Stac protein regulates release of neuropeptides.

Neuropeptides are important for regulating numerous neural functions and behaviors. Release of neuropeptides requires long-lasting, high levels of cytosolic Ca2+ However, the molecular regulation of neuropeptide release remains to be clarified. Recently, Stac3 was identified as a key regulator of L-type Ca2+ channels (CaChs) and excitation-contraction coupling in vertebrate skeletal muscles. There is a small family of stac genes in vertebrates with other members expressed by subsets of neurons in the central nervous system. The function of neural Stac proteins, however, is poorly understood. Drosophila melanogaster contain a single stac gene, Dstac, which is expressed by muscles and a subset of neurons, including neuropeptide-expressing motor neurons. Here, genetic manipulations, coupled with immunolabeling, Ca2+ imaging, electrophysiology, and behavioral analysis, revealed that Dstac regulates L-type CaChs (Dmca1D) in Drosophila motor neurons and this, in turn, controls the release of neuropeptides.

Congenital myopathy results from misregulation of a muscle Ca2+ channel by mutant Stac3.

Skeletal muscle contractions are initiated by an increase in Ca2+ released during excitation-contraction (EC) coupling, and defects in EC coupling are associated with human myopathies. EC coupling requires communication between voltage-sensing dihydropyridine receptors (DHPRs) in transverse tubule membrane and Ca2+ release channel ryanodine receptor 1 (RyR1) in the sarcoplasmic reticulum (SR). Stac3 protein (SH3 and cysteine-rich domain 3) is an essential component of the EC coupling apparatus and a mutation in human STAC3 causes the debilitating Native American myopathy (NAM), but the nature of how Stac3 acts on the DHPR and/or RyR1 is unknown. Using electron microscopy, electrophysiology, and dynamic imaging of zebrafish muscle fibers, we find significantly reduced DHPR levels, functionality, and stability in stac3 mutants. Furthermore, stac3NAM myofibers exhibited increased caffeine-induced Ca2+ release across a wide range of concentrations in the absence of altered caffeine sensitivity as well as increased Ca2+ in internal stores, which is consistent with increased SR luminal Ca2+ These findings define critical roles for Stac3 in EC coupling and human disease.

Transport of the alpha subunit of the voltage gated L-type calcium channel through the sarcoplasmic reticulum occurs prior to localization to triads and requires the beta subunit but not Stac3 in skeletal muscles.

Contraction of skeletal muscle is initiated by excitation-contraction (EC) coupling during which membrane voltage is transduced to intracellular Ca2+ release. EC coupling requires L-type voltage gated Ca2+ channels (the dihydropyridine receptor or DHPR) located at triads, which are junctions between the transverse (T) tubule and sarcoplasmic reticulum (SR) membranes, that sense membrane depolarization in the T tubule membrane. Reduced EC coupling is associated with ageing, and disruptions of EC coupling result in congenital myopathies for which there are few therapies. The precise localization of DHPRs to triads is critical for EC coupling, yet trafficking of the DHPR to triads is not well understood. Using dynamic imaging of zebrafish muscle fibers, we find that DHPR is transported along the longitudinal SR in a microtubule-independent mechanism. Furthermore, transport of DHPR in the SR membrane is differentially affected in null mutants of Stac3 or DHPRß, two essential components of EC coupling. These findings reveal previously unappreciated features of DHPR motility within the SR prior to assembly at triads.

RING finger protein 121 facilitates the degradation and membrane localization of voltage-gated sodium channels.

Following their synthesis in the endoplasmic reticulum (ER), voltage-gated sodium channels (NaV) are transported to the membranes of excitable cells, where they often cluster, such as at the axon initial segment of neurons. Although the mechanisms by which NaV channels form and maintain clusters have been extensively examined, the processes that govern their transport and degradation have received less attention. Our entry into the study of these processes began with the isolation of a new allele of the zebrafish mutant alligator, which we found to be caused by mutations in the gene encoding really interesting new gene (RING) finger protein 121 (RNF121), an E3-ubiquitin ligase present in the ER and cis-Golgi compartments. Here we demonstrate that RNF121 facilitates two opposing fates of NaV channels: (i) ubiquitin-mediated proteasome degradation and (ii) membrane localization when coexpressed with auxiliary NaVß subunits. Collectively, these results indicate that RNF121 participates in the quality control of NaV channels during their synthesis and subsequent transport to the membrane.

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.

Oxidative stress and successful antioxidant treatment in models of RYR1-related myopathy.

The skeletal muscle ryanodine receptor is an essential component of the excitation-contraction coupling apparatus. Mutations in RYR1 are associated with several congenital myopathies (termed RYR1-related myopathies) that are the most common non-dystrophic muscle diseases of childhood. Currently, no treatments exist for these disorders. Although the primary pathogenic abnormality involves defective excitation-contraction coupling, other abnormalities likely play a role in disease pathogenesis. In an effort to discover novel pathogenic mechanisms, we analysed two complementary models of RYR1-related myopathies, the relatively relaxed zebrafish and cultured myotubes from patients with RYR1-related myopathies. Expression array analysis in the zebrafish disclosed significant abnormalities in pathways associated with cellular stress. Subsequent studies focused on oxidative stress in relatively relaxed zebrafish and RYR1-related myopathy myotubes and demonstrated increased oxidant activity, the presence of oxidative stress markers, excessive production of oxidants by mitochondria and diminished survival under oxidant conditions. Exposure to the antioxidant N-acetylcysteine reduced oxidative stress and improved survival in the RYR1-related myopathies human myotubes ex vivo and led to significant restoration of aspects of muscle function in the relatively relaxed zebrafish, thereby confirming its efficacy in vivo. We conclude that oxidative stress is an important pathophysiological mechanism in RYR1-related myopathies and that N-acetylcysteine is a successful treatment modality ex vivo and in a vertebrate disease model. We propose that N-acetylcysteine represents the first potential therapeutic strategy for these debilitating muscle diseases.

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.

Loss of myotubularin function results in T-tubule disorganization in zebrafish and human myotubular myopathy.

Myotubularin is a lipid phosphatase implicated in endosomal trafficking in vitro, but with an unknown function in vivo. Mutations in myotubularin cause myotubular myopathy, a devastating congenital myopathy with unclear pathogenesis and no current therapies. Myotubular myopathy was the first described of a growing list of conditions caused by mutations in proteins implicated in membrane trafficking. To advance the understanding of myotubularin function and disease pathogenesis, we have created a zebrafish model of myotubular myopathy using morpholino antisense technology. Zebrafish with reduced levels of myotubularin have significantly impaired motor function and obvious histopathologic changes in their muscle. These changes include abnormally shaped and positioned nuclei and myofiber hypotrophy. These findings are consistent with those observed in the human disease. We demonstrate for the first time that myotubularin functions to regulate PI3P levels in a vertebrate in vivo, and that homologous myotubularin-related proteins can functionally compensate for the loss of myotubularin. Finally, we identify abnormalities in the tubulo-reticular network in muscle from myotubularin zebrafish morphants and correlate these changes with abnormalities in T-tubule organization in biopsies from patients with myotubular myopathy. In all, we have generated a new model of myotubular myopathy and employed this model to uncover a novel function for myotubularin and a new pathomechanism for the human disease that may explain the weakness associated with the condition (defective excitation-contraction coupling). In addition, our findings of tubuloreticular abnormalities and defective excitation-contraction coupling mechanistically link myotubular myopathy with several other inherited muscle diseases, most notably those due to ryanodine receptor mutations. Based on our findings, we speculate that congenital myopathies, usually considered entities with similar clinical features but very disparate pathomechanisms, may at their root be disorders of calcium homeostasis.

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.

Acetylcholine receptor clustering is required for the accumulation and maintenance of scaffolding proteins.

The maintenance of a high density of postsynaptic receptors is essential for proper synaptic function. At the neuromuscular junction, acetylcholine receptor (AChR) aggregation is induced by nerve-clustering factors and mediated by scaffolding proteins. Although the mechanisms underlying AChR clustering have been extensively studied, the role that the receptors themselves play in the clustering process and how they are organized with scaffolding proteins is not well understood. Here, we report that the exposure of AChRs labeled with Alexa 594 conjugates to relatively low-powered laser light caused an effect similar to chromaphore-assisted light inactivation (CALI) , which resulted in the unexpected dissipation of the illuminated AChRs from clusters on cultured myotubes. This technique enabled us to demonstrate that AChR removal from illuminated regions induced the removal of scaffolding proteins and prevented the accumulation of new AChRs and associated scaffolding proteins. Further, the dissipation of clustered AChRs and scaffold was spatially restricted to the illuminated region and had no effect on neighboring nonilluminated AChRs. These results provide direct evidence that AChRs are essential for the local maintenance and accumulation of intracellular scaffolding proteins and suggest that the scaffold is organized into distinct modular units at AChR clusters.

Dstac Regulates Excitation-Contraction Coupling in Drosophila Body Wall Muscles.

Stac3 regulates excitation-contraction coupling (EC coupling) in vertebrate skeletal muscles by regulating the L-type voltage-gated calcium channel (Cav channel). Recently a stac-like gene, Dstac, was identified in Drosophila and found to be expressed by both a subset of neurons and muscles. Here, we show that Dstac and Dmca1D, the Drosophila L-type Cav channel, are necessary for normal locomotion by larvae. Immunolabeling with specific antibodies against Dstac and Dmca1D found that Dstac and Dmca1D are expressed by larval body-wall muscles. Furthermore, Ca2+ imaging of muscles of Dstac and Dmca1D deficient larvae found that Dstac and Dmca1D are required for excitation-contraction coupling. Finally, Dstac appears to be required for normal expression levels of Dmca1D in body-wall muscles. These results suggest that Dstac regulates Dmca1D during EC coupling and thus muscle contraction.

The synaptic receptor Lrp4 promotes peripheral nerve regeneration.

Early during PNS regeneration, regenerating axons emerge from the proximal nerve stump, yet whether they extend simultaneously or whether pioneering axons establish a path for follower axons remains unknown. Moreover, the molecular mechanisms underlying robust regeneration are incompletely understood. Using live imaging, we demonstrate that in zebrafish pioneering axons establish a regenerative path for follower axons. We find this process requires the synaptic receptor lrp4, and in lrp4 mutants pioneers are unaffected while follower axons frequently stall at the injury gap, providing evidence for molecular diversity between pioneering and follower axons in regeneration. We demonstrate that Lrp4 promotes regeneration through an axon extrinsic mechanism and independent of membrane anchoring and MuSK co-receptor signaling essential for synaptic development. Finally, we show that Lrp4 coordinates the realignment of denervated Schwann cells with regenerating axons, consistent with a model by which Lrp4 is repurposed to promote sustained peripheral nerve regeneration via axon-glia interactions.

Dstac is required for normal circadian activity rhythms in Drosophila.

The genetic, molecular and neuronal mechanism underlying circadian activity rhythms is well characterized in the brain of Drosophila. The small ventrolateral neurons (s-LNVs) and pigment dispersing factor (PDF) expressed by them are especially important for regulating circadian locomotion. Here we describe a novel gene, Dstac, which is similar to the stac genes found in vertebrates that encode adaptor proteins, which bind and regulate L-type voltage-gated Ca2+ channels (CaChs). We show that Dstac is coexpressed with PDF by the s-LNVs and regulates circadian activity. Furthermore, the L-type CaCh, Dmca1D, appears to be expressed by the s-LNVs. Since vertebrate Stac3 regulates an L-type CaCh we hypothesize that Dstac regulates Dmca1D in s-LNVs and circadian activity.

The Zebrafish curly fry Is Required for Proper Centrosome and Mitotic Spindle Assembly.

The zebrafish curly fry (cfy) mutation leads to a dramatic increase in mitotic index and cell death starting during neural tube formation. The mutant phenotype is cell autonomous and does not result from defects in apical/basal polarity within the neuroepithelium. The increase in mitotic index could be due to increased proliferation or cell cycle arrest in mitosis. cfy embryos were analyzed to examine these two possibilities. By labeling embryos with a pulse of BrdU and anti-phospho-histone 3 and examining the DNA content by fluorescence activated cell sorting, we show that cfy mutants exhibit no increase in proliferation, but a significant increase in the number of cells arrested in mitosis. Furthermore, time-lapse microscopy in vivo confirmed that a great majority of dividing cells arrest during mitosis and that these mitotically arrested cells die in cfy embryos. Finally, immunostaining and confocal microscopy in cfy mutant embryos revealed that mitotic cells in mutants contain aberrant centrosomes and often exhibit monopolar spindles, thereby leading to mitotic cell cycle arrest. Our results suggest that the cfy gene is required for proper centrosome assembly and mitotic spindle formation, therefore critical for normal cell division.

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.

Chemokine signaling guides axons within the retina in zebrafish.

Chemokines are a large family of secreted proteins that play an important role in the migration of leukocytes during hematopoiesis and inflammation. Chemokines and their receptors are also widely distributed in the CNS. Although recent investigations are beginning to elucidate chemokine function within the CNS, relatively little is known about the CNS function of this important class of molecules. To better appreciate the CNS function of chemokines, the role of signaling by stromal cell-derived factor-1 (SDF-1) through its receptor, chemokine (CXC motif) receptor 4 (CXCR4), was analyzed in zebrafish embryos. The SDF-1/CXCR4 expression pattern suggested that SDF-1/CXCR4 signaling was important for guiding retinal ganglion cell axons within the retina to the optic stalk to exit the retina. Antisense knockdown of the ligand and/or receptor and a genetic CXCR4 mutation both induced retinal axons to follow aberrant pathways within the retina. Furthermore, retinal axons deviated from their normal pathway and extended to cells ectopically expressing SDF-1 within the retina. These data suggest that chemokine signaling is both necessary and sufficient for directing retinal growth cones within the retina.

The zebrafish shocked gene encodes a glycine transporter and is essential for the function of early neural circuits in the CNS.

shocked (sho) is a zebrafish mutation that causes motor deficits attributable to CNS defects during the first2dof development. Mutant embryos display reduced spontaneous coiling of the trunk, diminished escape responses when touched, and an absence of swimming. A missense mutation in the slc6a9 gene that encodes a glycine transporter (GlyT1) was identified as the cause of the sho phenotype. Antisense knock-down of GlyT1 in wild-type embryos phenocopies sho, and injection of wild-type GlyT1 mRNA into mutants rescues them. A comparison of glycine-evoked inward currents in Xenopus oocytes expressing either the wild-type or mutant protein found that the missense mutation results in a nonfunctional transporter. glyt1 and the related glyt2 mRNAs are expressed in the hindbrain and spinal cord in nonoverlapping patterns. The fact that these regions are known to be required for generation of early locomotory behaviors suggests that the regulation of extracellular glycine levels in the CNS is important for proper function of neural networks. Furthermore, physiological analysis after manipulation of glycinergic activity in wild-type and sho embryos suggests that the mutant phenotype is attributable to elevated extracellular glycine within the CNS.

Molecular cloning and expression of two small leucine-rich proteoglycan (SLRP) genes, dspg3l and optcl, in zebrafish.

Epiphycan (DSPG3) and opticin are two class III small leucine-rich proteoglycans (SLRP). We isolated two zebrafish cDNAs, dspg3l and optcl, that encode proteins homologous to epiphycan and opticin in other vertebrates. Like epiphycans in other species, dspg3l is exclusively expressed in the developing notochord and cartilage. optcl is expressed transiently in the developing nervous system, eyes and somites much like opticin. The zebrafish dspg3l and optcl genes are located in linkage group 4 and 11, respectively. The genomic locations for both genes in zebrafish are syntenic with the genomic locations of dspg3 and opticin (optc) in human and mouse. Synteny and the expression patterns of these genes suggest that the dspg3l and optcl are the orthologs to the mammalian dspg3 and optc genes, respectively.

Transmembrane sema4E guides branchiomotor axons to their targets in zebrafish.

Class 4 semaphorins are a large class of transmembrane proteins that contain a sema domain and that are expressed in the CNS, but their in vivo neural function is unknown. In zebrafish, the epithelial cells that line the pharyngeal arches express Sema4E. Extension of branchiomotor axons along the mesenchymal cells bounded by these epithelial cells suggests that Sema4E may act as a repulsive guidance molecule to restrict the branchiomotor axons to the mesenchymal cells. To test this hypothesis, Sema4E was misexpressed in hsp70 promoter-regulated transgenic zebrafish in which sema4E was heat-inducible, and Sema4E was knocked down by injection of antisense morpholino oligonucleotides that acted specifically against Sema4E. Ubiquitous induction of Sema4E retarded outgrowth by the facial and gill branchiomotor axons significantly. Furthermore, outgrowth by gill motor axons was specifically inhibited when Sema4E-expressing transgenic cells were transplanted to their pathway in nontransgenic host embryos. Morpholino knockdown of Sema4E caused facial motor axons to defasciculate and follow aberrant pathways. These results show that Sema4E is repulsive for facial and gill motor axons and functions as a barrier for these axons within the pharyngeal arches.

Semaphorin3D guides retinal axons along the dorsoventral axis of the tectum.

We examined the role of Sema3D, a semaphorin of previously unknown function, in guiding retinal ganglion cell (RGC) axons to the optic tectum in the developing zebrafish. Sema3D is expressed more strongly in the ventral versus dorsal tectum, suggesting that it may participate in guiding RGC axons along the dorsoventral axis of the tectum. Ubiquitous misexpression of Sema3D in transgenic zebrafish inhibits ventral but not dorsal RGC axon growth. In addition, ventral RGC axons avoid or stop at individual cells misexpressing Sema3D along their pathway. Sema3D ubiquitous misexpression at later stages also causes ventral RGC axon arbors to spread more widely along the dorsoventral axis of the tectum. Knock-down of Sema3D with morpholino antisense causes ventral RGC axons to extend aberrantly into the ventral tectum. These results suggest that Sema3D in the ventral tectum normally acts to inhibit ventral RGCs from extending into ventral tectum, ensuring their correct innervation of dorsal tectum.