Differential expression of unconventional myosins in apoptotic and regenerating chick hair cells confirms two regeneration mechanisms

Abstract

Hair cells of the inner ear are damaged by intense noise, aging, and aminoglycoside antibiotics. Gentamicin causes oxidative damage to hair cells, inducing apoptosis. In mammals, hair cell loss results in a permanent deficit in hearing and balance. In contrast, avians can regenerate lost hair cells to restore auditory and vestibular function. This study examined the changes of myosin VI and myosin VIIa, two unconventional myosins that are critical for normal hair cell formation and function, during hair cell death and regeneration. During the late stages of apoptosis, damaged hair cells are ejected from the sensory epithelium. There was a 4-5-fold increase in the labeling intensity of both myosins and a redistribution of myosin VI into the stereocilia bundle, concurrent with ejection. Two separate mechanisms were observed during hair cell regeneration. Proliferating supporting cells began DNA synthesis 60 hours after gentamicin treatment and peaked at 72 hours postgentamicin treatment. Some of these mitotically produced cells began to differentiate into hair cells at 108 hours after gentamicin (36 hours after bromodeoxyuridine (BrdU) administration), as demonstrated by the colabeling of myosin VI and BrdU. Myosin VIIa was not expressed in the new hair cells until 120 hours after gentamicin. Moreover, a population of supporting cells expressed myosin VI at 78 hours after gentamicin treatment and myosin VIIa at 90 hours. These cells did not label for BrdU and differentiated far too early to be of mitotic origin, suggesting they arose by direct transdifferentiation of supporting cells into hair cells. Indexing terms aminoglycoside ototoxicity; bromodeoxyuridine; transdifferentiation; unconventional myosinsHair cells are the mechanoreceptors in the inner ear responsible for the senses of hearing and balance. They have a bundle of 20-300 actin-rich membrane-encased projections on their apical surface called stereocilia. The stereocilia are embedded in a dense actin-rich meshwork below the luminal surface of the hair cell called the cuticular plate. Sound is transferred into fluid-waves in the inner ear that deflect the stereocilia, thereby opening transduction channels. The potassium-rich endolymph enters the hair cell, changing the potential of the cell and *Correspondence to: Douglas A. Cotanche, Children's Hospital Boston, ORL Research, Enders 4th Fl., 300 Longwood Ave., Boston, MA 02115. E-mail: douglas.cotanche@childrens.harvard.edu. NIH Public Access NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript triggering a neural impulse in the afferent nerves at the bases of the hair cells (reviewed in Hackney and Furness, 1995;Corey, 2003). The structure of the avian auditory sensory epithelium is different from the mammalian cochlea in that it lacks the distinct rows of inner and outer hair cells, distinguishable types of supporting cells, and characteristic spiral shape.The structure and function of the sensory hair cells, however, are highly conserved across species, making the avian ...

Supporting Cell Division Is Not Required for Regeneration of Auditory Hair Cells After Ototoxic Injury In Vitro

“…In organs dissected from control chicks and fixed without culturing, strong myosinVI labeling normally occurs in HC cytoplasm and stereocilia but not in nonsensory SCs (Duncan et al 2006; Fig. 4A, C).…”

“…In vivo, a few SCs in the BP enter S phase around 2 days after aminoglycoside treatment, and SC divi-sion peaks a day later (Bhave et al 1995(Bhave et al , 1998Stone et al 1999;Duncan et al 2006). To determine if SCs enter S phase on a similar schedule after streptomycin-induced HC damage in vitro, BrdU (1 µM) was added to culture media (DMEM/1% FBS) for 4 h at 1, 2, 3, 5, or 7 days in vitro, and cultures were fixed and immunolabeled for BrdU.…”

Comparison of activated caspase detection methods in the gentamicin-treated chick cochlea

“…Many studies have characterized changes in avian cochlear hair cells following treatment with gentamicin both in vivo and in vitro. Redistribution of F-actin labeling (Mangiardi et al, 2004) and changes in myosin VI and VIIa distribution and intensity (Duncan et al, 2006) have been reported to occur in chick cochlear hair cells prior to and during the process of ejection from the basilar papilla following gentamicin exposure. Additionally, recent morphological and biochemical evidence suggests that a significant amount of aminoglycoside-induced hair cell death occurs via apoptosis (Forge, 1985;Li et al, 1995;Nakagawa et al, 1997;Torchinsky et al, 1999;Forge and Li, 2000;Cunningham et al, 2002;Matsui et al, 2002Matsui et al, , 2003Matsui et al, , 2004Cheng et al, 2003;Mangiardi et al, 2004;Duncan et al, 2006;Sugahara et al, 2006).…”

“…Redistribution of F-actin labeling (Mangiardi et al, 2004) and changes in myosin VI and VIIa distribution and intensity (Duncan et al, 2006) have been reported to occur in chick cochlear hair cells prior to and during the process of ejection from the basilar papilla following gentamicin exposure. Additionally, recent morphological and biochemical evidence suggests that a significant amount of aminoglycoside-induced hair cell death occurs via apoptosis (Forge, 1985;Li et al, 1995;Nakagawa et al, 1997;Torchinsky et al, 1999;Forge and Li, 2000;Cunningham et al, 2002;Matsui et al, 2002Matsui et al, , 2003Matsui et al, , 2004Cheng et al, 2003;Mangiardi et al, 2004;Duncan et al, 2006;Sugahara et al, 2006). Apoptosis appears to be the main mechanism of hair cell death in the avian basilar papilla following a single large dose of aminoglycosides (Matsui et al, 2002(Matsui et al, , 2003(Matsui et al, , 2004Cheng et al, 2003;Mangiardi et al, 2004;Duncan et al, 2006).…”

“…A single systemic injection of a high dose of gentamicin (an aminoglycoside antibiotic) can create a consistent pattern of hair cell death in the avian cochlea, with cell loss beginning in the basal (proximal) end of the basilar papilla and progressing apically (distally) with time (Bhave et al, 1995;Janas et al, 1995;Park et al, 1998;Roberson et al, 2000;Mangiardi et al, 2004;Duncan et al, 2006). Many studies have characterized changes in avian cochlear hair cells following treatment with gentamicin both in vivo and in vitro.…”