Developmental consequences and molecular basis of cell-autonomous sex identity in the chicken

The mammalian model of sexual development is thought to apply to all vertebrates but we have shown that this is not the case. In mammals the SRY gene on the Y chromosome in males directs the path of gonad development toward making a testis rather than an ovary. Gonadal hormones then determine the secondary sexual characteristics of males and females. In birds, as in mammals, the sex chromosomes determine whether the individual is male or female but the secondary sex characteristics are not primarily determined by gonadal products circulating in the blood stream. We know this from our analysis of rare, naturally occurring birds known as gynandromorphs, which we have shown to be chimeras comprised of both male cells and female cells. These birds display a striking asymmetry that confers male characteristics on one side of the body (male feather colour, large musculature, large wattle) and female characteristics on the other - despite the fact that both sides of the body are exposed to the same levels of circulating hormones. We have carried out transplantation studies in normal chicken embryos at day 2 of development, before the gonads form. As the embryo develops, transplanted cells from one sex can integrate into tissues in the opposite sex, including gonad. If transplanted donor cells are from the same sex as the host, the donor cells will fully integrate into key compartments in the gonads and express genes characteristic of the embryonic testis or ovary. We have shown that cells from one sex will not integrate into the key functional compartment of the gonads of the opposite sex. It seems that transplanted presumptive gonadal cells can differentiate properly, but retain their sex identity regardless of environment. We maintain that bird somatic cells possess a 'cell-autonomous sex identity' (CASI). This project aims to i) establish the influence of CASI on phenotype at different stages of development and ii) establish the molecular basis underlying this phenomenon. i) We will extend our current transplantation studies to determine the extent to which cells are affected by CASI, to determine how CASI affects different cell types, and determine whether tissue grafted from a 2-day embryo retains its sex identity throughout the lifetime of the animal. ii) The sex chromosomes in birds are designated Z and W: males have two Z chromosomes and females have a Z and a W chromosome. Birds have an unusual mechanism for regulating expression of Z-chromosome genes which results in around 300 Z-chromosome genes that are expressed at different levels in male tissues than in female tissues (in addition to around 15 genes on the W-chromosome). We believe that there is a pattern of sex chromosome gene expression that establishes a 'ground state' that defines every cell in the body as male or female. To test our hypothesis and to identify the genes involved in this ground state, we will establish patterns of gene expression for the early male and female embryos and determine how differentiation and sex reversal affects these patterns.

Central to the model of mammalian sexual development are the concepts of a sexually 'indifferent' period early in development and that the adult sexual appearance depends on the type of gonad formed. This model is widely accepted and is considered to apply to all vertebrate species. We proposed that the secondary sexual characteristics in birds do not depend on the nature of the gonads formed and that somatic cells of male and female birds display a cell-autonomous sex identity. Our new data confirms that our hypothesis is correct and suggests that gonadal development is triggered by a constitutive property of the developing tissue rather than the transient action of a sex-determining gene as seen in mammals. Our finding of a cell-autonomous sex identity (CASI) in chickens will necessitate a re-evaluation of the established concept of sex differentiation in all vertebrates. The proposed experiments will fully characterise the developmental consequences of CASI and test the hypothesis we have developed on the molecular basis of this phenomenon. We will generate birds with male:female chimeric gonads and examine them at different stages of embryonic development and as adult birds, to determine how the presence of cells of the opposite sex affects the phenotype of the tissue, and how the cellular environment affects the function of the donor cells. Our hypothesis is that the molecular basis of CASI is dependent on sexually dimorphic gene expression resulting from the system of dosage compensation employed by birds. To test this hypothesis, we will; examine transcripts expressed in the very early male and female embryo to establish a 'ground state' of sex-chromosome gene expression in male and female cells, determine how lineage differentiation affects the male and female 'ground state' in gonad and muscle, and establish how sex-reversal affects the male and female 'ground state' of gene expression.

This is primarily a fundamental science project that aims to produce insights into the mechanisms of sex-determination, gonadal development and development of the sexual phenotype. The immediate impact will chiefly be upon the academic beneficiaries and the general public. Academic beneficiaries will gain new insights into the basis of sexual development and will be required to re-evaluate established concept of sex determination. Rather than the mammalian system being a model for sex-determination and sexual development for all vertebrates, it may be that the mammalian system is the exception. There will also be practical implications arising from this work. The poultry industry would prefer to raise all male broilers and only female birds of layer strains and have invested significant resources in efforts to permanently sex-reverse birds. These approaches have been largely based on findings gleaned from the mammalian system and have focused on manipulating hormone levels. Our findings explain why these efforts have not succeeded and suggest that such approaches are futile. There are significant welfare problems associated with alternative strategies to manipulate sex ratios that result in an annual commercial destruction of 250 million day-old male chicks in Europe alone. Our findings on the molecular basis of cell-autonomous sex identity should lead to the development of improved in ovo sexing assays and consequently reduce the number of day-old chicks slaughtered. Development of the techniques used in our current and proposed study should encourage the use of the chick as a model system for understanding early vertebrate development and lead to a reduction in the use of mammalian models.