【作者】 王敏；

【导师】 赵仲华；

【作者基本信息】 山西大学 ， 微生物学， 2020， 硕士

【摘要】 Gpr161蛋白是一种G蛋白偶联受体（G Protein Coupled Receptor,GPCR）^[9]。GPCR均是含有7个跨膜结构域的蛋白受体,是参与细胞信号传导的最大的细胞表面分子家族,参与调控多种不同的细胞功能,GPCR是目前各种疾病临床治疗的主要药物靶点^[15]。小鼠的研究提示,Gpr161在脊椎动物Hedgehog（Hh）信号通路中发挥重要的作用,Gpr161的缺失会导致小鼠胚胎致死。组织特异性敲除Gpr161导致小鼠出现多并趾、大脑发育不良和头骨闭合不全等畸形。研究人员发现GPR161是Hh通路的负向调控因子,通过调节细胞内cAMP的水平调控Hh通路下游的信号转导^[20]。然而,Gpr161在动物发育早期和Hh通路中功能在进化上是否具有保守性并不明确。特别是在人Hh通路相关癌症——髓母细胞瘤大规模测序中并没有发现Gpr161的突变与癌症的发生有关。此外,临床研究中仅有一例关于GPR161缺失后导致患者垂体柄发育异常的报道^[26]。这些结果使得在其它模式动物中对于GPR161功能的研究显得格外的重要。为了深入探索GPR161在脊椎动物早期发育和Hh通路中的功能,本研究以斑马鱼为模式动物,对Gpr161进行了克隆和研究,具体内容如下:1.我们通过生物信息学分析,克隆获得了斑马鱼两个gpr161同源基因,分别命名为gpr161a和gpr161b;通过全胚胎原位杂交技术（Whole mount in situ hybridization,WISH）以及实时定量PCR（qRT-PCR）分析了这两个基因的转录情况,发现它们在胚胎发育时期均存在遍在表达^[10]。但是gpr161a在神经管和近轴细胞（adaxial cell）中有富集,而gpr161b在神经管的中轴附近富集,提示其功能并不完全冗余,可能存在差异。2.为了进一步研究Gpr161在斑马鱼胚胎发育中的功能,我们应用CRISPR-Cas9基因编辑技术构建了斑马鱼gpr161基因的缺失突变体。初步表型分析显示,无论是gpr161a^-/-和gpr161b^-/-单突变纯合子还是gpr161a^-/-与gpr161b^-/-双纯合突变体,均可以正常发育,没有出现明显的异常表型。这一结果与小鼠Gpr161缺失突变体不一致。提示Gpr161在脊椎动物中的功能可能并不保守,或者斑马鱼在进化中获得了其他的补偿效应以应对gpr161缺失的效应。3.为了进一步研究Gpr161在Hh信号通路的功能是否保守。我们对斑马鱼gpr161突变体的Hh信号通路的活性进行了干预和分析。通过Hh通路特异性的分子标记ptch2,olig2与nkx2.2a的表达情况,以及慢肌纤维的分化情况对gpr161缺失突变体对不同信号活性干预的应答情况进行了分析,初步的结果显示gpr161缺失突变体本身并没有表现出Hh活性的异常,而且在上调和下调Hh活性的干预下,gpr161突变体与野生型也没有明显的差异,提示Gpr161可能不是斑马鱼Hh通路重要的调控因子,而Gpr161在Hh通路中的功能并不保守。综上所述,本研究基于斑马鱼动物模型,克隆了gpr161同源基因,分析了其表达情况;建立了斑马鱼gpr161缺失突变体模型,并对其表型进行了初步的分析;同时对于缺失突变体Hh通路活性进行了初步的分析,为深入研究gpr161基因的功能提供了重要的实验材料和前期数据的积累,为进一步研究脊椎动物Hh通路的分子机制提供了重要的证据和线索。更多还原

【Abstract】 Gpr161 Protein is one of the G Protein Coupled Receptors(G Protein Coupled Receptor,GPCR),typically sharing the structure of seven transmembrane domains.It is supposed that there are more than 800 GPCRs in human genome and they are involved in plenty cell behaviors,especially in cell signalling transduction.This makes that the GPCRs are the most common target of clinical drugs.Mouse lack of Gpr161 is embryonic lethal.Conditional knockout gpr161 in mice led to several Hedgehog related deforms,such combined digits,brain dysplasia and skull deformity.Mukhopadhyay et al has reported that Gpr161 is critical negative regulator of mouse Hedgehog(Hh)signalling pathway.It modulates the Hh signal transduction by regulating the c AMP level and PKA activity in primary cilia,the specific subcellular compartment.However,the conservation of the Gpr161 in Hh pathway in vertebrate is unclear.In addition,a deep sequencing study of medulloblastoma,a typical tumor with aberrant high Hh activity,failed to find any gpr161 homozygous mutation.Besides,there is only one reports that loss of gpr161 in human cause Pituitary Stalk Interruption Syndrome in Turkey.These data make the Gpr161 function in Hh pathway in debate.Thus,it is critical to dissect the Gpr161 fucntions in other model system rather than mouse.To examine the Gpr161 function in vertebrate embryogenesis and Hh pathway,taking the zebrafish as model,we have done the cloning,identification and preliminary function assay of Gpr161.1.Two gpr161 genes have been identified by using human GPR161 protein sequence blast against zebrafish genome.They were named as gpr161 a and gpr161 b,respectively.The expression pattern of these two genes have been examined by whole mount in situ hybridization and q RT-PCR.Although ubiquitous expression of both genes,gpr161 a accumulates at the spinal cord as well as adaxial cells,while gpr161 b accumulates at the midlineof the neural tube,suggesting their divergent function in embryogenesis.2.In order to dissect the function of Gpr161 in zebrafish,we have generated gpr161 mutants by CRISPR-Cas9 editing technique.Our preliminary analysis showed that both single homozygous mutants and double homozygous mutants are viable.Neither of them show obvious abnormal phenotype during embryo genesis.It could be interpreted that the Gpr161 function is not conserved in vertebrate,or zebrafish has evolved compensation pathway to overcome the loss of Gpr161.3.To further examine the function of zebrafish Gpr161 in Hh pathway,we have assay the Hh activity in all gpr161 mutant by perturbing the Hh pathway at different level.However,neither the targets of Hh,ptch2,olig2 and nkx2.2a,nor the differentiation of slow muscle firbres tested by Prox1 a expression showed differences compared to wild type embryos.In addition,no obvious changes has been detected no matter upregulation or downregulation of Hh activity in gpr161 mutants,suggesting that Gpr161 is not essential for vertebrate Hh pathway,at least in zebrafish.In summary,using zebrafish model,we have successfully identified and cloned zebrafish gpr161 homologs,and analyzed its expression;established zebrafish gpr161 deficient mutants and conducted a preliminary analysis of their phenotype;preliminarily analyzed Hh activity in gpr161 mutants.Our results provided important materials and preliminary data for gpr161 function assay and shed lights on the mechanism of vertebrate Hh signal transduction.更多还原