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Homotypic Fusion and Protein Sorting (HOPS) and Class C-core Vacuole/Endosome Tethering (CORVET) complexes regulate the correct fusion of endolysosomal bodies. Mutations in core proteins (VPS11, VPS16, VPS18, and VPS33) have been linked with multiple neurological disorders, including mucopolysaccharidosis (MPS), genetic leukoencephalopathy (gLE), and dystonia. Mutations in human Vacuolar Protein Sorting 16 (VPS16) have been associated with MPS and dystonia. In this study, we generated and characterized a zebrafish vps16(-/-) mutant line using immunohistochemical and behavioral approaches. The loss of Vps16 function caused multiple systemic defects, hypomyelination, and increased neuronal cell death. Behavioral analysis showed a progressive loss of visuomotor response and reduced motor response and habituation to acoustic/tap stimuli in mutants. Finally, using a novel multiple-round acoustic/tap stimuli test, mutants showed intermediate memory deficits. Together, these data demonstrate that zebrafish vps16(-/-) mutants show systemic defects, neurological and motor system pathologies, and cognitive impairment. This is the first study to report behavior abnormalities and memory deficiencies in a zebrafish vps16(-/-) mutant line. Finally, we conclude that the deficits observed in vps16(-/-) zebrafish mutants do not mimic pathologies associated with dystonia, but more align to abnormalities associated with MPS and gLE. [ABSTRACT FROM AUTHOR]

In mammals, photoreceptor loss causes permanent blindness, but in zebrafish (Danio rerio), photoreceptor loss reprograms Müller glia to function as stem cells, producing progenitors that regenerate photoreceptors. MicroRNAs (miRNAs) regulate CNS neurogenesis, but the roles of miRNAs in injury-induced neuronal regeneration are largely unknown. In the embryonic zebrafish retina, miR-18a regulates photoreceptor differentiation. The purpose of the current study was to determine, in zebrafish, the function of miR-18a during injury-induced photoreceptor regeneration. RT-qPCR, in situ hybridization, and immunohistochemistry showed that miR-18a expression increases throughout the retina between 1 and 5 days post-injury (dpi). To test miR-18a function during photoreceptor regeneration, we used homozygous miR-18a mutants (miR-18ami5012), and knocked down miR-18a with morpholino oligonucleotides. During photoreceptor regeneration, miR-18ami5012 retinas have fewer mature photoreceptors than WT at 7 and 10 dpi, but there is no difference at 14 dpi, indicating that photoreceptor regeneration is delayed. Labeling dividing cells with 5-bromo-2′-deoxyuridine (BrdU) showed that at 7 and 10 dpi, there are excess dividing progenitors in both mutants and morphants, indicating that miR-18a negatively regulates injury-induced proliferation. Tracing 5-ethynyl-2′-deoxyuridine (EdU) and BrdU-labeled cells showed that in miR-18ami5012 retinas excess progenitors migrate to other retinal layers in addition to the photoreceptor layer. Inflammation is critical for photoreceptor regeneration, and RT-qPCR showed that in miR-18ami5012 retinas, inflammatory gene expression and microglia activation are prolonged. Suppressing inflammation with dexamethasone rescues the miR-18ami5012 phenotype. Together, these data show that in the injured zebrafish retina, disruption of miR-18a alters proliferation, inflammation, the microglia/macrophage response, and the timing of photoreceptor regeneration. [ABSTRACT FROM AUTHOR]

Ageing is a significant risk factor for degeneration of the retina. Müller glia cells (MG) are key for neuronal regeneration, so harnessing the regenerative capacity of MG in the retina offers great promise for the treatment of age‐associated blinding conditions. Yet, the impact of ageing on MG regenerative capacity is unclear. Here, we show that the zebrafish retina undergoes telomerase‐independent, age‐related neurodegeneration but that this is insufficient to stimulate MG proliferation and regeneration. Instead, age‐related neurodegeneration is accompanied by MG morphological aberrations and loss of vision. Mechanistically, yes‐associated protein (Yap), part of the Hippo signalling, has been shown to be critical for the regenerative response in the damaged retina, and we show that Yap expression levels decline with ageing. Despite this, morphologically and molecularly altered aged MG retain the capacity to regenerate neurons after acute light damage, therefore, highlighting key differences in the MG response to high‐intensity acute damage versus chronic neuronal loss in the zebrafish retina. [ABSTRACT FROM AUTHOR]

Genetic Leukoencephalopathies (gLEs) are heritable white matter disorders that cause progressive neurological abnormalities. A founder mutation in the human endolysosomal trafficking protein VPS11 has been identified in Ashkenazi Jewish patients manifesting classic gLE symptoms of hypomyelination, developmental delay, motor and systemic deficits. In this study, we characterized the visual and sensorimotor function of two zebrafish vps11 mutant lines: the previously reported vps11(plt), and a new vps11(−/−) null mutant line, using behavioral analysis to track larval motor responses to visual and acoustic stimuli. We found that mutant larvae from both vps11(plt) and vps11(−/−) lines were able to visually distinguish light and dark, but showed a progressive loss of a normal sensorimotor response to visual stimuli from 5 days post fertilization (dpf) to 7dpf. Additionally, optokinetic response analysis performed at 5dpf indicated that the mutants were significantly visually impaired. Both mutant lines also displayed a progressively lower sensorimotor response to a singular acoustic stimulus from 5-7dpf. Next, we tested the habituation response of the mutant lines to series of acoustic taps. We found both mutant lines habituated faster than their siblings, and that vps11(plt) mutants habituated faster than the vps11(−/−) mutants. Together, these data suggest that loss of Vps11 function results in progressive visual and sensorimotor abnormalities in the zebrafish vps11(plt) and vps11(−/−) mutant lines. This is the first study to characterize behavioral deficits in a vertebrate model of Vps11-dependent gLE. The mutants and behavioral assays described here could be a valuable model system in which to test potential pharmacological interventions for gLE. [ABSTRACT FROM AUTHOR]

A founder mutation in human VPS11 (Vacuolar Protein Sorting 11) was recently linked to a genetic leukoencephalopathy in Ashkenazi Jews that presents with the classical features of white matter disorders of the central nervous system (CNS). The neurological deficits include hypomyelination, hypotonia, gradual loss of vision, and seizures. However, the cells expressing the mutation were not identified. Here we describe, using immunocytochemistry, the strong expression of Vps11 in mouse oligodendrocytes and, specifically, its localization with Myelin Associated Glycoprotein (MAG) in the inner tongue of myelin. In longitudinal sections of myelin, it forms a bead-like structure, alternating with Myelin Basic Protein (MBP). Immunofluorescent staining with Vps11 and neurofilament proteins indicates the absence of Vps11 in axons in vivo. Finally, changes in Vps11 expression are associated with altered proteolipid protein (PLP) levels based upon mice with duplications or deletions of the Plp1 gene. To determine potential functional contributions of Vps11, we combined Vps11 with Platelet Derived Growth Factor Receptor-α (PDGFRα) in vitro and in vivo : in both conditions, co-localization of the two proteins was frequently found in round vesicles of OPCs/oligodendrocytes, suggesting retrograde transport for degradation by the endolysosomal system. Neuron-to-glial communication has been invoked to explain degenerative changes in myelin followed by degenerative changes in axons, and vice versa; but to our knowledge, no specific proteins in retrograde transport from the myelin inner tongue to oligodendrocyte perikarya have been identified. The identification of mutations in VPS11 and its localization at the axon-myelin interface should open new avenues of research. [ABSTRACT FROM AUTHOR]

Zebrafish have the ability to regenerate damaged cells and tissues by activating quiescent stem and progenitor cells or reprogramming differentiated cells into regeneration-competent precursors. Proliferation among the cells that will functionally restore injured tissues is a fundamental biological process underlying regeneration. Midkine-a is a cytokine growth factor, whose expression is strongly induced by injury in a variety of tissues across a range of vertebrate classes. Using a zebrafish Midkine-a loss of function mutant, we evaluated regeneration of caudal fin, extraocular muscle and retinal neurons to investigate the function of Midkine-a during epimorphic regeneration. In wildtype zebrafish, injury among these tissues induces robust proliferation and rapid regeneration. In Midkine-a mutants, the initial proliferation in each of these tissues is significantly diminished or absent. Regeneration of the caudal fin and extraocular muscle is delayed; regeneration of the retina is nearly completely absent. These data demonstrate that Midkine-a is universally required in the signaling pathways that convert tissue injury into the initial burst of cell proliferation. Further, these data highlight differences in the molecular mechanisms that regulate epimorphic regeneration in zebrafish. [ABSTRACT FROM AUTHOR]

Summary: Anecdotal evidence indicating substantial silica accumulation in tissues of bryophytes suggests that silica (phytolith) deposition evolved early on in embryophytes. To test this hypothesis, we conducted the first survey of phytolith content representing the major liverwort, moss and hornwort clades. We also assessed the diagnostic value of bryophyte phytoliths.Silica extracted from bryophyte material through wet‐ashing was described, focusing on abundance, classifying taxa as nonproducers, light producers and higher producers; and phytolith morphotypes. Ancestral state reconstruction of these characters was performed for mosses and liverworts using published phylogenies.Phytoliths are present in multiple subclades within liverworts, mosses and hornworts, but these phyla were not ancestrally high silica‐producers. Higher deposition occurs in liverworts and mosses with specialized water‐conducting cells.We hypothesize that active, high silica accumulation was not ancestral for embryophytes, but became possible in clades with increased water conductance. Phytoliths of diagnostic structures (e.g. pegged rhizoids) could help track bryophyte clades or water conductance evolution in the fossil record. [ABSTRACT FROM AUTHOR]

Müller glia in the zebrafish retina respond to retinal damage by re-entering the cell cycle, which generates large numbers of retinal progenitors that ultimately replace the lost neurons. In this study we compared the regenerative outcomes of adult zebrafish exposed to one round of phototoxic treatment with adult zebrafish exposed to six consecutive rounds of phototoxic treatment. We observed that Müller glia continued to re-enter the cell cycle to produce clusters of retinal progenitors in zebrafish exposed to multiple rounds of phototoxic light. Some abnormalities were noted, however. First, we found that retinas exposed to multiple rounds of damage exhibited a greater loss of photoreceptors at 36 hours of light damage than retinas that were exposed to their first round of light damage. In addition, we found that Müller glia appeared to have an increase in the acute gliotic response in retinas exposed to multiple rounds of light treatment. This was evidenced by cellular hypertrophy, changes in GFAP cellular localization, and transient increases in stat3 and gfap expression. Finally, following the sixth round of phototoxic lesion, we observed a significant increase in mis-localized HuC/D-positive amacrine and ganglion cells in the inner plexiform layer and outer retina, and a decreased number of regenerated blue cone photoreceptors. These data add to recent findings that retinal regeneration in adult zebrafish occurs concomitant with Müller glia reactivity and can result in the generation of aberrant neurons. These data are also the first to demonstrate that Müller glia appear to modify their phenotype in response to multiple rounds of phototoxic lesion, exhibiting an increase in acute gliosis while maintaining a remarkable capacity for long-term regeneration of photoreceptors. [ABSTRACT FROM AUTHOR]

On the basis of mouse I-Ab-binding motifs, two sequences of the murine apolipoprotein B-100 (mApoB-100), mApoB-1003501-3515 (designated P3) and mApoB-100978-992 (designated P6), were found to be immunogenic. In this report, we show that P6 is also atherogenic. Immunization of Apoe-/- mice fed a high-fat diet (HFD) with P6 resulted in enhanced development of aortic atheroma as compared to control mice immunized with an irrelevant peptide MOG35-55 or with complete Freund's adjuvant alone. Adoptive transfer of lymph node cells from P6-immunized donor mice to recipients fed an HFD caused exacerbated aortic atheromas, correlating P6-primed cells with disease development. Finally, P6-specific T cell clones were generated and adoptive transfer of T cell clones into recipients fed an HFD led to significant increase in aortic plaque coverage when compared to control animals receiving a MOG35-55-specific T cell line. Recipient mice not fed an HFD, however, did not exhibit such enhancement, indicating that an inflammatory environment facilitated the atherogenic activity of P6-specific T cells. That P6 is identical to or cross-reacts with a naturally processed peptide of ApoB-100 is evidenced by the ability of P6 to stimulate the proliferation of T cells in the lymph node of mice primed by full-length human ApoB-100. By identifying an atherogenic T cell epitope of ApoB-100 and establishing specific T cell clones, our studies open up new and hitherto unavailable avenues to study the nature of atherogenic T cells and their functions in the atherosclerotic disease process. [ABSTRACT FROM AUTHOR]

Regeneration requires cells to regulate proliferation and patterning according to their spatial position. Positional memory is a property that enables regenerating cells to recall spatial information from the uninjured tissue. Positional memory is hypothesized to rely on gradients of molecules, few of which have been identified. Here, we quantified the global abundance of transcripts, proteins, and metabolites along the proximodistal axis of caudal fins of uninjured and regenerating adult zebrafish. Using this approach, we uncovered complex overlapping expression patterns for hundreds of molecules involved in diverse cellular functions, including development, bioelectric signaling, and amino acid and lipid metabolism. Moreover, 32 genes differentially expressed at the RNA level had concomitant differential expression of the encoded proteins. Thus, the identification of proximodistal differences in levels of RNAs, proteins, and metabolites will facilitate future functional studies of positional memory during appendage regeneration. [ABSTRACT FROM AUTHOR]

In this study we sought to identify genetic factors associated with the presenting white blood cell (WBC) count in B-precursor acute lymphoblastic leukemia (BP-ALL). Using ETV6-RUNX1-positive BP-ALL patient samples, a homogeneous subtype, we identified 16 differentially expressed genes based on the presenting WBC count (< 50,000/cumm vs > 50,000). We further confirmed that IL1R1, BCAR3, KCNH2, PIR, and ZDHHC23 were differentially expressed in a larger cohort of ETV6-RUNX1-negative BP-ALL patient samples. Statistical analysis demonstrated that expression levels of these genes could accurately categorize high and low WBC count subjects using two independent patient sets, representing positive and negative ETV6-RUNX1 cases. Further studies in leukemia cell line models will better delineate the role of these genes in regulating the white blood cell count and potentially identify new therapeutic targets. [ABSTRACT FROM AUTHOR]

Midkine is a heparin binding growth factor with important functions in neuronal development and survival, but little is known about its function in the retina. Previous studies show that in the developing zebrafish, Midkine-a (Mdka) regulates cell cycle kinetics in retinal progenitors, and following injury to the adult zebrafish retina, mdka is strongly upregulated in Müller glia and the injury-induced photoreceptor progenitors. Here we provide the first data describing Mdka protein localization during different stages of retinal development and during the regeneration of photoreceptors in adults. We also experimentally test the role of Mdka during photoreceptor regeneration. The immuno-localization of Mdka reflects the complex spatiotemporal pattern of gene expression and also reveals the apparent secretion and extracellular trafficking of this protein. During embryonic retinal development the Mdka antibodies label all mitotically active cells, but at the onset of neuronal differentiation, immunostaining is also localized to the nascent inner plexiform layer. Starting at five days post fertilization through the juvenile stage, Mdka immunostaining labels the cytoplasm of horizontal cells and the overlying somata of rod photoreceptors. Double immunolabeling shows that in adult horizontal cells, Mdka co-localizes with markers of the Golgi complex. Together, these data are interpreted to show that Mdka is synthesized in horizontal cells and secreted into the outer nuclear layer. In adults, Mdka is also present in the end feet of Müller glia. Similar to mdka gene expression, Mdka in horizontal cells is regulated by circadian rhythms. After the light-induced death of photoreceptors, Mdka immuonolabeling is localized to Müller glia, the intrinsic stem cells of the zebrafish retina, and proliferating photoreceptor progenitors. Knockdown of Mdka during photoreceptor regeneration results in less proliferation and diminished regeneration of rod photoreceptors. These data suggest that during photoreceptor regeneration Mdka regulates aspects of injury-induced cell proliferation. [ABSTRACT FROM AUTHOR]

Gene trapping is a high-throughput approach to elucidate gene functions by disrupting and recapitulating expression of genes in a target genome. A number of transposon-based gene-trapping systems are developed for mutagenesis in cells and model organisms, but there is still much room for the improvement of their efficiency in gene disruption and mutation. Herein, a gene-trapping system mediated by Sleeping Beauty (SB) transposon was developed by inclusion of three functional cassettes. The mutation cassette can abrogate the splice of trapped genes and terminate their translation. Once an endogenous gene is captured, the finding cassette independently drives the translation of reporter gene in HeLa cells and zebrafish embryos. The efficiency cassette controls the remobilization of integrated traps through inducible expression of SB gene. Analysis of transposon-genome junctions indicate that most of trap cassettes are integrated into an intron without an obvious 39 bias. The transcription of trapped genes was abrogated by alternative splicing of the mutation cassette. In addition, integrated transposons can be induced to excise from their original insertion sites. Furthermore, the Cre/LoxP system was introduced to delete the efficiency cassette for stabilization of gene interruption and bio-safety. Thus, this gene- trap vector is an alternative and effective tool for the capture and disruption of endogenous genes in vitro and in vivo. [ABSTRACT FROM AUTHOR]

In this study, we used a newly-created transgenic zebrafish, Tg(nrd:egfp)/albino, to further characterize the expression of neurod in the developing and adult retina and to determine neurod expression during adult photoreceptor regeneration. We also provide observations regarding the expression of neurod in a variety of other tissues. In this line, EGFP is found in cells of the developing and adult retina, pineal gland, cerebellum, olfactory bulbs, midbrain, hindbrain, neural tube, lateral line, inner ear, pancreas, gut, and fin. Using immunohistochemistry and in situ hybridization, we compare the expression of the nrd:egfp transgene to that of endogenous neurod and to known retinal cell types. Consistent with previous data based on in situ hybridizations, we show that during retinal development, the nrd:egfp transgene is not expressed in proliferating retinal neuroepithelium, and is expressed in a subset of retinal neurons. In contrast to previous studies, nrd:egfp is gradually re-expressed in all rod photoreceptors. During photoreceptor regeneration in adult zebrafish, in situ hybridization reveals that neurod is not expressed in Müller glial-derived neuronal progenitors, but is expressed in photoreceptor progenitors as they migrate to the outer nuclear layer and differentiate into new rod photoreceptors. During photoreceptor regeneration, expression of the nrd:egfp matches that of neurod. We conclude that Tg(nrd:egfp)/albino is a good representation of endogenous neurod expression, is a useful tool to visualize neurod expression in a variety of tissues and will aid investigating the fundamental processes that govern photoreceptor regeneration in adults. [ABSTRACT FROM AUTHOR]

Hox genes are re-expressed during regeneration in many species. Given their important role in body plan development, it has been assumed, but not directly shown, that they play a functional role in regeneration. In this paper we show that morpholino-mediated knockdown of either Hoxc13a or Hoxc13b during the process of zebrafish tail fin regeneration results in a significant reduction of regenerative outgrowth. Furthermore, cellular proliferation within the blastema is directly affected in both knockdowns. Hence, similar to the demonstration of unique functions of multiple Hox genes during limb formation, both Hoxc13 orthologs have distinct functions in regeneration. [ABSTRACT FROM AUTHOR]

Recent studies suggest that organophosphates and carbamates affect human fetal development, resulting in neurological and growth impairment. However, these studies are conflicting and the extent of adverse effects due to pesticide exposure warrants further investigation. In the present study, we examined the impact of the carbamate insecticide propoxur on zebrafish development. We found that propoxur exposure delays embryonic development, resulting in three distinct developmental stages: no delay, mild delay, or severe delay. Interestingly, the delayed embryos all physically recovered 5 days after exposure, but behavioral analysis revealed persistent cognitive deficits at later stages. Microarray analysis identified 59 genes significantly changed by propoxur treatment, and Ingenuity Pathway Analysis revealed that these genes are involved in cancer, organismal abnormalities, neurological disease, and hematological system development. We further examined hspb9 and hspb11 due to their potential roles in zebrafish development and found that propoxur increases expression of these small heat shock proteins in all of the exposed animals. However, we discovered that less significant increases were associated with the more severely delayed phenotype. This raises the possibility that a decreased ability to upregulate these small heat shock proteins in response to propoxur exposure may cause embryos to be more severely delayed. [ABSTRACT FROM AUTHOR]

Gram-positive bacteria remain the leading cause of endophthalmitis, a blinding infectious disease of the eye. Murine models have been widely used for understanding the pathogenesis of bacterial endophthalmitis. In this study, we sought to develop an alternative zebrafish (Danio rerio) model for Staphylococcus aureus and compare the disease pathobiology to a murine model. Endophthalmitis was induced in zebrafish and C57BL/6 mice through the intravitreal injection of S. aureus. Disease progression was monitored by assessing corneal haze, opacity, bacterial burden, and retinal histology. Our results demonstrated that, unlike the murine models, zebrafish maintained ocular integrity, corneal transparency, and retinal architecture. We found that the zebrafish was capable of clearing S. aureus from the eye via transport through retinal vessels and the optic nerve and by mounting a monocyte/macrophage response beginning at 8 hour post-infection (hpi). The bacterial burden increased up to 8 hpi and significantly decreased thereafter. An assessment of the innate retinal response revealed the induced expression of Il-1β and Il-6 transcripts. Collectively, our study shows that unlike the murine model, zebrafish do not develop endophthalmitis and rapidly clear the pathogen. Hence, a better understanding of the zebrafish protective ocular innate response may provide new insights into the pathobiology of bacterial endophthalmitis. [ABSTRACT FROM AUTHOR]