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

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.

Connexin 39.9 protein is necessary for coordinated activation of slow-twitch muscle and normal behavior in zebrafish.

In many tissues and organs, connexin proteins assemble between neighboring cells to form gap junctions. These gap junctions facilitate direct intercellular communication between adjoining cells, allowing for the transmission of both chemical and electrical signals. In rodents, gap junctions are found in differentiating myoblasts and are important for myogenesis. Although gap junctions were once believed to be absent from differentiated skeletal muscle in mammals, recent studies in teleosts revealed that differentiated muscle does express connexins and is electrically coupled, at least at the larval stage. These findings raised questions regarding the functional significance of gap junctions in differentiated muscle. Our analysis of gap junctions in muscle began with the isolation of a zebrafish motor mutant that displayed weak coiling at day 1 of development, a behavior known to be driven by slow-twitch muscle (slow muscle). We identified a missense mutation in the gene encoding Connexin 39.9. In situ hybridization found connexin 39.9 to be expressed by slow muscle. Paired muscle recordings uncovered that wild-type slow muscles are electrically coupled, whereas mutant slow muscles are not. The further examination of cellular activity revealed aberrant, arrhythmic touch-evoked Ca(2+) transients in mutant slow muscle and a reduction in the number of muscle fibers contracting in response to touch in mutants. These results indicate that Connexin 39.9 facilitates the spreading of neuronal inputs, which is irregular during motor development, beyond the muscle cells and that gap junctions play an essential role in the efficient recruitment of slow muscle fibers.

TRPM7 is required within zebrafish sensory neurons for the activation of touch-evoked escape behaviors.

Mutations in the gene encoding TRPM7 (trpm7), a member of the Transient Receptor Potential (TRP) superfamily of cation channels that possesses an enzymatically active kinase at its C terminus, cause the touch-unresponsive zebrafish mutant touchdown. We identified and characterized a new allele of touchdown, as well as two previously reported alleles, and found that all three alleles harbor mutations that abolish channel activity. Through the selective restoration of TRPM7 expression in sensory neurons, we found that TRPM7's kinase activity and selectivity for divalent cations over monovalent cations were dispensable for touch-evoked activation of escape behaviors in zebrafish. Additional characterization revealed that sensory neurons were present and capable of responding to tactile stimuli in touchdown mutants, indicating that TRPM7 is not required for sensory neuron survival or mechanosensation. Finally, exposure to elevated concentrations of divalent cations was found to restore touch-evoked behaviors in touchdown mutants. Collectively, these findings are consistent with a role for zebrafish TRPM7 within sensory neurons in the modulation of neurotransmitter release at central synapses, similar to that proposed for mammalian TRPM7 at peripheral synapses.

touché Is required for touch-evoked generator potentials within vertebrate sensory neurons.

The process by which light touch in vertebrates is transformed into an electrical response in cutaneous mechanosensitive neurons is a largely unresolved question. To address this question we undertook a forward genetic screen in zebrafish (Danio rerio) to identify mutants exhibiting abnormal touch-evoked behaviors, despite the presence of sensory neurons and peripheral neurites. One family, subsequently named touché, was found to harbor a recessive mutation which produced offspring that were unresponsive to light touch, but responded to a variety of other sensory stimuli. The optogenetic activation of motor behaviors by touché mutant sensory neurons expressing channelrhodopsin-2 suggested that the synaptic output of sensory neurons was intact, consistent with a defect in sensory neuron activation. To explore sensory neuron activation we developed an in vivo preparation permitting the precise placement of a combined electrical and tactile stimulating probe upon eGFP-positive peripheral neurites. In wild-type larva electrical and tactile stimulation of peripheral neurites produced action potentials detectable within the cell body. In a subset of these sensory neurons an underlying generator potential could be observed in response to subthreshold tactile stimuli. A closer examination revealed that the amplitude of the generator potential was proportional to the stimulus amplitude. When assayed touché mutant sensory neurons also responded to electrical stimulation of peripheral neurites similar to wild-type larvae, however tactile stimulation of these neurites failed to uncover a subset of sensory neurons possessing generator potentials. These findings suggest that touché is required for generator potentials, and that cutaneous mechanoreceptors with generator potentials are necessary for responsiveness to light touch in zebrafish.

Na(v)1.6a is required for normal activation of motor circuits normally excited by tactile stimulation.

A screen for zebrafish motor mutants identified two noncomplementing alleles of a recessive mutation that were named non-active (nav(mi89) and nav(mi130)). nav embryos displayed diminished spontaneous and touch-evoked escape behaviors during the first 3 days of development. Genetic mapping identified the gene encoding Na(V)1.6a (scn8aa) as a potential candidate for nav. Subsequent cloning of scn8aa from the two alleles of nav uncovered two missense mutations in Na(V)1.6a that eliminated channel activity when assayed heterologously. Furthermore, the injection of RNA encoding wild-type scn8aa rescued the nav mutant phenotype indicating that scn8aa was the causative gene of nav. In-vivo electrophysiological analysis of the touch-evoked escape circuit indicated that voltage-dependent inward current was decreased in mechanosensory neurons in mutants, but they were able to fire action potentials. Furthermore, tactile stimulation of mutants activated some neurons downstream of mechanosensory neurons but failed to activate the swim locomotor circuit in accord with the behavioral response of initial escape contractions but no swimming. Thus, mutant mechanosensory neurons appeared to respond to tactile stimulation but failed to initiate swimming. Interestingly fictive swimming could be initiated pharmacologically suggesting that a swim circuit was present in mutants. These results suggested that Na(V)1.6a was required for touch-induced activation of the swim locomotor network.

The zebrafish ennui behavioral mutation disrupts acetylcholine receptor localization and motor axon stability.

The zebrafish ennui mutation was identified from a mutagenesis screen for defects in early behavior. Homozygous ennui embryos swam more slowly than wild-type siblings but normal swimming recovered during larval stages and homozygous mutants survived until adulthood. Electrophysiological recordings from motoneurons and muscles suggested that the motor output of the CNS following mechanosensory stimulation was normal in ennui, but the synaptic currents at the neuromuscular junction were significantly reduced. Analysis of acetylcholine receptors (AChRs) in ennui muscles showed a marked reduction in the size of synaptic clusters and their aberrant localization at the myotome segment borders of fast twitch muscle. Prepatterned, nerve-independent AChR clusters appeared normal in mutant embryos and dispersed upon outgrowth of motor axons onto the muscles. Genetic mosaic analysis showed that ennui is required cell autonomously in muscle fibers for normal synaptic localization of AChRs. Furthermore, exogenous agrin failed to induce AChR aggregation, suggesting that ennui is crucial for agrin function. Finally, motor axons branched more extensively in ennui fast twitch muscles especially in the region of the myotome borders. These results suggest that ennui is important for nerve-dependent AChR clustering and the stability of axon growth.

Zebrafish relatively relaxed mutants have a ryanodine receptor defect, show slow swimming and provide a model of multi-minicore disease.

Wild-type zebrafish embryos swim away in response to tactile stimulation. By contrast, relatively relaxed mutants swim slowly due to weak contractions of trunk muscles. Electrophysiological recordings from muscle showed that output from the CNS was normal in mutants, suggesting a defect in the muscle. Calcium imaging revealed that Ca(2+) transients were reduced in mutant fast muscle. Immunostaining demonstrated that ryanodine and dihydropyridine receptors, which are responsible for Ca(2+) release following membrane depolarization, were severely reduced at transverse-tubule/sarcoplasmic reticulum junctions in mutant fast muscle. Thus, slow swimming is caused by weak muscle contractions due to impaired excitation-contraction coupling. Indeed, most of the ryanodine receptor 1b (ryr1b) mRNA in mutants carried a nonsense mutation that was generated by aberrant splicing due to a DNA insertion in an intron of the ryr1b gene, leading to a hypomorphic condition in relatively relaxed mutants. RYR1 mutations in humans lead to a congenital myopathy, multi-minicore disease (MmD), which is defined by amorphous cores in muscle. Electron micrographs showed minicore structures in mutant fast muscles. Furthermore, following the introduction of antisense morpholino oligonucleotides that restored the normal splicing of ryr1b, swimming was recovered in mutants. These findings suggest that zebrafish relatively relaxed mutants may be useful for understanding the development and physiology of MmD.

Non-sense mutations in the dihydropyridine receptor beta1 gene, CACNB1, paralyze zebrafish relaxed mutants.

Contractions by skeletal muscle require proper excitation-contraction (EC) coupling, whereby depolarization of the muscle membrane leads to an increase in cytosolic Ca(2+) and contraction. Changes in membrane voltage are detected by dihydropyridine receptors (DHPR) that directly interact with and activate ryanodine receptors to release Ca(2+) from the sarcoplasmic reticulum into the cytosol. A genetic screen for motility mutations isolated a new allele of the immotile zebrafish mutant relaxed. Muscles in relaxed embryos do not contract in response to potassium chloride (KCl) thus appear unresponsive to membrane depolarization, but do contract when stimulated by caffeine, an agonist of ryanodine receptors. This suggests that relaxed mutant muscles are defective in EC coupling. Indeed, immunohistochemical analysis demonstrated that mutants lack DHPRs in skeletal muscles. The mutant phenotype results from non-sense mutations in the zebrafish CACNB1 gene that encodes the DHPR beta1 subunit. The zebrafish CACNB1 gene is expressed in skeletal muscles, PNS and CNS. Electrophysiological recordings showed no obvious abnormalities in the motor output of relaxed mutants, presumably due to redundancy provided by other beta subunits. The structural and functional homology of CACNB1 in zebrafish and mammals, suggests that zebrafish can be useful for studying EC coupling and potential neuronal function of CACNB1.