Effect Of Stat1a/stat1b Double-gene Mutation Of Zebrafish On Skeletal Develpoment

Osteoporosis is a systemic metabolic bone disease,which is more common in postmenopausal women and older men.STAT1 is a downstream pathway of cytokine signaling,which utilizes complicated signaling cascade reactions to play an important role in its biological effects,such as promoting cell death,inhibiting cell growth and so on.Our research room found that there is a correlation between STAT1 gene and osteoporosis,and presented that STAT1 gene is closely related to human osteoporosis.STAT1 gene of hunman and stat1 gene of zebrafish had sufficient homology,so the model organism zebrafish was selected as the experimental organism in this study.The stat1a gene and stat1b gene of zebrafish had been knocked out by the CRISPR/Cas9 gene editing technology,and the corresponding mutant strains had been obtained.In this paper,the stat1a^-/-and stat1b^-/-mutation models were established by screening the mutated strains,and the zebrafish model of the stat1a/stat1b double-gene mutation was obtained by hybridization.And the phenotypic observation and the related functional experiments were carried out.It is found that the early embryo death rate of the homozygous single-gene mutant juvenile fish which was knocked out the stat1a and stat1b genes is higher,and the skeletal phenotype of the adult fish also changed significantly,mainly including of the length of the jaw bone,the curvature of the tail and the spine,and the disappearance of caudal fin.There was no significant change in the death rate of embryos and the phenotypic bones of adult fish between the stat1a/stat1b double-gene mutation and the wild type.In addition,micro-CT scanning showed the specific changes of the single-gene mutant zebrafish's bone clearly.When the embryos were 7 days old,the cord side lesions of the stat1a/stat1b double-gene mutant broader than the wild type and the single-gene mutant slightly by Alcian blue staining,and the ankle cartilage also increased slightly.The distribution of osteoblasts and osteoclasts were different obviously between the double-gene mutant of stat1a/stat1b and the single-gene mutant by ALP staining of osteoblasts and TRAP staining of osteoclasts,but the difference from the wild type was not obvious.This study shows that:1)Both the stat1a gene and the stat1b gene are involved in the early embryonic development of zebrafish and affects the osteoblasts and osteoclasts distribution of adult zebrafish.These two genes are important for maintaining the normal development of bones;2)Both the stat1a gene and the stat1b gene of the zebrafish are involved in the skeletal development,and the skeletal deformity is obvious in the zebrafish of knocking out the two genes respectively.3)The study of the double-gene mutant zebrafish shows that the skeletal phenotype of the double-gene mutant is not as obvious as that of the single-gene mutant,which indicates that maybe there are some interaction or some compensation mechanism between the stat1a gene and the stat1b gene.Or the deletion of these two genes affects the expression of other signaling pathways.This study provides an experimental basis for further clarification of the mechanism of stat1 gene regulating the zebrafish's skeletal development.