Study On The Role Of Cathepsin B Gene In Early Embryonic Development Of Zebrafish

The cysteine cathepsin B is mainly present in lysosomes and is an eosinophilic protease.The cathepsin B can hydrolyze damaged organelles and proteins in cells,and along with lysosomal vesicles,it can also secrete out of cells to degrade the components in extracellular matrix.There are two isoforms of cathepsin b genes in zebrafish,cathepsin ba(ctsba)and cathepsin bb(ctsbb).In this paper,the CRISPR/Cas9 technique was used to knock out the zebrafish cathepsin ba genes,and the heterozygous mutants were screened from the first generation.ctsba heterozygous mutants were crossed to obtain the second generation,some embryos development stopped at 50%epiboly and some of these embryos were dead,The deaths accounted for about 6.9 percent of all embryos.It was delayed 1-2 hours before another part of the embryos began to develop slowly.During the pharyngeal phase,these embryos developed abnormal phenotypes,pericardial cysts,folding and bending of the tail.Died after 2-3 days of development,these embryos accounted for 19.3%of the total number of embryos,the gene test results showed that these embryos are all homozygous.Embryonic mortality accounted for about a quarter of the total number of embryos,which was in accordance with Mendel's law of inheritance,indicating the existence of recessive lethal effects of ctsba gene.RT-PCR detected nonsense-mediated mRNA decay(NMD)inctsba-/-mutants,indicating that ctsba gene knockout was successful.Analysis by RT-PCR and in situ hybridization showed that the cathepsin ba was a maternal and zygotic gene,mainly expressed in the animal pole.It widely expressed in zebrafish embryos especially in yolk syncytial layer and neural tube.In situ hybridization using the chordin and gata2 as dorsal and ventral marker genes,it was found that abnormal expression of the chordin and gata2 did not occur in the ctsba heterozygous and homozygous mutants at shield stage.The expression of chordin was decreased in the dorsal midline of ctsba-/-mutants at 75%epiboly.The zebrafish gastrulation stage begins at 50%epiboly,and it is a critical period for the formation of the three germ layer,and the ctsba-/-mutants begin to develop abnormally at 50%epiboly.Therefore,in situ hybridization was carried out in the embryos by using marker genes from endoderm,mesoderm and ectoderm,they were sox17 evel and gsc.According to in situ hybridization and gene test analysis,sox 17,evel,and gsc all showed unusual expression in the ctsba-/-embryos:sox17 began to express the phenomenon of diffusion during the 75%epiboly,and the bud stage of the endoderm precursor cells decreased significantly;evel expressed disorder at the tail of the pharyngeal phase;the expression of gsc decreased markedly from the period of 75%epiboly.The results showed that ctsba may be involved in the back development of zebrafish,and ctsba may participate in the cell movement and the formation of three germ layers in the gastrulation stage of zebrafish embryos development.Ctsba gene defects may result in extensive embryo deformity and lethal effects.In this paper,the CRISPR/Cas9 technique was also used to knock out the zebrafish cathepsin bb genes,and screened for the cathepsin bb knock-out mutants.