Deciphering Gene Regulatory Networks governing Mammalian Sex Determination

Project Information

EnhanceSex

Grant agreement ID: 101039928

DOI

10.3030/101039928

EC signature date 17 February 2022

Start date 1 April 2022

End date 31 March 2027

Funded under

• European Research Council (ERC)

Total cost

€ 1 651 868,00

EU contribution

€ 1 651 868,00

Coordinated by

BAR ILAN UNIVERSITY
Israel

Periodic Reporting for period 1 - EnhanceSex (Deciphering Gene Regulatory Networks governing Mammalian Sex Determination)

Reporting period: 2022-04-01 to 2024-09-30

Summary of the context and overall objectives of the project

Mammalian sex is defined at three developmental stages: First, at fertilization, when either an X-bearing or Y-bearing sperm penetrates the haploid oocyte. XY embryos will develop as males whereas XX embryos will develop as females. Secondly, at E11.5 in the mouse, sex determination occurs with the bipotential gonad committing to either testicular or ovarian cell fates. Lastly, sex differentiation happens at both embryonic stages and puberty, relying on sex hormones secreted from the gonads. Discordance between these three stages can lead to Disorders of Sex Development (DSD) with an overall prevalence of 1 in 4000 newborns. Extensive genetic and molecular studies, in both mouse and human, indicate that the process of sex determination relies on a delicate balance of the expression and activity of several pro-male versus pro-female factors, most of which are transcription factors (TFs) and signalling pathways components. Although we now know many of these players, we still fail to understand the gene regulatory networks operated by these factors, the target genes they activate/repress, the genomic elements they exploit to exert this highly coordinated gene expression regulation, nor do we have an in vitro system to address these questions.

Two of the major drawbacks that prevented our ability to explore this delicate gene regulatory system, and understand cases of DSD patients, are the lack of an in vitro system that can closely model the gonads and the inability to isolate pure population of Sertoli and granulosa cells, the somatic cells of the gonads that determine the sex. In this proposal, we aim to characterize the gene regulatory networks operated by the key factors controlling testis and ovary development. We will identify the target genes activated and repressed by these key factors as well as the genomic elements they use to regulate their target genes. Furthermore, we will develop an in vitro system to model testis development. Altogether, this proposal will provide a systems biology view of the process of sex determination and the regulatory networks governing it. This will allow better diagnosis of DSD patients and modelling of these pathologies in vitro, in a human-related context. It will also provide wide implications for understanding spermatogenesis and potentially offer treatment for infertility. Insights we gain from this complex system can shed light on gene expression regulation and cell fate decisions in other developmental systems.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

In order to enhance the creation of stem cell-derived gonads, and as part of the ERC project, we developed a differentiation protocol that allows to generate early gonad progenitors from mouse embryonic stem cells (ESC). This was verified using bulk RNA-seq and single cell RNA-seq. We showed that the protocol developed for mouse ESC also applies for human ESC/ induced pluripotent cells (iPSCs). We used XY, XX and XY DSD iPSC, and for the first time demonstrated that we can model human DSD condition in a dish and understand the consequences of the variant on the ability of the cells to reach the proper gonadal somatic cell state. These findings were published in the prestige Science Advances journal. Next, since we show that neonatal somatic gonadal cells cannot be cultured in vitro in a standard 2D/ serum-based conditions, we set to develop testis organoid settings that could allow preservation of gonadal cells in vitro for prolonged periods. We screened both 2D and 3D settings and found that we can culture neonatal testicular cells on top of transwell inserts along with defined media to create testicular organoids. Remarkably, these organoids can be cultured in vitro for 9 weeks, all gonadal cell types are well maintained for the entire duration of the culture, and the gene expression profiles are highly similar to that of in vivo testis. Furthermore, we show first indications that these organoids can allow entry of germ cells into meiosis in vitro. These findings were recently published in the IJBS journal. These findings, together with the stem cell differentiation, pave the way for the creation of fully artificial gonads that can serve as a model to study sex determination, infertility, and maybe even provide therapeutic possibilities as in vitro gametogenesis.
When trying to explore the gene regulatory system controlling mammalian sex determination, we also wanted to focus on a previously identified enhancer of the Sox9 gene, called Enh13, and better understand its function. We show that mutations in two redundant transcription factor binding sites (TFBS) within Enh13 can fully phenocopy the phenotype of deleting the entire Enh13 and lead to XY male-to-female sex reversal. Furthermore, we show that different types of mutations in these TFBS can lead to completely different phenotypic outcomes, explaining the phenotypic variability seen in patients with different variants. These findings were recently published in the prestige Nucleic Acids Research (NAR) journal. Altogether, this suggest that even single nucleotide variants and INDELs within regulatory elements can be the cause of DSD.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

Based on the achievements accomplished during the project so far, we were able to make significant progress toward creation of testis organoids and, in the future, artificial gonads, that could significantly enhance the sex determination and reproduction fields. Our preliminary findings suggest that we are on the right path to meet the long-term goals of the project and opened new and promising directions of research that could lead to high impact publication. Using our systematic approach, we will be able to decipher the gene regulatory network controlling the process of sex determination, that would enable to better understand many unexplained cases of DSD patients. Altogether, we are confident that by the end of the project we will expand our knowledge about how mammalian sex determination occurs, the regulatory elements involved in its' regulation and how disturbance in these can lead to related pathologies.