Study of sex determination in mice lacking candidate genes identified by human exome sequencing

Disorders of sex development (DSD) comprise a heterogeneous group of conditions in which human sex development is atypical. The research described in this thesis aims at improving our understanding of DSDs, in particular those characterised by abnormal gonad development (gonadal dysgenesis, GD), using the mouse as a model of human sex development. I discuss four genes (TLE3, SEC31A, NCOR2 and ZNRF3) identified by exome sequencing screens of individuals with 46,XY GD and experiments aimed at assessing their candidacy as human testis-determining genes and, in the case of one (ZNRF3), exploring mechanisms that lead to mouse embryonic gonadal sex reversal in their absence.
Sexual development in the mouse starts at around 10 days post coitum (dpc), with the formation of the bipotential gonadal primordium on the surface of the mesonephros. At around 11.5 dpc, the gonad acquires a fate determined by the chromosomal sex of the individual – a process known as gonadal sex determination. In XY embryos, SRY expression from the Y chromosome results, by 14.5 dpc, in Sertoli cell differentiation and testis development. In XX embryos, in the absence of SRY, canonical WNT signals and FOXL2 cause granulosa cell specification and ovary development. Therefore, I examined mouse lines that lack function of the four genes discussed above during the period 11.5 to 14.5 dpc.
Loss of the transcriptional co-repressor TLE3 did not result in any overt gonadal phenotype. By contrast, loss of SEC31A, a component of the coat protein complex II (COPII), led to very early embryonic lethality, and I investigate the molecular and cellular basis of this lethality. NCOR2 encodes a nuclear receptor co-repressor that mediates transcriptional silencing of target genes. Exome sequencing identified sequence variants in unrelated 46,XY and 46,XX individuals with DSD, across 3 continents, making it the strongest of the candidate genes. I describe subtle but clear defects in testis and ovary development in a mouse line lacking NCOR2 function, dependent on the mouse genetic background in which the loss-of-function phenotype was analysed. These data support a role for NCOR2 in human sexual development and DSD, and warrant further investigations.
Finally, I describe experiments aimed at understanding the role of the anti-WNT ubiquitin ligase ZNRF3 in mouse testis determination, a role that was reported during the research period covered by this thesis. I show cell proliferation defects in the Znfr3 null XY gonad and discuss the relevance of these to the sex reversal phenotype. I also describe experiments aimed at being able to detect endogenous ZNRF3 protein, using antibody generation and CRISPR/Cas9-mediated epitope tagging of the endogenous gene. Finally, I describe data from an RNA sequencing (RNAseq) experiment aimed at identifying the transcriptional consequences of Znrf3 ablation at the sex-determining stage of gonadogenesis.
I conclude by discussing the benefits and limitations of using the mouse model for study of primary sex determination in humans, the importance of genetic background and suggest ways in which the success rate of this approach might be improved in future, by incorporating new models.

Advisor: Greenfield, A
Srinivas, S