Hedgehog Signaling Is Required In Pharyngeal Tooth Development And Regeneration In Danio Rerio

Teeth are an important organ of human body.Teeth play important roles in normal craniofacially development.Tooth defects and loss cased by developmenta deformity,dental caries,periodontal diseases,trauma,and tumor do harm to human physical and mental health.Tooth cannot repair and regenerate of itself,and dentists applied implants and crown prosthesis to recover the appearance and function of lost tooth.As regenerative medicine develops,tissue engineer-based tooth regeneration has received great progress.However,there are still some problems,like how to induce the odontogenic potential of stem cells to the full extent,how to accelerate the developmental process through manipulating associated genes.Tooth development is achieved by the reciprocal interactions between dental epithelium and mesenchyme.Hedgehog signaling is one of the pathways that mediate the interactions.Researches about the roles Hedgehog signaling plays in tooth development are almost performed in mouse,which demomstrate that Hedgehog signaling is essential for regulating early tooth initiation and morphogenesis.While report about its role in tooth regeneration is nearly none.Limitation of mammalian model animals makes deep study of this pathway during tooth development difficult.Zebrafish is an advantageous model in for having large similarity with mammalian teeth in structure and development,along with live image tracing and in situ research.Based on Mtz/NTR system and transgenic line Tg(dlx2b:Dendra2-NTR),zebrafish pharyngeal teeth in vivo injury and regeneration model is constructed.This project have done researches to further confirm the tooth position being injured and regenerated in this model,treating the fish with Hedgehog signaling inhibitor to explore its roles in pharyngeal tooth development and regeneration.We hope to provide experiment basis and support for tooth development regulating mechanisms and tissue engineer-based tooth regeneration.Objective To explore the potential roles for hedgehog signaling pathway during zebrafish pharyngeal teeth development and zebrafish teeth regeneration process.Methods Applying 50?M hedgehog signaling inhibitor cyclopamine to treat zebrafish in different time periods and observing alteration of signaling pathway-associated and pharyngeal tooth development-associate genes,and the development of zebrafish pharyngeal teeth with in situ hybridization,alizan red staining and confocal image.12 mM Mtz is used to treat Tg(dlx2b:Dendra2-NTR)3-5 dpf to obtain pharyngeal in vivo injury model.Pharyngeal teeth regeneration process is researched after Mtz retreatment.The tooth position is fully confirmed with in situ hybridization,alizan red staining,confocal image,antibody staining.Tg(dlx2b:Dendra2-NTR)fishes are treated with 50?M Cya after removal of Mtz,then teeth regeneration is observed with alizan red staining and confocal image.Results(1)After treatment with 50?M Cya from 30 hpf to 56 hpf,in situ results show that expression of ligand shha in pharyngeal region is found to decrease in intensity and range,of receptor smo is slightly reduced,and of receptors ptch1 and ptch2 is completely lost.Namely,pharyngeal Hedgehog signaling is inhibited after 50?M Cya treatment from 30 to 56 hpf.(2)After treatment with 50?M Cya from 30 hpf to 56 hpf,expression of dental epithelium maker fgf3 is disappeared,of scpp5 and fth1b is vanished,which means that early development of pharyngeal teeth is arrested.And the absence of pharyngeal teeth initiation marker pitx2 suggests that Hedgehog signaling is required in tooth initiation happens before 30 hpf.(3)After 36-100 hpf Cya treatment with 50?M Cya,mineralization of pharyngeal tooth can't be detected.Exposure to Cya from 48 hpf leads to small and aberrant teeth and arrest of branchial arch mineralization,and with confocal image,4V~1 is smaller than control and the base of the tooth is rounded,in addition,development of 3V~1 and 5V~1 is a little delayed.Exposure to Cya from 60 hpf results in smaller 4V~1 than control and the base of the tooth doesn't mineralize well,additionally,development of 5V~1 is delayed.Development of the three teeth is nearly normal after treatment from 72 hpf.(4)During the pharyngeal teeth injury and regeneration process of Tg(dlx2b:Dendra2-NTR),in situ exhits that expression marker scpp5 is lost after Mtz treatment and recovered during regeneration.Alizan red staining,confocal image of tooth structure and antibody staining all shows the injury and regeneration of pharyngeal teeth in ventral 3 and 5.(5)After application of 50?M Cya during tooth regeneration process,alizan red staing and confocal both cannot detect the regeneration of pharyngeal teeth in ventral 3 and 5.Conclusion Hedgehog signaling is involved in the early initiation stage before 30 hpf and morphogenesis stage of zebrafish pharyngeal teeth.Pharyngeal teeth ventral 3 and 5 are confirmed to be injured and regenerate in Tg(dlx2b:Dendra2-NTR)treaed with Mtz.What's more,hedgehog signaling is required in the pharyngeal teeth regeneration.