Zebrafish Muscle Research Articles

A Simple and Efficient Microinjection Protocol for Making Transgenic Sticklebacks

Gasterosteus aculeatus , the threespine stickleback, has been used for many years to study vertebrate behavior, physiology, evolution and ecology. Further studies of this organism at the molecular level would be greatly enhanced by methods to increase or decrease the activity of specific genes, or to transfer genes between fish with different morphologies. We have tested a variety of methods for microinjection of DNA into 1-2 cell stickleback embryos. Holding the embryos in place using a custom-made glass chamber made it possible to penetrate the chorion with a glass capillary needle, and inject small volumes of DNA solutions into the blastodisc region of the fertilized egg. To test for expression and maintenance of DNA following injection, we injected a DNA plasmid containing the zebrafish muscle specific (α) actin promoter fused to the coding region of a jellyfish green fluorescent protein (GFP) gene, together with the rare cutting endonuclease, ISceI . Injected sticklebacks expressed the GFP reporter gene in developing muscle, maintained expression for at least 6 months, and transmitted the GFP construct to their own progeny when raised to sexual maturity. Further use of this method should make it possible to introduce a variety of constructs into developing sticklebacks and to study the molecular basis of the interesting morphological, behavioral, and physiological differences among recently evolved stickleback forms.

Sarcoglycans of the zebrafish: orthology and localization to the sarcolemma and myosepta of muscle

The zebrafish is an established model of vertebrate development and is also receiving increasing attention in terms of human disease modelling. In order to provide experimental support to realize this modelling potential, we report here the identification of apparent orthologues of many critical members of the dystrophin-associated glycoprotein complex (DGC) that have been implicated in a diverse range of neuromuscular disorders. In addition, immunohistochemical studies show the localization of the DGC to the sarcolemma of adult zebrafish muscle and in particular the myosepta. Together, these data suggest that the DGC in adult zebrafish may play a highly conserved functional role in muscle architecture that, when disrupted, could offer insight into human neuromuscular disease processes.

A novel monoclonal antibody recognizes a previously unknown subdivision of the habenulo-interpeduncular system in zebrafish

The habenulo-interpeduncular system is an evolutionarily conserved structure found in the brain of almost all vertebrates. We prepared a monoclonal antibody (6G11) which very specifically recognizes only a part of this system. 6G11 is a monoclonal antibody prepared from a neuronal membrane protein in adult zebrafish brain. In western blot analysis of the adult zebrafish brain, the antibody recognized a 95 kDa protein, and the class of the antibody was determined to be IgM. The 6G11 antigen was not detected in zebrafish muscle, intestine, testis or ovary. A group of neurons stained by the 6G11 antibody was located in the caudomedial part of the zebrafish habenula. The 6G11-immunopositive neurons extended their axons into the fasciculus retroflexus (FR). One group of immunopositive neurons projected toward the interpeduncular nucleus (IPN), especially to the intermediate and the central subnucleus (type 1 neuron). The other group projected to the ventral midline at the level of the raphe nucleus; these axons passed ipsilaterally beside the IPN and converged in the ventral midline under the raphe nucleus (type 2 neuron). Both type 1 and type 2 fibers are relatively minor components of the FR. Little has previously been known about this topological pattern in any species. The 6G11 monoclonal antibody could be a useful tool for expanding knowledge of the habenulo-interpeduncular system.

Gli2 mediation of Hedgehog signals in slow muscle induction in zebrafish

Zebrafish skeletal muscles are composed of two major types of muscle fibers, broadly classified as fast or slow fibers. Recent studies have demonstrated that members of the Hedgehog (Hh) family induce the formation of slow muscle fibers. Hedgehog signals are secreted proteins that function through the transcription factor Glis. We report here the characterization of a zebrafish Gli2 expression in slow and fast muscle cells and the study of the roles of Hedgehogs and Gli2 in zebrafish muscle development using two mutant strains; sonic-you (syu) and you-too ( yot), respective for sonic hedgehog ( shh) and Gli2 mutation. We have demonstrated that Shh and Gli2 mutation causes similar defects in slow muscle formation. There is, however, a difference in the degree of defect between these two mutants. In yot mutant embryos, development of slow muscles was completely blocked, whereas in syu mutant embryos, a small number of slow muscle cells could still form, suggesting that other Hhs were also involved in slow muscle induction. Induction of slow muscles by other Hhs appeared to require Gli2, because ectopic expression of Echidna hedgehog (Ehh) and Tiggy-winkle hedgehog (Twhh) failed to induce slow muscles in yot mutant embryos. Together, these data suggest that further Hhs, other than Shh, are also involved in the induction and differentiation of slow muscle cells and that Gli2 is required by Shh, Twhh, and Ehh, thus playing a key role in the induction and differentiation of slow muscle cells.

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 βGal markers were compared, the GFP signal was visible in the cytoplasm of the living cell, whereas visualization of βGal 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.

The Zebrafish egr1 Gene Encodes a Highly Conserved, Zinc-Finger Transcriptional Regulator

The Egr family of transcriptional regulators comprises a group of genes which encode members of the Cys2-His2 class of zinc-finger proteins. We have isolated a zebrafish egr1 homologue by screening a zebrafish genomic library with a mouse Egr1 zinc finger probe. Southern blotting indicated the existence of a single zebrafish egr1 gene and, as in higher vertebrates, the presence of related members of a larger gene family. Sequence analysis of the zebrafish egr1 coding region revealed a high level of homology to the mouse, rat, and human Egr1 genes with the notable exception of a polymorphic, triplet nucleotide repeat sequence in the region coding for the amino terminus of the Egr1 protein. The predicted DNA-binding, zinc-finger domain protein sequence was strictly conserved. The 5' region of the zebrafish egr1 gene contained a variety of transcription factor binding sites, also present in the mouse gene, for serum response factor, CREB and c-Ets. The zebrafish egr1 transcript was approximately 3.4 kb in size and was expressed in adult zebrafish brain and muscle RNA, a pattern of expression similar to that observed in mice. The potential for zebrafish egr1 to function as a transcriptional regulator was tested by constructing an expression vector containing zebrafish egr1 coding sequences under the control of a cytomegalovirus promoter. This construct was found to activate transcription of a reporter plasmid bearing multiple Egr1 binding sites when transiently cotransfected into mouse 3T3 cells. Our results indicate that the structure, regulation, and function of the Egr1 gene have been highly conserved during vertebrate evolution and suggest an important role for this gene in growth and development.

The zebrafish egr1 gene encodes a highly conserved, zinc-finger transcriptional regulator.

The Egr family of transcriptional regulators comprises a group of genes that encode members of the Cys2-His2 class of zinc finger proteins. We have isolated a zebrafish egr1 homolog by screening a zebrafish genomic library with a mouse Egr1 zinc finger probe. Southern blotting indicated the existence of single zebrafish egr1 gene and, as in higher vertebrates, the presence of related members of a larger gene family. Sequence analysis of the zebrafish egr1 coding region revealed a high level of homology to the mouse, rat, and human egr1 genes with the notable exception of a polymorphic, triplet nucleotide repeat sequence in the region coding for the amino terminus of the Egr1 protein. The predicted DNA-binding, zinc finger domain protein sequence was strictly conserved. The 5' region of the zebrafish egr1 gene contained a variety of transcription factor binding sites, also present in the mouse gene, for serum response factor, CREB, and c-ets. The zebrafish egr1 transcript was approximately 3.4 kb in size and was expressed in adult zebrafish brain and muscle RNA, a pattern of expression similar to that observed in mice. The potential for zebrafish egr1 to function as a transcriptional regulator was tested by constructing an expression vector containing zebrafish egr1 coding sequences under the control of a cytomegalovirus promoter. This construct was found to activate transcription of a reporter plasmid bearing multiple Egr1 binding sites when transiently cotransfected into mouse 3T3 cells. Our results indicate that the structure, regulation, and function of the Egr1 gene have been highly conserved during vertebrate evolution and suggest an important role for this gene in growth and development.

Autonomous Expression of the nic1 Acetylcholine Receptor Mutation in Zebrafish Muscle Cells

The nic1 b107 ( nic1) mutation blocks expression of both functional and clustered acetylcholine receptors (AChRs) in zebrafish muscle. Normally, signaling between motoneurons and muscles regulates AChR clustering. To learn if signaling is affected and to identify the primary cellular target of the nic1 mutation, we made mosaic embryos by transplanting motoneurons and muscle precursors from wild-type to mutant embryos. Genotypically mutant muscle cells fail to cluster AChRs even when contacted by wild-type moteneurons, whereas genotypically mutant motoneurons induce AChR clustering on wild-type muscle cells. Moreover, mutant muscle cells fail to cluster AChRs under culture conditions that induce AChR clustering on wild-type cells. We conclude that the nic1 mutation acts autonomously in muscle cells rather than by affecting signaling between motoneurons and muscle. The wild-type nic1 gene is necessary in muscle for expression and clustering of AChRs.

The fub-1 mutation blocks initial myofibril formation in zebrafish muscle pioneer cells

The earliest muscle in zebrafish arises from iterated sets of two to six cells in each somite, the muscle pioneers (MP). MP develop synchronously in young trunk myotomes adjacent to the notochord, precisely where the horizontal myoseptum will form. They elongate without cell fusion and differentiate hours earlier than surrounding cells, thus providing a simple and accessible system for in vivo study of myogenesis and muscle patterning. Before the MP form definitive myofibrils they assemble long bundles of actin-containing filaments, similar to “stress-fiber-like structures” reported by others. In fub-1 mutants, in which myofibrils are disorganized in all skeletal muscle cells, the MP appear and elongate normally, but ordered actin filament bundles are not seen. This defect could underlie the later myofibrillar ones, consistent with the proposal that actin filament bundles are essential for proper formation of the muscle contractile apparatus.