How do the developmental networks that control sexual development function and evolve?
This work is funded in part by NSF IOS 0743284.
What renders TSD thermosensitive?
We use both candidate gene and global transcriptomic profiling approaches to address the crucial question of what molecular factor(s) renders TSD mechanisms thermosensitive. Potential candidates would be genes that express differentially by temperature prior to the onset of the thermosensitive period or TSP (genes organizing or activating the thermosensitive time window rather than those genes acting once the window has opened). Expression data early in development revealed such differential expression in TSD turtles in two early-acting genes (Sf1 and Wt1) involved in the formation of the bipotential gonad (prior to the gonadal commitment to the ovarian or testicular differentiation pathways) (Valenzuela et al. 2006, Valenzuela 2008). Because Wt1 regulates Sf1, Wt1 was a more likely candidate TSD master switch, and later data recapitulated these results and implicated Wt1 and Sf1 in the early activation of the thermosensitive period in a temperature-specific manner (Valenzuela et al. 2013). Transcriptomic profiling helped elucidate the full gene network regulating urogonadal development in turtles with contrasting sex determination (Radhakrishnan et al. 2017a; Gessler et al. 2023), confirming the thermosensitive transcription of many more genes in GSD turtles, despite the canalizing effect that sex chromosomes have on sex ratios.. Our studies also revealed epigenetic regulation via DNA methylation and alternative splicing of important genes such as Dmrt1 (Radhakrishnan et al. 2017b, 2018; Mizoguchi et al. 2020, 2022).
The flavors of temperature-dependent sex determination
Our data on six critical sex differentiation genes (Aromatase, Sf1, Wt1, Dmrt1, Sox9, and Dax1) in painted turtles (Chrysemys picta: TSD) and softshell turtles (Apalone mutica: GSD) indicated that multiple molecular pathways that differ in the regulation of common genes have evolved to produce ecologically-equivalent TSD systems, dispelling the assumption that TSD refers to a single trait (in contrast to GSD which encompasses distinct mechanisms such as XX/XY, ZZ/ZW, among others (Valenzuela et al. 2006; Valenzuela and Shikano 2007, Valenzuela 2008b, 2010a). Further, we have shown that the fundamental difference that sets TSD and GSD apart is not driven by Aromatase as previously proposed (Valenzuela and Shikano 2007). Still unknown is how many distinct systems of sexual polyphenism, i.e. TSD, exist in nature, how do they work, and why did they come into being? More recent work reveals that substantial transcriptional evolution has accrued across all vertebrates and is not unique to turtles (Valenzuela et al. 2013; Mizoguchi et al. 2020).
Evolutionary transcriptomics of gonadal development
We continue using a comparative transcriptomic approach to illuminate the regulation of the gene network underlying gonadal development at a genome-wide scale across TSD and GSD taxa and in an ecological context similar to that used for the candidate gene approach. This illuminates how this network responds to environmental perturbations at different time scales and what might be its evolutionary potential in the face of contemporary climate change.
Is gene expression totally thermoinsensitive in GSD taxa?
An additional evolutionary question is whether GSD species derived from TSD ancestors have lost all thermal sensitivity of the gonadal developmental network. Data from A. mutica indicate that this is not always the case, as this GSD turtle has retained its ancestral sensitivity in the expression of Wt1, the first such case ever to be reported (Valenzuela 2008). This result is paramount as it reveals that GSD taxa can harbor thermal sensitivity even when it is non-functional for sexual differentiation (temperature does not bias sex ratios in A. mutica). Such finding is critical because theoretical models for the evolution of TSD rely on the premise that GSD taxa posses an ubiquitous thermal sensitivity that can be co-opted during the evolution of phenotypic plasticity (TSD), and our data provide the first empirical evidence for its existence at the level of gene expression, not only in candidate genes but genome-wide, including epigenetic machinery genes (Radhakrishnan et al. 2017a,b; Gessler et al. 2023). Interestingly, some thermosensitive gene expression in Apalone spinifera is relic, some altered, and some de novo, present likely because the evolution of sex chromosomes in GSD turtles release thermosensitive transcription from purifying selection that affects TSD turtles (Radhakrishnan et al. 2017a; Gessler et al. 2023).
How are TSD males and females produced in nature, and how will TSD respond to climate change?
We are elucidating the effect of fluctuating temperature on gene expression underlying gonadogenesis in turtles, and testing the ecological relevance of our observations made under constant temperature. This approach is essential because most molecular studies of TSD have been carried out at constant temperatures, yet sex ratios produced under constant conditions often differ from sex ratios obtained in the field where temperature fluctuates daily (e.g. Valenzuela et al. 1997, Valenzuela 2001a). Our work has yielded developmental models that better predict how males and female TSD turtles are produced in natural nests, and shows that extreme thermal fluctuations predicted from climate change have the potential to unexpectedly accelerate demographic collapse of vertebrates with temperature-dependent sex determination (Valenzuela et al 2019).