Snx9 alignment, knockout efficiency of gRNA pools and CCV migration parameters. (a) Maximum projection of an Airyscan confocal image and ROIs of endothelial junctions in the CCV of a Tg(ve-cad:ve-cadTS) zebrafish embryo at 30 hpf. (b) Sequence homology alignment of zebrafish proteins Snx9a and Snx9b using Blast. SH3, PX, and BAR domains are indicated to indicate homology of these protein domains. (c and d) Validation of targeting efficiency by PCR analysis of each amplicon targeted by individual snx9a or snx9b gRNAs in the gRNA pool. Uninjected siblings from each experiment are compared against injected embryos showing indels causing the appearance of additional amplicons or loss of distinctive bands for lanes indicated with asterisks. (e) GFP image of the CCV of an uninjected Tg(kdrl:EGFP)^s843;Tg(fli1a:H2B-mCherry)^uq37bh zebrafish embryo. Lines indicate individual cell trajectories starting at 31 and ending at 40 hpf. Trajectories are color-coded based on starting cell location: leader cells (white), follower cells (magenta), cells at the dorsal side of the CCV with a double endothelial cell layer (followers high, orange), and cells at the lateral edges (edge, cyan). (f–h) Box-and-whisker plots representing single cell migration parameters separated by starting location in the CCV quantified for directionality (f), displacement (g), and cell velocity (h). n = 17, 28, 38, and 21 for leading cells, followers, dorsal followers, and lateral cells, respectively, from 4 to 5 replicate experiments. Data are presented as median with 25–75 percentiles, and whiskers indicate min-max values. Statistical analysis was performed by a one-way ANOVA test followed by Tukey HSD post hoc tests. P <0.05 = *, P < 0.01 = **, P < 0.001 = ***, and P < 0.0001 = ****. (i) Validation targeting efficiency by PCR analysis of each amplicon targeted by individual pacsin2 gRNAs in the gRNA pool. Uninjected siblings from each experiment are compared against injected embryos showing indels causing the appearance of additional amplicons or loss of distinctive bands for lanes indicated with asterisks. (j) Maximum projection of the CCV at 38 hpf in uninjected and pacsin2 crispants. (k and l) Dot plots representing quantification of maximum CCV length (k) and width (l) at 40 hpf for uninjected embryos compared with pacsin2 sibling crispants. n = 13, and 11 CCVs, respectively, from three independent experiments; from which one used a previously run control group in Fig. 7. (m–o) Dot plots representing single cell migration parameters acquired from tracks of follower cells for an 8-h period: directionality (m), displacement (n), and migration velocity (o) in pacsin2 crispants compared with their uninjected siblings. n = 18, 19 follower cells for uic and pacsin2 crispants, respectively, from three independent experiments; one of which used a previously run control group in Fig. 7. Data are presented as mean ± SD of experiment means. Circles indicate individual data points, and triangles indicate means of individual experiments with colors corresponding to the experimental replicates. Statistical analysis was performed by the Mann–Whitney U test. P-values are indicated on the graphs. Scale bars = 100 and 30 μm. Source data are available for this figure: SourceData FS5.