This years major accomplishments are in the following areas: 1) Genetic interactions support an inhibitory relationship between Bone morphogenetic protein 2 and Netrin 1 during semicircular canal formation. The three semicircular canals of the mammalian ear are the non-sensory components of the vestibular apparatus responsible for detecting angular acceleration. Malformations such as truncation or thinning of one of these canals will result in balancing deficits in mice. In humans, superior canal dehiscence is a syndrome associated with defects of the anterior canal during development, but the underlying genetic etiology is unknown. A better understanding of the molecular mechanisms underlying normal canal formation will provide insights into the genetics and pathology associated with the vestibular system in humans. Developmentally, the three semicircular canals are derived from two epithelial out-pockets of the otocyst. In these out-pockets, the opposing epithelia in each prospective canal move towards each other to form a fusion plate and the epithelial cells in the fusion plate resorb, leaving behind the rim of the out-pocket to form an arc-shaped canal. As a result, a canal will not form if the canal pouch fails to grow properly. Despite the presence of a canal pouch, extensive resorption in the fusion plate can chew away too much epithelial cells and leads to absence of a canal as well. In this study, we show that Bone morphogenetic protein 2 (Bmp2) expressed in both the center epithelia and the rim of the canal pouch is important for canal formation. In Bmp2 conditional knockout mouse mutants, all three semicircular canals are missing but the ampullae, which house the sensory tissue for the canals, the cristae, are normal. To investigate the cause of this phenotype, we conducted gene expression and genetic analyses. Our results suggest that Bmp2 has a dual function in canal formation. It functions to inhibit the resorption process by inhibiting the expression of a gene, Netrin1, which is important for mediating resorption. Concomitantly, it also functions to promote the growth of the canal rim. This work has been published in the journal, Development. doi:10.1242/dev.174748 2) Selectivity of afferent neurons by Emx2 in vestibular maculae of the inner ear All sensory end-organs need to be properly connected to the central nervous system by sensory neurons before sensory inputs can be interpreted in a meaningful manner. Understanding the mechanisms involved in this proper wiring during development is important from a functional standpoint. The lateral line system of aquatic animals, which functions to detect water pressure, is comprised of neuromasts that are studded along the surface of the animals body. Each neuromast consists of two groups of sensory hair cells, in which each group consists hair bundles that are oriented in opposite direction from the other group, organized along either the anterior-posterior or dorsal-ventral axis of the body. Notably, the neurons that innervate the two populations of hair cells within each neuromast are also segregated such that a single neuron only innervate hair cells that share the same bundle orientation. Previously, we have shown that the transcription factor Emx2 expressed only in hair cells that share the same orientation within a neuromast is important for mediating both the hair bundle orientation as well as the neuronal selectivity of the hair cells. Although both hair bundle orientation and selection of neuronal targets require Emx2, our results suggest that the two cellular processes are independently regulated by different transcription targets of Emx2. In the vestibular maculae of the mouse inner ear, the transcription factor Emx2 is also important for reversing hair bundle orientation from it default position by 180 degrees and thus establishes the line of polarity reversal (LPR) in the maculae. Interestingly, the neuronal innervation pattern in the two maculae appears to be segregated according to the LPR as well. Neurons that innervate hair cells in the Emx2-positive regions of the utricular and saccular macula, have central projections to the cerebellum, whereas neurons in the Emx2-negative regions project to the brainstem. Using lipophilic dye tracing and genetic mouse models, we are currently investigating the neuronal innervation pattern and the functional consequences of maculae in Emx2 knockout and gain-of-function mutants. 3) Cytochrome P450 26b1-mediated specification of vestibular striola and central zones is required for transient responses in linear acceleration The vestibular system of the inner ear is important for maintaining our sense of balance. Understanding how head movements and positional information are being coded by the vestibular sensory organs and relayed to the brain is important from both clinical and therapeutic perspectives. Two types of sensory organs are present in the vestibular system of the mammalian inner ear: cristae and maculae. The three cristae are responsible for detecting angular acceleration, whereas the two maculae, macula of the utricle and saccule, are responsible for detecting linear acceleration. A specialized region is present in each of the vestibular organ known as the central zone in the cristae and striola in the maculae. A defining feature of the striola/central zones is the calyceal nerve endings of afferent neurons that often encase multiple hair cells. The precise function of these complex calyces is not known but based on physiological properties of the hair cells and sensory neurons in these regions, it has been postulated that these regions are responsible of rapid signaling and important for mediating vestibular reflexes, which are extremely fast with response time of milliseconds in some species. Our study shows that formation of the striola/central zone is dependent on the presence of a Retinoic acid (RA) degradation enzyme, Cyp26b1, in the prospective striola/central zones during development, which functions to reduce the amount of RA emanating from the surrounding sensory tissues in the extrastriolar/peripheral zones. We generated Cyp26b1 conditional knockout (cKO) mice and based on the molecular, cellular and physiological analyses, we show that striola/central zones have adopted extrastriolar and peripheral zone-like properties in Cyp26b1 cKO mutants. To our surprise, behavioral and functional analyses of these Cyp26b1 cKO mice indicate that the striola/central zones are not required for mediating vestibular reflexes. Instead, our results show that the striola in the two maculae are responsible for generating the vestibular evoked potential, which is an assessment for macular functions. Although these mice exhibit no obvious deficits that are associated with vestibular dysfunctions such as circling, head bobbing or hyperactivity, they have difficulty in traversing the balance beam, suggesting that the striola/central zones are important for coordinating challenging vestibular and motor activities. A manuscript of this work is available on BioRxiv, doi: http://dx.doi.org/10.1101/726232.