Cloning And Identification Of Zebrafish Gpr161 Gene And Preliminary Functional Study

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.