AMMECR1基因家族起源及其在脊索动物神经嵴发育中的功能演化

It has been proven that AMMECR1 genes are related to the AMME syndrome in human. The genes are highly conserved throughout all chordate groups, hinting an important biological function in organisms. However, the origin and evolution of the gene family as well as their function are still unknown to date. During our pre-investigations on the gene family, we found that 1) the AMMECR1 gene family includes one member in invertebrates and two members in all vertebrate lineages except for cyclostomes and teleosts; 2) the AMMECR1 family genes probably have essencial roles in the development of chordate central neural system and nerual crest. Starting from these preliminary work, in the current proposal we will take full advantages of all publicly available genome resours (combining with gene cloning from selected animals), to clarify the evolution of the AMMECR1 gene family. Further, we will use amphioxus, zebrafish and frog as models and employ molecular tools, including RTqPCR, whole-mount in situ hybridization, gene knockdown and overexpression as well as Talen-knockout methods, to elucidate their roles in the developments of the central neural system and nerual crest in the above three species.By comparing with the nerual development molecular markers, we will disclose the mechanism performed by AMMECR1 in the formation of central development. Finally, we will summarise results from above experiments and deduce the origin and evolution of the gene family and their functional evolution in the development of chordate central neural system and nerual crest.

AMMECR1基因与人类AMME综合症有关，该基因在所有脊索动物中高度保守，表明具有重要的生物学功能，但至今人们对该基因家族的起源演化和功能却所知甚少。我们前期研究发现，1）该基因家族在非脊椎动物中包含一个成员，而在除硬骨鱼和圆口类之外的所有其他脊椎动物类群中则包含两个成员，2）该家族基因可能参与脊索动物中枢神经系统和神经脊的发育。本项目将在前期工作的基础上，利用现有公共数据资源（辅以基因克隆），明确AMMECR1基因家族起源及演化问题，并以文昌鱼、斑马鱼和爪蟾为研究模型，运用RTqPCR、原位杂交、mophorlino、Talen敲除基因、过表达及功能代偿等技术手段，通过比较发育生物学方法，深入探究AMMECR1基因家族在上神经系统发育过程中的功能，并通过神经发育标记分子，揭示其作用的分子机制。最后将综合以上数据，绘制AMMECR1家族基因在脊索动物神经系统和神经脊发育中的功能演化图。

AMMECR1基因最早发现于1998 年，是引起人类遗传病“邻近基因缺失综合症”(AMME)综合症的候选基因之一，然而该基因发现后，对其功能的后续研究报道不多。本项目以斑马鱼和头索动物文昌鱼为模型，用TALEN技术敲除该基因，构建基因缺失动物模型，研究该基因在斑马鱼/文昌鱼生长发育中的作用，取得以下主要结果：.1）组织原位杂交和RT-PCR实验结果表明斑马鱼中AMECR1基因的时空表达具有广泛性，隐喻着该基因在斑马鱼中具有广泛的生物学活性。.2）成功构建了AMMECR1基因敲除的斑马鱼模型，在对基因敲除斑马鱼培育过程中发现，AMMECR1属完全显性基因，杂合子与野生型个体生长发育完全相同，杂合子生长发育和繁殖均不受影响，但纯合突变个体难以存活，缺失突变的基因可以通过杂合子斑马鱼长期保存，以供长期研究用。.3）通过杂合子相互配对获得纯合突变的子代，以表征AMMECR1基因缺失斑马鱼的生长、发育及存活特性，结果发现该基因缺失后并不影响受精卵和早期幼鱼的发育，但严重影响随后的生长、发育、个体存活率和繁殖力。.4）对不同日龄段纯合突变体斑马鱼的解剖学和组织学检查发现，纯合突变体的生长较慢，性腺分化受阻，特别是雄性个体的精巢发育严重缺陷，不能产生有活力的精子，缺乏正常的繁殖行为。.5）少数雌性纯合突变体可以生长发育至性成熟，为进一步研究母源性AMMECR1基因表达产物在早期胚胎发育中的作用，用雌性纯合突变体与野生型雄性配对，结果发现，雌性纯合突变体所产卵的存活率显著降低，存活下来的后代在稚鱼阶段随着生长发育，畸形型率非常高。.6）构建文昌鱼AMMECR1同源基因敲除模型。.7）蛋白相互作用实验发现，在HEK293细胞中，人及斑马鱼的AMMECR1与人CLP1蛋白可以相互作用，提示AMMECR1蛋白可能参与到tRNA的加工成熟过程中。