Journal: bioRxiv

Article Title: In ovo electroporation of chicken limb bud ectoderm

doi: 10.1101/2020.11.18.388033

Figure Lengend Snippet: (A) The surface ectodermal cells in HH10 embryos were stained with PKH26 (a red fluorescent dye) marked with white arrowheads (upper panels). Stained regions at HH20 are indicated with white arrowheads (lower panels). (B) A schematic of results of the lineage tracing (n = 3 for each). HN stands for Hensen’s node. (C) Upper panels are schematics showing use of a parafilm well to target prospective forelimb ectoderm in HH10 embryos. The parafilm well was placed on an embryo, subsequently the well was filled with plasmid DNA solution, and then electric pulses were applied. Lower panels are actual views. (D) Electroporation of a plasmid expressing H2BmCherry-ires2-ZsGreen1 (H2BmCh-ZsG) in the forelimb ectoderm either without or with the parafilm well was performed. Note that only ZsG signals are shown. (E) Transverse sections of HH20 embryos electroporated with H2BmCh-ZsG using the parafilm well, and stained with antibodies against CD44. White arrowheads indicate electroporated AER cells while gray ones point to ZsG signals in the neural ectoderm. (F, G) Transverse sections of electroporated HH20 forelimbs stained with antibodies for CD44 (F) and Laminin-1 (G). White arrowheads mark ZsG signals in the limb ectoderm that are colocalized with the markers. Scale bars: 1 mm in (A, D), 400 μm in (E), 200 μm in (F, G).

Article Snippet: To obtain pT2A-CAGGS-Lifeact-mRuby2-ires2-ZsG, Lifeact-mRuby2 CDS was amplified by PCR from mRuby2-Lifeact-7 (addgene #54674; ), and subcloned into pT2A-CAGGS-ires2-ZsG. pCAGGS-mCherry was provided by Dr. Constance L. Cepko.

Techniques: Staining, Plasmid Preparation, Electroporation, Expressing

Journal: bioRxiv

Article Title: In ovo electroporation of chicken limb bud ectoderm

doi: 10.1101/2020.11.18.388033

Figure Lengend Snippet: (A) An HH20 chick embryo electroporated with Lifeact-mRuby expressing plasmids was placed in a glass-bottom dish, and subjected to time-lapse imaging analyses by confocal microscopy. (B) Selected frames from the time-lapse movie (Movie S1). F-actin signals were color-coded according to the intensity. (C, C’) Time-lapse imaging show that the wound epithelial cells bridged a lesion by pseudopodia, which is marked by white arrowheads (see also Movie S2). F-actin signals were depth-coded in (C’). Pseudopodia exist at a lower height, implying that these structures are crawling on non-labelled limb mesenchyme. (D-F) Rac1 activity is necessary for lamellipodia formation. (D) Control leading edge cells seal a lesion by extending lamellipodia (white arrowheads). Magnified stills are shown as lower panels. (E) dnRac1 transfected cells hardly display lamellipodia. See also Movies S3 and S4. (F) Quantitative representation of the number of the cells with lamellipodia per the electroporated cells at a wound edge. 63 cells out of 113 control-electroporated cells in 5 embryos showed lamellipodia, while 20 cells per 84 dnRac1 expressing cells in 5 embryos extended lamellipodia during time-lapse imaging. The P value was obtained using a 2-tailed, unpaired Student’s t test. Scale bars: 25 μm in (B, C, D, E), 15 μm in magnified images of (D, E).

Article Snippet: To obtain pT2A-CAGGS-Lifeact-mRuby2-ires2-ZsG, Lifeact-mRuby2 CDS was amplified by PCR from mRuby2-Lifeact-7 (addgene #54674; ), and subcloned into pT2A-CAGGS-ires2-ZsG. pCAGGS-mCherry was provided by Dr. Constance L. Cepko.

Techniques: Expressing, Imaging, Confocal Microscopy, Activity Assay, Transfection