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Fig. 6 The chloride channel SWELL1 promotes endothelial cell (EC) migration and sprouting angiogenesis. (A, B) UMAP plots showing co-expression of lrrc8aa and aqp1a.1 (A), and lrrc8aa and aqp8a.1 (B) in ECs from 24 hpf, 34 hpf, and 3 dpf embryos. (C–E) Detection of aqp1a.1, aqp8a.1, and lrrc8aa mRNA by RNAscope in situ hybridization in 22 hpf zebrafish embryo. Representative maximum intensity projection rendering of confocal z-stacks of tip and stalk cells (C). (D) and (E) show surface rendering of image (C) where only endothelial expression of lrrc8aa is shown after masking. Scale bar, 10 µm. (F, G) Representative maximum intensity projection confocal z-stacks of 26 hpf wildtype embryos treated with 0.05% EtOH (F) or 5 µM DCPIB (G) from 20 hpf to 26 hpf (control, n = 16 embryos; DCPIB, n = 18 embryos; two independent experiments). Scale bar, 50 µm. (H) Quantification of intersegmental vessel (ISV) length in 26 hpf wildtype embryos treated with 0.05% EtOH or 5 µM DCPIB (control, n = 133 vessels from 16 embryos; DCPIB, 157 vessels from 18 embryos; two independent experiments). (I) Quantification of tip cell leading edge displacement of wildtype embryos treated with 0.05% EtOH (n = 29 cells from seven embryos, two independent experiments) or 5 µM DCPIB (n = 29 cells from eight embryos, three independent experiments) at 25–30 hpf; data is presented as mean ± SD. (J) Quantification of tip cell migration velocity in wildtype treated with 0.05% EtOH (n = 29 cells from seven embryos, two independent experiments) or 5 µM DCPIB (n = 29 cells from eight embryos, three independent experiments) at 25–30 hpf. (K) Filopodia number in tip cells of wildtype embryos treated with 0.05% EtOH (n = 34 vessels from nine embryos, three independent experiments) or 5 µM DCPIB (n = 53 vessels from 16 embryos, three independent experiments). (L) Filopodia length in tip cells of wildtype embryos treated with 0.05% EtOH (n = 34 vessels from nine embryos, three independent experiments) or 5 µM DCPIB (n = 53 vessels from 12 embryos, three independent experiments). Statistical significance was determined with unpaired t-test; ****p<0.0001 (H, J, K, and L). (M, N) Still images from representative time-lapse movies of migrating tip cells in wildtype embryos treated with 0.05% EtOH (M, n = 7) or 5 µM DCPIB (N, n = 8). Movies were taken from 25 to 30 hpf (n = 3 independent experiments). Bracket, formation of stable protrusions. Scale bar, 20 µm.