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Files in this Data Supplement:
Fig. S1. Illustration of morphometry used for quantifying vessel geometry in wild-type and Nrp1 homozygous mutant yolk sacs. (A,C) Pecam1 staining of wild-type (A) and knockout (C) yolk sacs were used to quantitate the vascular phenotype. (B,D) Images were first thresholded to create binary images. Total vessel area was assessed by counting the number of red versus black pixels in the binary images. A vessel skeleton (blue lines) was extracted from the binary images using computer software (see Materials and methods). Points where the skeleton lines intersect (white dots) are counted as branchpoints. Segment length was assessed by dividing the total length of all skeleton lines by the number of intersections. If the total vessel area is constant, and the number of vessel branchpoints is reduced, one expects that the length of individual vessel segments increases. Vessel diameter was assessed indirectly, by measuring the average area of an avascular space (hole) between vessels. With constant vessel area and increased space between vessels, it follows that the individual segments must be thicker. Scale bar: 100 µm.
Movie 1. Flow through yolk sac of E8.5 wild-type embryo. Embryos were dissected at 5 somites and put into roller culture for 6 hours. Embryos were time-lapsed by placing them on a heated stage after culture and white light images were taken at 4× magnification and a rate of 2 Hz. Motion of erythroblasts is present throughout the yolk sac of wild-type embryos at this stage. The contraction of the heart is also clearly visible in the time-lapse movie.
Movie 2. Flow through yolk sac of E8.5 Nrp1-null embryo. As with wild type, embryos were dissected at 5 somites and put into roller culture for 6 hours. Time-lapse of erythroblast motion was performed on a heated stage after culture, with images taken at a rate of 2 Hz using white light and 4× magnification. At this stage, motion of erythroblasts is only observed in the distal part of the yolk sac (blood islands) of mutant embryos. The erythroblasts oscillate in place and circulating erythroblasts were not observed outside of the blood islands. Heart contraction, by contrast, appeared normal.
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