Extended Data Fig. 9 |. Anti-VCAM-1⁶⁴⁷ antibody labels usher cells without disrupting definitive haematopoiesis.

a, Injection of 1 nl (0.4 ng) of anti-VCAM-1⁶⁴⁷ antibody labels usher cells (arrows) in wildtype and itga4^cas010 mutants in the Tg(kdrl:eGFP) background, whereas injection of either control (non-specific) IgG⁶⁴⁷ antibody into wildtype cells or anti-VCAM-1⁶⁴⁷ antibody into vcam1^cas011 mutants in the Tg(kdrl:eGFP) background did not label any cells in the retention hotspots. Asterisk indicates nonspecific labelling on a chromatophore in the CHT. b, Anti-VCAM-1⁶⁴⁷ injection marginally influence definitive haematopoiesis. Statistical analysis shows the percentage of the three types of definitive haematopoietic phenotype in nine different conditions, including wild-type embryos without injection (#1), with 10 nl vehicle (#2) or 10 nl 0.4 mg ml⁻¹ IgG⁶⁴⁷ injection (#3), itga4^cas005 mutants (#4) or vcam1^cas011 mutants (#5) without injection, and wild-type embryos with 1–10 nl 0.4 mg ml⁻¹ anti-VCAM-1⁶⁴⁷ injection (#6-#9). c, d, Live imaging of HSPCs (c) or WISH analysis of the myb probe at 60 h.p.f. (d) of the wild-type CHT after vehicle or 1 nl anti-VCAM-1⁶⁴⁷ antibody injection. e, Schematic diagrams (left) and confocal imaging (right) show VCAM-1⁺ cells patrolling on a small scale in the CHT in itga4^cas010 mutant embryos. Cross indicates the original position at the initial time point. f, Statistical analysis of the duration of the interaction between HSPCs and usher cells, the HSPC retention time, the diameter of vessels and the dorsal-ventral relative location for HSPC retention in pre-type I (type 0), pre-type II (type 0), type I and type II. Duration, pre-I vs pre-II: ***P = 0.0001, t = 4.25, df = 41; duration, pre-I vs I: ****P< 0.0001, t = 5.31, df = 43; duration, pre-II vs II: P = 0.37, t = 0.93, df = 12; duration, I vs II: P = 0.46, t = 0.76, df = 14; retention: P = 0.16, t = 1.50, df = 14; diameter, pre-I vs pre-II: ****P < 0.0001, t = 8.80, df = 41; diameter, pre-I vs I: P = 0.68, t = 0.42, df = 43; diameter, pre-II vs II: P = 0.89, t = 0.14, df = 12; diameter, I vs II: **P = 0.006, t = 3.24, df = 14; location, pre-I vs pre-II: ****P < 0.0001, t = 7.64, df = 41; location, pre-I vs I: P = 0.6, t = 0.53, df = 43; location, pre-II vs II: **P = 0.005, t = 3.41, df = 12; location, I vs II: P = 0.41, t = 0.85, df = 14. g, In itga4^cas010 mutants, HSPCs encountered but failed to interact with usher cells and then went through the CHT within a few minutes (see Supplementary Video 10). h, The percentage of the type 0, type I and type II HSPC retention types in wild-type sibling and vcam1^cas011 mutants in the Tg(mpeg1:Gal4,kdrl:Dendra2) background with transient transgenesis of UAS:vcam1. None of the HSPCs in the vcam1 mutants could be classified into either type I or II retention types, or were comparable with the HSPCs in Extended Data Fig. 8h. Scale bars, 50 μm (a, c) and 20 μm (e, g).