Spatio-temporal Expression And Function Of Zebrafish Mast Cell Related Gene Cpα5 In Hematopoiesis

Background:Hematopoiesis is a subtle and ordered course,which give rise to diverse blood cells that participate in almost all physiological and pathological processes.A mass of molecules take part in this course,such as transcriptional factors,long-noncoding RNAs.Since hematopoiesis is a highly conserved process between zebrafish and mammalian,related researches have achieved significant advances with this novel model animal.Most kind of mammalian blood cells counterparts have been found and well-studied in zebrafish.However,mast cell study in zebrafish is very rare since lack of specific cell marker.In recent years,Jasonn.Berman et al.labeled cpα5+cells and sorted these cells out for morphological staining,suggest that cpα5 can serve as a specific marker of mast cells.In addition,Peter J.Lyons et al.confirmed that cpα5 was the only expressed gene in the zebrafish hematopoietic development site among cpa gene family with in situ hybridization experiments.The specificity of this expression pattern may suggest a particular role of cpα5 in zebrafish blood system.In order to verify whether cpα5 can be a specific mast cell marker from the molecular and functional aspects,we plan to detect the spatio-temporal expression pattern of cpα5 by synthesizing cpα5 probe and constructing cpα5 transgenic zebrafish line.Based on cpα5,single-cell sequencing and other techniques were proposed to further search for markers in different stages of mast cells.Meanwhile,the cpα5 mutant and the overexpressed zebrafish were constructed to analyze the role of cpα5 in zebrafish hematopoiesis.Objectives:1)to explore the cpα5+cell types of zebrafish;2)to detect the function of cpα5 in hematopoietic development of zebrafish;3)find specific marker for zebrafish mast cell.Methods:1)explore the cpα5+cell types through in situ hybrndization,co-localization and q-pcr;2)obtain cpα5+cells by constructing Tg(cpα5:eGFP)transgenic zebrafish lines for specific staining to study the cell types;3)construct cpα5 mutant by CRISPR/Cas9 and overexpress cpα5 in wildtype to study gene function.Results:1)cpα5 is expressed in the hematopoietic site of zebrafish,and can be colocalized with coro1a,lyz.The expression of cpα5 in c-mybhyper,pu.1G242D is consistent with the change of mature neutrophils in these mutants;2)the expression of cpα5 in the cpα5 mutant decreased,while the expression of the cpa family homologous genes increased.Among them,cpa1 ectopicly expressed at the hematopoietic site of mutant,and the ectopic expression of cpa1 could not be eliminated by upf3a morpholino;3)no obvious blood phenotype was observed in the cpα5 mutant,and the expression of macrophage-related markers increased after a transient overexpression of cpα5.Conclusions:1)most(about 80%)cpα5+cells are neutrophils,and they are relatively mature neutrophils,so it cannot be considered as a specific marker of mast cells;2)the cpα5 mutant shows no blood phenotype may attribute to genetic compensation effect,and this effect may be independent of the classical upf3a pathway;3)cpα5 may be related to the development of macrophages.