Eye Removal in Living Zebrafish Larvae to Examine Innervation-dependent Growth and Development of the Visual System

Summary

The article explains how to surgically remove eyes from living zebrafish larvae as the first step toward investigating how retinal input influences optic tectum growth and development. In addition, the article provides information about larval anesthetization, fixation, and brain dissection, followed by immunohistochemistry and confocal imaging.

Abstract

Zebrafish exhibit remarkable life-long growth and regenerative abilities. For example, specialized stem cell niches established during embryogenesis support continuous growth of the entire visual system, both in the eye and the brain. Coordinated growth between the retinae and the optic tectum ensures accurate retinotopic mapping as new neurons are added in the eyes and brain. To address whether retinal axons provide crucial information for regulating tectal stem and progenitor cell behaviors such as survival, proliferation, and/or differentiation, it is necessary to be able to compare innervated and denervated tectal lobes within the same animal and across animals.

Surgical removal of one eye from living larval zebrafish followed by observation of the optic tectum achieves this goal. The accompanying video demonstrates how to anesthetize larvae, electrolytically sharpen tungsten needles, and use them to remove one eye. It next shows how to dissect brains from fixed zebrafish larvae. Finally, the video provides an overview of the protocol for immunohistochemistry and a demonstration of how to mount stained embryos in low-melting-point agarose for microscopy.

Introduction

The goal of this method is to investigate how retinal input influences the growth and development of the optic tectum, the visual processing center in the zebrafish brain. By removing one eye and then comparing the two sides of the optic tectum, tectal changes within the same specimen can be observed and normalized, enabling comparison across multiple specimens. Modern molecular approaches combined with this technique will yield insights into the mechanisms underlying visual system growth and development, as well as axonal degeneration and regeneration.

Sensory systems - visual, auditory, and somatosensory - gather information from external....

Protocol

The methods in this paper were conducted in accordance with guidelines and approval of the Institutional Animal Care and Use Committees of Reed College and University College London. See the Table of Materials for details about zebrafish strains used in this study.

1. Prepare materials and tools

1. Make solutions.
   1. Make embryo medium (E3¹⁴) by diluting a 60x stock (300 mM NaCl, 10.2 mM KCl, 20 mM CaCl₂-dihydrate, and 20 mM MgCl₂-hexahydrate in deionized water, autoclaved, and stored at room temperature). Add 160 mL of 60x E3 to 10 L of deioni....

Results

To confirm whether eye removal was complete and assess how the optic tectum changes, surgeries were performed in the Tg[atoh7:RFP] strain, which labels all RGCs with a membrane-targeted RFP and, thus, all axons that project from the retina and form the optic nerve²⁴. Although using this strain is not absolutely necessary, it enables straightforward observation and visualization of the optic nerve termini in the optic tectum neuropil. Other approaches for labeling the optic nerve, such as .......

Discussion

The techniques described in this paper illustrate one of many approaches for studying vertebrate visual system development in zebrafish. Other researchers have published methods to dissect the embryonic retina and perform gene expression analyses¹⁹ or visualize neuronal activity in the optic tectum³⁰. This paper provides an approach for exploring how differential retinal input may influence cell behaviors in the optic tectum.

To ensure successful.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Equipment and supplies:
Breeding boxes	Aquaneering	ZHCT100
Dow Corning high vacuum grease	Sigma or equivalent supplier	Z273554
Erlenmeyer flasks (125 mL)			For making Marc's Modified Ringers (MMR) with antibiotics for post-surgery incubation.
Fine forceps - Dumont #5	Fine Science Tools (FST)	11252-20
Glass Pasteur pipettes	DWK Lifescience	63A53 & 63A53WT	For pipetting embryos and larvae.
Glass slides for microscopy	VWR or equivalent supplier	48311-703	Standard glass microscope slides can be ordered from many different laboratory suppliers.
Glassware including graduated bottles and graduated cylinders			For making and storing solutions.
2-part epoxy resin	ACE Hardware or other equivalent supplier of Gorilla Glue or equivalent	0.85 oz syringe	https://www.acehardware.com/departments/paint-and-supplies/tape-glues-and-adhesives/glues-and-epoxy/1590793
Microcentrifuge tube (1.7 mL)	VWR or equivalent supplier	22234-046
Nickel plated pin holder (17 cm length)	Fine Science Tools (FST)	26018-17	To hold tungsten wire while sharpening and performing surgeries/dissections.
Nylon mesh tea strainer or equivalent	Ali Express or equivalent		For harvesting zebrafish eggs after spawning; https://www.aliexpress.com/item/1005002219569756.html
Paper clip			For Tungsten needle sharpening device.
Petri dishes 100 mm	Fischer Scientific or equivalent supplier	50-190-0267
Petri dishes 35 mm	Fischer Scientific or equivalent supplier	08-757-100A
Pipette pump	SP Bel-Art or equivalent	F37898-0000
Potassium hydroxide (KOH)	Sigma	909122	For Tungsten needle sharpening device. Make a 10% w/v solution of KOH in the hood by adding pellets to deionized water.
Power supply (variable voltage)			For Tungsten needle sharpening device. Any power supply with variable voltage will work (even one used for gel electrophoresis).
Sylgard 184 Elastomer kit	Dow Corning	3097358
Tungsten wire (0.125 mm diameter)	World Precision Instruments (WPI)	TGW0515	Sharpen to remove eye and dissect larvae.
Variable temperature heat block	The Lab Depot or equivalent supplier	BSH1001 or BSH1002	Set to 40-42 °C ahead of experiments.
Wide-mouth glass jar with lid (e.g., clean jam or salsa jar)			For Tungsten needle sharpening device.
Wires with alligator clip leads			For Tungsten needle sharpening device.
Microscopes:
Dissecting microscope			Any type will work but having adjustable transmitted light on a mirrored base is preferred.
Laser scanning confocal microscope			High NA, 20-25x water dipping objective lens is recommended. Microscope control and image capture software (NIS-Elements by Nikon) is used here but any confocal microscope will work.
Reagents for surgeries and dissections:
Calcium chloride dihydrate	Sigma	C7902	For Marc's Modified Ringers (MMR) and embryo medium (E3).
HEPES	Sigma	H7006	For Marc's Modified Ringers (MMR).
Low melting point agarose	Invitrogen	16520-050	Make 1% in embryo medium (E3) or Marc's Modified Ringers (MMR).
Magnesium chloride hexahydrate	Sigma	1374248	For embryo medium (E3).
Magnesium sulfate	Sigma	M7506	For Marc's Modified Ringers (MMR).
Paraformaldehyde	Electron Microscopy Sciences	19210	Dilute 8% (w/v) stock with 2x concentrated PBS (diluted from 10x PBS stock).
Penicillin/Streptomycin	Sigma	P4333-20ML	Dilute 1:100 in Marc's Modified Ringers.
Phosphate buffered saline (PBS) tablets	Diagnostic BioSystems	DMR E404-01	Make 10x stock in deionized water, autoclave and store at room temperature. Dilute to 1x working concentration.
Potassium chloride	Sigma	P3911	For Marc's Modified Ringers (MMR) and embryo medium (E3).
Sodium chloride	Sigma	S9888	For Marc's Modified Ringers (MMR) and embryo medium (E3).
Sodium hydroxide	Sigma	S5881	Make 10 M and use to adjust pH of MMR to 7.4.
Sucrose	Sigma	S9378
Tricaine-S	Pentair	100G #TRS1	Recipe: https://zfin.atlassian.net/wiki/spaces/prot/pages/362220023/TRICAINE
Reagents for immunohistochemistry:
Alexafluor 568 tagged Secondary antibody to detect rabbit IgG	Invitrogen	A-11011	Use at 1:500 dilution for wholemount immunohistochemistry.
DAPI or ToPro3	Invitrogen	1306 or T3605	Make up 1 mg/mL solutions in DMSO; 1:5,000 dilution for counterstaining.
Dimethyl sulfoxide (DMSO)	Sigma	D8418	A component of immunoblock buffer.
Methanol (MeOH)	Sigma	34860	Mixing MeOH with aqueous solutions like PBST is exothermic. Make the MeOH/PBST solutions at least several hours ahead of time or cool them on ice before using.
Normal goat serum	ThermoFisher Scientific	50-062Z	A component of immunoblock buffer. Can be aliquoted in 1-10 mL volumes and stored at -20 °C.
Primary antibody to detect phosphohistone H3	Millipore	06-570	Use at 1:300 dilution for wholemount immunohistochemistry.
Primary antibody to detect Red Fluorescent Protein (RFP; detects dsRed derivatives)	MBL International	PM005	Use at 1:500 dilution for wholemount immunohistochemistry.
Proteinase K (PK)	Sigma	P2308-10MG	Make up 10 mg/mL stock solutions in PBS and use at 10 µg/mL.
Triton X-100	Sigma	T8787	Useful to make a 20% (v/v) stock solution in PBS.
Software for data analysis
ImageJ (Fiji)			Freeware for image analysis; https://imagej.net/software/fiji/
RStudio			Freeware for statistical analysis and data visualization; https://www.rstudio.com/products/rstudio/download/
Adobe Photoshop or GIMP			Proprietary image processing software (Adobe Photoshop and Illustrator) are often used to compose figures). A freeware alternative is Gnu Image Manipulation Program (GIMP; https://www.gimp.org/)
Zebrafish strains. This study used the AB, TU, Tg[atoh7:RFP] strains.			Available from the  Zebrafish International Resource Centers in the US (https://zebrafish.org/home/guide.php) or in Europe (https://www.ezrc.kit.edu/). Specialized transgenic strains that have not yet been deposited in either resource center can be requested from individual labs after publication.