The discovery of avian cochlear hair cell regeneration in the late 1980's and the concurrent development of new techniques in molecular and developmental biology generated a renewed interest in understanding the genetic mechanisms that regulate hair cell development in the embryonic avian and mammalian cochlea and regeneration in the mature avian cochlea. Research from many labs has demonstrated that the development of the inner ear utilizes a complex series of genetic signals and pathways to generate the endorgans, specify cell identities, and establish innervation patterns found in the inner ear. Recent studies have shown that the Notch signaling pathway, the Atoh1/Hes signaling cascade, the stem cell marker Sox2, and some of the unconventional myosin motor proteins are utilized to regulate distinct steps in inner ear development. While many of the individual genes involved in these pathways have been identified from studies of mutant and knockout mouse cochleae, the interplay of all these signals into a single systemic program that directs this process needs to be explored. We need to know not only what genes are involved, but understand how their gene products interact with one another in a structural and temporal framework to guide hair cell and supporting cell differentiation and maturation. KeywordsCochlea; Hair Cell; Supporting Cell; Genetic Regulation; Notch Pathway; Development; Regeneration Hearing impairment affects almost 49 million people in the US and over 249 million worldwide. Approximately 17% of children under age 18 have a hearing loss and the incidence increases with age: roughly 30% of people over 65 and over 90% of people over 80 have a substantial hearing loss. Although rarely life-threatening, hearing loss affects more people than epilepsy, multiple sclerosis, spinal injury, stroke, Huntington's and Parkinson's diseases combined (Hudspeth, 1997) and has a huge financial impact on our economy and lifestyle. Hearing impairment is mainly caused by damage to the hair cells, the sensory cells in the cochlea (Figure 1). In mammals, the absence of these cells results in permanent hearing loss

Objectives/Hypothesis-The avian cochlea regenerates hair cells following aminoglycoside treatment through supporting cell proliferation. Immunocytochemical labeling of BrdU, a thymidine analog, is a popular nonradioactive marker for identifying cells in the DNA Synthesis (S phase) of the cell cycle. However, it requires harsh treatments to denature double-stranded DNA for the antibody to bind BrdU. We explored a new method using EdU as a thymidine analog and a non-antibody azide/alkyne reaction between the EdU and the fluorescent probe. We propose that EdU is as effective as BrdU but without the requirement for harsh denaturation or the use of antibodies for detection.Study Design-Two week-old chicks received a single gentamicin injection followed by a single EdU injection 72h later. Cochleae were extracted 4-8h later, fixed, and processed for fluorescent detection of EdU.Methods-Cochleae were processed for detection of incorporated EdU using the Click-iT™ Imaging Kit (Invitrogen) and co-labeled with Sox2, myosin VI, or myosin VIIa antibodies. Whole-mount cochlear preparations were examined with confocal microscopy.Results-Supporting cells incorporated EdU into their newly synthesized DNA during the 4-8h following the EdU injection and were readily detected with little background signal. The intensity and quantity of cells labeled were similar to or better than that seen for BrdU. Conclusions-TheEdU method is as effective as BrdU without requiring harsh denaturation or secondary antibodies to identify proliferating cells. Thus, the non-antibody EdU system allows more flexibility by enabling co-labeling with multiple antibodies to other cellular proteins involved in regeneration.

Aminoglycoside antibiotics induce caspase-dependent apoptotic death in cochlear hair cells. Apoptosis, a regulated form of cell death, can be induced by many stressors, which activate signaling pathways that result in the controlled dismantling of the affected cell. The caspase family of proteases is activated in the apoptotic signaling pathway and is responsible for cellular destruction. The initiator caspase-9 and the effector caspase-3 are both activated in chick cochlear hair cells following aminoglycoside exposure. We have analyzed caspase activation in the avian cochlea during gentamicin-induced hair cell death to compare two different methods of caspase detection: caspase antibodies and CaspaTag kits. Caspase antibodies bind to the cleaved activated form of caspase-9 or caspase-3 in specific locations in fixed tissue. CaspaTag is a fluorescent inhibitor that binds to a reactive cysteine residue on the large subunit of the caspase heterodimer in unfixed tissue.To induce cochlear hair cell loss, 1-2 week-old chickens received a single injection of gentamicin (300 mg/kg). Chicks were sacrificed 24, 30, 42, 48, 72, or 96 h after injection. Cochleae were dissected and labeled for activated caspase-9 or caspase-3 using either caspase-directed antibodies or CaspaTag kits. Ears were co-labeled with either phalloidin or myosin VI to visualize hair cells and to determine the progression of cochlear damage. The timing of caspase activation was similar for both assays; however, caspase-9 and caspase-3 antibodies labeled only those cells currently undergoing apoptotic cell death. Conversely, CaspaTag-labeled all the cells that have undergone apoptotic cell death and ejection from the sensory epithelium, in addition to those that are currently in the cell death process. This makes CaspaTag ideal for showing an overall pattern or level of cell death over a period of time, while caspase antibodies provide a snapshot of cell death at a specific time point.