Fig. 9: Rab35 is required for blood vessel development in zebrafish.

a CRISPR-mediated knockout of Rab35A/B confirmation by sequencing. Four random fish were sequenced following CRISPR/guide injections. b Zebrafish morphology at 48 h post fertilization (hpf) post injection of scramble (Scram) and Rab35A/B CRISPR guides. c Representative images of intersomitic blood vessels (ISVs) of Scram and Rab35A/B knockout, as well as CK-666 (Arp Inhibitor), treated zebrafish at 48 hpf expressing endothelial specific LifeAct-GFP. Red arrowheads indicate abnormal aggregates of actin. d Quantification of actin aggregates between groups. n = number of ISVs. A minimum of 5 fish were used per group. Error bars represent standard deviation, middle bars are the mean. e Representative images of mosaic expression of Tag-RFP-Rab35 WT (top row), CA (second row), DN (third row), and WT with CK-666 treatment in zebrafish at 48 hpf. Red arrowheads depict excess of Rab35 at the plasma membrane. f Percentage of non-lumenized vessels at 48 hpf between groups mentioned in panel (e). n = number of ISVs. g Cartoon representation of microangiography in zebrafish larvae using quantum dots 647 (Qdot647) at 48 hpf. h Representative images of ISVs after transplantation of Tg(kdrl:GFP) donor (D) into Tg(kdrl:mCherry) host (H) (top panels). Bottom panels- representative images of ISVs after transplantation of Rab35A/B knockout donor cells from Tg(kdrl:GFP) line into Tg(kdrl:mCherry) host. Red arrowheads indicate lumen failure. NS = non-significant. Error bars represent standard deviation. Statistical significance was assessed with an unpaired t-test or a 1-way ANOVA followed by a Dunnett multiple comparisons test. Insets are areas of higher magnification. All experiments were done in triplicate.