Goals of this project

Some background information on hair cell regeneration

The process of hair cell regeneration has been characterized most completely in the inner ears of birds. Both the avian cochlea and vestibular organs possess a robust capacity to quickly replace lost sensory cells. Although several distinct mechanisms may be responsible for the production of new hair cells hair cell regeneration in the avian ear appears to involve the proliferation of epithelial supporting cells. Although the undamaged cochlea is mitotically quiescent, numerous studies have shown that the loss of hair cells from the avian ear stimulates renewed mitosis of supporting cells, and that the progeny of those divisions go onto differentiate as replacement hair cells and supporting cells. A more limited regenerative response also occurs in the vestibular organs of mammals, but sensory regeneration does not occur in the normal mammalian cochlea.

Many of the cellular events that occur during regeneration in the avian ear have been described, but the precise signaling events that initiate regenerative proliferation are not known. Results obtained from studies of cultured avian cochleae have shown that the death of hair cells triggers the renewed proliferation of supporting cells within 8-16 hours after injury. Regenerative proliferation is confined to areas within or near (<200 µm) the lesion site, suggesting that hair cell death triggers the release of a difusible mitogen. Consistent with this suggestion, cochlear supporting cells express mRNA for FGF-2 and are immunoreactive for FGF-2 after the loss of hair cells. Other studies have shown that the proliferation of supporting cells can be enhanced by treatment with FGF-2, GGF-2, IGF-1, insulin, TGF-_, or TNF-_. Present knowledge of downstream signaling pathways in regeneration is more limited, but it has been shown that supporting cell proliferation can be induced by activation of the cAMP pathway. Still, it is not known which mitogen(s) and signaling pathways are involved in the normal process of hair cell regeneration.

In addition to regeneration after injury, hair cells in the vestibular organs of birds also display a unique pattern of postnatal survival and replacement. In most vertebrates, hair cells appear to be capable of surviving for the lifetime of the animal. Interestingly, vestibular (but not cochlear) hair cells in mature birds have a relatively short lifetime, estimated at 1-2 months. Dying hair cells are quickly replaced by new sensory cells that are produced by ongoing proliferation of epithelial supporting cells. Significant levels of cell death and cell proliferation are observed in the undamaged chick utricle, suggesting that a sizable fraction of hair cells in that organ are either in an immature state or are near-death. The factors that are responsible for the limited lifetime of these hair cells are not known, but their identification may produce insights in the mechanisms of age related hearing and balance disorders.

The cochlea and vestibular organs of mature birds exhibit very different patterns of hair cell production and survival. Hair cells in the cochlea are produced during a short period in embryonic development. Cell proliferation in the sensory regions of the cochlea terminates on about embryonic day ten as hair cells and supporting cells begin to differentiate. Under normal conditions, cochlear hair cells appear to be capable of surviving for the lifetime of the animal, and the normal (undamaged) cochleae of mature birds contains very few proliferative cells. In contrast, hair cells in the vestibular sensory organs (which are initially produced during a similar period of embryonic development) have a very short normal lifetime. Hair cells in the mature vestibular organs appear to die spontaneously, and are then replaced by new cells that arise from the ongoing proliferation of epithelial supporting cells. Estimates of the lifespan of vestibular hair cells in chickens range from 2-6 weeks.

Few studies to-date have examined the genetic basis for hair cell regeneration. Studies based on differential display of mRNA following acoustic trauma have described 70 cDNA bands that are observed in the avian cochlea after injury. The identities of most of these bands were not determined, although it was shown that genes for parathyroid hormone-related protein, a neuron-specific CaM-kinase, the GTPase Cdc42, and UBE3B (a ubiquitin ligase) were expressed within 48 hours of cochlear injury. Also, as noted above, expression of certain members of the FGF family of growth factors and their receptors are observed after cochlear injury. Finally, a recent study has shown ototoxic insult to the avian utricle results in increased gene expression for certain cytoskeletal proteins.

Another strategy for determining the role of specific genes in the process of hair cell regeneration is to study the regenerative capabilities of mice with identified genetic deficits. Such techniques have demonstrated that disruption of the gene for the cyclin dependent kinase inhibitor p27Kip1 appears to permit the proliferation of cochlear supporting cells in mature mice. These are the first results that have conclusively demonstrated postnatal proliferation in the mammalian cochlea, and suggest that the inability of the normal mammalian cochlea to regenerate hair cells may be due (in part) to the expression of genes that inhibit renewed proliferation. Additional studies have shown an age-related decrease in expression of the CDK inhibitor p57Kip2 in the developing vestibular organs of mice.

All previously published studies of gene expression after cochlear injury have suffered from two key limitations: (1) the inability to simultaneously monitor changes in the expression of large numbers (>100) of genes, and (2) the inability to positively identify many of genes that are expressed early in the process of regeneration. The recent development of cDNA arrays, which can measure the expression levels of thousands of genes at once, provides an exciting opportunity to investigate this process in enormous detail.

In the past numerous methods have been used to detect or quantitate mRNAs. Most of these only work on one or a few genes at once. More recently, two global methods of analyzing gene-expression have been developed and are being used increasingly by many research groups. These are chip or array-based methods of spotting genes onto solid supports and the Serial Analysis of Gene Expression (SAGE) which is a form of molecular "bar coding". Of the array- based approaches, one relies on spotting multiple cDNA clones onto modified class microscope slides (cDNA microarrays). The other array-based approach is to covalently attach oligonucleotides chemically synthesized in a highly combinatorial fashion onto a solid substrate. This latter method has been pioneered by Affymetrix and is a powerful "off the shelf" method for measuring gene expression. Unfortunately, Affymetrix chips are not useful for measuring gene expression across species because the entire readout is predicated by the ability of a cRNA to make a perfect match with the relatively short oligonucleotides that are on the chip. This will rarely occur if a human gene chip is used to interrogate chick gene expression. In our studies we employ our own custom cDNA microarrays in combination with some modifications to SAGE

For information on the incidence of deafness and what is being done to research its causes please look at the following links: