| Fish teeth are important feeding organs.Its main function is to prey and prevent food escape,some also have the effect of tearing and biting food.Fish teeth originate from the skin teeth(placoid sacle)and are mainly divided into jaw teeth and pharyngeal teeth.Jaw teeth are widely found in chondrichthys and Osteichthyes,but pharyngeal teeth are more limited in Cyprinidae,Leuciscinae and so on.Pharyngeal teeth play an important role in fish feeding and have a very high frequency of replacement.At present,most of the research on fish teeth focuses on the development regulation of jaw teeth,but there is a lack of research on the specific genes of pharyngeal teeth development and the corresponding molecular regulation mechanism.In this study,We use RNA-seq to compare the gene expression of the gill arches without pharyngeal teeth and the gill arches with pharyngeal teeth of medaka and zebrafish.The result was compared with the RNA-seq of the gill arches without pharyngeal teeth and the gill arches with pharyngeal teeth of zebrafish to screen out the key genes regulating the development of pharyngeal teeth in fish.Zebrafish mutant strains with functional deletion of key genes for pharyngeal tooth development were constructed.The growth,development and number of pharyngeal teeth of wild type and mutant strains were compared by alizarin red staining and histological analysis,so as to verify the regulatory functions of key genes on pharyngeal tooth development in fishes.The main results are as follows:1.Development characteristics and excavation of key regulatory genes of pharyngeal teeth in medaka and zebrafishTranscriptome sequencing was performed on the gill arches without pharyngeal teeth and the gill arches with pharyngeal teeth of 90 dpf medaka using RNA-seq technique.The results showed that a total of 1576 differentially expressed genes were identified in the gill arch with pharyngeal teeth compared with the gill arch without pharyngeal teeth.Differential expression gene analysis showed that panx3,scpp5,sp7,scpp9,cts12 and so on were highly expressed in the gill arches with pharyngeal teeth,but were less expressed in the gill arches without pharyngeal teeth.Bmp family genes,such as bmp5,bmp6 and bmp10,were highly expressed in the gill arches without pharyngeal teeth,but were less expressed in the gill arches with pharyngeal teeth.KEGG enrichment analysis showed that the up-regulated differentially expressed genes were mainly concentrated in glutathione metabolism pathway,starch and sucrose metabolism pathway,glycolysis and gluconeogenesis pathway,ECM-receptor interaction pathway,calcium signaling pathway,amino acid biosynthesis pathway,etc.The down-regulated differentially expressed genes were mainly concentrated in sphingolipid metabolic pathway,signaling pathways such as cytokine-cytokine receptor interaction pathway,cell adhesion molecular pathway,and cell apoptosis.The RNA-seq data of the gill arches with and without pharyngeal teeth were compared with those of the gill arches with and without pharyngeal teeth of zebrafish.The results showed that a total of 187 genes were the same in the gill arch with pharyngeal teeth upregulation gene of medaka and zebrafish and a total of 21 genes were the same in the gill arch with pharyngeal teeth downregulation gene of medaka and zebrafish.The third and fifth gill arches of medaka had similar differentially expressed genes and KEGG enrichment pathway to the fourth and fifth gill arches of zebrafish.The up-regulated differentially expressed genes in the gill arch with pharyngeal teeth were enriched to the same 9 signaling pathways,while the down-regulated differentially expressed genes were enriched to the same 3 signaling pathways.A total of 15 genes(odam,sp7,panx3,cts12,col1a1 b,entpd5b,spp1,scpp1,scpp5,scpp9,bmp2 a,bmp5,bmp6,bmp10,bmp16)were used for q RT-PCR.The q RT-PCR results were consistent with the RNA-seq.2.Regulation research of bmp5 and bmp6 genes on the development of pharyngeal teeth in zebrafishIn situ hybridization analysis of bmp5 and bmp6 genes in 2dpf,3dpf and 5dpf zebrafish showed that bmp5 and bmp6 were strongly expressed in the gill arch of 2dpf,3dpf and 5dpf zebrafish.The knockout lines of bmp5 and bmp6 genes of zebrafish were constructed using CRISPR/Cas9 technology.Compared with wild-type zebrafish,an 2bp deletion occurred in bmp5 mutant sequence,resulting in a frameshift mutation.Protein translation was prematurely terminated at the 172 nd amino acid position.An19 bp deletion occurred in bmp6 mutant sequence,resulting in a frameshift mutation.Protein translation was prematurely terminated at the 216 nd amino acid position.Alizarin red staining was performed on 90 dpf wild type,bmp5 and bmp6 mutant zebrafish.The results showed that compared with wild-type zebrafish,bmp5 and bmp6 mutant zebrafish showed a significant reduction in the number of pharyngeal teeth in the fifth pair of gill arches.Different from the arrangements of 5-4-2 and2-4-5 in wild-type zebrafish,the arrangements of pharyngeal teeth in bmp5 mutant zebrafish were 4-3-1 and 1-3-4,missing 6 pharyngeal teeth.The arrangements of pharyngeal teeth in bmp6 mutant zebrafish were 4-2 and 2-4,missing 10 pharyngeal teeth.The tissue slices of pharyngeal teeth of wild-type bmp5 and bmp6 homozygous zebrafish showed that,compared with wild-type zebrafish,the number of pharyngeal odonts of bmp5 and bmp6 homozygous zebrafish decreased,and there were more holes in the gill raker.The pharyngeal tooth tissue structure of bmp5 and bmp6 homozygous zebrafish was not tight.The growth data of wild type and bmp5 mutant zebrafish at each growth stage(30dpf,60 dpf,90dpf)were measured.The results showed that the length and weight of bmp5 mutant zebrafish were higher than those of wild-type zebrafish at 30 dpf.But at 60 dpf and 90 dpf,the length and weight of bmp5 mutant zebrafish were significantly lower than those of wild-type zebrafish.The wild type and bmp5 mutant zebrafish juveniles at 10 dpf and 15 dpf were stained of alizarin red.The results showed that compared with wild-type zebrafish,bmp5 mutant zebrafish developed faster in head bones and pharyngeal teeth at 10 dpf and 15 dpf.bmp5 and bmp6 double homozygous mutants were screened out from bmp5 and bmp6 mutants.Alizarin red staining showed that the number of pharyngeal teeth of bmp5 and bmp6 double homozygous mutants was significantly reduced,and the pharyngeal teeth were arranged in 4-2 and 2-4.The bmp5-bmp6 double homozygous mutants showed enlarged gills,deformed ribs,short fins,low bone salinity,and gradually died at75 dpf.3.Regulation research of scpp9 gene on the development of pharyngeal teeth in zebrafishIn situ hybridization analysis of scpp9 gene in 2dpf,3dpf and 5dpf zebrafish showed that scpp9 was strongly expressed in pharyngeal teeth of 5dpf zebrafish.The knockout lines of scpp9 gene of zebrafish were constructed using CRISPR/Cas9 technology.Compared with wild-type zebrafish,an 13 bp deletion occurred in scpp9 mutant sequence,resulting in a frameshift mutation.Protein translation was prematurely terminated at the 115 nd amino acid position.Alizarin red staining was performed on 90 dpf wild type and scpp9 mutant zebrafish.The results showed that compared with wild-type zebrafish,scpp9 mutant zebrafish showed a significant reduction in the number of pharyngeal teeth in the fifth pair of gill arches.Different from the arrangements of 5-4-2 and 2-4-5 in wild-type zebrafish,the arrangements of pharyngeal teeth in scpp9 mutant zebrafish were 4-4-1 and 2-4-5,missing 2pharyngeal teeth.The wild type and scpp9 mutant zebrafish juveniles at 10 dpf and15dpf were stained of alizarin red.The results showed that compared with wild-type zebrafish,scpp9 mutant zebrafish did not form gill rakers and mineralized only 4pharyngeal teeth at 10 dpf.The tissue slices of pharyngeal teeth of wild-type and scpp9 homozygous zebrafish showed that,compared with wild-type zebrafish,the number of pharyngeal odonts of scpp9 homozygous zebrafish decreased,and there were more holes in the gill raker.The pharyngeal tooth tissue structure of scpp9 homozygous zebrafish was not tight.In the research,the key genes regulating the development of pharyngeal teeth,such as bmp5,bmp6 and scpp9,were screened by RNA-seq.The homozygous mutants of bmp5,bmp6 and scpp9 of zebrafish were constructed by CRISPR/Cas9 technique.The quantity and arrangement characteristics of pharyngeal teeth of bmp5,bmp6,scpp9 and bmp5-bmp6 of zebrafish were studied.This study will provide a theoretical basis for the subsequent development of pharyngeal teeth in fishes and lay a foundation for the development and regeneration of teeth in vertebrates. |
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