Yes相关蛋白（YAP）在脊椎动物斑马鱼胚胎发育中的作用

【作者】 蒋璆；

【导师】 宋后燕； 汤其群； 马端； 于敏； 王跃祥；

【作者基本信息】 复旦大学 ， 生物化学与分子生物学， 2009， 硕士

【摘要】 最近,在果蝇中的研究描绘了一个新的信号通路——Hippo通路。这一通路能同时抑制细胞增殖和促进凋亡,在生长调节方面起重要作用。Hippo激酶级联效应最终拮抗转录共激活因子Yorkie(Yki),从而正向调控参与细胞生长、增殖和存活的靶基因转录。这一系统的进化保守性已由多个实验室证实。哺乳动物YAP(Yki的同源物)能够在体挽救果蝇突变体。YAP在小鼠肝脏过度表达会引起肝脏尺寸大幅度增加,在肠道过度表达会扩大祖细胞池。YAP在鸡胚过度表达导致神经祖细胞池显著扩大。所有这些发现提示:脊椎动物YAP在调控细胞增殖和存活方面有着相当保守的功能。细胞培养和生化实验已经证实YAP能结合并/或共激活一些蛋白和蛋白家族,如Yes、Runx、EBP50、p73、TBX5、p53BP2、TEFs/TEADs、14-3-3、ErbB4、Smad7和hnRNPU等。但是,缺乏一个生理性的实验系统来更好地在体研究YAP的功能。新兴模式生物斑马鱼胚胎透明、体外发育、个体小、繁殖率高、产卵周期长以及鱼种发育遗传背景清晰等优点使其正成为研究脊椎动物胚胎发育过程中基因功能的理想模式系统。2000年,Nasevicius等首次通过注射吗啡啉修饰性寡核苷酸(MO)获得了目的基因特异表达下调的斑马鱼。此方法正作为快速、高效的基因表达下调方法成功应用于斑马鱼,加快了基因功能研究的步伐。利用斑马鱼研究胚胎发育中yap基因的功能具有独特优势。本文第一部分:利用生物信息学的方法证明在脊椎动物进化过程中YAP分子的功能结构域保守。用反义RNA探针-全胚胎原位杂交和RT-PCR的方法研究yap基因在斑马鱼胚胎发育早期的时空表达谱。本文第二部分:用靶向yap mRNA的MO(yap-MO)干扰YAP翻译起始,建立了yap基因表达下调的斑马鱼模型。为了验证yap-MO的有效性,我们将其与编码yap-EGFP蛋白质的质粒DNA共注射,结果显示yap-MO抑制了yap-EGFP蛋白的表达,从而也验证了yap-MO能够有效抑制内源性的YAP表达。本文第三部分:在细胞水平上,用原位免疫(荧光)和流式细胞分析方法检测yap基因表达下调后胚胎24 hpf前各个阶段的细胞凋亡和增殖状态。结果显示:在YAP抑制组胚胎的头部和尾部细胞凋亡显著增加,胚胎细胞增殖减少。本文第四部分:在胚胎整体发育水平上,我们主要运用胚胎整体原位杂交和组织化学方法分析yap基因表达下调对背腹轴模式、体节发生、神经系统、血液系统和心血管系统发育的影响:1).在原肠期,yap基因表达下调导致背腹轴标记基因整体表达延迟。同时,背部标记基因goosecoid(gsc)表达减少,腹部标志基因bmp2b表达增强。2).在体节发生期,YAP抑制组胚胎myoD表达显著增强,形成‘u’型体节。3).yap基因表达下调后导致斑马鱼脑部神经前体细胞显著减少,而这种改变是由于相应细胞凋亡增加引起的。4).yap基因表达下调干扰了斑马鱼软骨发生。在YAP抑制组胚胎中,咽弓软骨的缺失是由相应神经嵴细胞丢失引起的。5).yap基因表达下调后,斑马鱼血液系统发生、心脏发生出现了延迟表型,但血管发育并没有明显异常和延迟。更多还原

【Abstract】 Recent studies in Drosophila have delineated a novel signaling pathway, the Hippo pathway, which is extremely powerful in regulating growth due to its ability to simultaneously inhibit cell proliferation and promote apoptosis. This Hippo kinase cascade ultimately antagonizes the transcriptional coactivator Yki, which positively regulates the transcription of target genes involved in cell growth, proliferation, and survival. The evolutionary conservation is underscored by the demonstration that the mammalian YAP (the Yki homologue) can functionally rescue its Drosophila mutant in vivo. Overexpression of YAP in the mouse liver dramatically increases liver size; and its overexpression in the intestine expands the progenitor pool. In chick embryo, overexpression of YAP causes a marked expansion of the neural progenitor pool. All these findings suggest that YAP has conserved functions in regulating cell proliferation and survival in vertebrates.Previous studies showed that YAP binds and/or coactivates several proteins/protein families in biochemical assays and in cell culture model systems, including Yes, Runx, EBP50, p73, TBX5, p53BP2, TEFs/TEADs, 14-3-3, ErbB4, Smad7 and hnRNPU. But the lack of a physiological assay system has made it difficult to investigate the YAP function in vivo. Owing to its optical clarity, genetics, and ease of manipulation, zebrafish may be an ideal model system to obtain a comprehensive understanding of YAP in vivo. In order to investigate in vivo roles for yap, we knocked down the yap in zebrafish embryos by using antisense morpholino oligonucleotides. Further studies indicate that yap is required for zebrafish embryogenesis.Section 1. Bioinformatics analysis and expression pattern of yap during embryogenesisBioinformatics analysis indicated that zebrafish YAP shares significant sequence similarities with representatives from the fruit fly, chicken, mouse, and human (31%, 59%, 76%, 62% identity respectively). Homology is especially high in the WW domains that are known to interact with PPXY-containing polypeptides, in the TEF/TEAD-binding (TB) domain which affects TEF/TEAD binding to YAP family members, in the 14-3-3 binding site (containing the third HXRXXS motif) which is identified as the consensus phosphorylation sequences for LATS/Ndr kinase (known as a central player of Hippo signaling pathway) substrates, and in the Proline-rich sequence which is implicated in binding YAP to the SH3 domain of Yes proto-oncogene product and other signaling molecules. Expression analysis revealed that yap maternal transcripts are ubiquitous and endogenous yap is chronologically expressed in the notochord, brain, eyes, branchial arches and pectoral fins.Section 2. Confirmation of the yap morphant phenotypeTo determine the role of yap during zebrafish embryogenesis, we knocked down yap using a MO against the 5’ untranslated region (yap-MO) of the yap cDNA . To confirm the efficacy of the MO approach, yap-MO was co-injected with a GFP reporter containing partial 5’untranslated region and the start site sequence of yap. yap-MO specifically knocked down expression of GFP from this RNA transcript. In comparison, there was no detectable knockdown of GFP when co-injected with a random control MO. This revealed the ability of the yap-MO to inhibit protein production from its target sequence.Section 3. Effect of yap on regulating cell proliferation and survival in zebrafishAt the cellular level, whole-mount in situ immnofluorescence and FACS were done to examine the state of cell death and cell proliferation in yap morphant embryos. We found that cell proliferation is decreased and apoptosis is increased in yap-depleted embryos. Moreover, a significant increase in cell death is localized to the head and tail of yap-MO-injected embryos at 18 and 24 hpf.Section 4. Effect of yap on zebrafish embryogenesisAt the developmental level, whole-mount in situ hybridization and histochemical staining were done to analyze the dorsoventral patterning at gastrulation, the somitogenesis, the neurogenesis, the blood system, and the cardiovascular system in yap morphant embryos. We found that reduction of YAP function causes developmental delay, a phenotype similar to that of Drosophila yki mutants or zebrafish matsl morphants. Moreover, proneural and neuronal gene expression in yap morphant brain is significantly reduced. The expression of crestin is also markedly reduced in all recognizable arch-associated regions of yap morphants. To our surprise, the gsc expression might not be delayed, but the expression area is reduced at gastrulation stage, yap morphants have expanded expression of myoD and display u-shaped somite.更多还原