利用斑马鱼模型研究DNAjc17调控毛细胞发育与再生的机制

DNAjc17 is a member of HSP40 family proteins. It can coordinate with other proteins and widely affect the expression and function of its target proteins by regulating the process of mRNA splicing, protein synthesis and folding. In previous study, we found that mutated DNAjc17 was a potential causable reason for deafness via performing exome sequencing and linage analysis on a family with non-syndromic hereditary hearing loss. Furthermore, our initial experimental results demonstrated that DNAjc17 was highly expressed in zebrafish hair cells, and interestingly, the downregulation of its expression led to a significant decrease of hair cells. Therefore, we infer that DNAjc17 is a new deafness gene of which the functional mechanism remains unclear. This project aims to further study the functions and mechanisms of DNAjc17 in hair cell development and regeneration. First, DNAjc17 expression during hair cell development and regeneration will be detected using in situ hybridization and immunohistochemical analysis and other methods. Then we will construct different zebrafish strains with knocking out, knocking down and overexpression of DNAjc17 respectively. The strains will be analyzed for their phenotypes regarding the hair cell development, regeneration and hearing behavior to investigate the functions and the cellular mechanisms of DNAjc17. Transcriptome sequencing will also be utilized to find the downstream target genes of DNAjc17 and further reveal the molecular mechanisms. The study will deepen our understanding of the mechanisms of hair cell development and regeneration, and also provide new ideas for the treatment of deafness-associated diseases.

DNAjc17是HSP40家族成员，可与其他蛋白共同作用于mRNA的剪接、蛋白的合成、折叠等过程来调节靶标蛋白的表达与功能。前期工作中，我们通过对一个非综合征型耳聋家系的外显子测序和连锁分析发现DNAjc17突变可诱导耳聋的发生。预实验表明该基因在斑马鱼毛细胞中表达水平较高，而下调后却导致毛细胞数量的显著减少。据此推断DNAjc17是一个新的致聋基因，但其机制尚无研究。本项目拟对DNAjc17在毛细胞发育和再生中的功能和机制进行研究，首先利用原位杂交和免疫组化等方法研究其在毛细胞发育过程中的表达动态，再构建该基因敲除、下调和上调的斑马鱼品系，通过对毛细胞发育和再生及听力行为等表型的分析来研究DNAjc17的功能并进一步揭示其细胞机制。利用转录组测序等技术鉴定DNAjc17的下游靶标分子，阐明其分子机制。本研究将加深人们对毛细胞发育和再生机制的理解，同时也为耳聋相关疾病的治疗研究提供新思路。

DNAjc17是HSP40家族成员，可与其他蛋白共同作用于mRNA的剪接、蛋白的合成、折叠等过程来调节靶标蛋白的表达与功能。前期工作中，我们通过对一个非综合征型耳聋家系的外显子测序和连锁分析发现DNAjc17突变可诱导耳聋的发生。在本项目中，我们首先利用原位杂交技术对DNAjc17基因在斑马鱼体内的表达情况进行了检测，发现该基因在斑马鱼耳石和神经丘中均有特异性的高表达；我们在毛细胞特异性标记的转基因斑马鱼上利用Morpholino下调DNAjc17的基因表达，显微镜观察发现被下调的斑马鱼中毛细胞数量显著减少，挽救实验进一步证实了该表型的发生是由DNAjc17的下调所导致。为研究DNAjc17的功能即其对斑马鱼听力的影响，我们利用Crispr-cas9技术构建DNAjc17功能缺失的斑马鱼品系，但在后续的筛选过程中，未能获得纯合子突变体；同时，我们利用行为学实验，对DNAjc17下调后的斑马鱼的听力应激行为进行了检测，结果表明该基因的下调使得斑马鱼对噪音的反应能力显著下降。为阐明DNAjc17在毛细胞中的功能机制，我们对基因下调后的斑马鱼进行了转录组测序分析，发现差异表达基因在声光信号传导通路中有显著富集，表明该基因对斑马鱼声音信号的生物传导有直接调控的作用。我们进一步挑选了部分与声音传导有关的功能基因，并再次利用基因下调和挽救实验对其功能进行验证，最终发现一个与神经递质传导有关的基因，cplx4a是潜在的受DNAjc17调控进而影响听力的下游基因。本研究在临床水平发现了一个新的导致耳聋的基因，并从遗传和分子生物学水平对其致聋的分子机制进行了研究探讨，相关结果对未来耳聋和听力损伤疾病的检测和治疗提供了重要的理论参考。