Our major accomplishments are in the following areas: 1) Specification of vestibular and auditory neuronal fates in the mouse inner ear (manuscript published) The vestibular and auditory neurons, which innervate the inner ear, originate from the otic epithelium at an early stage of embryogensis. The molecular mechanisms that specify these two types of neurons are not known. From the analyses of the mouse mutant, dreher, in which the Lmx1a gene is mutated, we showed that Lmx1a is important for specifying the auditory and vestibular neural fates. Based on gene expression analyses, we showed that the normal boundary that separates the vestibular and auditory neurogenic regions is likely to be disrupted in dreher inner ears. Consequently, there is an increase in the size of the vestibular ganglion and no obvious change in the auditory ganglion in these mutants. This change in neuronal fate could also be related to the vestibular-like hair cells observed in the proximal region of the cochlear duct in dreher. Additionally, the lack of Lmx1a also affects the normal boundaries between sensory and non-sensory tissues in these mutants. Thus Lmx1a functions to maintain boundaries among neural, sensory and non-sensory fates in the inner ear. 2) Sensory organ separation in the mouse inner ear: analyses of Foxg1-/- mouse mutants (manuscript accepted) The anterior and lateral cristae of the mouse inner ear are thought to arise from a common prosensory region, which splits to form two separate cristae. In Foxg1-/- inner ears, there is only a single sensory crista in the anterior region, and it is not clear whether this sensory organ represents the anterior crista only or a fused anterior and lateral cristae that fail to separate. Using a genetic fate mapping strategy, we showed that while some lateral cristae fail to develop in Foxg1-/- mutants, many of the single crista represents a fusion of the anterior and lateral cristae. Our results also showed that most of the cristae in Foxg1 mutants including the posterior crista, are malformed with a defective septum cruciatum, which is a non-sensory region bisecting the anterior or posterior crista into two equal halves normally. These combined results indicate that Foxg1 normally functions to maintain sensory and non-sensory domains within the sensory crista. 3) Differential contributions from the hindbrain and mesenchyme to inner ear morphogenesis (manuscript submitted) Using microsurgical ablations and tissue transplantation techniques in ovo, we showed that ectoderm/mesenchyme adjacent to the developing inner ear are required for shaping the vestibular canals and ampullae. In contrast, the rhombomeres adjacent to the developing inner ear are important for the proper growth and morphogenesis of the cochlear duct. These results provide a basis for future studies in identifying the signals from these tissues, which pattern the inner ear. 4) Role of Bone morphogenetic proteins in cochlear hair cell formation: analyses of Noggin-/- and Bmp2 conditional null mutants (manuscript in preparation) The organ of Corti of the mammalian cochlear duct is a highly organized structure consisting of one row of inner hair cells and three rows of outer hair cells, as well as several specialized supporting cell types such as pillar cells and Deiters cells. Many genetic pathways are involved in the formation of the organ of Corti, which develops from a prosensory domain within the cochlear duct. We showed that Noggin, an antagonist of Bone morphogenetic proteins (Bmp) is required for sensory hair cell formation in the organ of Corti. In Noggin-/- cochleae, there is an increase in the numbers of inner and outer hair cells, suggesting that proper levels of Bmps are normally required for organ of Corti formation. We showed that Bmp2 is transiently expressed in the nascent sensory hair cells within the organ of Corti. To address the potential role of Bmp2 in regulating hair cell number, we deleted Bmp2 in the prosensory domain using two cre strains, Foxg1-cre and Gfi1-cre. Our results indicate that Bmp2 expression in the nascent hair cells is not required for normal hearing and hair cell formation. The combined results of Noggin null embryos and Bmp2 conditional mutants suggest that Bmp4 expression adjacent to the prosensory domain is more important for hair cell formation in the mammalian cochlea.
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