Clcs Chloride Channels Regulate Chicken Mandibular Development In Early Embryo Stage

During embryonic mandibular development, mesenchymal cell recruitment and migration, proliferation and condensation and undifferentiated mesenchymal cells differentiate to chondrocytes to bone, and secrete matrix.Complex regulatory and signaling networks involved in the early stage of mandibular development.Chloride channels (ClCs) are ubiquitously expressed in almost all eukaryotic cells and may play an important role in the regulation of intracellular pH, cell volume homeostasis, organic solute transport, and cell migration, proliferation, differentiation and apoptosis?The member potential of rabbit cultured articular chodrocytes was mainly determined by the voltage-dependent Cl- channels. Mutations of these genes result in genetic diseases with abnormal bone deformation and body size. Loss of ClC-7 leads to osteopetrosis in mice and humans. Clcn5 gene knock mice have an abnormal dorsal spine and backward growth of teeth. Clcn3 gene knockout mice exhibit a complex phenotype including poor growth and kyphosis. These data indicate that chloride channel genes can play an important role in mandibular growth. The relationship between ClCs and bone development led us to question whether ClCs may also contribute to chondrogenesis of mandible development .Little is known about members of the CLC family might be involved in the regulation of the process of mandibular formation. In terms of this context we designed and carried out the experiment below in order to investigated the effect of ClCs regulate proliferation and chondrocyte differentiation , to help to know the abnormal mandibular deformation, and find the new concept to cure the abnormal mandibular deformation.1. The histological study and the expression of ClCs in mandibular processesMandibular processes were isolated in different embryos stage and stained using haematoxylin and eosin (HE) or alcian blue .And we used RT-PCR to study expression of Voltage gated chloride channels(ClCs). We observed histological changes of mandibular processes in different embryos stage and the expression of ClCs in mandibular processes.2. The bioresearch study in chicken mandibular mesenchymal cellsMesenchymal cells were derived from Hamburger-Hamilton (HH) stage 26(4.5day) embryos mandibular processes by treated with a mixture of trypsin and pancreatin. We stimulated chicken mandibular mesenchymal cells (CMMC)proliferation and differentiation using ascorbic acid and?-glycerophosphate (AA-BGP). CMMC proliferation were analyzed using MTT. Micromass culture cells were staining with alcian blue to analyze cartilage matrix. We used real-timePCR to study expression of chondrocyte differentiation marker mRNA after treated with AA-BGP in micromass culture . We study the AA-BGP induced the CMMC proliferation and differentiation.3. The study of NPPB influence CMMCWe assessed the effects of the chloride channel inhibitor NPPB on ClCs in micromass culture cells using real-time-PCR. In AA-BGP and AA-BGP-free culture, NPPB inhibiting the growth of CMMC were analyzed using MTT. Micromass culture cells were staining with alcian blue to analyze cartilage matrix inhibited by NPPB. We used real-timePCR to study expression of chondrocyte differentiation marker mRNA after treated with NPPB in micromass culture. We study the NPPB induced the CMMC proliferation and differentiation.Result:1. At 26 stage embryos, the mandibular process consisted of undifferentiation mesenchyme covered by a simple cuboidal epithelium. At 32 stage, chondrocytes become mature and cartilage staining prevails. At 14 day, the beak shows generous bone growth . Beak shapes are similar to adult chickenn. At 17 day, most of the beak is bone. Only a few remain as permanent cartilages.To date, We first detected the expression of chicken CLCNmRNA in chicken mandibular using RT-PCR,but CLCNmRNA expression level is different.2. We performed RT-PCR to analyze mRNA expression of cartilage marker (type II collagen , aggrecan ,Sox9, type X collagen )found that they were expressed in CMMC. Using MTT and real-time-PCR,We found that AA-BGP as a stimulator can induce the CMMC proliferation (p<0.05) and differentiation. AA-BGP increased the expression of type II collagen, aggrecan and Sox9(p<0.01), except type X collagen(p>0.05). Alcian blue staining confirmed that AA-BGP can induce the increased cartilaginous matrix formation.3. Real-timePCR analysis of CLCN gene mRNA expression revealed that NPPB suppressed the expression levels of CLCN3, CLCN5, and CLCN7 mRNA compared to control (p < 0.05), whereas no significant changes in the expression of CLCN1, CLCN4, and CLCN6 compared to controls (p > 0.05). NPPB inhibited the growth of mandibular mesenchymal cells in AA-BGP and AA-BGP-free cultures (p < 0.05). NPPB treatment inhibited cartilage matrix formation. NPPB treatment inhibited the expression of type X collagen mRNA in AA-BGP and AA-BGP-free culture and inhibited the expression of type II collagen and aggrecan mRNA in AA-BGP culture (p < 0.05). NPPB treatment did not affect the expression of Sox9 mRNA in AA-BGP and AA-BGP-free cultures(p > 0.05).Conclution:1. During mandible development, the mesenchymal cells differentiate into chondroblasts and then membrane bones form to become a mandibular. Only a few remain as permanent cartilages. CLCN1 and CLCN3-7 mRNA were aboundently expressed in chicken CMMC.2. Cartilage marker (type II collagen, aggrecan,Sox9, type X collagen mRNA ) were expressed in CMMC, but expression level is different. AA-BGP increased the growth embryonic CMMC and cartilaginous matrix formation. We speculate that alterations in pH inside cell can contribute to cells proliferation and differentiation. AA-BGP increased the expression of type II collagen, aggrecan, except type X collagen. Sox9 is an regulator of type II collagen and aggrecan. AA-BGP also increased the expression of Sox9. We speculate AA may play an important role in cell proliferation and differentiation ,at least in part, by increasing the expression level of Sox9.3. NPPB was found to suppress CLCN3, CLCN5, and CLCN7 mRNA expression. NPPB inhibited the growth of embryonic CMMC and cartilaginous matrix formation. NPPB antagonized the effects of AA-BGP on the expression of these genes (except Sox9) in these cells. NPPB inhibited the expression of type X collagen mRNA but the levels of type II collagen, aggrecan, and Sox9 mRNA were not significantly altered by NPPB.We conclude that CLCN3, CLCN5, and CLCN7 plays a major role in the progression of chondrocyte differentiation into hypertrophic cartilage. Type X collage might be a sensitive target gene. Sox9 is not involved in the regulation of chondrogenesis by ClCs. CLCN3, CLCN5, and CLCN7 are nCl?/H+ exchange, and we speculate that alterations in pH inside cell can contribute to cells proliferation and differentiation.