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Cyclin-dependent kinase-like-5 (CDKL5) deficiency disorder (CDD) is a severe X-linked neurodegenerative disease characterised by early-onset epileptic seizures, low muscle tone, progressive intellectual disability and severe motor function. CDD affects ∼1 in 60,000 live births, with many patients experiencing a reduced quality of life due to the severity of their neurological symptoms and functional impairment. There are no effective therapies for CDD, with current treatments focusing on improving symptoms rather than addressing the underlying causes of the disorder. Zebrafish offer many unique advantages for high-throughput preclinical evaluation of potential therapies for neurological diseases, including CDD. In particular, the large number of offspring produced, together with the possibilities for in vivo imaging and genetic manipulation, allows for the detailed assessment of disease pathogenesis and therapeutic discovery. We have characterised a loss-of-function zebrafish model for CDD, containing a nonsense mutation in cdkl5. cdkl5 mutant zebrafish display defects in neuronal patterning, seizures, microcephaly, and reduced muscle function caused by impaired muscle innervation. This study provides a powerful vertebrate model for investigating CDD disease pathophysiology and allowing high-throughput screening for effective therapies. This article has an associated First Person interview with the first author of the paper.

Cyclin-dependent kinase-like-5 (CDKL5) Deficiency Disorder (CDD) is a severe X-linked neurodegenerative disease characterized by early-onset epileptic seizures, low muscle tone, progressive intellectual disability, severe motor function and visual impairment. CDD affects approximately 1 in 60,000 live births with many patients dying by early adulthood. For many patients, quality of life is significantly reduced due to the severity of their neurological symptoms and functional impairment. There are no effective therapies for CDD with current treatments focusing on improving symptoms rather than addressing the underlying causes of the disorder. Zebrafish offer a number of unique advantages for high-throughput pre-clinical evaluation of potential therapies for human neurological diseases including CDD. In particular, the large number of zebrafish that can be produced, together with the possibilities for in vivo imaging and genetic manipulation, allows for the detailed assessment of disease pathogenesis and therapeutic discovery. We have characterised a loss of function zebrafish model for CDD, containing a nonsense mutation in cdkl5. cdkl5 mutant zebrafish display defects in neuronal patterning, microcephaly, and reduced muscle function caused by impaired muscle innervation. This study provides a powerful vertebrate model to investigate CDD disease pathophysiology and allow high-throughput screening for effective therapies.

International audience; The mechanisms that modulate the kinetics of muscle relaxation are critically important for muscle function. A prime example of the impact of impaired relaxation kinetics is nemaline myopathy caused by mutations in KBTBD13 (NEM6). In addition to weakness, NEM6 patients have slow muscle relaxation, compromising contractility and daily life activities. The role of KBTBD13 in muscle is unknown, and the pathomechanism underlying NEM6 is undetermined. A combination of transcranial magnetic stimulation-induced muscle relaxation, muscle fiber- and sarcomere-contractility assays, low-angle x-ray diffraction, and superresolution microscopy revealed that the impaired muscle-relaxation kinetics in NEM6 patients are caused by structural changes in the thin filament, a sarcomeric microstructure. Using homology modeling and binding and contractility assays with recombinant KBTBD13, Kbtbd13-knockout and Kbtbd13R408C-knockin mouse models, and a GFPlabeled Kbtbd13-transgenic zebrafish model, we discovered that KBTBD13 binds to actin - a major constituent of the thin filament - and that mutations in KBTBD13 cause structural changes impairing muscle-relaxation kinetics. We propose that this actin-based impaired relaxation is central to NEM6 pathology.

Purpose To compare the effects of reduced inhibitory neuron function in the retina across behavioral, physiological, and anatomical levels. Methods Inhibitory neurons were ablated in larval zebrafish retina. The Ptf1a gene, which determines inhibitory neuron fate in developing vertebrates, was used to express nitroreductase. By exposing larvae to the prodrug metronidazole, cytotoxicity was selectively induced in inhibitory neurons. Visual phenotypes were characterized at behavioral, physiological, and anatomical levels using an optomotor response (OMR) assay, electroretinography (ERG), and routine histology, respectively. Nonvisual locomotion was also assessed to reveal any general behavioral effects due to ablation of other nonvisual neurons that also express Ptf1a. Results Injured larvae showed severely reduced OMR relative to controls. Locomotor assessment showed unaltered swimming ability, indicating that reduced OMR was due to visual deficits. For ERG, injured larvae manifested either reduced (type-I) or absent (type-II) b-wave signals originating from bipolar interneurons in the retina. Histologic analysis showed altered retinal morphology in injured larvae, with reductions in synaptic inner plexiform layer (IPL) thickness and synaptic density more pronounced in type-II than type-I larvae; type-II larvae also had smaller retinae overall. Conclusions The consequences of inhibitory neuron ablation corresponded closely across behavioral, physiological, and anatomical levels. Inhibitory neuron loss likely increases the ratio of neural excitation to inhibition, leading to hyperexcitability. In addition to modulating visual signals, inhibitory neurons may be critical for maintaining retinal structure and organization. This study highlights the utility of a multidisciplinary approach and provides a template for characterizing other zebrafish models of neurological disease.

Laminins comprise structural components of basement membranes, critical in the regulation of differentiation, survival and migration of a diverse range of cell types, including skeletal muscle. Mutations in one muscle enriched Laminin isoform, Laminin alpha2 (Lama2), results in the most common form of congenital muscular dystrophy, congenital muscular dystrophy type 1A (MDC1A). However, the exact cellular mechanism by which Laminin loss results in the pathological spectrum associated with MDC1A remains elusive. Here we show, via live tracking of individual muscle fibres, that dystrophic myofibres in the zebrafish model of MDC1A maintain sarcolemmal integrity and undergo dynamic remodelling behaviours post detachment, including focal sarcolemmal reattachment, cell extension and hyper-fusion with surrounding myoblasts. These observations imply the existence of a window of therapeutic opportunity, where detached cells may be “re-functionalised” prior to their delayed entry into the cell death program, a process we show can be achieved by muscle specific or systemic Laminin delivery. We further reveal that Laminin also acts as a pro-regenerative factor that stimulates muscle stem cell-mediated repair in lama2-deficient animals in vivo. The potential multi-mode of action of Laminin replacement therapy suggests it may provide a potent therapeutic axis for the treatment for MDC1A.

Purpose The human PDZK1 gene is located in a genomic susceptibility region for neurodevelopmental disorders. A genome-wide association study identified links between PDZK1 polymorphisms and altered visual contrast sensitivity, an endophenotype for schizophrenia and autism spectrum disorder. The PDZK1 protein is implicated in neurological functioning, interacting with synaptic molecules including postsynaptic density 95 (PSD-95), N-methyl-d-aspartate receptors (NMDARs), corticotropin-releasing factor receptor 1 (CRFR1), and serotonin 2A receptors. The purpose of the present study was to elucidate the role of PDZK1. Methods We generated pdzk1-knockout (pdzk1-KO) zebrafish using CRISPR/Cas-9 genome editing. Visual function of 7-day-old fish was assessed at behavioral and functional levels using the optomotor response and scotopic electroretinogram (ERG). We also quantified retinal morphology and densities of PSD-95, NMDAR1, CRFR1, and serotonin in the synaptic inner plexiform layer at 7 days, 4 weeks, and 8 weeks of age. Standard RT-PCR and nonsense-mediated decay interference treatment were also performed to assess genetic compensation in mutants. Results Relative to wild-type, pdzk1-KO larvae showed spatial frequency tuning functions with increased amplitude (likely due to abnormal gain control) and reduced ERG b-waves (suggestive of inner retinal dysfunction). No synaptic phenotypes, but possible morphological retinal phenotypes, were identified. We confirmed that the absence of major histological phenotypes was not attributable to genetic compensatory mechanisms. Conclusions Our findings point to a role for pdzk1 in zebrafish visual function, and our model system provides a platform for investigating other genes associated with abnormal visual behavior.

L-tyrosine supplementation may provide benefit to nemaline myopathy (NM) patients, however previous studies are inconclusive, with no elevation of L-tyrosine levels in blood or tissue reported. We evaluated the ability of L-tyrosine treatments to improve skeletal muscle function in all three published animal models of NM caused by dominant skeletal muscle α-actin (ACTA1) mutations. Highest safe L-tyrosine concentrations were determined for dosing water and feed of wildtype zebrafish and mice respectively. NM TgACTA1D286G-eGFP zebrafish treated with 10 μM L-tyrosine from 24 hours to 6 days post fertilization displayed no improvement in swimming distance. NM TgACTA1D286G mice consuming 2% L-tyrosine supplemented feed from preconception had significant elevations in free L-tyrosine levels in sera (57%) and quadriceps muscle (45%) when examined at 6–7 weeks old. However indicators of skeletal muscle integrity (voluntary exercise, bodyweight, rotarod performance) were not improved. Additionally no benefit on the mechanical properties, energy metabolism, or atrophy of skeletal muscles of 6–7 month old TgACTA1D286G and KIActa1H40Y mice eventuated from consuming a 2% L-tyrosine supplemented diet for 4 weeks. Therefore this study yields important information on aspects of the clinical utility of L-tyrosine for ACTA1 NM.

Nemaline myopathies are heterogeneous congenital muscle disorders causing skeletal muscle weakness and, in some cases, death soon after birth. Mutations in nebulin, encoding a large sarcomeric protein required for thin filament function, are responsible for approximately 50% of nemaline myopathy cases. Despite the severity of the disease there is no effective treatment for nemaline myopathy with limited research to develop potential therapies. Several supplements, including L-tyrosine, have been suggested to be beneficial and consequently self-administered by nemaline myopathy patients without any knowledge of their efficacy. We have characterized a zebrafish model for nemaline myopathy caused by a mutation in nebulin. These fish form electron-dense nemaline bodies and display reduced muscle function akin to the phenotypes observed in nemaline myopathy patients. We have utilized our zebrafish model to test and evaluate four treatments currently self-administered by nemaline myopathy patients to determine their ability to increase skeletal muscle function. Analysis of muscle pathology and locomotion following treatment with L-tyrosine, L-carnitine, taurine, or creatine revealed no significant improvement in skeletal muscle function emphasizing the urgency to develop effective therapies for nemaline myopathy. Electronic supplementary material The online version of this article (10.1186/s40478-018-0546-9) contains supplementary material, which is available to authorized users.

The lack of a mutant phenotype in homozygous mutant individuals’ due to compensatory gene expression triggered upstream of protein function has been identified as genetic compensation. Whilst this intriguing process has been recognized in zebrafish, the presence of homozygous loss of function mutations in healthy human individuals suggests that compensation may not be restricted to this model. Loss of skeletal α-actin results in nemaline myopathy and we have previously shown that the pathological symptoms of the disease and reduction in muscle performance are recapitulated in a zebrafish antisense morpholino knockdown model. Here we reveal that a genetic actc1b mutant exhibits mild muscle defects and is unaffected by injection of the actc1b targeting morpholino. We further show that the milder phenotype results from a compensatory transcriptional upregulation of an actin paralogue providing a novel approach to be explored for the treatment of actin myopathy. Our findings provide further evidence that genetic compensation may influence the penetrance of disease-causing mutations., Author summary Many healthy individuals carry loss of function mutations in essential genes that would normally be deleterious for survival. Intriguingly, it may be the presence of the genomic lesion itself in these individuals that triggers the compensatory pathways. It is not known how widespread this phenomenon is in vertebrate populations and how genetic compensation is activated. We have shown that knockdown of actin causes nemaline myopathy as indicated by the formation of nemaline bodies within the skeletal muscle and reduced muscle function which, remarkably, we did not observe in an actin genetic mutant. We have identified that protection from the disease phenotype results from transcriptional upregulation of an actin paralogue restoring actin protein in the skeletal muscle. This study demonstrates that genetic compensation may be more prevalent than previously anticipated and highlights phenotypic differences resulting from genetic mutations versus antisense knockdown approaches. Furthermore, we suggest that activating compensatory pathways may be exploited as a potential novel therapeutic approach for human disorders caused by loss of function mutations.

Nemaline myopathy (NM) is a skeletal muscle disorder with no curative treatment. Although L-tyrosine administration has been indicated to provide benefit to patients, previous studies have been limited due to sample size or not testing for raised L-tyrosine levels. We evaluated the efficacy of L-tyrosine treatment to improve skeletal muscle function in three animal models of NM caused by skeletal muscle α-actin (ACTA1) mutations. Firstly we determined the maximum safest L-tyrosine concentration for inclusion in the water of wildtype zebrafish. We then treated NM TgACTA1D286G-eGFPzebrafish from 24 hours post fertilization with the highest safe L-tyrosine dose (10 µM). At 6 days post fertilization, no significant improvement was detected in skeletal muscle function (swimming distance). We also determined the highest safe L-tyrosine dose for dietary L-tyrosine supplementation to wildtype mice. Next we treated the NM TgACTA1D286Gmouse model continuously from preconception with 2% L-tyrosine supplemented to regular feed. We examined skeletal muscles at 6–7 weeks using indicators of skeletal muscle integrity: bodyweight, voluntary running wheel and rotarod performance, all parameters previously shown to be reduced in TgACTA1D286Gmice. The L-tyrosine treatment regime did not result in any improvement of these parameters, despite significant elevation of free L-tyrosine levels in sera (57%) and quadriceps muscle (45%) of treated TgACTA1D286Gmice. Additionally, we assessed the effects of 4 weeks of 2% L-tyrosine dietary supplementation on skeletal muscle function of older (6-7 month old) NM TgACTA1D286Gand KIActa1H40Ymice. This dosing regime did not improve decreased bodyweight, nor the mechanical properties, energy metabolism, or atrophy of skeletal muscles in these NM models. Together these findings demonstrate that with the treatment regimes and doses evaluated, L-tyrosine does not therapeutically modulate dysfunctional skeletal muscles in NM animal models with dominantACTA1mutations. Therefore this study yields important information on aspects of the clinical utility of L-tyrosine forACTA1NM.Summary statementDespite previous encouraging reports, this study utilising zebrafish and mouse models of nemaline myopathy shows no therapeutic benefit on skeletal muscle functionality in response to L-tyrosine supplementation.

The zebrafish is an excellent vertebrate model system to investigate skeletal muscle development and disease. During early muscle formation the small size of the developing zebrafish allows for the characterization of gene expression in whole embryos. However, as the zebrafish develops, access to the underlying skeletal muscle is limited, requiring the skeletal muscle to be sectioned for a more detailed examination. Here, we describe a straightforward and effective method to prepare adult zebrafish skeletal muscle sections, preserving muscle morphology, to characterize gene expression in the zebrafish adult skeletal muscle.

Zebrafish muscle development is highly conserved with mammalian systems making them an excellent model to study muscle function and disease. Many myopathies affecting skeletal muscle function can be quickly and easily assessed in zebrafish over the first few days of embryogenesis. By 24 hr post-fertilization (hpf), wildtype zebrafish spontaneously contract their tail muscles and by 48 hpf, zebrafish exhibit controlled swimming behaviors. Reduction in the frequency of, or other alterations in, these movements may indicate a skeletal muscle dysfunction. To analyze swimming behavior and assess muscle performance in early zebrafish development, we utilize both touch-evoked escape response and locomotion assays. Touch-evoked escape response assays can be used to assess muscle performance during short burst movements resulting from contraction of fast-twitch muscle fibers. In response to an external stimulus, which in this case is a tap on the head, wildtype zebrafish at 2 days post-fertilization (dpf) typically exhibit a powerful burst swim, accompanied by sharp turns. Our method quantifies skeletal muscle function by measuring the maximum acceleration during a burst swimming motion, the acceleration being directly proportional to the force produced by muscle contraction. In contrast, locomotion assays during early zebrafish larval development are used to assess muscle performance during sustained periods of muscle activity. Using a tracking system to monitor swimming behavior, we obtain an automated calculation of the frequency of activity and distance in 6-day old zebrafish, reflective of their skeletal muscle function. Measurements of swimming performance are valuable for phenotypic assessment of disease models and high-throughput screening of mutations or chemical treatments affecting skeletal muscle function.

This study establishes PYROXD1 variants as a cause of early-onset myopathy and uses biospecimens and cell lines, yeast, and zebrafish models to elucidate the fundamental role of PYROXD1 in skeletal muscle. Exome sequencing identified recessive variants in PYROXD1 in nine probands from five families. Affected individuals presented in infancy or childhood with slowly progressive proximal and distal weakness, facial weakness, nasal speech, swallowing difficulties, and normal to moderately elevated creatine kinase. Distinctive histopathology showed abundant internalized nuclei, myofibrillar disorganization, desmin-positive inclusions, and thickened Z-bands. PYROXD1 is a nuclear-cytoplasmic pyridine nucleotide-disulphide reductase (PNDR). PNDRs are flavoproteins (FAD-binding) and catalyze pyridine-nucleotide-dependent (NAD/NADH) reduction of thiol residues in other proteins. Complementation experiments in yeast lacking glutathione reductase glr1 show that human PYROXD1 has reductase activity that is strongly impaired by the disease-associated missense mutations. Immunolocalization studies in human muscle and zebrafish myofibers demonstrate that PYROXD1 localizes to the nucleus and to striated sarcomeric compartments. Zebrafish with ryroxD1 knock-down recapitulate features of PYROXD1 myopathy with sarcomeric disorganization, myofibrillar aggregates, and marked swimming defect. We characterize variants in the oxidoreductase PYROXD1 as a cause of early-onset myopathy with distinctive histopathology and introduce altered redox regulation as a primary cause of congenital muscle disease.

Laminins form essential components of the basement membrane and are integral to forming and maintaining muscle integrity. Mutations in the human Laminin-alpha2 (LAMA2) gene result in the most common form of congenital muscular dystrophy, MDC1A. We have previously identified a zebrafish model of MDC1A called candyfloss (caf), carrying a loss-of-function mutation in the zebrafish lama2 gene. In the skeletal muscle, laminins connect the muscle cell to the extracellular matrix (ECM) by binding either dystroglycan or integrins at the cell membrane. Through epistasis experiments, we have established that both adhesion systems individually contribute to the maintenance of fibre adhesions and exhibit muscle detachment phenotypes. However, larval zebrafish in which both adhesion systems are simultaneously genetically inactivated possess a catastrophic failure of muscle attachment that is far greater than a simple addition of individual phenotypes would predict. We provide evidence that this is due to other crucial laminins present in addition to Lama2, which aid muscle cell attachments and integrity. We have found that lama1 is important for maintaining attachments, whereas lama4 is localized and up-regulated in damaged fibres, which appears to contribute to fibre survival. Importantly, our results show that endogenous secretion of laminins from the surrounding tissues has the potential to reinforce fibre attachments and strengthen laminin-ECM attachments. Collectively these findings provide a better understanding of the cellular pathology of MDC1A and help in designing effective therapies.

One of the central questions of developmental biology is how cells of equivalent potential—an equivalence group—come to adopt specific cellular fates. In this study we have used a combination of live imaging, single cell lineage analyses, and perturbation of specific signaling pathways to dissect the specification of the adaxial cells of the zebrafish embryo. We show that the adaxial cells are myogenic precursors that form a cell fate equivalence group of approximately 20 cells that consequently give rise to two distinct sub-types of muscle fibers: the superficial slow muscle fibers (SSFs) and muscle pioneer cells (MPs), distinguished by specific gene expression and cell behaviors. Using a combination of live imaging, retrospective and indicative fate mapping, and genetic studies, we show that MP and SSF precursors segregate at the beginning of segmentation and that they arise from distinct regions along the anterior-posterior (AP) and dorsal-ventral (DV) axes of the adaxial cell compartment. FGF signaling restricts MP cell fate in the anterior-most adaxial cells in each somite, while BMP signaling restricts this fate to the middle of the DV axis. Thus our results reveal that the synergistic actions of HH, FGF, and BMP signaling independently create a three-dimensional (3D) signaling milieu that coordinates cell fate within the adaxial cell equivalence group., Author Summary How specific genes and signals act on initially identical cells to generate the different tissues of the body remains one of the central questions of developmental genetics. Zebrafish are a useful model system to tackle this question as the optically clear embryo allows direct imaging of forming tissues, tracking individual cells in a myriad of different genetic contexts. The zebrafish myotome, the compartment of the embryo that gives rise to skeletal muscle, is subdivided into a number of specific cell types—one of which, the adaxial cells, gives rise exclusively to muscle of the “slow twitch” class. The adaxial cells give rise to two types of slow muscle cell types, muscle pioneer cells and non-muscle pioneer slow cells, distinguished by gene expression and different cellular behaviours. In this study we use lineage tracing live imaging and the manipulation of distinct genetic pathways to demonstrate that the adaxial cells form a cell fate “equivalence group” that is specified using separate signaling pathways that operating in distinct dimensions.

Adult zebrafish show a remarkable capacity to regenerate their spinal column after injury, an ability that stands in stark contrast to the limited repair that occurs within the mammalian CNS post-injury. The reasons for this interspecies difference in regenerative capacity remain unclear. Here we demonstrate a novel role for Fgf signaling during glial cell morphogenesis in promoting axonal regeneration after spinal cord injury. Zebrafish glia are induced by Fgf signaling, to form an elongated bipolar morphology that forms a bridge between the two sides of the resected spinal cord, over which regenerating axons actively migrate. Loss of Fgf function inhibits formation of this “glial bridge” and prevents axon regeneration. Despite the poor potential for mammalian axonal regeneration, primate astrocytes activated by Fgf signaling adopt a similar morphology to that induced in zebrafish glia. This suggests that differential Fgf regulation, rather than intrinsic cell differences, underlie the distinct responses of mammalian and zebrafish glia to injury.

Laminins are essential components of all basement membranes and are fundamental to tissue development and homeostasis. Humans possess at least 16 different heterotrimeric laminin complexes formed through different combinations of alpha, beta, and gamma chains. Individual chains appear to exhibit unique expression patterns, leading to the notion that overlap between expression domains governs the constitution of complexes found within particular tissues. However, the spatial and temporal expression of laminin genes has not been comprehensively analyzed in any vertebrate model to date. Here, we describe the tissue-specific expression patterns of all laminin genes in the zebrafish, throughout embryonic development and into the "post-juvenile" period, which is representative of the adult body form. In addition, we present phylogenetic and microsynteny analyses, which demonstrate that the majority of our zebrafish sequences are orthologous to human laminin genes. Together, these data represent a fundamental resource for the study of vertebrate laminins.

Congenital myopathies are characterized by progressive muscle weakness and low muscle tone. Mutations in ACTA1 are responsible for up to 20% of all congenital myopathies ranging in severity from long-term survival with minor muscle weakness to lethality prior to or shortly after birth. To date over 200 different ACTA1 mutations have been described. At the cellular level ACTA1 mutations result in multiple pathologies including; nemaline bodies, intranuclear rods, actin aggregates, congenital fibre type disproportion, or cores, however, the exact pathophysiology of muscle weakness remains unknown. To investigate the mechanism by which mutations results in the distinct pathologies we have generated stable transgenic zebrafish strains expressing human ACTA1 tagged with GFP. We show, that expression of ACTA1 variants in zebrafish recapitulates the histo-pathological hallmarks of their respective diseases. Using the advantages of the zebrafish model system we followed the progression of disease in vivo and identified distinct mechanisms of muscle weakness. We show that in ACTA1D286G-GFP fish, nemaline bodies initiate as punctate actin aggregations at the muscle attachment sites before elongating to form the characteristic rod shape. In addition to its inclusion in rods, ACTA1D286G-GFP is incorporated into sarcomeres, which is the likely cause of muscle weakness. By contrast the formation of actin aggregates in ACTA1D154N myopathy occurs in the cytoplasm without a clearly defined site of origin and aggregates remain punctate, never elongating to form rods. The accumulation of aggregates triggers cell death leading to muscle weakness. The generation of zebrafish models and identification of distinct mechanisms of disease provides an experimental platform for the development, testing and specific targeting of potential therapies to combat Actin myopathies.

Evidence suggests a proinflammatory role of lysophosphatidic acid (LPA) in various pathologic abnormalities, including in the central nervous system. Herein, we describe LPA as an important mediator of inflammation after spinal cord injury (SCI) in zebrafish and mice. Furthermore, we describe a novel monoclonal blocking antibody raised against LPA that potently inhibits LPA's effect in vitro and in vivo. This antibody, B3, specifically binds LPA, prevents it from interacting with its complement of receptors, and blocks LPA's effects on the neuronal differentiation of human neural stem/progenitor cells, demonstrating its specificity toward LPA signaling. When administered systemically to mice subjected to SCI, B3 substantially reduced glial inflammation and neuronal death. B3-treated animals demonstrated significantly more neuronal survival upstream of the lesion site, with some functional improvement. This study describes the use of anti-LPA monoclonal antibody as a novel therapeutic approach for the treatment of SCI.