Sex-determining mechanisms in the chick

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This research project is aimed at understanding the mechanism of sex determination in the chicken i.e. the series of molecular events that determine whether the embryonic gonad develops as a testis or as an ovary. It is widely recognized that such primary sex determining mechanisms evolve rapidly, as exemplified by the marked differences between mammals and other vertebrates. Gonadogenesis in mammals is envisaged as a linear process that is dependent on a 'switch' mechanism based on the Y-chromosome gene Sry. If Sry is expressed appropriately, then the developing gonad becomes a testis: without Sry the gonad becomes an ovary. With only limited exceptions, extensive studies to identify similar master 'switch' genes in other vertebrate species have been unsuccessful. In our studies, we have demonstrated that chicken cells acquire an inherent sex identity at fertilisation or shortly thereafter and believe that this is key to avian sex determination; i.e. the testis forms because the genital ridge is composed of male cells or the ovary forms because the genital ridge is composed of female cells. This suggests that, in birds, rather than gonadal sex determination depending on a sex-specific switch mechanism, testis and ovary differentiation represent two separate pathways. It was widely accepted that once gonadal fate had been determined, it was permanent, but some surprising recent findings suggest that this is not the case. In fact, the adult mammalian gonad displays a great degree of plasticity and testicular and ovarian identity has to be maintained throughout life. It appears that this maintenance depends on the expression of two genes, DMRT1 in males or FOXL2 in female. Dmrt1 has also been shown to be necessary for the proper development and survival of male germ cells. Dmrt1 and Foxl2 are not thought to be important for primary sex determination events in the mouse embryo, but they have been shown to play key roles in gonadogenesis in several vertebrate species including the chick and some mammals. It may be that the requirement for DMRT1 and FOXL2 to maintain adult mammalian gonads represent an evolutionary residue of their major roles in primary sex determining mechanisms in lower vertebrates, where plasticity is often evident during embryonic stages. It is also possible that, unlike the mouse, a number of mammalian species retain elements or this earlier primary sex determining system.We will investigate the possibility that Dmrt1 and Foxl2 balance of expression determines the sexual fate of the embryonic gonads in birds. To do this, we will use cutting edge methods of genetic manipulation to delete copies of Dmrt1 and Foxl2 from the genome of chicken germ cells (PGC) and use these germ cells to derive birds with these genetic mutations.We will assess the effects of these deletions on PGC growth in culture and on germ cell development after injection of PGCs into embryos. Injected embryos will be hatched and raised to sexual maturity and crossed with wild-type birds: by selective crossing we can generate birds with either one or no copies of Dmrt1 or Foxl2. We will compare the development of the gonads and germ cells in these manipulated birds with that in wild-type male and female birds, and so determine the effect of Dmrt1 and Foxl2 on primary sex determination and germ cell development in birds.We will also carry out a series of molecular analyses to determine the networks of genes regulated by Dmrt1 and Foxl2, and identify the genes affected by manipulating the expression levels/ balance of these transcription factors.