| TGF-β superfamily is composed of TGF-β, ACTIVIN and BMP. They play a very important role in embryonic development, organofaction, regulating cell growth, differentiation, apoptosis. TGF-β receptor and Smads proteins take part in their signal transductions. Co-mediator Smads (Smad4) functions in TGF-β signaling pathways. Receptor-regulated Smads form heterodimers with Smad4, and then translocate into the nucleus to induce or repress the expression of TGF-β target genes. Smad4 inactivation could influence TGF-β signaling pathway. To explore the role of Smad4 in the inner ear development, chondrocyte specific Smad4 conditional knockout mice were made by Cre-LoxP system bypassing the hindrance of embryonic death in Smad4 gene knockout mice. In this study we explored the roles of Smad4 in the inner ear development by different methods of hearing genotype and inner ear morphology.Part one: Evaluation of mouse model and cochlear expression of Smad4 in miceWe analyzed the expression pattern of Cre recombinase in the Col2a1-Cre transgenic mouse, utilizing the ROSA26 Cre reporter mouse strain. LacZ staining was performed in the Col2a1-Cre and ROSA26 doubly transgenic mice. In the skeleton of the neonatal mouse positive for LacZ staining, the cartilages locating at the temporal bones were stained blue. All these data showed that the Col2a1-Cre transgenic mice we generated could be used to achieve Cre-mediated recombination in chondrocytes. By Southern blot, it confirmed that the exon 8 of the Smad4 was deleted effectively in cartilage tissues by Cre-mediated recombination. These results indicated that the Smad4 gene has been disrupted efficiently in chondrocytes. Itshowed there were strong expression in chondrocytes of Smad4+/+ mice while there were no fluorescence detected in chondrocytes of Smad4-/- mice by immunohistochemistry fluorescence. All these demonstrated the mouse model has been successfully established.To study whether Smad4 gene exists in mice cochlea or not and detect how the gene expresses and distributes in cochlea. By immunohistochemistry we indicated Smad4 gene expressed throughout cochlea of three genotypes. Positions of expression concentrated on stria vascularis and hair cells. Expression in stria vascularis and organs of corti were strongest in all structures. The data confirmed that Smad4 gene conditional knockout in chondrocytes does not influence this gene expression in inner ear sensory epithelium.Part two: Evaluation of hearing function in mouse modelTo explore the role of Smad4 in inner ear development, we learn about the hearing phenotype of mouse model after Smad4 knockout in chondrocytes. It helps to explore the role of Smad4 in inner ear development to learn about the hearing phenotype of Smad4 knockout model firstly. In this section we used auditory brainstem response (ABR), cochlear microphonic (CM), compound action potential (CAP) to evaluate mice hearing function. We found that Smad4-/- mice had severe or profound severe hearing loss after conditional knockout Smad4 gene in chondrocytes by chance. In the ABR examination, none of the Smad4-/- mice could been induced recognizable waveforms at 120 dB SPL sound pressures in given conditions (click,4kHz, 8kHz, 16kHz,32,kHz). But Smad4+/+ and Smad4+/- mice could been induced recognizable and reduplicative waveforms. Significant difference between smad4-/- and the other two genotypes (P<0.01) was found after determining ABR threshold and analyzing the data. The results from CAP were similar with those of ABR. Smad4-/- mice could not been induced any waveforms at highest sound pressure in given condition (0.5Hz, 1kHz, 2kHz,4kHz, 8kHz, 16kHz,32kHz). By CM, It showed that non-linear tendency with increasing sound pressures in Smad4+/+ and Smad4+/- mice while it showed linear tendency in Smad4-/- mice. All these indicated that Smad4-/- mice had severe sensorineural hearing loss after Smad4 gene conditional knockout in chondrocytes. This mouse is one kind of Smad4 gene mutant deafness mouse model with stable phenotype.Part Three: Evaluation of cochlea morphology in mouse modelWe had to make some experiment to know what pathological changes causes these hearing phenctypes after learning about the information of hearing function. The newborn Smad4-/- mice were smaller than Smad4+/+ and Smad4+/- littermates. The inner ears dissected from Smad4-/- mice were smaller than those of Smad4+/- and Smad4+/+ littermates. At the same time the osseous cochlear of Smad4-/- mice showed obvious malformations, with introcession on the surface of osseous cochlea apex whereas the osseous cochlea of Smad4+/- and Smad4+/+ mice showed normal size and morphology. Comparing to Smad4+/+ mice, Auditory ossicles dissected from Smad4-/- and Smad4+/- mice showed obvious malformation of incus. Semithin sections showed that disparities between Smad4+/+ and Smad4-/- in gross appearance of cochlea and osseous spiral lamina. The angle in consecutive osseous spiral lamina in Smad4-/- mice was different with the other genotype mice. There was statistic significant between Smad4-/- and the other genotype mice (p<0.05). Smad4+/+ and Smad4+/- mice showed normal morphology of inner and outer hair cells without obvious cell absence. In Smad4-/- mice, we observed punctiform absence in inner and outer hair cells occasionally. No obvious morphological disparities could be observed in tectorial membrane and spiral ligament between three genotype mice. Surprisingly we observed that spiral prominence in Smad4-/- mice were remarkable swelling comparing with the other two genotype mice. Spiral ganglion cells in three genotypes were observed no significant difference in numbers of spiral ganglion cells (P>0.05) by serial sections counting.In scanning electron microscope examination, it was found that malformations of osseous spiral lamina and morphologic abnormality of basal membrane in the samples from smad4 -/- mice. The morphologic abnormality of basal membrane showed abnormal curve comparing with the other two genotype then it cound not provide a perfect view to observe stereocilia of outer and inner hair cells. Smad4+/+ and smad4+/- mice showed normal appearance and well developed stereocilia of outer and inner hair cells. Only in the apex could we observe the stereocilia of outer hair cells in smad4 -/- mice. It showed that the stereocilia of outer hair cells were in poor development and in disorganization, then rotation of stereocilia of outer hair cells could also been seen.Based on data from SEM, we couldn't assess the comprehensive informations about stereocila of outer and inner hair cells because of the malformation of osseous spiral lamina. To bypass this hindrance, we made the whole cochlear tissue preparation and double stained with hoechst and phalloidin. Surface preparation showed that Smad4 +/+ and Smad4+/- mice had few absence in inner and outer hair cells and Smad4-/- mice had also a few scattered absence in inner and outer hair cells. Phalloidin stain could show stereocilia's appearance and organization. In smad4+/+ and smad4+/- mice, stereocilia were in normal shapes and cells were in normal organization while those in smad4-/- mice were in unhealthy shapes and in irregular organization at all turns. Surface preparation stained with Hoechst could show nucleus of hair cell, so we counted and compared the numbers of inner and outer hair cells between three genotypes of mice. It showed there was no statistical significance in the numbers of inner and outer hair cells between three genotypes of mice (P>0.05).The whole cochlea tissue preparation stained with MyosinVI and Neurofilamen antibody could demonstrated hair cell body, nerve synapsis and nerve plexus adhering to synapsis. Inner hair cells in Smad4-/- mice showed irregular and different morphology, in irregular organization. The nerve synapsis had intumescentiae and were in abnormal organization, nerve fibers adhering to synapsis diminished and even disappeared. Oppositely, Smad4+/+ and Smad4+/- mice were in normal condition.The results from transmission electron microscope showed organelle in outer hair cells had normal appearance and numbers, the efferent and afferent never endings adhered to outer hair cells in Smad4+/+ and Smad4+/- mice seemed larger than those in Smad4-/- mice, at the same time the numbers of the efferent and afferent never synapsis adhered to outer hair cells in Smad4+/+ and Smad4+/- were much more than those in Smad4-/- mice. Comparing to Smad4+/+ and Smad4+/- mice, the afferent nerve synapsis had obvious vacuolization in Smad4-/- mice, the boundary between inner hair cells and the afferent nerve synapsis was in unsharpness, the number of nerve fibers reduced.All these evidence indicated that Smad4 is essential for inner ear development and maintaining normal hearing function. Smad4 is one kind of deafness gene and Smad4 mutant mice could suffer from sensorineural hearing loss. Chondrocyte specific Smad4 conditionalknockout in mice could lead to abnormal cochlear sensory cells development, this mouse model provided genetic evidence that there are the epithelial - mesenchymal tissue interactions in embryonic development, which control the differentiation and maturity of the cochlear sensory cells originating from sensory epithelium.
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