Panel a shows a fluorescence image of a zebrafish embryo at 48 hours post fertilization, highlighting the Common cardinal vein in Tg(kdrl:EGFP)s843 and Tg(fli1a:H2B-mCherry)uq37bh, with a 200 micrometer scale bar. Panel b shows merged and individual fluorescence images of the Common cardinal vein at 31 hours post fertilization in Tg(kdrl:EGFP)s843 and Tg(fli1a:H2B-mCherry)uq37bh, with a 100 micrometer scale bar. Panel c shows a schematic of the experimental workflow, including zebrafish crosses, CAS9-gRNP pool injection at 0 hours, the start time of time-lapse imaging at 24 hours, and Common cardinal vein development until 48 hours post fertilization. Panel d shows fluorescence images of Tg(kdrl:EGFP)s843 comparing Uninjected and snx9ab crispant embryos at approximately 38 hours post fertilization, with a 100 micrometer scale bar. Panel e shows a schematic illustrating measurements of Maximum Common cardinal vein length and Width (leading edge). Panel f shows a dot plot of Maximum Common cardinal vein length (micrometers) comparing UIC and snx9ab crispant, with p equals 0.2804. Panel g shows a dot plot of Maximum Common cardinal vein width (micrometers) comparing UIC and snx9ab crispant, with p equals 0.0156. Panel h shows a dot plot of Directionality comparing UIC and snx9ab crispant, with p equals 0.0219. Panel i shows a dot plot of Displacement (micrometers) comparing UIC and snx9ab crispant, with p equals 0.0050. Panel j shows a dot plot of Migration velocity (micrometers per hour) comparing UIC and snx9ab crispant, with p equals 0.0284. Panel k shows fluorescence images of Tg(ve-cad:ve-cadTS)uq11bh comparing Uninjected and snx9ab crispant embryos, with a 30 micrometer scale bar. Panel l shows a dot plot of Junction straightness comparing UIC and snx9ab crispant, with p equals 0.0046.
SNX9 controls endothelial collective migration within the CCV of zebrafish. (a) Zebrafish 48 hpf embryo with Tg(kdrl:EGFP)s843 blood vasculature in magenta and Tg(fli1a:H2B-mCherry)uq37bh endothelial cell nuclei in cyan. (b) Images from a time-lapse sequence showing EGFP from Tg(kdrl:EGFP)s843 (blood vasculature, magenta) and mCherry (nuclei, cyan) from Tg(fli1a:H2B-mCherry)uq37bh expression in the CCV at 31 hpf. (c) Schematic representation of experimental set-up for CRISPR/CAS9-gRNP injections into the yolk of 1-cell stage embryos from Tg(kdrl:EGFP)s843 and Tg(fli1a:H2B-mCherry)uq37bh crosses and microscopy pipeline to image cell migration dynamics during the development of the CCV between ∼24–48 hpf. (d) Maximum projection of the CCV at 38 hpf in uninjected and snx9ab crispants. (e) Schematic of quantification set-up with maximum CCV length and width. (f and g) Dot plots representing quantification of maximum CCV length (f) and width (g) at 40 hpf for uninjected embryos compared with snx9 sibling crispants. n = 11, and 12 CCVs, respectively, from three independent experiments. (h–j) Dot plots representing single-cell migration parameters acquired from tracks of follower cells for an 8-h period: directionality (h), displacement (i), and migration velocity (j) in snx9ab crispants compared with their uninjected siblings. n = 22, 28 follower cells for uic and snx9ab crispants, respectively, from 4 independent experiments. (k) Representative timeframe of a confocal Airyscan maximum projection time-lapse sequence of the leading edge of the CCV of uninjected or snx9ab crispant TgBAC(ve-cad:ve-cadTS)uq11bh siblings at ∼30 hpf. (l) Dot plot of quantification of junctional straightness for uninjected and snx9ab crispant siblings. Graph is clipped at 0.6, close to 0.637, which is the straightness of a semicircle, to more accurately visualize the distribution of the data. A semicircle, curved line (straightness = 0.96) and straight line (straightness = 1) are indicated on the graph as reference points. n = 58 and 49 junctions for uic and snx9ab crispant embryos, respectively, from four independent experiments. 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 Mann–Whitney U test in f–j and l. P values are indicated on the graphs. Scale bars = 200, 100, and 30 μm.