wu et  (Addgene inc)

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

Journal: medRxiv

Article Title: Somatic RIT1 indels identified in arteriovenous malformations hyperactivate RAS-MAPK signaling and are amenable to MEK inhibition

doi: 10.1101/2023.11.13.23298448

Figure Lengend Snippet: A. Experimental layout: The plasmid containing human wildtype RIT1 or RIT1 variants, under the control of a UAS element and linked to GFP with a P2A sequence is mixed with transposase mRNA and injected into the one-cell stage of Tg(fli1a:Gal4; UAS:RFP) embryos. Thereafter, embryos are either examined at 48 hpf or treated with trametinib from 14 to 48 hpf followed by an examination. B. Vascular network (red) in the tail of an uninjected Tg(fli1a:Gal4; UAS:RFP) embryo; the dashed white box is enlarged in B’ ; arrows represent the direction of arterial and venous blood flow (pink and blue arrow, respectively). C. Tail of a fli1a:RIT1 wt G0 mosaic embryo with normal vasculature (red) despite overexpression of the construct, as indicated by the eGFP expression (cyan). D. Tail of fli1a:RIT1 P1 G0 mosaic embryos. Scale bar 50 µm. E. Tail of fli1a:RIT1 P2 G0 mosaic embryos. Scale bar 50 µm. F. Tail of fli1a:RIT1 P3 G0 mosaic embryos. Note the malformed vasculature with a fusion of the dorsal aorta and the caudal vein as well as dilation of the vessel (in D, E and F). Scale bar 50 µm. G. Quantification of the vascular anatomy at 48 hpf following the injection of plasmids containing the indicated RIT1 variants, with and without treatment. n =2. Fisher’s exact test, two-tailed. ****P(WT vs P1) = 0.00002, ***P(WT vs P2) = 0.0007, ****P(WT vs P3) = 0.00001. Data are presented as mean ± SEM.

Article Snippet: Homo sapiens RIT1 sequence was obtained from the online database Ensembl (Transcript ID: ENST00000368323.8), minimally codon optimized for Danio rerio and ordered as a plasmid including Tol2 sites, a UAS promoter, RIT1 P2 , and P2A-GFP from Twist Bioscience (South San Francisco, CA, USA).

Techniques: Plasmid Preparation, Sequencing, Injection, Over Expression, Construct, Expressing, Two Tailed Test