Research in my laboratory focuses on the cellular and molecular processes underlying differentiation of the mechanosensory hair cells of the inner ear, and on the innervation of these cells by axons of the eighth cranial nerve. The aquatic amphibians, Xenopus laevis and Xenopus tropicalis, are being used as a model system for these investigations. The broad objective of this research is to gain an integrated view of the development and proliferation of sensory hair cells of the inner ear by using multidisciplinary approaches that draw on techniques from biophysics, anatomy, tissue culture, and molecular biology. A major long term goal of my research is to understand the genetic basis of hair cell function, differentiation, and regeneration. As part of this effort we seek to gain an integrated view of sensory organ formation during inner ear development, and to identify novel genes expressed in the developing auditory and vestibular system.

Experiments underway in the laboratory are testing several hypotheses about the expression of ion channel genes during development, and about the mechanisms that produce functionally heterogeneous hair cells in mature inner ears. For example, in some experiments, we are determining whether endorgans of the inner ear that are responsive to stimuli of different frequencies have hair cells with different types and complements of ion channels for calcium and potassium ions. In complementary experiments, we are studying the innervation of hair cells by efferent and afferent axons, and the ontogeny of hair cell and sensory ganglion cell proliferation during inner ear development. We have recently initiated a series of experiments that will use the Gravitational Biology facilities at NASA Ames Research Center to provide a hypergravity stimulus during Xenopus development. This study may be useful in identifying genes that are responsive to a gravitational stimulus, and may provide insight into the mechanisms that underlie hair cell differentiation and axon guidance during inner ear and central nervous system development.

Our research uses molecular approaches to clone the genes for calcium and potassium channels and cytoskeletal proteins expressed in the ear. An emerging body of data indicate that these types of proteins may interact in novel and complex ways in cells of the nervous system. We rely on RT-PCR methods to clone genes expressed in the inner ear, and use antibodies to confirm protein expression using methods that rely on immunodetection. We have developed techniques that amplify sequences from small amounts of brain and inner ear tissue and have constructed cDNA libaries from inner ear and brain that are presently being evaluated. We intend to use these libraries to prepare DNA microarrays that can be used to determine global changes in gene expression during inner ear development. As part of this effort, gene expression patterns in the developing auditory and vestibular system are visualized in sectioned and whole mount tissue with in situ hybridization and immunohistohemical techniques. Furthermore, multi-photon and confocal fluorescence microscopy are being used to gather digitized information about cell structure and gene expression that can be processed to render tridimensional images of developing inner ear endorgans. Presently an in vitro culture system is being developed that will be used to test hypotheses about hair cell differentiation and regeneration. We hope to use our recently acquired a multiphoton microscope to develop imaging methods that will allow us to monitor inner ear development in living tissue.

The knowledge gained from this research should prove useful in developing treatments for hearing and balance disorders based on hair cell and eighth nerve dysfunction, particularly those caused by trauma, or those with a genetic basis. Results also will be directly applicable to investigations of the effects, adverse or otherwise, of an altered gravity environment on the vestibular system.
Laboratory expertise: primary neuronal and glial culture, histology, immunohistochemistry, SEM, TEM, confocal microscopy, multiphoton microscopy, patch clamp, 2 microelectrode voltage clamp, DNA, RNA and protein isolation, cDNA library construction and screening, Northern and Southern blots, RT-PCR, westerns

Lab members prepare to load a sequencing gel

Publications that represent the work I do: