Hedgehog 信号通路调控文昌鱼口发育的机制研究

Unlike vertebrates, amphioxus exhibits a unique pattern of mouth formation, in which the mouth initially opens on the left lateral side of body. Therefore, it has been debatable if the amphioxus mouth is homologous to its vertebrate counterpart. Histological examination can not fully explain that issue. If the regulatory mechanism of amphioxus mouth formation is elucidated, the molecular evidence will be provided to understand that issue. In our recent study, it has been found that the loss of Hedgehog(Hh) function results in the failure of mouth formation in amphioxus, indicating that the Hh signal plays a key role in amphioxus mouth development. To further probe the functions and regulatory mechanism of Hh signalling in amphioxus mouth formation, we will make comprehensive analysis of mouth development in Smo knockout and Hh signal upregulated amphioxus using the methods of in situ hybridization, histological sections and immunofluorescence. In addition, we will also examine the specific genes expression of mouth，the basement membrane dissolution and the regulatory relationship of Hh signal and Nodal signal in amphioxus mouth formation, the results will be significant for the full understanding of molecular regulation mechanism of amphioxus mouth development and providing molecular evidence to explain if the mouths of amphioxus and vertebrates are homologous organs.

与脊椎动物不同，文昌鱼的口形成于身体左侧，因此文昌鱼与脊椎动物的口是否为同源器官一直存在争议。仅靠组织学方法不能全面解释文昌鱼与脊椎动物口的同源关系问题，如能阐明口发育过程分子调控机制，将从发育机制角度回答上述问题。前期研究中我们发现敲除Hedgehog（Hh）基因导致文昌鱼口发育异常，说明Hh信号通路对文昌鱼口的发育是必须的。为深入研究Hh信号通路在文昌鱼口发育过程中的功能与机制，本项目将进一步构建Smo基因敲除突变体，利用组织切片、透射电镜等方法检测Smo敲除和Hh信号通路上调对文昌鱼口发育的影响，确定Hh信号通路在文昌鱼口发育过程中的功能。在此基础上分析口特异基因表达、基膜溶解以及Hh信号通路与Nodal信号通路作用关系，阐明Hh信号通路参与文昌鱼口发育调控的机制。项目成果将对全面理解文昌鱼口发育过程分子调控机制具有重要意义，同时也将为回答文昌鱼口与脊动物口是否同源提供理论依据。

在整个脊索动物中文昌鱼口的形成最为特殊，最初开口于身体左侧。由于口发生位置的特殊性，关于文昌鱼口是否与脊椎动物口同源一直争议不断。如能阐明文昌鱼口发育调控的分子机制，将从发育机制角度回答上述问题。前期研究已经证明Hedgehog（Hh）信号通路与文昌鱼口发育相关，但其具体扮演的角色和调控机制尚不明确。本项目采用基因敲除、原位杂交和过表达等多种手段对Hh信号通路在文昌鱼口发育过程的功能与机制开展研究，取得以下研究成果：1）获得了Smo基因敲除突变体文昌鱼，分析了Smo敲除突变体文昌鱼的表型，证明Smo基因敲除影响口的发育，而对早期左右体轴的建立无影响。2）发现上调Hh信号通路导致文昌鱼口变大，下调Hh信号通路文昌鱼口变小，说明Hh信号通路参与文昌鱼口大小的调控并存在剂量效应。3）发现在口形成早期，敲除Smo基因对口标记基因的表达没有影响，说明Hh信号通路可能不参与口原基的形成而只参与后期开口过程的调控。4）发现敲除Hh基因可导致Frizb1基因在口处的表达消失，说明Hh信号通路参与文昌鱼口的发育调控可能是通过与Wnt信号通路的互作实现的。上述研究结果为文昌鱼口发育机制研究提供了新的思路，为回答文昌鱼口是否与脊椎动物口同源提供了理论依据。