domain: sdv sdv.bv sde

text:
Nabil Girollet

Bernadette Rubio

Céline Lopez-Roques

Sophie Valière

Nathalie Ollat

Pierre-François Bert
email: pierre-francois.bert@u-bordeaux.fr

De novo phased assembly of the Vitis riparia grape genome

Grapevine is one of the most important fruit species in the world. In order to better understand genetic basis of traits variation and facilitate the breeding of new genotypes, we sequenced, assembled, and annotated the genome of the american native Vitis riparia, one of the main species used worldwide for rootstock and scion breeding. A total of 164 Gb raw DNA reads were obtained from Vitis riparia resulting in a 225X depth of coverage. We generated a genome assembly of the V. riparia grape de novo using the PacBio long-reads that was phased with the 10x Genomics Chromium linked-reads. At the chromosome level, a 500 Mb genome was generated with a scaffold N50 size of 1 Mb. More than 34% of the whole genome were identified as repeat sequences, and 37,207 protein-coding genes were predicted. This genome assembly sets the stage for comparative genomic analysis of the diversification and adaptation of grapevine and will provide a solid resource for further genetic analysis and breeding of this economically important species.

Background & Summary

Since few decades and the development of sequencing technologies, the number of species whose genome has been totally sequenced has increased exponentially. There is a large variability for the quality of all the sequences assemblies. In 2017, 72 plant reference quality genome assemblies were reported in NCBI [START_REF] Peterson | Sequencing Plant Genomes[END_REF] . For plant breeding, the availability of a contiguous genome sequence provides a tool to better identify genes underlying traits and how they may be regulated by various environmental parameters in different genetic backgrounds. At the simplest, it allows for association of genetic markers for selection and introgression of traits across germplasm to enable the development of novel products for consumers 2,3 .

As an important crop, Vitis vinifera was one of the first higher plant species whose genome was sequenced by a French-Italian consortium 4 . The consortium decided to sequence a near homozygous V. vinifera cultivar related to Pinot Noir (PN40024) in order to facilitate the sequence assembly by limiting sequence variability. To date, this genome still stands as the reference for the grapevine community, but grapevine intra species and interspecies diversity makes using a single reference genome inadequate for studying the function of other genotypes. In order to address the variations in a cultivated V. vinifera variety, the Pinot Noir genome was sequenced using Sanger sequencing providing a high quality draft of the genome with about 10X coverage [START_REF] Velasco | A High Quality Draft Consensus Sequence of the Genome of a Heterozygous Grapevine Variety[END_REF] . Next Generation Sequencing reads are too short to resolve abundant repeats in particular in plants genome, leading to incomplete or ambiguous assemblies 6 . Few attempts to produce high quality grapevine genomes were undertaken in grapevine and produced valuable data to study the genetic variations of V. vinifera cv. Tannat 7 and cv. Thompson seedless 8 through comparison with the reference genomes.

The last few years have seen rapid innovations in sequencing technologies and improvement in assembly algorithms that enabled the creation of highly contiguous genomes. The development of third generation sequencing technologies that deliver long reads from single molecules and carry the necessary information to phase haplotypes over several kilobases have greatly improved the feasibility of de novo assemblies [9][START_REF] Li | Genome Sequencing and Assembly by Long Reads in Plants[END_REF][11] . Sequences of V. vinifera cv. Cabernet Sauvignon were first released 12 using PacBio sequencing and FALCON, and FALCON-Unzip pipeline 12 . This generated a 591 Mbp haplotype genome from a set of 718 primary contigs, and a set of correlated 2,037 haplotigs spanning 367 Mbp. The total p-contig size was larger than the estimated genome size of V. vinifera (~500 Mbp) suggesting that in some cases FALCON-Unzip underestimated the alternative haplotype sequences because of high heterozygosity between homologous regions, which is common in grapevine [START_REF] Roach | Purge Haplotigs: allelic contig reassignment for third-gen diploid genome assemblies[END_REF][START_REF] Vinson | Assembly of polymorphic genomes: algorithms and application to Ciona savignyi[END_REF] . Later, the PacBio assembly and annotation of V. vinifera cv Chardonnay variety provided after curation of artefactual contig assignment, 854 p-tigs and 1883 h-tigs, totaling 490 Mb and 378 Mb 15 . More recently, another version of the Chardonnay genome was proposed with a different level of curation at 605 Mb 16 .

An evaluation of genetic diversity based on a panel of 783 V. vinifera varieties using 10 K SNPs revealed a high level of diversity (He = 0.32) and confirmed the close pedigree relationship within the cultivated grapevine due to the wide use of the most interesting parents during domestication and early selection by humans [START_REF] Laucou | Extended diversity analysis of cultivated grapevine Vitis vinifera with 10 K genome-wide SNPs[END_REF] . Considering that grape cultivation currently faces severe pathogen pressures and climate change, we assume that the exploitation of the natural genetic diversity may ensure the long-term sustainability of the grape and wine industries [START_REF] Myles | Genetic structure and domestication history of the grape[END_REF] . Grapes belong to the genus Vitis, which includes over 60 inter-fertile species. The most common grape cultivars derive their entire ancestry from the species V. vinifera, but wild relatives have also been exploited to create hybrid cultivars, often with increased disease resistances [START_REF] Migicovsky | Genomic ancestry estimation quantifies use of wild species in grape breeding[END_REF] .

To date, no wild Vitis genomes have been released so far and the only whole genome sequences for grape are from V. vinifera varieties and yet there is a clear need for genetic resources [START_REF]FAO Commission on genetic resources for food and agriculture assessment[END_REF] . Here, we report the first de novo assembly and genome annotation of the North American native grape V. riparia. Using the latest sequencing technologies, we show that 10x Genomics Chromium data can be combined with long read PacBio sequencing to effectively determine genome phasing. The phased haplotypes of V. riparia genome will greatly contribute to give more insight into the functional consequences of genetic variants.

Methods

Sample collection, library construction and whole genome sequencing. The Vitis riparia Gloire de Montpellier (RGM) selection was obtained in 1880 by L. Vialla and R. Michel from North American collections and is the only commercially available pure V. riparia stock. RGM clone #1030 and the European native Vitis vinifera Cabernet sauvignon (CS) clone #15 were grown at INRA, Bordeaux (France). A F1 segregating population of 114 individuals named CSxRGM1995-1 was derived from the cross between CS and RGM [START_REF] Marguerit | Genetic dissection of sex determinism, inflorescence morphology and downy mildew resistance in grapevine[END_REF] . This population was genotyped using the GBS approach [START_REF] Elshire | A robust, simple genotyping-by-sequencing (GBS) approach for high diversity species[END_REF] to create a high resolution genetic map to assist in anchoring and orienting the assembled V. riparia genome scaffolds.

Total DNA was isolated and extracted using QIAGEN Genomic-tips 100/G kit (Cat No./ID: 10243) following the tissue protocol extraction. Briefly, 1 g of young leaf material was ground in liquid nitrogen with mortar and pestle. After 3 h of lysis and one centrifugation step, the DNA was immobilized on the column. After several washing steps, DNA is eluted from the column, then desalted and concentrated by alcohol precipitation. The pellet is resuspended in TE buffer.

Three PacBio libraries with a 20-kb insert size were also constructed and sequenced on RSII platforms (97.71 Gb data; ~118-fold covering), following the standard PacBio protocol of Sequencing Kit 1.2.1 (Pacific Biosciences, USA). Four 10x Chromium Genomics libraries were constructed using the Chromium TM Genome Solution (10X Genomics, USA), and 2 × 150 bp sequenced on Illumina HiSeq3000, producing ~350 million paired-end linked-reads (~ 107-fold covering). Finally, 2 libraries for 2 × 100 bp sequencing were built with different insert sizes: 500 bp for paired-end (PE) and 6 kb for mate-pair (MP), based on the standard Illumina protocol and sequenced on the Illumina HiSeq2500. The raw reads were trimmed before being used for subsequent genome assembly. For Illumina HiSeq sequencing, the adaptor sequences, the reads containing more than 10% ambiguous nucleotides, as well as the reads containing more than 20% low-quality nucleotides (quality score less than 5), were all removed. After data cleaning and data preprocessing, we obtained a total of 164 Gb of clean data (52 Gb PacBio data, 59 Gb 10X Genomics, 33 Gb PE reads and 20 Gb MP reads,), representing 331X coverage of the V. riparia genome (Table 1).

Genome size and heterozygosity estimation. Lodhi and Reisch 23 estimated the genome size in grape to be approximately 475 Mb based on measurements using flow cytometry for 19 species including wild Vitis species, V. vinifera and V. labrusca cultivars. The measurements showed intraspecific variation in genome size between different varieties of Vitis vinifera ranging from 1C = 415 to 511 Mb, and between different North America Vitis species ranging from 1C = 411 to 541 Mb, with V. riparia around 470 Mb. Genome sequencing of different V. vinifera varieties gave values in the same range or greater depending on the methods of sequencing and assembly. In order to verify these values, we estimated genome size of V. riparia by the k-mer method [START_REF] Liu | Estimation of genomic characteristics by analyzing k-mer frequency in de novo genome projects[END_REF][START_REF] Marcais | A fast, lock-free approach for efficient parallel counting of occurrences of k-mers[END_REF] using data from pair-end and mate-pair Illumina sequencing. By analyzing the 21-mers depth distribution, a total of ~50 billion k-mers were estimated with a peak frequency of 100, corresponding to a genome size of 494 Mb and the estimated repeat sequencing ratio was 33.74%. In this study, V. riparia heterozygosity was estimated to be 0.46% (mean distance 1 SNP each 217 bp between heterozygous SNPs) from 10x Chromium Genomics data processing.

De novo Genome assembly and scaffolding of the Vitis riparia genome. We employed a hybrid de novo whole-genome assembly strategy, combining both short linked-reads and PacBio long reads data. Genome assembly was first performed on full PacBio cleaned reads using FALCON v0.3.0 26 . Error correction and pre-assembly were carried out with the FALCON/FALCON Unzip pipeline after evaluating the outcomes of using different parameters in FALCON during the pre-assembly process. Based on the contig N50 results, a length_cutoff of 5 kb and a length_cutoff_pr of 8 kb for the assembly step were ultimately chosen. The draft assembly was polished using Quiver [START_REF]SMRT tools[END_REF] , which mapped the PacBio reads to the assembled genome with the BLASER pipeline [START_REF] Chaisson | Mapping single molecule sequencing reads using basic local alignment with successive refinement (BLASR): application and theory[END_REF] .

Haplotypes were separated during assembly using FALCON-Unzip and the preliminary genome assembly was approximately 530 Mb (1,964 primary-contigs) and 317 Mb (3,344 haplotigs). A summary of the assembly statistics can be found in Table 1. Assembly was then processed with Purge Haplotigs [START_REF] Roach | Purge Haplotigs: allelic contig reassignment for third-gen diploid genome assemblies[END_REF] to investigate the proper assignment of contigs, followed by 2 rounds of polishing to correct residual SNP and INDELs errors with Pilon v1.22 software 29 using high-coverage (~106X) Illumina paired-end and mate pair data. The 10x Chromium Genomics linked-reads were used to produce a separate V. riparia assembly using the Supernova assembler option-style = pseudohap2 and created two parallel pseudohaplotypes [START_REF] Weisenfeld | Direct determination of diploid genome sequences[END_REF] . The mean input DNA molecule length reported by the Supernova assembler was 45 kb and the assembled genome size was 424 Mb with a N50 scaffold of 711 kb.

Subsequently, the PacBio assembly was scaffolded with the 10x Chromium Genomics one using the hybrid assembler LINKS 31 with 7 iterations, producing 870 scaffolds spanning 500 Mb with N50 = 964 kb and L50 = 255 (Table 2). Finally, genome phasing was reconstituted using Long Ranger analysis pipeline that processes Chromium sequencing output to align reads and call and phase SNPs, indels, and structural variants on the basis of molecular barcodes information.

Genotyping by Sequencing and genetic mapping. Two 96-plex GBS libraries (Keygene N.V. owns patents and patent applications protecting its Sequence Based Genotyping technologies) were constructed for the two parents (two replicates for each) and the 114 F1 plants of the cross CS × RGM. Raw reads were checked with FastQC 32 , demultiplexed with a custom script and cleaned with CutAdapt [START_REF] Martin | Cutadapt removes adapter sequences from high-throughput sequencing reads[END_REF] . Cleaned reads were then mapped to the V. riparia RGM scaffolds previously obtained, the V. vinifera Cabernet Sauvignon contigs 12 and V. vinifera PN40024 genome assemblies 4 for SNP calling. Aligned on these genomes were performed using BWA [START_REF] Li | The Sequence Alignment/Map format and SAMtools[END_REF] , SAMtools [START_REF] Li | Fast and accurate long-read alignment with Burrows-Wheeler transform[END_REF] and Picard tools [START_REF]Available from[END_REF] and SNP genotypes were detected with GATK 37 using the hardfilter parameters [START_REF] Van Der Auwera | From FastQ Data to High-Confidence Variant Calls: The Genome Analysis Toolkit Best Practices Pipeline[END_REF] . In the variant call format (VCF) output file only sites with less than 20% missing data and a minimum allele frequency (MAF) ≥ 0.2 were retained. The SNP set was parsed into two data sets based on a pseudo-test cross mapping strategy [START_REF] Grattapaglia | Genetic mapping of QTLs controlling vegetative propagation in Eucalyptus grandis and E. urophylla using a pseudo-testcross mapping strategy and RAPD markers[END_REF] using major_minor and get_pseudo_test_cross scripts from Hetmapps [START_REF] Hyma | Heterozygous mapping strategy (HetMappS) for high resolution Genotyping-By-Sequencing markers: a case study in grapevine[END_REF] . The segregation ratios of markers in the population were examined by Chi-square analysis. Markers with segregation ratios that differed from expected 1:1 at P < 0.05 were classified as segregation distortion markers and discarded. The RGM and CS sets contain 1591 and 2359 SNPs respectively. Linkage groups (LGs) were determined using software JoinMap ® 4.1 41,42 and Rqtl 43 .

LG were formed with a logarithm of odds (LOD) threshold of 6 and a maximum recombination frequency of 0.45. The 19 LGs that corresponded to the 19 chromosomes of grapevine were reconstructed and leaded to a total genetic map length of 2,268 cM and 2,514 cM for RGM and CS respectively.

Pseudo-molecule construction. The PacBio/10x Chromium Genomics hybrid scaffolding was organized into pseudo-molecules using GBS markers information from the CS × RGM genetic map. Scaffolds were anchored and oriented SNP using AllMaps [START_REF] Tang | ALLMAPS: robust scaffold ordering based on multiple maps[END_REF] with the unequal weights2 parameters for a single run for the entire genome. Final pseudo-molecules were named according to Vitis vinifera PN40024 reference genome using SNP identification through SNP calling on this reference. Since PN40024 genome is the only one available who has been scaffolded into pseudo-molecules, collinearity with V. riparia was evaluated using D-GENIES [START_REF] Cabanettes | dot plot large genomes in an interactive, efficient and simple way[END_REF] and showed extremely high conservation along the 19 chromosomes of the species (Fig. 1) even if the North American and Eurasian Vitis species diverged approximately 46.9 million years ago [START_REF] Ma | Phylogenomics, biogeography, and adaptive radiation of grapes[END_REF] .

Genome annotation and gene prediction.

Consistent with observations that long reads sequencing technologies are a better solution for resolving repeat sequences, we found that known repetitive elements accounted for 170 Mb (33.94%) of the genome in V. riparia. This is a lower proportion among grape genomes when comparing published values to date. However, when comparisons are performed with the same analysis workflow and tools [START_REF] Tarailo-Graovac | Using RepeatMasker to identify repetitive elements in genomic sequences[END_REF][START_REF] Bao | Repbase Update, a database of repetitive elements in eukaryotic genomes[END_REF] , the percentages obtained between the two genotypes were in the same range (Online-only After repeat masking, the genome was ab initio annotated using MAKER-P pipeline [START_REF] Campbell | Genome Annotation and Curation Using MAKER and MAKER-P[END_REF][START_REF] Campbell | MAKER-P: a tool kit for the rapid creation, management, and quality control of plant genome annotations[END_REF] , SNAP [START_REF] Korf | Gene finding in novel genomes[END_REF] and Augustus 52 gene finder with 3 rounds of Maker and an Augustus prediction. Structural annotation was then followed with an Interproscan functional annotation and putative gene function assignation using BLAST on UniProtKB. MAKER-P quality metrics with a threshold of AED < 0.5 were chosen to retain the set of predicted genes. We finally generated a gene set of 37,207 protein-coding genes (11,434_AED < 0.1; 8,638_0.1 ≤ AED < 0.2; 5,748_0.2 ≤ AED < 0.3; 5,418_0.3 ≤ AED < 0.4; 5,969_0.4 ≤ AED < 0.5) with 31,240 of them coupled with an evidence of protein function.

To facilitate genomic investigations for the community, a JBrowse Genome Browser 53 was set up for V. riparia pseudo-molecules and is available from https://www6.bordeaux-aquitaine.inra.fr/egfv/.

Data Records

The V. riparia genome project was deposited at NCBI under BioProject number PRJNA512170 and BioSample SAMN10662253. The DNA sequencing data from Illumina, PacBio and 10x Genomics have been deposited in the Sequence Read Archive (SRA) database under accession SRP174866 54 from SRX5189632 to SRX5189680. This Whole Genome Shotgun project has been deposited at DDBJ/ENA/GenBank under the accession SJAQ00000000 [START_REF] Bert | Vitis riparia cultivar Riparia Gloire de Montpellier isolate 1030, whole genome shotgun sequencing project[END_REF] . The versions described in this paper are version SJAQ01000000 and genome ID 13150 (https:// www.ncbi.nlm.nih.gov/genome/13150). Genetic mapping data and structural and functional annotation file of the Vitis riparia assembly are available on figshare [START_REF] Girollet | De novo phased assembly of the Vitis riparia grape genome[END_REF] .

technical Validation

To evaluate the accuracy and completeness of the V. riparia assembly, genome features were compared to those of V. vinifera (Table 2). We found that both contig and scaffold N50 lengths of Vitis riparia reached considerable continuity. The Guanine-Cytosine content (GC = 34.32%) was similar to those of V. vinifera Chardonnay (34.43%). To further assess the accuracy of the V. riparia genome assembly, the NGS-based short reads from whole-genome sequencing data were also aligned against the genome assembly using BWA mem [START_REF] Li | Aligning sequence reads, clone sequences and assembly contigs with BWA-MEM[END_REF] . We found that 98.4% of the reads were reliably aligned to the genome assembly, and 95.8% of the reads were properly aligned to the genome with their mates. Paired-end reads data were not used during the contig assembly, thus the high alignment ratio demonstrated the high quality of contig assembly.

The assembled genome was also subjected to Benchmarking Universal Single-Copy Orthologs [START_REF] Simão | BUSCO: assessing genome assembly and annotation completeness with single-copy orthologs[END_REF] , which quantitatively assesses genome completeness using evolutionarily informed expectations of gene content from near-universal single-copy orthologs, using the genes in the embryophyta release 9 dataset (embryophyta.odb9). The BUSCO results showed that 96.5% of conserved BUSCO proteins were detected in the V. riparia assembly, including 1.1% of fragment BUSCO proteins (Table 2). Overall, these metrics compare well with other recently published grape genomes, providing a high quality genome sequences for the following functional investigations. 17. Interproscan functional annotation and putative gene function assignation Download protein DB from http://www.uniprot.org makeblastdb -in protein_db.fasta -input_type fasta -dbtype prot blastp -db protein_db.fasta -query maker3.proteins.fasta -out maker3.proteins.blastp -evalue 0.000001 -outfmt 6 -max_hsps 1 maker_functional_gff protein_db.fasta maker3.proteins.blastp maker3.gff3 ≫ maker3.putative.gff3 maker_functional_fasta protein_db.fasta maker3.proteins.blastp maker3.proteins.fasta ≫ maker3.putative.proteins.fa maker_functional_fasta protein_db.fasta maker3.proteins.blastp maker3.transcripts.fasta ≫ maker3.putative. transcripts.fa Run Interproscan interproscan.sh -iprlookup -goterms -f tsv -i maker3.putative.proteins.fa -pa -b RGM.annotated.proteins 18. Assembly validation using WBA mem bwa mem -M -t 20 VitRiparia.fasta reads_pe.R1.fastq reads_pe.R2.fastq > aln_pe_reads.sam samtools view -bS aln_pe_reads.sam -o aln_pe_reads.bam #| samtools sort -aln_reads.sorted.bam samtools sort -o aln_pe_reads.sorted.bam aln_pe_reads.bam bamtools stats -in aln_pe_reads.sorted.bam > bamstat_pe.reads

Fig. 1

 1 Fig. 1 Comparison of Vitis riparia hybrid scaffolds with the reference PN40024 assembly. Hybrid scaffolds (Y-axis) were aligned to all 19 PN40024 chromosomes (X-axis) using D-GENIES and alignments were subsequently filtered for 1-on-1 alignments and rearrangements with a 20 Kbps length cutoff.

Table 1 .

 1 Data count and library informations for Vitis riparia genome sequencing.

	Sequencing			Number of	Number of	Sequence
	platform	Insert size (bp)	Read length (bp)	sequences (million)	bases (billion)	depth	Application
	PacBio	NA	7054	8.3	59	118X	Genome assembly
	10X Chromium 400	2 × 150	350	52	107X	Genome scaffolding and phasing
	Illumina	400 (pair end) 6,000 (mate pair) 2 × 100 2 × 100	331 200	33 20	66X 40X	Genome survey and genomic base correction
	Total				164	331X

Table 2 .

 2 Summary of the V. riparia genome assembly and comparison with with V. vinifera varieties.

Table 1 )

 1 . Similar to other grape genomes, long terminal repeat (LTR) elements constituted the highest proportion of all repeated elements in V. riparia, (21.44%) with Copia and Gypsy families accounting for 8.33% and 12.66% respectively. The Long Interspersed Nuclear Elements (LINEs) and Miniature Inverted-repeat Transposable Elements (MITEs) represented 3.61% and 6.02% of the whole genome respectively.

Scientific Data | (2019) 6:127 | https://doi.org/10.1038/s41597-019-0133-3

acknowledgements

We thank Dr. Gregory Gambetta for critical review of the manuscript. We thank the INRA Genotoul bioinformatics platform (http://genotoul.toulouse.inra.fr) for providing computational resources and support and INRA-CNRGV (http://cnrgv.toulouse.inra.fr/) for V. riparia HMW DNA extraction. Sequencing was performed at the GeT core facility, Toulouse, France, supported in part by France Génomique National infrastructure (contract ANR-10-INBS-09). We thank Dr. Timothée Flutre and Amandine Launay for their help in coordinating the FruitSelGen project and in acquiring GBS data. This work was supported by a grant overseen by the French National Research Agency (ANR) as part of the ANR-09-GENM-024 "Vitsec" program and by the University of Bordeaux.

Code availability

1. GBS demultiplexing

additional Information

Competing Interests: The authors declare no competing interests.

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S P I Vidal

E P Sacco

Grégoire

Eric Wilhelm

Pailhoux

M-C De Cian

E Pauper

R Bandiera

Vpi Vidal

S Sacco

E P Gregoire

A-A Chassot

C Panzolini

D Wilhelm

E Pailhoux

S A Youssef

A De Bruin

K Teerds

A Schedl

Dr I Gillot
email: gillot@unice.fr

Dr M-C Chaboissier

Amplification of R-spondin1 signaling induces granulosa cell fate defects and cancers in mouse adult ovary

Amplification of R-spondin1 signaling induces granulosa cell fate defects and cancers in mouse adult ovary

INTRODUCTION

WNT/β-catenin (CTNNB1) signaling is involved in numerous biological processes from embryogenesis to stem cell activation. Deregulation of this pathway is responsible for many diseases, most notably colorectal cancer. Indeed, 90% of colorectal cancers result from APC mutations leading to the activation of this pathway. [START_REF] Clevers | Wnt/beta-catenin signaling and disease[END_REF] WNT signaling is tightly controlled by negative and positive modulators like APC and R-spondins (RSPO), a family of four members of secreted proteins. Fusions of RSPO2 and RSPO3 activate WNT signaling in colorectal cancers, [START_REF] Seshagiri | Recurrent R-spondin fusions in colon cancer[END_REF] and amplifications of RSPO1 have been associated with 8% of ovarian epithelial cancers. [START_REF] Kuchenbaecker | Identification of six new susceptibility loci for invasive epithelial ovarian cancer[END_REF][START_REF] Gao | Integrative analysis of complex cancer genomics and clinical profiles using the cBioPortal[END_REF] However, functional evidence for a direct involvement of RSPO1 in ovarian tumor development is still missing.

Depending on the cell type, Rspo1 may act either as a mitogen or a tumor suppressor: [START_REF] Kim | Mitogenic influence of human R-spondin1 on the intestinal epithelium[END_REF][START_REF] Parma | R-spondin1 is essential in sex determination, skin differentiation and malignancy[END_REF] variants of the RSPO1 locus have been reported in ovarian cancers highlighting its role as an oncogene. [START_REF] Kuchenbaecker | Identification of six new susceptibility loci for invasive epithelial ovarian cancer[END_REF][START_REF] Gao | Integrative analysis of complex cancer genomics and clinical profiles using the cBioPortal[END_REF] By contrast, RSPO1 loss-of-function mutations predispose to squamous cell carcinoma. [START_REF] Parma | R-spondin1 is essential in sex determination, skin differentiation and malignancy[END_REF] On the molecular level, RSPO1 binds to the recruitment receptors LGR4, LGR5 or LGR6 leading to stabilization of CTNNB1, and in turn CTNNB1 interacts with transcription factors such as LEF/TCF in the nucleus and trans-activates target genes such as Axin2 (for a model, see ref. 7).

CTNNB1 is not only a key factor of the WNT signal-transduction pathway, but is also a component of adherens junctions linking cadherins via α-catenin to the actin cytoskeleton at the plasma membrane of epithelial cells. [START_REF] Kemler | From cadherins to catenins: cytoplasmic protein interactions and regulation of cell adhesion[END_REF] The membrane function of CTNNB1 can be partially substituted by the related protein JUP (Plakoglobin/ γ-catenin), an important component of desmosomes that anchors epithelial cells to intermediate filaments and can also act as a signaling molecule at least in vitro. [START_REF] Lyashenko | Differential requirement for the dual functions of beta-catenin in embryonic stem cell self-renewal and germ layer formation[END_REF][START_REF] Maeda | Plakoglobin (gamma-catenin) has TCF/LEF family-dependent transcriptional activity in betacatenin-deficient cell line[END_REF] Several studies support a role for JUP in cancer. [START_REF] Chidgey | Desmosomes: a role in cancer?[END_REF] Although a direct link between RSPO1 and JUP has not yet been established, skin cells from patients affected by RSPO1 loss-of-function mutations exhibit defects reminiscent of desmosome abnormalities. [START_REF] Parma | R-spondin1 is essential in sex determination, skin differentiation and malignancy[END_REF] In addition to the skin diseases, such as palmoplantar hyperkeratosis, patients carrying mutations in RSPO1 exhibit female-to-male sex reversal, and predisposition to squamous cell carcinoma and seminoma. [START_REF] Parma | R-spondin1 is essential in sex determination, skin differentiation and malignancy[END_REF][START_REF] Tomaselli | Syndromic true hermaphroditism due to an R-spondin1 (RSPO1) homozygous mutation[END_REF] Using functional analysis in mice, the role of RSPO1 and canonical WNT signaling has been unraveled in female ovarian development. [START_REF] Maatouk | Stabilization of beta-catenin in XY gonads causes male-to-female sex-reversal[END_REF][START_REF] Chassot | Activation of beta-catenin signaling by Rspo1 controls differentiation of the mammalian ovary[END_REF][START_REF] Tomizuka | R-spondin1 plays an essential role in ovarian development through positively regulating Wnt-4 signaling[END_REF] Prior to sex determination, Rspo1 is involved in the proliferation of progenitor cells that are located within the surface epithelium of the undifferentiated gonads. [START_REF] Karl | Sertoli cells of the mouse testis originate from the coelomic epithelium[END_REF][START_REF] Chassot | WNT4 and RSPO1 together are required for cell proliferation in the early mouse gonad[END_REF] Following sex determination, cells of the ovarian surface epithelium (OSE) continue to proliferate and enter the gonads to give rise to precursors of the granulosa cells. [START_REF] Mork | Temporal differences in granulosa cell specification in the ovary reflect distinct follicle fates in mice[END_REF][START_REF] Zheng | Two classes of ovarian primordial follicles exhibit distinct developmental dynamics and physiological functions[END_REF] At this stage, Rspo1 is required to maintain granulosa precursors in the undifferentiated state. [START_REF] Chassot | Activation of beta-catenin signaling by Rspo1 controls differentiation of the mammalian ovary[END_REF][START_REF] Maatouk | Disruption of mitotic arrest precedes precocious differentiation and transdifferentiation of pregranulosa cells in the perinatal Wnt4 mutant ovary[END_REF] Accordingly, LGR4-and LGR5-expressing precursor cells contribute to the formation of the follicle pool from which follicles are recruited during reproductive life. [START_REF] Rastetter | Marker genes identify three somatic cell types in the fetal mouse ovary[END_REF] In mice, Rspo1 expression becomes significantly downregulated in the first week post partum, [START_REF] Chassot | Loss of R-spondin1 and Foxl2 amplifies female-to-male sex reversal in XX mice[END_REF] suggesting that RSPO1 is not required for the recruitment and maturation of follicles, and consequently proper ovarian functioning.

From the onset of puberty, the ovary undergoes successive cycles of follicular recruitment, maturation, OSE exfoliation, subsequent ovulation and corpus luteum (CL) formation. [START_REF] Hirshfield | Overview of ovarian follicular development: considerations for the toxicologist[END_REF] Progesterone synthetized by the CL is necessary to prepare the uterine wall for implantation. LGR5-positive OSE cells are presumed to contribute to both post-ovulatory repair of the OSE [START_REF] Ng | Lgr5 marks stem/progenitor cells in ovary and tubal epithelia[END_REF] and, together with LGR4-positive lutein cells, progesterone production. [START_REF] Pan | Lgr4 gene regulates corpus luteum maturation through modulation of the WNT-mediated EGFR-ERK signaling pathway[END_REF][START_REF] Sun | Ovarian LGR5 is critical for successful pregnancy[END_REF] Activation of canonical WNT signaling must therefore be tightly controlled in the adult ovary. Consequently premature activation of canonical WNT signaling in differentiating granulosa cells leads to the appearance of precancerous follicle-like lesions around 6 weeks post partum, and 57% of these become granulosa cell tumors (GCTs) before the age of 7.5 months. [START_REF] Boerboom | Misregulated Wnt/beta-catenin signaling leads to ovarian granulosa cell tumor development[END_REF] In women, ovarian cancers derive from three major histotypes: surface epithelia, sex-chord stromal cells and germ cells. [START_REF] Mullany | Minireview: Animal Models and Mechanisms of Ovarian Cancer Development[END_REF] Approximately 3% of ovarian cancers are derived from germ cells, 7% come from sex-chord stromal cells and 90% arise from OSE or fallopian tube epithelium. [START_REF] Mullany | Minireview: Animal Models and Mechanisms of Ovarian Cancer Development[END_REF] GCTs are categorized into two distinct subtypes, the juvenile and the adult form. [START_REF] Jamieson | Molecular pathogenesis of granulosa cell tumors of the ovary[END_REF] Although less common than OSE-derived cancers in women, GCT frequently occurs in primates and domestic animals. [START_REF] Mullany | Minireview: Animal Models and Mechanisms of Ovarian Cancer Development[END_REF] Moreover, in mouse models adult granulosa cells originate from two distinct waves of migration of progenitor cells of the OSE. [START_REF] Mork | Temporal differences in granulosa cell specification in the ovary reflect distinct follicle fates in mice[END_REF] Given that (i) ectopic activation of canonical WNT signaling within the ovary has oncogenic effects, [START_REF] Boerboom | Misregulated Wnt/beta-catenin signaling leads to ovarian granulosa cell tumor development[END_REF] (ii) RSPO1 is an activator of canonical WNT signaling during ovarian development [START_REF] Chassot | Activation of beta-catenin signaling by Rspo1 controls differentiation of the mammalian ovary[END_REF] and (iii) RSPO1 is amplified in ovarian cancers, [START_REF] Kuchenbaecker | Identification of six new susceptibility loci for invasive epithelial ovarian cancer[END_REF] we hypothesized that RSPO1 misexpression could be a cause of ovarian defects and eventually cancer development. Using a gain-of-function mouse model (Sf1:Cre Tg/+ ; R26 Rspo1/+ ), we show that post partum maintenance of Rspo1 expression in somatic cells of the mouse ovary impairs homeostasis of the healthy ovary.

RESULTS

Rspo1 expression is downregulated in adult follicles RSPO1/Rspo1 is highly expressed in somatic cells of the ovary during embryogenesis. [START_REF] Parma | R-spondin1 is essential in sex determination, skin differentiation and malignancy[END_REF][START_REF] Tomaselli | Human RSPO1/R-spondin1 is expressed during early ovary development and augments beta-catenin signaling[END_REF] In the 2 weeks following birth, however, Rspo1 expression is downregulated as evidenced by qRT-PCR analysis. [START_REF] Chassot | Loss of R-spondin1 and Foxl2 amplifies female-to-male sex reversal in XX mice[END_REF] To obtain a more detailed expression profile and identify the cell types that express Rspo1 late in development and during the postnatal period, we performed in situ hybridization experiments. Strong expression of Rspo1 was found within the fetal ovary at 16.5 days post coitum (dpc), which significantly decreased after birth and remained at low levels in granulosa cells of cortical follicles in adult ovaries (Figures 1a-c). In contrast, Rspo1 was robustly expressed in the OSE at all stages (Figures 1a-c). To understand whether RSPO1 was able to activate canonical WNT signaling, we next analyzed whether the Axin2 +/LacZ reporter of this pathway [START_REF] Lustig | Negative feedback loop of Wnt signaling through upregulation of conductin/axin2 in colorectal and liver tumors[END_REF] was expressed in the adult ovary. β-Galactosidase staining was detected in the OSE and in the ovarian stroma as well as in oocytes (Figures 1e-g). By contrast, granulosa cells were weakly stained after birth, indicating that RSPO1/WNT signaling is downregulated in this compartment of the follicle.

Increased RSPO1 expression activates canonical WNT signaling in adult ovaries

To assess the importance of Rspo1 repression in the postnatal ovary, we next investigated the effect of induced Rspo1 expression on ovarian homeostasis. To this end, we generated Sf1-Cre Tg/+ ; R26 Rspo1/+ mice (Supplementary Figure S1). We first established the ability of female Sf1-Cre Tg/+ ; R26 Rspo1/+ mice to ectopically express Rspo1 throughout the ovary (Figures 1c andd). Next, we assessed that induced expression of Rspo1 led to a strong, although variable, follicular activation of canonical WNT signaling by detecting β-galactosidase in ovaries from Sf1-Cre Tg/+ ; R26 Rspo1/+ ; Axin2 +/LacZ mice (Figures 1g andh). This confirmed that sustained expression of Rspo1 activates canonical WNT signaling in ovaries. Rspo1 is expressed at low level in the R26 Rspo1/+ mouse adult ovary, mainly in the epithelial surface and in granulosa cells of some follicles in the ovarian cortex, whereas adult Sf1-Cre Tg/+ ; R26 Rspo1/+ ovaries display Rspo1 expression throughout the whole organ (d). Activation of the canonical WNT signaling by RSPO1 visualized by β-galactosidase staining of ovaries expressing the Axin2-LacZ reporter. Axin2-LacZ is expressed at 2.5 and 12 months in pre-antral follicles and the stroma (e and g) and strongly expressed in oocytes and the epithelial surface surrounding the ovary (e and f, arrow) in R26 Rspo1/+ mice. Induced Rspo1 expression leads to Axin2-LacZ expression in the whole Sf1-Cre Tg/+ ; R26 Rspo1/+ ovary (h) with strong staining in granulosa cells (arrow) and weaker staining in aberrant follicular structures lesions (*). Scale bars: 300 μm.

Rspo1

Rspo1 activation induces ovarian defects, subfertility and cancer To evaluate the effect of RSPO1 expression on ovarian function we first assessed the fertility of Sf1-Cre Tg/+ ; R26 Rspo1/+ female mice. Litter sizes were significantly reduced with only 1.25 pup/female (±1.58) compared with 7.25 pups/female (±1.16) for R26 Rspo1/+ littermates (n = 8 plugged female per genotype). By the age of 6 months double-transgenic mice were all sterile (n = 8). Macroscopic observation of dissected ovaries revealed a marked increase in the size of mutant ovaries when compared with those of the control littermates (Figures 2a-c). The size of the transgenic ovaries increased over time (data not shown).

To gain insides into the ovarian phenotype, we examined 170 haematoxylin and eosin-stained slides containing serial sections from 14 murine ovaries from Sf1-Cre Tg/+ ; R26 Rspo1/+ mice. Histological analysis revealed a variety of abnormalities, ranging from 100% penetrance of dense follicle-like lesions to tumors in about 10% of ovaries (Figure 2). The ovarian tissue presented multifocal aggregates of neoplastic cells that arrange either in discrete solid aggregates, solid sheets or in microfollicular pattern (Figures 2f-h) reminiscent to granulosa cells. They are bland, ovoid, have distinct borders, scant amount of cytoplasm and ovoid nuclei. On the basis of these observations, we conclude that GCTs accumulate in Sf1-Cre Tg/+ ; R26 Rspo1/+ mouse ovaries. None of these abnormalities were detected in control ovaries (Figure 2d). A proportion of these follicle-like structures developed into blood-filled cysts (Figures 2i andj) surrounded by cuboidal cells (Figure 2j) that were never observed in ovaries from control animals. In addition, fewer corpora lutea (CLs) were observed which is in agreement with the reduced fertility of the Sf1-Cre Tg/+ ; R26 Rspo1/+ females (Figure 2e). A proportion of regressing CLs contained erythrocytes infiltration (Figures 2l andm). One-year-old animals carried bigger cysts (Figures 2k andn), which, in some cases, contained layered keratinocytes in the cavity (Figure 2n). In addition to the accumulation of GCT, solid tumors were observed in 12-18-month-old females with 1 tumor/13 Sf1-Cre Tg/+ ; R26 Rspo1/+ and 3/19 Sf1-Cre Tg/+ ; R26 Rspo1/Rspo1 females, whereas 0/22 control littermates were scored (Figure 2c). The severity of the different defects varied between animals and was not linked to whether they were heterozygous or homozygous for the Rosa26-Rspo1 transgene.

We next analyzed whether GCTs were proliferative or undergoing apoptosis in 40 days post partum (dpp) Sf1-Cre Tg/+ ; R26 Rspo1/+ ovaries using immunostaining for MKI67 and active CASP3, respectively, two markers of these processes. Only occasionally, we observed a cell expressing one of these markers in the GCT, implying that cell proliferation and apoptosis were negligible in these structures (Supplementary Figures S2A-D). Interestingly, increased proliferation was detected in the stromal compartment of Sf1-Cre Tg/+ ; R26 Rspo1/+ ovaries (Supplementary Figures S2A andB). Consistent with this proliferative activity and clear staining for the theca cell marker SF1 (Supplementary Figures S2E andF), the theca cell layer seems to be expanded in size as indicated by SF1 staining (Supplementary Figure S2F).

We conclude that Rspo1 acts as an oncogene in the ovary because its upregulation leads to ovarian tumor development.

Ovarian defects induced by RSPO1 expression occur from the onset of puberty

We next examined the development of the ovarian phenotype in Sf1-Cre Tg/+ ; R26 Rspo1/+ mice before and soon after pubertal age. Immunostaining revealed that follicle-like structures are composed of FOXL2-positive granulosa cells (Figures 3e andf) indicating they are indeed aberrant follicles. To investigate when these abnormal follicular structures first appear, we analyzed histological sections at different stages. Pre-pubertal ovaries (21 dpp) appeared normal and no follicular lesions were detected (Figures 5a andb). The first phenotypic changes became apparent in 28 dpp ovaries (n = 6; Figures 3a andb). At this age most of the follicles contained an oocyte. By 40 dpp, serial histological sections analysis revealed an increased abundance of aberrant follicular structures with the majority of follicles being devoid of oocytes (Figures 3c andd). To further investigate the status of the aberrant follicles, we stained sections of ovaries from 40 dpp mice for AMH (anti-Müllerian hormone), a specific marker of early granulosa cells in primary to early antral follicles. [START_REF] Durlinger | Regulation of ovarian function: the role of anti-Mullerian hormone[END_REF] AMH-positive follicles were observed at this stage in Sf1-Cre Tg/+ ; R26 Rspo1/+ mice, however, the abnormal follicular structures were negative for this marker (Figures 3g andh). Vimentin (VIM), a marker for mural granulosa cells of antral follicles, was expressed in both R26 Rspo1/+ and Sf1-Cre Tg/+ ; R26 Rspo1/+ ovaries (Figures 3i andj) and was also readily detectable in aberrant follicles (Figure 3j). Thus, GCT in Sf1-Cre Tg/+ ; R26 Rspo1/+ ovaries originate from maturing antral follicles and appear around the onset of puberty, suggesting that their formation is sensitive to gonadotrophins, two specifications of adult GCT. [START_REF] Jamieson | Molecular pathogenesis of granulosa cell tumors of the ovary[END_REF] RSPO1 expression impairs ovulation leading to the formation of GCT As the Sf1-Cre transgene is also expressed in the pituitary that produces multiple hormones and especially growth hormone, we examined Sf1-Cre Tg/+ ; R26 Rspo1/+ pups. Female and male mice were healthy and their body size was normal (n = 6; data not shown), indicating that activating Rspo1 mutations do not hamper the development and functions of this organ. We next investigated serum levels of gonadotropins, estradiol and progesterone in adult Sf1-Cre Tg/+ ; R26 Rspo1/+ females (Figure 4a). Luteinizing Hormone (LH) and progesterone levels were considerably lower in Sf1-Cre Tg/+ ; R26 Rspo1/+ mice, compared with R26 Rspo1/+ females. This observation was consistent with the low level of LH receptors observed in the ovaries (Figures 4c ande) and the lack of corpora lutea (Figure 2e). In contrast, follicle-stimulating hormone (FSH) levels were three times higher in Sf1-Cre Tg/+ ; R26 Rspo1/+ mice (Figure 4a).

In laboratory mouse strains, puberty occurs between 30-40 dpp. [START_REF] Ahima | Leptin accelerates the onset of puberty in normal female mice[END_REF] Although GCT accumulation arises at the onset of puberty, we hypothesized that gonadotropins are involved in GCT formation in Sf1-Cre Tg/+ ; R26 Rspo1/+ ovaries. Thus, we stimulated follicular maturation by injecting PMSG to pre-pubertal females (21-24 dpp). Fortyeight hours after PMSG administration, numerous large antral follicles in both Sf1-Cre Tg/+ ; R26 Rspo1/+ and R26 Rspo1/+ ovaries were observed (Figures 5c andd), whereas they were absent in the untreated females (Figures 5a andb). This indicates that RSPO1 does not impair follicular growth upon FSH stimulation. However, most of these large antral follicles in Sf1-Cre Tg/+ ; R26 Rspo1/+ mice exhibited structural abnormalities ranging from asymmetric repartition of granulosa cells to naked or abnormal oocytes (Figures 5c-g) as described in ref. 34.

In order to test whether the antral follicles present in PMSGtreated pre-pubertal Sf1-Cre Tg/+ ; R26 Rspo1/+ females are competent for ovulation, superovulations were induced by injecting human chorionic gonadotropin (hCG) 48 h after PMSG administration. Ovulation takes place about 12 h post-hCG injection. We first examined the expression of Fshr and Foxo1, two factors involved in follicular development, 4 h after LH/hCG injection and found no significant differences between the two genotypes (Figure 6a). Twenty hours after hCG injection, control ovaries were mainly constituted by post-ovulatory CLs, but none or only a few were identified in Sf1-Cre Tg/+ ; R26 Rspo1/+ females (Figures 6b andc). Strikingly, in Sf1-Cre Tg/+ ; R26 Rspo1/+ ovaries, follicular lesions began to emerge and many large antral follicles were still apparent, some of which still contained oocytes (Figure 6c), suggesting that ovulation did not process efficiently. To confirm this observation, we quantified ovulated oocytes in superovulated females. Indeed, the number of ovulated oocytes was significantly reduced in Sf1-Cre Tg/+ ; R26 Rspo1/+ females (Figure 6d) and when ovulated, the oocytes were devoid of a cumulus oophorus (data not shown). Moreover, we observed a dramatic reduction of the level of expression of Amphiregulin (Areg) and Epiregulin (Ereg), two EGF-like factors involved in cumulus cell-oocyte complex expansion and thus required for ovulation (Figure 6a), which could at least partially explain the improper ovulation phenotype in Sf1-Cre Tg/+ ; R26 Rspo1/+ females. The lack of CLs in Sf1-Cre Tg/+ ; R26 Rspo1/+ ovaries (Figure 6c) suggests that RSPO1 expression impairs the terminal differentiation of antral follicles. Indeed, the expression of Lhr, Cyp11a1 and Star, key genes of luteinization process of the granulosa cells which occurs upon LH/ hCG surge, was significantly downregulated in Sf1-Cre Tg/+ ; R26 Rspo1/+ (Figure 6a). As a result, although R26 Rspo1/+ ovaries were filled with CLs expressing CYP11A1, only few CYP11A1-positive CL were observed in Sf1-Cre Tg/+ ; R26 Rspo1/+ ovaries (Figures 6f andg). Interestingly, the newly formed follicular lesions were devoid of CYP11A1 (Figure 6g). In addition, 21-24 dpp superovulated Sf1-Cre Tg/+ ; R26 Rspo1/+ ovaries contained aberrant follicles including degenerating oocytes as observed in 40 dpp non-superovulated Sf1-Cre Tg/+ ; R26 Rspo1/+ ovaries (Figures 3d and6e), suggesting that follicular lesions become apparent in the later stages of follicular development.

Altogether our results demonstrate that follicular abnormalities observed in Sf1-Cre Tg/+ ; R26 Rspo1/+ ovaries are driven by puberty, that Sf1-Cre Tg/+ ; R26 Rspo1/+ ovaries are unable to properly respond to LH and that GCT formation is linked to FSH stimulation. These results show that superovulation experiments induce and accelerate the formation of GCT and that Rspo1 downregulation within adult ovary is required for the proper occurrence of the ovulation process and the subsequent formation of CLs.

Induced expression of RSPO1 promotes cell junction adherence and epithelial traits in GCT Next, we searched for an explanation for the persistence of these abnormal follicular structures. RSPO1 is typically associated with canonical WNT signaling as corroborated by the expression of the Axin2 +/LacZ reporter 31 (Figures 1g andh). However, CTNNB1 is also a component of adherens junctions, where it associates with cadherins (CDH). [START_REF] Kemler | From cadherins to catenins: cytoplasmic protein interactions and regulation of cell adhesion[END_REF][START_REF] Rowlands | Cadherins: crucial regulators of structure and function in reproductive tissues[END_REF] Maintaining adherence junctions has been shown previously to inhibit apoptosis of granulosa cells thus preventing follicular degeneration. [START_REF] Makrigiannakis | N-cadherin-mediated human granulosa cell adhesion prevents apoptosis: a role in follicular atresia and luteolysis?[END_REF] Therefore, the presence of adherens junctions was investigated in ovaries of 6-month-old Sf1-Cre Tg/+ ; R26 Rspo1/+ and R26 Rspo1/+ mice by immunostainings using the markers CTNNB1 and cell-adhesion molecule N-cadherin (CDH2). CTNNB1 was mostly detected at the membrane of granulosa cells of preantral follicles and in granulosa cells surrounding the antrum in larger antral follicles in control ovaries (Figures 7a andc). CTNNB1 was enriched at the membrane of the cells in the GCT in Sf1-Cre Tg/+ ; R26 Rspo1/+ ovaries (Figures 7b andd). The presence of CDH2 was evident at all stages during maturation in both genotypes (Supplementary Figures S3A,B). GCT also strongly expressed membrane-bound plakoglobin (JUP), a component of desmosomes and intermediate junctions (Figures 7e andf), and the epithelial markers KRT18 and pan-cytokeratin (Figures 7g andh and Supplementary Figures S3C-F) that are absent or weakly expressed in follicles in control ovaries. These data reveal the presence of strong intercellular junctions in GCT in Sf1-Cre Tg/+ ; R26 Rspo1/+ ovaries, indicating that induced expression of Rspo1 increases cellular adhesion, which in turn may promote epithelialisation of granulosa cells and the persistence of the follicular remnants.

DISCUSSION

Although RSPO1 is crucial for ovarian development, [START_REF] Chassot | Activation of beta-catenin signaling by Rspo1 controls differentiation of the mammalian ovary[END_REF] here we show that its postnatal downregulation is critical for the maintenance of a healthy adult ovary. When RSPO1 expression is maintained, GCT appear at the onset of puberty in 100% of the cases. Prolonged RSPO1 expression allows follicular maturation until the late antral stage. Most follicles are capable of responding to FSH stimulation as previously reported for induced CTNNB1 expression, [START_REF] Fan | Beta-catenin (CTNNB1) promotes preovulatory follicular development but represses LHmediated ovulation and luteinization[END_REF] and fail to respond to LH. It has been shown that FSH increases the occurrence and onset of GCT formation. [START_REF] Liu | FOXO1/3 and PTEN Depletion in Granulosa Cells Promotes Ovarian Granulosa Cell Tumor Development[END_REF] These GCT are classified as adult GCT. [START_REF] Jamieson | Molecular pathogenesis of granulosa cell tumors of the ovary[END_REF] Accordingly, we observed high FSH levels that could contribute to the adult GCT formation in Sf1-Cre Tg/+ ; R26 Rspo1/+ mice.

Endocrine functionality of CL involves LGR receptors. [START_REF] Pan | Lgr4 gene regulates corpus luteum maturation through modulation of the WNT-mediated EGFR-ERK signaling pathway[END_REF][START_REF] Sun | Ovarian LGR5 is critical for successful pregnancy[END_REF] However, neither Rspo1 nor the CTNNB1 target Axin2 is expressed in these structures, which suggests that LGRs may act through an RSPO1/ CTNNB1 independent pathway in this cell type. In contrast, RSPO1, stabilized CTNNB1 and LGR5 are expressed in the OSE stem cells that contribute to post-ovulatory repair, [START_REF] Ng | Lgr5 marks stem/progenitor cells in ovary and tubal epithelia[END_REF] putting forward that these factors act together in the ovulation process. However, the ovulatory defects observed in our model prevented us from analyzing whether induced Rspo1 affects CL physiology and post-ovulatory OSE repair. In addition to the granulosa cell phenotype, we observed an enriched population of SF1-positive theca cells suggesting a role of CTNNB1 signaling in differentiation of steroidogenic lineages. The steroidogenic precursors of adrenal and gonadal cells have a common origin [START_REF] Hatano | Identical origin of adrenal cortex and gonad revealed by expression profiles of Ad4BP/SF-1[END_REF] and both arise from a pool of WT1-positive cells. [START_REF] Bandiera | WT1 maintains adrenal-gonadal primordium identity and marks a population of AGP-like progenitors within the adrenal gland[END_REF][START_REF] Liu | Lineage specification of ovarian theca cells requires multicellular interactions via oocyte and granulosa cells[END_REF] In adrenals, CTNNB1 is required for the formation and maintenance of the steroidogenic cells of the adrenal cortex, [START_REF] Kim | Targeted disruption of beta-catenin in Sf1-expressing cells impairs development and maintenance of the adrenal cortex[END_REF][START_REF] Huang | Progenitor cell expansion and organ size of mouse adrenal is regulated by sonic hedgehog[END_REF] but the role of this signaling pathway in the fate of the theca cells has not been investigated to date.

21-24dpp + PMSG + 48h

Ablation of Rspo1 induces a precocious differentiation of granulosa cells during fetal development. [START_REF] Maatouk | Disruption of mitotic arrest precedes precocious differentiation and transdifferentiation of pregranulosa cells in the perinatal Wnt4 mutant ovary[END_REF] This demonstrates that Rspo1 is required to maintain an undifferentiated state of the granulosa cell progenitors during early development, before Rspo1 expression decreases within the ovary around birth. Here, we show that when Rspo1 expression is maintained, it alters the differentiation of granulosa cells via their epithelialization. First, CTNNB1 and JUP are maintained at the plasma membrane of aberrant follicular structures and contribute to cell junction formation. Although follicular atresia is promoted by oocytes disruption, [START_REF] Osman | Rate and course of atresia during follicular development in the adult cyclic rat[END_REF] the maintenance of intercellular junctions can prevent degeneration of the granulosa cells. [START_REF] Rowlands | Cadherins: crucial regulators of structure and function in reproductive tissues[END_REF] This suggests that stabilization of cell-cell contacts promote survival of the GCT in Sf1-Cre Tg/+ ; R26 Rspo1/+ mice. Interestingly, in a granulosa cell dominant-stable CTNNB1 mutant mouse model, GCTs develop via a mechanism that mainly implies canonical WNT signaling. [START_REF] Boerboom | Dominant-stable beta-catenin expression causes cell fate alterations and Wnt signaling antagonist expression in a murine granulosa cell tumor model[END_REF] Here, we show that RSPO1 activation in adult ovaries induces formation of GCTs by regulating not only canonical WNT signaling activation but also intercellular junction homeostasis in granulosa cells. Second, GCT exhibit not only granulosa cell identity as highlighted by FOXL2 and Vimentin expression, but also epithelial cell traits are reactivated as shown by the concomitant expression of epithelial markers like pan-cytokeratin and KRT18. Granulosa cells originate from surface epithelial progenitors. [START_REF] Albrecht | Evidence that Sry is expressed in pre-Sertoli cells and Sertoli and granulosa cells have a common precursor[END_REF] Our results suggest that adult granulosa cells maintain the potential to reactivate their epithelial program if properly stimulated, and that RSPO1 is sufficient to induce this program. Moreover, FOXO1/3 and PTEN factors mutations in granulosa cells also promoted epithelial traits reactivation in GCTs. [START_REF] Liu | FOXO1/3 and PTEN Depletion in Granulosa Cells Promotes Ovarian Granulosa Cell Tumor Development[END_REF] The mixed identity of GCT as granulosa cells and epithelial progenitors is likely a susceptibility factor for ovarian pathologies including ovarian cancers. Indeed, our results evidence that afterbirth maintenance of RSPO1 expression induces GCT but also severe ovarian pathologies and eventually cancers in mice. In human RSPO1 has been reported as a susceptibility factor of ovarian epithelial cancers and is amplified in 8% of them, [START_REF] Kuchenbaecker | Identification of six new susceptibility loci for invasive epithelial ovarian cancer[END_REF][START_REF] Gao | Integrative analysis of complex cancer genomics and clinical profiles using the cBioPortal[END_REF] but so far its exact role in ovarian cancers was not clear. Here, we show for the first time that the overexpression of RSPO1 in mouse ovarian tissues is sufficient to lead to GCT formation with 100% penetrance. Our results highlight RSPO1 as a main player in the ovarian oncogenic process and provide new insights into the molecular roles of RSPO1 in the pathology.

MATERIALS AND METHODS

Mouse strains and genotyping

The experiments were carried out in compliance with the relevant institutional and French animal welfare laws, guidelines and policies and have been approved by the French ethics committee (CIEPAL: NCE/2011-12).

Rspo1 gain-of-function model (Supplementary Figure S1) carries an inducible Rspo1 gene knocked into the Rosa26 locus. The construct is similar to the one reported in ref. 46 with the exception of the promoter. Here, the Rosa26 promoter controls Rspo1 expression. R26 Rspo1/+ mice were mated with the Sf1-Cre high (Sf1-Cre Tg/+ ) strain, which drives expression of the Cre recombinase in somatic cells of the gonad from 11.5 dpc onward. [START_REF] Bingham | Development of a steroidogenic factor 1/Cre transgenic mouse line[END_REF] Axin2-LacZ mice were described previously, [START_REF] Lustig | Negative feedback loop of Wnt signaling through upregulation of conductin/axin2 in colorectal and liver tumors[END_REF] and crossed with Sf1-Cre Tg/+ ; R26 Rspo1/+ males to generate Sf1-Cre Tg/+ ; R26 Rspo1/+ ; Axin2 +/LacZ animals. Fertility tests were performed using 3-4-month-old Sf1-Cre Tg/+ ; R26 Rspo1/+ females mated with R26 Rspo1/+ males (Supplementary Figure S1). Genotyping was performed using DNA extracted from tail tips or ear biopsies of mice. The presence of the Y chromosome and the Axin2-LacZ transgene was determined as described previously. [START_REF] Chassot | Activation of beta-catenin signaling by Rspo1 controls differentiation of the mammalian ovary[END_REF]

Histological analysis

Ovaries were dissected, fixed in Bouin's solution overnight, and embedded in paraffin. Five-micrometer-thick sections were stained with hematoxylin and eosin. To determine the ovarian phenotype in Rspo1 expressing ovaries, consecutive sections were analyzed. Pictures were taken with an Axioscope 2 (Zeiss, Oberkochen, Germany) or MZ9.5 (Leica, Mannheim, Germany) microscope coupled with an Axiocam MRc5 (Zeiss) or DHC490 (Leica) camera and Axiovision 4.8 (Zeiss) or application suite V3.3.0 (Leica) software, and processed with Adobe Photoshop (San Jose, CA, USA).

Serum collection and hormone assays

Blood was collected from adult mice in the same stage of estrous cycle and allowed to clot for 90 min at room temperature. Samples were then centrifuged and the serum was collected and stored at -20 °C. Hormone analyses were performed by the Ligand Assay and Analysis Core Laboratory at The Center for Research and Reproduction, University of Virginia (Charlottesville, VA, USA). LH, FSH, estradiol and progesterone measurements were run in duplicates using specific assays (n = 5 for each genotype).

Superovulations

Immature 3-4-week-old mice were injected intraperitoneally (i.p.) with 5 IU/ mouse pregnant mare serum gonadotropin (PMSG) (Sigma-Aldrich, St Louis, MO, USA) to promote follicle maturation, followed by 5 IU/mouse hCG (Sigma-Aldrich) 48 h later to induce ovulation. Mice were humanely killed 48 h after the first hormone injection for follicle maturation studies or 4 h and 20 h after the second injection for ovulation and luteinization studies.

Ovaries, including fat pad, oviduct and partial uterine tube, were fixed in 4% paraformaldehyde or Bouin's solution overnight. For qPCR analyses, ovaries were carefully isolated from the genital tract and quickly frozen in liquid nitrogen. For oocyte counting, immature females (n = 8 (R26 Rspo1/+ ) and 10 (Sf1-Cre Tg/+ ; R26 Rspo1/+ ) ovaries) were similarly superovulated and killed 20 h after the hCG injection. Oviducts were isolated from ovary and uterus and opened to collect the oocytes.

Gomez for critical reading of the manuscript. This work was supported by grants from the Fondation ARC pour la Recherche sur le Cancer (PJA 20131200236) and Agence Nationale de la Recherche (ANR-09-GENM-009-03 and ANR-11-LABX-0028-01). EP was supported by a fellowship from INRA and Ligue Nationale contre le Cancer.

Figure 1 .

 1 Figure 1. Pattern of Rspo1 expression and activation of WNT signaling in ovaries. Expression of Rspo1 revealed by in situ hybridization in R26 Rspo1/+ mouse ovaries at 16.5 dpc (a), 4 dpp (b) and 12 months (c).Rspo1 is expressed at low level in the R26 Rspo1/+ mouse adult ovary, mainly in the epithelial surface and in granulosa cells of some follicles in the ovarian cortex, whereas adult Sf1-Cre Tg/+ ; R26 Rspo1/+ ovaries display Rspo1 expression throughout the whole organ (d). Activation of the canonical WNT signaling by RSPO1 visualized by β-galactosidase staining of ovaries expressing the Axin2-LacZ reporter. Axin2-LacZ is expressed at 2.5 and 12 months in pre-antral follicles and the stroma (e and g) and strongly expressed in oocytes and the epithelial surface surrounding the ovary (e and f, arrow) in R26 Rspo1/+ mice. Induced Rspo1 expression leads to Axin2-LacZ expression in the whole Sf1-Cre Tg/+ ; R26 Rspo1/+ ovary (h) with strong staining in granulosa cells (arrow) and weaker staining in aberrant follicular structures lesions (*). Scale bars: 300 μm.

Figure 2 .

 2 Figure 2. Ovarian defects and tumors in Sf1-Cre Tg/+ ; R26 Rspo1/+ ovaries. Macroscopic view of 18-month-old R26 Rspo1/+ (a) and Sf1-Cre Tg/+ ; R26 Rspo1/+ ovaries (b and c). The size of the Sf1-Cre Tg/+ ; R26 Rspo1/+ ovaries is increased in comparison with the control ovary owing to the presence of prominent tumor tissue (c) and/or blood-filled cysts (b). Magnification 10 × . Histological sections (haematoxylin and eosin staining) analysis of 12-month-old ovaries. Sf1-Cre Tg/+ ; R26 Rspo1/+ ovaries (e) contain numerous multifocal aggregates of neoplastic cells that arrange either in discrete solid aggregates (*) or in microfollicular pattern (arrows) in comparison with R26 Rspo1/+ ovaries (d). Scale bars, 500 μm. Magnifications of structural abnormalities in Sf1-Cre Tg/+ ; R26 Rspo1/+ ovaries (f-n): neoplastic cells arranged in microfollicular or rosette-like pattern (f; *) with central pale eosinophilic globule (Call-Exner body; f and g arrow); neoplastic cells arranged in solid sheets composed of bland, ovoid, scant amount of cytoplasm and ovoid nuclei granulosa cells. (h, arrow): hematocysts (i and j), with columnar cells (j, arrow), CL or regressing CL with erythrocytes infiltration (l and m, arrow), Ovarian epithelial cyst composed of cystic and papillary structures lined with tall columnar ciliated epithelium. (k, arrow) and ovarian epithelial inclusion cyst composed of keratinized squamous epithelium (n, arrow). Scale bars: 100 μm. a, antral follicle; cl, corpus luteum.

Figure 3 .

 3 Figure 3. RSPO1 promotes GCT. Histological analysis (haematoxylin and eosin staining) of R26 Rspo1/+ and Sf1-Cre Tg/+ ; R26 Rspo1/+ ovaries at 28 dpp (a and b) and 40 dpp (c and d). Follicle-like structures (*) consisting of nests of disorganized, pleiomorphic granulosa cells become evident at 28 dpp (b) in Sf1-Cre Tg/+ ; R26 Rspo1/+ ovaries, whereas they are not observed in R26 Rspo1/+ ovaries (a). They enclose oocytes at 28 dpp (b), whereas they lack an antrum and contain no visible or a degenerating oocyte at 40 dpp (d). Scale bar: 300 μm. Immunolabelling of FOXL2 (e and f), AMH (g and h) and Vimentin (VIM) (i and j) in R26 Rspo1/+ (left panel) and Sf1-Cre Tg/+ ; R26 Rspo1/+ ovaries (right panel) at 40 dpp. FOXL2 staining in the GCT (f; *) confirms the presence of granulosa cells in these follicle-like structures in R26 Rspo1/+ ovaries (e). The absence of AMH staining (h) and the presence of VIM staining (j) in GCT (*) suggest that follicular maturation can occur up to the antral stages. Nuclei are labeled with DAPI (blue). Scale bars: 100 μm. a, antral follicle; pa, pre-antral follicle, cl, corpus luteum.

Figure 4 .

 4 Figure 4. Induced Rspo1 expression impairs LH-mediated luteinization. (a) Serum levels of FSH, LH, estradiol and progesterone in R26 Rspo1/+ (black bars) and Sf1-Cre Tg/+ ; R26 Rspo1/+ ovaries (white bars) from adult females (n = 5 for each genotype). Bars represent mean ± s.e.m. with significance set at Po0.05 (*). Expression of LH receptor revealed by in situ hybridization in adult (P40) R26 Rspo1/+ and Sf1-Cre Tg/+ ; R26 Rspo1/+ ovaries (b-e). Lhr is strongly expressed in mural granulosa cells of mature antral (a) follicles (band d) and in the theca cells of R26 Rspo1/+ ovaries, whereas Lhr expression is clearly downregulated in antral follicles (c and e) of Sf1-Cre Tg/+ ; R26 Rspo1/+ ovaries and is absent from GCT (*). Scale bars: 100 μm. ns, not significant.

Figure 5 .Figure 6 .

 56 Figure5. Sf1-Cre Tg/+ ; R26 Rspo1/+ ovaries respond to hormonal stimulation. Histological analysis (haematoxylin and eosin staining) of R26 Rspo1/+ (a, c and e) and Sf1-Cre Tg/+ ; R26 Rspo1/+ (b, d, f and g) ovaries of 21 dpp (a and b) and at 21-24 dpp stimulated by PMSG injection and collected 48 h later (c-g). When stimulated by PMSG, ovaries from both genotypes contain large antral (a) follicles (c and d) but in Sf1-Cre Tg/+ ; R26 Rspo1/+ mice, most of these follicles exhibit dissymmetric mural granulosa (f and g, arrowheads) when compared with follicles of R26 Rspo1/+ ovaries (e). Aberrant follicles are also observed containing naked (*) or degenerating (**) oocytes (g). Scale bar: 300 μm (a-d) and 100 μm (e-g).

Figure 7 .

 7 Figure 7. RSPO1 expression maintains intercellular junctions and epithelial identity in GCT. Immunostaining for CTNNB1 (a-d), JUP (e and f), and KRT18 (g and h) in 6-month-old R26 Rspo1/+ (left panel) and Sf1-Cre Tg/+ ; R26 Rspo1/+ (right panel) ovaries. Membrane staining for CTNNB1 and JUP underline cellular junctions in these follicle-like structures (h). Nuclei are labeled with DAPI (blue). Scale bars: 100 μm.

RSPO1 and ovarian disorders M-C De Cian et al 209 © 2017 Macmillan Publishers Limited, part of Springer Nature. Oncogene (2017) 208 -218

Oncogene (2017) 208 -218 © 2017 Macmillan Publishers Limited, part of Springer Nature.

© 2017 Macmillan Publishers Limited, part of Springer Nature.Oncogene (2017) 208 -218

RSPO1 and ovarian disorders M-C De Cian et al

ACKNOWLEDGEMENTS

We thank Mireille Cutajar-Bossert for her work and management of funding and also Fariba Jianmotamedi, Aurélie Charlet and Béatrice Polo for their punctual help in this work. We are thankful to Walter Birchmeier (Berlin, Germany) for the Axin2 +/LacZ mice and Ken Morohashi (Fukuoka, Japan) for the SF1 antibody. We thank Aitana Perea-

X-gal staining and immunological analyses

Tissue samples were fixed in 4% paraformaldehyde overnight and then processed for paraffin embedding or equilibrated in sucrose and embedded in Cryomount (Histolab, Göteburg, Sweden) for cryosectioning. Cryostat or microtome sections of 10 μm and 5 μm thickness respectively were processed for X-Gal and immunostaining. Samples for X-Gal staining were processed as described previously. [START_REF] Chassot | Activation of beta-catenin signaling by Rspo1 controls differentiation of the mammalian ovary[END_REF] Immunohistochemical experiments were performed as described in. [START_REF] Chassot | Activation of beta-catenin signaling by Rspo1 controls differentiation of the mammalian ovary[END_REF] The following dilutions of primary antibodies were used: AMH (C-20, cat sc6886, Santa Cruz Biotechnology, Dallas, TX, USA) 1:200, CTNNB1 (cat 610153, BD Biosciences, San Jose, CA, USA) 1:250, active CASP3 (cat AF835, R&D Systems) 1:200, CDH2 (cat 33-3900, Invitrogen) 1:200, CYP11A1 [START_REF] Svingen | Three-dimensional imaging of Prox1-EGFP transgenic mouse gonads reveals divergent modes of lymphangiogenesis in the testis and ovary[END_REF] 1:300, FOXL2 [START_REF] Rastetter | Marker genes identify three somatic cell types in the fetal mouse ovary[END_REF] 1:400, JUP (cat 610254, BD Biosciences) 1:250, KRT18 (cat ab52948, Abcam, Cambridge, UK) 1:300, LHR (LHR-29, ATCC-CRL-2685, ATCC) 1:150, MKi67 (clone SP6, cat 9106, Thermo Fisher Scientific, Waltham, MA, USA) 1:200, SF1 (kindly provided by Professor Morohashi) 1:1000 and Vimentin (cat ab92547, Abcam) 1:200. Slides were counterstained with DAPI diluted in the mounting medium at 10 μg/ml (Vectashield, Vector laboratories, Burlingame, CA, USA) to detect nuclei. Imaging was performed with a motorized Axio ImagerZ1 microscope (Zeiss) coupled with an Axiocam Mrm camera (Zeiss) and processed with Axiovision LE (Zeiss) and Adobe Photoshop.

In situ hybridization

Tissue samples were fixed in 4% paraformaldehyde in 1 × PBS overnight at 4 °C. Seven-micrometer sections were cut for in situ hybridization experiments using Rspo1 and Lhr digoxigenin-labeled riboprobes as described previously. [START_REF] Chassot | Activation of beta-catenin signaling by Rspo1 controls differentiation of the mammalian ovary[END_REF] Each experiment was repeated on at least two gonads. Posthybridization washes were done in 100 mM maleic acid Ph7.5, 150 mM NaCl, 0.1% (v/v) tween-20 (MABT). Imaging was performed on a MZ9.5 microscope (Leica) as described above and processed with Adobe Photoshop.

Quantitative PCR analysis

Individual ovaries from either adult or 21-24 dpp superovulated mice were dissected in 1 × PBS and quickly frozen in liquid nitrogen. RNA was extracted using the RNeasy Qiagen kit and reverse transcribed using the RNA RT-PCR kit (Stratagene). Primers and probes were designed by Roche Assay Design Center (http://qpcr.probefinder.com/organism.jsp). Primer sequences are available upon request to the authors. All real-time PCR assays were carried out using LightCycler Taqman Master kit (Roche, Basel, Switzerland). QPCR was performed on cDNA from one gonad and compared with a standard curve. Relative expression levels of each gene was determined in the same run and normalized on the levels of endogenous Sdha1 cDNA. QPCR were repeated at least twice. For adult mice, n = 4 (R26 Rspo1/+ ) and 8 (Sf1-Cre Tg/+ ; R26 Rspo1/+ ) gonads. For 21-24 dpp superovulated mice, n = 6 gonads for each genotype.

Statistical analysis

Data are shown as mean ± s.e.m.

All the data were analyzed by unpaired one-sided Student's t-test using Microsoft Excel (Redmond, WA, USA). Asterisks highlight the pertinent comparisons and indicate levels of significance: *Po0.05, **Po 0.01 and ***P o0.001.

CONFLICT OF INTEREST

The authors declare no conflict of interest.