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First published online 3 September 2003
doi: 10.1242/dev.00733

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Angiogenic network formation in the developing vertebrate trunk

Sumio Isogai1,2, Nathan D. Lawson1,*, Saioa Torrealday1,{dagger}, Masaharu Horiguchi2,{ddagger} and Brant M. Weinstein1,§

1 Laboratory of Molecular Genetics, NICHD, NIH, Bethesda, MD 20892, USA
2 Department of Anatomy, School of Medicine, Iwate Medical University, Morioka, Japan



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  		Fig. 1. Anatomy of the zebrafish trunk and its blood vessels at ~3 days post-fertilization. At this stage, there is active flow through the dorsal aorta, (DA), posterior cardinal vein (PCV) and most intersegmental arteries (ISA) and intersegmental veins (ISV). The ISA and ISV are linked together dorsally via paired dorsal longitudinal anastomotic vessels (DLAV). All of these vessels are shown relative to adjacent tissues and structures in the mid-trunk including the gut (G), myotomes (M), notochord (N), neural tube (NT), left pronephric duct (P) and yolk mass (Y). In addition to the functioning vessels noted above, parachordal vessels (PAV) run longitudinally to either side of the notochord, along the horizontal myoseptum. The parachordal vessels are linked to the posterior cardinal vein and intersegmental veins (arrows), but generally not to intersegmental arteries. At 3 dpf, the parachordal vessels do not yet carry flow. Anterior is towards the left and above the plane of the page, and dorsal is upwards.

 


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  		Fig. 8. (A) Steps leading to assembly of the trunk angiogenic vascular network and (B) a proposed model for determination of secondary sprout fate and intersegmental vessel AV identity. For clarity, both diagrams show the vessels on only one side of the trunk. (A, part i) Primary sprouts emerge bilaterally exclusively from the dorsal aorta (red). (A, part ii) Primary sprouts grow dorsally, branching cranially and caudally at the level of the dorsolateral roof of the neural tube. (A, part iii) Branches interconnect on either side of the trunk to form two dorsal longitudinal anastomitic vessels (DLAV). (A, part iv) Secondary sprouts begin to emerge, exclusively from the posterior cardinal vein (blue). (A, part v) Some secondary sprouts connect to the base of primary segments, while others do not. (A, part vi) Primary segments with patent connections to secondary segments become intersegmental veins (blue), while primary segments that remain connected only to the dorsal aorta become intersegmental arteries (red). Most of the secondary sprouts that do not connect to primary segments serve instead as ventral roots for the parachordal vessels. Intersegmental veins form additional connections to the parachordal vessels at the level of the horizontal myoseptum. (B) How flow dynamics might help to guide the patterning of vessel connections (see Discussion for details). Primary segments without blood flow are shown in gray, while those carrying arterial or venous blood flow are shown in red and blue, respectively. Unconnected (growing) secondary sprouts are shown in black. Flow through a primary segment inhibits connection to the segment by an adjacent secondary sprout (inhibitory cues are shown as sideways `T' symbols).

 


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  		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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  		Fig. 3. Emergence and fate of secondary angiogenic sprouts. Images shown are lateral views of vessels at single intersomitic positions in the mid-trunk of TG(fli-egfp)y1 zebrafish embryos. Sequential image stacks were collected by multiphoton confocal microscopy. Time-lapse movies of the sequences in A, C, E are available as Movies 6, 7 and 9 at http://dev.biologists.org/supplemental/ The images shown in these panels are from selected frames of these movies, labeled with the time in hours:minutes from the first frame (arbitrarily designated time zero). (A) A secondary sprout (arrow) emerges from the posterior cardinal vein immediately adjacent to a primary segment (arrowhead). After an extended time it fuses with the primary vessel, lumenizes and begins to carry blood. Images are from ~1.5-2.5 dpf. The dorsal aorta (DA) and posterior cardinal vein (PCV) are noted. (B) A secondary sprout (arrow) emerges from the posterior cardinal vein slightly away from the vertical myoseptum. It elongates towards and reaches the proximal part of the primary segment (arrowhead), forming a patent connection and lumenizing. Images are from ~1.5-2.0 dpf. (C) A nonfunctional vestigial ventral segment connecting a primary segment to the DA (arrows) thins and then regresses completely adjacent to a large robustly patent secondary connection to the posterior cardinal vein. This vessel is thus an intersegmental vein. Images are from ~1.7-2.5 dpf. (D) Explanatory diagram showing the vessels in C. (E) A secondary sprout elongates next to a primary segment, partially lumenizing but failing to connect. Images are from ~1.6-2.2 dpf. Anterior is towards the right in A and B, and to the left in C-E. Primary (arrowhead) and secondary (arrowhead) intersegmental vascular segments are noted. Scale bar: 25 µm. 3D reconstructions of these images are available at http://dir.nichd.nih.gov/lmg/uvo/ISV3_D.html

 


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  		Fig. 4. Formation of the parachordal vessels. Multiphoton confocal images (B,C) show lateral (top) and ventrolateral (bottom, image tilted upwards -40 to -50 degrees relative to the top images) views of vessels on one side of the mid-trunk of fli-egfp transgenic zebrafish embryos at ~1.8-2.2 dpf. (A) Parachordal vessels form from sprouts derived from both the posterior cardinal vein (small arrow) and from future intersegmental veins (large arrow). (B) Parachordal segment connected to an intersegmental vein at one end (small arrow), and connected to the posterior cardinal vein at the other end via a ventral root (large arrow) adjacent to an intersegmental artery (arrowheads). A vestigial connection to the dorsal aorta persists on the intersegmental vein (asterisk). (C) Intersegmental vein connected to the adjacent parachordal vessel (arrow). Anterior is towards the left in all panels. Scale bar: 25 µm. 3D reconstructions of these images are available at http://dir.nichd.nih.gov/lmg/uvo/ISV3_D.html

 


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  		Fig. 5. Primary network formation proceeds normally in embryos that have no blood circulation. (A-D) Lateral views of the mid-trunk of Tg(fli1:EGFP)y1/Tg(fli1:EGFP)y1, sih/sih or Tg(fli1:EGFP)y1/Tg(fli1:EGFP)y1 (E,F) embryos. (A) The primary vascular lattice appears normal at 1.5 dpf. (B) Secondary sprouts (arrows) are apparent by 2 dpf. Their appearance is also normal. (C) Secondary sprouts (arrows) contribute to the parachordal system by 2.5 dpf, as they do in wild-type embryos. (D) By ~3.5 dpf primary segments display dorsal enlargement and ramification (arrows), but lack obvious vessel lumenization. (E) Control morpholino and (F) anti-Vegf morpholino injected transgenic at 1.5 dpf. Anti-Vegf morpholino-injected animals do not form the primary angiogenic network, although initial sprouting is sometimes observed (arrows in F). Images were collected by multiphoton confocal microscopy. Anterior is towards the left in all panels. Scale bar: 50 µm. 3-D reconstructions of these images are available at http://dir.nichd.nih. gov/lmg/uvo/ISV3_D.html

 


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  		Fig. 6. Secondary sprouts contribute to either intersegmental veins or parachordal vessels. Blood vessels on the left side of the trunk were imaged in two separate Tg(fli1:EGFP)y1 animals at ~1.8 and 2.2 dpf (A) or 1.9 dpf (B) and the final AV identity of each intersegmental vessel was determined at 7 dpf. The data for trunk vascular wiring are presented schematically. Horizontal lines show the dorsal aorta (red), posterior cardinal vein (blue) and parachordal segments (gray). Vertical lines show primary segments (red), secondary segments connecting to form intersegmental veins (blue), and secondary segments forming ventral parachordal roots or whose fate has not yet been determined (gray). Connections to intersegmental vessels are noted with black rings; vessels depicted as crossing one another without a black ring are adjacent, but not connected. Asterisk notes the lone ISA that did form a connection to the parachordal system. See text for additional details.

 


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  		Fig. 7. Circulation through segments with functional connections to both the dorsal aorta and the posterior cardinal vein. (A) The ventral half of a primary segment connected to both the dorsal aorta (red) and the posterior cardinal vein (blue). Blood flow through the vessels is noted (gray arrows). Numbers shown are fraction of 21 vessels examined that initially (at 2 dpf) had functioning dual connections that later resolved in favor of either an intersegmental artery (top, 11/21) or an intersegmental vein (bottom, 10/21) between 2 and 4 dpf (see text for details). (B-D) Representative examples of `dual connection' segments in which blood is flowing from dorsal aorta to posterior cardinal vein. Images shown (top) are lateral views of the trunks and tails of fli-egfp transgenic zebrafish embryos at approximately 2-2.2 dpf. (C,D) The vessels on opposite sides of the tail of at the same anterior-posterior position. Images were collected by multiphoton microscopy. Accompanying illustrative diagrams (bottom) show blood flow patterns through the dual-connected intersegments (flow direction is noted with red arrows). 3D reconstructions of these images are available at http://dir.nichd. nih.gov/lmg/uvo/ISV3_D.html Movies showing blood flow through these vessels are available at http://dev.biologists.org/supplemental/.

 

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© The Company of Biologists Ltd 2003