Sexual Phenotype Differences in zic2 mRNA Abundance in the Preoptic Area of a Protogynous Teleost, Thalassoma bifasciatum

The highly conserved members of the zic family of zinc-finger transcription factors are primarily known for their roles in embryonic signaling pathways and regulation of cellular proliferation and differentiation. This study describes sexual phenotype differences in abundances of zic2 mRNA in the preoptic area of the hypothalamus, a region strongly implicated in sexual behavior and function, in an adult teleost, Thalassoma bifasciatum. The bluehead wrasse (Thalassoma bifasciatum) is a valuable model for studying neuroendocrine processes because it displays two discrete male phenotypes, initial phase (IP) males and territorial, terminal phase (TP) males, and undergoes socially-controlled protogynous sex change. Previously generated microarray-based comparisons suggested that zic2 was upregulated in the brains of terminal phase males relative to initial phase males. To further explore this difference, we cloned a 727 bp sequence for neural zic2 from field-collected animals. Riboprobe-based in situ hybridization was employed to localize zic2 signal in adult bluehead brains and assess the relative abundance of brain zic2 mRNA across sexual phenotypes. We found zic2 mRNA expression was extremely abundant in the granular cells of the cerebellum and widespread in other brain regions including in the thalamus, hypothalamus, habenula, torus semicircularis, torus longitudinalis, medial longitudinal fascicle and telencephalic areas. Quantitative autoradiography and phosphorimaging showed zic2 mRNA hybridization signal in the preoptic area of the hypothalamus was significantly higher in terminal phase males relative to both initial phase males and females, and silver grain analysis confirmed this relationship between phenotypes. No significant difference in abundance was found in zic2 signal across phenotypes in the habenula, a brain region not implicated in the control of sexual behavior, or cerebellum.

锌指转录因子（zinc-finger transcription factors）zic家族的高度保守成员，其功能最初主要被认为与胚胎信号通路、细胞增殖与分化调控相关。本研究针对成年硬骨鱼双带锦鱼（Thalassoma bifasciatum，蓝头锦鱼）的下丘脑视前区，分析了该区域内zic2 mRNA丰度的性表型差异——而下丘脑视前区这一脑区与性行为及性功能密切相关。蓝头锦鱼是研究神经内分泌过程的理想模型：它存在两种独立的雄性表型，即初始阶段（IP）雄鱼与领地性终末阶段（TP）雄鱼，且可经历社会调控的雌性先熟性转变。此前基于微阵列的比较分析显示，相较于初始阶段雄鱼，终末阶段雄鱼的脑部zic2表达会上调。为进一步探究这一表达差异，我们从野外采集的样本中克隆了神经源性zic2的727 bp序列。我们采用基于核糖探针的原位杂交技术，对成年蓝头锦鱼脑部的zic2信号进行定位，并评估不同性表型个体的脑部zic2 mRNA相对丰度。结果显示，zic2 mRNA在小脑颗粒细胞中表达量极高，且广泛分布于其他脑区，包括丘脑、下丘脑、缰核、半环形隆起、纵隆起、内侧纵束以及端脑区域。定量放射自显影与磷光成像结果表明，相较于初始阶段雄鱼与雌性个体，终末阶段雄鱼下丘脑视前区的zic2 mRNA杂交信号显著更高，银粒分析也验证了不同表型间的这一差异。而在未参与性行为调控的脑区缰核以及小脑中，不同表型个体的zic2信号丰度无显著差异。