The Roles Of ClC-7 During Mouse Tooth Development

Teeth are vertebrate-specific structures which develop through a series of reciprocal interactions between two adjacent tissues : Epithelium and mesenchyme. Odontogenesis involves three fundamental processes: first, positional information is provided and interpreted to initiate organ formation at the right place (initiation); second, cells build up an organ rudiment (morphogenesis); finally, cells form organ-specific structures (differentiation). A number of transcription factors, signaling molecules, growth factor receptors, signaling molecules and calciotropic hormones regulate these processes.Ion channels are ubiquitous transmembrane proteins that confer selective ionic permeability to cell surface and intracellular membranes in virtually every cell in every known organism. Recent studies demonstrated that several chloride channels in tooth can directly or indirectly influence calcium phosphorus metabolism. Furthermore, a few Cl- channels deficient such as CFTR and ClC-5 showed abnormal tooth structure and tooth growth. ClC-7, a member of the ClC family of voltage-gated chloride channels, is a late endosomal/lysosomal Cl channel that is expressed ubiquitously, expecially highly in osteoclasts where it can be inserted into the ruffled bord.Clcn7 deficient mice showed severe osteopetrosis,retinal degeneration and no teeth eruption. Recently there are increasing reports about the physiological function of Cl- channels and ClC-7 in many organs. But it is not clear about the expression pattern of ClC-7 in tooth tissue and how ClC-7 affects tooth development. Therefore, we conducted the following experiments to examine the expression of ClC-7 in teeth and to find out the possible functions of ClC-7 during the tooth development. The present study consists of three parts.1. Expression of ClC-7during mouse tooth developmentIn this part we first confirmed the expression of ClC-7 in mouse tooth germ by reverse transcription-polymerase chain reaction(RT-PCR) and then detected the temporal-spatial expression patterns of ClC-7 in mouse tooth development. Clcn7 mRNA was observed in the tooth germ of new born mouse. With the analysis of immunohistochemistry and in situ hybridization(ISH), we found that ClC-7 is ubiquitously expressed in the bell stage of mouse tooth, ClC-7 located in outer enamel epithelium cells, inner enamel epithelium cells, stellate reticulum cells, stratum intermedium cells, ameloblasts and odontoblasts.2. Expression of ClC-7 in functional cells of toothWe confirmed that both MDPC-23 and LS8 expressed Clcn7 mRNA by RT-PCR methods. Immunohistochemical showed that ClC-7 protein was expressed in both MDPC-23 and LS8 cell line, and strong immunoreactivity was observed in the odontoblast-like cell line MDPC-23. These results provide us a good research basis for ClC-7 function in tooth germ.3. Study of ClC-7 in dental papilla cellDental papillas from new born mice were dissected and dental papilla cells were primarily cultured in vitro. The expression of mRNA and protein of ClC-7 in dental papilla cell by immunohistochemistry and in situ hybridization.Stimulation factors M-CSF and RANKL were added to the culture medium in vitro.After 48 hours, Clcn7 mRNA and ClC-7 protein were analyzed and compared with the control. Clcn7 mRNA was inceased after the stimulation of M-CSF and RANKL.In conclusion, ClC-7 has a spatial and temporal distribution during mouse tooth development and might contribute to the mouse tooth development; Clcn7 mRNA was inceased after the stimulation of M-CSF and RANKL, indicating that ClC-7 may play a role during dental papilla cells differentiation.