Additional file 1 of The cytoskeleton adaptor protein Sorbs1 controls the development of lymphatic and venous vessels in zebrafish
Creators: name: Veloso, Alexandra
name: Bleuart, Anouk
name: Conrard, Louise
name: Orban, Tanguy
name: Bruyr, Jonathan
name: Cabochette, Pauline
name: Germano, Raoul F. V.
name: Schevenels, Giel
name: Bernard, Alice
name: Zindy, Egor
name: Demeyer, Sofie
name: Vanhollebeke, Benoit
name: Dequiedt, Franck
name: Martin, Maud
Publisher: name: figshare
Year: 2024
Subjects: subject: Biochemistry
subject: Space Science
subject: Medicine
subject: Cell Biology
subject: Genetics
subject: FOS: Biological sciences
subject: Molecular Biology
subject: Physiology
subject: Developmental Biology
subject: Cancer
subject: Hematology
subject: Infectious Diseases
subject: FOS: Health sciences
subject: Computational Biology

description: Additional file 1:Supplemental Figure 1. Characterization of Sorbs1 depletion in vivo and its expression in endothelial cells. (A) Phylogenetic tree was constructed from human, mouse and potential zebrafish mRNA of Sorbs1, Sorbs2 and Sorbs3 using ClustalW software. One zebrafish ortholog was identified for sorbs1 (Dr sorbs1) and sorbs3 (Dr sorbs3) and two for sorbs2 (Dr sorbs2a and Dr sorbs2b). (B) DNA and amino-acid sequences of the region around the CRISPR/Cas9 target site of the wild-type sorbs1 allele (WT) and its 14-bp deletion (-14) following CRISPR/ Cas9-based editing. (C) Western blotting analysis of protein extracts from wild-type (WT) and Sorbs1 mutant (sorbs1−/−) embryos, using anti-Sorbs1 antibody. GAPDH was used as loading control. (D) Phase-contrast imaging of 48 hpf embryos (upper panel) and zooms on the somites from trunk regions (down panel) of wild-type (WT) and sorbs1−/− Tg(fli1a:eGFP) embryos used to quantify the antero-posterior (AP) body length (white line), the brain size (dashed white line) and the somite angle (white circular arc) and AP length (white line) (n = number of embryos, ns = non-significant, Mann–Whitney U-test). Scale bars represent 500 μm. (E) Expression of Sorbs1 in various human tissues assessed by immunohistochemistry. Typical Sorbs1 staining in endothelial cells is illustrated for the indicated tissues. Boxes correspond to the enlarged area showing expression of Sorbs1 in blood vessels (arrows). Scale bars represent 50 μm and 500 μm respectively in large and zoomed picture. (F) Western blotting analysis of Sorbs1 expression in various human endothelial cells: HDMECs (Human Dermal Microvascular Endothelial Cells), HMECs (Human Mammary Epithelial Cells), HUAECs (Human Umbilical Artery Endothelial Cells), HUVECs (Human Umbilical Endothelial Cells), HMVEC-dLyAd (Human Dermal Lymphatic Microvascular Endothelial Cells), HEK293 (Human Embryonic Kidney 293) and Hela cells. HSP90 was used as a loading control. (G) Whole mount in situ hybridization using a digoxigenin-labeled antisense sorbs1 probe at the different indicated time points. Scale bars represent 100 μm. Supplemental Figure 2. Sorbs1 knockdown recapitulates sorbs1−/− phenotypes. (A) Maximal intensity projection of a confocal z-stack of the cranial vasculature of Tg(fli1a:eGFP)y1 wild-type (WT) and sorbs1 mutant (sorbs1−/−) embryos at 60 hpf in dorsal views (anterior to the left) and wire diagram of the brain vasculature in dorso-lateral view. Red vessels in the 3D renderings represent the intra-cerebral central arteries (CtAs) and gray vessels represent the perineural vessels (primordial hindbrain channels: PHBC and basilar artery: BA). Quantification of the corresponding hindbrain CtAs in wild-type (WT) and sorbs1 homozygous (sorbs1−/−) embryos at 60 hpf. Error bars represent median ± interquartile range; (n = number of embryos; ns = non-significant, Kruskal–Wallis test). Scale bar represents 100 μm. (B) Analysis of sorbs1−/− embryo survival over time (4–10 dpf) in relation to their TD defects. TD defects were classified based on the percentage of its development through 10 somites (total number of analyzed embryos: 70). (C) RT-PCR analysis on total RNA from embryos injected with 5 pg control morpholino (Ctl Mo) or with a splice-blocking morpholino against sorbs1 (sorbs1 Mo). (D) Western blotting analysis of total protein extracts from 48 hpf embryos injected with control (Ctl), ATG- and splice-blocking morpholino against sorbs1, using an anti-Sorbs1 antibody. GAPDH was used as loading control. (E) Quantification of PLs was performed at 48 hpf between 10 somites in Ctl and sorbs1 morphant embryos (n = number of embryos; ****P &lt; 0.001, ***P &lt; 0.001, *P &lt; 0.05; two-tailed Mann–Whitney U-test). Decreasing doses of two types of morpholino were injected at one cell stage, one that blocks sorbs1 splicing (splic.) and one that blocks mRNA translation (ATG). (F) Quantification of thoracic duct (TD) formation was performed at 4 and 6 dpf between 10 somites in embryos injected with Ctl or decreasing doses of the sorbs1 splice-blocking morpholino (n = number of embryos; ****P &lt; 0.001, ***P &lt; 0.001, *P &lt; 0.05; ns = non-significant, two-tailed Mann–Whitney U-test). (G) Z-maximum projections of confocal images of the trunk vasculature of 54 hpf Tg(fli1a:eGFP)y1 embryos injected with ctl or sorbs1 morpholino and expressing transgenic endothelial constructs coding for human Sorbs1 or not were used to quantify the number of PLs in 10 somites. BFP is used as transgenesis marker. Scale bar represents 50 μm (n = number of embryos; **P &lt; 0.01, *P &lt; 0.05; Mann–Whitney U-test). Supplemental Figure 3.Sorbs1 depletion impairs secondary sprouting from the PCV. (A) Frames (Z-maximum projections) from time-lapse confocal imaging of sorbs1−/− Tg(fli1a:eGFP)y1 embryos during venous secondary sprouting. Scale bar represents 50 μm. Numbered black arrows follow sprouting events from the PCV, one of them being illustrated with zooms spanning the time indicated with the dashed arrows in the last boxes. (B) Quantification of the time (in minutes) between the emergence of a venous sprout to its connection to an pre-existent aISV in WT and sorbs1−/− Tg(fli1a:GFP) embryos imaged as described in A. n = number of embryos; *P &lt; 0.05; two-tailed Mann–Whitney U-test). (C) Quantification of secondary sprouts in control and sorbs1-morphant embryos (splice-blocking morpholino) at 36 hpf was established using the five indicated categories (n = number of embryos; **P &lt; 0.1; two-tailed Mann–Whitney U-test). (D) Percentages of vISVs and aISVs were quantified at 48 hpf in a 10 somite region in the trunk of embryos injected with control or sorbs1 splice-blocking morpholino (n = number of embryos; ***P &lt; 0.001; χ2 without Yates correction). Supplemental Figure 4.Sorbs1 expression in zebrafish embryos. Illustration of the methodology used to quantify Sorbs1 FISH signals in Figure 4A. Confocal images of sorbs1 expression (magenta/white dots) and of the vessels (green) of Tg(fli1a:GFP)y1 embryos were used to quantify the distribution of sorbs1 transcripts in the DA and the PCV. The FISH signal was filtered using a wavelet decomposition method, the spots were detected and their cumulative area was quantified in the DA and the PCV. Supplemental Figure 5. Sorbs1 functions independently of Vegfc signaling during in vivo lymphangiogenesis. (A) Frames (Z-maximum projections) from time-lapse lightsheet imaging of Prox1 expressing ECs sprouting from the posterior cardinal vein in WT and sorbs1−/− TgBAC(prox1a:KalTA4-4xUAS-ADV.E1b:TagRFP)nim5 embryos (the green cell in the scheme on the left). Scale bars represent 25 μm. ISVs: intersegmental vessels; DA: dorsal aorta; PCV: posterior cardinal vein. (B) Quantification of PL extent within the trunk region of 54 hpf Tg(fli1a:eGFP)y1 embryos from the indicated genotype resulting from the incross of sorbs1+/− with vegfc+/− embryos (n = number of embryos; ns = non-significant). (C) Quantification of the trunk TD extent (4dpf) in WT or sorbs1−/− Tg(fli1a:eGFP)y1 embryos expressing or not a transgenic constructs coding for human VEGFC under the shh promoter (n = number of embryos; **P &lt; 0.01; Mann–Whitney U-test). (D) Confocal images of FISH staining used to determine flt4 expression (magenta) in WT and sorbs1−/− Tg(fli1a:eGFP)y1 embryos at 30 hpf. Quantification of the fluorescent flt4 signal intensity was performed in the PCV (n = 5 embryos; ns = non-significant; Mann–Whitney U-test). Scale bar represents 50 μm. DA: dorsal aorta; PCV: posterior cardinal vein. Supplemental Figure 6.Sorbs1 depletion leads to defects in ventral sprouting from the PCV. (A) Quantification of subintestinal plexus phenotypes in embryos injected with control or sorbs1 Mo (n = number of embryos; ***P &lt; 0.001; two-tailed Mann–Whitney U-test). (B) Quantification of tip cell numbers were performed at 28 hpf in embryos injected with control or sorbs1 Mo (n = number of embryos; ***P &lt; 0.001; two-tailed Mann–Whitney U-test). Supplemental Figure 7. Sorbs1 deletion affects the migratory and adhesive properties of endothelial cells. (A) HUVECs were transfected with control or Sorbs1-targeting siRNA. Efficiency of RNA silencing was assessed by Western blotting analysis using Sorbs1 specific antibody 48 h after transfection. Actin was used as loading control. (B) HUVECs were transfected as in (A), harvested 24 h after transfection and plated in triplicate at a defined density. Cell number was then assessed by semi-automatic counting at 48 h and 72 h after transfection. Results are presented as the average ± SD increase in cell number, from 3 independent experiments (ns = non-significant, Student’s t test). (C) Micrographs representing HUVECs transfected with control siRNA or with siRNA against Sorbs1 submitted to a scratch-wound assay were taken before (oh) and 16 h after making the scratch to quantify their migratory abilities. Scale bar represents 100 μm. Histogram represents mean ± sd of 3 independent experiments (*: P &lt; 0.05, Student’s t test). (D) Rac1 activity in HUVECs transfected with control (Ctl) or Sorbs1 siRNA was assessed by Western blot analysis of PAK2 and PAK4 phosphorylation using phospho-specific antibodies. Total amounts of PAK2 and PAK4 were used as loading controls. (E) Co-localization of Sorbs1 and adhesion complexes was analyzed in HUVECs by confocal microscopy after immuno-staining using antibodies specific to paxillin and sorbs1. Scale bar represents 20 μm. (F) Adhesion complexes were analyzed by confocal microscopy after immunostaining of paxillin and phospho-paxillin in HUVECs transfected with control or Sorbs1 siRNA as in Figure 6C. Nascent adhesions (NA) and focal complexes (Fx) were classified into two size categories and their distribution in each condition was quantified (n = 21; *: P &lt; 0.05, ns = non-significant, Student’s t test). (G-I) Western blotting was used to assess phosphorylation of Paxillin in Ctl and Sorbs1-depleted cells using a phospho-specific antibody (G), Sorbs1 expression after seeding onto fibronectin for the indicated time (H) and FAK-Src-ERK signaling in Ctl and Sorbs1-depleted cells using phospho-specific antibodies for activated FAK and ERK and inactivated Src (I). Total amounts of the corresponding proteins or actin level were used as loading controls. (J) WT and sorbs1−/− embryos were treated or not with RhoA inhibitor at 26 hpf and the number of sprouting cells at the edge of developing CVP at 28 hpf was quantified (n = number of embryos; ***P &lt; 0.001, ns = non-significant; Mann–Whitney U-test).