Gene expression in regenerating hair cells of avian sensory epithelia: implications for human hearing and balance disorders

Dora Oroian¹, Michael Lovett, David Hawkins, Stavros Baschiardes², Biology Department, Washington University, St. Louis, MO¹, Department of Genetics, Washington University School of Medical, St. Louis, MO²

The vertebrate ear is a complex structure that is responsible for the hearing, balance, and movement control of the organisms that possess it. Therefore, if something goes wrong in the ear, the consequences are grave. The multifarious disorders that result from ear damage have impelled many scientists to look very carefully at the nature and cause of inner ear hair cell deterioration in order to potentially find a way to cure hearing and balance disorders. The hearing mechanism is complicated. Sound travels from the outside environment to the middle ear where it causes vibrations that pass through the coiled cochlea. It is in the Organ of Corti (in the cochlea) that these vibrations are converted to impulses that are then transmitted to the brain. It is the specialized sensory hair cells that make this conversion; they are also the cells that are found in the utricle (a vestibular organ), where balance and movement are detected.

In humans and other mammals, these hair cells are made during the embryonic stage of development. After that stage, mammalian species lose the ability to make these cells. Therefore, if hair cells are damaged, they will not be replaced. Since humans only have a total of 8000 to 10000 hair cells, even slight damage will cause permanent, irremediable hearing and equilibrium defects. About 28 million people in the U.S. have some degree of reduced hearing sensitivity, and 54% of the population over the age of 65 has hearing loss. Hearing loss is the 3rd most prevalent chronic condition in the older population. However, about 20 years ago, it was found that lower vertebrates have the ability to regenerate these hair cells by converting the epithelial supporting cells in the cochlea and utricle into hair cells, if the latter have been damaged or completely destroyed. The hair cells in the cochlea (responsible for hearing) are called quiescent because they only grow back when damaged, while the hair cells in the utricle (responsible for balance and movement detection) are called proliferative because they are in a constant cycle of apoptosis and regeneration.

In the present study, utricle sensory epithelia hair cells and supporting cells were extracted from chickens and grown in cultures. A laser is then passed over the culture to kill the hair cells but not the surrounding supporting cells. The cells are then allowed to regenerate for 30 minutes, 1, 2, and 3 hours. After these four different samples are produced, they are in turn compared to control samples (cells that were not damaged) in order to detect and assess the differences in gene expression (which genes are strongly expressed at the respective time points). Gene expression profiling on custom microarrays was used in order to determine the differences in gene expression throughout the course of recovery. The process required us to print an 8000 gene microarray that these samples could later be hybridized onto. 8000 human genes were amplified by PCR from plasmid vectors containing each gene and then were spotted on polylysine-coated glass slides. To detect the differences in gene expression, amplified mRNA from the cells is converted to fluorescently labeled cDNA and hybridized to the microarray. The amount of fluorescence is then measured in each sample to determine the levels of gene expression. By inserting a modified base (uracil amino allyl) during this process, fluorescent dyes can be attached to the cDNA samples. The samples are labeled with either a Cy3 or a Cy5 fluorescent dye, one dye in one sample, the other dye to the sample that the first one is being compared to. After the different dyes are coupled to their respective samples, I combined the two samples and hybridized them on the same slide. The slides were then scanned under the green laser (lights up the Cy3 labeled cDNA) and the red laser (lights up the Cy5 labeled cDNA). These two images were then overlaid to see whether the image was mostly red, green, or yellow, in which case the gene is equally expressed in the two samples. Two types of experiments were performed: the “selfs”, comparing a laser time point of one biological sample to the same time point of a second biological sample (in order to check the consistency of the RNA amplification), and the laser vs. control, where samples from the laser damaged cells were compared to samples from chicks whose hair cells were not damaged. We also performed dye switch experiments in order to check for consistencies in dye intensity. This insures that the changes seen in gene expression are not caused by one dye being brighter than the other. In the laser vs. control we are checking for the differences between the genes that are normally expressed in the sensory epithelia (control) and the genes that turn on during hair cell regeneration. We are looking at these early stages of regeneration to identify the primary steps for this process. By finding out which are the genes that turn on during hair cell regeneration in chicks we gain insight that will in the future allow us to turn on the equivalent genes in humans in order to regenerate hair cells that have been destroyed.