Supplementary Material for: Developmental Profiles and Thyroid Hormone Regulation of Brain Transcripts in Frogs: A Species Comparison with Emphasis on Physalaemus pustulosus

In amphibians, thyroid hormones (THs) are considered key regulators of brain remodeling during metamorphosis, while sex steroids (estrogens and androgens) control sexual differentiation and gonadal development. However, these two endocrine axes can interact during tadpole brain development. Previously, we demonstrated that THs affect sex steroid-related gene expression in the developing brain of Silurana tropicalis and Rana pipiens; however, the gene expression changes differed between species. We chose to study a third anuran species, Physalaemuspustulosus, to test new hypotheses about the role of THs in the regulation of brain gene expression. We first established developmental transcript profiles of TH- and sex steroid-related genes in the brain of P. pustulosus. Then, following the same protocols as in our previous studies, we investigated triiodothyronine (T3) regulation of brain transcripts in premetamorphic P.pustulosus and then compared the results with our previous two studies. In the case of TH-related genes, TH receptor beta (trbeta) and deiodinase type 3 (dio3), mRNA developmental profiles were similar in the three species and with respect to other species in the published literature. However, the profiles of TH receptor alpha (tralpha) and deiodinase type 2 (dio2) mRNA revealed differences between anuran species. Among the three anurans we have studied, the direction of the T3 regulation of TH-related genes was overall similar, but the magnitude of gene expression change differed depending on the rate of metamorphosis in a given species. For the sex steroid-related genes, each species exhibited similar developmental profiles but differed in their response to T3. In P. pustulosus, T3 reduced the expression of aromatase (cyp19) while increasing mRNA levels of androgen and estrogen receptors. These results are similar to previous research in R. pipiens but differ from data for S. tropicalis, for which we found an increase in androgen synthesis enzymes but no effect on cyp19. Together, we propose that T3 has the potential to induce the brain androgen system in anurans. This could be achieved by increasing androgen synthesis enzymes (S. tropicalis) or by decreasing estrogen synthesis (due to a decrease in cyp19 in P. pustulosus and R. pipiens). In conclusion, we demonstrated that mechanisms of hormone interactions differ between anuran species, but in all cases T3 appears to affect the balance of sex steroids in the brain, stimulating the androgen system. We have shown that the regulation of sex steroid-related genes by T3 is more similar among closely related species than species with similar reproductive and developmental characteristics.在两栖动物中，甲状腺激素（thyroid hormones, THs）被认为是变态过程中大脑重塑的关键调控因子，而性类固醇激素（sex steroids，包括雌激素与雄激素）则控制性分化与性腺发育。然而，这两条内分泌轴在蝌蚪大脑发育阶段可发生相互作用。此前本团队已证实，甲状腺激素可影响热带爪蟾（Silurana tropicalis）与豹蛙（Rana pipiens）发育中大脑内与性类固醇相关的基因表达，但不同物种间的基因表达变化模式存在差异。 我们选取第三种无尾目物种——纹胸狭口蛙（Physalaemus pustulosus），以检验关于甲状腺激素在大脑基因表达调控中作用的全新假说。本研究首先构建了纹胸狭口蛙大脑内甲状腺激素及性类固醇相关基因的发育转录本谱；随后沿用此前研究的相同实验方案，探究了三碘甲状腺原氨酸（triiodothyronine, T3）对变态前纹胸狭口蛙大脑转录本的调控作用，并将结果与本团队此前两项研究的数据进行对比。 针对甲状腺激素相关基因，甲状腺激素受体β（thyroid hormone receptor beta, trβ）与Ⅲ型脱碘酶（deiodinase type 3, dio3）的mRNA发育谱在三个物种间，以及与已发表文献中的其他物种均较为相似；但甲状腺激素受体α（thyroid hormone receptor alpha, trα）与Ⅱ型脱碘酶（deiodinase type 2, dio2）的mRNA谱则展现出无尾目物种间的差异。 在本团队已研究的三种无尾目动物中，三碘甲状腺原氨酸对甲状腺激素相关基因的调控方向整体相似，但基因表达变化的幅度因特定物种的变态速率而异。对于性类固醇相关基因，各物种均展现出相似的发育谱，但对三碘甲状腺原氨酸的响应存在差异。 在纹胸狭口蛙中，三碘甲状腺原氨酸可降低芳香化酶（aromatase, cyp19）的表达，同时提升雄激素与雌激素受体的mRNA水平。该结果与此前在豹蛙中的研究相似，但与热带爪蟾的实验数据存在差异：在热带爪蟾中本团队发现雄激素合成酶表达上调，但未观察到对cyp19的影响。 综上，我们提出三碘甲状腺原氨酸具备诱导无尾目动物大脑雄激素系统的潜力，这一效应可通过两种途径实现：上调雄激素合成酶（如热带爪蟾），或降低雌激素合成能力（因纹胸狭口蛙与豹蛙中cyp19表达下调）。 本研究证实，无尾目物种间的激素相互作用机制存在差异，但在所有案例中，三碘甲状腺原氨酸似乎均可影响大脑内性类固醇激素的平衡，进而激活雄激素系统。此外，我们的研究表明，相较于具备相似生殖与发育特征的物种，亲缘关系较近的物种间，三碘甲状腺原氨酸对性类固醇相关基因的调控模式更为相似。