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DVDY_22303_sm_suppfig1.tif3442KSupporting Figure 1. Cranial neural crest cells (NCCs)fail to completely invade the anterior portion of the third branchial arch (ba3) microenvironment, but invade the fourth branchial arch microenvironment normally when neuropilin-1 function is knocked down. A: Static confocal image of a typical embryo in which postotic cranial NCCs were transfected with a control enhanced green fluorescent protein (EGFP) construct, n=8 for 24 hr and n=5 for 36 hr after electroporation. B: Static confocal image of an embryo in which postotic cranial NCCs were transfected with Np-1 siRNA, 36 hr after electroporation, n=10 for 24 hr and n=6 for 36 hr after electroporation. The Np-1 siRNA transfected cranial NCCs (green) migrated out of the neural tube; however, the cranial NCCs failed to invade the anterior portion of ba3. Untransfected cranial NCCs (red) were seen throughout the developing arches. Dotted lines represent how quantitative measurements were calculated. C: Quantitative measurements of the distance cranial NCCs migrated from the neural tube into the ba3 microenvironment, as a percentage of the distance from the neural tube to the distal end of ba3. D: Quantitative measurements of the anterior–posterior width of the ba3 microenvironment that cranial NCCs populated, as a percentage of the total ba3 width. E: Quantitative measurements of the distance cranial NCCs migrated from the neural tube into the ba4 microenvironment, as a percentage of the distance from the neural tube to the distal end of ba4. F: Quantitative measurements of the anterior–posterior width of the ba4 microenvironment that cranial NCCs populated, as a percentage of the total ba4 width. Scale bars=100 μm. r, rhombomere; ba, branchial arch; *significantly different, P < 0.005. r7 NCCs transfected with Np-1 siRNA migrate when transplanted into r4. G: A schematic representation of the ba2 to r4 transplantation technique. H: A static confocal image of a host embryo, 24 hr after a subpopulation of DiI (1,1′, di-octadecyl-3,3,3′,3′,-tetramethylindo-carbocyanine perchlorate) -labeled (red), EGFP-transfected (green) migratory r4 NCCs from the ba2 microenvironment entrance were transplanted heterochronically into r4, n=8. Both the transfected (green) and untransfected (red) cranial NCCs were able to migrate from the transplant site toward the ba2 microenvironment. I: A static confocal image of a host embryo, 24 hr after a subpopulation of DiI-labeled (red), Np-1 siRNA-transfected (green) migratory r4 NCCs from the ba2 microenvironment entrance were transplanted heterochronically into r4, n=10. Both the transfected (green) and untransfected (red) cranial NCCs were able to migrate from the transplant site toward the ba2 microenvironment. r, rhombomere; ba, branchial arch. Scale bars=100 μm.

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