This years major accomplishments are in the following areas: 1) Conserved role of Sonic Hedgehog in tonotopic organization of the avian basilar papilla and mammalian cochlea Mutations in Sonic Hedgehog (Shh) causes holoprosencephaly and cyclopia in humans. The cyclopia phenotype is attributed to the loss of Shh secreted by the ventral midline structures - floor plate and notochord - during embryogenesis. This same source of Shh has also been shown to be important for the ventral patterning of the inner ear. The cochlea is tonotopically organized such that the base of the cochlear duct is most sensitive to high frequency sounds and the apex to low frequencies. Many cellular and physiological features facilitated this property such as changes in the length of hair cells as well as the height and number of stereocilia of hair cells along the cochlear duct. The molecular mechanisms that give rise to these important cellular features are not clear. Here, we show that Shh signaling from the ventral midline induces differential expression of various genes along the apical-basal axis of the cochlear anlage in both chicken and mice. The expression domain of genes associated with the cochlear apex are expanded in the presence of ectopic Shh signaling. More importantly, these gene expression changes resulted in hair cells in the basal region of the chicken basilar papilla acquiring cellular features that are typical of apical hair cells. Hence, these results suggest that the early regional patterning of the cochlear anlage by Shh pre-stages the tontopic organization of the cochlea. 2) The specification of neural and macular fates within the chicken inner ear are temporally coupled The cochleo-vestibular ganglion (CVG), which relays sensory information from the inner ear to the brain, is derived from the same placodal epithelium that gives rise to the inner ear. During inner ear development, the neural-fated cells exit from the otic epithelium and coalesce to form the CVG. These neural-fated cells originate from the neural-sensory competent region of the otic cup, which also gives rise to some of the sensory organs of the inner ear in later stages. Deciphering the molecular pathways that mediate the neural versus sensory fate during normal development may provide insights into alternatives for hair cell replacement and therapy in the long run. Previously published results using genetic fate mapping techniques in mice suggest that the neuronal fates and the two vestibular sensory organs, the maculae, share a common lineage. Based on the physical locality of these sensory organs within the mature inner ear and the positions where neuroblasts initially delaminate from the otic cup, we hypothesize that specification of maculae of the utricle and saccule occur early during development coupled in timing to the specification of the vestibular and auditory neurogenic fates, respectively. We tested this hypothesis by inverting the position of the vestibular and auditory neurogenic regions in ovo. This was accomplished by surgically inverting the medial-lateral axis of the otic cup relative to the body axis at the time when neuroblasts started to delaminate from the epithelium. We then asked whether formation of these various neuronal and sensory fates were affected as a result of this surgical manipulation. We reasoned that cell fates that were already specified at the time of transplantation should maintain their identities though their positions might be displaced within the body. Our results showed that the formation of the vestibular ganglion was not affected by the surgery and appeared specified at the time of transplantation. Coincidently, the identity of the utricular macula, the sensory organ postulated to be related to the vestibular ganglion was also maintained in the transplanted ears. In contrast, both the auditory ganglion and its presumed-associated sensory organ, the saccular macula, only maintained their identities partially in the transplants. The correlation in the timing of specification among the two postulated ganglion - macula pairs supported our hypothesis that specifications of the neuronal and macular fates are temporally coupled during inner ear development. 3) Stereocilia polarity of sensory hair cells The stereociliary bundle on the apical surface of a sensory hair cell is comprised of a kinocilium and a number of specialized microvilli arranged in a staircase pattern. The asymmetric positioning of the staircase dictates the directional sensitivity of its hair cell; only deflections of the stereociliary bundle towards the kinocilium open the mechanotransduction channels located on the tip of the stereocilia and lead to activation of the hair cell. The proper positioning and formation of the kinocilium are essential for normal hair cell functions and these failings are associated with deafness in humans. As predicted by the importance of stereocilia orientation in hair cell function, each sensory organ of the inner ear displays a unique and defined pattern of stereocilia orientation. Thus, understanding the molecular mechanisms that establish stereocilia pattern in each sensory organ is important from both a functional and clinical perspective. A knockout mouse mutant of Emx2, which encodes a homeodomain transcription factor, was reported to show disrupted stereocilia pattern in the maculae of the inner ear. Both of these vestibular sensory organs lack the normal line of polarity reversal (LPR) and all the stereocilia are pointing unidirectionally in the mutants, in contrast to the wildtype maculae, each consists of two regions of hair cells with stereocilia that are arranged in opposite orientation across the two regions. We investigated these mutant phenotypes in more detail and we showed that the unidirectional orientation of stereocilia in the mutant maculae is caused by 180 degrees inversion of stereocilia in the region that normally expresses Emx2. To test the role of Emx2 in mediating stereocilia polarity, we generated several gain-of-function mouse models, in which Emx2 can be activated in nascent hair cells. Our preliminary results indicate that hair cells that do not normally express Emx2 show a 180 degrees reversal of their stereocilia orientation in the presence of ectopic Emx2. Taken together, these results suggest that Emx2 is necessary and sufficient to invert stereocilia orientation in sensory hair cells. 4) Functions of striola of the maculae and central zone of the cristae within the inner ear Reflexes initiated from the vestibular organs of the inner ear such as vestibule-ocular reflex (VOR) and vestibular-colic reflex (VCR) are extremely quick responses operating in millisecond range to help us maintain gaze and gait during head movements, respectively. While the neuronal circuits for these reflexes are well defined, the type of sensory hair cells that are responsible for initiating these reflexes in the vestibular organs are not clear. Presumably, type I hair cells residing in the central zone of the cristae and the striola of the maculae are most likely to play a direct role in mediating these reflexes since they are more sensitive to frequency changes. To test this hypothesis, we are in the process of generating mouse mutants that are devoid of the central zones and striola. Generation of these mouse mutants will serve as good models to study vestibular disorders in humans.
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