This years major accomplishments are in the following areas: 1) Transcription factor Emx2 controls stereociliary bundle orientation in sensory hair cells. The sensory hair cells are mechanotransducers, transducing sensory information such as sound, head movements and water pressure in the form of vibrations into chemical signals, which are then propagated to specific regions of the brain via electrical signals of connecting neurons. These vibrations are detected by the stereociliary bundle (hair bundle) erected on the apical surface of sensory hair cells. Only deflection of the hair bundle towards a specific direction opens the mechanotransduction channels on the tips of the stereocilia and results in activating of its hair cell. Hence, orientation of the hair bundle is fundamental in providing the directional sensitivity of any given hair cell, and hair bundle pattern is well defined for each sensory hair cell organ. Notably, hair bundles in the two vestibular sensory organs of the vertebrate inner ear, the utricle and saccule, are oriented such that an imaginary line, line of polarity reversal (LPR), can be drawn which divides each organ into two regions of hair bundles with opposite orientation. A similar bundle pattern is present in neuromasts of the lateral line system of fish, in which hair cells are generated as pairs with opposite polarity. The mechanism(s) that establishes these mirror-images of hair bundle orientation is not known. In the past year, we published a paper showing that the transcription factor, Emx2, is responsible for establishing this bundle polarity pattern in the inner ear and the neuromast. We show that Emx2 is only expressed in hair cells of which the hair bundles are at approximately 180 degrees from the default orientation. Using gain- and loss-of-function mutants in both mice and fish, we demonstrated that Emx2 is sufficient and necessary to alter the hair bundle orientation. Currently, we are using RNAseq approaches to identify the downstream targets of Emx2 that mediate hair bundle orientation. 2) Inner ear source rather than hindbrain source of Lmx1a is important for inner ear information. The mammalian inner ear is a structurally complex organ responsible for detecting two important sensory inputs: sound and head position. Proper detection of these sensory inputs is heavily dependent on the three-dimensional structure of the inner ear. Understanding the molecular mechanisms underlying the formation of this complex structure during development will aid in the design of strategies to alleviate deafness and balance disorders caused by structural defects of the inner ear. The inner ear acquires its positional information from the surrounding tissues during development. Namely, the developing hindbrain provides many signals such as Fibroblast growth factors, Mafb, and Sonic hedgehog that are instructive for inner ear formation. However, most of these factors are also expressed in the developing inner ear as well. Therefore, to ascribe gene function to its tissue source is important for understanding ear development. For example, Lmx1a is LIM-domain transcription factor expressed in both the developing hindbrain and inner ear. The mouse dreher mutants, which lack functional Lmx1a, have defects in both the hindbrain and inner ear. The inner ears of dreher mutants are rudimentary in structure that lack the endolymphatic duct. However, most of the sensory organs of the inner ear, though fused, are identifiable within the rudimentary inner ear. To address the question whether the inner ear phenotype in dreher is caused by the lack of Lmx1a in the inner ear or indirectly caused by defects in the hindbrain, we conducted inner ear specific knockout of Lmx1a by generating a Lmx1a-lox allele. Inner ear phenotypes obtained from two independent conditional knockout of Lmx1a in the inner ear are indistinguishable from the dreher mutants, suggesting that the inner ear source of Lmx1a rather than its source from the hindbrain is more important for inner ear formation. 3) Reduction of retinoic acid signaling specifies the striola and central zone of vestibular sensory organs. Reflexes that involve the vestibular sensory organs of the inner ear such as vestibular ocular reflex and vestibular colic reflex are quick with response time of milliseconds. While the neuronal circuitry that is required to mediate each of these reflexes is known, it is not clear how these responses are initiated within the vestibular sensory organs and whether a specific type of hair cells is dedicated to process these acute sensory inputs. A better understanding of how various sensory inputs are qualitatively being integrated at the sensory organ level may help to alleviate incidences of falling, which is the number one cause of accidental deaths in the elderly population. Because of the fast response time of vestibular type I hair cells residing in the striola of the maculae and the central zone of the cristae, it has been proposed that these hair cells are particularly important for mediating vestibular reflexes. We found that these specialized regions, striola and central zones, are generated by a low levels of retinoic acid (RA) during development by the expression of the retinoic acid degradation enzyme, Cyp26b1. Using conditional knock out of Cyp26b1 in the inner ear, we showed that the striola and central zones in the vestibule were markedly reduced in these mutants, based on the expression of Type I hair cell-specific marker, oncomodulin, and the striolar supporting cell-marker, -tectorin. These conditional knockout mice will allow us to test directly effects of loss of striola and central zone on vestibular-mediated reflexes. 4) Delivery of Sonic Hedgehog from the spiral ganglion neurons to the cochlea of the mouse inner ear. The spiral ganglion (SG) of the mouse inner ear spirals along the length of the cochlear duct and it is comprised of otic-derived neurons that innervate the cochlear hair cells and neural crest-derived glial cells. During development, only a subpopulation of cells within the SG expresses the secreted molecule Sonic hedgehog (Shh). Accumulative studies from our lab show that this source of Shh is important for mediating the growth of the cochlear duct, timing of terminal mitosis and differentiation of cochlear hair cells. These developmental events are thought to pre-stage the tonotopic organization of the cochlea such that high frequency sound is detected by hair cells at the base of the cochlea and low frequency sound at the apex. Given the importance of Shh in mediating cochlear development, we further investigated the regulation of Shh expression in the SG. Our lineage analyses using Shh-creER mice showed that despite the restricted Shh expression consistently in a subpopulation of SG from mid- to late-gestation during mouse embryogenesis, this expression represents a transient stage of nascent SG neurons. As neuroblasts become postmitotic, they express Shh and as these nascent neurons continue to differentiate, Shh expression is downregulated. In the past year, we focused on asking whether Shh in the SG is being delivered to the cochlear epithelium via filopodia-like processes. In other systems, Shh can be transported to target cells at a distance via these types of processes. Our preliminary live imaging results using the Shh-creER and a cre reporter mouse, Rosa-tdTomato, suggest that Shh-positive SG neurons indeed extend processes towards the cochlea. We are currently investigating the specificity of these processes and whether this is also a mechanism whereby Shh is being transported to the cochlear epithelium at a distance.
Showing the most recent 10 out of 18 publications
