The Effects Of Conditional Deletion Of Sox10Using Wntl-cre On The Development Of Spiral Ganglion In The Inner Ear Of Mouse And The Possible Mechanisms Underlying Such Actions

Part lSox10but not MMP-24regulates migration of spiral ganglion neurons in the early embros of mouseObjectives/Hypothesis:The development of the mammalian auditory system is affected by the internal genes and external environment whose abnormality will cause the congenital deafness. The structures in the inner ear may be from the different stem cells migrating and differentiating at different time points, and then connecting together into a functional organ and delivering auditory information. Unlike most neural crest derived sensory neurons, the spiral ganglion neurons of inner ear are of placodal origin and the glial cells are neural crest derivatives. In mouse embros, a group of neuronal precursors become postmitotic and migrate across the basilar membrane otic sensory epithelial of cochlear duct to the spiral ganglion location and then send fibers to innervate hair cells in the organ of Corti. However, little is known about the exact molecular mechanisms that regulate the precisely organized development of spiral ganglion neurons (SGNs). It has been certified that Sox10is crucial to the survival of neural crest derivatives. The absence of Sox10can cause Waarndenburg syndrome characterized by sensorineural hearing loss and other symptoms. It has been reported that metalloproteinase (MMPs) are involved in the proteolytic remodeling of extracellular matrix (ECM), metastasis of tumors, the development of embryo as well as the regeneration of nerves. However, there has bee still no information about the function of MMP-24during the development of spiral ganglion in the inner ear and the effects of Sox10deletion on the expression pattern of MMP-24to date.Methods:The MMP-24-/-, Wnt1-cre; Sox10f/f and the corresponding control littermate mice were genotyped. The mice were perfused with4%paraformaldehyde and dissected. The MMP-24and Sox10RNA probe were made to do in situ hybridization for the whole tissue. The tissue was embedded in the plastic block and cutted. Lipophilic dyes were applied to trace the peripheral and central fibers of spiral ganglion neurons.Results:Here we used in situ hybridization to show the expression of MMP-24mRNA in the delaminating of eighth sensory neurons in the early stages of embros. Using MMP-24as a marker of neurons and Sox10as the marker of neural crest derivates, we found eliminating neural crest derived cells without loss of otic placode derived cells in Sox10deficient mice. Moreover, we demonstrated that the absence of glial cells does not largely affect the survival but contribute to aberrant migration of spiral ganglion neurons (SGNs). Using lipophilic dye tracing for efferent nerve of spiral ganglia and its central projection in the hindbrain, we found no difference between the MMP-24mutant and control mice at P1.Conclusions:The finding suggests that although MMP-24can be used as a marker for early delaminating neurons, it is dispensable for the development of SGNs in the early stages. There may have other genes that can make compensation to the functions of MMP-24. Sox10plays an important role in the neural crest derivatives, which may provide stop signal to the migration of spiral ganglion neurons. Part IITargeted deletion of Sox10by Wnt1-cre defects neuronal migration and projection in the mouse inner earObjectives/Hypothesis:Spiral ganglion neurons and most structures in the inner ear are originated from otic placode and the Schwann cells around the nerve fibers are neural crest derivatives. It was reported that in the tyrosine kinase receptor ErbB2knockout mice, abnormal migration of spiral ganglion neurons were found. There were still innervations to the peripheral and central regions although the nerve fibers grew messy and sparse. But, ErbB2gene is not only expressed in the Schwann cells but also in the supporting cells in the inner ear, so it can not be sorted out which type of cells provides the guidance function to the neurons and nerve fibers. In the previous study, we used the neural crest specific Wnt1-cre to knock out the transcription factor Sox10in order to eliminate Sox10gene in the neural crest derived cells, without affecting the otic placode derived structures such as sensory epithelial cells in which Sox10is positive. We found out a new model to discover the function of Schwann cells to the development of spiral ganglion.Methods:The Wnt1-cre; Sox10f/f and the corresponding control littermate mice were genotyped. The mice were perfused with4%paraformaldehyde and the cochlea was dissected. P75NTR and NT-3RNA probe were used to do in situ hybridization for the whole cochlea. The tissue was embedded in the plastic block and sliced. The sections were treated by the Stevenel’s blue staining. Immunochemistry was done by using various antibodies:MyoⅧ labeling hair cells, Neurofilament labeling efferent fibers, Tubulin labeling afferent fibers,Caspase3and PSVue labeling apoptosis cells, Hoechst labeling cell nucleus. Lipophilic dye was applied to trace the peripheral and central fibers of spiral ganglion neurons.Results:In the absence of Schwann cells, spiral ganglion neurons migrated into the center of the cochlea and even out of the ear toward the brain. In these five E18.5day old ears, we found that spiral ganglia in the middle turn was about4times further away (green lines in Fig.2A’, B’) from the medial wall of the cochlear duct in Sox10mutants (Mean=125μm; St.Dev=28; N=5) compared to control littermates (Mean=34μm; St.Dev=14; N=5). This indicates that absence of Schwann cells causes an approximately3-4times additional migration of spiral ganglion neurons. Spiral ganglion neuron afferent processes reached the organ of Corti, but many afferent fibers bypass the organ of Corti to enter the lateral wall of the cochlea. In contrast to this peripheral disorganization, the central projection to cochlear nuclei was normal. Compared to ErbB2mutants, conditional Sox10mutants had limited cell death in spiral ganglion neurons, indicating that the absence of Schwann cells alone contributed little to the embryonic survival of neurons.Conclusions:These data suggests that neural crest-derived cells are dispensable for all central and some peripheral targeting of inner ear neurons. The loss of Schwann cells can not directly induce the death of spiral ganglion neurons. However, Schwann cells provide a stop signal for migratory spiral ganglion neurons and facilitate proper targeting of the organ of Corti by spiral ganglion afferents. Sox10may be crucial to the differentiation and survival of Schwann cells by regulating the expression of ErbB2.