Gene expression of male pathway genes sox9 and amh during early sex differentiation in a reptile departs from the classical amniote model.

BACKGROUND: Sex determination is the process whereby the bipotential embryonic gonads become committed to differentiate into testes or ovaries. In genetic sex determination (GSD), the sex determining trigger is encoded by a gene on the sex chromosomes, which activates a network of downstream genes; in mammals these include SOX9, AMH and DMRT1 in the male pathway, and FOXL2 in the female pathway. Although mammalian and avian GSD systems have been well studied, few data are available for reptilian GSD systems. RESULTS: We conducted an unbiased transcriptome-wide analysis of gonad development throughout differentiation in central bearded dragon (Pogona vitticeps) embryos with GSD. We found that sex differentiation of transcriptomic profiles occurs at a very early stage, before the gonad consolidates as a body distinct from the gonad-kidney complex. The male pathway genes dmrt1 and amh and the female pathway gene foxl2 play a key role in early sex differentiation in P. vitticeps, but the central player of the mammalian male trajectory, sox9, is not differentially expressed in P. vitticeps at the bipotential stage. The most striking difference from GSD systems of other amniotes is the high expression of the male pathway genes amh and sox9 in female gonads during development. We propose that a default male trajectory progresses if not repressed by a W-linked dominant gene that tips the balance of gene expression towards the female trajectory. Further, weighted gene expression correlation network analysis revealed novel candidates for male and female sex differentiation. CONCLUSION: Our data reveal that interpretation of putative mechanisms of GSD in reptiles cannot solely depend on lessons drawn from mammals.

Corticosterone does not have a role in temperature sex reversal in the central bearded dragon (Pogona vitticeps).

Environmental sex determination (ESD) is common among ectothermic vertebrates. The stress axis and production of stress hormones (corticosteroids) regulates ESD in fish, but evidence of a similar influence in reptiles is sparse and conflicting. The central bearded dragon (Pogona vitticeps) has a system of sex determination involving the interplay between sex chromosomes (ZZ/ZW female heterogamety) and the thermal environment. High egg incubation temperatures induce sex reversal of the ZZ genotype, feminizing chromosomally male individuals. Here we show that corticosterone elevation is not associated with sex reversal in the central bearded dragon, either during embryonic development or adulthood. We also demonstrate experimentally that sex determination is not affected by corticosterone injection into the yolk. This strongly suggests that stress axis upregulation by high temperature during incubation does not cause sex reversal in P. vitticeps. Our work is in general agreement with other research in reptiles, which suggests that the stress axis does not mediate sex in reptiles with ESD. Alternative biological systems may be responsible for capturing environmental conditions during reptile development, such as cellular calcium and redox regulation or the action of temperature-sensitive splicing factors.

Temperature-Induced Sex Reversal in Reptiles: Prevalence, Discovery, and Evolutionary Implications.

Sex reversal is the process by which an individual develops a phenotypic sex that is discordant with its chromosomal or genotypic sex. It occurs in many lineages of ectothermic vertebrates, such as fish, amphibians, and at least one agamid and one scincid reptile species. Sex reversal is usually triggered by an environmental cue that alters the genetically determined process of sexual differentiation, but it can also be caused by exposure to exogenous chemicals, hormones, or pollutants. Despite the occurrence of both temperature-dependent sex determination (TSD) and genetic sex determination (GSD) broadly among reptiles, only 2 species of squamates have thus far been demonstrated to possess sex reversal in nature (GSD with overriding thermal influence). The lack of species with unambiguously identified sex reversal is not necessarily a reflection of a low incidence of this trait among reptiles. Indeed, sex reversal may be relatively common in reptiles, but little is known of its prevalence, the mechanisms by which it occurs, or the consequences of sex reversal for species in the wild under a changing climate. In this review, we present a roadmap to the discovery of sex reversal in reptiles, outlining the various techniques that allow new occurrences of sex reversal to be identified, the molecular mechanisms that may be involved in sex reversal and how to identify them, and approaches for assessing the impacts of sex reversal in wild populations. We discuss the evolutionary implications of sex reversal and use the central bearded dragon (Pogona vitticeps) and the eastern three-lined skink (Bassiana duperreyi) as examples of how species with opposing patterns of sex reversal may be impacted differently by our rapidly changing climate. Ultimately, this review serves to highlight the importance of understanding sex reversal both in the laboratory and in wild populations and proposes practical solutions to foster future research.

Cellular calcium and redox regulation: the mediator of vertebrate environmental sex determination?

Many reptiles and some fish determine offspring sex by environmental cues such as incubation temperature. The mechanism by which environmental signals are captured and transduced into specific sexual phenotypes has remained unexplained for over 50 years. Indeed, environmental sex determination (ESD) has been viewed as an intractable problem because sex determination is influenced by a myriad of genes that may be subject to environmental influence. Recent demonstrations of ancient, conserved epigenetic processes in the regulatory response to environmental cues suggest that the mechanisms of ESD have a previously unsuspected level of commonality, but the proximal sensor of temperature that ultimately gives rise to one sexual phenotype or the other remains unidentified. Here, we propose that in ESD species, environmental cues are sensed by the cell through highly conserved ancestral elements of calcium and redox (CaRe) status, then transduced to activate ubiquitous signal transduction pathways, or influence epigenetic processes, ultimately to drive the differential expression of sex genes. The early evolutionary origins of CaRe regulation, and its essential role in eukaryotic cell function, gives CaRe a propensity to be independently recruited for diverse roles as a 'cellular sensor' of environmental conditions. Our synthesis provides the first cohesive mechanistic model connecting environmental signals and sex determination pathways in vertebrates, providing direction and a framework for developing targeted experimentation.

Sex determination mode does not affect body or genital development of the central bearded dragon (Pogona vitticeps).

BACKGROUND: The development of male- or female-specific phenotypes in squamates is typically controlled by either temperature-dependent sex determination (TSD) or chromosome-based genetic sex determination (GSD). However, while sex determination is a major switch in individual phenotypic development, it is unknownhow evolutionary transitions between GSD and TSD might impact on the evolution of squamate phenotypes, particularly the fast-evolving and diverse genitalia. Here, we take the unique opportunity of studying the impact of both sex determination mechanisms on the embryological development of the central bearded dragon (Pogona vitticeps). This is possible because of the transitional sex determination system of this species, in which genetically male individuals reverse sex at high incubation temperatures. This can trigger the evolutionary transition of GSD to TSD in a single generation, making P. vitticeps an ideal model organism for comparing the effects of both sex determination processes in the same species. RESULTS: We conducted four incubation experiments on 265 P. vitticeps eggs, covering two temperature regimes ("normal" at 28 °C and "sex reversing" at 36 °C) and the two maternal sexual genotypes (concordant ZW females or sex-reversed ZZ females). From this, we provide the first detailed staging system for the species, with a focus on genital and limb development. This was augmented by a new sex chromosome identification methodology for P. vitticeps that is non-destructive to the embryo. We found a strong correlation between embryo age and embryo stage. Aside from faster growth in 36 °C treatments, body and external genital development was entirely unperturbed by temperature, sex reversal or maternal sexual genotype. Unexpectedly, all females developed hemipenes (the genital phenotype of adult male P. vitticeps), which regress close to hatching. CONCLUSIONS: The tight correlation between embryo age and embryo stage allows the precise targeting of specific developmental periods in the emerging field of molecular research on P. vitticeps. The stability of genital development in all treatments suggests that the two sex-determining mechanisms have little impact on genital evolution, despite their known role in triggering genital development. Hemipenis retention in developing female P. vitticeps, together with frequent occurrences of hemipenis-like structures during development in other squamate species, raises the possibility of a bias towards hemipenis formation in the ancestral developmental programme for squamate genitalia.