Initiation and Maintenance of Temperature-Dependent Sex Determination in the Red-Eared Slider Turtle

Abstract

The vertebrate gonad is an excellent model to study organogenesis due to its unique ability to form two distinct organs from a common bipotential primordium. No single factor is responsible for activation of ovary or testis development in all vertebrate species, but these developmental pathways tend to converge on the same cohort of genetic regulators. The structures of testes and ovaries are extremely similar across vertebrates, and this high level of conservation is also observed in the gene regulatory processes underlying their differentiation. In heterogametic species such as mice and chickens, genes on the sex chromosomes activate the genes that drive differentiation of the testis of ovary. However, not all vertebrate species have sex chromosomes, and it’s unknown how the many genetic and cellular processes that direct gonad development are activated in the absence of a clear genetic signal. Temperature-dependent sex determination (TSD) is one of the primary sex determination strategies found in reptiles and has repeatedly evolved in multiple reptilian lineages. During TSD, the fate of the gonad is driven by nest temperatures experienced during embryonic development. In the decades since TSD was first described, the molecular processes underlying this phenomenon have remained a mystery. The Red-Eared Slider turtle, Trachemys scripta elegans (T. scripta), is a widely-studied model for temperature-dependent sex determination. When eggs are incubated at a constant 26˚C, 100% of embryos will develop testes. Incubating eggs at a constant 31˚C produces only embryos with ovaries. Prior work has focused the regulation of aromatase, which is crucial to estrogen synthesis, but it is expressed relatively late in the sex determination window. A transcriptome analysis of T. scripta gonads through sex determination revealed a group of early, male-biased genes, including the H3K27 demethylase Kdm6b. In many vertebrates, the epigenetic state of key sex determining genes appears to be critical in the activation of testis or ovary specific-signaling. We investigated whether KDM6B mediates the effect of temperature on gene expression in T. scripta and we found that it activates a conserved regulator of male sex development, DMRT1. One of the few identified transcriptional regulators of Kdm6b, the transcription factor STAT3, is only phosphorylated at the warmer, female-producing temperature (FPT). We show that pSTAT3 binds the Kdm6b locus to repress transcription and inhibition of pSTAT3 is sufficient to induce female-to-male sex reversal. Using primary cells derived from T. scripta gonads, we found that a heat-mediated influx of calcium at FPT promotes phosphorylation of STAT3. From these data we propose the model that heat-mediated influx of calcium at FPT promotes activation of STAT3, a transcriptional repressor of the male pathway. Our model is the first proposed mechanism of temperature-dependent sex determination supported by direct experimental evidence. It is unknown how the gonad interprets environmental signals and coordinates cell fates across the tissue. The embryonic gonad coelomic epithelium is a common feature of many vertebrate gonads, and its development is critical to placement of the germ cells in the appropriate stem cell niche, which is required for germ cell survival and maturation. Previous studies of testis morphogenesis in T. scripta show that invaginations of the coelomic epithelium move germ cells into the gonad medulla to form the seminiferous tubules. We show that these invaginations only occur below germ cells, express the conserved Steroli cell marker SOX9, and are sensitive to the hormone environment of the gonad. These data suggest that signals between the germ cell, somatic cells in the coelomic epithelium, and somatic cells of the primordial cords collectively participate in the morphogenetic changes underlying testis development in T. scripta.Our findings provide a framework for future investigations into the mechanism underlying temperature-dependent sex determination by identifying the initial signaling events that regulate the epigenetic state of sex-specific genes and describing how cellular fates are maintained during the sex determination window. STAT3 signaling can be activated by many inputs and have numerous downstream impacts, only some of which have been experimentally tested, providing direction and future lines of investigation for the field. The data presented here has laid the groundwork for identifying how temperature-sex determination operates in the turtle and how pieces of this process may be conserved among many animal phyla.