利用基因敲除小鼠研究FCHSD1在听觉中的功能

The auditory hair cells are sensory cells responsible for hearing. Stereocilia and cuticular plate are actin-rich subcellular structures in auditory hair cells, which are very important for hearing transduction. Our previous study showed that FCHSD1 is expressed in cuticular plate and stereocilia, and could promote WASP-Arp2/3-dependent F-actin polymerization in vitro through binding to SNX9. We hypothesize that FCHSD1 might regulate the development and maintenance of stereocilia and cuticular plate, and hence play an important role in hearing transduction. In order to examine our hypothesis and further explore the function of FCHSD1 in hearing transduction, we plan to establish inner ear conditional knockout mice. We plan to introduce two LoxP sites flanking the 3rd exon of mouse FCHSD1 gene, and cross the genome-modified mice with inner ear-specific Cre mice, to get inner ear-specific FCHSD1 knockout mice. Using the knockout mice, we will examine whether the disruption of FCHSD1 expression affects development and/or maintenance of stereocilia and cuticular plate, and ultimately, affects hearing transduction. In addition, we will examine the expression of F-actin regulatory proteins in auditory hair cells in wild type and FCHSD1 knockout mice to explore the molecular mechanisms of FCHSD1 in F-actin dynamics of stereocilia and cuticular plate.

听毛细胞是负责感受声音的感受器细胞，而听纤毛和表皮板是听毛细胞中以肌动蛋白纤维（F-actin）为主体的亚细胞结构，对听觉传导的发生至关重要。我们发现FCHSD1在表皮板和听纤毛表达，并且能够通过与SNX9的结合在体外促进WASP和Arp2/3蛋白复合物依赖的F-actin聚合，因此可能参与调控听纤毛和表皮板的发育和维持，从而在听觉传导中发挥重要作用。为了深入研究FCHSD1在听觉传导中的功能，我们计划在小鼠FCHSD1第三个外显子两侧引入LoxP位点，并与内耳特异的Cre小鼠交配，从而得到在内耳中特异性敲除FCHSD1表达的基因敲除小鼠。我们会检测FCHSD1基因敲除小鼠的听纤毛和表皮板发育是否异常，以及听力水平是否发生改变。此外我们会对与FCHSD1相关的F-actin调控蛋白在敲除小鼠中的表达进行研究，对FCHSD1调控听纤毛和表皮板F-actin的分子机制进行深入探讨。

听毛细胞是负责感受声音的感受器细胞，而听纤毛和表皮板是听毛细胞中以肌动蛋白纤维（F-actin）为主体的亚细胞结构，对听觉传导的发生至关重要。我们发现FCHSD1在表皮板和听纤毛表达，并且能够通过与SNX9的结合在体外促进WASP和Arp2/3蛋白复合物依赖的F-actin聚合，因此可能参与调控听纤毛和表皮板的发育和维持，从而在听觉传导中发挥重要作用。在本项目中，为了深入研究FCHSD1在听觉传导中的功能，我们在小鼠FCHSD1第三个外显子两侧引入LoxP位点，通过与不同的Cre小鼠交配，获得了全身性及内耳特异性的FCHSD1基因敲除小鼠。HE染色结果显示敲除小鼠各组织器官无明显生理变化，统计结果显示敲除小鼠的生殖状态也未受影响。整封免疫荧光结果及扫描电镜结果显示敲除小鼠的听觉系统发育基本正常，毛细胞没有发生严重的缺失，听纤毛形态正常、没有缺损及凌乱的现象，FM1-43FX染色结果显示敲除小鼠听纤毛上的离子通道活性也没有受到影响。与以上结果一致，ABR测听结果显示FCHSD1的敲除并没有影响小鼠的听力。我们推测这可能与小鼠中存在FCHSD1的同源基因FCHSD2有关，它的存在有可能代偿了FCHSD1的功能，使小鼠的听觉系统保持正常发育。因此，我们计划对小鼠进行FCHSD1和FCHSD2的双基因敲除，来进一步研究FCHSD1的功能，以及它在小鼠听觉系统发育过程中与其他相关基因之间的联系。