Regulation Of Scpp5 Gene On Tooth Development Of Zebrafish

The dentition of teleost fishes is the most representative of vertebrates,and for different species,the distribution of tooth in the oral cavity,tooth arrangement,tooth number,shape and size all show a complex diversity.All bony fish have the phenomenon of continuous replacement of tooth.After the tooth in the oral cavity or pharynx have passed the functional period,a tooth is replaced by a new tooth at the same position(site).This tooth replacement lasts for life.The molecular mechanism of odontoid development is not well understood.The Secretory Calciumbinding Phosphoprotein(SCPP)gene family encodes a variety of phosphoproteins,many of which are involved in bone ossification and have been reported in the development of mammalian bones,enamel and dentin played a vital role.However,its expression regulation effect on fish tooth development is not very clear.In this study,the aquatic model animal zebrafish was used as the research object.On the basis of comparing the differences in gene expression of the transcriptome of the branchial arch transcriptome with and without tooth,the scpp5 gene was focused on,and a mutant strain of zebrafish scpp5 gene function was constructed.Observation methods The growth and tooth development of the mutant lines were compared,and the regulatory function of the scpp5 gene on tooth development was analyzed.At the same time,the gene expression differences in the fifth pair of branchial arch(with tooth)tissues of the mutant and wild-type zebrafish were compared by transcriptome sequencing.Molecular regulation of SCPP family genes on tooth development.Below are key research findings.1.Transcriptome comparative analysis of wild-type zebrafish with and without dentate branchial archesTranscriptome sequencing of the fourth branchial arch(no tooth)and fifth branchial arch(dentate)tissues of 3-month-old wild-type zebrafish adult fish using transcriptome sequencing technology,wild-type zebrafish fifth branchial arch and fifth branchial arch Compared with the four branchial arches,there were 3416 differentially expressed genes,of which 2272 genes were up-regulated and 1144 genes were down-regulated.The results of differential gene expression analysis showed that among the key genes of SCPP family,ODAM,sparcl1,spp1,scpp1,scpp5,scpp6,scpp7,scpp8 and scpp9,except for sparcl1 gene,the expression levels of the other 8 genes in the fifth branchial arch were significantly higher than The fourth branchial arch(P<0.05).Pathway enrichment analysis of differentially expressed genes in wild-type fifth and fourth branchial arch tissues revealed cytokine-cytokine receptor interaction pathway,phagosome pathway,TGF-β signaling pathway,ECM-receptor interaction pathway,Signaling pathways such as calcium signaling play an important role in zebrafish tooth development.2.Construction and phenotypic analysis of scpp5 knockout zebrafish strainsThrough CRISPR/Cas9 gene editing technology,a scpp5 knockout zebrafish line was constructed,and a mutant line with a deletion of 16 bp in the first exon of scpp5 made the amino acid coding terminated earlier.Alizarin red skeletal staining was performed on 90 dpf adult fish of wild-type and scpp5 mutant lines.The results showed that the skeletal phenotype of scpp5 was not significantly different from that of wild-type.The vertebrae,ribs,skulls and intermuscular bones belonged to normal mineralized growth.The number of ribs and intermuscular bones were not significantly different,but the mutant strains had obvious missing tooth compared with the wild ones.The tooth of wild-type zebrafish are usually divided into three layers,which are distributed in the dorsal,middle and ventral sides,and the arrangement is 5-4-2,while the tooth arrangement of the scpp5 gene mutant strain is not particularly fixed compared with the wild type,and after 90 dpf the adult mutants mostly exhibit a 2-2-2 arrangement.Furthermore,we observed the tooth development of the mutant strains from 10 dpf to 90 dpf,the results showed that the mutant strains had tooth loss at the beginning of 20 dpf,and this phenomenon became gradually obvious at 30 dpf,until the tooth could be clearly observed at 90 dpf lack of phenomenon.3.Preliminary study on the molecular regulation mechanism of scpp5 gene on zebrafish tooth developmentTranscriptome sequencing analysis of the fourth and fifth branchial arch tissues of 3-month-old scpp5 knockout line zebrafish adult fish,a total of 2365 branchial arches of the fourth pair of branchial arches of scpp5 homozygotes and wild type Among the differentially expressed genes,877 genes were significantly up-regulated genes,and 1488 genes were significantly down-regulated genes.Compared with the homozygous fifth pair of branchial arches and the homozygous fourth pair of branchial arches,there were a total of 6263 differentially expressed genes.Among these genes,3376 genes were significantly up-regulated genes,and 2887 genes were significantly down-regulated genes.There were 1255 differentially expressed genes in the fifth pair of branchial arches of homozygotes and wild type.Among these genes,645 genes were significantly up-regulated genes,and 610 genes were significantly down-regulated genes.The differentially expressed genes of the scpp5 gene mutant line and the wild-type fifth branchial arch are mainly concentrated in the cytokine-cytokine receptor interaction pathway,adipocytokine signaling pathway,amino acid biosynthesis pathway,herpes simplex infection pathway,and arginine biosynthesis pathway.Signal pathways such as synthetic pathway,cysteine.and methionine metabolism pathway.The expression of ODAM,sparcl1,fa93e10,spp1,scpp1,scpp5,scpp6,scpp7,scpp8 and scpp9 in the fourth and fifth branchial arches of wild-type and mutant lines was analyzed by q RT-PCR.The expression levels of 9genes in scpp5 gene mutation homozygotes were lower than those in wild type(P<0.05).