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.