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Adult mammalian tissues such as heart, brain, retina, and the sensory structures of the inner ear do not effectively regenerate, although a latent capacity for regeneration exists at embryonic and perinatal times. We explored the epigenetic basis for this latent regenerative potential in the mouse inner ear and its rapid loss during maturation. In perinatal supporting cells, whose fate is maintained by Notch-mediated lateral inhibition, the hair cell enhancer network is epigenetically primed (H3K4me1) but silenced (active H3K27 de-acetylation and trimethylation). Blocking Notch signaling during the perinatal period of plasticity rapidly eliminates epigenetic silencing and allows supporting cells to transdifferentiate into hair cells. Importantly, H3K4me1 priming of the hair cell enhancers in supporting cells is removed during the first post-natal week, coinciding with the loss of transdifferentiation potential. We hypothesize that enhancer decommissioning during cochlear maturation contributes to the failure of hair cell regeneration in the mature organ of Corti.

Proliferation and transdifferentiaton of supporting cells in the damaged auditory organ of birds lead to robust regeneration of sensory hair cells. In contrast, regeneration of lost auditory hair cells does not occur in deafened mammals, resulting in permanent hearing loss. In spite of this failure of regeneration in mammals, we have previously shown that the perinatal mouse supporting cells harbor a latent potential for cell division. Here we show that in a subset of supporting cells marked by p75, EGFR signaling is required for proliferation, and this requirement is conserved between birds and mammals. Purified p75 + mouse supporting cells express receptors and ligands for the EGF signaling pathway, and their proliferation in culture can be blocked with the EGFR inhibitor AG1478. Similarly, in cultured chicken basilar papillae, supporting cell proliferation in response to hair cell ablation requires EGFR signaling. In addition, we show that EGFR signaling in p75 + mouse supporting cells is required for the down-regulation of the cell cycle inhibitor p27 Kip1 (CDKN1b) to enable cell cycle re-entry. Taken together, our data suggest that a conserved mechanism involving EGFR signaling governs proliferation of auditory supporting cells in birds and mammals and may represent a target for future hair cell regeneration strategies.

SummaryThe organ of Corti, the auditory organ of the inner ear, contains two types of sensory hair cells and at least seven types of supporting cells. Most of these supporting cell types rely on Notch-dependent expression of Hes/Hey transcription factors to maintain the supporting cell fate. Here, we show that Notch signaling is not necessary for the differentiation and maintenance of pillar cell fate, that pillar cells are distinguished by Hey2 expression, and that—unlike other Hes/Hey factors—Hey2 expression is Notch independent. Hey2 is activated by FGF and blocks hair cell differentiation, whereas mutation of Hey2 leaves pillar cells sensitive to the loss of Notch signaling and allows them to differentiate as hair cells. We speculate that co-option of FGF signaling to render Hey2 Notch independent also liberated pillar cells from the need for direct contact with surrounding hair cells, and enabled evolutionary remodeling of the complex cellular mosaic of the inner ear.

The sensory hair cells and supporting cells of the organ of Corti are generated by a precise program of coordinated cell division and differentiation. Since no regeneration occurs in the mature organ of Corti, loss of hair cells leads to deafness. To investigate the molecular basis of hair cell differentiation and their lack of regeneration, we have established a dissociated cell culture system in which sensory hair cells and supporting cells can be generated from mitotic precursors. By incorporating a Math1-GFP transgene expressed exclusively in hair cells, we have used this system to characterize the conditions required for the growth and differentiation of hair cells in culture. These conditions include a requirement for epidermal growth factor, as well as the presence of periotic mesenchymal cells. Lastly, we show that early postnatal cochlear tissue also contains cells that can divide and generate new sensory hair cells in vitro.

Mice that lack caspase-3, which functions in apoptosis, were generated by gene targeting and shown to undergo hearing loss. The ABR threshold of the caspase-3(-/-) mice was significantly elevated compared to that of caspase-3(+/+) mice at 15 days of age and was progressively elevated further by 30 days. Distortion product otoacoustic emissions were not detectable in caspase-3(-/-) mice at 15 days of age. Caspase-3(-/-) mice exhibited marked degeneration of spiral ganglion neurons and a loss of inner and outer hair cells in the cochlea at 30 days of age, although no such changes were apparent at 15 days. The degenerating neurons manifested features, including cytoplasmic vacuolization, distinct from those characteristic of apoptosis. Spiral ganglion neurons and cochlear hair cells thus appear to require caspase-3 for survival but not for initial development. The mapping of both the human caspase-3 gene and the locus responsible for an autosomal dominant, nonsyndromic form of hearing loss (DFNA24) to chromosome 4q35 suggests that the caspase-3(-/-) mice may represent a model of this human condition.

The larynx of adult South African clawed frogs, Xenopus laevis, is larger in males than in females and hypertrophies in adult females and juveniles in response to androgen. Sexual dimorphism and androgen sensitivity suggest that the larynx is a testosterone target tissue. Saturation analysis of androgen (R1881) binding in laryngeal cytosol revealed an approximately threefold quantitative difference between male and female androgen-binding levels (36.4 vs 11.5 fm/mg protein). By contrast, as measured by one-point assay, androgen-binding levels in thigh muscle of either males or females were between 0 and 4 fm/mg protein with no apparent sex difference. Competition studies indicated that dihydrotestosterone was the most effective competitor for R1881 binding activity in the larynx. Saturation analysis showed the binding activity to be saturable and of high affinity (apparent Kd 0.46 nM in the male and 0.38 nM in the female). After 1 month of testosterone treatment, female binding levels averaged 16.6 fm/mg protein with a Kd of 0.49 nM, within the range for normal females. In males castrated for 4 months, binding levels were 52 fm/mg protein. After 1 year of castration, binding levels were 25 fm/mg protein. We conclude that laryngeal muscle is an androgen target tissue with sexually dimorphic levels of binding in adults.

1. 1. The kinetic characteristics of pyruvate kinase isozymes from oocytes, embryos, liver and skeletal muscle from the clawed frog Xenopus laevis were measured in cell extracts. 2. 2. The muscle and liver isozymes display Michaelis-Menten kinetics with Km s for phosphoenolpyruvate (PEP) of 0.02 and 0.05 mM, respectively. 3. 3. Pyruvate kinase from oocytes and embryos displays cooperative kinetics for PEP with a Km of about 0.15 mM; the kinetics become hyperbolic and the Km for PEP is reduced to 0.05 mM in the presence of μM concentrations of fructose-1,6-bisphosphate. 4. 4. These data serve to characterize pyruvate kinase activity in oocytes and embryos and the kinetics are compared to mammalian pyruvate kinase isozymes.

Whole pituitary glands, as well as equivalent-sized pieces of kidney, liver, and adrenal tissue, were implanted into the anterior chamber of the adult newt eye. Secondary lens formation was present in 16 out of 17 cases of the pituitary implantation experiments in which the pituitary tissues were exposed to the anterior chamber fluid. In only one case out of 23, did another implanted tissue (liver) stimulated secondary lens formation. The extent of secondary lens formation was correlated with the position (closeness) of the implant in relationship to the dorsal iris. In all cases of secondary lens formation from the dorsal iris stimulated by tissue implants, the polarity of the developing lens was reversed so that the pole of the fiber axis was directed toward the implant rather than the retina. It appears that the ectopic pituitary produces a diffusible substance capable of initiating lens formation from the dorsal margin of the iris and capable of determining the orientation of the fiber axis pole of the developing lens.

Development of the sexually dimorphic larynx in African clawed frogs is controlled by secretion of androgenic steroids (D. Sassoon and D. Kelley, 1986, Amer. J. Anat. 177, 457-472). Adult laryngeal muscle shows high levels of androgen binding relative to other skeletal muscles and binding activity in males is three times that in females (N. Segil, L. Silverman, and D. Kelley, 1987, Gen. Comp. Endocrinol. 66, 95-101). To determine when androgen sensitivity and sex differences arise, we assayed [3H]dihydrotestosterone (DHT) binding activity in larynges from metamorphic and postmetamorphic male and female frogs. Scatchard analyses indicate that DHT binds to a saturable component with high affinity. At metamorphosis, male and female juveniles have average binding levels of 262 and 269 fmoles/mg protein, respectively, approximately 7 to 20 times their adult values. At 3 months postmetamorphosis (PM), sexually dimorphic binding levels are observed. Binding activity declines gradually in females from metamorphosis to 9 months PM. In males, levels of binding activity remain high throughout the first 6 months PM and then decrease to near adult levels by 9 months PM. Administration of exogenous DHT to 3 months PM juveniles decreases average binding activity from 180 (male) or 74 fmoles/mg (female) to 33.5 fmoles/mg in both sexes. Testosterone has a less pronounced effect on binding activity in males than DHT and is ineffective in females. We conclude that sexually dimorphic adult levels of androgen binding in larynx arise by differential decrease from initially high, sexually monomorphic levels and that high titers of circulating androgens normally present by 6 months PM in males are responsible for the marked decrease in binding activity observed during laryngeal development.

We investigated a possible role for testosterone-induced cell proliferation in the development of sexual dimorphism in the larynx of South African clawed frogs, Xenopus laevis. Androgen-induced cell proliferation was studied using [3H]thymidine autoradiography. Nuclei of cartilage, perichondrium, and muscle were labeled in the larynx of sexually immature frogs of both sexes but not in adults. Cell proliferation did not occur with estradiol treatment nor was it seen in nonlaryngeal muscle or cartilage. Electron microscopic/autoradiographic studies of laryngeal muscle indicate that testosterone stimulates satellite cell division which later results in formation of myonuclei. We conclude that testosterone induces both chondrogenesis and myogenesis in juvenile larynx and that this process may contribute to the pronounced sexual dimorphism of the adult vocal organ.