Journal: bioRxiv

Article Title: Hand2 represses non-cardiac cell fates through chromatin remodeling at cis- regulatory elements

doi: 10.1101/2023.09.23.559156

Figure Lengend Snippet: (A) Schematic representation of the hand2enh reporter construct. Construct contains a putative enhancer 28Kb upstream of the hand2 TSS (blue rectangle; hand2enh ), an e1b minimal promoter (orange) and eGFP (green), flanked by minimal Tol2 cis sequences to permit integration by Tol2 transposase. (B) Confocal images of reporter expression in the LPM of a 14hpf Tg(hand2enh:eGFP ) embryo (dorsal view, anterior to the top), and in the heart of a 48 hpf Tg(hand2enh:eGFP) ; Tg(myl7:cherry-Ras) embryo (ventral view). (C) Depiction of the Danio rerio hand2 locus with the CRISPR-Cas9 target site used to generate hand2 crispants at 260bp into exon 1, marked with a yellow lightning bolt. PCR primers used for fragment analysis to confirm CRISPR-induced mutagenesis are shown as red arrowheads. (D) Epifluorescence images of Tg(hand2enh:eGFP) embryos at 14 (dorsal view, anterior to the top) and 48 hpf (lateral view, anterior to the left): wildtype (WT), hand2 mutant ( hanS6 ), and hand2 crispant ( hand2 -CRP). White arrows indicate residue myocardial tissue in hand2 mutant and crispant. (E) Representative capillary electrophoresis traces of WT and hand2 -CRP embryos, plotted as the number of bases from the start of the amplified fragment. The percentage of cells with indel mutations for 9 representative embryos is indicated at right. (F) Confocal images of control (uninjected) and crispant Tg(myl7:nucGFP) embryos at 20-ss (19 hpf), ventral view. hand2 crispants have significantly fewer myocardial cells than wildtype embryos (unpaired two-tailed t-test; *p < 0.0001). (G) Confocal images, ventral view, of whole mount Tg(myl7:nucGFP) embryos at 19 hpf. WT and hand2 crispant transgenic embryos, immunostained with an antibody against aPKCs (purple) and nuclear-counterstained with DAPI (cyan); myocardial cells are labeled by myl7-driven nuclear GFP expression (green). Scale bars: 50um in B, D; 30um in F; 20um in G.

Article Snippet: Upstream cis -regulatory elements were amplified from wildtype zebrafish genomic DNA and cloned into the e1b-eGFP-Tol2 vector (available from Addgene, Plasmid #37845) by restriction cloning at the XhoI and BglII sites.

Techniques: Construct, Expressing, CRISPR, Mutagenesis, Residue, Electrophoresis, Amplification, Control, Two Tailed Test, Transgenic Assay, Labeling

Journal: bioRxiv

Article Title: Hand2 represses non-cardiac cell fates through chromatin remodeling at cis- regulatory elements

doi: 10.1101/2023.09.23.559156

Figure Lengend Snippet: (A) Confocal images of wildtype and hanS6 14 hpf embryos injected with the eDAR1031-e1b-eGFP reporter construct, showing reporter gene expression in the LPM (yellow dashed region) only in the hand2 mutant embryo (lower panel). Scale bars are 50um. (B) Quantification of wildtype and hanS6 embryos injected with eDAR1031-e1b-eGFP that exhibit eGFP expression within the LPM. “N” denotes the number of independent experimental replicate. “n” denotes the total number of embryos quantified. eGFP expression from embryos injected with the e1b-eGFP construct are shown as a control (*, p-value < 0.05, ns = not significant). (C) Putative Hand2-regulated gene network compiled based on the analysis of genes that are downregulated (blue text) or upregulated (red text) in the absence of Hand2. Lines with arrowheads signify an activator function for Hand2 in the indicated biological process while lines with a block arrow signify a repressive function for Hand2 in the indicated biological process. Dashed lines indicate Hand2 may either directly or indirectly regulate list genes. (D) Proposed mechanism of Hand2-dependent regulation of non-LPM and LPM-derived tissues based on the results of this study. Schematic of a 14 hpf zebrafish embryo is shown in the middle with the LPM colored in green. The embryo proper is colored blue while the yolk is colored beige. In the LPM, Hand2 positively regulate expression of LPM genes and limit the accessibility of non-LPM genes. In non-LPM tissues where Hand2 is not expressed, enhancers of non-LPM genes are accessible and can act on target non-LPM genes.

Article Snippet: Upstream cis -regulatory elements were amplified from wildtype zebrafish genomic DNA and cloned into the e1b-eGFP-Tol2 vector (available from Addgene, Plasmid #37845) by restriction cloning at the XhoI and BglII sites.

Techniques: Injection, Construct, Gene Expression, Mutagenesis, Expressing, Control, Blocking Assay, Derivative Assay

Journal: Molecular & Cellular Proteomics : MCP

Article Title: A Toolbox for Efficient Proximity-Dependent Biotinylation in Zebrafish Embryos

doi: 10.1016/j.mcpro.2021.100128

Figure Lengend Snippet: Inducible transgenic expression of TurboID-LMNA or miniTurbo-LMNA identifies LMNA proximal proteins. A , cassettes compatible with the Tol2 trangenesis system were generated that express the selected fusion proteins (TurboID-LMNA and GFP, miniTurbo-LMNA and GFP) downstream of the heat shock inducible Hsp70 promoter. The cassettes also contain GFP under the control of a cardiomyocyte specific Cmlc2 promoter to allow selection of integration-positive embryos. B , TurboID-GFP and miniTurbo-GFP transgenic embryos were heat shocked at 60 hpf for 1 h and imaged at 72 hpf. Integration-positive embryos were selected prior to heat shock based on expression of GFP in the heart ( yellow arrow ). GFP expression 12 h following heat shock is indicated by white arrowheads , and GFP expression in the eye by red arrows (magnification 16×, Scale Bar = 1 mm). C , immunofluorescence microscopy on embryos collected at 72 hpf following 1 h heat shock at 60 hpf time point was performed as detailed in . FLAG staining to detect the bait expression and streptavidin-fluorophore (Strep) to detect biotinylation. DAPI was used to stain the nucleus. ( Upper panels Scale bar 50 μm, magnification 20×; Lower panels scale bar 5 μm, magnification 40×). D , the scatterplot shows all proteins detected with a SAINTexpress Bayesian FDR ≤1% with all least one of the baits (miniTurbo on the y-axis, TurboID on the x-axis); the dashed lines represent the different fold changes listed at the top . Preys are color-coded based on uniquely scoring as high-confidence interactors with either miniTurbo ( green ) or TurboID ( blue ) while preys scoring as high-confidence with both baits are coded in red . E , list of the 27 common partners. F , the 25 most abundant proteins (by spectral counts) with a BFDR ≤1% from the TurboID-LMNA experiment are listed, alongside their abundance across both conditions.

Article Snippet: To generate transgenic zebrafish lines, 30 pg of the final Tol2 destination vectors (purified using the QIAprep Spin Miniprep Kit; Qiagen cat. 27106) was injected into single cell stage zebrafish embryos together with 75 pg of Tol2 transposase mRNA (purified using the MEGAclear Transcription Clean-Up Kit; Thermo Fisher Scientific cat. AM1908).

Techniques: Transgenic Assay, Expressing, Generated, Control, Selection, Immunofluorescence, Microscopy, Staining

Journal: bioRxiv

Article Title: Dynamic interactions between epithelial skin cells and a sensory cavity sculpt the growing olfactory orifice

doi: 10.1101/2025.07.25.666761

Figure Lengend Snippet: a. Live imaging on a Tg(krt4:lynGFP) embryo, in which the membrane of peridermal skin cells are labelled (black). Dorsal view, anterior on the top, lateral on the left, maximum projections. Asterisk = orifice. Black line = orifice contour. Scale bar = 20 µm. a’ . Segmentation of the skin cell contours. Asterisk = orifice. Pink = first neighbor skin cells (i.e. cells located on the orifice edge, or bordering cells). Purple = second neighbors. b, b’, b’’, b’’’ . Quantification of orifice area (b), orifice perimeter (b’), number of bordering cells (b’’), and average border length (b’’’), defined as the length of the portion of bordering skin cell membranes contributing to the orifice edge, as depicted with double headed arrows in the images (right panel) extracted from the segmented movie shown in a’ (n = 9 movies from 2 independent experiments). c. Examples of cell divisions occurring in the bordering skin cells (pink) or second neighbors (purple), images extracted from the segmented movie shown in a’. Asterisk = orifice. The mother cell and the two daughter cells are indicated with colored circles. c’ . Map of cell divisions occurring in the field of view during the time lapse sequence (one embryo). Grey lines = contours of the growing orifice, the time is color coded. For each division, a segment links the centers of the two daughter cells, which are represented by the dots. Pink segments = bordering skin cell divisions, purple segments = 2nd neighbor cell divisions, grey segments = divisions in the rest of the skin epithelium (for those, time is color coded). c’’ . Quantification of the rate of cell divisions in the bordering cells and 2nd neighbors versus in the rest of the epithelium. Mann- Whitney test. c’’’ . Rose plots showing the distribution of the absolute cell division angles (alpha) with respect to the edge of the orifice (as depicted on the schematic view, see also Methods) in the bordering cells and 2nd neighbors (pink) and the rest of the epithelium (grey). A permutation test was performed (see Methods). d. Images of Tg(cldnb:meGFP) embryos treated with DMSO or HUA from 32 hpf, fixed at 48 hpf and immunostained for GFP (grey) and ZO-1 (cyan). Top images = skin seen with the Tg(cldnb:meGFP) transgene and extracted with the SurfCut plugin (see Methods). Asterisk = orifice. White line = orifice contour. Bordering cells are outlined with a pink line. Bottom images = cavity seen with ZO-1 immunostaining, maximum projections. Dotted white line = cavity contour. Scale bar = 20 µm. d’ . Quantification of orifice area, cavity area, number of bordering cells and mean border length in HUA-treated embryos and DMSO-treated controls at 48 hpf (3 independent experiments, n = 39 HUA- treated embryos, n = 37 DMSO controls). Mann-Whitney tests for orifice and cavity area, Welch’s t-tests for number of bordering cells and mean of border length. e. Images of Tg(krt4:KalTA4; uas:cdkn1b-E2A-GFP) embryos, in which cell divisions are inhibited in the skin, and control siblings fixed at 48 hpf, immunostained for ZO-1 (cyan) and stained with phalloidin-rhodamine (grey), GFP in green. Top images = skin extracted with the SurfCut plugin (see Methods). Asterisk = orifice. White line = orifice contour. Bordering cells are outlined with a pink line. Bottom images = cavity seen with ZO-1 immunostaining, maximum projections. Dotted white line = cavity contour. Scale bar = 20 µm. e’ . Quantification of orifice area, cavity area, number of bordering cells and mean border length in Tg(krt4:KalTA4; uas:cdkn1b-E2A-GFP) embryos and control siblings at 48 hpf (3 independent experiments, n = 30 double transgenic embryos, n = 25 control siblings). Unpaired, two-tailed t-test for number of bordering cells, Welch’s t-tests for the other graphs.

Article Snippet: The Tol2 uas:cdkn1b-E2A-meGFP plasmid was designed by the authors and synthesized by Twist Bioscience.

Techniques: Imaging, Membrane, Sequencing, MANN-WHITNEY, Immunostaining, Control, Staining, Transgenic Assay, Two Tailed Test