Peter D Currie Thomas F Schilling and Philip W Ingham 1 Introduction

We describe a standardized mutagenic protocol and a methodology for small-scale directed screening of the zebrafish genome for mutations in specific developmental processes. The methods are based primarily on those developed for large-scale screens in Tubingen, Germany; Boston, MA; and Eugene, OR as well as our experiences with a smaller facility. By combining a marker-based screening protocol with both haploid and diploid screening methods, one can efficiently recover mutants in specific processes.

Random mutagenesis provides the ability to survey the genome of an organism, without bias, for genes that function in particular processes. For many years, geneticists have been reaping the rich harvest of mutations produced by such a mutagenic approach, directed against particular developmental processes of the fruit fly Drosophila melanogaster (1). Analysis of the genes uncovered by this approach has revolutionized our understanding of the genetic control of animal development.

Researchers eager to see a similar mutagenic approach applied to the vertebrate genome have been stymied by the genetic intractability of classical vertebrate developmental models. The mouse is the only vertebrate organism in which large-scale screens for mutations have been performed. These screens have, in the main, been limited to identification of defects in visible morphological traits after birth, since screening for embryonic mutant phenotypes is difficult because development occurs in utero (2). Biologists interested in using a mutagenic approach to study early aspects of vertebrate development have been forced to search for an alternative.

From: Methods in Molecular Biology, Vol. 97: Molecular Embryology: Methods and Protocols Edited by: P. T. Sharpe and I. Mason © Humana Press Inc., Totowa, NJ

The fishes represent the largest group of vertebrate taxa and perhaps the largest uncharted waters in terms of vertebrate developmental studies. Until recently, debates on the molecular mechanisms that govern vertebrate development have largely ignored fish. However, with the rediscovery that certain teleosts allow the application of both sophisticated embryological manipulations and classical genetic analysis, the stock of fish as a developmental system has risen. Specifically, studies on the zebrafish, Brachydanio rerio, have indicated its promise as both a genetic and an embryological model (3,4).

Zebrafish are small and have a short life cycle, reaching sexual maturity in three months. Females produce a large brood size, typically hundreds of eggs, which are fertilized externally. They are inexpensive to maintain and can be bred to great numbers easily. Embryonic stages are completely transparent, allowing most structures of the developing fish to be viewed without the aid of sophisticated microscopy. This last attribute has allowed researchers to gain an intricate knowledge of cell movements and behavior during early development. This information can now be coupled with detailed fate maps and a precise staging series, making zebrafish a sophisticated embryological model (5-7). As with some other teleosts, zebrafish can be induced to undergo either gyno-genetic or haploid development, abilities that greatly enhance its stock as a genetically manipulable model and make it unique among animal developmental systems. These attributes have attracted a number of laboratories to undertake large-scale mutagenic screens in an attempt to reach saturation for lethal mutations within the zebrafish genome (8,9, and Note 1).

The data published by these laboratories describe the general classes of mutations found using this approach (8,9). These include general necrosis, edema, brain necrosis, and general retardation. Mutations of these common classes account for up to two-thirds of the mutations uncovered and have, for the most part, been discarded by the large screens, since they represent an unwieldy task in the determination of individual complementation groups. Small-scale directed screening may reveal that a significant number of such mutations disrupt in specific developmental processes that can only be revealed by the use of gene or protein-specific markers, that is, marker-based screening.

The efficiency of different mutagenic agents in inducing mutations within the germline of zebrafish has also been assessed. These studies have relied largely on the vast literature concerning the efficient induction of mutations in mice by ethyl nitrosourea (ENU). Similarly, Mullins et al. (8) and Solnica-Krezel et al. (9) demonstrated that ENU induces mutations within male sper-matogonial cells at rates between 1/450 and 1/1000 for specific pigmentation genes in zebrafish. This range is typical for mutagenic rates at given loci for a number of mutations, and the rate is significantly higher than that produced in similar treatments with the other most frequently used alkylating agent, EMS

(8,9). Large-scale mutagenic screens have tended to focus on the use of chemical mutagens as their action is not site-specific and they usually induce lesions that are limited to single genes. However X-and y-rays are also efficient in inducing mutations within the zebrafish germline and are more applicable to other screening rationales (see ref. 10).

The two large scale mutagenic screens have both used a similar general methodology (see Fig. 1). Mutations are induced by ENU in G0 males and detected in the diploid offspring of the intercrossed F2, where the mutation has been driven to homozygosity. A "family" of F2 fish, half of which is heterozygous for a given mutation, is a significant advantage of a F2 diploid screen. It provides an immediate working stock from which to rescreen and recover mutants. Its major disadvantage is the large numbers of fish that have to be maintained and screened. The large screens have chosen not to use F1 screening via the induction of haploid or gynogenetic diploids because: (1) some strains do not consistently produce eggs for fertilization in vitro, and (2) the manipulations produce a high background of developmental defects.

Although these disadvantages may preclude the detection of every mutable locus within the zebrafish genome via F1 screening, those laboratories not concerned with such a goal can still effectively screen for mutations in a given developmental process.

Large-scale diploid screens require facilities and support beyond the scope of most laboratories. The flexibility of zebrafish as a genetic model makes it suitable for use in small-scale mutagenic screens directed to specific developmental processes. In particular, these attributes include:

1. The ability to screen for defects in haploid or gynogenetic diploid embryos. As outlined below, this greatly reduces the number of fish needed, since the offspring of F1 founders can be screened directly. Thus, even small facilities can survey a significant amount of the genome for particular types of mutations (3).

2. The optical transparency of the embryo allows the detection of gene-specific protein or mRNA markers in whole mounts and, therefore, allows a greater efficiency of screening by the direct detection of mutations that are involved in a given developmental process. The three major advantages of such an approach are:

a. The detection of subtle defects not visible by morphological screening.

b. The ability to direct a screen toward mutations that affect a process of interest. Thus, the use of tank space can be maximized by immediately discarding mutations in which phenotype is unrevealing, and there are no alterations to a given marker's distribution.

c. The ability to interpret better mutants identified by morphological inspection in which phenotype is unrevealing regarding gene function, but developmental markers are affected.

Generation Flowchart

Fig. 1. Flowchart of procedures for F1- and F2-based screening. Screening the progeny of F1 females saves the researcher one generation time and the space required for large families of F2 fish in diploid screening. Single mutations (*) are generated in the spermatogonia of G0 males, and these are mated (X) to wild-type females. Similar pairwise matings of F1 or F2 offspring are used to identify fish, male or female, carrying the mutation, and these fish must be kept separate during the screening. Time intervals are indicated alongside arrows, which are not drawn to scale.

Fig. 1. Flowchart of procedures for F1- and F2-based screening. Screening the progeny of F1 females saves the researcher one generation time and the space required for large families of F2 fish in diploid screening. Single mutations (*) are generated in the spermatogonia of G0 males, and these are mated (X) to wild-type females. Similar pairwise matings of F1 or F2 offspring are used to identify fish, male or female, carrying the mutation, and these fish must be kept separate during the screening. Time intervals are indicated alongside arrows, which are not drawn to scale.

Was this article helpful?

0 0

Post a comment