Analysis of Embryonic and Larval Zebrafish Skeletal Myofibers from Dissociated Preparations

The zebrafish has  proven to be a valuable model system for exploring skeletal muscle function and  for studying human muscle diseases. Despite  the many ...

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

The zebrafish model is an emerging system for the study of neuromuscular disorders.  In the study of neuromuscular diseases, the integrity of the muscle ...

University of Michigan 

Eunice Kennedy Shriver National Institute of Child Health and Human Development

University of Michigan

Vascular endothelial growth factor (VEGF) is a major mediator of pathologic angiogenesis, a process necessary for the formation of new blood vessels to support tumor growth. Historically, VEGF has been thought to signal via receptor tyrosine kinases, which are not typically considered to be G protein dependent. Here, we show that targeted knockdown of the G protein gng2 gene (Ggamma2) blocks the normal angiogenic process in developing zebrafish embryos. Moreover, loss of gng2 function inhibits the ability of VEGF to promote the angiogenic sprouting of blood vessels by attenuating VEGF induced phosphorylation of phospholipase C-gamma1 (PLCgamma1) and serine/threonine kinase (AKT). Collectively, these results demonstrate a novel interaction between Ggamma2- and VEGF-dependent pathways to regulate the angiogenic process in a whole-animal model. Blocking VEGF function using a humanized anti-VEGF antibody has emerged as a promising treatment for colorectal, non-small lung cell, and breast cancers. However, this treatment may cause considerable side effects. Our findings provide a new opportunity for cotargeting G protein- and VEGF-dependent pathways to synergistically block pathologic angiogenesis, which may lead to a safer and more efficacious therapeutic regimen to fight cancer.

Zebrafish G protein gamma2 is required for VEGF signaling during angiogenesis.

Here, we report the identification and expression analysis of the zebrafish G protein gammaT1 subunit gene (gngT1) during development. Similar to its human and mouse homologs, we confirm zebrafish gngT1 is expressed in the developing retina, where its transcription overlaps with the photoreceptor cell-specific marker, rhodopsin (rho). Surprisingly, we also show zebrafish gngT1 is expressed in the dorsal diencephalon, where its transcription overlaps with the pineal specific markers, arylalkylamine N-acetyltransferase-2 (annat-2) and extra-ocular rhodopsin (exorh). Analysis of the proximal promoter sequence of the zebrafish gngT1 gene identifies several conserved binding sites for the cone-rod homeobox/orthodenticle (Crx/Otx) homeodomain family of transcription factors. Using a morpholino anti-sense approach in zebrafish, we show that targeted knockdown of otx5 potently suppresses gngT1 expression in the pineal gland, whereas knockdown of crx markedly reduces gngT1 expression in the retina. Taken together, these data indicate that pineal- and retinal-specific expression of the gngT1 gene are controlled by different transcription factors and exogenous signals.

Expression of the G protein gammaT1 subunit during zebrafish development.

Excitation-contraction coupling, the process that regulates contractions by skeletal muscles, transduces changes in membrane voltage by activating release of Ca(2+) from internal stores to initiate muscle contraction. Defects in excitation-contraction coupling are associated with muscle diseases. Here we identify Stac3 as a novel component of the excitation-contraction coupling machinery. Using a zebrafish genetic screen, we generate a locomotor mutation that is mapped to stac3. We provide electrophysiological, Ca(2+) imaging, immunocytochemical and biochemical evidence that Stac3 participates in excitation-contraction coupling in muscles. Furthermore, we reveal that a mutation in human STAC3 is the genetic basis of the debilitating Native American myopathy (NAM). Analysis of NAM stac3 in zebrafish shows that the NAM mutation decreases excitation-contraction coupling. These findings enhance our understanding of both excitation-contraction coupling and the pathology of myopathies.

Stac3 is a component of the excitation-contraction coupling machinery and mutated in Native American myopathy.

Couplage, le processus qui r&eacute;gule les contractions de muscles squelettiques, excitation-contraction transduit changements de tension membranaire en activant la lib&eacute;ration de Ca(2+) des r&eacute;serves internes pour initier la contraction musculaire. D&eacute;fauts dans le couplage excitation-contraction sont associ&eacute;s aux maladies musculaires. Ici, nous identifions Stac3 comme un nouveau composant de la machinerie du couplage excitation-contraction. &Agrave; l&#39;aide d&#39;un d&eacute;pistage g&eacute;n&eacute;tique du poisson-z&egrave;bre, nous g&eacute;n&eacute;rons une mutation locomotrice qui est mapp&eacute;e &agrave; stac3. Nous fournissons &eacute;lectrophysiologiques, Ca(2+) imagerie, immunocytochimiques et indices biochimiques que Stac3 participe &agrave; l&#39;excitation-contraction de couplage dans les muscles. En outre, nous r&eacute;v&egrave;lent qu&#39;une mutation dans la STAC3 humaine est la base g&eacute;n&eacute;tique de la myopathie d&eacute;bilitante am&eacute;rindienne (NAM). Analyse de NAM stac3 chez le poisson z&egrave;bre montre que la mutation de NAM diminue couplage excitation-contraction. Ces r&eacute;sultats d&#39;am&eacute;liorer notre compr&eacute;hension du couplage excitation-contraction de fois et la pathologie des myopathies.

Stac3 est un composant de la machinerie du couplage excitation-contraction et une mutation dans la myopathie am&eacute;rindien.

A new and exciting phase of muscle disease research has recently been entered. The application of next generation sequencing technology has spurred an unprecedented era of gene discovery for both myopathies and muscular dystrophies. Gene-based therapies for Duchenne muscular dystrophy have entered clinical trial, and several pathway-based therapies are doing so as well for a handful of muscle diseases. While many factors have aided the extraordinary developments in gene discovery and therapy development, the zebrafish model system has emerged as a vital tool in these advancements.

Swimming into prominence: the zebrafish as a valuable tool for studying human myopathies and muscular dystrophies.

Une phase nouvelle et passionnante de recherche sur les maladies musculaires a r&eacute;cemment &eacute;t&eacute; conclue. L&#39;application de la prochaine g&eacute;n&eacute;ration de technologies de s&eacute;quen&ccedil;age a stimul&eacute; une &egrave;re sans pr&eacute;c&eacute;dent de la d&eacute;couverte de g&egrave;nes pour les myopathies et les dystrophies musculaires. Les th&eacute;rapies g&eacute;niques pour la dystrophie musculaire de Duchenne sont entr&eacute;s essai clinique, et plusieurs th&eacute;rapies ax&eacute;es sur la voie font de m&ecirc;me pour une poign&eacute;e de maladies musculaires. Alors que de nombreux facteurs ont aid&eacute; les d&eacute;veloppements extraordinaires dans le d&eacute;veloppement de d&eacute;couverte et de la th&eacute;rapie g&eacute;nique, le syst&egrave;me de mod&egrave;le de poisson-z&egrave;bre est devenu un outil essentiel dans ces progr&egrave;s.

Natation en &eacute;vidence : le poisson-z&egrave;bre comme un outil pr&eacute;cieux pour l&#39;&eacute;tude des myopathies humaines et dystrophies musculaires.

An essential step in muscle fiber maturation is the assembly of highly ordered myofibrils that are required for contraction. Much remains unknown about the molecular mechanisms governing the formation of the contractile apparatus. We identified an early embryonic motility mutant in zebrafish caused by integration of a transgene into the pseudophosphatase dual specificity phosphatase 27 (dusp27). Dusp27 mutants exhibit near complete paralysis at embryonic and larval stages, producing extremely low levels of spontaneous coiling movements and a greatly diminished touch response. Loss of dusp27 does not prevent somitogenesis but results in severe disorganization of the contractile apparatus in muscle fibers. Sarcomeric structures in mutants are almost entirely absent and only rare triads are observed. These findings are the first to implicate a functional role of dusp27 as a gene required for myofiber maturation and provide an animal model for analyzing the mechanisms governing myofibril assembly.

Eric J. Horstick

Departments of Pediatrics and Neurology

University of Michigan

Analysis of Embryonic and Larval Zebrafish Skeletal Myofibers from Dissociated Preparations

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

Eric J. Horstick

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Departments of Pediatrics and Neurology

University of Michigan

Analysis of Embryonic and Larval Zebrafish Skeletal Myofibers from Dissociated Preparations

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

Departments of Pediatrics and Neurology