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

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.

Dmrt1 induces the male pathway in a turtle with temperature-dependent sex determination

“…Recent transcriptomes of TSD taxa show a series of genes exhibiting earlier differential expression between MPT and FPT, before the onset of TSP or even the formation of a gonad, such as Sf1 (Valenzuela et al, 2006;Valenzuela, 2008), Igf1r, Insr (Radhakrishnan et al, 2017), Fdxr, Pcsk6, Kdm6b, Twist1, Hsp6b and TRP channel genes (Czerwinski et al, 2016). These genes might be involved in regulating temperature-induced activation or repression of putative sex-determining genes.…”

“…The idea that TSD and GSD systems share common genetic components is strongly supported by recent transcriptomes of TSD taxa from the red-eared slider turtle (Czerwinski et al, 2016), the painted turtle (Radhakrishnan et al, 2017) and the alligator (Yatsu et al, 2016). Although RNA interference has been applied on in vitro gonads of TSD turtles to knockdown Sox9 expression (Sifuentes-Romero et al, 2013;Shoemaker-Daly et al, 2010), a functional analysis of candidate sex-determining genes has not yet been performed in vivo in any reptile with TSD, which is largely due to lack of efficient genetic manipulation techniques in TSD species.…”

Chromosomal Rearrangements during Turtle Evolution Altered the Synteny of Genes Involved in Vertebrate Sex Determination

“…For instance, chromosomal rearrangements can impact gene expression [Hare- Grafodatskaya et al [2007]; 9, Radhakrishnan et al [2017]; 10, Chassot et al [2012]; 11, Tomaselli et al [2011]; 12, Valenzuela [2008b]; 13, Valenzuela et al [2006];14, Valenzuela [2008a]. wood and Fraser, 2014], either by direct physical disruption, by changing regulatory elements, or by creating positional effects, as in the case of many mendelian disorders in humans, some of which are associated with sex determination [Harewood et al, 2012].…”

“…Examples include the translocation breakpoint of SOX9 which is associated with male-to-female sex reversal in XY patients [Velagaleti et al, 2005], inversions linked to the aromatase ( CYP19 ) excess syndrome that place this gene under the control of the promoter of other expressed genes [Demura et al, 2007], and the translocation of SRY from the human Y to the X chromosome in XX hermaphrodite patients [Margarit et al, 2000]. Whether chromosomal rearrangements alter the relative location of genes and syntenic groups of the gene regulatory network of sexual development in turtles remains unknown [Valenzuela, 2008a;Czerwinski et al, 2016;Radhakrishnan et al, 2017]. We address this question here.…”

Effects of Incubation Temperature on the Expression of Sex-Related Genes in the Chinese Pond Turtle, <b><i>Mauremys reevesii</i></b>

“…Despite widespread occurrence of TSD in reptiles, it is still unclear how the molecular network responds to temperature and drives the sexual fate. Our understanding of the molecular network in reptiles is mostly based on previous studies that investigated sex determination in mammals, although recent transcriptomic approaches have examined the alligator ( Alligator mississippiensis ) [Yatsu et al, 2016], slider turtle ( Trachemys scripta ) [Czerwinski et al, 2016], and painted turtle ( Chrysemys picta ) [Radhakrishnan et al, 2017] genomes directly. Earlier studies suggested that in therian mammals the high expression of S ry upregulates the expression of Sox9 and downstream genes such as Dmrt1 [Bullejos and Koopman, 2001].…”