From Phage to Fish

In the late 1960s George Streisinger began working with zebrafish at the University of Oregon with the goal of devel oping a vertebrate genetic model system comparable to the fly. Streisinger had worked with bacteriophage at Caltech during the critical foundation period of the molecular era, when the basic DNA-RNA-protein story was being established. His studies at Caltech involved a mutational approach to evaluate the genetic code, including its structure and the process of translation in the phage. These studies helped show that in vitro proposed codons did translate to in vivo specification of amino acid sequences based on specific base sequences. The success of genetic studies such as these in phage led Streisinger and others to wonder whether more complex genetic and biological processes could also be deciphered through mutational analysis. Taking this idea forward, Streisinger and some of his Caltech colleagues decided to attempt to study the nervous system using genetic analysis. Sydney Brenner turned to Caenorhabditis elegans for this purpose, while Seymour Benzer turned to the fruit fly to address the questions of nervous system function and behavior. Streisinger, however, was determined to find a vertebrate genetic model and set upon the zebrafish as a vertebrate organism in which it would be feasible to use mutational analysis to dissect biological (neurological in particular) development. It was joked that he wanted to find a phage with a backbone.

Why Zebrafish?

The zebrafish possesses some of the genetic and experimental accessibility of the fly and nematode worm, but is much more closely related to mammals. Some of the general

1970

Development of mapping and linkage methods initiated.

Mid-1970's

Techniques for producing homozygous diploid offspring derived only from the maternal genome are deve|oPed. 1980's

Methods for performing mutagenesis regimes reported.

1982

Kimmel characterizes the morphology and arrangement of embryonic neurons.

1988

First transgenic germline is reported in the zebrafish.

1990

Kimmel publishes the zebrafish fate map.

1994

no tail is the first mutant identified molecularly and found to be the ortholous to the mouse gene for the Brachyury mutant.

1998

one-eyed pinhead is the first mutant to be positionally cloned.

1996

Results of the "Big Screen" published in historical issue #123 of Development.

2000

Morpholinos, antisense modified oligonucleotides, are used to knock down gene expression levels.

Late 1960's

George Streisinger begins working with the zebrafish.

Mid-1970

The development of methods for mapping genes begins.

1981

Streisinger publishes the genetic procedures for generating homozygous diploid clones of zebrafish, the first vertebrate clone!

1989

First embryonic lethal mutant, spadetail, is published

1990

The first zebrafish conference is held in Eugene, OR.

1993

Christiane Nusslein-Volhard, Mark Fishman, and Wolfgang Driever begin the "Big Screen," and the systematic production of embryonic lethal mutations.

1997

The Trans-NIH Zebrafish Initiative is launched.

2000

Sanger Institute initiates the zebrafish genome sequencing project.

Figure 1 Major events that contributed to the success of the zebrafish. The zebrafish has been around for a number of years, but was not considered an established model for biological study until the past decade. This timeline highlights some of the major events that have contributed to the establishment of the zebrafish as a model organism. (see color insert)

features of the zebrafish that initially attracted Streisinger were the large numbers of adult fish that could be maintained in a relatively small space (adult zebrafish are only an inch long) and their year-round breeding in the laboratory. Zebrafish mature in 3 months' time, are capable of breeding every 1 to 2 weeks, and can produce hundreds of eggs per clutch. The external fertilization of the eggs permits in vitro manipulation of ploidy and fertilization for genetic analysis. Streisinger also realized the tremendous advantage afforded by the optically clear embryo of the zebrafish, facilitating screening for developmental phenotypes of interest even in organs and tissues deep within the animal.

Streisinger's Success

Streisinger's efforts to establish the zebrafish as a genetic model were not without risk. In the 1970s little was known about the conservation of regulatory pathways, and the question of translation from the fish to other vertebrates was a serious concern. Fortunately, the environment at the University of Oregon was such that Streisinger had liberty to pursue his interests without the risk of losing the support and resources provided by the "community" at Oregon, and he slowly began to make progress. He developed methods to produce haploid and diploid gynogenotes, permitting recessive mutations to be uncovered in a single generation without elaborate and time-consuming inbreeding schemes. He established a breeding program to remove lethal background mutations from strains, and developed methods to efficiently induce and recover new germ-line mutations. In 1981, Streisinger published a paper in Nature [1] describing the production of homozygous diploid clones of zebrafish. Meanwhile, work in other model organisms was leading to growing acceptance for forward-genetic analysis as a means of dissecting developmental processes. Arguably the most notable of these was the report published by Christiane Nüsslein-Volhard and Eric Wieschaus describing large-scale genetic analysis of Drosophila development [2]. As Streisinger's work with the zebrafish progressed, other faculty members at the University of Oregon also became interested in the zebrafish. In 1990 Charles Kimmel and colleagues published a cellular fate map for the zebrafish, a project that took nearly 10 years to complete [3]. This report laid the groundwork and provided tools for further studies of cell lineage and cell fate in the zebrafish. Monte Westerfield helped open the door further to other groups interested in working with zebrafish when he generated a protocol book covering the housing, handling, and laboratory use of zebrafish [4]. In 1988, Westerfield and colleagues also published the first report of stable germ-line transmission of foreign DNA in the zebrafish [5], laying the foundations for the subsequent very fruitful use of transgenic technologies in the fish.

Two additional events helped to solidify the fish as a vertebrate genetic model, by demonstrating its "relevance" to mammals and other "higher" vertebrates. The first was the molecular cloning of the zebrafish no tail mutant, which was shown to result from a mutation in the zebrafish homolog of the mouse T (Brachyury) gene and to cause phenotypically similar defects [6]. This demonstrated the fundamental conservation of developmental processes between these divergent vertebrate species. The second was publication of the results of the "Big Screens" carried out by two groups in Tübingen, Germany, and in Boston. Some 4,000 mutant phenotypes were identified in these screens, whose results were published in a single, historic volume of Development [7]. Mutants were uncovered affecting a wide variety of vertebrate developmental processes, including many modeling human congenital diseases, and analysis of these mutants has led to many insights into the genetic pathways regulating early development. These screens demonstrated the feasibility of large-scale forward-genetic analysis of vertebrate development. Today the process of setting up and running a zebrafish screen is almost routine, and numerous genetic screens are being carried out targeting a wide variety of developmental processes.

The Postscreen Era

Interest in the use of the zebrafish greatly increased after publication of the results of the first large-scale screens in 1996, and development of additional tools and financial investment accelerated. Efforts began to develop dense genetic and physical maps of the genome, work that has led to a much more rapid turnaround time between identification of an interesting mutant phenotype and identification of the gene responsible for the phenotype. In 1997 the NIH established the Trans-NIH Zebrafish Coordinating Committee (TZCC) to help provide funding for zebrafish as a model organism for the study of vertebrate development, physiology, and disease. The efforts of this committee have been instrumental in facilitating increased and better-targeted channeling of NIH resources to the zebrafish community. A whole-genome sequencing project was initiated by the Sanger Institute, a project that has reached approximately fourfold coverage of the genome to date. Anther important advance was the development of targeted gene "knockdown" technology using antisense, morpholino-modified oligonucleotides (morpholinos, MOs) to inhibit translation or splicing of target genes of interest [8]. Suspected mutant sequences can now be confirmed with MO injections, conservation of gene function between fish and mice can be easily tested, and single and multiple gene knockdowns open the door to epistasis experiments and serve as a valuable complementary method to mutants for studying vertebrate development and disease.

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