nesly6和negaly6在斑马鱼侧线原基迁移和分化中的功能与机制研究

The posterior lateral line primordium (pLLp) of zebrafish is a well-established in vivo model for insights into collective cell migration. pLLp migration depends on the activity of CXCR4 and CXCR7, which are both regulated by FGF, Wnt and TGFβ signaling pathways. However, the factors regulated by these signaling pathways and those involved in pLLp migration and lateral line development are poorly defined. Recently, we have found two FGF signaling pathway regulated genes of lymphocyte antigen 6 (Ly6) superfamily, called neuromast-expressed secreted lymphocyte-6 （nesly6）and neuromast-expressed gpi-anchored lymphocyte antigen-6 (negaly6), respectively, which are specifically expressed in pLLp and neuromasts. In addition, knockdown of nesly6 resulted in a conspicuous reduction of neuromast number and aberrant inter-neuromast distance. However, the modes of action of Nesly6 and Negaly6 in pLLp migration and lateral line development remain elusive. We plan to first examine the expression pattern of negaly6 and nesly6 and their regulation by signal pathways in detail. We will then perform a systematic analyses using knockdown or knockout techniques to investigate their roles in pLLp migration, deposit and proliferation. We will also explore whether nesly6 and negaly6 is implicated in cell migration by cell transfection. Finally, we will identify the interacting partners of Nesly6 and Negaly6 using yeast two hybrid and co-immunoprecipitation approaches. Collectively, these studies will certainly shed light on how Nesly6 and Negaly6 regulates pLLp migration and neuromast formation, enriching our understanding of the mechanisms of lateral line development in fish.

斑马鱼后端侧线原基（pLLp）是研究群体细胞迁移一个好的活体模型。pLLp细胞迁移受趋化因子控制，并受FGF和Wnt等信号通路调节，但受这些通路调控的因子在pLLp迁移、分化等发育过程中的作用亟需深入研究，更多参与调控pLLp迁移的因子尚有待鉴定。我们发现多功能淋巴抗原6基因家族nesly6和negaly6在斑马鱼侧线原基和神经丘特异表达，且受FGF信号通路调控，功能研究显示其功能受抑制时会导致侧线发育异常，但具体作用机制不明。我们拟在确定这两个基因表达、调控基础上，利用反向遗传学等方法探索它们在pLLp迁移分化以及神经丘沉积的作用；采取体外分析方法确定nesly6和negaly6是否直接影响细胞迁移；利用酵母双杂交等方法鉴别与其互作蛋白，确定互作蛋白在侧线发育中的作用，阐明nesly6和negaly6在侧线发育调节通路中的地位、功能与机制，丰富对pLLp迁移与侧线发育调节机理的认识。

斑马鱼后端侧线原基（pLLp）是研究群体细胞迁移一个好的活体模型。pLLp细胞迁移 受趋化因子控制，并受FGF和Wnt等信号通路调节，但受这些通路调控的因子在pLLp迁移、 分化等发育过程中的作用亟需深入研究，更多参与调控pLLp迁移的因子尚有待鉴定。我们发现多功能淋巴抗原6基因家族nesly6和negaly6在斑马鱼侧线原基和神经丘特异表达，且受FGF信号通路调控，功能研究显示其功能受抑制时会导致侧线发育异常，但具体作用机制不明。我们发现了nesly6和negaly6这两个基因表达在侧线原基上，并且nesly6主要表达在定位的神经丘上，利用MO敲降nesly6可以影响侧线原基的迁移和神经丘的定位和数量。我们发现nesly6敲降的斑马鱼的侧线原基细胞的死亡增加，增殖减少。体外构建瞬时表达的nesly6的HEK293细胞系，我们也发现了过表达nesly6可以显著增加细胞的增殖和数量。说明nesly6可以调控细胞增殖和凋亡。我们又构建了nesly6启动子驱动EGFP表达的斑马鱼，发现nesly6表达在神经丘的所有细胞，包括毛细胞和支持细胞。我们检测下游的效应因子，发现p-ERK减少，p-MLC2减少，通过免疫沉淀我们发现nesly6可以和cxcr7和cxcr4相互作用，调节cxcr信号，进而调节侧线原基的迁移和细胞增殖。