Daniel F. Eberl - US grants

The long-term objectives of this proposal are to understand the molecular and cellular mechanisms of auditory mechanosensation. These goals will be achieved by using a new genetic model system for hearing in Drosophila. The public availability of almost the entire genome sequence, together with the genetic, developmental and molecular tools for manipulating Drosophila make this a very powerful model. Hearing in insects is mediated by chordotonal organs, which are related to vertebrate auditory and vestibular hair cells because they are developmentally specified by homologs of the same gene, atonal. The first approach will be to identify mutations that specifically disrupt chordotonal organ function, to clone the corresponding genes and to elucidate the cellular location and molecular function of their gene products. Mutations in three genes, beethoven, smetana and touch-insensitive-larva-B will be subjected to this analysis. The second approach will make use of enhancer trap strains, whose engineered transposon inserts express a reporter gene specifically in chordotonal organs. Four enhancer trap strains identified by this criterion will be used as starting points to clone the flanking sequences to identify candidate chordotonal-specific genes. If the transposon does not disrupt the gene, imprecise excision derivatives will be generated as a way to introduce mutations in the gene. The transposons therefore act not only as reporters, but also as molecular tags and as a mutagen. The third approach will use known human genes associated with deafness as a starting point to identify Drosophila homologs and then to use reverse genetics to identify mutations in these genes to test for function. Methods for this reverse genetic approach will include characterization of nearby transposon insertions or their imprecise excision derivatives as well as recently described gene replacement strategies. Identifying auditory genes by any of these approaches and elucidating the molecular roles of their products will provide very important insight into the fundamental but poorly understood process of mechanosensation.

[unreadable] DESCRIPTION (provided by applicant): The long term objectives of this proposal are to understand the development and function of Johnston's organ (JO), which is the auditory organ in the genetic model organism, the fruit fly Drosophila. The Drosophila JO is homologous to the mammalian inner ear because they both rely for their specification on the activity of highly conserved transcription factors of the atonal family. The mouse atonal homolog 1 (Math1) and the fly atonal genes can substitute for each other's function in reciprocal transgenic rescue experiments. Furthermore, the human atonal homolog 1 (Atoh1) can mediate regeneration of auditory hair cells in pharmacologically deafened mammals. Thus, the fly JO represents a powerful gene discovery resource for hearing. JO differs from other chordotonal organs in several ways that specialize it for hearing. At early pupal stages when many critical events of JO development occur, including asymmetric divisions of precursor cells, specification of sense organ cell lineages, and cell shape changes essential for correct differentiation, the JO is obscured within the puparium and very fragile to dissect. Our general strategy is to devise methods to better visualize the developing JO, and to use these methods as the basis for systematic expression microarray analysis for gene discovery. First we will characterize the roles of several JO genes at these early pupal stages. To characterize the medical relevance of these genes, we will screen for associations of their human homologs with families segregating deafness. Second, we will culture dissected antennal disks to image cell lineages and marker expression dynamically in vivo, away from the obscuring puparium. We will determine the fidelity of development in culture with several markers, and define the salient events at these stages. Third, we will exploit the higher throughput of this approach to recover sufficient RNA from wild-type and mutant antennal disks at these critical stages to compare gene expression using microarray analysis. We plan to use cut mutants initially as a paradigm. The cut transcription factor is required for normal JO development, and the mammalian homolog, CDP/Cux1 is expressed in the inner ear. Thus, we expect that the results of our experiments will permit us to identify target genes of cut that act at these critical stages. Overall, these studies will inform future research on the developmental and functional biology of the mammalian inner ear, and accelerate our understanding of human auditory disorders. [unreadable] [unreadable] [unreadable]

Program Summary The Interdisciplinary Graduate Program in Genetics at the University of Iowa has a 45-year history of training PhD students in Genetics, with well over 100 graduates in that time. The program currently serves 71 faculty and 43 students in four colleges and 17 academic departments across our campus. Since the time of the last competitive submission of this application, our interdisciplinary faculty has grown by 4 with an average of 7 new students entering the program each year. Our Computational Genetics subtrack was one of the first programs in the country to offer in-depth PhD training at the interface between genetics, genomics and bioinformatics, and 15 students have now graduated from the subtrack, four of which are already in faculty positions. We have an enviable record of program completion, on-time graduation rates, publications and awards, as well as career advancement to postdoctoral fellowships and faculty positions at research-intensive universities. We have made continuing progress in diversifying our student population, and incorporated several strategies to build on a nucleus of minority students, as well as to enhance the retention of these and indeed all students in the program. In this renewal application we request a continuation of the six fellowship slots we currently have available to serve our cadre of high quality and productive students. This application will outline our successes over the last 40 years of program existence, our current structure, and plans for future improvements in diversity, enhanced career development initiatives for versatility in employment, as well as student and faculty mentoring. We have a cohesive group of students working with a broad and diverse collection of faculty who represent the genetics community in all its facets. Finally we have stable program leadership at both the faculty and administrative level and very strong support within the graduate college and the institution that helps to ensure not only the continued success of the program but its further strengthening and diversification at a time when scientific careers are proving increasingly challenging.

Auditory; Auditory system; base; Bioinformatics; Biology; Candidate Disease Gene; Collaborations; comparative genomics; Custom; Data; Data Analyses; Data Collection; density; Ensure; experience; Funding; Gene Expression; Gene Expression Profile; Genes; genome-wide; Genomics; Goals; Hearing; Human Resources; instrumentation; Iowa; Labyrinth; Methodology; Methods; Modeling; Molecular; Molecular Biology; Molecular Biology Techniques; Molecular Genetics; National Institute on Deafness and Other Communication Disorders; Neurosciences; next generation; Pattern; Procedures; Protocols documentation; Research; Research Personnel; Research Project Grants; Sampling; Services; Technical Expertise; Techniques; Time; Training; Universities; Work

DESCRIPTION (provided by applicant): Our understanding of Noise-Induced Hearing Loss is rather rudimentary, despite the importance of this problem to human occupational and recreational health. The damage is permanent because mammals cannot regenerate their auditory hair cells once they die. Studies of the underlying causes of noise-induced hearing loss have focused on mechanical damage to cells, and molecular studies centered around oxidative damage and cytoskeletal disruptions. Mammalian studies are challenged by difficult access to the inner ear inside the temporal bone, and expensive animal care costs. In this proposal we seek to capitalize on the genetic model organism, Drosophila, to systematically characterize gene expression changes induced by acute or chronic over-exposure to sound. Our preliminary results show that there are strong immediate effects on auditory function after acute exposure. We propose first to study the immediate and longer-term morphological and physiological consequences of these forms of noise trauma. Second, we will identify genome-wide responses to acoustic stress using microarray analysis. These studies will provide insight into possible pathways through which to increase resistance to noise damage as a preventative measure, or to reduce the deleterious effects of noise damage after the fact as a therapeutic measure. They will also inform the individual genetic differences in susceptibility to noise damage. PUBLIC HEALTH RELEVANCE: Over-exposure to noise is a serious problem in today's industrial and technological world, especially with increasing age structure in human populations. The proposed project uses the genetic model organism Drosophila to investigate functional and structural consequences to the auditory organ upon acute or chronic over-exposure to sound, and to understand gene expression changes evoked by this treatment. The results of our studies will facilitate identification of putative preventative and therapeutic targets for human noise-induced hearing loss and age-related hearing loss.

DESCRIPTION (provided by applicant): The Predoctoral Training Program in Genetics at the University of Iowa has a 40-year history during which more than 80 students have graduated with a Ph.D. The program currently serves 67 faculty and 44 students in four colleges and 18 academic departments across our campus. Since the time of the last competitive submission of this application, our interdisciplinary faculty has grown by 12 with an average of 8 new students entering the program each year. We have an enviable record of student retention, on-time graduation rates, publications and awards, as well as career advancement to postdoctoral fellowships and faculty positions at research-intensive universities. Of particular note is the central role that our program has played in building graduate programs in comparative genomics and bioinformatics on campus. We have seen growth and solidification of the nascent Computational Genetics subtrack option in the Genetics Ph.D., with now seven students that have already graduated from the track, four of which are already in faculty positions. We have made recent progress in diversifying our student population, and incorporated several strategies to build on a nucleus of minority students, as well as to enhance the retention of these and indeed all students in the program. In this renewal application we request a continuation of the six fellowship slots we currently have available to serve our cadre of high quality and productive students. This application will outline our successes over the last 40 years of program existence, our current structure, and plans for future improvements in diversity, graduation times and mentoring. We have a cohesive group of students working with a broad and diverse collection of faculty who represent the genetics community in all its facets. Finally we have stable program leadership at both the faculty and administrative level and very strong support within the graduate college that helps to ensure not only the continued success of the program but its further strengthening and diversification at a time when scientific careers are proving increasingly challenging.