Lens Transplantation in Zebrafish and its Application in the Analysis of Eye Mutants

Summary

Lens development involves interactions with other tissues. Several zebrafish eye mutants are characterized by an abnormally small lens size. Here we demonstrate a lens transplantation experiment to determine whether this phenotype is due to intrinsic causes or defective interactions with tissues that surround the lens.

Abstract

The lens plays an important role in the development of the optic cup^[1,2]. Using the zebrafish as a model organism, questions regarding lens development can be addressed. The zebrafish is useful for genetic studies due to several advantageous characteristics, including small size, high fecundity, short lifecycle, and ease of care. Lens development occurs rapidly in zebrafish. By 72 hpf, the zebrafish lens is functionally mature ^[3]. Abundant genetic and molecular resources are available to support research in zebrafish. In addition, the similarity of the zebrafish eye to those of other vertebrates provides basis for its use as an excellent animal model of human defects^[4-7]. Several zebrafish mutants exhibit lens abnormalities, including high levels of cell death, which in some cases leads to a complete degeneration of lens tissues ^[8].

To determine whether lens abnormalities are due to intrinsic causes or to defective interactions with the surrounding tissues, transplantation of a mutant lens into a wild-type eye is performed. Using fire-polished metal needles, mutant or wild-type lenses are carefully dissected from the donor animal, and transferred into the host. To distinguish wild-type and mutant tissues, a transgenic line is used as the donor. This line expresses membrane-bound GFP in all tissues, including the lens. This transplantation technique is an essential tool in the studies of zebrafish lens mutants.

Protocol

Part 1: Preparing the embryos

In this protocol, we will use the jj^xy symbol to denote a hypothetical zebrafish lens mutant.

1. Zebrafish strains are maintained in standard fish facility conditions at 28.5 C on a 14h light/10h dark cycle.
2. In the evening, place males and females of the zebrafish strain jj^xy:AB/TU; tg(mGFP) in a tank with a divider to separate them from each other. The progeny of this cross will express the GFP transgene in all tissues, and will be used as donors. In parallel, use the same procedure to set up crosses between non-transgenic wild-type animals. Dep....

Discussion

Several steps require special attention during lens transplantations.

1. Sharpened needles: It’s better to prepare several needles before carrying out lens transplantation, and the tip of the needles should be as thin as possible. A thicker needle will tear more tissue because of its wider diameter, causing a failure in the eye to heal.
2. Arranging the embryos: Right before transplantation you must position the embryos so they are lying on their sides. When positioning, the 1.2% agarose can .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.1mm diameter tungsten wire	A Johnson Matthey	45086
Agarose(low melting point)	Sigma-Aldrich	39346
Borosilicate glass capillaries	World Precision Instruments, Inc.	TW 100-4
Forceps	Dumont	11252-30
Pasteur pipette	Fisher Scientific	13-678-20C
Pipette pump	Fisher Scientific	13-683C
Petri Dish	Cardinal Health	D1906
Penicillin-streptomycin	GIBCO, by Life Technologies	15140-122