斑马鱼突变体34c脑发育异常的分子机理研究

The zebrafish is becoming a more and more important model organism in genetics and developmental biology, including the analysis of brain development and disorders, since this organism share conserved neurochemistry and brain organization with their mammalian counterparts. In the forward genetics screen for mutations affecting zebrafish organgenesis, we have isolated a mutant called 34c, who has smaller head and smaller eyes as compared to the wild type embryos. Small head, also called microcephaly in human, is a type of autosomal recessive disease of brain development. This brain disorder is caused by the reduction in the proliferation of neural progenitors during the development of central nervous system. Some genes encoding centrosomal proteins are associated with microcephaly, but the molecular and cellular mechanisms of the cell dividing defect are still poorly understood. The affected gene in 34c is identified as kctd10, belonging to a very conserved gene family. Kctd10 encodes a tetramerization domain containing protein, whose function in the neurogenesis is totally unknown. We hypothesize that Kctd10 plays essential role in DNA replication or centrosome duplication to regulate neural cell proliferation. Kctd10 might also regulate expression of the genes essential for mitosis. In this proposal, we will check the brain regionalization of 34c, examine the proliferation and apoptosis of the neural progenitor cells in this mutant, and analysis the behavior of 34c by conditional targeting kctd10. More importantly, we will focus on the mechanism that kctd10 regulates the proliferation of neural cells. The role of Kctd10 protein in DNA replication, centrosome duplication and elongation will be examined. Kctd10 binding protein and the proteins regulated by Kctd10 will also be isolated to unravel the molecular mechanisms of Kctd10 in the regulation of neural cell proliferation. The fulfillment of this project will shed light on the role of Kctd10 in neurogenesis, clarify the molecular mechanism underlying neural cell proliferation, and identify the new candidate causing genes of microcephaly. Moreover, we will also establish the technique of conditional gene targeting in zebrafish.

斑马鱼是研究神经发育及发育异常的遗传机制的理想模式生物。我们利用正向遗传筛选，分离到一株头和眼睛显著缩小的斑马鱼突变体34c，其表型与人类的小头症非常类似。小头症的病因是神经祖细胞分裂受阻，但其分子机制还不太清楚。我们鉴定到34c突变体的变异基因为kctd10，这个基因在脑发育中的功能人们还一无所知。我们推测，Kctd10可能与DNA复制或中心体的复制与定位有关，也可能调控细胞周期相关基因的表达，从而调控神经祖细胞的增殖。本课题将深入分析34c突变体脑神经细胞的分裂与凋亡，并在此基础上研究Kctd10与DNA复制、中心体定位及复制的关系，鉴定受到Kctd10调控的下游基因，最终阐明Kctd10调控脑发育的遗传机制。本项目的完成，将首次明确Kctd10在神经发育中的功能，深化人们对神经细胞增殖调控机理的认识，并鉴定导致小头症的新候选基因。我们还将在斑马鱼中建立基因条件敲除技术。

斑马鱼是研究脊椎动物早期发育及其分子机制的理想模式生物。我们利用正向遗传筛选，分离到一株头和眼睛显著缩小、心脏发育畸形的斑马鱼突变体34c。我们鉴定到34c突变体的变异基因为kctd10，此基因在脑和心脏发育中的功能人们还缺乏了解。我们采用了原位杂交的手段检测大脑的发育和脑区的分化是否出现异常，结果表明kctd10的功能缺失不造成脑区分化的异常，但34c突变体的大脑在发育较晚时期的细胞凋亡显著增加。通过对蛋白质相互作用的解析，我们注意到Kctd10与PCNA存在蛋白-蛋白相互作用，而且二者在斑马鱼大脑中有共同表达，说明Kctd10有可能对神经祖细胞增殖有重要作用。.因为kctd10的突变既影响脑的发育，又影响心脏的发育，为排除两种器官之间的相互影响，我们尝试在斑马鱼中建立基因条件敲除技术，而这种遗传学技术也是研究斑马鱼所必需的重要方法。通过靶向插入方法，我们成功构建了kctd10条件性等位基因，并实现了条件性等位基因的种系传递。我们又构建了Tg(cmlc2: DD-Cre-2A-CFP)转基因鱼，以实现心肌特异的Cre表达。在Tg (cmlc2: EGFP) 34c携带者杂交的子代胚胎中通过对条件敲除基因纯合体胚胎注射Cre mRNA，可以得到与34c突变体相同的表型，不注射的胚胎均正常，说明条件性等位基因在Cre的作用下可以实现基因失活。.利用Tg(cmlc2: DD-Cre-2A-CFP)在心脏中进行kctd10的条件性敲除，子代未出现类似34c突变体的表型，这说明其他组织器官对心脏表型的产生也有影响。我们又利用Tg(HuC: Cre-2A-td Tomato) 转基因鱼对中枢神经系统进行kctd10条件敲除，结果发现部分子代有类似34c突变体的表型，其心脏中也发生了kctd10的重组，说明HuC在斑马鱼早期胚胎中可能广泛表达，无法达到在神经组织条件性敲除kctd10的目的。目前我们尝试使用特异性地在神经上皮干细胞表达的nestin基因启动子，进行中枢神经系统的条件敲除。.综上所述，我们的研究证明，kctd10对斑马鱼脑和心脏的发育起着重要作用，而心脏的正常发育，还依赖于其他组织器官中kctd10的功能。同时，我们在斑马鱼中初步建立于一种基因条件敲除技术，对研究基因在特定组织和器官发育中的作用奠定了基础。