National Centre for Biological Sciences, Tata Institute of Fundamental Research; The University of Trans-Disciplinary Health Sciences and Technology.
National Centre for Biological Sciences, Tata Institute of Fundamental Research.
J Vis Exp. 2022 Jun 16(184). doi: 10.3791/64172.
The inner ear perceives sound and maintains balance using the cochlea and vestibule. It does this by using a dedicated mechanosensory cell type known as the hair cell. Basic research in the inner ear has led to a deep understanding of how the hair cell functions, and how dysregulation can lead to hearing loss and vertigo. For this research, the mouse has been the pre-eminent model system. However, mice, like all mammals, have lost the ability to replace hair cells. Thus, when trying to understand cellular therapies for restoring inner ear function, complementary studies in other vertebrate species could provide further insights. The auditory epithelium of birds, the basilar papilla (BP), is a sheet of epithelium composed of mechanosensory hair cells (HCs) intercalated by supporting cells (SCs). Although the anatomical architecture of the basilar papilla and the mammalian cochlea differ, the molecular mechanisms of inner ear development and hearing are similar. This makes the basilar papilla a useful system for not only comparative studies but also to understand regeneration. Here, we describe dissection and manipulation techniques for the chicken inner ear. The technique shows genetic and small molecule inhibition methods, which offer a potent tool for studying the molecular mechanisms of inner ear development. In this paper, we discuss in ovo electroporation techniques to genetically perturb the basilar papilla using CRIPSR-Cas9 deletions, followed by dissection of the basilar papilla. We also demonstrate the BP organ culture and optimal use of culture matrices, to observe the development of the epithelium and the hair cells.
内耳通过耳蜗和前庭感知声音并维持平衡。它通过使用一种称为毛细胞的专门机械感觉细胞类型来实现这一点。内耳的基础研究使我们深入了解了毛细胞的功能,以及调控失调如何导致听力损失和眩晕。为此,老鼠一直是卓越的模式系统。然而,老鼠和所有哺乳动物一样,已经失去了替换毛细胞的能力。因此,当试图了解用于恢复内耳功能的细胞治疗时,在其他脊椎动物物种中的互补研究可能会提供进一步的见解。鸟类的听觉上皮,即基底乳头(BP),是由机械敏感的毛细胞(HCs)和支持细胞(SCs)穿插而成的上皮片。尽管基底乳头的解剖结构和哺乳动物的耳蜗不同,但内耳发育和听力的分子机制是相似的。这使得基底乳头不仅成为比较研究的有用系统,而且还可以理解再生。在这里,我们描述了鸡内耳的解剖和操作技术。该技术展示了遗传和小分子抑制方法,为研究内耳发育的分子机制提供了有力的工具。在本文中,我们讨论了在鸡胚中用电穿孔技术对基底乳头进行基因干扰的 CRISPR-Cas9 缺失,然后对基底乳头进行解剖。我们还展示了基底乳头器官培养和最佳使用培养基质,以观察上皮和毛细胞的发育。