Wolfgang Driever

Zebrafish: genetic tools for studying vertebrate development

Zebrafish have entered the arena of vertebrate biology as a mainstream model system, and the use of genetic tools in this tropical fish should enhance our understanding of vertebrate development. The zebrafish system allows genetic experiments that are not possible in other vertebrates, and the mutations isolated thus far attest to its usefulness, complementing knowledge obtained from other model organisms.

Characterization of a cell line derived from zebrafish (Brachydanio rerio) embryos

SummaryDuring the last decade, zebrafish (Brachydanio rerio) have emerged as a novel and attractive system to study embryogenesis and organogenesis in vertebrates. The main reason is that both extensive genetic studies and detailed embryologic analysis are possible using this small tropical fresh water teleost. However, in vitro analysis using cell culture or molecular genetics are still far less advanced than in other vertebrate systems. Here we report the generation and characterization of a fibroblast like cell line, ZF4, derived from 1-day-old zebrafish embryos. The hyperploid cell line has been stable in multiple passages for more than 2 yr now and is the first zebrafish cell line that can be maintained in conventional medium containing mammalian serum. Using a series of plasmids for expression of a marker gene, we evaluate in ZF4 cells the relative strength of expression from several different viral, fish, and mammalian promoters. Stable integration can be obtained by using G418 selection. We hope that our cell line will be a useful tool for the analysis of gene regulation in zebrafish.

Axis formation in zebrafish.

Recent advances in our understanding of axis formation and patterning in zebrafish relate the developmental mode of this aspiring genetic model organism to higher vertebrates. The effect of UV irradiation and lithium treatment, as well as detailed early lineage analyses, have shed some light on dorsoventral axis formation. However, the molecular mechanism of axis formation, as well as the identity of a fish Nieuwkoop center, are still open issues. A Vg1 homolog is expressed in zebrafish, and activin as well as the mouse nodal gene product have been demonstrated to induce mesoderm and ectopic axes, respectively, in zebrafish. The zebrafish organizer is defined by the expression domains of goosecoid, axial, and lim1. The cyclops gene is involved in maintaining goosecoid expression in axial mesoderm of the head. Large mutagenesis screens provide the basis for a genetic analysis of axis formation.

Green fluorescent protein marks skeletal muscle in murine cell lines and zebrafish

The green fluorescent protein (GFP) acts as a vital dye upon the absorption of blue light. When the gfp gene is expressed in bacteria, flies or nematodes, green fluorescence can be directly observed in the living organism. We inserted the cDNA encoding this 238-amino-acid (aa) jellyfish protein into an expression vector containing the rat myosin light-chain enhancer (MLC-GFP) to evaluate its ability to serve as a muscle-specific marker. Transiently, as well as stably, transfected C2C12 cell lines produced high levels of GFP distributed homogeneously throughout the cytoplasm and was not toxic through several cell passages. Expression of MLC-GFP was strictly muscle-specific, since Cos 7 fibroblasts transfected with MLC-GFP did not fluoresce. When GFP and beta Gal markers were compared, the GFP signal was visible in the cytoplasm of the living cell, whereas visualization of beta Gal required fixation and resulted in deformation of the cells. When the MLC-GFP construct was injected into zebrafish embryos, muscle-specific gfp expression was apparent within 24 h of development. gfp expression was never observed in non-muscle tissues using the MLC-GFP construct. Transgenic fish continued to express high levels of gfp in skeletal muscle at 1.5 months, demonstrating that GFP is an effective marker of muscle cells in vivo.

Zebrafish: tools for investigating cellular differentiation

Two recent large-scale genetic screens in zebrafish have identified many mutations that affect differentiation in a variety of organ systems, particularly the notochord, the neural crest and the blood. The combination of these newly identified mutations and well established embryological methods makes zebrafish uniquely suited among vertebrate experimental systems to simultaneously address the roles of specific genes and specific cell-cell interactions during differentiation.