Transcriptomic responses to environmental temperature by turtles with temperature-dependent and genotypic sex determination assessed by RNAseq inform the genetic architecture of embryonic gonadal development

Abstract

<p>Vertebrate sexual fate is decided primarily by the individual’s genotype (GSD), by the environmental temperature during development (TSD), or both. Turtles exhibit TSD and GSD, making them ideal to study the evolution of sex determination. Here we analyze temperature-specific gonadal transcriptomes (RNA-sequencing validated by qPCR) of painted turtles (<em>Chrysemys picta</em> TSD) before and during the thermosensitive period, and at equivalent stages in soft-shell turtles (<em>Apalone spinifera—</em>GSD), to test whether TSD’s and GSD’s transcriptional circuitry is identical but deployed differently between mechanisms. Our data show that most elements of the mammalian urogenital network are active during turtle gonadogenesis, but their transcription is generally more thermoresponsive in TSD than GSD, and concordant with their sex-specific function in mammals [e.g., upregulation of <em>Amh</em>, <em>Ar</em>, <em>Esr1</em>, <em>Fog2</em>, <em>Gata4</em>, <em>Igf1r</em>, <em>Insr</em>, and <em>Lhx9</em> at male-producing temperature, and of <em>β-catenin</em>, <em>Foxl2</em>, <em>Aromatase</em> (<em>Cyp19a1</em>), <em>Fst</em>, <em>Nf-kb</em>, <em>Crabp2</em> at female-producing temperature in <em>Chrysemys</em>]. Notably, antagonistic elements in gonadogenesis (e.g., <em>β-catenin</em> and <em>Insr</em>) were thermosensitive only in TSD early-embryos. <em>Cirbp</em> showed warm-temperature upregulation in both turtles disputing its purported key TSD role. Genes that may convert thermal inputs into sex-specific development (e.g., signaling and hormonal pathways, RNA-binding and heat-shock) were differentially regulated. <em>Jak-Stat</em>, <em>Nf-κB</em>, <em>retinoic-acid</em>, <em>Wnt</em>, and <em>Mapk-</em>signaling (not <em>Akt</em> and <em>Ras</em>-signaling) potentially mediate TSD thermosensitivity. Numerous species-specific ncRNAs (including <em>Xist</em>) were differentially-expressed, mostly upregulated at colder temperatures, as were unannotated loci that constitute novel TSD candidates. <em>Cirbp</em> showed warm-temperature upregulation in both turtles. Consistent transcription between turtles and alligator revealed putatively-critical reptilian TSD elements for male (<em>Sf1</em>, <em>Amh</em>, <em>Amhr2</em>) and female (<em>Crabp2</em> and <em>Hspb1</em>) gonadogenesis. In conclusion, while preliminary, our data helps illuminate the regulation and evolution of vertebrate sex determination, and contribute genomic resources to guide further research into this fundamental biological process.</p>