Sign In

A subscription to JoVE is required to view this content. Sign in or start your free trial.

In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

The chick is a cost-effective, accessible, and widely available model organism for a variety of studies. Here, a series of protocols is detailed to understand the molecular mechanisms underlying avian inner ear development and regeneration.

Abstract

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.

Introduction

The inner ear of all vertebrates is derived from a simple epithelium known as the otic placode1,2. This will give rise to all the structural elements and the cell types necessary to transduce the mechanosensory information associated with hearing and balance perception. Hair cells (HCs), the ciliated sensor of the inner ear, are surrounded by supporting cells (SCs). HCs relay information to the auditory hindbrain through the neurons of the eighth cranial nerve. These are also generated from the otic placode3. The primary transduction of sound is achieved at the apical surface of the aud....

Protocol

Protocols involving the procurement, culture, and use of fertilized chicken eggs and unhatched embryos were approved by the Institutional Animal Ethics Committee of the National Centre for Biological Sciences, Bengaluru, Karnataka.

1. In ovo electroporation of chick auditory precursors

  1. sgRNA design and cloning for CRISPR/Cas9 gene knockout
    1. For creating gene knockouts, design guide RNAs to disrupt the exon regions of the gene, preferably closer .......

Representative Results

In the electroporation setup, electrode positioning can play a role in the domain of transfection. The positive electrode is placed under the yolk, and the negative above the embryo (Figure 1A). This results in higher GFP expression in much of the inner ear and both vestibular organs (Figure 1B), and auditory basilar papilla (Figure 1C,D), confirming transfection.

In assessing the phenoty.......

Discussion

The chick is a cost-effective and convenient addition to the model organisms that a lab may use to research the inner ear. The methods described here are routinely used in our lab and complement ongoing research in the mammalian inner ear. In ovo electroporation is used to introduce genetic manipulations into the chick genome. Electroporation can also be used to introduce constructs that encode fluorescent proteins targeted to particular organelles or subcellular structures35,<.......

Acknowledgements

We gratefully acknowledge support from NCBS, TIFR, Infosys-TIFR Leading Edge Research Grant, DST-SERB, and the Royal National Institute for the Deaf. We would like to thank Central Poultry Development Organization and Training Institute, Hesaraghatta, Bengaluru. We are grateful to CIFF and EM facility and lab support at NCBS. We thank Yoshiko Takahashi and Koichi Kawakami for the Tol2-eGFP and T2TP constructs, and Guy Richardson for HCA and G19 Pcdh15 antibody. We are grateful to Earlab members for their constant support and valuable feedback on the protocol.

....

Materials

NameCompanyCatalog NumberComments
Alexa Fluor 488 PhalloidinThermo Fisher ScientificA12379
Alexa Fluor 647 PhalloidinThermo Fisher ScientificA22287
Alt-R S.p. HiFi Cas9 Nuclease V3Integrated DNA Technologies1081061High fidelity Cas9 protein
Anti-GFP antibodyAbcamab290Rabbit polyclonal to GFP
Bovine Serum AlbuminSigma-AldrichA9647
Calcium Chloride DihydrateThermo Fisher ScientificQ12135
Collagen I, rat tailThermo Fisher ScientificA1048301
Critical Point Dryer Leica EM CPD300Leica
CUY-21 ElectroporatorNepagene
Dimethyl sulfoxide (DMSO)Sigma-AldrichD8418
DM5000B Widefield MicroscopeLeica
DMEM, high glucose, GlutaMAX Supplement, pyruvateThermo Fisher Scientific10569010
Dumont #5 ForcepsFine Science Tools11251-20
Dumont #55 ForcepsFine Science Tools11255-20
Fast Green FCFSigma-AldrichF7252
FluoroshieldSigma-AldrichF6182
FLUOVIEW 3000 Laser Scanning MicroscopeOlympus
Glutaraldehyde (25 %)Sigma-Aldrich340855
Goat anti-Mouse IgG Secondary Antibody, Alexa Fluor 488Thermo Fisher ScientificA-11001
Goat anti-Mouse IgG Secondary Antibody, Alexa Fluor 594Thermo Fisher ScientificA-11032
Goat anti-Rabbit IgG Secondary Antibody, Alexa Fluor 488Thermo Fisher ScientificA-11008
Goat Serum Sterile filteredHiMediaRM10701Heat inactivated
Hanks' Balanced Salt Solution (HBSS)Thermo Fisher Scientific14025092
LSM980 Airyscan MicroscopeZeiss
Millicell Cell Culture Insert, 30 mm, hydrophilic PTFE, 0.4 µmSigma-AldrichPICM03050
MVX10 Stereo MicroscopeOlympus
MYO7A antibodyDSHB138-1Mouse monoclonal to Unconventional myosin-VIIa
MZ16 Dissecting microscopeLeica
N-2 Supplement (100X)Thermo Fisher Scientific17502048
Noyes Scissors, 14cm (5.5'')World Precision Instruments501237
Osmium tetroxide (4%)Sigma-Aldrich75632
ParaformaldehydeSigma-Aldrich158127
PC-10 PullerNarishige
pcU6_1sgRNAAddgene92395Mini vector with modified chicken U6 promoter
Penicillin G sodium saltSigma-AldrichP3032
Phosphate Buffered Saline (PBS)Thermo Fisher Scientific10010023
ProLong Gold Antifade MountantThermo Fisher ScientificP36934
SMZ1500 Dissecting microscopeNikon
Sodium Cacodylate Buffer, 0.2MElectron Microscopy Sciences11652
Sodium chlorideHiMediaGRM853
Sputtre Coater K550XEmitech
Standard Glass Capillaries 3 in, OD 1.0 mm, No FilamentWorld Precision Instruments1B100-3
SucroseSigma-Aldrich84097
The MERLIN Compact VPZeiss
ThiocarbohydrazideAlfa AesarL01205
TWEEN 20Sigma-AldrichP1379

References

  1. Sai, X., Ladher, R. K. Early steps in inner ear development: induction and morphogenesis of the otic placode. Frontiers in Pharmacology. 6, 19 (2015).
  2. Groves, A. K., Fekete, D. M. Shaping sound in space: ....

Explore More Articles

Avian Inner EarHair Cell RegenerationChick EmbryoOtotoxicityGene KnockoutOtic VesicleElectroporationT7 Endonuclease Assay

This article has been published

Video Coming Soon

JoVE Logo

Privacy

Terms of Use

Policies

Research

Education

ABOUT JoVE

Copyright © 2024 MyJoVE Corporation. All rights reserved