Signal Transducation Mechanism Of Smads In The Development Of Mouse Inner Ear

Hearing loss has a very high prevalence. Remarkable progress has been made in the cloning of deafness genes. These findings offer the promise to significantly expand our knowledge on the molecular mechanisms underlying the auditory and vestibular functions and on the pathophysiological mechanisms for hearing loss. But sometimes it is difficult to identify these genes because of lack clinical characteristics, genetic heterogeneity and small family constellation. In addition, some related genes are difficult to discover because there is no suitable family constellation which can be used for linkage analysis. The inner ear structure, development and pathology of auditory sensation shift of mouse are extremely similar with human being. Thus, the study of mouse deafness related genes offer another method to identify possible mankind deaf locus. Smad4 and Smad5 are signal transduction member of transforming growth factor-β superfamily, and mainly mediate BMP4 signal transduction. TGF-β superfamily members participate regulating the blastodermal development, organic formation, epithelial tissular accrementition, extracellular metrical synthesis, immune reaction and other biological process. It has been reported that BMP family members and their signal transduction molecule express in the inner ear of chicken, mouse, zebrafish, Xenopus. Our previous data show that Smad4, Smad5 gene knock-out can result in mouse serious hearing loss and the inner ear hearing organ including of hair cells, supporting cells, spiral ganglion damaged. So Smad4, Smad5 genes probably are auditory function correlation genes. But where, when and how they play roles has not been reported. Therefore, this study use frozen sections and HE staining to observe the morphological change of mouse cochlea and immunohistochemistry to determinethe expression of BMP4 and Smad4, Smad5 proteins in the development of mouse cochlea, and to investigate the signal transduction mechanism in the inner ear development.part one: the development of mouse cochleaObjective: To explore the developmental process of mouse inner ear and offer a choice for select suitable mouse model in studying inner ear development. Methods: The inner ears were obtained from C57BL/6 mice and Kunming mice, with ages ranging from gestational day 9 through postnatal day 14. Gestational age was determined by the vaginal plug technique considering the day for the vaginal plug as day one. Frozen 6 μ m sections for embryos were processed for HE staining to observe the morphological change of mouse cochlea. MyosinVI was used as Hair Cell Marker. Results: The inner ear developmental processes of the two strains mice are similar on the whole, while the developmental process of Kunming mouse was later one day than that of C57BL/6 mouse. For C57BL/6 mice, at the tenth day of gestation otocyst was a closed ovoid sac. The cochlear duct started to develop at the twelfth day of gestation as an extension of the ventral part of the otocyst. At the seventeenth day the inner hair cells and the outer hair cells were identified. Cochlea was maturely shaped at P1, but organ of Corti was matured at P10. Positive staining of MyosinVI immunofluorescence became obvious at E17. Conclusion : The development of the mouse inner ear followed the regular pattern, and hair cells shape at E17. The developmental process of Kunming mouse is later one day than C57BL/6 mouse.part two: signal transduction mechanism of Smad4, Smad5 in the development of mouse cochleaObjective: To explore the roles of BMP4, Smad4, Smad5 in the development process of mouse cochlea and investigate the possible mechanism. Methods: Embryos were isolated from timed mating of C57BL/6 mice. The morning that vaginal plugs were detected was designated E1. Frozen 6 μm sections for embryos were processed for HE staining to observe the morphological change of mouse cochlea and for immunohistochemistry to determine the expression of BMP4, Smad4, Smad5. Results: The expression of BMP4 and Smad5 were almost same. They were expressed wildly in cochlea throughout whole stage of embryo. From fifteenth day to seventeenth day their expression was concentrated in the part to be acoustic perceptive organ on the basal membrane. Later they were expressed in supporting cell, hair cell, spiral ganglion, vestibular membrane and epithelium of stria vascularis. While the expression of Smad4 was different from BMP4 and Smad5, it began expressed at E15. At first, it expressed in the parts to be cochlear axis, sensory cells and supporting cells. Smad4 expression was concentrated in supporting cell, hair cell, spiral ganglion and epithelium of stria vascularis during later stage. From E15, BMP4, Smad4 and Smad5 expressed at equal pace. Conclusion : BMP4, Smad4 and Smad5 might play important roles in the development of mouse inner ear, and they are necessary for inner ear development. After E14, there might be a signal pathway of BMP4 ligand to BMP receptor, and BMP receptor to Smad5 and Smad4 transcriptional activator. Before E14, BMP4 can be a key signal by Smad5 independent pathways or can operate in concert with other key factors unknown.