Sex-determining mechanisms in the chick

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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.