Post-Transcriptional Mechanisms Respond Rapidly to Ecologically Relevant Thermal Fluctuations During Temperature-Dependent Sex Determination

Abstract

An organism’s ability to integrate transient environmental cues experienced during development into molecular and physiological responses forms the basis for adaptive shifts in phenotypic trajectories. During temperature-dependent sex determination (TSD), thermal cues during discrete periods in development coordinate molecular changes that ultimately dictate sexual fate and contribute to patterns of inter- and intra-sexual variation. How these mechanisms interface with dynamic thermal environments in nature remains largely unknown. By deploying thermal loggers in wild nests of the American alligator (Alligator mississippiensis) over two consecutive breeding seasons, we observed that 80% of nests exhibit both male- and female-promoting thermal cues during the thermosensitive period, and of these nests, all exhibited both male- and female-promoting temperatures within the span of a single day. These observations raise a critical question— How are opposing environmental cues integrated into sexually dimorphic transcriptional programs across short temporal scales? To address this question, alligator embryos were exposed to fluctuating temperatures based on nest thermal profiles and sampled over the course of a daily thermal fluctuation. We examined the expression dynamics of upstream genes in the temperature-sensing pathway and find that post-transcriptional alternative splicing and transcript abundance of epigenetic modifier genes JARID2 and KDM6B respond rapidly to thermal fluctuations while transcriptional changes of downstream effector genes, SOX9 and DMRT1, occur on a delayed timescale. Our findings reveal how the basic mechanisms of TSD operate in an ecologically relevant context. We present a hypothetical hierarchical model based on our findings as well as previous studies, in which temperature-sensitive alternative splicing incrementally influences the epigenetic landscape to affect the transcriptional activity of key sex-determining genes.

Incubation Temperature and Maternal Resource Provisioning, but Not Contaminant Exposure, Shape Hatchling Phenotypes in a Species with Temperature-Dependent Sex Determination

“…Nest thermal environments in nature are often more dynamic than those implemented in artificial incubation experiments (Paitz et al, 2010;Bowden et al, 2014;Bock et al, 2020a). In this study, we found that fluctuating thermal regimes yielded different outcomes from the constant temperature control for a subset of hatchling traits.…”

“…Mounting evidence suggests that thermal fluctuations in wild nests play key roles in shaping offspring sex ratios in other TSD species, such as Trachemys scripta (Carter et al, 2018); yet we currently lack an empirically validated model to predict crocodilian sex ratios under fluctuating thermal regimes. This is in part because few experiments have implemented incubation treatments that incorporate thermal variability in these taxa (Simoncini et al, 2019;Bock et al, 2020a) and because the molecular mechanisms underlying TSD remain elusive, despite recent advances in this area (Czerwinski et al, 2016;Yatsu et al, 2016;Deveson et al, 2017;Ge et al, 2018). Predicting the impacts of thermal fluctuations on crocodilian sex ratios is further complicated by their unique temperature-bysex reaction norm, in which females are produced at extreme temperatures and males at intermediate temperatures (F-M-F pattern) (Lang and Andrews, 1994).…”

“…1; Lang and Andrews, 1994;Gonzalez et al, 2019). The shape of the fluctuating treatments was designed based on empirically derived nest temperature profiles to mirror natural fluctuations of wild nests, as described in Bock et al (2020a); and the constant intermediate temperature was based on the overall mean nest temperature at Tom Yawkey Wildlife Center in 2015. The constant female-and male-promoting temperatures were selected because each of these temperatures reliably produces unisexual or near-unisexual outcomes (∼100% females and ∼100% males, respectively), with very little variation observed across studies or populations (Lang and Andrews, 1994;Gonzalez et al, 2019).…”

Brief exposure to warm temperatures reduces intron retention in Kdm6b in a species with temperature-dependent sex determination

“…Recent research suggests that temperature-responsive epigenetic regulators initiate sex-specific gene expression in reptiles [4–7] and may drive sex determination in species with TSD. The histone demethylase Kdm6b has received specific attention in this regard, as it is among the earliest genes to respond to temperature [8,9]; in T. scripta , cool, male-producing temperatures result in increased expression of Kdm6b [5].…”

“…The effect of temperature on Kdm6b expression is regulated by signal transducer and activator of transcription 3 (STAT3), where levels of phosphorylated STAT3 increase at warm temperatures, repressing Kdm6b transcription [6]. For several species with TSD, including T. scripta , an intron-containing transcript of Kdm6b accumulates at cool incubation temperatures [7,8,10]. More generally, intron retention has been demonstrated for a number of genes in the developing gonads of species with TSD [7–12], including the American alligator ( Alligator mississippiensis ), where Jarid2 exhibits intron retention that can change over the course of hours when incubation temperatures fluctuate [7].…”