Morphology Of Zebrafish Research Articles

NMJ Analyser: a novel method to quantify neuromuscular junction morphology in zebrafish.

Neuromuscular junction (NMJ) structural integrity is crucial for transducing motor neuron signals that initiate skeletal muscle contraction. Zebrafish has emerged as a simple and efficient model to study NMJ structural morphology and function in the context of developmental neurobiology and neuromuscular diseases. However, methods to quantify NMJ morphology from voluminous data of NMJ confocal images accurately, rapidly, and reproducibly are lacking. We developed an ImageJ macro called "NMJ Analyser" to automatically and unbiasedly analyse NMJ morphology in zebrafish. From the Z-stack of a zebrafish hemisomite, both presynaptic and postsynaptic fluorescently labeled termini at NMJs are extracted from background signal, with larger clusters of termini being segmented into individual termini using an unbiased algorithm. The program then determines whether each presynaptic terminus is co-localized with a postsynaptic terminus and vice versa, or whether it is orphaned, and tabulates the number of orphan and co-localized pre- and postsynaptic termini. The usefulness of this ImageJ macro plugin will be helpful to quantify NMJ parameters in zebrafish, particularly during development and in disease models of neuromuscular diseases. It can enable high-throughput NMJ phenotypic screens in the drug discovery process for neuromuscular diseases. It could also be further applied to the investigation of NMJ of other developmental systems. NMJ Analyser is available for download at https://github.com/PattenLab/NMJ-Analyser.git.

A Novel Cis-Regulatory lncRNA, Kalnc2, Downregulates Kalrn Protein-Coding Transcripts in Mouse Neuronal Cells

The KALRN gene encodes several multi-domain protein isoforms that localize to neuronal synapses, conferring the ability to grow and retract dendritic spines and shaping axonal outgrowth, dendrite morphology, and dendritic spine re-modeling. The KALRN genomic locus is implicated in several neurodevelopmental and neuropsychiatric diseases, including autism, schizophrenia, bipolar disease, and intellectual disability. We have previously shown that a novel brain-specific long non-coding RNA (lncRNA) arising from the 5' end of the kalrna gene, called durga, regulates neuronal morphology in zebrafish. Here, we characterized mammalian Kalrn loci, annotating and experimentally validating multiple novel non-coding RNAs, including linear and circular variants. Comparing the mouse and human loci, we show that certain non-coding RNAs and Kalrn protein-coding isoforms arising from the locus show similar expression dynamics during development. In humans, mice, and zebrafish, the 5' end of the Kalrn locus gives rise to a chromatin-associated lncRNA that is present in adult ovaries, besides being expressed during brain development and enriched in certain regions of the adult brain. Ectopic expression of this lncRNA led to the downregulation of all the major Kalrn mRNA isoforms. We propose that this lncRNA arising from the 5' end of the Kalrn locus is functionally the mammalian ortholog of zebrafish lncRNA durga.

In vivo exposure to pyriproxyfen causes ovarian oxidative stress and inhibits follicle maturation in zebrafish.

We analyzed the effects of pyriproxyfen (PPF) on oxidative stress and ovarian morphology in zebrafish. PPF (10-9 M) exposure increased reactive oxygen species generation in ovaries, in association with a decrease in glutathione content. The activities of glutathione S-transferase, superoxide dismutase, and catalase were increased, while γ-glutamyltransferase activity was not altered by pesticide treatment. The histology of ovarian tissue showed an increase in the number of previtellogenic oocytes I, and a decrease in the rate of vitellogenic oocyte (VIT) count, suggesting inhibition of follicular maturation. An increase in the thickness of the vitelline envelope was observed in VIT, as was a tendency toward an increase in atresia in the ovary of the PPF-treated group. These findings indicate that the deleterious effect of PPF on ovarian maturation is mediated by a redox imbalance and oxidative damage. So, PPF acts as an endocrine disruptor chemical and may compromise fish reproduction by reducing female fertility.

Selective Histone Deacetylase 6 Inhibitors Restore Cone Photoreceptor Vision or Outer Segment Morphology in Zebrafish and Mouse Models of Retinal Blindness.

Blindness arising from retinal or macular degeneration results in significant social, health and economic burden. While approved treatments exist for neovascular (‘wet’) age-related macular degeneration, new therapeutic targets/interventions are needed for the more prevalent atrophic (‘dry’) form of age-related macular degeneration. Similarly, in inherited retinal diseases, most patients have no access to an effective treatment. Although macular and retinal degenerations are genetically and clinically distinct, common pathological hallmarks can include photoreceptor degeneration, retinal pigment epithelium atrophy, oxidative stress, hypoxia and defective autophagy. Here, we evaluated the potential of selective histone deacetylase 6 inhibitors to preserve retinal morphology or restore vision in zebrafish atp6v0e1–/– and mouse rd10 models. Histone deacetylase 6 inhibitor, tubastatin A-treated atp6v0e1–/– zebrafish show marked improvement in photoreceptor outer segment area (44.7%, p = 0.027) and significant improvement in vision (8-fold, p ≤ 0.0001). Tubastatin A-treated rd10/rd10 retinal explants show a significantly (p = 0.016) increased number of outer-segment labeled cone photoreceptors. In vitro, ATP6V0E1 regulated HIF-1α activity, but significant regulation of HIF-1α by histone deacetylase 6 inhibition in the retina was not detected. Proteomic profiling identified ubiquitin-proteasome, phototransduction, metabolism and phagosome as pathways, whose altered expression correlated with histone deacetylase 6 inhibitor mediated restoration of vision.

Short Exposure to Moderate Concentration of Alcohol During Embryonic Development Does Not Alter Gross Morphology in Zebrafish.

Several studies have demonstrated translational potential of the zebrafish in modeling fetal alcohol spectrum disorders (FASDs), including the less severe forms of this disease. Short exposure to even low doses of alcohol during embryonic development has been shown to disrupt behavior, alter neurochemistry, and expression of neuronal markers and glial cell phenotypes in zebrafish. However, no study to date has systematically analyzed the potential morphological effects of the short- and low-dose embryonic alcohol exposure regimen used before with zebrafish to model milder forms of human FASD. In this study, we use this previously used embryonic alcohol exposure regimen. We immerse intact zebrafish eggs of AB strain and of a genetically variable wild-type population for 2 h into 1% or 0% (vol/vol) ethanol bath at one of five developmental stages (8, 16, 24, 32, or 40 h postfertilization). At 8 days postfertilization, we quantify body length and width and eye diameter of the larvae. We report nonsignificant effects of embryonic alcohol exposure used at all developmental stages in both populations of zebrafish. Our results confirm that visual perception or motor function is unlikely to have contributed to previously reported behavioral abnormalities resulting from embryonic alcohol exposure in zebrafish.

Effects of NDRG1 family proteins on photoreceptor outer segment morphology in zebrafish.

Rods and cones are functionally and morphologically distinct. We previously identified N-myc downstream-regulated gene 1b (ndrg1b) in carp as a cone-specific gene. Here, we show that NDRG1b and its paralog, NDRG1a-1, contribute to photoreceptor outer segment (OS) formation in zebrafish. In adult zebrafish photoreceptors, NDRG1a-1 was localized in the entire cone plasma membranes, and also in rod plasma membranes except its OS. NDRG1b was expressed specifically in cones in the entire plasma membranes. In a developing retina, NDRG1a-1 was expressed in the photoreceptor layer, and NDRG1b in the photoreceptor layer plus inner nuclear layer. Based on our primary knockdown study suggesting that both proteins are involved in normal rod and cone OS development, NDRG1a-1 was overexpressed or NDRG1b was ectopically expressed in rods. These forced-expression studies in the transgenic fish confirmed the effect of these proteins on the OS morphology: rod OS morphology changed from cylindrical to tapered shape. These taper-shaped rod OSs were not stained with N,N’-didansyl cystine that effectively labels infolded membrane structure of cone OS. The result shows that rod OS membrane structure is preserved in these taper-shaped OSs and therefore, suggests that tapered OS morphology is not related to the infolded membrane structure in cone OS.

Transcriptional enhancement of Smn levels in motoneurons is crucial for proper axon morphology in zebrafish.

An unresolved mystery in the field of spinal muscular atrophy (SMA) is why a reduction of the ubiquitously expressed Smn protein causes defects mostly in motoneurons. We addressed the possibility that this restricted vulnerability stems from elevated Smn expression in motoneurons. To explore this, we established an ex vivo zebrafish culture system of GFP-marked motoneurons to quantitatively measure Smn protein and smn mRNA levels as well as promoter activity in motoneurons versus other cell types. Importantly, we uncovered that Smn levels are elevated in motoneurons by means of transcriptional activation. In addition, we identified the ETS family transcription factor Etv5b to be responsible for increased smn transcription in motoneurons. Moreover, we established that the additional supply of Smn protein in motoneurons is necessary for proper axonogenesis in a cell-autonomous manner. These findings demonstrate the reliance of motoneurons on more Smn, thereby adding a novel piece of evidence for their increased vulnerability under SMA conditions.

Histo-architectural changes of intestinal morphology in Zebra fish (Danio rerio) exposed to Sumithion

Organophosphorous pesticide sumithion, the O, O Dimethyl O- (3-methyl-4-nitrophenyl), is a widely used pesticide in agricultural land and in aquaculture to control some harmful pests. A study was conducted in laboratory condition with aquaria (36 inch × 10 inch × 12 inch) to evaluate the effects of sumithion on histo-architecture of intestine in zebrafish (Danio rerio). The experiment was carried out with three treatments (T1: 0.5 ppm, T2: 1.0 ppm, T3: 2.0 ppm) and a control (T0: 0ppm), each having three replications. Zebra fishes (Danio rerio) (4±1cm and 0.9±0.2g) were stocked for the experiment and sacrificed after 7 days of exposure of sumithion. During the study period, the temperature was almost constant (21- 22OC) but dissolved oxygen, pH and total alkalinity values were tended to decrease with the increase in concentrations of test chemicals. The histo-architectural changes in intestine suggested that the intestinal epithelial cells, lumen, villi and intestinal folding were varied significantly (P<0.05) in treatment groups rather than the control groups (T0). Disappeared mucosa (DM) along with abnormal lumen (AL) were found in case of T1, while destructed intestinal villi (DV), sloughing of superficial epidermal cells (SEC) and uneven intestinal folding (UF) were found in T2 and T3. The obtained result supports the toxic potentiality of sumithion. Therefore, the use of sumithion must be evaluated carefully in agriculture and aquaculture.Res. Agric., Livest. Fish.2(3): 499-506, December 2015

Effects of propranolol on heart rate and development in Japanese medaka (Oryzias latipes) and zebrafish (Danio rerio)

Propranolol is a β-adrenergic receptor antagonist (β-blocker) that is frequently used to treat hypertension and other cardiovascular conditions in humans. Detected in surface waters due to discharge of domestic wastewater, propranolol has demonstrated significant species differences in toxicity between fish. The aim of this study was to investigate the effects of propranolol on heart rate and development in embryos of two species of fish; Japanese medaka (JM) Oryzias latipes and zebrafish (ZF) Danio rerio. Parents and fertilized embryos of each species were exposed to nominal (measured) concentrations of 0.1 (0.09), 1 (1.1) and 10 (8.3) μg/L of propranolol. Heart rate was monitored during subsequent exposure in embryos at incremental developmental periods (44, 54, 64h post-fertilization (hpf) for ZF and 68, 116, 164 hpf for JM). Heart development and morphology was examined using whole mount immunostaining with distance measurements between the sinus venosus (SV) and bulbus arteriosis (BV). Morphological measurements were made at 44 hpf for ZF and 164 hpf for JM. In ZF, a significant reduction in heart rate was observed at 0.08μg/L propranolol, along with an increase in the SV–BA distance at 44 hpf. Significant reductions in heart rate were also observed in ZF at 54 and 64 hpf at all concentrations of propranolol. For JM, heart rates generally decreased at all developmental timepoints (68, 116 and 164 hpf) after propranolol treatment, with concentration dependent decreases observed at 164 hpf and a lowest observed effect concentration (LOEC) of 0.09μg/L propranolol at each timepoint. However, significant alterations in cardiac morphology were not observed in JM at 164 hpf. In contrast, heart rates and morphology in ZF were affected with a non-monotonic concentration response in morphology and a LOEC of 0.09μg/L propranolol for morphological alterations at 44 hpf and for heart rate at each timepoint. These data indicated unique developmental stages of susceptibility between species and that combined parental and embryo exposures may lead to greater impairment of cardiac development and function in offspring than separate exposures of adults and embryos.

Ontogeny of form and function: Locomotor morphology and drag in zebrafish (Danio rerio)

Many fish species transform in body shape during growth, but it remains unclear how this influences the mechanics of locomotion. Therefore, the present study focused on understanding how drag generation during coasting is affected by ontogenetic changes in the morphology of zebrafish (Danio rerio). The shapes of the body and fins were measured from photographs of fish ranging in size from small larvae to mature adults and these morphometrics were compared to drag coefficients calculated from high-speed video recordings of routine swimming. We found that the viscous drag coefficient of larval and juvenile fish increased by more than an order of magnitude during growth and the inertial drag coefficient decreased at a comparable rate in adults. These hydrodynamic changes occurred as zebrafish disproportionately increased the span of their fins and their body changed shape from elongated to streamlined, as reflected by the logistic growth of a newly defined streamlining index, SL. These results suggest that morphological changes incur a performance cost by generating greater drag when larvae and juveniles operate in the viscous regime, but later provide a performance benefit by reducing pressure drag in the inertial regime of the adult stage.