| Eosinophil play an important role in immune response and allergic reaction in mammals,mainly involved in the process of resisting parasitic infection and asthma response.The differentiation and maturation of eosinophil,as well as the role and mechanism in infection,have always been fundamental questions of concern.Recent studies have shown that zebrafish eosinophil has similar functional characteristics with mammals,suggesting the study of zebrafish eosinophil may also have guiding significance for higher animals.However,zebrafish eosinophil transgene line which have been reported to be unable to specifically mark eosinophil in larva,so there is still a lack of specific molecular markers which can mark the eosinophil both in adult and larva zebrafish for subsequent studies.In this study,the KM of wild-type zebrafish and cebp1smu1 mutant which with increase number of eosinophils in KM were used to sequence and analyze the expression through RNA-Seq to further screen and identify eosinophils specific molecular markers in zebrafish.first of all,according to previous study we known adult zebrafish eosinophils show positive in PAS staining,so we confirmed the existence of eosinophil in 3-5dpf zebrafish larva by PAS staining and morphological observation,preliminary concluded that the generation of eosinophils in zebrafish larva.Secondly,we found that the number of eosinophil significantly increased in adult cebp1smu1 mutant zebrafish KM and the morphology was normal.Because cebp1smu1 mutant had been previously reported that other blood cells are defective or unaffected,it means that the eosinophil related gene expression level might increase specifically in cebp1smu1 mutant.We collected the KM of wild-type zebrafish and cebp1smu1 mutant,sequenced them by RNA-Seq and analyze their expression to obtain candidate specific eosinophil molecular markers in zebrafish,then we get five candidate markers with tissue and lineage specificity through RT-qPCR for further screening.Finally,two marker genes,si:dkeyp-75b4.10 and si:dkey-203a12.3,their expression pattern and cebp1smu1 mutant phenotype were confirmed by WISH,and the specificity was further confirmed by co-staining.Our results will contribute to the further exploration of the cytological mechanism of eosinophil development in early stage,the molecular mechanism of eosinophil development in embryo and the function of eosinophil,these subsequent studies may provide a molecular basis for novel therapies of eosinophil related diseases. |
