Analysis on the function of a protein encoded by a thyroid hormone responsive gene, ZAKI-4, in order to elucidate the molecular mechanizm of thyroid hormone action on brain development

众所周知，甲状腺激素(T_3)在脑发育中起着关键作用，但其分子机制尚不清楚。我们之前发现了一个甲状腺激素反应基因，Zaki-4。Zaki-4编码一个相对分子质量为26kd的蛋白质(Zaki-4蛋白)。最近有报道称，Zaki-4属于体内抑制钙调神经磷酸酶活性的家族蛋白。Sicne Calcineurin在脑发育和功能中起重要作用，我们假设Zaki-4蛋白是在脑发育过程中介导T_3作用的关键因子之一，并设计了本研究项目来阐明Zaki-4蛋白在体内的功能。为此，我们试图培育出完全消除Zaki-4表达的小鼠。作为敲除Zaki-4基因的第一步，我们确定了小鼠Zaki-4基因的基因组结构，并构建了靶向载体。我们目前正在进行胚胎…的同源重组更多的干细胞来源于小鼠系(129/Sv)。本研究还检测了Zaki-4mRNA在啮齿类动物脑内的空间和个体表达，以及甲状腺功能减退对其表达的影响。在大脑皮层，早在胚胎第18天就可观察到Zaki-4mRNA的表达，并随着大脑发育逐渐增强。这种增加在甲状腺功能减退大鼠身上明显减弱。另一方面，它的表达是恒定的，不受小脑甲状腺功能减退的影响。原位杂交结果显示，Zaki-4mRNA在整个脑组织中广泛表达。研究发现，Zaki-4在嗅球、海马区、大脑皮层和小脑等钙调神经磷酸酶含量丰富的区域高表达。这些结果表明，Zaki-4mRNA的表达确实受到体内甲状腺状态的调节，并提示甲状腺激素在脑发育过程中发挥其生物学功能，至少部分是通过调节Zaki-4的表达，从而控制Calcineruin的活性。较少

It is well known that thyroid hormone (T_3) plays a pivotal role in brain development, however, its molecular mechanism remains to be elucidated. We previously identified a thyroid hormone-responsive gene, ZAKI-4. ZAKI-4 encodes a protein (ZAKI-4 protein) with a molecular mass of 26kd. Recently, it has been reported that ZAKI-4 belongs to a family protein that inhibits calcineurin activity in vivo. Sicne calcineurin is known to play important roles in brain development and function, we hypothesized, that ZAKI-4 protein is one of key factors to mediate T_3 action during brain development and designed the present research projects to elucidate the in vivo function of ZAKI-4 protein. For this purpose we tried to produce mice in which the expression of ZAKI-4 is completely eliminated. As the first step to knockout ZAKI-4 gene, we determined the genomic organization of mouse ZAKI-4 gene and a targeting vector was constructed. We are currently carrying out homologous recombination in embryon … More ic stem (ES) cells derived from a mouse train (129/Sv). In the present study, the spatial and ontogenic expression of ZAKI-4 mRNA and the effect of hypothyroidism on the expression were also examined in brain of rodents. In cerebral cortex, the expression of ZAKI-4 mRNA was observed as early as embryonic day 18 and the expression increased during the brain development. This increase was significantly blunted tn hypothyroid rats. On the other hand the expression was constant and was not affected by hypothyroidism in cerebellum. In situ hybridization revealed that ZAKI-4 mRNA was widely expressed in the entire brain. It was noted that ZAKI-4 was highly expressed in the area such as olfactory bulb, hipocampus, cerebral cortex and cerebellum, where calcineurin is abundt. These results demonstrated that the expression of ZAKI-4 mRNA was indeed regulated by thyroid status in vivo and would suggest that thyroid hormone excerts its biological function during brain development at least in part by regulating ZAKI-4 expression and thus by controling calcineruin activity. Less