蚋类气管鳃早期发育及初级分支的分子机制研究

Ramified tubular systems, which developed out from epithelia through a process known as branching morphogenesis, are commonly existed in animals. In recent years, it is found that fibroblast growth factor (FGF) plays a critical role in branching morphogenesis of the principal model system, the tracheal system of Drosophila, and many genes involved in branching morphogenesis are identified. However, there are many problems stayed in the mechanisms of branching morphogenesis. One of the reasons exists in the complicated interaction between the branched structures and their surrounding tissues. Another reason is that lethal mutations are difficulty to identify out. Here we take Simuliidae tracheal gills as a model to research the mechanisms of primary branching process, because this model probably does not exist the aforementioned problems. We have established the determination of larval instars for two species of Simuliidae insects, that as the essential reference time scales for researching the developmental processes of tracheal gill. We have also described morphological development processes of tracheal gills on late larval stages of these species, and set up a series of artificial Simuliidae rearing procedures applied to gene silencing. In this project, we employ immunohistochemistry techniques to describe the cellular and morphological changes during the development of tracheal gills. After screening out the candidate genes related to branching morphogenesis with bioinformatics, we can detect the temporal and spatial distribution of expression of these genes by double-label in situ hybridization protocols, and observe the effects on the branching of tracheal gills following RNA interference on these genes. With approval of this project, we are intending to elucidate the branching development processes of the tracheal gill of one or two Simuliidae species, and to identify and find out the functions of about 10 genes regulating the branching morphogenesis of Simuliidae, finally to clarify the mechanisms involving in the primary branching of tracheal gill.

分支化管道系统普遍存在于动物中，其发育过程称为分支形态发生。相关研究主要以果蝇气管为模型，发现FGF在分支形态发生中起主导作用，并鉴定了众多相关基因。迄今研究的分支化系统与周围组织具有复杂关系、致死性突变较难发现，导致对分支形态发生的机制尚存在许多问题。我们拟以蚋蛹气管鳃为对象研究初级分支产生的分子机制，因为气管鳃可能不存在上述问题。前期我们研究了2个蚋种的幼虫龄期划分，提供了必要的时间坐标；初步阐明了气管鳃晚期发育的形态学过程；建立了适应基因沉默的蚋类人工饲养成套方法。本项目拟利用免疫组化技术深入阐述气管鳃发育过程中的细胞学和形态学变化；用生物信息学技术筛选分支化候选基因；用双标记原位杂交技术研究候选基因表达的时空分布，并以基因沉默技术研究候选基因对气管鳃初级分支化的影响。拟阐明1-2个蚋种的气管鳃分支形态发育过程，鉴定10个左右的相关基因，初步阐明气管鳃初级分支化的分子机制。

分支化管道系统是普遍存在于动物中的一种基本结构，是呼吸、循环、神经等功能的基础；蚋类气管鳃是一种分支化结构，本项目对其分支化机制开展初步研究。.. 我们用传统的免疫组化方法，阐明了Simulium xingyiense（兴义维蚋）、S. ornatum、S. quinquestriatum、S. bidentatum、S. aureohirtum、S. takahasii和S. guiyangense等7个蚋种的气管鳃在形态发育过程分支化发育及呼吸丝卷曲的详细过程，建立了蚋类气管鳃形态发育的共同模式；并通过与其他物种的比较，明确蚋类气管鳃起源很可能与附肢相同。我们以果蝇基因组为线索，用RACE技术获得Sbtl、Sbnl、Scda4、Shh和Swg等5个兴义维蚋基因的cDNA全长序列，并确定了这5个基因的结构；用FISH发现Sbtl、Sbnl、Scda4和Shh在气管鳃发育过程中表达；用RNAi技术发现Sbtl参与呼吸丝管壁形态构建，其被沉默后可使呼吸丝卷曲模式的对称性被破坏、卷曲杂乱；Shh被沉默后，可使个别呼吸丝变短。未发现上述基因影响气管鳃的分支化。我们建立了兴义维蚋连续传代的体系，在自行设计的羽化、喂血、产卵等系列装置支持下，使用最佳饲养条件（喂血培养基温度为34℃，添加ATP的牛血清培养基），可在实验室内连续饲养两代。我们测定了兴义维蚋全基因组序列，获得scaffold级别的全基因组序列，scaffold N50为20.6 Mb；注释得到24833个基因，其中96.98%得到功能注释。.. 本项目阐明了蚋类气管鳃形态发育的共同模式，明确蚋类气管鳃的起源与附肢类似、应该从附肢发育方向研究其分支化，建立了开展气管鳃发育研究的实验饲养条件，测定了兴义维蚋全基因组序列，为下一步大规模开展蚋类气管鳃发育分子机制研究奠定了必要且充分的基础。.. 通过对模式生物的研究，发现生命规律，寻找人类疾病机制线索，是生物学研究的一个重要主题。我们在蚋类气管鳃研究上开展的基础工作，有可能最终让气管鳃成为一个新的研究模型。