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

Lead Research Organisation: University of Edinburgh
Department Name: The Roslin Institute

Abstract

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.

Technical Summary

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.

Planned Impact

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.

Publications

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Clinton M (2012) Evidence for avian cell autonomous sex identity (CASI) and implications for the sex-determination process? in Chromosome research : an international journal on the molecular, supramolecular and evolutionary aspects of chromosome biology

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Clinton M (2016) Real-Time Sexing of Chicken Embryos and Compatibility with in ovo Protocols. in Sexual development : genetics, molecular biology, evolution, endocrinology, embryology, and pathology of sex determination and differentiation

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Garcia-Morales C (2015) Cell-autonomous sex differences in gene expression in chicken bone marrow-derived macrophages. in Journal of immunology (Baltimore, Md. : 1950)

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Guioli S (2014) Gonadal asymmetry and sex determination in birds. in Sexual development : genetics, molecular biology, evolution, endocrinology, embryology, and pathology of sex determination and differentiation

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Yang X (2016) A Window of MHM Demethylation Correlates with Key Events in Gonadal Differentiation in the Chicken. in Sexual development : genetics, molecular biology, evolution, endocrinology, embryology, and pathology of sex determination and differentiation

 
Description We have established that male and female cells respond in different ways to extracellular signals (developmental, hormonal etc.)

We have identified molecular signatures that are inherent features of male and female cells.

We have shown that cells of the female immune system are primed to respond more readily to infection than cells of the male immune system.

We have shown that the sexually dimorphic characteristics that we have identified are not dependent on gonadal hormones.

We have identified key differences in the developing muscle of male and female embryos.

Our findings suggest that differences between the male and female phenotypes are a direct result of non-compensated expression of sex chromosome genes.
Exploitation Route It may be possible to select for larger female birds and thereby increase levels of poultry meat production
Sectors Agriculture, Food and Drink

 
Description Male meat poultry have a greater food conversion efficiency than female meat birds and are approximately 33% heavier at the time of marketing. A number of commercial enterprises and academic researchers have attempted to increase meat production from female birds by essentially sex-reversing these birds. These efforts have focused on adjusting the hormonal profiles of the birds either by the direct administration of hormones, or by altering the developmental fate of the gonads. To date, these efforts have been unsuccessful. Our findings that the sexual phenotype is essentially cell autonomous should permanently curtail such efforts. Our data demonstrates that differences in muscle mass between male and female birds is established early in embryonic development and that these differences result from the differential expression of particular sex chromosome genes. Our findings may enable the development of a breeding strategy to produce larger female birds and thereby increase meat production.
First Year Of Impact 2015
Sector Agriculture, Food and Drink
Impact Types Societal

 
Description AMH expression 
Organisation University of Nottingham
Country United Kingdom 
Sector Academic/University 
PI Contribution discussion molecular biology
Collaborator Contribution discussions material biology
Impact publication
 
Description avian gonadal development 
Organisation Guangxi University
Country China 
Sector Academic/University 
PI Contribution discussions and laboratory work
Collaborator Contribution discussions and laboratory work
Impact publications
Start Year 2014
 
Description sex determination in birds 
Organisation Francis Crick Institute
Country United Kingdom 
Sector Charity/Non Profit 
PI Contribution discussions, laboratory work, funding applications
Collaborator Contribution discussions, laboratory work, funding applications
Impact publications
Start Year 2012
 
Description Cell autonomous sex identity 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact Favourable feedback

none
Year(s) Of Engagement Activity 2010
 
Description Contributed to online article 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact Contributed to an article on BBC Earth
Year(s) Of Engagement Activity 2015,2016
URL http://www.bbc.com/earth/uk
 
Description Institute open day 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact discussion with members of public

none
Year(s) Of Engagement Activity 2012,2013,2014
 
Description contributed to online article 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact Contributed to article in The Natural History magazine
Year(s) Of Engagement Activity 2017
URL http://www.naturalhistorymag.com/