Five degrees Celsius separates a male leopard gecko from a female one

Incubate a leopard gecko egg at 26.5 degrees Celsius, and you will get a female. Raise the temperature to 31.5 degrees, and you will almost certainly get a male. This much has been known for years. What has not been known - at least not in any systematic detail for lizards and snakes - is precisely when temperature locks in that decision, what genes drive it, and how the molecular events unfold before anything visible changes in the developing embryo.

A team led by Professor Shinichi Miyagawa at Tokyo University of Science has now filled that gap with the first comprehensive histological and transcriptomic analysis of gonadal development in a squamate reptile with temperature-dependent sex determination.

The shift experiments

The researchers incubated leopard gecko (Eublepharis macularius) eggs at either 26.5 degrees Celsius (a female-producing temperature) or 31.5 degrees (male-producing). Then they performed a series of shift experiments, moving eggs between the two temperatures at different developmental stages to determine exactly when the temperature signal becomes irreversible.

The results were clean. Incubation at the cooler temperature produced 100% females. The warmer temperature produced 91% males. And the critical window - the temperature-sensitive period - closed at embryonic stage 36. Before that stage, switching temperatures could still flip the outcome. After it, the sex was set regardless of subsequent temperature changes.

Genes that diverge before bodies do

Early in development, embryos from both temperature groups looked identical under the microscope. No external differences, no obvious structural changes in the developing gonads. The first visible divergence came later, when ovaries became more spherical and testes elongated into the characteristic tubular structures.

But gene expression analysis told a different story. The molecular programs for male and female development had already begun to diverge well before any structural differences appeared. At male-producing temperatures, testis-related genes - AMH, DMRT1, and SOX9 - activated earlier. At female-producing temperatures, ovarian genes FOXL2 and CYP19A1 ramped up instead.

This temporal gap between molecular commitment and visible differentiation means the embryo's fate is decided at a level invisible to the eye, days before the gonads take shape. The temperature signal works through gene regulation, not through direct physical reshaping of tissue.

A familiar gene behaves differently

One finding sets the leopard gecko apart from other reptiles with temperature-dependent sex. The gene KDM6B, a chromatin modifier that plays a key role in male determination in certain turtles, showed a different regulatory pattern in geckos. Where turtle KDM6B responds directly and early to temperature cues, the gecko version appears to follow a different regulatory logic.

The study also identified early temperature-responsive genes involved in RNA splicing and cell adhesion - molecular processes that operate upstream of the sex-determination cascade itself. These genes responded to temperature before the canonical sex-determining genes activated, suggesting they may represent the initial molecular events that translate a thermal signal into a developmental decision.

Shared toolkit, different wiring

The broader picture that emerges is one of conserved parts but divergent assembly. The genes responsible for forming gonads - AMH, DMRT1, SOX9, FOXL2, CYP19A1 - are largely shared across reptiles. But the way temperature controls these genes has evolved differently in different lineages. Turtles, alligators, and geckos use many of the same molecular components, but the regulatory circuitry connecting temperature to gene activation is not identical.

This evolutionary plasticity is itself significant. Temperature-dependent sex determination has arisen independently multiple times across the reptile family tree, and the fact that different groups have wired the same genes differently suggests there are multiple viable solutions to the problem of translating an environmental cue into a developmental outcome.

The researchers note some caveats. The mother's body temperature before egg-laying may influence early development and could vary between laboratory colonies, introducing a source of variation that is difficult to control. The shift experiments, while informative, involve abrupt temperature changes that do not perfectly replicate the gradual thermal fluctuations eggs experience in natural nests.

Still, the study addresses a major gap. Temperature-dependent sex determination has been studied extensively in turtles and crocodilians but barely explored in squamates - the largest and most diverse reptile group. This work provides a molecular and developmental baseline that future studies of lizards and snakes can build on.