|
Fig. 5 Changes in blood flow trigger vessel pruning.
(A–G) Changes in blood flow before and during vessel pruning. (A–C) Data obtained from the same vessel segments. (A) Representative of simultaneous serial imaging and axial line scanning of midbrain vessels in a 2-dpf Tg(kdrl:eGFP,PU.1:gal4-uas-GFP) larva. Red and blue lines in the first panel indicate the site where axial line scanning was performed on a pruned (red arrow) and its adjacent unpruned segments, respectively. The numbers on the top of each panel represent the blood flow velocity and segment diameter of the pruned vessel segment. The white dashed arrows indicate blood flow direction. White signals in vessels were originated from moving blood cells that expressed GFP. The dashed square in the first panel marks the position from which blood flow is shown in Video S5. (B) Example showing changes with time of the blood flow velocity (filled circle) and diameter (open circle) of pruned (red) and adjacent unpruned (blue) segments before and during vessel pruning. The arrow and arrowhead mark the time point when the blood flow velocity showed an irreversible drop or the segment exhibited an obvious reduction in diameter, respectively. (C) Representative of kymographs showing bi-directional flow in the pruned segment (right) and uni-directional flow in an adjacent unpruned segment (left). The data were obtained from the pruned (red line) and unpruned (blue line) segments in (A) at the time point of 80 min. Green arrows indicate forward movement of blood cells, and yellow ones (right) indicate reverse flow in the pruned segment. (D and E) Average magnitude (D) and coefficient of variation (E) of flow velocities among different time points in pruned and adjacent unpruned segments before the time when an irreversible drop of flow velocity in pruned segments occurred (as indicated by the shadow region in B). The numbers on the bars represent the numbers of segments examined. (F and G) Normalized average magnitude (F) and coefficient of variation (G) of shear stress among different time points in pruned and adjacent unpruned segments before the time when an irreversible drop of flow velocity in pruned segments occurred (as indicated by the shadow region in B). (H and I) Effects of blood flow manipulation on vessel pruning. (H) Representative of serial imaging showing that the obstruction of blood flow by beads triggered vessel pruning. The beads were loaded several minutes before the time zero via the duct of Cuvier microinjection. The red and blue circles mark the beads that were successful or failed to block blood flow, respectively. The red and blue arrows mark the pruned and unpruned segments, respectively. The yellow arrowheads represent moving blood cells in the vessel segment in which a bead (blue circle) failed to block the blood flow. (I) Effects of norepinephrine bitartrate (NB) treatment at 2.0 dpf for 24 h on the occurrence of vessel pruning per larva. Each symbol represents data obtained from one larva. Scales, 10 μm in (A), 5.89 µm (x-axis) and 143 ms (y-axis) in (C) and 10 μm in (H). * p<0.05; ** p<0.01 (Student′s t test). Error bars, ± SEM.