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The muscular dystrophies encompass a broad range of pathologies with varied clinical outcomes. In the case of patients carrying defects in fukutin-related protein (FKRP), these diverse pathologies arise from mutations within the same gene. This is surprising as FKRP is a glycosyltransferase, whose only identified function is to transfer ribitol-5-phosphate to α-dystroglycan (α-DG). Although this modification is critical for extracellular matrix attachment, α-DG’s glycosylation status relates poorly to disease severity, suggesting the existence of unidentified FKRP targets. Here we reveal that FKRP directs sialylation of fibronectin, a process essential for collagen recruitment to the muscle basement membrane. Thus, our results reveal that FKRP simultaneously regulates the two major muscle-ECM linkages essential for fibre survival, and establishes a new disease axis for the muscular dystrophies., FKRP mutations cause muscular dystrophies with varied clinical presentations. The target of FKRP is α-dystroglycan, but here the authors show that FKRP also directs sialylation of fibronectin, a process that is essential for recruitment o collagen to the muscle basement membrane.

Retinal regeneration research offers hope to people affected by visual impairment due to disease and injury. Ongoing research has explored many avenues towards retinal regeneration, including those that utilizes implantation of devices, cells or targeted viral-mediated gene therapy. These results have so far been limited, as gene therapy only has applications for rare single-gene mutations and implantations are invasive and in the case of cell transplantation donor cells often fail to integrate with adult neurons. An alternative mode of retinal regeneration utilizes a stem cell population unique to vertebrate retina - Müller glia (MG). Endogenous MG can readily regenerate lost neurons spontaneously in zebrafish and to a very limited extent in mammalian retina. The use of adenosine triphosphate (ATP) has been shown to induce retinal degeneration and activation of the MG in mammals, but whether this is conserved to other vertebrate species including those with higher regenerative capacity remains unknown. In our study, we injected a single dose of ATP intravitreal in zebrafish to characterize the cell death and MG induced regeneration. We used TUNEL labelling on retinal sections to show that ATP caused localised death of photoreceptors and ganglion cells within 24 h. Histology of GFP-transgenic zebrafish and BrdU injected fish demonstrated that MG proliferation peaked at days 3 and 4 post-ATP injection. Using BrdU labelling and photoreceptor markers (Zpr1) we observed regeneration of lost rod photoreceptors at day 14. This study has been undertaken to allow for comparative studies between mammals and zebrafish that use the same specific induction method of injury, i.e. ATP induced injury to allow for direct comparison of across species to narrow down resulting differences that might reflect the differing regenerative capacity. The ultimate aim of this work is to recapitulate pro-neurogenesis Müller glia signaling in mammals to produce new neurons that integrate with the existing retinal circuit to restore vision.

Since the use of the zebrafish Danio rerio genetic model organism within the scientific research community continues to grow rapidly, continued procedural refinement to support high-quality, reproducible research and improve animal welfare remains an important focus. As such, anesthesia remains one of the most frequent procedures conducted. Here, we compared the effectiveness of clove oil (active ingredient eugenol) and AQUI-S (active ingredient iso-eugenol) with the currently most commonly used tricaine/MS-222 (ethyl 3-aminobenzoate methanesulfonate) and benzocaine anesthesia. We focused on embryos (1 day postfertilization), larvae (5 days postfertilization), and adults (9-11 months) and for the first time used exposure times that are the most relevant in research settings by using zebrafish as a genetic model system. For each age, tricaine and benzocaine achieved the most reproducible, robust anesthesia with the quickest induction and recovery. For some experimental procedures, specific clove oil concentrations in embryos and larvae may represent suitable alternatives. Although different aquatic species at specific ages respond differentially to these agents, the systematic study of comparable effective dosages for procedures most commonly employed represent an important step toward refinement.

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.

During neurogenesis, progenitors balance proliferation and cell cycle exit together with expression of fate determinant genes to ensure that the correct number of each of these neuron types is generated. Although intrinsic gene expression acting cell autonomously within each progenitor drives these processes, the final number of neurons generated is also influenced by extrinsic cues, representing a potential avenue to direct neurogenesis in developmental disorders or regenerative settings without the requirement to change intrinsic gene expression. Thus, it is important to understand which of these stages of neurogenesis are amenable to such extrinsic influences. Additionally, all types of neurons are specified in a highly conserved histogenic order, although its significance is unknown. This study makes use of conserved patterns of neurogenesis in the relatively simple yet highly organized zebrafish retina model, in which such histogenic birth order is well characterized. We directly visualize and quantify birth dates and cell fate determinant expression in WT vs. environments lacking different neuronal populations. This study shows that extrinsic feedback from developing retinal neurons is important for the temporal expression of intrinsic fate determinants but not for the timing of birth dates. We found no changes in cell cycle exit timing but did find a significant delay in the expression of genes driving the generation only of later- but not earlier-born cells, suggesting that the robustness of this process depends on continuous feedback from earlier-formed cell types. Thus, extrinsic cues selectively influence cell fate determinant progression, which may explain the function of the retinal histogenic order observed. J. Comp. Neurol. 524:2553-2566, 2016. © 2016 Wiley Periodicals, Inc.

Calcium binding proteins show stereotypical expression patterns within diverse neuron types across the central nervous system. Here, we provide a characterization of developmental and adult secretagogin-immunolabelled neurons in the zebrafish retina with an emphasis on co-expression of multiple calcium binding proteins. Secretagogin is a recently identified and cloned member of the F-hand family of calcium binding proteins, which labels distinct neuron populations in the retinas of mammalian vertebrates. Both the adult distribution of secretagogin labeled retinal neurons as well as the developmental expression indicative of the stage of neurogenesis during which this calcium binding protein is expressed was quantified. Secretagogin expression was confined to an amacrine interneuron population in the inner nuclear layer, with monostratified neurites in the center of the inner plexiform layer and a relatively regular soma distribution (regularity index > 2.5 across central-peripheral areas). However, only a subpopulation (~60%) co-labeled with gamma-aminobutyric acid as their neurotransmitter, suggesting that possibly two amacrine subtypes are secretagogin immunoreactive. Quantitative co-labeling analysis with other known amacrine subtype markers including the three main calcium binding proteins parvalbumin, calbindin and calretinin identifies secretagogin immunoreactive neurons as a distinct neuron population. The highest density of secretagogin cells of ~1800 cells / mm2 remained relatively evenly along the horizontal meridian, whilst the density dropped of to 125 cells / mm2 towards the dorsal and ventral periphery. Thus, secretagogin represents a new amacrine label within the zebrafish retina. The developmental expression suggests a possible role in late stage differentiation. This characterization forms the basis of functional studies assessing how the expression of distinct calcium binding proteins might be regulated to compensate for the loss of one of the others.

Background Unlike mammals, zebrafish exhibits extensive neural regeneration after injury in adult stages of its lifetime due to the neurogenic activity of the radial glial cells. However, the genes involved in the regenerative neurogenesis response of the zebrafish brain are largely unknown. Thus, understanding the underlying principles of this regeneration capacity of the zebrafish brain is an interesting research realm that may offer vast clinical ramifications. Results In this paper, we characterized the expression pattern of cxcr5 and analyzed the function of this gene during adult neurogenesis and regeneration of the zebrafish telencephalon. We found that cxcr5 was upregulated transiently in the RGCs and neurons, and the expression in the immune cells such as leukocytes was negligible during both adult neurogenesis and regeneration. We observed that the transgenic misexpression of cxcr5 in the ventricular cells using dominant negative and full-length variants of the gene resulted in altered proliferation and neurogenesis response of the RGCs. When we knocked down cxcr5 using antisense morpholinos and cerebroventricular microinjection, we observed outcomes similar to the overexpression of the dominant negative cxcr5 variant. Conclusions Thus, based on our results, we propose that cxcr5 imposes a proliferative permissiveness to the radial glial cells and is required for differentiation of the RGCs to neurons, highlighting novel roles of cxcr5 in the nervous system of vertebrates. We therefore suggest that cxcr5 is an important cue for ventricular cell proliferation and regenerative neurogenesis in the adult zebrafish telencephalon. Further studies on the role of cxcr5 in mediating neuronal replenishment have the potential to produce clinical ramifications in efforts for regenerative therapeutic applications for human neurological disorders or acute injuries.