Fig. 2. Formation of the primary angiogenic network. Primary sprouts emerge bilaterally from the dorsal aorta at each vertical myoseptal boundary, then elongate dorsally, ramify and interconnect along the dorsolateral roof of the neural tube to form paired dorsal longitudinal anastomotic vessels. Images shown are lateral (A-F,H) or dorsolateral (G,I) views of the trunk vasculature of different TG(fli-egfp)^y1 embryos at 0.8-1.5 dpf. Images were collected by standard confocal microscopy. (A) Primary sprouts (arrow) just beginning to emerge from the dorsal aorta. (B) Paired primary sprouts appear bilaterally at or adjacent to each vertical myoseptum. The dorsal aorta (arrowhead) and posterior cardinal vein (arrow) are noted. (C) Close-up high-contrast image of filopodia extend from a growing primary sprout (see Movie 4 at http://dev.biologists.org/supplemental/). (D) Paired primary sprouts extending in the anterior trunk. (E) Primary sprouts in the posterior trunk. The dorsal aorta (DA) and posterior cardinal vein (PCV) are labeled, and end of the yolk extension is noted with an arrow. Increased numbers of filopodia are observed as sprouts approach the dorsolateral surface of the neural tube (arrowhead). (F) Primary sprouts split into rostral (arrowhead) and caudal (arrow) branches at the level of the dorsolateral surface of the neural tube. (G) Rostral and caudal branches from adjacent primary sprouts interconnect to form the paired dorsal longitudinal anastomotic vessels (arrows). (H) Completed primary network. Venous sprouts are still absent. (I) The two dorsal longitudinal anastomotic vessels are separate and only sparsely linked by filododial connections (arrow). The dorsal aorta and posterior cardinal vein are more ventral and are not imaged in this confocal stack. Anterior is towards the left. Scale bar: 50 µm in A,B,D-I; 25 µm in C. 3D reconstructions of these images are available at http://dir.nichd.nih.gov/lmg/uvo/ISV3_D.html

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