14 research outputs found

    Intrinsic myocardial defects underlie an Rbfox-deficient zebrafish model of hypoplastic left heart syndrome

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    Hypoplastic left heart syndrome (HLHS) is characterized by underdevelopment of left sided structures including the ventricle, valves, and aorta. Prevailing paradigm suggests that HLHS is a multigenic disease of co-occurring phenotypes. Here, we report that zebrafish lacking two orthologs of the RNA binding protein RBFOX2, a gene linked to HLHS in humans, display cardiovascular defects overlapping those in HLHS patients including ventricular, valve, and aortic deficiencies. In contrast to current models, we demonstrate that these structural deficits arise secondary to impaired pump function as these phenotypes are rescued when Rbfox is specifically expressed in the myocardium. Mechanistically, we find diminished expression and alternative splicing of sarcomere and mitochondrial components that compromise sarcomere assembly and mitochondrial respiration, respectively. Injection of human RBFOX2 mRNA restores cardiovascular development in rbfox mutant zebrafish, while HLHS-linked RBFOX2 variants fail to rescue. This work supports an emerging paradigm for HLHS pathogenesis that centers on myocardial intrinsic defects

    Thalamic neuron models encode stimulus information by burst-size modulation

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    Thalamic neurons have been long assumed to fire in tonic mode during perceptive states, and in burst mode during sleep and unconsciousness. However, recent evidence suggests that bursts may also be relevant in the encoding of sensory information. Here, we explore the neural code of such thalamic bursts. In order to assess whether the burst code is generic or whether it depends on the detailed properties of each bursting neuron, we analyzed two neuron models incorporating different levels of biological detail. One of the models contained no information of the biophysical processes entailed in spike generation, and described neuron activity at a phenomenological level. The second model represented the evolution of the individual ionic conductances involved in spiking and bursting, and required a large number of parameters. We analyzed the models' input selectivity using reverse correlation methods and information theory. We found that n-spike bursts from both models transmit information by modulating their spike count in response to changes to instantaneous input features, such as slope, phase, amplitude, etc. The stimulus feature that is most efficiently encoded by bursts, however, need not coincide with one of such classical features. We therefore searched for the optimal feature among all those that could be expressed as a linear transformation of the time-dependent input current. We found that bursting neurons transmitted 6 times more information about such more general features. The relevant events in the stimulus were located in a time window spanning ~100 ms before and ~20 ms after burst onset. Most importantly, the neural code employed by the simple and the biologically realistic models was largely the same, implying that the simple thalamic neuron model contains the essential ingredients that account for the computational properties of the thalamic burst code. Thus, our results suggest the n-spike burst code is a general property of thalamic neurons

    Spatial and Temporal Control of Transgene Expression in Zebrafish

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    <div><p>Transgenic zebrafish research has provided valuable insights into gene functions and cell behaviors directing vertebrate development, physiology, and disease models. Most approaches use constitutive transgene expression and therefore do not provide control over the timing or levels of transgene induction. We describe an inducible gene expression system that uses new tissue-specific zebrafish transgenic lines that express the Gal4 transcription factor fused to the estrogen-binding domain of the human estrogen receptor. We show these Gal4-ERT driver lines confer rapid, tissue-specific induction of UAS-controlled transgenes following tamoxifen exposure in both embryos and adult fish. We demonstrate how this technology can be used to define developmental windows of gene function by spatiotemporal-controlled expression of constitutively active Notch1 in embryos. Given the array of existing UAS lines, the modular nature of this system will enable many previously intractable zebrafish experiments.</p></div

    The Gal4-ERT system provides rapid induction of gene expression.

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    <p>(<b>A–D</b>) Kinetics of EGFP expression in <i>Tg(krt5:Gal4-ERT-VP16; UAS:EGFP)</i> animals upon administration of 2 μM 4-OHT at 24 hpf for 0–4.5 hours. (<b>A′–D′</b>) High magnification images demonstrating EGFP expression for each treatment. White arrowheads point to epidermal expression of EGFP; blue arrows indicate bleed-through from the heart muscle-specific ECFP transgenesis marker.</p

    Inducible transgene expression in adult zebrafish.

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    <p>(<b>A–D</b>) Expression of EGFP in the eye of a single adult <i>Tg(krt5:Gal4-ERT-VP16; UAS:EGFP)</i> zebrafish prior to (A and B) and after (C and D) treatment with 1 μM tamoxifen for one hour per day for three consecutive days. Green arrows point to EGFP expression in the eye. The areas bounded by the dashed red box in A and C are shown at high magnification in B and D, respectively. (<b>E–H</b>) Immunostaining of paraffin sections with anti-EGFP antibodies (shown in green) in eyes from DMSO- (E and F) and tamoxifen-treated (G and H) <i>Tg(krt5:Gal4-ERT-VP16; UAS:EGFP)</i> fish. Panels F and H show overlays with anti-EGFP antibody staining in green, Hoechst-stained nuclei in blue, and auto-fluorescence in red. Fish were drug treated as in A–D. Green arrows indicate EGFP expression in photoreceptors and asterisks (*) denote auto-fluorescence in photoreceptor outer segments. (<b>I</b>) In situ hybridization for <i>EGFP</i> mRNA in a paraffin section from a 4-OHT treated <i>Tg(krt5:Gal4-ERT-VP16; UAS:EGFP)</i> animal. The blue arrow shows <i>EGFP</i> expression in photoreceptors.</p

    Using inducible expression to define temporal roles of Notch1 signaling in notochord development.

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    <p>(<b>A–E</b>) Expression of myc-tagged NICD by immunostaining with myc antibodies on 24 hpf <i>Tg(dusp6:Gal4-ERT-VP16; UAS:NICD)</i> embryos treated with ethanol (A) or 4 μM 4-OHT at the indicated times (B–E). Blue arrows indicate myocardial ECFP expression to mark transgenic animals and magenta arrows show expression of myc-tagged NICD. Panel insets display high magnification images of boxed regions where red arrows indicate the notochord (A and E) and the missing notochord (B–D). (<b>F</b>) Normalized penetrance of notochord defects in <i>Tg(dusp6:Gal4-ERT-VP16; UAS:NICD)</i> animals treated with 4-OHT at the indicated stages of development. Each data point represents the normalized fraction of affected animals in sets of treated embryos from four independent clutches. The data is normalized to the average fraction of abnormal fish in the four 4–24 h sets. The double asterisk indicates a significant difference between 4–24 h and 10–24 h 4-OHT treated fish (P<0.005). (<b>G–I</b>) Expression of <i>shha</i> in the floor plate of animals treated with ethanol (G) or with 4-OHT for the indicated times (H–I). Boxed regions are shown at higher magnification in the panel insets. Red arrows indicate <i>shha</i> expressing floor plate cells.</p

    Overexpression of the Notch1 intracellular domain using the <i>dusp6:Gal4-ERT</i> driver disrupts notochord development.

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    <p>(<b>A and B</b>) EGFP expression at the 10-somite stage in control (A) and 2 μM 4-OHT treated <i>Tg(dusp6:Gal4-ERT-VP16; UAS:EGFP)</i> fish. The green arrow points to EGFP expression at the midline. (<b>C–F</b>) DIC images of control (C and D) and 4-OHT treated (4 μM from 2–24 hpf, E and F) <i>Tg(dusp6:Gal4-ERT-VP16; UAS:NICD)</i> animals at 24 hpf. Regions bounded by the dashed red box in panels C and E are shown in high magnification in panels D and F, respectively. In panel D, the red arrow indicates the floor plate (fp) and the blue arrow indicates the notochord (nc); in panel E, the magenta arrow highlights the reduced notochord and disorganized floor plate.</p

    Transgene expression levels depend upon 4-OHT dosage.

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    <p>(<b>A–D</b>) EGFP expression upon treatment of <i>Tg(krt5:Gal4-ERT-VP16; UAS:EGFP)</i> zebrafish with ethanol or the indicated dose of 4-OHT from 4–24 hpf. The blue arrow indicates <i>myl7:ECFP</i> expression. (<b>A′–D′</b>) High-magnification images of ventral epidermis from fish in each treatment group. (<b>E</b>) Normalized EGFP intensity (to the 0.5 μM 4-OHT treated group) of fish treated with ethanol or 4-OHT. Error bars represent standard deviations.</p

    Temporally and spatially controlled transgene expression in zebrafish.

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    <p>(<b>A</b>) Schematic of transgenic constructs used in the Gal4-ERT system. A tamoxifen-responsive Gal4-ERT-VP16 construct is expressed from a tissue-specific promoter that activates any UAS-linked responder line (shown here as a <i>5xUAS:EGFP</i> reporter) upon tamoxifen or 4-OHT exposure (orange squares). The <i>myl7:ECFP</i> cassette serves as a transgenesis marker for the Gal4-ERT lines. (<b>B–D</b>) Visualization of EGFP expression in <i>Tg(krt5:Gal4-ERT-VP16; UAS:EGFP)</i> animals treated with ethanol (B) or 2 μM 4-OHT (C and D) from 4–24 hpf. The white arrow in panel D highlights expression of EGFP in the epidermis. (<b>E–I</b>) EGFP expression in <i>Tg(dusp6:Gal4-ERT-VP16; UAS:EGFP)</i> animals treated with vehicle (E) or 2 μM 4-OHT (F-I) from 4–24 hpf. In panels G and H, the white arrow indicates EGFP expression in the hindbrain and midbrain-hindbrain boundary. In panel I, arrowheads mark dorsal spinal cord neurons and the arrow points to EGFP expression in the floor plate. (<b>J–M</b>) Expression of EGFP in control (J) and 4-OHT treated (2 μM, K-M) <i>Tg(ef1α:Gal4-ERT-VP16; UAS:EGFP)</i> animals in a variety of cell types throughout the embryo including skeletal muscle (K, white arrow), the eye (L, white arrow), and the midbrain/midbrain-hindbrain boundary (M, white arrow). In panels B, E, and J, blue arrows point to myocardial ECFP expression, which represents the marker for transgenesis and serves as an internal control.</p
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