Roles And Mechanisms Of NELL1during The Regression Of Meckel’s Cartilage In Mice

Meckel’s cartilage, a hyaline cartilage derived from the first branchial arch, provides mechanical strength for the embryonic mandibular development and degenerates almost immediately after birth. The degeneration of extracellular matrices and the cell death of chondrocytes occur during the regression of Meckel’s cartilage; however, the mechanism remains unclear. NELL1is preferentially expressed in craniofacial tissues which are originated from neural crest. It is involved in the proliferation and differentiation of chondrocytes. However, roles and mechanisms of NELL1on chondrocytes are still controversial. According to detect the expression of NELL1and other factors related with NELL1, and construct adenovirus-mediated NELL1to transfect primary chondrocytes, this study aimed to explore roles and possible mechanisms of NELL1during the regression of Meckel’s cartilage. In addition, we detect the expression of autophagy and apoptosis-related markers during the regression of Meckel’s cartilage, to probe the possible mechanism of chondrocyte cell death during the regression process.Part Ⅰ:Role of NELL1during the regression of Meckel’s cartilageNELL1plays an important role during craniofacial skeletal development and regulates the proliferation and differentiation of chondrocytes. However, the role of NELL1during the regression of Meckel’s cartilage has not been reported. Objective: To detect the expression of NELL1and other factors related with NELL1during the regression of Meckel’s cartilage, to explore possible roles and mechanisms on chondrocytes during this process. Methods:In mice of embryonic day from15.5to17.5, we applied Alizarin red S and Alcian blue staining and Toluidine blue staining to detect histological changes of Meckel’s cartilage, applied immunohistochemistry or Realtime PCR to detect the expression of Col2, Co110, BMP2, RUNX2, NELL1, NFATc1, ERK, MMP9, MMP13in Meckel’s cartilage. Results:Alizarin red S and Alcian blue staining and Toluidine blue staining showed that Meckel’s cartilage was a rod-shaped cartilage, consisting of mature chondrocytes, without degeneration of extracellular matrices in E15.5. In E16.5, chondrocytes in the central portion of Meckel’s cartilage became hypertrophic, and some extracellular matrices started to degenerate. In E17.5, the degeneration progressed anteriorly and posteriorly, some terminal hypertrophic chondrocytes died and disappeared, and the central portion of Meckel’s cartilage became discontinuous. The expressions of BMP2, NELL1,NFATc1, ERK, MMP9were mainly observed in hypertrophic chondrocytes from E16.5. The mRNA expression of Col2decreased, and the mRNA expressions of Col10. NELL1, NFATc1, MMP9. MMP13increased significantly from E15.5toE17.5. There was no significant change in the expression of ERK. Conclusions:The degeneration in the central portion of Meckel’s cartilage is initiated from E16.5. NELL1may be involved in the degeneration of extracellular matrices of Meckel’s cartilage by regulating the expressions of MMP9and MMP13through the upregulation of NFATcl or ERK pathway.Part Ⅱ:Effects of adenovirus-mediated NELLl on chondrocytesEffects of NELL1on chondrocytes are quite controversial. The first part of this study demonstrated that NELL1was expressed in hypertrophic chondrocytes during the regression of Meckel’s cartilage; however, the mechanism remains unclear.Objective:To construct adenovirus-mediated NELL1overexpression vector, transfect primary chondrocytes, and detect the effect and mechanism of NELL1on chondrocytes. Methods:We constructed adenovirus-mediated NELL1, cultured primary chondrocytes transfected with adenovirus-mediated NELL1, and then applied Western blotting or Realtime PCR to detect the expressions of NFATcl, ERK, MMPs and TIMPs. Results:Adenovirus-mediated NELL1was successfully constructed. After the transfection with adenovirus-mediated NELL1on primary chondrocytes, the mRNA expressions of NFATc1, ERK, MMP9, and MMP13and the protein expression of ERK and MMP9increased significantly with dose-response relationship. In addition, TIMP3and TIMP4decreased significantly. Conclusions:NELL1regulates the expression of MMPs by upregulating the expression of NFATc1or ERK, and may be involved in the degeneration of extracellular matrices. Part Ⅲ:Role of autophagy during the regression of Meckel’s cartilageAlthough molecular mechanisms between the regression of Meckel’s cartilage and endochondral ossification are not the same, histological features between them are very similar. The hypertrophy of chondrocytes, the degeneration of extracellular matrices, and the cell death of hypertrophic chondrocytes can be observed in both processes. Autophagy is involved in the cell death of terminally differentiated chondrocytes during endochondral ossification, however, whether autophagy is involved in the regression of Meckel’s cartilage remains unclear. Objective:To explore the role of autophagy and apoptosis in chondrocyte cell death during the degeneration of Meckel’s cartilage according to detect the expression of autophagy and apoptosis-related factors. Methods:We applied immunohistochemistry or Realtime PCR to detect the expressions of Beclinl, LC3, Caspase3and Bcl2during the regression of Meckel’s cartilage in embryonic mice from E15.5to E17.5, and applied TUNEL assay to detect DNA fragments in chondrocytes. Results:Beclinl and LC3were mainly observed in hypertrophic chondrocytes and terminally differentiated chondrocytes. Beclinl and LC3mRNA expressions increased significantly from E15.5to E17.5. TUNEL and Caspase3positive signals were mainly expressed in the lateral part of terminal hypertrophic chondrocytes, along the degeneration area of Meckel’s cartilage, instead of in chondrocytes of Meckel’s cartilage. In addition, Bc12expression increased significantly from E15.5to E17.5. Conclusions:These results indicate that autophagy is involved in hypertrophic chondrocytes during the regression of Meckel’s cartilage and occurs prior to chondrocyte cell death during this process. Apoptosis is not involved in the chondrocyte cell death.