK, Lutsch G, Leheste JR, Landrock D, Ziegler U, Gubler MC, Schedl A. Two splice variants of the Wilms' tumor 1 gene have distinct functions during sex determination and nephron formation. Cell. 2001; 106: 319-329. [PubMed: 11509181]

21. Bradford ST, Wilhelm D, Bandiera R, Vidal V, Schedl A, Koopman P. A cell-autonomous role for WT1 in regulating Sry in vivo. Hum Mol Genet. 2009; 18: 3429-3438. [PubMed: 19549635]

22. Wang ZY, Madden SL, Deuel TF, Rauscher FJ. The Wilms' tumor gene product, WT1, represses transcription of the platelet-derived growth factor A-chain gene. J Biol Chem. 1992; 267: 21999-22002. [PubMed: 1429549]

23. Bandiera R, Vidal VPI, Motamedi FJ, Clarkson M, Sahut-Barnola I, von Gise A, Pu WT, Hohenstein P, Martinez A, Schedl A. WT1 Maintains Adrenal-Gonadal Primordium Identity and Marks a Population of AGP-like Progenitors within the Adrenal Gland. Developmental Cell. 2013; 27: 5-18. [PubMed: 24135228]

24. Ward A, Pooler JA, Miyagawa K, Duarte A, Hastie ND, Caricasole A. Repression of promoters for the mouse insulin-like growth factor II-encoding gene (Igf-2) by products of the Wilms' tumour suppressor gene wt1. Gene. 1995; 167: 239-243. [PubMed: 8566784]

25. Bouma GJ, Hudson QJ, Washburn LL, Eicher EM. New candidate genes identified for controlling mouse gonadal sex determination and the early stages of granulosa and Sertoli cell differentiation. Biol Reprod. 2010; 82: 380-389. [PubMed: 19864314]

26. Cory AT, Boyer A, Pilon N, Lussier JG, Silversides DW. Presumptive pre-sertoli cells express genes involved in cell proliferation and cell signalling during a critical window in early testis differentiation. Mol Reprod Dev. 2007; 74: 1491-1504. [PubMed: 17410545]

27. Swain A, Zanaria E, Hacker A, Lovell-Badge R, Camerino G. Mouse Dax1 expression is consistent with a role in sex determination as well as in adrenal and hypothalamus function. Nat Genet. 1996; 12: 404-409. [PubMed: 8630494]

28. Eicher EM, Washburn LL. Inherited sex reversal in mice: identification of a new primary sex-determining gene. J Exp Zool. 1983; 228: 297-304. [PubMed: 6663259]

29. Bullejos M, Koopman P. Delayed Sry and Sox9 expression in developing mouse gonads underlies B6-Y(DOM) sex reversal. Dev Biol. 2005; 278: 473-481. [PubMed: 15680364]

30. Hiramatsu R, Matoba S, Kanai-Azuma M, Tsunekawa N, Katoh-Fukui Y, Kurohmaru M, Morohashi K-I, Wilhelm D, Koopman P, Kanai Y. A critical time window of Sry action in gonadal sex determination in mice. Development. 2009; 136: 129-138. [PubMed: 19036799]

31. Rhen T, Fagerlie R, Schroeder A, Crossley DA, Lang JW. Molecular and morphological differentiation of testes and ovaries in relation to the thermosensitive period of gonad development in the snapping turtle, Chelydra serpentina. Differentiation. 2015; 89: 31-41. [PubMed: 25662229]

32. Schaub S, SebastienSchaub/DICHisto: Gregoireetal2023. 2023; doi: 10.5281/ZENODO.8329139

33. Lachambre, Nuclei count in gonad. 2023; doi: 10.5281/ZENODO.8345079

34. Lachambre, Intensity measurement in gonad. 2023; doi: 10.5281/ZENODO.8345091

35. Hacker A, Capel B, Goodfellow P, Lovell-Badge R. Expression of Sry, the mouse sex determining gene. Development. 1995; 121: 1603-1614. [PubMed: 7600978]

36. Wilhelm D, Englert C. The Wilms tumor suppressor WT1 regulates early gonad development by activation of Sf1. Genes Dev. 2002; 16: 1839-1851. [PubMed: 12130543]

37. Polanco JC, Wilhelm D, Davidson T-L, Knight D, Koopman P. Sox10 gain-of-function causes XX sex reversal in mice: implications for human 22q-linked disorders of sex development. Hum Mol Genet. 2010; 19: 506-516. [PubMed: 19933217]

38. Gregoire EP, Stevant I, Chassot A-A, Martin L, Lachambre S, Mondin M, de Rooij DG, Nef S, Chaboissier M-C. NRG1 signalling regulates the establishment of Sertoli cell stock in the mouse testis. Mol Cell Endocrinol. 2018; 478: 17-31. [PubMed: 30040984]
39. Schmidt, U, Weigert, M, Broaddus, C, Myers, G. Medical Image Computing and Computer Assisted Intervention-MICCAI 2018. Frangi, AF, Schnabel, JA, Davatzikos, C, Alberola-López, C, Fichtinger, G, editors. Vol. 11071. Springer International Publishing; Cham: 2018. 265–273. Lecture Notes in Computer Science

40. Dobin A, Davis CA, Schlesinger F, Drenkow J, Zaleski C, Jha S, Batut P, Chaisson M, Gingeras TR. STAR: ultrafast universal RNA-seq aligner. Bioinformatics. 2013; 29: 15–21. [PubMed: 23104886]

41. Li B, Dewey CN. RSEM: accurate transcript quantification from RNA-Seq data with or without a reference genome. BMC Bioinformatics. 2011; 12: 323. [PubMed: 21816040]

42. Quinlan AR, Hall IM. BEDTools: a flexible suite of utilities for comparing genomic features. Bioinformatics. 2010; 26: 841–842. [PubMed: 20110278]

43. Hao Y, Hao S, Andersen-Nissen E, Mauck WM, Zheng S, Butler A, Lee MJ, Wilk AJ, Darby C, Zager M, Hoffman P, Stoeckius M, et al. Integrated analysis of multimodal single-cell data. Cell. 2021; 184: 3573–3587. e29 [PubMed: 34062119]

44. McGinnis CS, Murrow LM, Gartner ZJ. DoubletFinder: Doublet Detection in Single-Cell RNA Sequencing Data Using Artificial Nearest Neighbors. Cell Systems. 2019; 8: 329–337. e4 [PubMed: 30954475]

45. Chalmel F, Primig M. The Annotation, Mapping, Expression and Network (AMEN) suite of tools for molecular systems biology. BMC Bioinformatics. 2008; 9: 86. [PubMed: 18254954]

46. Stévant I, Neirijnck Y, Borel C, Escoffier J, Smith LB, Antonarakis SE, Dermitzakis ET, Nef S. Deciphering Cell Lineage Specification during Male Sex Determination with Single-Cell RNA Sequencing. Cell Rep. 2018; 22: 1589–1599. [PubMed: 29425512]

47. Luo X, Ikeda Y, Parker KL. A cell-specific nuclear receptor is essential for adrenal and gonadal development and sexual differentiation. Cell. 1994; 77: 481–490. [PubMed: 8187173]

48. Miyabayashi K, Katoh-Fukui Y, Ogawa H, Baba T, Shima Y, Sugiyama N, Kitamura K, Morohashi K. Aristaless Related Homeobox Gene, Arx, Is Implicated in Mouse Fetal Leydig Cell Differentiation Possibly through Expressing in the Progenitor Cells. Plos One. 2013; 8 e68050 [PubMed: 23840809]

49. Mayère C, Regard V, Perea-Gomez A, Bunce C, Neirijnck Y, Djari C, Bellido-Carreras N, Sararols P, Reeves R, Greenaway S, Simon M, et al. Origin, specification and differentiation of a rare supporting-like lineage in the developing mouse gonad. Sci Adv. 2022; 8 eabm0972 [PubMed: 35613264]
39. Schmidt, U, Weigert, M, Broaddus, C, Myers, G. Medical Image Computing and Computer Assisted Intervention-MICCAI 2018. Frangi, AF, Schnabel, JA, Davatzikos, C, Alberola-López, C, Fichtinger, G, editors. Vol. 11071. Springer International Publishing; Cham: 2018. 265–273. Lecture Notes in Computer Science

40. Dobin A, Davis CA, Schlesinger F, Drenkow J, Zaleski C, Jha S, Batut P, Chaisson M, Gingeras TR. STAR: ultrafast universal RNA-seq aligner. Bioinformatics. 2013; 29: 15–21. [PubMed: 23104886]

41. Li B, Dewey CN. RSEM: accurate transcript quantification from RNA-Seq data with or without a reference genome. BMC Bioinformatics. 2011; 12: 323. [PubMed: 21816040]

42. Quinlan AR, Hall IM. BEDTools: a flexible suite of utilities for comparing genomic features. Bioinformatics. 2010; 26: 841–842. [PubMed: 20110278]

43. Hao Y, Hao S, Andersen-Nissen E, Mauck WM, Zheng S, Butler A, Lee MJ, Wilk AJ, Darby C, Zager M, Hoffman P, Stoeckius M, et al. Integrated analysis of multimodal single-cell data. Cell. 2021; 184: 3573–3587. e29 [PubMed: 34062119]

44. McGinnis CS, Murrow LM, Gartner ZJ. DoubletFinder: Doublet Detection in Single-Cell RNA Sequencing Data Using Artificial Nearest Neighbors. Cell Systems. 2019; 8: 329–337. e4 [PubMed: 30954475]

45. Chalmel F, Primig M. The Annotation, Mapping, Expression and Network (AMEN) suite of tools for molecular systems biology. BMC Bioinformatics. 2008; 9: 86. [PubMed: 18254954]

46. Stévant I, Neirijnck Y, Borel C, Escoffier J, Smith LB, Antonarakis SE, Dermitzakis ET, Nef S. Deciphering Cell Lineage Specification during Male Sex Determination with Single-Cell RNA Sequencing. Cell Rep. 2018; 22: 1589–1599. [PubMed: 29425512]

47. Luo X, Ikeda Y, Parker KL. A cell-specific nuclear receptor is essential for adrenal and gonadal development and sexual differentiation. Cell. 1994; 77: 481–490. [PubMed: 8187173]

48. Miyabayashi K, Katoh-Fukui Y, Ogawa H, Baba T, Shima Y, Sugiyama N, Kitamura K, Morohashi K. Aristaless Related Homeobox Gene, Arx, Is Implicated in Mouse Fetal Leydig Cell Differentiation Possibly through Expressing in the Progenitor Cells. Plos One. 2013; 8 e68050 [PubMed: 23840809]

49. Mayère C, Regard V, Perea-Gomez A, Bunce C, Neirijnck Y, Djari C, Bellido-Carreras N, Sararols P, Reeves R, Greenaway S, Simon M, et al. Origin, specification and differentiation of a rare supporting-like lineage in the developing mouse gonad. Sci Adv. 2022; 8 eabm0972 [PubMed: 35613264]
**One-Sentence Summary**

The -KTS splice variant of WT1 has a dual function in mediating gonadal sex determination
**One-Sentence Summary**

The -KTS splice variant of WT1 has a dual function in mediating gonadal sex determination
<figure><img src="image_1.png" /><figcaption>Fig. 1. Dynamic distribution of +KTS and -KTS transcripts and single-cell transcriptomic analysis of -KTS KO and +KTS KO during early mouse gonad development.</figcaption></figure>

(A) Representative area distribution of -KTS (magenta) and +KTS (cyan) transcripts from Basescope in situ hybridizations on XY gonad sections at E11.5 (21ts (tail somites)) in µm² per nucleus measured using DicHysto protocol. Data from both gonads are representative of biological and technical duplicates. (B) -KTS and +KTS mRNA ratio in E11.5 (21ts) XY wildtype individual cells. (C) +KTS and -KTS transcript levels in single-cell transcriptomic dataset of differentiating supporting cells. (D) UMAP projection of the 75,360 cells colored
<figure><img src="image_2.png" /><figcaption>Fig. 1. Dynamic distribution of +KTS and -KTS transcripts and single-cell transcriptomic analysis of -KTS KO and +KTS KO during early mouse gonad development.</figcaption></figure>

(A) Representative area distribution of -KTS (magenta) and +KTS (cyan) transcripts from Basescope in situ hybridizations on XY gonad sections at E11.5 (21ts (tail somites)) in µm² per nucleus measured using DicHysto protocol. Data from both gonads are representative of biological and technical duplicates. (B) -KTS and +KTS mRNA ratio in E11.5 (21ts) XY wildtype individual cells. (C) +KTS and -KTS transcript levels in single-cell transcriptomic dataset of differentiating supporting cells. (D) UMAP projection of the 75,360 cells colored
by clusters or (E) by associated cell types. (F) Association of cell clusters with genotypes. (G-I) UMAP projection by genotypes with XY (blue) and XX wildtype (pink) (G), XY (purple) and XX -KTS KO (-KTS/-KTS~) (brown) (H), and XY (green) and XX +KTS KO (+KTS/+KTS~) gonads (orange) (I).
by clusters or (E) by associated cell types. (F) Association of cell clusters with genotypes. (G-I) UMAP projection by genotypes with XY (blue) and XX wildtype (pink) (G), XY (purple) and XX -KTS KO (-KTS/-KTS) (brown) (H), and XY (green) and XX +KTS KO (+KTS/+KTS) gonads (orange) (I).
<figure><img src="image_2.png" /><figcaption>Fig. 2. -KTS is necessary for sex differentiation of the supporting cells.</figcaption></figure>

Immunodetection of (A) the pre-supporting cell marker RUNX1 (magenta) and the progenitor marker NR2F2 (green) at E12.5 (scale bars: 50 µm), (B) the Sertoli cell marker SOX9 (green) and pre-granulosa cell marker FOXL2 (magenta) (scale bars: 100 µm, 10 µm), and (C) SOX9 (green) and AMH (magenta) (scale bars: 50 µm) in the indicated genotypes. (A-C) Data are representative of triplicate biological replicates. Nuclei labelled with DAPI are shown in white. (D) Quantification of *Foxl2*, *Sox9*, and *Amh* transcripts
<figure><img src="image_3.png" /><figcaption>Fig. 2. -KTS is necessary for sex differentiation of the supporting cells.
Immunodetection of (A) the pre-supporting cell marker RUNX1 (magenta) and the progenitor marker NR2F2 (green) at E12.5 (scale bars: 50 µm), (B) the Sertoli cell marker SOX9 (green) and pre-granulosa cell marker FOXL2 (magenta) (scale bars: 100 µm, 10 µm), and (C) SOX9 (green) and AMH (magenta) (scale bars: 50 µm) in the indicated genotypes. (A-C) Data are representative of triplicate biological replicates. Nuclei labelled with DAPI are shown in white. (D) Quantification of *Foxl2*, *Sox9*, and *Amh* transcripts</figcaption></figure>
after normalization to *Gapdh* by RT-qPCR. Data are shown as means ± SEM. *-KTS KO: -KTS/-KTS*.
after normalization to *Gapdh* by RT-qPCR. Data are shown as means ± SEM. *-KTS KO: -KTS/-KTS*.
<figure><img src="image_3.png" /><figcaption>Fig. 3. Early pre-granulosa cell differentiation occurs in *XY* and *XX +KTS KO* gonads.</figcaption></figure>

(A) Immunofluorescence of the Sertoli cell marker SOX9 (green) and the pre-granulosa cell marker FOXL2 (magenta) in indicated genotypes at E12.5 (n=4). Scale bars: 100 µm. (B) Immunostaining of SRY (green) at E11.5 (21±1ts) in *XY, XY +KTS KO (+KTS-/ +KTS*)*, and *XY compound heterozygotes (+KTS KO/Δ: +KTS/Wt1-)*. Scale bars: 50 µm. Quantification of SRY+ cells normalized to DAPI+ cells labeled in white in the upper panel. n=4, 2 sections/embryo. Data are shown as means ± SEM. (C) Immunodetection of the pre-granulosa cell marker FOXL2 in indicated genotypes of triplicate biological replicates at 20-21ts. Scale bars: 50 µm. (D) Relative mRNA expression of *Foxl2* normalized to *Gapdh* at 20-21ts. Data are shown as means ± SEM.
<figure><img src="image_4.png" /><figcaption>Fig. 3. Early pre-granulosa cell differentiation occurs in XY and XX +KTS KO gonads.</figcaption></figure>

(A) Immunofluorescence of the Sertoli cell marker SOX9 (green) and the pre-granulosa cell marker FOXL2 (magenta) in indicated genotypes at E12.5 (n=4). Scale bars: 100 µm. (B) Immunostaining of SRY (green) at E11.5 (21±1ts) in XY, XY +KTS KO (+KTS-/+KTS-), and XY compound heterozygotes (+KTS KO/Δ: +KTS/Wt1-). Scale bars: 50 µm. Quantification of SRY+ cells normalized to DAPI+ cells labeled in white in the upper panel. n=4, 2 sections/embryo. Data are shown as means ± SEM. (C) Immunodetection of the pre-granulosa cell marker FOXL2 in indicated genotypes of triplicate biological replicates at 20-21ts. Scale bars: 50 µm. (D) Relative mRNA expression of *Foxl2* normalized to *Gapdh* at 20-21ts. Data are shown as means ± SEM.
<figure><img src="image_4.png" /><figcaption>Fig. 4. *-KTS* induces pre-granulosa cell differentiation in *XY* transgenic gonads.</figcaption></figure>

(A) Immunofluorescence of the pre-granulosa cell marker FOXL2 (magenta) and WT1 (green) in the indicated genotypes at E12.5. Scale bars: 50 µm. (B) Immunostaining of the Sertoli cell markers SOX9 (green) and AMH (magenta) in indicated genotypes. Scale bars: 50 µm. (C) Model of supporting cell differentiation in wild-type and *KTS* mutant gonads: absence of *-KTS* in *-KTS KO* gonads promotes the maintenance of *Rspol* transcripts and
<figure><img src="image_5.png" /><figcaption>Fig. 4. *-KTS* induces pre-granulosa cell differentiation in *XY* transgenic gonads.</figcaption></figure>

(A) Immunofluorescence of the pre-granulosa cell marker FOXL2 (magenta) and WT1 (green) in the indicated genotypes at E12.5. Scale bars: 50 µm. (B) Immunostaining of the Sertoli cell markers SOX9 (green) and AMH (magenta) in indicated genotypes. Scale bars: 50 µm. (C) Model of supporting cell differentiation in wild-type and *KTS* mutant gonads: absence of *-KTS* in *-KTS KO* gonads promotes the maintenance of *Rspol* transcripts and
impairs SOX9 and FOXL2 expression leading to gonadal dysgenesis. Increasing -KTS in
+KTS KO gonads results in ovarian differentiation in both genetic sexes.
impairs SOX9 and FOXL2 expression leading to gonadal dysgenesis. Increasing -KTS in
+KTS KO gonads results in ovarian differentiation in both genetic sexes.