How Turning Up The Heat Turns Male Dragons Into Females
Australian scientists may have figured out how reptiles change sex under the stress of extreme temperatures
If you’re a reptile keeper, you’re no doubt familiar with the phenomenon of temperature-dependent sex determination. Essentially, the sex of many reptiles — and even the sex of a variety of fishes — is determined by environmental temperatures experienced during sensitive stages of development rather than by the presence of a particular combination of sex chromosomes. Temperature-dependent sex determination differs from mammals and birds, which rely almost exclusively upon chromosomal sex determination. Yet, despite decades of research, we still don’t know exactly how temperature-dependent sex differences come about. But it appears that a group of Australian scientists have finally figured it out: it’s all about RNA editing.
Australian bearded dragons rely either on chromosomal or temperature-dependent sex determination
Sex in almost all mammals is dependent upon sex chromosomes. Mammalian sex is the result of their XX/XY sex determination system, where males are the heterogametic (XY) sex. But in many reptiles, their sex is the result of temperatures experienced during embryonic development: males result from exposure to some temperatures, whereas females result from other temperatures.
But bearded dragons rely on both sex chromosomes and environmental temperatures experienced during embryonic development to determine sex. At normal temperatures, their chromosomal make-up dictates which sex they are. But at high temperatures, dragons with male chromosomes undergo sex reversal and develop as females. So far, dragons are oddballs because they are the only reptiles known to undergo sex reversal at high temperatures — other reptiles are sensitive to cold temperatures (except snakes, which rely solely on sex chromosomes).
There are eight species of bearded dragons, all of which occur exclusively in Australia. One of these species, the Australian central bearded dragon, Pogona vitticeps, is an especially popular pet and zoo specimen because it is hardy and easy to care for. Additionally, this species is a powerful model organism that is providing scientists with a clearer understanding of the molecular events associated with temperature-dependent sex determination.
The bearded dragon relies on a ZZ/ZW sex chromosome system to guide sexual differentiation. In dragons, males are the ancestral homogametic sex, possessing two Z chromosomes, and females are heterogametic, with ZW sex chromosomes. This is opposite to the mammalian XX/XY sex chromosome system, where being female is the ancestral “default” sex.
Bearded dragons are a unique model system because high temperatures override their chromosomal sex determination system. When eggs are incubated below 32° Celsius (89.6° Fahrenheit), their sex chromosomes dictate their sex, but for temperatures above 32° Celsius, increasing numbers of eggs develop into females, regardless of their chromosomal make-up (ref). When temperatures reach 36° Celsius (96.8° Fahrenheit), 100% of genetic males develop into sex-reversed (ZZf) females.
Further, when a normal (ZZm) male dragon is mated with a sex-reversed ZZf dragon, this pairing necessarily yields only ZZ offspring. But the sex of the offspring resulting from this particular pairing is determined solely by incubation temperature, suggesting that some sort of permanent genetic change has occurred. In fact, according to previous research, this heritable genetic change is the complete loss of the W chromosome (ref).
High temperatures trigger over-expression of stress genes and release of stress hormones
To better understand the molecular mechanisms that control development of sex in bearded dragons, a team of Australian researchers at the Garvan Institute of Medical Research in Sydney, the University of Canberra and the Commonwealth Scientific and Industrial Research Organisation (CSIRO), examined and compared RNAs that are produced in a variety of tissues that they collected from adult dragons.
When the researchers compared sex-reversed ZZf female dragons to normal (ZWf) females, they found unique RNA expression profiles for 17 genes in their brain tissues. Most pronounced was the dramatic temperature-triggered over-expression (327-fold) of the environmental stress gene, Pro-opiomelanocortin (POMC, pronounced “pom-c”). POMC is 241 amino acid residues long. It is synthesized in the pituitary and is a precursor to the peptide hormone adrenocorticotropin (ACTH), which triggers the release of stress hormones in vertebrates. So dragons exposed to warm temperatures during embryonic development became stressed.
In addition to this dramatic over-expression of POMC, the researchers also found that sex-reversed ZZf dragons have female-like expression of male-biased genes, even though these animals do show some male-like behaviors and morphologies.
The most interesting finding is that two over-expressed genes, JARID2 and JMJD3, are members of the Jumonji gene family. Jumonji proteins are best known for their role both in development and in cancer: they control the identity of stem cells, and they are essential to normal organ development and sex differentiation in animals. At this point, we still don’t know much about the precise actions of individual Jumonji genes, but we do know that the mammalian version of JARID2 interacts with SRY, a gene on the mammalian Y chromosome that initiates the development of testes (ref). Further, dysfunction in this gene causes male-to-female sex reversal in mice.
The hidden importance of “junk DNA”
The researchers found that in adult dragons, JARID2 and JMJD3 were expressed more highly in ZZf tissues than in either ZWf of ZZm tissues. Not only were these two genes over-expressed, but the researchers were surprised to discover that JARID2 and JMJD3 produced a unique alternative transcript in sex-reversed ZZf dragons — a transcript that is not seen in the tissues of normal ZWf and ZZm dragons. Each gene’s alternative RNA transcript retained one intron. Introns are stretches of DNA that guide gene expression patterns instead of encoding proteins, and these regions are spliced out (or edited out), of the mature RNA message. These non-coding stretches were long known as “junk DNA” because, until recently, we didn’t understand their essential roles in gene expression.
But what was the result of these unedited introns? A careful examination of sequence data showed that JARID2 and JMJD3 each retained an intron that contained a stop codon. These premature stop codons either halt production of the protein or they cause smaller proteins to be constructed. Such abbreviated proteins don’t work normally: their functionality is either reduced, altered, or completely abolished.
We know that Jumonji genes control the expression of a suite of genes, at least some of which are involved in sex determination. So when Jumonji genes are altered by environmental stress, the downstream genes that they control are not turned on or off appropriately, and thus, they too are rendered sensitive to environmental stress — high temperatures in this case. Since these downstream genes orchestrate developmental processes involved in sex determination, environmental stress is linked to sex determination through these two Jumonji genes in dragons.
The researchers wondered whether alternative JARID2 and JMJD3 transcripts may be associated with temperature-sensitive sex determination in other reptiles? How universal is this molecular mechanism amongst reptiles?
To answer these questions, they compared the sequences of their newly-identified intron-retaining JARID2 and JMJD3 transcripts to RNAs from alligators and turtles, both of which are very distant relatives to dragons, and both of which show temperature-dependent sex determination. Turtles, which have an XX/XY system, undergo low-temperature masculinization, whereas alligators, which have a ZZ/ZW system, experience low-temperature femininization.
The researchers found similar intron-retaining JARID2 and JMJD3 transcripts in sex-reversed alligators and turtles, which makes these genes the most compelling candidates for being the molecular “switch” that controls sex reversals in reptiles.
Sex (reversal) is all about dosage
It is important to point out that the intron-retaining versions of these two genes are associated with temperature but not with a particular sex because sex determination is more subtle than this. The researchers propose that some reptile lineages, such as alligators and dragons, evolved from female heterogametic sex determination systems (ZZ/ZW) whilst others, such as turtles, evolved from male heterogametic systems (XX/XY). So intron-retaining JARID2/JMJD3 genes cause sex reversals by overriding development of the heterogametic sex by reducing the overall amount of vital proteins manufactured during sensitive stages of development. For example, in bearded dragons, males are the homogametic sex, so they get a double dose of all genes located on the Z chromosome. Loss of expression of some of these genes due to environmental stress would result in a reduction or loss of the proteins they encode, and this smaller dosage of key proteins could cause a genetic male to develop as a female.
Since this research was done in adult dragons, the team is currently working with embryonic dragons to identify when these temperature-sensitive RNA editing differences first appear. They also are removing either JARID2 or JMJD3 genes from dragon DNA to see how embryonic development is affected and whether this genetic loss may prevent sex reversal at high temperatures.
Seeing that “intron retention” in these two Jumonji genes was documented in dragons, alligators and turtles, which are evolutionarily distant reptilian lineages (Figure A), the researchers suggest this phenomenon is an ancient, conserved mechanism controlling reptilian temperature-dependent sex determination. Further, because the environmental stress gene, POMC, is dramatically over-expressed in sex-reversed individuals, these genetic events provide yet another compelling link between environmental stress and sex determination in reptiles.
Extrapolating outward, this means that global warming poses a serious threat to the continued existence of dragons because it will alter the sex ratios of these species’ populations. But now that we’ve got an idea of how to reverse the sex of reptiles, it will become possible to manipulate the sex ratios of these animals to help conserve them for future generations to enjoy.
Ira W. Deveson, Clare E. Holleley, James Blackburn, Jennifer A. Marshall Graves, John S. Mattick, Paul D. Waters, and Arthur Georges (2017). Differential intron retention in Jumonji chromatin modifier genes is implicated in reptile temperature-dependent sex determination, Science Advances, 3:e1700731, published online on 14 June 2017 ahead of print | doi:10.1126/sciadv.1700731
Shunsuke Kuroki, Shogo Matoba, Mika Akiyoshi, Yasuko Matsumura, Hitoshi Miyachi, Nathan Mise, Kuniya Abe, Atsuo Ogura, Dagmar Wilhelm, Peter Koopman, Masami Nozaki, Yoshiakira Kanai, Yoichi Shinkai and Makoto Tachibana (2013). Epigenetic Regulation of Mouse Sex Determination by the Histone Demethylase Jmjd1a, Science 341(6150):1106–1109 | doi:10.1126/science.1239864
Clare E. Holleley, Denis O’Meally, Stephen D. Sarre, Jennifer A. Marshall Graves, Tariq Ezaz, Kazumi Matsubara, Bhumika Azad, Xiuwen Zhang and Arthur Georges (2015). Sex reversal triggers the rapid transition from genetic to temperature-dependent sex, Nature, 523:79–82 | doi:10.1038/nature14574
Originally published at Forbes on 19 June 2017.