Thermal Response of Epigenetic Genes Informs Turtle Sex Determination with and without Sex Chromosomes

Abstract

Vertebrate sexual fate can be established by environmental cues (e.g., temperature-dependent sex determination, TSD) or by genetic content (genotypic sex determination, GSD). While methylation is implicated in TSD, the influence of broader epigenetic processes in sexual development remains obscure. Here, we investigated for the first time the embryonic gonadal expression of the genome-wide epigenetic machinery in turtles, including genes and noncoding RNAs (ncRNAs) involved in DNA/histone acetylation, methylation, ubiquitination, phosphorylation, and RNAi. This machinery was active and differentially thermosensitive in TSD versus GSD (ZZ/ZW) turtles. Methylation and histone acetylation genes responded the strongest. The results suggest these working hypotheses: (i) TSD might be mediated by epigenetically controlled hormonal pathways (via acetylation, methylation, and ncRNAs), or by (ii) hormonally controlled epigenetic processes, and (iii) key epigenetic events prior to the canonical thermosensitive period may explain differences between TSD and GSD. Novel epigenetic candidate regulators other than methylation were identified, including previously unknown ncRNAs that could potentially mediate gonadogenesis. These findings illuminate the molecular ecology of reptilian sex determination and permitted hypothesis building to help guide future functional studies on the epigenetic transduction of external cues in TSD versus GSD systems.

Turtle Insights into the Evolution of the Reptilian Karyotype and the Genomic Architecture of Sex Determination

“…However, no master sex-determining gene(s) has been discovered in reptiles. Some candidates exist in reptiles, including in chelonians [72][73][74][75][76][77], and the list is growing thanks to the advent of third generation sequencing and advanced molecular cytogenetics (e.g.,) [28,61,[74][75][76][77][78]. Namely, the XY of S. triporcatus turtles contains Dmrt1 [28,78], a gene whose molecular evolution is linked to transitions in sex determination in reptiles [79] and which displays sexually dimorphic expression in TSD turtles ( [80] and references therein).…”

“…Candidate gene approaches, along with transcriptome and methylome analyses, plus initial functional assays have provided a number of candidates in TSD turtles for an upstream function in sexual development (e.g., [74][75][76][77], ruling out elements whose sexually dimorphic expression occurs too late in development to act as top master regulators (e.g., [73,76,[82][83][84][85][86][87][88][89]). To complicate matters further, this body of evidence has uncovered significant divergence in the transcriptional patterns of genes in this regulatory network [73,80].…”

Embryonic Temperature Programs Phenotype in Reptiles

“…Epigenetic mechanisms have been shown to play a role in environmental regulation of several traits in animals. In recent years, attempts have been made to survey epigenetic mechanisms to understand how temperature programs phenotype including studies of DNA methylation and histone modifications in several reptiles with TSD (Navarro-Martin et al, 2011;Matsumoto et al, 2013Matsumoto et al, , 2016Czerwinski et al, 2016;Yatsu et al, 2016;Fan et al, 2017;Radhakrishnan et al, 2017Radhakrishnan et al, , 2018Ge et al, 2018;Martín-del-Campo et al, 2019).…”

“…Radhakrishnan et al (2018) tried to correlate temperature with epigenetic mechanisms by studying differences in expression of genes known to be involved in DNA methylation and histone modification along with some small RNAs in TSD (Chrysemys picta) and GSD (Apalone spinifera) turtle species. Several genes involved in DNA methylation (Dnmt3b) and histone methylation (Nsd1, Setd1a, Carm1, Prmt1, Ash1l, and Prdm2) were found to be differentially regulated between male and female producing temperatures in Chrysemys picta (Radhakrishnan et al, 2018). These results together underscore the importance of studying mechanisms involved in epigenetic regulation of gene expression and TSD.…”

The neurogenetics of sexually dimorphic behaviors from a postdevelopmental perspective

“…This program, in turn, supports sex-specific cellular functions via sex-biased transcriptional and physiological states. [27][28][29][30][31][32][33][34] Because of their genetic tractability, the neuronal sexdetermination pathways of the fruit fly Drosophila melanogaster and the lab mouse Mus musculus are particularly well described. Both species produce sexually dimorphic male and female forms, which exhibit multiple sex-dependent morphological, neuroanatomical and behavioral traits.…”