Diphtheria Toxin-Induced Cell Death Triggers Wnt-Dependent Hair Cell Regeneration in Neonatal Mice.
Cochlear hair cells (HCs), the sensory cells that respond to sound, do not regenerate after damage in adult mammals, and their loss is a major cause of deafness. Here we show that HC regeneration in newborn mouse ears occurred spontaneously when the original cells were ablated by treatment with diphtheria toxin (DT) in ears that had been engineered to overexpress the DT receptor, but was not detectable when HCs were ablated in vivo by the aminoglycoside antibiotic neomycin. A variety of Wnts (Wnt1, Wnt2, Wnt2b, Wnt4, Wnt5a, Wnt7b, Wnt9a, Wnt9b, and Wnt11) and Wnt pathway component Krm2 were upregulated after DT damage. Nuclear β-catenin was upregulated in HCs and supporting cells of the DT-damaged cochlea. Pharmacological inhibition of Wnt decreased spontaneous regeneration, confirming a role of Wnt signaling in HC regeneration. Inhibition of Notch signaling further potentiated supporting cell proliferation and HC differentiation that occurred spontaneously. The absence of new HCs in the neomycin ears was correlated to less robust Wnt pathway activation, but the ears subjected to neomycin treatment nonetheless showed increased cell division and HC differentiation after subsequent forced upregulation of β-catenin. These studies suggest, first, that Wnt signaling plays a key role in regeneration, and, second, that the outcome of a regenerative response to damage in the newborn cochlea is determined by reaching a threshold level of Wnt signaling rather than its complete absence or presence.



Sensory HCs of the inner ear do not regenerate in the adult, and their loss is a major cause of deafness. We found that HCs regenerated spontaneously in the newborn mouse after diphtheria toxin (DT)-induced, but not neomycin-induced, HC death. Regeneration depended on activation of Wnt signaling, and regeneration in DT-treated ears correlated to a higher level of Wnt activation than occurred in nonregenerating neomycin-treated ears. This is significant because insufficient regeneration caused by a failure to reach a threshold level of signaling, if true in the adult, has the potential to be exploited for development of clinical approaches for the treatment of deafness caused by HC loss.
Ear Institute, Shanghai Key Laboratory of Translational Medicine on Ear and Nose Diseases, Department of Otolaryngology-Head and Neck Surgery, Xinhua Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China, Department of Otology and Laryngology, Harvard Medical School, Boston, Massachusetts 02115, and Eaton-Peabody Laboratory, Massachusetts Eye and Ear Infirmary, Boston, Massachusetts 02114.
Lingxiang
L
Hu
Ear Institute, Shanghai Key Laboratory of Translational Medicine on Ear and Nose Diseases, Department of Otolaryngology-Head and Neck Surgery, Xinhua Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China, Department of Otology and Laryngology, Harvard Medical School, Boston, Massachusetts 02115, and Eaton-Peabody Laboratory, Massachusetts Eye and Ear Infirmary, Boston, Massachusetts 02114.
Jingrong
J
Lu
Department of Otology and Laryngology, Harvard Medical School, Boston, Massachusetts 02115, and Eaton-Peabody Laboratory, Massachusetts Eye and Ear Infirmary, Boston, Massachusetts 02114.
Hao
0000-0002-5919-3586
H
Chiang
Ear Institute, Shanghai Key Laboratory of Translational Medicine on Ear and Nose Diseases, Department of Otolaryngology-Head and Neck Surgery, Xinhua Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China, albert_edge@meei.harvard.edu wuhao622@sina.cn.
Hao
H
Wu
Department of Otology and Laryngology, Harvard Medical School, Boston, Massachusetts 02115, and Eaton-Peabody Laboratory, Massachusetts Eye and Ear Infirmary, Boston, Massachusetts 02114 albert_edge@meei.harvard.edu wuhao622@sina.cn.
Albert S B
0000-0001-8641-755X
AS
Edge
Department of Otology and Laryngology, Harvard Medical School, Boston, Massachusetts 02115, and Eaton-Peabody Laboratory, Massachusetts Eye and Ear Infirmary, Boston, Massachusetts 02114.
Fuxin
F
Shi
D000367:Age Factors; D000818:Animals; D000831:Animals, Newborn; D017209:Apoptosis / Q000187:drug effects / Q000235:genetics; D052160:Benzothiazoles / Q000494:pharmacology; D016923:Cell Death / Q000187:drug effects* / Q000235:genetics; D002454:Cell Differentiation / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D004167:Diphtheria Toxin / Q000633:toxicity*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D018507:Gene Expression Regulation, Developmental / Q000187:drug effects*; D006198:Hair Cells, Auditory / Q000187:drug effects*; D066257:Heparin-binding EGF-like Growth Factor / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008822:Mice, Transgenic; D009355:Neomycin / Q000494:pharmacology; D011042:Poisons / Q000633:toxicity*; D012038:Regeneration / Q000187:drug effects* / Q000235:genetics; D055748:SOXB1 Transcription Factors / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D051153:Wnt Proteins / Q000378:metabolism*; D051176:beta Catenin / Q000235:genetics / Q000378:metabolism
D052160:Benzothiazoles; D004268:DNA-Binding Proteins; D004167:Diphtheria Toxin; D004791:Enzyme Inhibitors; C079873:Gfi1 protein, mouse; D066257:Heparin-binding EGF-like Growth Factor; C000611186:IWP-2 compound; D011042:Poisons; D055748:SOXB1 Transcription Factors; C527783:Sox2 protein, mouse; D014157:Transcription Factors; D051153:Wnt Proteins; D051176:beta Catenin; D009355:Neomycin
Wnt; apoptosis; hair cell; necrosis; regeneration