In Ovo and Ex Ovo Methods to Study Avian Inner Ear Development

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 placode^1,2. 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 .......

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 structures^35,<.......

Materials

Name	Company	Catalog Number	Comments
Alexa Fluor 488 Phalloidin	Thermo Fisher Scientific	A12379
Alexa Fluor 647 Phalloidin	Thermo Fisher Scientific	A22287
Alt-R S.p. HiFi Cas9 Nuclease V3	Integrated DNA Technologies	1081061	High fidelity Cas9 protein
Anti-GFP antibody	Abcam	ab290	Rabbit polyclonal to GFP
Bovine Serum Albumin	Sigma-Aldrich	A9647
Calcium Chloride Dihydrate	Thermo Fisher Scientific	Q12135
Collagen I, rat tail	Thermo Fisher Scientific	A1048301
Critical Point Dryer Leica EM CPD300	Leica
CUY-21 Electroporator	Nepagene
Dimethyl sulfoxide (DMSO)	Sigma-Aldrich	D8418
DM5000B Widefield Microscope	Leica
DMEM, high glucose, GlutaMAX Supplement, pyruvate	Thermo Fisher Scientific	10569010
Dumont #5 Forceps	Fine Science Tools	11251-20
Dumont #55 Forceps	Fine Science Tools	11255-20
Fast Green FCF	Sigma-Aldrich	F7252
Fluoroshield	Sigma-Aldrich	F6182
FLUOVIEW 3000 Laser Scanning Microscope	Olympus
Glutaraldehyde (25 %)	Sigma-Aldrich	340855
Goat anti-Mouse IgG Secondary Antibody, Alexa Fluor 488	Thermo Fisher Scientific	A-11001
Goat anti-Mouse IgG Secondary Antibody, Alexa Fluor 594	Thermo Fisher Scientific	A-11032
Goat anti-Rabbit IgG Secondary Antibody, Alexa Fluor 488	Thermo Fisher Scientific	A-11008
Goat Serum Sterile filtered	HiMedia	RM10701	Heat inactivated
Hanks' Balanced Salt Solution (HBSS)	Thermo Fisher Scientific	14025092
LSM980 Airyscan Microscope	Zeiss
Millicell Cell Culture Insert, 30 mm, hydrophilic PTFE, 0.4 µm	Sigma-Aldrich	PICM03050
MVX10 Stereo Microscope	Olympus
MYO7A antibody	DSHB	138-1	Mouse monoclonal to Unconventional myosin-VIIa
MZ16 Dissecting microscope	Leica
N-2 Supplement (100X)	Thermo Fisher Scientific	17502048
Noyes Scissors, 14cm (5.5'')	World Precision Instruments	501237
Osmium tetroxide (4%)	Sigma-Aldrich	75632
Paraformaldehyde	Sigma-Aldrich	158127
PC-10 Puller	Narishige
pcU6_1sgRNA	Addgene	92395	Mini vector with modified chicken U6 promoter
Penicillin G sodium salt	Sigma-Aldrich	P3032
Phosphate Buffered Saline (PBS)	Thermo Fisher Scientific	10010023
ProLong Gold Antifade Mountant	Thermo Fisher Scientific	P36934
SMZ1500 Dissecting microscope	Nikon
Sodium Cacodylate Buffer, 0.2M	Electron Microscopy Sciences	11652
Sodium chloride	HiMedia	GRM853
Sputtre Coater K550X	Emitech
Standard Glass Capillaries 3 in, OD 1.0 mm, No Filament	World Precision Instruments	1B100-3
Sucrose	Sigma-Aldrich	84097
The MERLIN Compact VP	Zeiss
Thiocarbohydrazide	Alfa Aesar	L01205
TWEEN 20	Sigma-Aldrich	P1379