Mapping the Learning Pathways of Larval Zebrafish through Positively Stimulating Their Reward Pathways Using Paramecium

Zebrafish rely on visual acuity to hunt for prey all of their lives, for this reason, their eyesight develops when they are embryos. The zebrafish in this experiment are between 5 and 20 days old. Once their egg yolks are completely reabsorbed the zebrafish have a need to eat, and only then will they have an interest in eating Paramecium. The zebrafishes’ eyes will be able to see clearly by 5 days post fertilization as well, so when food is introduced to them, a certain colored light will be simultaneously shined in the same direction as the one the food is coming from. The purpose of this experiment is to use different colored visual cues to train larval zebrafish into expecting food whenever they are shone. The usage of lights also tests their extraordinary visual abilities. Red lights will be associated with a food reward in one group of larvae, and in the second group, green lights will be associated with a food reward, demonstrating associative learning. The zebrafishes’ unconditioned response (eating Paramecium when they appear) will be trained into a conditioned response (looking to eat Paramecium when the light is shined). After they have been conditioned to respond to these signals, the zebrafishes’ brains will be studied to find changes in their neural pathways. The expected results of this experiment should lead to the fish thinking Paramecium are coming at just the glimpse of a light

Bridging the gap: functional healing of embryonic small intestine ex vivo.

The ability to grow embryonic organs ex vivo provides an opportunity to follow their differentiation in a controlled environment, with resulting insights into normal development. Additionally, similar strategies can be used to assess effects on organogenesis of physical and chemical manipulations. This study aimed to create an organ culture model with which to test physical manipulations to enhance healing of gut segments, thus generating a single functional organ. Embryonic mouse jejunum was isolated and cut into 2–3 mm tubes, which were placed in pairs, separated by a small gap, on semi‐permeable supports. Each pair was linked by a nylon suture threaded through their lumens. After 3 days in organ culture fed by defined serum‐free media, the rudiments differentiated to form tubes of smooth muscle surrounding a core of rudimentary villi. Of 34 such pairs, 74% had touching and well aligned proximate ends. Of these joined structures, 80% (59% of the total pairs) had a continuous lumen, as assessed by observing the trajectories of fluorescent dextrans injected into their distal ends. Fused organ pairs formed a single functional unit, as assessed by spontaneous contraction waves propagated along their lengths. In these healed intestines, peripherin(+) neurons formed a nexus in the zone of fusion, linking the rudiment pairs. In future, this system could be used to test whether growth factors enhance fusion. Such results should in turn inform the design of novel treatments for short bowel syndrome, a potentially fatal condition with a currently limited and imperfect range of therapies. ©2015. The Authors Journal of Tissue Engineering and Regenerative Medicine Published by John Wiley & Sons, Lt

The Lowe syndrome protein OCRL1 is required for endocytosis in the zebrafish pronephric tubule.

Lowe syndrome and Dent-2 disease are caused by mutation of the inositol 5-phosphatase OCRL1. Despite our increased understanding of the cellular functions of OCRL1, the underlying basis for the renal tubulopathy seen in both human disorders, of which a hallmark is low molecular weight proteinuria, is currently unknown. Here, we show that deficiency in OCRL1 causes a defect in endocytosis in the zebrafish pronephric tubule, a model for the mammalian renal tubule. This coincides with a reduction in levels of the scavenger receptor megalin and its accumulation in endocytic compartments, consistent with reduced recycling within the endocytic pathway. We also observe reduced numbers of early endocytic compartments and enlarged vacuolar endosomes in the sub-apical region of pronephric cells. Cell polarity within the pronephric tubule is unaffected in mutant embryos. The OCRL1-deficient embryos exhibit a mild ciliogenesis defect, but this cannot account for the observed impairment of endocytosis. Catalytic activity of OCRL1 is required for renal tubular endocytosis and the endocytic defect can be rescued by suppression of PIP5K. These results indicate for the first time that OCRL1 is required for endocytic trafficking in vivo, and strongly support the hypothesis that endocytic defects are responsible for the renal tubulopathy in Lowe syndrome and Dent-2 disease. Moreover, our results reveal PIP5K as a potential therapeutic target for Lowe syndrome and Dent-2 disease

Electron microscopy analysis of endocytic compartments in OCRL1 deficient pronephros.

<p>A. Block face scanning electron microscopy (SEM) images of transverse sections through the zebrafish proximal pronephric tubule of wild-type and <i>ocrl^-/-</i> mutant 72 hpf embryos. The apical membrane, identified by numerous microvilli, lines the central lumen of the pronephric tubule. Vacuolar endosomes are false coloured in green. B and D. Block face SEM showing apical endocytic vesicles at the apical pole of pronephric proximal tubule cells (false coloured in orange in top row) (B) and vacuolar endosomes (false coloured in green in top row) (D). C and E. Quantification of endocytic compartments. Numbers of apical endocytic vesicles were counted per region of interest (C), and vacuolar endosome number, size and total area were counted per entire section (E). Data are presented as the mean ± SD. Statistical analysis was performed using the unpaired t-test. ***p < 0.0001. Scale bars represent 5 μm (A), 2 μm (D) or 1 μm (B).</p

Megalin transcript and protein analysis in OCRL1-deficient zebrafish embryos.

<p>A. Transverse confocal images of the proximal pronephric region of wild-type (WT) and <i>ocrl^-/-</i> mutant 72 hpf embryos labelled with anti-megalin antibodies. The white dashed lines indicate the outline of pronephric tubules. Arrowheads indicate sub-apical punctate and vacuolar megalin staining. B. Transverse confocal images of the proximal pronephric region of 72 hpf <i>ocrl^-/-</i> embryos labelled with antibodies to megalin (green in left panel, red in right panel) and EEA1 (red) or GFP (gfp-, green) to detect ectopically expressed Rab5 or Rab7. mApple (a-) tagged Rab11 is in red. Arrowheads indicate colocalisation. C. Quantification of the relative fluorescence levels of megalin in confocal transverse sections of the indicated embryo types. D. Western blot of 72 hpf wild-type (WT) or <i>ocrl^-/-</i> embryos with antibodies to megalin and tubulin. Three equivalent samples for genotype are analyzed. E. In situ hybridisation of megalin transcript in 48 hpf (top) and 72 hpf (bottom) wild-type (WT) or <i>ocrl^-/-</i> embryos. F. Quantitative RT-PCR (qPCR) of megalin transcript levels in wild type and <i>ocrl^-/-</i> embryos at 72 hpf. Data are presented as the mean ± SD. Statistical analysis was performed using the unpaired t-test. ***p < 0.0001. Scale bars in A, B and E represent 10, 2 and 20 μm respectively.</p

Covalent Modification and Regulation of the Nuclear Receptor Nurr1 by a Dopamine Metabolite

Nurr1, a nuclear receptor essential for the development, maintenance, and survival of midbrain dopaminergic neurons, is a potential therapeutic target for Parkinson's disease, a neurological disorder characterized by the degeneration of these same neurons. Efforts to identify Nurr1 agonists have been hampered by the recognition that it lacks several classic regulatory elements of nuclear receptor function, including the canonical ligand-binding pocket. Here we report that the dopamine metabolite 5,6-dihydroxyindole (DHI) binds directly to and modulates the activity of Nurr1. Using biophysical assays and X-ray crystallography, we show that DHI binds to the ligand-binding domain within a non-canonical pocket, forming a covalent adduct with Cys566. In cultured cells and zebrafish, DHI stimulates Nurr1 activity, including the transcription of target genes underlying dopamine homeostasis. These findings suggest avenues for developing synthetic Nurr1 ligands to ameliorate the symptoms and progression of Parkinson's disease

Rescue of the pronephric uptake defect in OCRL1 deficient embryos by suppression of PIP5K.

<p>A. RT-PCR detection of PIP5Kαb and eIF1α in wild-type and <i>ocrl^-/-</i> embryos at the indicated developmental timepoints. B, left. RT-PCR of PIP5Kαb and eIF1α in 3 dpf zebrafish embryos injected with the indicated amount of PIP5Kαb splice morpholino. The asterisk indicates morpholino-induced abnormally spliced PIP5Kαb transcript. Right, mortality of PIP5Kαb morpholino-injected embryos at 24 hpf. C. PtdIns(4,5)P₂ levels in untreated wild-type or <i>ocrl^-/-</i> embryos or embryos injected with 2 ng PIP5Kαb morpholino. Data are presented as the mean ± SE (n = 6–13). Statistical analysis was performed using the one-way ANOVA with a post-hoc Dunnett’s multiple comparisons test. *p < 0.05. D. Images of pronephric uptake of Alexa 488-10 kDa dextran (green) in wild type (WT) or <i>ocrl^-/-</i> embryos or WT or <i>ocrl^-/-</i> embryos injected with 2 ng PIP5Kαb morpholino. The pronephric tubules are indicated with a green dashed line. E. Quantification of pronephric uptake of Alexa 488-10 kDa dextran in each of the indicated embryo types. F. Transverse confocal images showing megalin labelling in the proximal pronephric region of 72 hpf wild-type (WT), <i>ocrl^-/-</i> or <i>ocrl^-/-</i> embryos injected with 2 ng PIP5Kαb morpholino (top) and quantitation of megalin fluorescence (bottom). G. Transverse confocal images showing EEA1 labelling in the proximal pronephric region of 72 hpf wild-type (WT), <i>ocrl^-/-</i> or <i>ocrl^-/-</i> embryos injected with 2 ng PIP5Kαb morpholino. H. Block face scanning electron microscopy images of transverse sections through the proximal pronephric tubule of wild-type (WT), <i>ocrl^-/-</i> or <i>ocrl^-/-</i> embryos injected with 2 ng PIP5Kαb morpholino. The bottom row is a colour-coded version of the top row, with vacuaolar endosomes false coloured in green. I. Quantification of vacuolar endosome number, size and total area. Data in E, F and I are presented as the mean ± SEM. Statistical analysis was performed using the Pearson’s chi-squared test. ***p < 0.0001, **p < 0.001, *p < 0.01. Scale bars represent 10 μm (F, G) and 2 μm (H).</p

Pronephric cilia in <i>ocrl^-/-</i> zebrafish.

<p>A. Confocal images of pronephric cilia, detected using anti-acetylated tubulin antibody, in wild-type, <i>ocrl^-/-</i> mutant, control morphant or OCRL1 morphant zebrafish embryos (26hpf). B. Fluorescence dissecting microscope image of excretion of Alexa 488-10 kDa dextran from the cloacae of zebrafish embryos (72hpf). Bottom panels show cloacae immediately after injection (left) and excreting dextran 30–60s after injection (wild-type middle, <i>ocrl^-/-</i> right). Dextran excretion was identical in control and <i>ocrl^-/-</i> embryos (20 embryos of each genotype, 2 independent experiments). C. Brightfield images of wild-type (WT), <i>ocrl^-/-</i> mutant or IFT88/polaris morphant (MO) embryos. The morphants were injected with different concentrations of morpholino as indicated. Embryos were imaged using brightfield microscopy. Bottom panel shows <i>ocrl^-/-</i> mutant and polaris morphant (injected with 4 ng MO) and zoom of boxed area. The arrowhead indicates a pronephric cyst in the polaris morphant. D. Confocal images of pronephric cilia, detected using anti-acetylated tubulin antibody, in wild-type (WT), <i>ocrl^-/-</i> mutant or IFT88/polaris morphant (MO) embryos. E. Wild-type (WT), <i>ocrl^-/-</i> mutant and IFT88/polaris morphant embryos were injected with Alexa 488-10 kDa dextran (green) and pronephric accumulation after 2.5 h monitored by fluorescence microscopy. The pronephric tubules are indicated with a dashed line. Uptake was quantitated as indicated. Data are presented as the mean ± SEM. Statistical analysis was performed using the Pearson’s chi-squared test. ***p < 0.0001, **p < 0.001, *p < 0.01. F. Confocal transverse sections of the zebrafish proximal pronephric tubule of 72 hpf wild type and <i>double bubble (dbb</i>) cilia mutant showing 10 kDa-FD uptake into endocytic compartments in pronephric cells 2h after injection. Scale bars represent 10 μm (A and D).</p