Microdissection of Zebrafish Embryonic Eye Tissues

Summary

This article describes an approach to microdissect zebrafish retinas with and without retinal pigment epithelium attached, from one to three days postfertilization embryos.

Abstract

Zebrafish is a popular animal model for research on eye development because of its rapid ex utero development and good fecundity. By 3 days post fertilization (dpf), the larvae will show the first visual response. Many genes have been identified to control a proper eye development, but we are far from a complete understanding of the underlying genetic architecture. Whole genome gene expression profiling is a useful tool to elucidate genetic regulatory network for eye development. However, the small size of the embryonic eye in zebrafish makes it challenging to obtain intact and pure eye tissues for expression analysis. For example, the anterior-posterior length of the eye between day 2 and 3 is only approximately 200-300 μm, while the diameter of the lens is less 100 μm. Also, the retinal pigment epithelium (RPE) underlying the retina is just a single-layer epithelium. While gene expression profiles can be obtained from the whole embryo, they do not accurately represent the expression of these tissues. Therefore pure tissue must be obtained for a successful gene expression profiling of eye development. To address this issue, we have developed an approach to microdissect intact retina and retina with RPE attached from 1-3 dpf, which cover major stages of eye morphogenesis. All procedures can be done with fine forceps and general laboratory supplies under standard stereomicroscopes. For retinal dissection, the single-layer RPE is removed and peeled off by brushing action and the preferential adherence of the RPE remnants to the surface of the culture plate for dissection. For RPE-attached retinal dissection, the adherence of RPE to the dissection plate is removed before the dissection so that the RPE can be completely preserved with the retina. A careful lifting action of this tissue can efficiently separate the presumptive choroid and sclera. The lens can be removed in both cases by a chemically etched tungsten needle. In short, our approach can obtain intact eye tissues and has been successfully utilized to study tissue-specific expression profiles of zebrafish retina^{1, 2} and retinal pigment epithelium³.

Protocol

Part 1: Preparations before microdissection

1. Solutions
   1. E3 medium⁴ (5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl₂ and 0.33 mM MgSO₄).
   2. Ringer's solution^{1, 5} (116 mM NaCl, 2.9 mM KCl, 1.8 mM CaCl₂, 5 mM HEPES, pH7.2), filter sterilized.
   3. 5N NaOH.
2. Chemical etching of tungsten needles
   1. Secure a small beaker containing 5N NaOH on a Petri dish by clay.
   2. Attach a paper clip on the side of the beaker, so that it comes in contact with the NaOH solution.
   3. Connect the negative electrode....

Discussion

Microdissection of zebrafish eye tissues can effectively obtain intact retinas and RPE-attached retinas. This substantially assists expression studies pertaining to a specific eye tissue (i.e. retina or RPE). In fact, we have successfully utilized these procedures to obtain RNA expression profiles of the whole retina¹ and RPE³. The utility of these profiles is strongly supported by our recent identification of pathways and gene families that are perturbed in a retinal differentiation mutant²

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Cordless pestle motor	VWR international	47747-370
DC power supply	Lascar	PSU130	Any DC supply would work. The specific voltage of a different machine will need further optimization.
Disposable pestle & microtube, 1.5 mL (DNase, RNase and pyrogen-free)	VWR international	47747-366	These are used for tissue collection in TRIzol for expression analysis.
Dumont #5 forceps, Tips: 0.05 x 0.01mm, Inox	World Precision Instruments, Inc.	500341	Fine tip dimension is desirable but is not inflexible, as one may need to sharpen the tip from time to time.
Dumont #5SF forceps, Tips: 0.025 x 0.005mm, Inox	Fine Science Tools	11252-00	Fine tip dimension is desirable but is not inflexible, as one may need to sharpen the tip from time to time.
Falcon polystyrene culture plates, 60 X 15 mm	BD Biosciences	351007	These plates are used as dissection plates.
Olympus SZX16 Stereomicroscope	Olympus Corporation	SZX16	Any stereomicroscope would work. We used Leica stereomicroscope in previous studies1-3 without any issues. We also use the 1X objective exclusively for the dissection even though we have a 2X objective installed.
Sharpening stone	Fine Science Tools	29008-01	Use this to sharpen the tip of the forceps if necessary
Thermo plate	Tokai Hit	MATS-U55SZX2B	This is used to maintain the temperature of the tissue throughout dissection and minimize the influence of temperature fluctuation on gene expression. We also put the whole microscope in an environmentally controlled room at 28°C during dissection in previous studies1-3 with good success.
Trizol, 100 mL	Invitrogen	15596-026
tungsten wire, 0.015 inch diameter	World Precision Instruments, Inc.	TGW1510
Wooden Applicator	Puritan	807	This is used for holding the chemically-etched tungsten needle.