The number of NRP1-positive tip cells in blood vessels with mosaic expression of NRP1 is a good predictor of branching frequency. (A-C) Live cells from the indicated genotypes were FACS-sorted with a PECAM-APC antibody (y axis) and for YFP fluorescence (x axis); (A) Cells from a control embryo lacking YFP and not stained for PECAM; this FACS profile was used to determine gating parameters to identify YFP-positive, PECAM-stained cells in (B,C). (D) Percentage of total PECAM-positive (Q1 + Q2 quadrants) and PECAM-negative (Q3 + Q4 quadrants) cells in (B and C). (E) Percentage of YFP-positive, PECAM-positive cells (Q2 quadrant) and YFP-negative, PECAM-positive cells (Q1 quadrant) in (B,C). (F,G) The number of vessel branchpoints in three littermate mutants lacking NRP1 in the Tie2-Cre lineage correlates inversely with the number of YFP-positive (F) and positively with the number of NRP1-positive tip cells (G). (H) Schematic representation of NRP1 localization in wild-type (top) and chimeric (bottom) vessel sprouts; NRP1 is present on both tip and stalk cells in wild-types (bright red), whereas NRP1 is high on mutant tip cells (bright red) and low on mutant stalk cells (faded red) of chimeric vessels.