Teresa Nicolson, Ph.D. - US grants

DESCRIPTION (provided by applicant): Deafness is one of the most common hereditary diseases, affecting one out of 1000 children. Human geneticists have made remarkable progress in identifying genes responsible for deafness, yet in many cases, we do not clearly understand either the function these genes or the pathology caused by the mutations. Moreover, our understanding of the sense of hearing lags behind our knowledge of other senses such as vision, taste, and touch. The molecules that directly mediate mechanotransduction in hair cells have yet to be been identified. In order to gain insight into the molecular basis of mechanotransduction and the function of deafness genes, we will take advantage of both forward and reverse genetics in the model vertebrate organism, the zebrafish. We have identified a total of 24 genes required for larval auditory and vestibular function from large scale mutagenesis screens, and have cloned four zebrafish genes thus far. Orthologues of all four genes are responsible for deafness in either humans or mice, demonstrating the relevance of our screen and a high degree of conservation of gene function. We will continue our cloning efforts by candidate or positional cloning approaches. In addition, our reverse genetic studies have proven fruitful, identifying a potential candidate gene for the mechanotransduction channel, NompC. As further evidence for a direct role in transduction, we will localize the NompC channel in zebrafish hair cells. One particular challenge will be to characterize and pinpoint the nature of the defects in our mutants or morpholino-injected animals. We will therefore create a transgenic calcium indicator line that will enable us to determine whether transduction, synaptic transmission, or central processing of auditory signals is affected in our auditory/vestibular mutants. The data from these experiments will help to increase our understanding of the biology of deafness genes and may lead to potential therapies for deafness patients.

DESCRIPTION (provided by applicant): The identification of molecules relevant to human health not only improves the chances for new therapeutic approaches, but can also reveal critical insight into the etiology underlying disease. Forward genetic screens in vertebrates have greatly aided in our understanding of biological processes and disease, and we aim to expand the ability of the zebrafish community to screen for components critical to development and function. We propose to adapt two new screening methods 'BioID' and 'Isolation of Nuclei TAgged in specific Cell Types' (INTACT) for use with zebrafish. Both techniques involve tissue-specific biotinylation of nuclei or proteins that occurs in vivo before processing of samples, and both techniques offer several advantages over current methods. For the BioID method, we will generate versions of the Tol2 Gateway vector that can accommodate any promoter and/or protein of interest. For the INTACT method, our aim is to generate stable transgenic lines that would enable the community to use Gal4 lines already available as a means of achieving tissue specificity. We will also generate a stable line that ubiquitously expresses the biotin ligase for those laboratories that wish to use a specific promoter. For both approaches, we will develop simple protocols for post-in vivo expression steps using zebrafish embryos and larvae as the source of material for purification and identification of novel factors.

? DESCRIPTION (provided by applicant): Mutations in Protocadherin 15 (PCDH15) cause deafness in fish, mice, and humans. As a central and conserved component of the mechanotransduction complex in sensory hair cells, this unusual cadherin forms part of the extracellular filaments at the tips of stereocilia. These so-called `tip links' are thought to gate mechanically sensitive channels. To gain a better understanding of how PCDH15 is coupled to the mechanotransduction machinery, we performed an unbiased molecular screen using zebrafish Pcdh15a as bait in a membrane-based yeast two-hybrid screen. We identified a positive interaction with Tmc2a, an orthologue of mammalian TMC2. Tmc2 was recently implicated in deafness and vestibular dysfunction in mice, and its closely related gene, Tmc1, is associated with both recessive and dominant forms of hearing loss in mice and humans (DFNA36 and DFNB7/11). Our preliminary results recapitulate the protein interactions found in the screen among both the zebrafish and mouse TMC and PCDH15 orthologues. In addition, we have discovered both loss-of-function and gain-of-function effects on hair-cell mechanosensitivity upon overexpression of fragments of Tmc2a in wild-type fish. Together, the link to Pcdh15 and the dominant negative or activating effects in hair cells provide compelling evidence that Tmc1/2 proteins are central players of the mechanotransduction complex in hair cells. We will characterize the interaction of Pcdh15 with Tmc1/2 proteins with genetic and biochemical methods, and study structure/function aspects of the complex in vivo. This work will increase our knowledge of the molecular basis of mechanotransduction and the understanding of how lesion of Tmc1 leads to hearing loss.

PROJECT SUMMARY Transmembrane inner ear-expressed gene (TMIE) encodes a transmembrane protein that essential for hearing and vestibular function in vertebrates. A recent study of Tmie-/- mice has provided strong evidence for a central role of TMIE in mechanotransduction in cochlear hair cells. As a member of the transduction complex, mouse TMIE can bind to a specific isoform of Protocaderin 15 and/or another membrane protein, protein Lipoma HMGIC Fusion Partner-Like 5 (LHFPL5). Despite these seminal findings, it is not well understood how TMIE functions as a member of the transduction complex, and to date, its role in vestibular hair cells is unexplored. The aim of the present proposal is to conduct a comprehensive study of zebrafish Tmie in terms of identifying the protein motifs that are required for (i) localization of Tmie to stereocilia, (ii) modulation of mechanotransduction, and (iii) genetic and biochemical interactions with other members of the transduction complex. The proposed experiments will take advantage of our collection of mechanotransduction mutants and tools we have developed in zebrafish to investigate the function of Tmie in hair cells. Our preliminary data indicate that zebrafish Tmie is required the localization of Transmembrane channel like 1 (Tmc1) and Tmc2b to the stereocilia of hair cells in the inner ear and lateral line organ. This observation is surprising in light of experiments with Myc tagged TMC2 in mouse outer hair cells, however, the results suggest that TMIE may play different roles in different cell types. The proposed experiments will expand upon these novel findings and identify the amino acid motifs in Tmie that promote interaction with Lhfpl5a and the localization of the Tmcs to the site of mechanotransduction in hair cells. This work will provide mechanistic insights into the role of Tmie in hair cells and provide a better understanding the molecular determinants of Tmie that are critical for vertebrate hearing and balance.

PROJECT SUMMARY Approximately one quarter of patients with vertigo or dizziness have central vestibular disorders. In addition, hearing loss or tinnitus can have central origins. Despite the prevalence of central deficits in the auditory/vestibular system in patients, our understanding of central dysfunction at the molecular or cellular level in vertebrates is lacking. Here, we propose to characterize a novel class of zebrafish mutants that have central auditory/vestibular deficits to gain insights into this understudied area of research. This proposal focuses on two mutants: raumschiff and starliner, which were isolated from chemical mutagenesis screens for hearing and balance defects. Unlike our previously characterized mutants, raumschiff and starliner mutants have normal vestibular induced eye movements despite presenting with an obvious balance defect while swimming or at rest. In addition, a defect in hearing is present in both mutants. We have identified mutations in two genes: in starliner mutants, the split ends (spen) gene harbors a nonsense mutation and in raumschiff we identified a missense mutation in vacuolar protein sorting 4a (vps4a). RNAseq anaylsis of mutant and sibling transcripts indicate that both mutants have striking misregulation of gene expression in the hindbrain and midbrain regions, yet their development and gross brain morphology is normal. These results suggest that the defects are functional in nature and may involve circuit level or synaptic changes. To gain a better understanding of the central defects, we will take advantage of imaging whole fish expressing relevant transgene markers and use newly developed methods for brain-wide imaging of cellular responses to auditory and vestibular stimuli. These experiments will focus on the regions or cell types where misregulation of gene expression is most prevalent. Collectively our studies will enhance our understanding of the genes and regulatory networks involved in central function.