Sex-determining mechanisms in the chick

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.

The mechanism of primary sex determination in the chicken differs from that seen in mammalian embryos, but seems to involve elements that are required to maintain gonadal fate in adult mammals. The available evidence suggests that a balance between the transcription factors Dmrt1 and Foxl2, determines the sexual fate of the embryonic gonads in birds. To investigate this, we will use PGC culture and a CRISPR/Cas9 based approach to produce targeted mutations of both the Foxl2 and Dmrt1 loci. We have already cloned CRISPR oligo guides for each gene and produced targeted cell lines. We will generate GFP-expressing PGC lines heterozygous for a null mutation in Dmrt1 and lines heterozygous or homozygous for a null mutation in Foxl2 and derive birds with these genetic mutations. Birds will be raised to sexual maturity and assessed as to sexual phenotype, fertility, and morphology and function of the gonads. Blood samples from sexually mature birds will be used to determine circulating levels of steroids.
We will assess the effects of altering the Dmrt1/Foxl2 balance and of deleting these genes on gonadal development and on germ cell differentiation. We will examine different embryonic stages and young/adult birds and embryos at different stages of development and compare to wild-type siblings.
We will assess i) germ cell development and ii) somatic gonadal differentiation at the morphological and cellular level by histology and by expression analysis for markers of i) mitosis and meiosis, and ii) male supporting & steroidogenic cells and female supporting & steroidogenic cells. Immunostaining and/or RNA in situ techniques will be performed on cryosections to assess the pattern of these markers at protein and/or RNA level; qPCR will be performed on extracted RNA to quantify levels of expression.
We will define the network of genes regulated by Dmrt1 and Foxl2 by Chip-seq and RNA-seq analysis of gonads from embryos with genetic mutations and from male and female siblings

Impact Summary
Scientists.
The project aims to uncover mechanisms of sex determination in the chick. We have chosen to use new methods of genome editing (specifically the CRISPR/Cas9 system) to inactivate two genes thought likely to have opposing roles in testis and ovary determination and differentiation. The choice of using a genetic approach to test the current model of sex determination in the chick will provide a clear answer to long unresolved questions in the field, opening new ways forward for scientists working directly on the chicken system, and presumably that of other birds. This in itself will have an immediate impact on scientists working in the field of sex determination, within the UK and internationally. However, the development of the genome editing methods in birds will also have a broad impact on both basic and applied research in birds generally.
Moreover by testing the degree of conservation of the pathways or networks uncovered in the chick with those known to occur in mammals, the work will be of interest to scientists working in evolutionary biology and in disorders of sex differentiation in humans. The data obtained will help us to construct the networks of gene activity required for testis versus ovary development and reveal how certain genes take on critical roles within these networks in a species-specific manner.

Societal Impact
Our work will be relevant to the differentiation of both somatic cells and germ cells within the gonads, and will therefore be beneficial to researchers working directly in more applied fields of reproductive biology in chicken, but also probably in other birds and in mammals, including humans. The work is therefore likely to be of relevance to gender-based healthcare in humans and to the pharmaceutical industry for gender-based drug development. A greater understanding of gonadogenesis and germ cell development in birds may also be of benefit to conservation programmes. This knowledge may also lead to the development of improved in ovo sexing tests and could have significant consequences for commercial poultry breeding and meat production.
The differences between the sexes, how these arise during development, and their consequences are of widespread public interest. Similarly the use of genome editing techniques in farm animals is of current widespread interest and debate and this interest is likely to continue. Consequently it is envisaged that opportunities will arise throughout the period of the grant (and beyond) to highlight our research findings as well as the technologies involved and their use in wider contexts. This will also be important for policies affecting both research and application using the methods. Both the Roslin Institute and the Francis Crick Institute are actively involved in public engagement, as are the two main PIs.

Research staff
The project will also provide excellent opportunities for training at two of the UK's leading Institutes. Notably the work will require skills of working with chick embryos and hatched birds, fostering the development of in vivo skills. It will also involve the development and use of cutting edge methods of genetic manipulation and molecular analysis. This will enhance career opportunities for staff employed on the grant and for others associated with the work.
All the benefits and impacts of the research will accumulate over the period of the grant and beyond.