Regulation of sex determination and ovarian differentiation: implications for disorders of sexual development – SexDiff

We proposed to use the genetic tools and experimental models mastered by each consortium

partner to identify new factors in sex determination and understand their role in the

emergence of differences in sexual development. Partner 1 contributed her unique collection

of mutant mice for the genes encoding the players in this process. Partner 2, an expert in

bioinformatics, analysed some of the transcriptomic data generated by the consortium and

compared it with publicly available data. Partner 3, an expert in human gonadal

differentiation, contributed her skills in the in vitro differentiation of human pluripotent

stem cells. The gonadal phenotypes of patients and mutant mouse models were analysed

using histology, immunohistochemistry, in situ hybridisation techniques and gene

expression analysis using analytical methods such as quantitative PCR or high-throughput

RNA sequencing.

We have identified important new factors in gonadal differentiation. In particular, we have

discovered the mammalian ovarian determinant, a specific protein or isoform of the WT1 gene. A mutation that prevents the production of this isoform leads to a block in the sexual

differentiation of the bipotential gonad, while its overexpression induces male-to-female sex

reversal, demonstrating that WT1 is necessary and sufficient for ovarian differentiation.

The results of the SexDiff project have been presented at national/international conferences

and published in 14 articles in peer-reviewed scientific journals (such as Science, Science

Advances, etc.). The discovery of the ovarian determinant was covered by mainstream

newspapers such as Le Monde

(https://www.lemonde.fr/sciences/article/2023/11/22/differenciation-sexuelle-la-proteine-quilance-

la-formation-des-ovaires-chez-l-embryon-enfin-identifiee_6201635_1650684.html), and

selected as scientific breakthrough of the year 2023 by Inserm

(https://www.calameo.com/read/005154450f2be2999145b).

Disorders of sex development (DSD) are very heterogeneous and despite intensive research over the last 30 years, only about 50% of DSDs can be explained on the molecular level. This highlights that our knowledge on mechanisms governing sex determination is still fragmented. In the project SexDiff, we will use mouse and human genetics in combination with transcriptomic analyses and bio-informatics to clarify how sex determination is driven.

Sex determination is a developmental process allowing the differentiation of a bipotential precursor in two completely different organs, the testis or the ovary. This decision is driven by the paternal transmission of the Y-linked gene SRY which eventually initiates testicular development by up-regulating the transcription factor SOX9. In absence of SRY, R-spondin1 (RSPO1), an activator of the WNT/?-catenin signalling pathway, initiates ovarian differentiation. Both pathways antagonize each other, and sexual differentiation is determined by the dominant pathway. We have a long-standing interest in mechanisms of mammalian sex determination and have contributed to understanding of function of key genes such as SOX9 and RSPO1 and how these genes promote one sexual fate and repress the other. Despite recent advances our understanding of genetic components and mechanisms of sex determination remain limited. Discovery of novel factors and mechanisms involved in the process is of vital interest as mutations in yet to be discovered genes may be a cause of human reproductive pathologies, particularly DSD.
The Wilms’ tumour suppressor WT1 is essential for the differentiation of the gonad. Different variants of WT1 exist and the imbalance of the ratio between the alternative spliced isoforms +KTS/-KTS are the cause of the male-to-female sex reversal in the Frasier syndrome. Recently discovered mutations impacting specifically the 4th DNA binding Zinc finger promote female-to-male sex reversal. This positions WT1 at the crossroad of the cell fate decision during sexual development. In this project we aim to decipher the role of these isoforms/variant in the differentiation of the gonad using mouse models and modified induced pluripotent stem cells (iPSC) and to uncover novel signalling networks regulated by WT1 during gonadal differentiation. We believe that it is important because these newly identified factors may provide a valuable diagnostic tool to understand the aetiology of idiopathic cases of human disorders of sex development.
In contrast to testis, much less is known about the mechanisms of ovarian development in mammals. We have recently established that ovarian differentiation is decided before the first signs of sexual differentiation, a concept that breaks with the present view of sex determination. During the course of SexDiff, we will further explore this and characterize the molecular function of Rspo1 and identify new actors of ovarian differentiation using our Rspo1 loss-of-function mutants. These newly identified factors will then be further characterised to establish their causality and contribution to errors of ovarian development in human using the combinatorial approach involving genomics, in-silico, in-vitro, ex-vivo and cellular reprogramming approaches.
To achieve these goals, we will employ a whole range of state-of the art techniques including single-cell RNA-Sequencing, cellular reprogramming (using iPSCs) and targeted mutagenesis by CRISPR/Cas9. Using the concerted approaches involving mouse models, human patients, cellular models and in–silico analyses, SexDiff will contribute to an in-depth understanding of the normal and pathogenic development of the gonads. This is envisaged to contribute and enhance our understanding of the aetiology of errors in gonad development in human and pave way for a better diagnosis. SexDiff, therefore, is of interest not only for enhancing our knowledge of fundamental biology but has an implicit clinical application.