Expression And Localization Of Yap And Taz During Molar Development And Jaw Defect Regeneration In Rats

Yap (Yes-associated protein) as an effective downstream transcription factors of the Hippo signaling pathway, which was discovered in Drosophila in 1995. Previous studies demonstrated that Yap was expressed in a variety of tissues and organs, such as intestine, liver and neural tube etc. Taz (transcriptional coactivator with PDZ-binding motif) is a 14-3-3 binding protein, containing a conserved WW domain. Taz is another important transcription factor in Hippo signaling pathway and is homologous with Yap. Hippo signaling pathway can manage organ size through regulating cell proliferation and apoptosis. Research has confirmed that Yap/Taz played an important role in regulating organ size and stem cells self-renewal, proliferation and differentiation. There are relevant reports about Yap in the development of early tooth germ and mouse incisor. However, the research in rat molar remains elusive. In addition, it has not been reported that Taz as a homologous of Yap was involved in tooth development. We hypothesized that Yap/Taz may be involved in the process of molar development. The development of cranio maxillofacial bone and tooth are derived from the neural crest and the gene expression may have some similarities. The jaw is one of the important anatomic structures in craniofacial bone. TAZ can interact with Runx2, and then enhance the transcriptional activity of Runx2. What’s more, it can promote the differentiation of ostoblasts and the formation of bone tissue. What we speculated that Taz and Runx2 may take part in the jaw repair and regeneration. This study was divided into two parts and we used Sprague Dawley rats as the experimental animals and detected the spatial-temporal expression and the relative expression level of Yap/Taz through immunohistochemistry and qRTPCR in the first part. The second part, we constructed model of jaw tissue defect and tested the expression of Taz and Runx2 through immunohistochemistry and Western blotting. Our study may provide corresponding experimental basis for researching the role of Yap/Taz in tooth development and jaw tissue regeneration.Part I Expression and localization of Yap and Taz during the development of mandibular first molar in ratsObjective:The purpose of this study was to observe the spatial-temporal expression pattern of Yap/Taz through establishing the experimental animal model of tooth development in Sprague Dawley rats.Methods:10w Sprague Dawley rats (male and female with1:1) were mated to obtain tissue samples of embryonic and postnatal rats. Noon of the day on which the vaginal plug was found was regarded as embryonic day 0.5 (E0.5). Tissue samples include embryonic rat heads (E14.5, E16.5, E18.5) and postnatal rat mandibles from new birth to postnatal 4 weeks (PNO, PN3, PN7, PN14, PN21, PN28). All samples were treated with conventional fixed, decalcified, dehydration and paraffin embedded. Hematoxylin-eosin staining (HE staining) was used to observe the development morphology of mandibular first molar in rats and immunohistochemistry was used to observe the spatial-temporal expression and localization of Yap/Taz. In addition, qRTPCR was used to validate the relative expression of Yap/Taz gene at PNO, PN4, PN7 and PN10.Results:(1) The results of immunohistochemistry staining:Yap/Taz were found in oral epithelium and mesenchyme at E14.5 and E16.5.At E18.5, Yap/Taz was detected in dental papilla and whole enamel organ. At PNO, PN3 and PN7, positive expression existed in ameloblasts, odontoblasts and stratum intermedium. Meanwhlie, Yap/Taz were expressed in Hertwig’s epithelial root sheath (HERS) distinctly at PN7. At PN3, PN7 and PN14, Yap was detected in enamel matrix. From PN21 to PN28, parts of odontoblast which owned secretory function still remained Yap/Taz expression weakly during this period. However, Taz was highly detected in endothelial cells of blood vessels, periodontal ligament and alveolar bone.(2) The results of qRTPCR:To assess the Yap/Taz gene expression during the development of mandibular first molar in rats, we performed qRTPCR analyses in tooth germ at PNO, PN4, PN7 and PN10. The result of qRTPCR is that the expression of Yap/Taz in tooth germ of PNO was low. The level of Yap/Taz expression at PN4 and PN7 increased significantly than PN10. The level of Yap/Taz expression at PN4 was similar to PN7 and they were higher than PN0 and PN10.Conclusion:(1) Yap/Taz appeared spatial-temporal expression during the development of mandibular first molar in rats.(2) Yap/Taz might be involved in differentiation of ameloblasts and odontoblasts, enamel mineralization, crown morphogenesis, root formation and periodontium development.Part Ⅱ Preliminary study of Taz during the process of jaw regeneration in rats Objective:The purpose of this study was to construct the experimental animal model of jaw tissue damage in Sprague Dawley rats and to explore the role of Taz in the healing process of jaw tissues in rats.Methods:We pruchased 20 male Sprague Dawley rats (8 weeks) from Laboratory Animal Center of Shandong University. We constructed jaw defect model on the first and second molar buccal of mandible in rats. Samples were obtained from 3 day,7 day,14 day and 21 day after operation respectively. The expression and localization of Taz and Runx2 during the process of tissue wound healing was observed through immunohistochemical staining. Western blotting was used to detect the protein expression level during the process of damage healing.Results:(1)The results of immunohistochemistry staining:On the third days of jaw tissue defect healing, immunohistochemical staining showed the positive expression of Taz and Runx2 in fibroblasts attached to biological membranes and positive staining in the muscle fiber was obvious. In addition, the endothelial cells of blood in alveolar bone was Taz positive expression. The seventh days in tissue healing, the expression of Taz and Runx2 in endothelial cells and fibroblasts was positive. On the fourteenth days of healing, bone cell in the. new bone was not mature, irregular arrangement and lack of canalicular system. What’s more, the collagen fibers were disordered. On the 21st day of the healing tissue, bone tissue area which was newly formed was significantly larger than before, and bone mineral density was increase. Taz and Runx2 expression were detected in endothelial cells of blood and osteoid tissue. In addition, the positive expression of Taz was detected in bone lacuna.(2) Western blotting results:Taz and Runx2 were expressed during the wound healing of jaw. With GAPDH as an internal reference, TAZ and Runx2 were standardized analysis. The expression of Taz and Runx2 were gradually increased during the wound healing.Conclusions:(1) Taz and Runx2 were specific expression during the wound healing of jaw in rats.(2) Taz and Runx2 might play an important role in the regeneration of jaw in rats.