Robin Lovell-Badge

 

Regulatory mechanisms responsible for the maintenance of granulosa and ovarian cell fate

While we know much about mechanisms of sex determination, much less is understood about the maintenance of the differentiated state of cell types in the ovaries and testes. The gonads begin to develop in an identical manner in XX and XY embryos. They contain a population of bipotential precursor cells (supporting cells, steroidogenic cells, and primordial germ cells) that can follow either the male or female pathway. When the Y-linked gene Sry is expressed, its protein product, SRY, activates Sox9 expression, which is required for the differentiation of the supporting cells into Sertoli cells. These produce signaling molecules that trigger differentiation of the other testicular cell types, including Leydig cells and prospermatogonia, and coordinate their organisation into the testis-specific pattern. In XX embryos, supporting cells differentiate as granulosa cells, steroidogenic cells become theca cells and germ cells give oocytes. Ovary development depends at least in part on the action of anti-testis/ovary promoting genes that function to repress Sox9. One pathway involves -catenin, which is stabilised by WNT4 and R-spondin1. However loss-of-function mutations in these genes result in only partial XX male sex reversal, arguing that other factors are involved. One such factor, FOXL2, is expressed specifically in the ovary as it forms, however the Foxl2-/- null mutant phenotype (ovary dysgenesis) is only observed postnatally. But critically, when Foxl2 is deleted from adult mouse ovaries, these are reprogrammed into testes:  granulosa cells transdifferentiate into Sertoli-like cells, seminiferous tubule-like structures develop and Leydig cells differentiate. Molecular data suggested that FOXL2 acts by binding to a Sox9 testis-specific enhancer (TES) to directly repress its transcription in granulosa cells. The ovarian pathway therefore requires continuous repression of Sox9 via FOXL2 throughout life. The plasticity of ovarian cells to transdifferentiate into testicular cells has significant implications for reproductive biology with relevance for understanding and perhaps treating sex differentiation disorders, premature ovarian failure and menopause; and in improving gender reassignment procedures.

This proposal aims to explore in more detail the mechanisms involved in the repression of Sox9 by FOXL2. We recently showed that the TES enhancer is responsible for about 55% of Sox9 expression in the embryonic XY gonad, but the remainder is still sufficient to give testis development. This led us to search for additional enhancers for Sox9 and we have now defined several that appear to act at different stages of testis development. Interestingly, some of the new enhancers give expression of reporter genes in the ovary and not the testis; perhaps these have a repressive role in their normal genomic context. The project will address the relative importance of all these enhancers in the maintenance of both the adult testis and ovary, using molecular and genetic techniques. The former will involve the use of methods such as ChIPseq and conformation capture to look for binding of FOXL2 and its partners or DNA looping, respectively. The latter will include asking whether the ovary to testis transdifferentiation after Foxl2 deletion depends on TES or on one or more of the new enhancers, where these can be deleted using genome editing.

1. Sekido, R. and Lovell-Badge, R. (2008)
Sex determination involves synergistic action of SRY and SF1 on a specific Sox9 enhancer.
Nature 453: 930-934. PubMed abstract

2. Uhlenhaut, N. H., Jakob, S., Anlag, K., Eisenberger, T., Sekido, R., Kress, J., Treier, A.-C., Klugmann, C., Klasen, C., Holter, N. I., Riethmacher, D., Schütz, G., Cooney, A. J., Lovell-Badge, R. and Treier, M. (2009)
Somatic sex reprogramming of adult ovaries to testes by FOXL2 ablation.
Cell 139: 1130-1142. PubMed abstract

3. Sutton, E., Hughes, J., White, S., Sekido, R., Tan, J., Arboleda, V., Rogers, N., Knower, K., Rowley, L., Eyre, H., Rizzoti, K., McAninch, D., Goncalves, J., Slee, J., Turbitt, E., Bruno, D., Bengtsson, H., Harley, V., Vilain, E., Sinclair, A., Lovell-Badge, R. and Thomas, P. (2011)
Identification of SOX3 as an XX male sex reversal gene in mice and humans.
Journal of Clinical Investigation 121: 328-341. PubMed abstract

4. Jakob, S. and Lovell-Badge, R. (2011)
Sex determination and the control of Sox9 expression in mammals.
FEBS Journal 278: 1002-1009. PubMed abstract

5. Gonen, N., Quinn, A., O'Neill, H. C., Koopman, P. and Lovell-Badge, R. (2017)
Normal levels of Sox9 expression in the developing mouse testis depend on the TES/TESCO enhancer, but this does not act alone.
PLOS Genetics 13: e1006520. PubMed abstract