FUNCTION OF GLI GENES IN SHH SIGNALING

hedgehog （hh）信号通路涉及无脊椎动物和脊椎动物胚胎的模式。果蝇的遗传分析表明，cubitus interruptus （ci）是信号通路中最下游的部分。在脊椎动物中有Gli1、Gli2和Gli3三个同源蛋白。该建议的总体目标是通过敲入方法确定这三个Gli基因在hh信号通路中的不同而又重叠的功能。具体来说，Gli2的内源性编码序列将被Gli1和Gli3的编码序列所取代。这个项目将分三步完成。首先，分析Gli2基因组区域的5′区，选择敲入位点。同时，Gli1和Gli3的敲入构建将在Sonic hedgehog反应细胞系MNS70中进行测试。第二步，敲入结构将被引入胚胎干细胞。将产生嵌合胚胎和种系突变小鼠。最后，利用脊髓、肺和骨骼中的各种标记基因分析杂合子和纯合子突变小鼠的相关表型。Gli基因的功能研究与人类发育和疾病有直接关系，因为这三个基因在人类中存在同源性，并且这些基因的突变与癌症和发育缺陷有关。

The hedgehog (hh) signaling pathway is implicated in patterning both invertebrate and vertebrate embryos. Genetic analysis in Drosophila established that the cubitus interruptus (ci) is the most downstream component of the signaling pathway. There are three ci homologs in vertebrate, Gli1, Gli2 and Gli3. The overall goal of the proposal is to determine the distinct and yet overlapping function of these three Gli genes in hh signaling pathway by using the knock-in approach. Specifically, the endogenous coding sequence of Gli2 will be replaced by that of Gli1 and Gli3. This project will be done in three steps. First, the 5' region of Gli2 genomic region will be analyzed to select a knock-in site. In the meantime, Gli1 and Gli3 knock-in construct will be made and tested in Sonic hedgehog responsive cell line MNS70. In the second step, knock-in constructs will be introduced into embryonic stem cells. Chimeric embryos and germline mutant mice will be generated. Finally, the phenotypes associated with heterozygous and homozygous mutant mice will be analyzed by using various marker genes in the spinal cord, lung and skeleton. Studies on the function of Gli genes have direct relevance to human development and disease since homolog of these three genes exist in humans and mutations in these genes have been implicated in cancers and developmental defects.