摘要
斑马鱼是优良的模式脊椎动物，其胚胎的早期发育受到许多基因的调控，突变体筛选是鉴定具有特定功能的基因的重要手段。为寻找参与调控斑马鱼胚胎早期发育的基因，本研究利用Tol2转座子介导的基因捕获技术筛选早期发育异常的突变体，并通过对突变体的研究阐明相关基因在胚胎发育中的作用及机理。
本次筛选共建立了219个表达EGFP的转基因鱼品系，其中绝大部分品系的荧光只在母源期表达或者在母源期和合子期均表达，并且大部分品系的荧光是广泛表达的。本研究共鉴定出了8种突变体，克隆了其中3个品系中Tol2转座子捕获到的基因，并对其中一个T086/grhl2b品系进行了详细的研究。
grhl2b品系转基因鱼的荧光在内耳和侧线等多种器官中表达，纯合体胚胎的内耳表现出耳腔增大、耳石变小甚至消失、半规管发育畸形等缺陷，使突变体的听力降低并且不能维持身体的平衡。在该品系中，Tol2转座子插入到grhl2b基因的第一个内含子中使其表达明显减少。抑制grhl2b的表达可使野生型胚胎产生类似grhl2b突变体的表型，而过表达grhl2b能挽救突变体内耳的缺陷，表明其表型确实是由于grhl2b基因被捕获造成的。进一步的分析表明，grhl2b基因突变不影响内耳的诱导及图式形成，也不影响毛细胞和位听神经节的发育和成熟，而是使内耳上皮的屏障功能降低并使耳腔内淋巴液的稳态失衡，最终导致内耳的发育异常。在这一过程中，Grhl2b直接与细胞连接和黏附蛋白基因cldnb和epcam的启动子或增强子结合促进它们在内耳中的转录，进而促进内耳上皮细胞紧密连接的生长以维持内耳上皮的屏障功能。转基因实验分析表明，cldnb启动子上的Grhl2结合位点对于cldnb在内耳中的表达是必需的。
斑马鱼Grhl2b与小鼠及人类Grhl2蛋白的序列有很高的保守性，过表达小鼠和人类Grhl2mRNA可以促进斑马鱼cldnb和epcam的表达，并且可以挽救grhl2b突变体的缺陷，表明Grhl2在功能上也是保守的。鉴于人类GRHL2基因突变会导致常染色体非综合征型耳聋DFNA28发生，本研究筛选出的斑马鱼grhl2b突变体可以作为DFNA28型耳聋的动物模型来研究其病因并寻找可以缓解或者治愈这一疾病的药物。
本课题证实了Tol2转座子介导的基因捕获技术可用于筛选斑马鱼突变体，并且建立了首个人类DFNA28型耳聋的动物模型，阐明了grhl2b基因调控内耳发育的机制。
Zebrafish is an excellent vertebrate model organism to explore the molecular mech-anisms of embryonic development, in which many genes involve. Generating geneticmutants is an effective approach to identify new functional genes implicated in specificdevelopmental processes. In this study, the Tol2transposon-mediated gene trap methodisutilizedtoisolatenewzebrafishmutantsandtoelucidatetherolesandmolecularmech-anisms of the trapped genes in embryonic development.
This study generates219transgenic zebrafish lines, most of which express ubiq-uitous EGFP maternally with or without zygotic expression. From these lines, eightmutants are isolated. The trapped genes in three mutant lines are identified, includinggrhl2b gene from the T086line.
The transgenic embryos of the T086/grhl2b line express EGFP in many organs in-cluding the inner ear and lateral line neuromasts. Mutant ears exhibit severe defectssuch as expanded otocysts, decreased or eliminated otoliths, malformed semicircularcanals, etc., resulting in hearing impairment and swimming imbalance. In this line, theTol2transposon element is inserted into the first intron of thegrhl2b locus, which causesa sharp reduction of the amount of normal transcripts. The mutant phenotype can bemimicked in wild-type embryos by knockdown of grhl2b using specific morpholinosand can be rescued by injecting grhl2b mRNA, indicating that the mutant phenotype isascribed to the loss of normal grhl2b transcripts. Further studies reveal that grhl2b isdispensable for otic induction and patterning as well as for hair cell and statoacousticganglion development. Instead, grhl2b plays an crucial role in the maintenance of theotic epithelial barrier and the endolymph homeostasis. Mechanistically, Grhl2b proteinbinds to the promoter or enhancer and regulates the transcription of cldnb and epcam,which encode junctional proteins essential for tight junction formation in the otic epithe-lial cells. Promoter assay by transgenic approach confirms that the Grhl2b binding sitein the cldnb promoter is required for the Grhl2b-dependent expression of cldnb in theinner ear.
Zebrafish Grhl2b protein shares a highly conserved sequence with mouse and hu-man Grhl2proteins. Overexpression of mouse or human Grhl2mRNA can rescue theotic defects of grhl2b mutant embryos and promote the transcription of cldnb and ep- cam in wild-type embryos, indicating that Grhl2is also functionally conserved. Sincehuman GRHL2mutation leads to autosomal non-syndromic hearing loss DFNA28, thezebrafish grhl2b mutants may serve as the first animal model of DFNA28to analyze itsetiology and develop drugs that can alleviate or cure the disease.
This study substantiates the application of Tol2transposon-mediated gene trap ingenerating zebrafish mutants. Meanwhile, this study provides the first animal model ofDFNA28and clarifies the essential roles and the molecular mechanisms of grhl2b ininner ear development.
引文
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