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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.
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.
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....
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
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.......
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,<.......
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.
....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 |
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