Abstract
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 (Chrysemys picta TSD) before and during the thermosensitive period, and at equivalent stages in soft-shell turtles (Apalone spinifera—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 Amh, Ar, Esr1, Fog2, Gata4, Igf1r, Insr, and Lhx9 at male-producing temperature, and of β-catenin, Foxl2, Aromatase (Cyp19a1), Fst, Nf-kb, Crabp2 at female-producing temperature in Chrysemys]. Notably, antagonistic elements in gonadogenesis (e.g., β-catenin and Insr) were thermosensitive only in TSD early-embryos. Cirbp 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. Jak-Stat, Nf-κB, retinoic-acid, Wnt, and Mapk-signaling (not Akt and Ras-signaling) potentially mediate TSD thermosensitivity. Numerous species-specific ncRNAs (including Xist) were differentially-expressed, mostly upregulated at colder temperatures, as were unannotated loci that constitute novel TSD candidates. Cirbp showed warm-temperature upregulation in both turtles. Consistent transcription between turtles and alligator revealed putatively-critical reptilian TSD elements for male (Sf1, Amh, Amhr2) and female (Crabp2 and Hspb1) 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.
Highlights
Organisms vary wildly in how they determine sex [1]
The commitment of the bipotential gonad to differentiate into testes or ovaries is triggered by the genotype in genotypic sex determination (GSD), and by temperatures experienced during the thermosensitive period of embryonic or larval development in temperature-dependent sex determination (TSD) [4,5,6]
RNAseq data was obtained from Chrysemys embryonic tissues before the thermosensitive period when the gonads are not yet discernible, at the onset of the TSP when gonads are hard to separate from surrounding tissue, and during the mid and late TSP, from male-producing temperature (MPT = 26 ̊C), and female-producing temperature (FPT = 31 ̊C) [42]
Summary
Organisms vary wildly in how they determine sex [1]. Vertebrate sex-determining mechanisms range between genotypic sex determination (GSD) and environmental sex determination (ESD) [2, 3]. TSD is ancestral to turtles, reptiles, and perhaps even amniotes [11,12,13,14] such that the TSD of turtles and crocodilians may be considered homologous traits [11, 14] This diversity has defied scientific explanation [1], impeding a full understanding of sex ratio evolution and of population dynamics and diversification both in the past and in the face of contemporary environmental change [15,16,17,18]. Unlike GSD models such as mammals [19,20,21,22] and chicken [23] whose gonadal developmental pathways are well understood (albeit not fully), our knowledge for GSD reptiles and TSD species is incipient, and prevents deciphering the genetic architecture of vertebrate sex determination and its evolution
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