Journal: eLife

Article Title: Basement membranes are crucial for proper olfactory placode shape, position and boundary with the brain, and for olfactory axon development

doi: 10.7554/eLife.92004

Figure Lengend Snippet:

Article Snippet: To generate the Tg(UAS:lyn-tagRFP ) line, a 10XUAS:lyn-tagRFP plasmid was synthesised by Gateway cloning (Multisite Gateway system kit, Invitrogen, 12537–023, final destination vector: pDest:tol2) and co-injected with Tol2 mRNA (25 ng/µL of plasmid and 25 ng/µL of Tol2 mRNA) in 1 cell-stage wild type embryos.

Techniques: Recombinant, Plasmid Preparation, Clone Assay, Sequencing, Amplification, Software

Journal: bioRxiv

Article Title: A novel auxin-inducible degron system for rapid, cell cycle-specific targeted proteolysis

doi: 10.1101/2021.04.23.441203

Figure Lengend Snippet: Schematic representation of the design for Regulated OsTIR1 Levels of Expression based on the Cell Cycle Status (ROLECCS) variants. (A) OsTIR1-mEmerald protein (asynchronous ROLECCS, ROLECCS AS, 92 KDa) is stably expressed throughout the cell cycle. Upon auxin treatment, OsTIR1 enzymatic activity elicits the degradation of the mAID-tagged protein of interest (POI) in any cell, independently of the cell cycle status. (B) The expression of the ROLECCS G1 variant (OsTIR1-mEmerald-Cdt1, 103 KDa) is restricted to the G1/early S phase by the presence of the Cdt1 tag, when the SCF Skp E3 ligase activity is off. This, in turn, leads to auxin-dependent ubiquitylation and proteasome degradation of mAID-tagged POIs. In cells transitioning during S, G2 and M phases, SCF Skp activity is naturally restored, leading to ROLECCS G1 degradation by ubiquitylation, and stabilization of the POI even in the presence of auxin. (C) The Geminin tag of the ROLECCS G2 variant (OsTIR1-mEmerald-GEM, 105 KDa) ensures its restricted expression during the late S-G2-M phase, as APC Cdh -mediated ubiquitylation and degradation is rapidly triggered during M/G1 transition. Therefore, auxin treatment induces degradation of the POI exclusively in cells going through the late S-G2-M phase of the cell cycle.

Article Snippet: To construct pAAVS1-ROLECCS AS, pAAVS1-ROLECCS G1, and pAAVS1-ROLECCS G2 plasmids, multiple fragments were PCR amplified from different donor plasmids and assembled as follow: pMK232 CMV-OsTIR1-PURO (Addgene #72834 ) was used as donor plasmid for the expression of OsTIR1 from the AAVS1 locus, the mEmerald tag was PCR amplified from mEmerald-PLK1-N-16 vector (Addgene #54234; http://n2t.net/addgene:54234 ; RRID:Addgene_54234), while pEN435 - pCAGGS-TagBFP-hGeminin-2A-mCherry-hCdt1- rbgpA-Frt-PGK-EM7-PuroR-bpA-Frt Tigre targeting (Addgene #92139 ) was used as template for both hGeminin and hCdt1 tags.

Techniques: Expressing, Stable Transfection, Activity Assay, Variant Assay

Journal: bioRxiv

Article Title: A novel auxin-inducible degron system for rapid, cell cycle-specific targeted proteolysis

doi: 10.1101/2021.04.23.441203

Figure Lengend Snippet: The ROLECCS system performs a Boolean logic computation. The contemporary presence of auxin and appropriate phase of the cell cycle are both simultaneously required to lead to targeted protein degradation. ROLECCS G1 and G2 are stable only through specific phases of the cell cycle (G1/early S for ROLECCS G1, late S/G2/M for ROLECCS G2), therefore their biological activity is restricted to those phases. However, auxin is required to trigger OsTIR1- mediated protein ubiquitylation, allowing proteasomal degradation of the POI only “on demand”, and only in the appropriate phase of the cell cycle.

Article Snippet: To construct pAAVS1-ROLECCS AS, pAAVS1-ROLECCS G1, and pAAVS1-ROLECCS G2 plasmids, multiple fragments were PCR amplified from different donor plasmids and assembled as follow: pMK232 CMV-OsTIR1-PURO (Addgene #72834 ) was used as donor plasmid for the expression of OsTIR1 from the AAVS1 locus, the mEmerald tag was PCR amplified from mEmerald-PLK1-N-16 vector (Addgene #54234; http://n2t.net/addgene:54234 ; RRID:Addgene_54234), while pEN435 - pCAGGS-TagBFP-hGeminin-2A-mCherry-hCdt1- rbgpA-Frt-PGK-EM7-PuroR-bpA-Frt Tigre targeting (Addgene #92139 ) was used as template for both hGeminin and hCdt1 tags.

Techniques: Activity Assay

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