Manuscript in Preparation

The following manuscript is an ongoing study that is not fully achieved. We plan to finalise the analysis and to submit the manuscript on bioRXiv and Cell Reports next April.

3.2.2 Contribution

This study was conceived by Serge Nef and Isabelle Stévant. Isabelle performed the collection of the XX mouse gonads, the somatic cell purification prior to the single-cell captures, the cDNA libraries, the mapping and the analysis of the resulting sequencing data. She wrote the manuscript with the support of all the authors and prepared the main and the supplementary figures.

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Supporting cell lineage specification and sex-specific differentiation into Sertoli or Granulosa cells occurs sequentially during gonad sex determination

Authors: Isabelle Stévant^1,2,3, Emmanouil T. Dermitzakis^1,2,3, Serge Nef^1,2,*.

Affiliations:

1Department of Genetic Medicine and Development, University of Geneva, 1211 Geneva, Switzerland;

2iGE3, Institute of Genetics and Genomics of Geneva, University of Geneva, 1211 Geneva, Switzerland;

3SIB, Swiss Institute of Bioinformatics, University of Geneva, 1211 Geneva, Switzerland;

Contact information:

*Corresponding Author: Serge.Nef@unige.ch

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Summary:

Gonadal sex determination is a unique and complex process ideally suited to study cell commitment and how multipotent progenitors acquire sex-specific fates as the organ differentiates as a testis or an ovary. Using time-series single-cell RNA sequencing on XX Nr5a1-GFP⁺ somatic cells, we found a lineage specification process similar to the one found during testis development, with the presence of a single Nr5a1-GFP ⁺ cell population of early progenitors giving rise to the supporting cell lineage at the origin of pre-granulosa cells and steroidogenic precursor cells potentially at the origin of theca cells. When comparing expression data of time-series scRNA-Seq on both XX and XY mouse gonads during sex determination, we found that supporting cell commitment and sex-specific differentiation into either Sertoli or pre-granulosa cells occurs sequentially. Moreover, we demonstrated that the remaining XY interstitial cells and XX stromal cells are transcriptionally similar and express set of genes that specify their fate as steroidogenic cell precursors at the origin of fetal Leydig cells and theca cells.

Keywords:

Single-cell RNA-Seq, sex determination, ovary, testis, granulosa cell, Sertoli cells, supporting cells, progenitors, differentiation, lineage specification, cell fate decision, gene expression

3 Introduction

Testis and ovary derive from a common primordium, the genital ridges, that start developing around embryonic day (E)9.5 in mice through thickening and proliferation of the coelomic epithelium on the ventromedial surface of the mesonephroi (Byskov, 1986). The bipotential gonads are composed of somatic progenitor cells and migrating primordial germ cells. The gonads operate sex determination around E11.5 with the differentiation of the supporting cell progenitors as Sertoli cells in XY with the expression of Sry (Albrecht and Eicher, 2001; Sekido et al., 2004) or as granulosa cells in XX with the activation of the WNT/β-catenin signalling (Chassot et al., 2008, 2012). Following sex determination, the germ cells and the other somatic cells adopt their respective sex-specific cell fate.

While the origin of germ cells is well established, the origins of somatic cell lineages of both the XX and XY gonads is not as well understood. Regarding the supporting cells, lineage tracing experiments indicate that Sertoli cells and pre-granulosa cells have a common origin and their progenitors originate from the coelomic epithelium (Albrecht and Eicher, 2001; Karl and Capel, 1998; Mork et al., 2012). In the developing ovary, the pre-granulosa cell population does not expand by mitotic division but rather by differentiation of entering coelomic epithelial cells until E14.5 (Mork et al., 2012). Two pre-granulosa cell populations have been observed based on the mutually exclusive expression of FOXL2 and LGR5 (Rastetter et al., 2014). The LGR5 positive cells are located in the cortical zone and the FOXL2 positive cells are in the medullary zone of the developing ovaries and give rise to the cortical and the medullary follicles respectively (Rastetter et al., 2014).

Concerning the steroidogenic cell lineage, the fetal Leydig cells arise from precursor cells present in the interstitial compartment of the testis, however, the origin the theca cells, the female counterpart of the Leydig cells, is still unclear. A third ovarian somatic cell population have been described and express Maf and Mafb (DeFalco et al., 2011; Jameson et al., 2012) and Nr2f2 (Rastetter et al., 2014), and is suspected to be a precursor of the steroidogenic theca cells (Rastetter et al., 2014; Takamoto et al., 2005). Moreover, a recent study showed that steroidogenic theca cells derive mainly from E10.5 WT1 positive progenitor cells and from a small fraction from GLI1 positive mesonephric cells (Liu et al., 2015). The link between these cells has to be made to resolve the female steroidogenic cell lineage.

In this study, we first performed time-series single-cell RNA sequencing (scRNA-seq) on XX Nr5a1-GFP somatic cells to identify the different cell types composing the developing ovary and we reconstructed the cell lineages and the expression dynamics controlling cell differentiation during early ovary development. Then, by combining expression data for time-series scRNA-Seq on both XX and XY mouse gonads during sex determination, we were able to reconstitute the fates and sex-specific differentiation of the Nr5a1-GFP cell lineage. We identified a single population of XX and XY somatic multipotent progenitor at E10.5, that give rise through cell fate restriction to the supporting cell lineage at the origin of granulosa and Sertoli cells and to the steroidogenic precursor cells at the origin of fetal Leydig cells and theca cells. Interestingly, we found that supporting cell commitment and sex-specific differentiation into either Sertoli or granulosa cells occurs sequentially. Initially both XX and XY multipotent progenitors that adopt a supporting cell fate and share a similar transcriptomic identity before initiating the sex-dependent genetic program leading to their differentiation into Sertoli and granulosa cells. In addition, we found that XX and XY remaining precursors are transcriptionally similar and evolve to express steroidogenic

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precursor cell markers at the origin of fetal Leydig cells and theca cells. This study provides the most refined and complete characterization of the genetic programs mediating cell fate specification and represent a rich resource for the field.

Results

Ovarian somatic single-cell RNA-sequencing and classification

Somatic cells of the developing ovary were purified from XX Tg(Nr5a1-GFP) animals (Nef et al., 2005; Pitetti et al., 2013; Stallings et al., 2002) at six different developmental stages of ovarian differentiation (E10.5, E11.5, E12.5, E13.5, E16.5 and PND6) (Figure 1A & Methods). Briefly, at each relevant embryonic stages, ovaries from Tg(Nr5a1-GFP) animals were dissociated and the Nr5a1-GFP⁺ cells were sorted by FACS (fluorescent active cell sorting, Figure S1). GFP⁺ cells were isolated and processed with the Fluidigm C1 Autoprep system (small size IFC). Doublet cells were excluded after careful visual inspections of the chips. A total of 653 individual cells were collected and sequenced (full-length RNA-seq, 100bp paired-end reads, 10M reads per cell), from which 563 passed the quality filters (see Supplemental experimental procedures). Cells expressed a total of 16,765 protein-coding genes (RPKM>0), with a median of 4,909 genes per cell (Figure S2).

To classify the different somatic cell populations present in the developing ovary, we selected the highly variable genes (Brennecke et al., 2013), performed a hierarchical clustering on the significant principal components (Figure 1B) (Chung and Storey, 2015; Lê et al., 2008) and visualized the results with t-SNE (Maaten et al., 2008) (Figure 1C&D, Methods). We obtained four cell clusters mixing together different embryonic stages. The dendrogram (Figure 1B) reveals that cells from Cluster 1 (C1) and C2 are similar and so are cells from C3 and C4. The cluster C1 contains 90 cells from E13.5 to E16.5, the cluster C2 contains 240 cells from E10.5 to E16.5, the cluster C3 contains 190 cells from E11.5 to P6, and the cluster C4 contains 43 cells from E16.5 and P6. We found 4,435 genes differentially expressed between the four cell clusters (q-value<0.5, Supplementary Data 1).

A combination of parameters including embryonic stages, marker genes differentially expressed between the cell clusters (Figure 1E) and GO terms (Figure 1F) allowed us to assign an identity to each of the cell clusters. We noticed that the clusters C1 and C2 highly express Nr2f2, Maf, and to a lesser extent but still significantly Mafb (DeFalco et al., 2011; Kilcoyne et al., 2014). The GO term associated with the genes over-expressed in these two cell clusters are related to urogenital system development, DNA replication, and reproductive system development (Figure 1F and Supplementary Data 2). The cluster C1 specifically express genes related to extracellular organization, whereas the cluster C2 display genes related to mitotic division. As Nr2f2 is a known marker of the interstitial progenitor cells in foetal testis, we hypothesize these two cell populations represent the XX counterpart of the XY progenitor cells. We called cluster C2 the early progenitors as it contains early embryonic stages, and cluster C1 the stromal progenitors, even though the stroma is not defined morphologically before folliculogenesis.

We identified the clusters C3 and C4 as granulosa cells as they both express the markers Foxl2, Runx1, Kitl, and Fst. However, the cluster C3 that contains mostly foetal cells and express genes related to pre-granulosa cells, such as Rspo1, Lgr5, and Bmp2. In contrast, the cluster C4 contains the post-natal P6 cells and expresses Amh, which is associated with granulosa cells from primordial

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follicles, and steroid-related genes Esr2, Hsd17b1 and Hsd3b1. Thereby, we called the C3 cluster the pre-granulosa, and the C4 cluster the granulosa to distinguish these two populations.

To summarize, the time-series single-cell RNA-seq of the Nr5a1-GFP⁺ of the developing ovary from E10.5 to PND6 identified four cell populations, including early and stromal progenitor cells, foetal pre-granulosa cells and post-natal granulosa cells. The absence of other detected cell type at PND6 indicates that we did not capture differentiated theca cells.

Female somatic cell lineage specification

We next sought to reconstitute the Nr5a1-GFP⁺ cell lineage specification in XX gonads during the process of sex determination with the aim of analysing the cell transition states leading to their differentiation. We used diffusion map to reconstruct the lineages based on gene expression, from which we calculated the cell trajectories and ordered the cells along a pseudotime in each of the obtained trajectories (Figure 2A-B, Supplementary experimental procedures).

We obtained one lineage starting from the E10.5 cells that bifurcates around E11.5 to give rise subsequently to the granulosa cell lineage and the progenitor cell lineage. For the two cell trajectories, we performed differential expression analysis over the pseudotime to identify the genes that are dynamically expressed during the lineage progression. We identified 2,290 genes dynamically expressed in the progenitor lineage, and 2,785 genes in the granulosa lineage (q-value<0.05) (Supplementary Data 3 & 4).

To compare how progenitor and granulosa cell lineages acquire their identity, we classified these genes by expression profile (P) and looked at their expression dynamics along the predicted pseudotime in a single heatmap (Figure 2C) (Supplementary data 3 & 4).

The profiles P1 to P3 represent genes that are over-expressed in the common cells in the undifferentiated gonad, prior to sex determination. These genes are related to mitotic cell division, mesonephros development, positive regulation of stem cell development and epithelium morphogenesis. The profiles P4 to P10 contains genes that exhibit a dynamic expression profile during granulosa cell differentiation. Of interest, the P5 profile contains 100 genes that are transiently over-expressed at the onset of granulosa cell differentiation. Among these genes, we found Dmrt1 (Lindeman et al., 2015; Minkina et al., 2014), Cyp11a1, Lgr4 (known to act as a receptor of RSPO1 and promotes Wnt/beta-catenin signaling(Koizumi et al., 2015)), Oca2 and Tshr (reported to be expressed in granulosa cells (Aghajanova et al., 2009; Sun et al., 2010)). The profiles P6 and P7 contain genes over-expressed in two successive waves in pre-granulosa cells. In P6 we found Wt1, Gata6 (Padua et al., 2014), Lhx9 (Mazaud et al., 2002), Nr0b1, Numb. In P7 we found Axin2, Wnt2b, Irx5 (Nef et al., 2005), Fgfr2. The profile P8 contains genes expressed in both foetal and PND6 granulosa cells. Among them, we found Foxl2, Lhcgr (Edson et al., 2009), Pde8a (Bergeron et al., 2017; Petersen et al., 2015), Cdkn1b (Gustin et al., 2016). And finally the profiles P9 and P10 represent genes that are over-expressed in the post-natal granulosa cells. It includes genes such as Aard, Cited1 (Perlman et al., 2006), Inha, Inhba, Inhbb (Findlay, 1993; Mather et al., 1997), Amh, Nr5a2 (Meinsohn et al., 2018).

In contrast to the highly dynamic program mediating granulosa cell differentiation, the progenitor cell lineage displays much less variation in gene expression during the process of ovarian development. The gene group P11 regroups genes that are expressed in the progenitor cells with various expression profiles. Among them, we found genes known as markers of steroidogenic cell precursors such as Wnt5a (Stévant et al., 2018), Pdgfra (Brennan et al., 2003), Tcf21 (Bhandari et

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al., 2012; Cui et al., 2004), Gli2 (Barsoum and Yao, 2011) and the secreted negative regulators of the Wnt signalling pathway Sfrp1 (Warr et al., 2009). The group P12 is composed of genes that are over-expressed in post-natal progenitors and includes genes known to be present in steroidogenic lineage of gonads such as Ptch1 (Wijgerde et al., 2005), Bgn (Miqueloto and Zorn, 2007), Arx (Miyabayashi et al., 2013), Acta2 (Hatzirodos et al., 2015), Ar. These results suggest that the progenitor cell lineage undergo transcriptional changes that restrict their competence towards a steroidogenic fate required for the differentiation of theca.

Overall, the reconstruction of the Nr5a1-GFP⁺ cell lineage revealed that before female sex determination, we detected a single highly proliferative progenitor cell population. A subset of this population differentiates as pre-granulosa cells, driven by a highly dynamic transcriptional program composed of waves of expression of hundreds of genes, including genes implicated in the Wnt signalling pathway. Conversely, the remaining progenitor cell population operates transcriptomic changes that restrict their competence towards a steroidogenic fate during foetal ovarian development ultimately expressing genes known as makers of steroidogenic cell precursors in the testis. If we set aside the absence of theca, we observed striking similarities between the XX and XY differentiation process of Nr5a1⁺ cell lineage. In both cases, a single Nr5a1⁺ cell population of early progenitors gives rise through cell fate restriction to the supporting cell lineage at the origin of granulosa and Sertoli cells and steroidogenic precursor cells at the origin of foetal Leydig cells and theca cells.

Male and female Nr5a1+ cells comparison

By combining scRNA-seq data from 400 XY Nr5a1-GFP⁺ cells (Stévant et al., 2018) together with the present data from 563 XX Nr5a1-GFP⁺ cells, we undertook to investigate the precise moment of sex determination and characterize the sex-specific differences in each lineage. We merged the two datasets and applied the same clustering method to classify the cells. We selected the highly variable genes and projected them with PCA, and selected the significant PCs to perform hierarchical clustering (Figure 3A) and visualize the cells with t-SNE (Figure 3B&C, Methods).

We noticed that the XX and XY progenitor cell population from E10.5 to P6 cluster together, as well as the E11.5 pre-Sertoli and the pre-granulosa cells. However, the foetal Leydig cells do not constitute a separated cell cluster. This is probably due to the low number of these cells (seven cells) relatively to the entire dataset.

To understand the cell lineage specification we projected the cells on a diffusion map and predicted the lineage trajectories (Figure 3D). The lineage reconstruction predicted one common origin starting from the E10.5 cells irrespective of the genetic sex. We then observed two sequential branching in the Nr5a1-GFP cell lineage. The first bifurcation occurs around E11.5-E12.5 where a branch containing the E11.5 pre-Sertoli cells and the pre-granulosa cells separates from the progenitor cell. The second bifurcation occurs with the separation of the Sertoli cells from the pre-granulosa cells. The two-step branching of the supporting lineage suggests that the transcriptome of pre-Sertoli cells and pre-granulosa cells are similar prior to their differentiation into Sertoli and granulosa cells. The progenitor branch mixes XX and XY cells from the same embryonic stages, suggesting the interstitial and the stromal cells evolve the same way and do not exhibit sexual dimorphisms.

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Supporting cell commitment occurs before sex-dependent differentiation into Sertoli and granulosa cells

To better characterize how the supporting cell lineage commits and differentiate into Sertoli and granulosa cells, we selected XX and XY cells from supporting cell branch as defined in the previous lineage and analyzed the dynamic of gene expression that drives their sex-specific differentiation.

First, we looked at the differentially expressed genes between XX and XY supporting cells at each embryonic stages (Figure 4A). At E11.5, only 10 genes were significantly differentially expressed between XX and XY cells, two over-expressed in XX cells (Gbp2, Gbp5), and eight over-expressed in XY (Ddx3y, Gatm, Hmgcs2, Mamld1, Mmd2, Nfil3, Sox9, Uty). Sry upregulation has not been detected in our analysis probably due to the small number of XY pre-Sertoli cell expressing it (11 cells). By E12.5, the XY supporting cells are acquiring sex dimorphism and the number of over-expressed genes relative to the XX cells increases until E16.5. Sexual dimorphism in XX supporting cells appears delayed and start to be significant from E13.5 (Supplementary Data 4).

In parallel, to compare how the supporting cell lineage acquires either the Sertoli or granulosa identity, we reconstructed the supporting cell lineage and ordered the cells along a pseudotime (Figure 4B&C). As expected, XX and XY supporting cell share initially a common transcriptomic profile and then bifurcate toward either the Sertoli or the granulosa branch. We then performed a differential expression analysis along the pseudotime on the whole transcriptome during the process of supporting cell commitment into either Sertoli or granulosa cells. We identified 1,449 genes presenting a dynamic expression over the pseudotime and classified them according to their expression pattern (P1 to P10; Figure 4D and Supplementary Data 5). The profile P1 contains 182 genes that are over-expressed in both XX and XY early supporting cells. Among them, we found Cyp11a1, Nr5a1, Gadd45g. Of interest, the profile P2 display 79 genes that are expressed in the common supporting cells and are up-regulated in XY committing Sertoli cells but are down-regulated in the XX cells during pre-granulosa commitment. Among them, we found Cited2, Cyp26b1, Ptgr1, and Eno1. Conversely, the profile P6 shows genes that are expressed commonly in the early supporting cells and later stay over-expressed in the granulosa cells only. Among these genes, we found Lhx9, Kitl, Numb, Sall3, Nr0b1, and Wnt4.

With this analysis, we demonstrate that the supporting cells initiate their differentiation from the progenitor cells with a common genetic program that is not sex-independent.

This indicates also that supporting cell commitment is initially sex independent and is disconnected from the sex-specific differentiation into either Sertoli or granulosa cells.

Discussion

Unlike in the testis, foetal ovarian development is characterized by slight and late morphological changes. Consequently, the ovarian development was considered as less active than the testis development (Smith et al., 2014). Transcriptomic analysis showed however that the ovary engages a strong genetic programme as early as E11.5 to initiate its differentiation (Jameson et al., 2012;

Munger et al., 2013; Nef et al., 2005). The pre-granulosa cells are the first female-specific cell type to differentiate. Their differentiation starts from E11.5 from the supporting cells originating from the coelomic epithelium of the genital ridge (Albrecht and Eicher, 2001; Karl and Capel, 1998). A second foetal ovarian cell type expressing Nr2f2 and Maf has been identified and called stromal cells and are suspected to be the theca precursor cells (Jameson et al., 2012; Rastetter et al., 2014;

Takamoto et al., 2005), but their origin remains unclear.

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Herein, we aimed to reconstitute the cell lineage specification of the ovarian somatic cells by analysing the transcriptomes of individual Nr5a1-GFP⁺ cells, from the bipotential gonads at E10.5, to post-natal ovaries at PND6. We found a similar lineage specification process than what we previously observed in the foetal testis (Stévant et al., 2018), with the presence of a common progenitor cell population at E10.5, from which differentiate the pre-granulosa cells with a strong and dynamic genetic program. The remaining progenitor cells that escape pre-granulosa differentiation highly express Nr2f2 and persist in the ovary until as late as PND6 and display progressive transcriptomic changes specifying their identity as potential steroidogenic cell precursors.

We saw that the differentiation of the pre-granulosa cells is driven by the dynamic expression of

We saw that the differentiation of the pre-granulosa cells is driven by the dynamic expression of