Data supporting the analysis of lymphatic endothelial cell junctions and shape

Data in support of: Schoofs H, Daubel N, Schnabellehner S, Grönloh M, Palacios Martínez S, Halme A, Barcos S, Brakebusch C, Benedito R, Engelhardt B, Vestweber D, Gängel K, Linsenmeier F, Schürmann S, Saharinen P, van Buul JD Friedrich O, Smith RS, Majda M, and Mäkinen T. Resilience of lymphatic endothelium through .... (manuscript). DATASET A: Annotated cell-cell junction types in lymphatic capillaries of wild type mouse ear skin at different ages __________________________________________________________________________________________________________ Contents SOURCE DATA Fig1 FINAL. xlsx 3w animal 1 animal 2 animal 3 animal 4 animal 5 sprouts 5w animal 1 animal 2 animal 3 animal 4 animal 5 diaphragm trachea 25w animal 1 animal 2 animal 3 animal 4 animal 5 diaphragm trachea File legends C1 images: inverted LYVE1 signal (.tif) C2 images: inverted VE-cadherin signal (.tif) MAX images: RGB merge of LYVE1 (cyan) and VE-cadherin (red) (.tif) "NAME".roi: Regions of interest (ROI) of annotated junctions can be imported in ImageJ Methods Junctional classification: Analysis of junction morphology was done on blunt-ended initial lymphatic capillaries in the segment between the intial tip and the first valve. Junction types were quantified in Z-stack projection by numbering of individual lobes of LYVE1 and VE-cadherin-stained LECs and subsequent categorizing of lobe-associated junctions based on VE-cadherin signal. Four categories were defined: 1) Button junction – a punctate VE-cadherin+ deposit at the neck of LYVE1+ lobe/overlap, with no detectable VE-cadherin at the borders of the overlap, 2) Curvilinear junction – continuous or discontinuous distribution of VE-cadherin within one border of LYVE1+ lobe/cellular overlap, 3) Double junction – continuous or discontinuous distribution of VE-cadherin within both borders of LYVE1+ lobe/cellular overlap, and 4) Zipper junction – continuous linear VE-cadherin distribution at cell-cell contacts in the absence of LYVE1. Wild-type C57BL/6J mice were used for analysis of junction types, and 4-5 blunt ended vessels per mouse from five mice per age group and condition were analysed; in total 1785 junctions were annoted Imaging: Confocal images were obtained using a Leica Stellaris 5 confocal microscope equipped with 405 nm and white light lasers, 63x/1.3 HC PL APO CORR CS2 Glycerol immersion objective, and Leica LAS X software. Images were aquired at 1.51 digital zoom using a 2048x2048 resolution Tissue processing and staining: Tissues were fixed in 4% paraformaldehyde for 2 h at RT and permeabilized in 0.3% Triton X-100 in PBS (PBST) for 10 min. After blocking in PBST with 2% bovine serum albumin, 1% FBS for 2 h, tissues were incubated with primary antibodies in blocking buffer overnight, followed by PBST washing and incubation with fluorescent dye-conjugated secondary antibodies for 2 h. All incubation steps were carried out at RT. Prior to mounting in Mowiol, samples were repeatedly washed in PBST and water. Antibodies used: Goat anti-mouse VE-cadherin (R&D Systems, AF1002; 1:200), Rat anti-mouse LYVE1 (R&D Systems, MAB2125; 1:200) DATASET B: Finite element method (FEM) simulations of cellular stresses _______________________________________________________________ The FEM simulations were performed with MorphoMechanX using available models adapted from Sapala et al, eLife 7, e32794 (2018). A regular cylindrical grid 45 µm wide and 200 µm long was created and outlines from the cells of a lymphatic vessel were projected onto it and smoothed. These cells were then extruded inward to make 3D volumetric cells with a depth of 2 µm and triangulated using a threshold area of 4 µm. The template was then used as the reference configuration for triangular 3 node membrane elements which were given a thickness of 0.1um. An isotropic St. Venant material model (linear, large deformation) was used with the Young's modulus set to 100 kPa to match a 10 kPa cell level Young's modulus estimated from the literature (ignoring the cell ends, the 2 x 0.1 µm membrane thickness occupied roughly 1/10th the cross-sectional area of the cell that were 2 µm deep). A uniform internal pressure was applied normal to the inside faces of the elements, which cancels out on the shared walls between cells. For simulations with a lower pressure inside the vessel, the inside faces were assigned a higher pressure. Stresses were visualized as the trace of the stress tensor.