Evidence for avian cell autonomous sex identity (CASI) and implications for the sex-determination process?
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For the majority of animals, males and females are obviously different in terms of appearance, behaviour and physiology, and until recently, these differences were considered to be the result of hormone actions. However, there is now considerable evidence that the development of some sexually dimorphic structures/behaviours is a function of properties inherent to male and female cells (hormone independent). The relative contribution of hormones and cellular identity to the development of the phenotype is not clear and is likely to vary from species to species. The study of gynandromorph birds and chimeric embryos has greatly assisted efforts to distinguish between the effects of hormones and inherent cellular factors on phenotype. It is now clear that in birds, male/female differences are not primarily the result of hormone action and that male and female somatic cells possess a cell autonomous sex identity (CASI). Here, we review evidence for CASI in birds and discuss the implications for the process of sex determination.
KeywordsCASI gynandromorph dosage avian chicken Z chromosome W chromosome gonad dimorphic sex sexual chimeras
Green fluorescent protein
Cell autonomous sex identity
Sex determination region Y-chromosome gene
The DM-domain gene on the Y chromosome
Sry-box containing gene 9
Doublesex and Mab-3-related transcription factor 1
Mega base pairs
Kilo base pairs
Male hypermethylated region
Previously, the general view of vertebrate sex determination equated gonadal differentiation with sexual development: it was thought that the action of a ‘master regulator’ gene located on a sex chromosome determines whether the genital ridge develops as a testis or ovary, and then the male or female phenotype of other somatic tissues is imposed by gonadal hormones (regardless of genotype). However, it is now widely accepted that hormone-independent characteristics that are inherent to male and female cells, also play a role in determining the final phenotype (reviewed in Arnold 2003; Davies and Wilkinson 2006; Blecher and Erickson 2007; Arnold and Chen 2009; Ngun, et al. 2011). This raises the question about the extent of the contribution that these cell autonomous properties make to structural and functional differences between males and females, and the answer seems to be that the effect varies from species to species.
In mammals, hormones are still considered to play a dominant, but not exclusive, role in establishing the phenotype of non-gonadal tissues, while there is growing evidence that the inherent sex identity of individual male and female cells plays a much more significant role in defining sexual dimorphisms in birds, and possibly other vertebrates. Here, we will review the evidence for cell autonomous sex identity (CASI) and consider both the molecular basis of CASI and the possible implications of CASI for sex determination in birds.
Evidence for CASI
For some time, it has been clear that the development of sex differences in the avian brain are not solely due to sex differences in gonadal hormone secretion (reviewed in Arnold 1997). Recent studies of gynandromorph birds have demonstrated that this is also true for other somatic tissues and have suggested that this CASI of individual cells plays a major role in defining the phenotype of non-gonadal tissues (Agate et al. 2003; Zhao et al. 2010).
A long-standing theory on the etiology of gynandromorphs proposed that these birds arose because of the loss of a single sex-chromosome on one side of the animal at the two-cell stage of development (Cock 1955). The gynandromorph zebrafinch analysis, although not establishing a formal karyotype, suggested that this was not the case (Agate et al. 2003), and the study by Zhao et al. (2010) has now established, unequivocally, that these birds are not anueploids: tissues are composed of normal diploid male and female cells. A thorough analysis of the cellular composition of multiple tissues established that the majority of cells on the side that appears female contain one Z and one W sex chromosome, while the majority of cells on the side that appears male contain two Z sex chromosomes: these analyses have demonstrated that gynandromorph birds are undoubtedly male:female chimeras. The presence of both ZZ- and ZW-containing cells suggests that it is highly unlikely that these birds arise as a consequence of mutation at the two-cell stage of development and would support the hypothesis that gynandromorphs arise as a result of failure of extrusion of a polar body during meiosis and subsequent fertilisation of both a Z- and W-bearing female pronucleus (Hollander 1975).
The demonstration that gynandromorphs are male/female chimeras and that the phenotype of non-gonadal tissues reflects cellular genotype rather than circulating hormones is at odds with the classical view of vertebrate sex determination. These results strongly suggest that the avian phenotype is dependent on the nature of the cells comprising the individual tissue rather than being imposed by the type of gonad formed: both sides of these animals are exposed to an identical profile of gonadal products yet each side responds differently to these stimuli (Agate et al. 2003; Zhao et al. 2010). These analyses led to the conclusion that male and female chicken somatic cells possess a cell-autonomous sex identity (CASI). An analysis of the gonads present in the three gynandromorph chickens studied also supports the concept of CASI. Of the three birds, one carried a testis-like gonad on the left side, a second carried an ovary-like gonad on the left side, and the third carried an ovo-testis on the left side. Detailed analyses demonstrated that the morphological appearance of the gonads conformed to the cellular composition in that the structures which appeared to be testis or ovary were composed principally of ZZ- or ZW-containing cells, respectively, while the ‘ovo-testis’ comprised a mixture of ZZ- and ZW-containing cells.
Molecular basis of CASI
As regards the molecular basis underlying male and female CASI, clearly the most obvious difference between male and female somatic cells is the sex chromosomes. In birds, of course, the sex chromosomes are designated Z and W, with the females the heteromorphic sex (ZW) and the males the homomorphic sex (ZZ). Although the Z and W chromosomes share a common ancestry, a lack of meiotic pairing along most of the length of the female-specific W chromosome has resulted in its degeneration in both size and sequence content (Marshall Graves and Shetty 2001). The chicken Z chromosome is around 85 Mb long and encodes approximately 1,000 genes (Bellott et al. 2010), while the small and highly heterochromatic W chromosome is poorly represented with only 0.26 Mb of sequence available and only a handful of possible protein-coding genes identified (http://www.ensembl.org/index.html).
In species with heteromorphic sex chromosomes, it is thought that as genes are lost from the degenerating sex chromosome, a genetic regulatory mechanism must evolve to equalise the phenotypic expression of characteristics determined by genes on the Z chromosome. The shorthand description of this mechanism is ‘dosage compensation’, and this has been accomplished by a variety of different mechanisms in different species. These include, elevating X-chromosome-linked gene expression in male drosophila, lowering X-chromosome-linked gene expression in female Caenorhabditis elegans, and the random inactivation of one X chromosome in mammals (reviewed in Zarkower 2001; Straub and Becker 2007).
The differences between male and female cells that underlie CASI are obviously a function of the sex chromosomes, and we believe that the sex identity evident in avian male and female somatic tissues is a result of the type of dosage compensation system utilised in birds.
Surprisingly, until relatively recently, it was widely accepted that avian Z chromosomes were not subjected to the process of ‘dosage compensation’. This is surprising because the consequence of no dosage compensation would be that all Z-chromosome genes would be expressed at relatively higher levels in males than in females. This belief was mainly based on limited evidence regarding sex differences in the activity of the Z-linked aconitase enzyme (Baverstock et al. 1982). However, in 2001, this view was overturned when we established that the levels of expression of some Z chromosome genes were equalised in male and female birds, suggesting some form of dosage compensation (McQueen et al. 2001). This study examined transcript levels of only nine Z chromosome genes in male and female chick embryos prior to the development of the gonads (very few genes had been mapped to the Z chromosome by 2001). Although we observed very heterogeneous levels of expression between individuals of the same sex, similar averaged levels of expression were found in males and in females for six of these genes. This led to the conclusion that these genes must be subject to some form of dosage compensation but that this regulation did not affect all Z chromosome genes. These studies established for the first time that a significant proportion of avian Z chromosome genes were likely to be subject to dosage compensation but gave no real indication as to the extent of this compensation or to the nature of the mechanism involved. Since then, a large number of studies have been carried out that have extended and confirmed our initial findings, in that the majority of Z chromosome genes were examined and a significant proportion shown to be compensated. It is also clear from these studies that a large number of genes across the avian Z chromosome are expressed at a higher level from the two male Z chromosomes than from the single female Z chromosome (e.g., Itoh et al. 2007; Ellegren et al. 2007; reviewed in McQueen and Clinton 2009). This strongly argues against a global all-inclusive system of dosage compensation across the avian Z chromosome and suggests that many genes are not compensated or only partially compensated.
We believe that the male and female cellular identity evident in avian tissues is a direct result of a large number of sex chromosome genes being expressed at different levels in male and female cells. These CASI genes will include W-chromosome specific genes and non-compensated Z chromosome genes. We predict that CASI genes will be expressed in a sexually dimorphic fashion in all cells/tissues at all stages of development (including adult tissues).
While published studies would appear to support the existence of a large number of CASI genes, the majority of these male:female comparisons were performed on tissues from either adult birds or from embryos at stages after the development of the gonads, making it impossible to determine whether the sexually dimorphic expression is due to a lack of dosage compensation or a result of hormone action. However, a small number of studies have been carried out on tissues from stages before gonadal differentiation, and these strongly support the concept of CASI genes. Most notably, the study performed by Zhang et al. (2010) identified over 200 sexually dimorphic transcripts in H&H Stage 4 male and female embryos (Scholz et al. 2006; Lee et al. 2009; Zhang et al. 2010). On the basis of these and other studies (Melamed and Arnold 2007; Itoh et al. 2007; Mank and Ellegren 2009; McQueen and Clinton 2009), we believe that as many as 300 CASI genes exist.
At this stage, it is not clear whether CASI is likely to be manifested by the actions of a small number of genes with significant effects or by the cumulative activity of a large number of genes with small effects—or indeed, both.
Implication for sex determination
Sex determination is often equated solely with gonadal differentiation but is actually a life-long process. For species with genetic sex determination, sex determination per se obviously occurs at fertilisation when one sex acquires a pair of homomorphic sex chromosomes and the other, a pair of heteromorphic sex chromosomes. Gonadal development occurs later in development when the action of a sex chromosome gene(s) determines whether the genital ridge differentiates into a testis or an ovary, and, finally, hormones produced by the gonad then affect the phenotype of non-gonadal tissues.
Here, we review current understanding of sex-determination in birds and consider how an effective CASI may influence the development of the gonads and the sexual phenotype.
Sex-determining mechanisms and gonad-determining genes
During gonadal development, a specific trigger is thought to initiate a hierarchical cascade of regulatory gene activity in the indifferent genital ridge that induces the development of testes in males and ovaries in females. Vertebrate gonads are highly conserved structures, so it is not surprising that the genes regulating the morphological differentiation of the gonads appears to be largely conserved across different vertebrate species (Morrish and Sinclair 2002; Ferguson-Smith 2007; Marshall Graves and Peichel 2010; Chue and Smith 2011). However, the ‘trigger’ that initiates testis or ovary development is clearly not conserved. In mammals, this trigger is a localised, short burst of expression of a gene encoded on the male-specific Y chromosome (Sekido and Lovell-Badge 2009; Kashimada and Koopman 2010). This gene, Sry, is expressed in the male genital ridge for a short period immediately before the first appearance of male/female differences. Sry represented a ‘master regulator’ gene that was originally thought to divert the developing gonad onto the path of testis differentiation and off the ‘default’ ovarian path. Although the discovery of Sry represented a major breakthrough, in many ways, it also heralded a false dawn: attempts to identify ‘sex-determining genes’ in other species focused on matching the Sry-template, and this may not always have been appropriate. Despite major efforts in many species, only one other example quite like Sry has been identified: this gene is known as DMY, it is encoded on the Y chromosome and its expression can initiate testis development in either genetically male or female fish (Matsuda et al. 2007). However, DMY appears only in one fish species, Medaka, and may have only recently evolved (Kondo, et al. 2003).
Although the mammalian model is seen as a template for all vertebrate sex-determination systems, it may well be that some vertebrate gonad-determining genes conform to a mechanism that is more akin to that found in invertebrates. For example, in both flies and worms, the sex determination mechanism is cell autonomous and is based on the ratio between sex chromosomes and sets of autosomes (the X:A ratio) (Cline and Meyer 1996; Zarkower 2006). In Drosophila, an X:A ratio of 1 results in female (and ovary) differentiation while an X:A ratio of 0.5 generates males (and testes). In birds, it has not yet been established whether the sex (gonadal)-determination mechanism is dependent on a W-chromosome element or a Z-chromosome element (or both!) (Clinton 1998; Smith 2007). A W-chromosome ovary determining mechanism is simpler in principle and would be analogous to the Sry gene: each is sex specific and only requires to be ‘turned on’ at the appropriate time. A Z-chromosome gene on the other hand would be present in both sexes and would either require an upstream sex-specific signal (which would then be the gonad-determining factor) or would need to function differently in both sexes.
Although no obvious W-chromosome candidate ovary-determining genes have been identified, a gene known as DMRT1 (doublesex and Mab-3-related transcription factor 1) is a likely Z-chromosome testis-determining gene (Raymond et al. 1999; Ferguson-Smith 2007; Nanda et al. 2008; Smith 2010). In chickens, DMRT1 is expressed at higher levels in male (ZZ) gonads than in female (ZW) gonads, both at and after the suspected point of gonadal differentiation (Smith et al. 1999). Most significantly, DMRT1 expression is elevated in embryonic female gonads ‘masculinised’ by the aromatase inhibitor, fadrozole (Smith et al. 2003), and experimentally reducing levels of the DMRT1transcript in male chick embryos resulted in, at least partial, feminisation of the left gonad into an ovary-like structure (Smith et al. 2009). In the latter study, expression of the male-specific marker, SOX9, was significantly reduced, and expression of the female marker, aromatase, was ectopically activated in male gonads. These findings suggested that the DMRT1 gene product is required for testis development in the chicken and that the effect is dose dependent. The presence of DMRT1 on the ratite Z chromosome and its absence from the near homomorphic W chromosome (Shetty et al. 2002) supports the suggestion that DMRT1 is required for testis development in birds.
The mechanism of action of DMRT1 in avian gonadal differentiation is unclear, although a theoretical threshold is often proposed: levels above this threshold produce testes while levels below the threshold produce ovaries. It seems unlikely that this theoretical threshold is represented by an absolute level of DMRT1—with the variation in gene expression seen between different individual birds, one might expect high ZW expressers to exceed the threshold and low ZZ expressers to fail to achieve the required level. It seems more likely that the threshold is defined by the interaction of DMRT1 and some autosomal factor along the lines of the X:A ratio system seen in flies and worms. Under this scenario, the product of two copies of DMRT1 in males would initiate testis differentiation while the product of the single copy in females would not initiate testis differentiation.
The W chromosome?
While it seems clear that DMRT1 is required for testis differentiation, it may well be that ovary differentiation is not simply due to the lack of DMRT1 but is a process that requires the presence of a W chromosome. In support of this concept, a triploid line of birds produce 3A:ZZW hatchlings that appear phenotypically female and are described as intersex with a right testis and a left ovo-testis (Lin et al. 1995). Although the ovarian component of the left gonad is gradually lost as these birds mature, these observations suggest that the W chromosome may influence normal ovarian development. In this context, it is interesting to note that no avian aneuploids such as 2A:Z0 (which would be extremely informative), have ever been identified, and it has been suggested that such genotypes may be lethal (Marshall Graves 2003).
It has been suggested that W-chromosome gene products may promote ovarian development by regulating the methylation status and expression of the MHM gene (male hypermethylation) on the Z chromosome, which in turn effects expression of DMRT1 (Teranishi et al. 2001; Yang et al. 2011).
CASI and sex determination
Of course, it is possible that gonadal differentiation in birds does not simply depend on sex-specific expression differences at a single locus but rather on sexually dimorphic expression at multiple loci resulting from the different sex chromosome constitutions. However, regardless of which basic mechanism may be involved, the relationship, if any, between CASI and the genes regulating gonadal differentiation is unclear. DMRT1 is a Z-chromosome gene that does not appear to be dosage-compensated, but it is not a CASI gene because it is not expressed prior to the initiation of gonadal differentiation, and DMRT1 expression is restricted to a single tissue. While it is possible that male and female CASI regulates DMRT1 expression, this seems unlikely and would require some indirect mechanism as transcription of DMRT1 occurs in both sexes, and the differences in levels of DMRT1 product seem to simply reflect the difference in gene copy number. It seems more likely that transcription of the DMRT1 gene is directly regulated by some common autosomal factor.
While CASI is clearly involved in establishing and maintaining the tissue phenotype, it is not clear what role, if any, that CASI plays in initiating the development of any tissue, including the gonads.
We gratefully acknowledge support from the BBSRC and DEFRA.
- Barnard CJ (1983) Animal behaviour: ecology and evolution. J. Wiley, New YorkGoogle Scholar
- Gilbert SF (2006) Developmental biology, 8th edn. Sinauer Associates, Sunderland MassGoogle Scholar
- Morgan TH (1991) The physical basis of heredity. Lippincott, PhiladelphiaGoogle Scholar
- Teranishi M, Shimada Y, Hori T, Nakabayashi O, Kikuchi T, Macleod T, Pym R, Sheldon B, Solovei I, Macgregor H, Mizuno S (2001) Transcripts of the MHM region on the chicken Z chromosome accumulate as non-coding RNA in the nucleus of female cells adjacent to the DMRT1 locus. Chromosome Res 9:147–165PubMedCrossRefGoogle Scholar
- Wolpert L, Smith J, Jessell T, Lawrence P, Robert-son E, Meyerowitz E (2006) Principles of development, 3rd edn. Oxford University Press, OxfordGoogle Scholar
- Zarkower D (2006) Somatic sex determination. WormBook 10:1–12Google Scholar