Role Of Smad4 Gene In Vestibular Development Of Inner Ear In Mice

It is well known that mouse is the most suitable species used to study morphogenesis and cell differentiation and hereditary disease in mammalian inner ear. Vestibule, an important part of inner ear, has the same origin as cochlea and earlier development than cochlea. Mice have mature vestibule at birth. During development of vestibule in mammalian inner ear, there are more than eighty genes differential co-expressions in precisely time and spaces, which decides the fate of sense organs and cells. BMP4, member of TGF-β superfamily, and Smad4, downstream regulation gene of BMP4, may play a key role in development of inner ear, but it is unknown what is the role and what is the mechanism of action.Gene knockout mice are often used to study genic functions. According to animal experiment, Smad4 knockout homozygote mouse can't survive in earlier period embryo, and hearing loss and inner ear defects is obvious in heterozygote. Nevertheless, Haploinsufficiency exists in heterozygote. So, Smad4 conditional knockout mice were generated using the Cre-loxP approach and it were expected to reveal the role of Smad4 in vestibular development.To clarify the role of Smad4 in vestibular development, we investigated as follows: 1.Changes of balance functions in Smad4 conditional knockout mice (including wild type Smad4+/+, heterozygote Smad4+/- and homozygote Smad4-/-, so as below), 2.Pathological and morphologic changes of periphery vestibular organs in inner ear of Smad4 conditional knockout mice, 3.Morphological development process of periphery vestibular organs in C57BL/6 mice, 4. Expression patterns of BMP4/Smad4 in vestibule of C57BL/6 mice on different development stage.Part One: Evaluation of vestibular functions in Smad4 conditional knockout miceVestibular functions in Smad4 conditional knockout mice were evaluated through series tests which consist of general behavioral observation, air righting reflex, swimming test, VOR and VEMP. Our findings are as follows: behaviors, gaits, air righting reflex and swimming test in Smad4-/- mice were normal, and all the three genotypes mice can be induced identical nystagmus through rotation. Latency of VEMP had no difference among the three genotypes mice but the response threshold of VEMP in Smad4-/- mice were obviously elevated. These results indicated that only partial functions of saccule were damaged to some extent in Smad4 conditional knockout mice, and other vestibular endorgans had normal functions.Part Two: Pathological and morphologic changes of periphery vestibular organs in inner ear of Smad4 conditional knockout micePathological and morphologic changes of periphery vestibular organs in inner ear of Smad4 conditional knockout mice were investigated through frozen sections, semithin sections, ultrathin sections, toluidine blue stain, HE stain, immunohistochemistry, immunofluorescence, Southern blot, Confocal microscopy, scanning electron microscope and transmission electron microscope. Our findings are as follows: 1. By southern blot and immunohistochemistry we find that Smad4 were efficiently knocked out in periotic vestibular cartilage capsule but not in vestibular sensory epithelium. 2. Body and internal auditory volume of Smad4-/-mice were obviously smaller than that of Smad4+/+ or Smad4+/- mice, and so did semicircular canal and crista ampullaris, utricle and macula utriculi, saccule and macula sacculi, endolymphatic duct and sac in the same manner. Outlines of vestibulum auris internae in Smad4-/- were normal but morphous had local malformation, and Semicircular canals in Smad4-/-, without morphous malformation, had different track compared with in Smad4+/+ or Smad4+/-. 3. Crista neglecta in posterior ampulla canalis semicircularis of Smad4-/- was obviously enlargement and distance from crista neglecta to crista ampullaris was shorten. The vacuole-like defects occasionally appeared inside the sensoryepithelium of posterior ampulla canalis semicircularis of Smad4-/-. Studying at scanning electron microscope and transmission electron microscope levels showed that no pathological changes were observed in all three genotype mice. Anti-filament immunofluorescence indicated the distribution of primary vestibular neuron endings in vestibular sensory epithelium and the contacts between it and hair cells were normal.These results indicated that Smad4 conditional knockout mice were generated successfully, whose morphological and pathological changes in vestibulum auris internae were slightly.Part Three: Morphological development process of periphery vestibular organs in C57BL/6 mice inner ear and expression patterns of BMP4/Smad4 in vestibule on different development stageMorphological development process of periphery vestibular organs in C57BL/6 mice inner ear and expression patterns of BMP4/Smad4 in vestibule on different development stage were investigated through frozen serial sections, HE stain, immunohistochemistry and immunofluorescence. Our findings are as follows: 1. Mice vestibular development initiated early and the process is short. All macula and crista ampullaris were formed at E15, which Myosin VI was expressed in hair cells at this time but not expressed in support cells. 2. There was BMP4 expression at E10 in epithelium of otic capsule. Later, BMP4 expressions were affluent in condensed mesenchyme, vestibular cartilage capsule and sense epithelium. 3. The expression pattern of Smad4 in vestibule is different from BMP4. It was not conspicuous until E15 that Smad4 expressed in macula and crista ampullaris and until E16 in periotic cartilage capsule.These results indicate that E15 is the critical period during generation of vestibular hair cells, and BMP4 is involved in vestibular morphogenesis, hair cell differentiation and function acquiring. The expression temporal spatial pattern of Smad4 in vestibule during each development stage is different from BMP4.The above results imply that Smad4 may don't play a key role during earlystage of vestibular development in mice inner ear, but it may play a role in morphous modification and functional development during late stage of vestibular development. More experiments are required to determine whether Smad4 affects the development of vestibular sense epithelium and whether there exists a Smad-independent pathway for transduction of BMP signals in inner ear.