Figure 2.
Core Planar Cell Polarity Proteins, Celsr1 and Vangl2, Are Recruited from Membrane Filopodia to Cell-Cell Contacts in the Valve-Forming Endothelial Cells
(A–C) Whole-mount immunofluorescence for Celsr1 (green) and Prox1 (red) at indicated stages of development. Note punctuate Celsr1 staining in areas of Prox1high valve-forming cells. Dotted lines outline lymphatic vessels.
(D) Localization of Celsr1 (green) and Vangl2 (red) in mature valves.
(E–G′) Visualization of Celsr1 (purple, E and F) or Vangl2 (purple, G) in individually labeled lymphatic endothelial cells (marked by mGFP fluorescence (green) in Prox1-CreERT2;R26-mTmG vessels) at E16.5. (E and E′) Tile scan of E16.5 lymphatic vessel showing Celsr1 at areas of developing valves (arrowheads). (F–G′) Higher magnification images of vessels showing localization of Celsr1 (F and F′) and Vangl2 (G and G′) to the tips of membrane filopodia (arrowheads) at an early stage of valve development.
(H–I) Whole-mount immunofluorescence of E16.5 wild-type mesenteric lymphatic vessel for Celsr1 (purple), Claudin-5 (green, H–H″), or VE-cadherin (green, I). Boxed areas in (H) are magnified in (H′) and (H″). The direction of flow is indicated by an arrow. Note the localization of Celsr1 to the tips of Claudin-5+ and VE-cadherin+ filopodia (arrow in H′ and I) and membrane protrusions (H″).
(J–M′) Visualization of Celsr1 (purple) in individually labeled lymphatic endothelial cells (green) at indicated stages. Celsr1 is localized to cell-cell contacts (arrowheads in J–M′) and junctions (arrows in J–M′) in reorienting cells. (L and M) Magnifications of areas shown by arrowhead and arrow, respectively, in (K). Dotted lines in (M) and (M′) outline individual endothelial cells. Single channel images for indicated stainings are shown.
Scale bars represent 40 μm (A–D), 100 μm (E–E″), 40 μm (F–G′ and J–M′), 30 μm (H), 7.5 μm (H′ and H″), and 20 μm (I).
See also Figure S2.