Spatial and Temporal Control of Transgene Expression in Zebrafish

<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

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

<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

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

<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

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

<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.

<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

Transgene expression levels depend upon 4-OHT dosage.

<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.

<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

Aerobic Copper-Catalyzed Alkene Oxyamination for Amino Lactone Synthesis

A convenient alkene oxyamination compatible with a wide range of alkenoic acids and electron-rich amines is accomplished via aerobic copper catalysis. The synthetic value of this protocol is highlighted with the stereoselective formation of complex amino lactone products. In addition, product derivatizations to privileged nitrogen heterocycles have also been demonstrated via simple reduction methods. The reaction is proposed to proceed through a copper-catalyzed iodolactonization process

Tandem affinity purification of chromatin-remodeling complex protein Baf57c using SGTAP.

<p>(A) Schematic of LR recombination reaction used to create pEpic CMV:Baf57c-SGTAP. (B) Schematic of steps for TAP of Baf57-SGTAP. Step 1: Baf57c with a C-terminally conjugated SBP, TEV protease cleavage site, and tandem copies of protein G is first isolated by affinity purification using IgG-sepharose beads; Step 2: Baf57c-SBP is cleaved from protein G bound to IgG-sepharose beads by the addition of TEV protease; Step 3: Baf57c-SBP is further isolated by affinity purification using streptavidin-sepharose beads; Step 4: Baf57c-SBP is finally eluted from streptavidin by the addition of biotin. (C) Western blot for SBP at various stages of Baf57c purification from nuclear extracts of HEK293T cells expressing pEpic CMV-Baf57c-SGTAP. 10% of each indicated fraction was used for immunoblotting. Lane 1: the crude nuclear extract; Lane 2: nuclear extract after incubation with IgG beads; Lane 3: post-TEV protease cleavage of proteins bound to IgG beads; Lane 4: SDS elution of proteins from beads following Streptavidin purification. The asterisk indicates Baf57c-SGTAP fusion proteins; the arrow indicates the cleaved Baf57c-SBP fusion; molecular weights in kilodaltons are shown at the right.</p

Effective dual protein expression through N-terminal P2A conjugation to HA-Neuroligin1.

<p>(A) Schematic of LR recombination reaction used to create pEpic_Lite mCMV:memGFP-P2A-HA-Neuroligin1. (B) Dual fluorescent western blot of COS7 cell lysate 24 hours after transfection with mCMV:memGFP-P2A-HA-Neuroligin1. Immunoblotting was performed with antibodies against GFP and HA. (C) Immunocytochemistry for GFP and HA in COS7 cells 24 hours after transfection with mCMV:memGFP-P2A-HA-Neuroligin1. Cells were fixed with paraformaldehyde and surface stained for HA, then permeabilized and stained for GFP. (D) Immunocytochemistry for GFP, HA and the synaptic vesicle-associated protein Synapsin1 in cultured rat hippocampal neurons. Cells were transduced with lentivirus carrying mCMV:memGFP-P2A-HA-Neuroligin1at 2DIV and fixed for immunolabeling at 14DIV. Cells were surface stained for HA, then permeabilized and stained for GFP and Synapsin1. Inset is of an individual basal dendrite segment; the GFP mask is a binarized image of the dendrite using intensity thresholding of the GFP signal. Arrowheads mark dendritic spines containing HA and co-localized Synapsin1 puncta. Scale bar = 10 μm.</p