RESUMO
To build a functional inner ear, hair cell morphology must be precisely controlled along the proximo-distal axis. A new paper in Development shows that differential mitochondrial dynamics in proximal versus distal cells impacts on the apical cell surface area - a key aspect of morphology. To find out more about this work, we spoke to first author James O'Sullivan and senior author Zoë Mann, both at King's College London, UK.
Assuntos
Biologia do Desenvolvimento , Animais , Humanos , Biologia do Desenvolvimento/história , Células Ciliadas Auditivas/citologia , História do Século XXI , História do Século XX , Orelha Interna/embriologia , Orelha Interna/citologia , Dinâmica MitocondrialRESUMO
In vivo gene delivery to tissues using adeno-associated vector (AAVs) has revolutionized the field of gene therapy. Yet, while sensorineural hearing loss is one of the most common sensory disorders worldwide, gene therapy applied to the human inner ear is still in its infancy. Recent advances in the development recombinant AAVs have significantly improved their cell tropism and transduction efficiency across diverse inner ear cell types to a level that renders this tool valuable for conditionally manipulating gene expression in the context of developmental biology studies of the mouse inner ear. Here, we describe a protocol for in utero micro-injection of AAVs into the embryonic inner ear, using the AAV-PHP.eB and AAV-DJ serotypes that respectively target the sensory hair cells and the supporting cells of the auditory sensory epithelium. We also aimed to standardize procedures for imaging acquisition and image analysis to foster research reproducibility and allow accurate comparisons between studies. We find that AAV-PHP.eB and AAV-DJ provide efficient and reliable tools for conditional gene expression targeting cochlear sensory and supporting cells in the mouse inner ear, from late embryonic stages on.
Assuntos
Dependovirus , Orelha Interna , Técnicas de Transferência de Genes , Vetores Genéticos , Animais , Dependovirus/genética , Camundongos , Orelha Interna/metabolismo , Orelha Interna/embriologia , Orelha Interna/citologia , Vetores Genéticos/genética , Vetores Genéticos/administração & dosagem , Feminino , Transdução Genética/métodos , Gravidez , Terapia Genética/métodos , HumanosRESUMO
Death of mechanosensory hair cells in the inner ear is a common cause of auditory and vestibular impairment in mammals, which have a limited ability to regrow these cells after damage. In contrast, non-mammalian vertebrates, including zebrafish, can robustly regenerate hair cells after severe organ damage. The zebrafish inner ear provides an understudied model system for understanding hair cell regeneration in organs that are highly conserved with their mammalian counterparts. Here, we quantitatively examine hair cell addition during growth and regeneration of the larval zebrafish inner ear. We used a genetically encoded ablation method to induce hair cell death and we observed gradual regeneration with correct spatial patterning over a 2-week period following ablation. Supporting cells, which surround and are a source of new hair cells, divide in response to hair cell ablation, expanding the possible progenitor pool. In parallel, nascent hair cells arise from direct transdifferentiation of progenitor pool cells temporally uncoupled from supporting cell division. These findings reveal a previously unrecognized mechanism of hair cell regeneration with implications for how hair cells may be encouraged to regenerate in the mammalian ear.
Assuntos
Transdiferenciação Celular , Orelha Interna , Células Ciliadas Auditivas , Regeneração , Células-Tronco , Peixe-Zebra , Animais , Regeneração/fisiologia , Orelha Interna/citologia , Células-Tronco/citologia , Células Ciliadas Auditivas/citologia , Células Ciliadas Auditivas/fisiologia , Proteínas de Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genética , Animais Geneticamente Modificados , Larva/citologiaRESUMO
Mammalian inner ear hair cell loss leads to permanent hearing and balance dysfunction. In contrast to the cochlea, vestibular hair cells of the murine utricle have some regenerative capacity. Whether human utricular hair cells regenerate in vivo remains unknown. Here we procured live, mature utricles from organ donors and vestibular schwannoma patients, and present a validated single-cell transcriptomic atlas at unprecedented resolution. We describe markers of 13 sensory and non-sensory cell types, with partial overlap and correlation between transcriptomes of human and mouse hair cells and supporting cells. We further uncover transcriptomes unique to hair cell precursors, which are unexpectedly 14-fold more abundant in vestibular schwannoma utricles, demonstrating the existence of ongoing regeneration in humans. Lastly, supporting cell-to-hair cell trajectory analysis revealed 5 distinct patterns of dynamic gene expression and associated pathways, including Wnt and IGF-1 signaling. Our dataset constitutes a foundational resource, accessible via a web-based interface, serving to advance knowledge of the normal and diseased human inner ear.
Assuntos
Regeneração , Análise de Célula Única , Transcriptoma , Humanos , Animais , Regeneração/genética , Camundongos , Sáculo e Utrículo/metabolismo , Sáculo e Utrículo/citologia , Neuroma Acústico/genética , Neuroma Acústico/metabolismo , Neuroma Acústico/patologia , Orelha Interna/metabolismo , Orelha Interna/citologia , Fator de Crescimento Insulin-Like I/metabolismo , Fator de Crescimento Insulin-Like I/genética , Masculino , Células Ciliadas Vestibulares/metabolismo , Feminino , Perfilação da Expressão GênicaRESUMO
The planar polarized organization of hair cells in the vestibular maculae is unique because these sensory organs contain two groups of cells with oppositely oriented stereociliary bundles that meet at a line of polarity reversal (LPR). EMX2 is a transcription factor expressed by one hair cell group that reverses the orientation of their bundles, thereby forming the LPR. We generated Emx2-CreERt2 transgenic mice for genetic lineage tracing and demonstrate Emx2 expression before hair cell specification when the nascent utricle and saccule constitute a continuous prosensory domain. Precursors labeled by Emx2-CreERt2 at this stage give rise to hair cells located along one side of the LPR in the mature utricle or saccule, indicating that this boundary is first established in the prosensory domain. Consistent with this, Emx2-CreERt2 lineage tracing in Dreher mutants, where the utricle and saccule fail to segregate, labels a continuous field of cells along one side of a fused utriculo-saccular-cochlear organ. These observations reveal that LPR positioning is pre-determined in the developing prosensory domain, and that EMX2 expression defines lineages of hair cells with oppositely oriented stereociliary bundles.