Role Of Retinoic Acid Signal In The Development And Regeneration Of Pharyngeal Teeth In Zebrafish

The loss of teeth caused by caries,periodontitis and trauma will greatly reduce people's quality of life and even affect their overall health.At present,the clinical measures for dentition defects are mainly partial denture repair or dental implant repair.These repair methods have a good repair effect,but some adverse consequences are unavoidable.Tooth regeneration is a safe and ideal method for the restoration of missing teeth.At present,the exploration of tooth regeneration has made great breakthroughs and progress in tissue engineering teeth,it has achieved the regeneration of roots,pulp and other tissues and even whole teeth.However,endogenous regeneration is still in the early stages of exploration.Current researches on endogenous tooth regeneration are limited because there is no suitable animal model.In this experiment,we used the advantages of live imaging and real-time dynamic observation of zebrafish to study the molecular mechanism of tooth development and regeneration,and mainly explored the role of the retinoic acid signal pathway in the development and regeneration of pharyngeal teeth of zebrafish.The role of this signal pathway was initially explored through the treatment of pharyngeal teeth with exogenous retinoic acid(RA)and its inhibitor diethylaminobenzaldehyde(DEAB)during the development and regeneration process of zebrafish,respectively.In addition,the transgenic line Tg(dlx2b:Dendra2-NTR)constructed by our members early marked the early development of pharyngeal teeth.After exploration,in this study,we successfully constructed another transgenic line Tg(scpp5:Dendra2-NTR).In this line,life-time specific marking of zebrafish pharyngeal teeth and high specificity can make the study of zebrafish pharyngeal teeth not limited by the development time,which is conducive to further exploring the mechanism of tooth development.Objective:To explore the effect of retinoic acid signal on the development and regeneration of pharyngeal teeth in zebrafish.Methods:(1)Zebrafish embryos at different developmental stages were treated with 3×10~-7 M RA and 1×10~-7 M DEAB,the embryos were collected for in situ hybridization,alizarin red,and confocal microscopy to observe the effects of retinoic acid signals on the development of zebrafish pharyngeal teeth;(2)The effect of retinoic acid on the regeneration of pharyngeal teeth in zebrafish was explored by constructing over-expressing lines hsp:cyp26b1-GFP and hsp:aldh1a2-GFP,which is the retinoic acid degrading enzymes and synthetases,and hybridize with Tg(dlx2b:Dendra2-NTR),respectively.After cross-breeding,the zebrafish in the process of pharyngeal tooth regeneration was heat-shocked after MTZ treatment,and embryos were collected at 24 hours and 48 hours after withdraw the MTZ to detect the pharyngeal tooth regeneration.(3)Construction of transgenic fish Tg(scpp5:Dendra2-NTR),we cloned a 4.9 kb promoter sequence of the scpp5 gene from the zebrafish genome,and successfully constructed the pBluescript2KS-scpp5-Dendra2-NTR recombinant plasmid.After injection of the purified plasmid pBluescript2KS-scpp5-Dendra2-NTR into single-cell embryos by microscopy,zebrafish embryos were screened for three consecutive generations of forward genetic screening for stable hereditary transgenic fish Tg(scpp5:Dendra2-NTR).Results:(1)After treatment of 24-36 hpf zebrafish embryos with 1×10~-7 M DEAB,the development of pharyngeal teeth was detected at 48 hpf,56hpf and 72 hpf.The results of in situ hybridization showed that the expression of pitx2 and fth1b was lost,in the 3×10~-7 M RA treatment group,the expressions of pitx2 and fth1b were both stronger than the control;And embryos were collected and stained with alizarin red at 5 dpf,the results showed that in the 1×10~-7 M DEAB-inhibited group,no mineralized teeth were formed,but it can be observed the unmineralized teeth germ by confocal microscope,in 3×10~-7 M RA treatment group the teeth mineralization was slightly stronger than that in the control group,there's stronger mineralization of 3V~1,4V~1 and 5V~1 observed by confocal microscope,indicating that retinoic acid signal plays a role in the early development of pharyngeal teeth of zebrafish.Inhibition or enhancement of retinoic acid signal will both affect the normal development of pharyngeal teeth.(2)After treatment of 24-36 hpf zebrafish embryos with retinoic acid inhibitors DEAB,the expression of cranial neural crest cell-related genes were affected,the expression of the gene snail1a,which marks the epithelial-mesenchymal transition process,is slightly decreased,and the expression of cranial neural crest cells proliferation and migration gene cad is also reduced,indicating that retinoic acid mainly plays a role in affecting the normal activities of cranial neural crest cells in the early stage of pharyngeal tooth development,especially the proliferation and migration of cranial neural crest cells.(3)Transgenic fish Tg(dlx2b:Dendra2-NTR)was used to hybridize with hsp:cyp26b1-GFP and hsp:aldh1a2-GFP,respectively,to heat-shock the recovery embryos after pharyngeal teeth damaged.After 24hours and 48 hours of recovery,alizarin red staining and confocal microscopy observations revealed that the regenerated pharyngeal teeth were weakly mineralized in the hsp:cyp26b1-GFP group,and stronger in the hsp:aldh1a2-GFP group than in the control group,while there was no difference in the shape and number of regenerated pharyngeal teeth.That is,during the regeneration of pharyngeal teeth in zebrafish,the retinoic acid signal plays a role in the mineralization without affecting the shape and number of regenerated pharyngeal teeth.(4)In situ hybridization showed that gene scpp5 was specifically expressed in the zebrafish pharyngeal teeth at 3 dpf-13 dpf.Based on this,the group successfully constructed Tg(scpp5:Dendra2-NTR)transgenic line.At 3 dpf,green fluorescence can be observed at the pharyngeal teeth.Tg(scpp5:Dendra2-NTR)embryos were detected by antibody staining at 5 dpf,it was found that the gene scpp5 fusion green fluorescent protein Dendra2 was expressed around the zebrafish pharyngeal teeth and surrounded the zebrafish teeth,secreting calcium-related proteins to promote mineralization of pharyngeal teeth.Conclusion:The retinoic acid signal pathway plays an important role in the early development of pharyngeal teeth of zebrafish.Activation or inhibition of retinoic acid signal can affect the development of pharyngeal teeth of zebrafish,cause it can regulate the proliferation and migration of cranial neural crest cell which is the initiation of tooth germ.However,it only slightly affects the mineralization of regenerated pharyngeal teeth.Transgenic line Tg(scpp5:Dendra2-NTR)has optimized the zebrafish pharyngeal teeth development and injury regeneration model,which can be used to explore the development and regeneration mechanism of zebrafish pharyngeal teeth in later stages.