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doi: 10.1242/10.1242/dev.00516

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Semaphorin3a1 regulates angioblast migration and vascular development in zebrafish embryos

¹ Department of Cell Biology, Institute of Development, Aging and Cancer, Tohoku University, Sendai 980-8575, Japan
² Department of Anatomy, School of Medicine, Iwate Medical University, Morioka 020-8505, Japan
³ Department of Molecular, Cellular and Developmental Biology, University of Michigan, Ann Arbor, MI 48109-1048, USA

View larger version (38K): [in a new window]   Fig. 1. The hsp70:gfpsema3a1myc transgene is heat inducible in transgenic embryos. (A) On the right, transgenic embryos (18 hpf) after heat induction at 15 hpf exhibit ubiquitous GFP fluorescence, indicating that the transgene was induced. On the left, transgenic embryos not heat induced exhibit no GFP fluorescence. (B) After heat induction of transgenic embryos, the GFPSema3a1myc fusion protein was detected by western blotting using an anti-Myc antibody. No band corresponding to the fusion protein is detectable in wild-type embryos (lane 1), nor non-heat induced transgenic embryos (lane 2). An approximately 150 kDa band corresponding to the fusion protein is labeled immediately after the heat treatment (lane 3), 1 hour (lane 4), 3 hours (lane 5), 7 hours (lane 6) and 15 hours (lane 7) after heat treatment of transgenic embryos. In lanes 3-7, heat treatment consisted of raising the water temperature from 28.5°C to 38°C over a period of 15 minutes, then holding at 38°C for 30 minutes and then decreasing to 28.5°C over the next 15 minutes in a programmable water bath.

View larger version (101K): [in a new window]   Fig. 2. The expression patterns of sema3a1/sema3a2 and neuropilin 1 in the trunk region correlate with the migration pathway of angioblasts. Expression of sema3a1 (B,C,G,H), sema3a2 (A), their receptor subunit neuropilin 1 (D-F,I-K), and co-expression of neuropilin 1 and fli1 (L-N) were assayed by in situ hybridization. Unless otherwise noted, embryos are oriented with rostral towards the left and dorsal upwards. (A) At 17 hpf, sema3a2 is expressed by the posterior part of newly segmented somites as seen in a dorsal view. (B) sema3a1 is also initially expressed by the posterior regions of each somite, as seen in a lateral view of a 20 hpf embryo. (C) Subsequently, sema3a1 expression changes so that it is expressed by the dorsal and ventral region of each somite, but not the horizontal myoseptal region as seen at 23 hpf. (D) At 18 hpf neuropilin 1 is expressed by gut endoderm ventral to the somites and by a strip of cells that appear to be extending dorsally (arrow). These strips of cells were adjacent to the anterior regions of the somites as judged by comparison with the borders of the somites that are out of the plane of focus. neuropilin 1 is also expressed by subsets of cells within the spinal cord dorsal to the notochord. (E) By 22 hpf many more strips of neuropilin 1expressing cells were evident, with the anterior strips generally extending more dorsally compared with more posterior strips (arrows). (F) The neuropilin 1-expressing strips are missing in 22 hpf hsp70:gfpsema3a1myc transgenic embryos following ubiquitous induction of sema3a1 at 15 hpf, whereas expression in the ventral spinal cord is unperturbed. (G) Transverse section of the trunk of a 23 hpf wild-type embryo showing expression of sema3a1 in the dorsal and ventral but not horizontal myoseptal region of the somites. (H) In the U-type mutant, you-too (yot), sema3a1 is expressed throughout the entire somite as seen in a transverse section. (I) Transverse section of the trunk of an 18 hpf wild-type embryo showing expression of neuropilin 1 in the strip of cells that appears to be extending along the medial surface of the somites (arrow). neuropilin 1 is also expressed by hypochord cells just ventral to the notochord. (J) At 22 hpf, cells expressing neuropilin 1 (arrow) are seen extending to the dorsal aortic region beneath the hypochord. (K) Transverse section of a 22 hpf hsp70:gfpsema3a1myc transgenic embryo heat induced at 15 hpf showing that the neuropilin 1-expressing cells normally found between the notochord and the yolk tube are missing. (L-N) Transverse sections of a 22 hpf wild-type embryo double-labeled for neuropilin 1 (green, L) and the endothelial marker fli1 (red, M) showing that many of the neuropilin 1-positive cells (arrow) located between the notochord and yolk tube express fli1 (red and green merged, N). Scale bars: 20 µm.

View larger version (99K): [in a new window]   Fig. 3. Dorsal migration by putative angioblasts is interrupted by overexpression of sema3a1. In all panels, Nomarski and fluorescence images are overlaid. (A) DiI-labeled cells ventrolateral to the somites immediately following diI injection in an 18 hpf wild-type embryo. (B) Some of the diI-labeled cells have migrated dorsally by 22 hpf in the same embryo shown in A. Broken line designates the ventral boundary of the notochord. (C) Transverse section at 24 hpf of another embryo showing that some of the diI-labeled cells (arrowhead) labeled at 18 hpf had migrated to a position just ventral to the notochord. (D) DiI-labeled cells ventrolateral to the somites immediately following diI injection in an 18 hpf hsp70:gfpsema3a1myc transgenic embryo following heat induction at 15 hpf. (E) The diI-labeled cells failed to migrate dorsally and remained in ventrolateral positions at 22 hpf in the same embryo shown in D. Broken line designates the ventral boundary of the notochord. (F) Transverse section of a hsp70:gfpsema3a1myc transgenic embryo at 24 hpf showing that diI-labeled cells have not migrated dorsally and remain very ventral. Scale bars: 20 µm.

View larger version (105K): [in a new window]   Fig. 4. Induced ubiquitous expression of sema3a1 adversely affects the distribution of cells expressing the angioblast marker fli1, but not the hematopoietic marker, gata1. All panels show the trunk of whole-mount embryos following in situ hybridization for fli1 (A-D) or gata1 (E-H) in wild-type embryos (A,B,E,F), yot embryos that express sema3a1 through the entire somites (C,G), and hsp70:gfpsema3a1myc transgenic embryos (D,H) following heat induction at 15 hpf. In wild-type embryos, fli1 is expressed diffusely between the notochord and yolk tube at 20 hpf (A), but by 25 hpf fli1 is expressed most strongly by presumptive dorsal aorta cells immediately ventral to the notochord (B, arrowhead). At 25 hpf the pattern of fli1 is disrupted with aberrant patches of cells (C, asterisk) between the notochord and yolk tube in yot embryos, and with diffused pattern in transgenic embryos following heat induction (D). fli1 expression can also be seen in the intersegmental arteries (arrows in B-D). gata1 is expressed by many cells between the notochord and yolk tube in wild-type embryos at 20 (E) and 25 hpf (F). gata1 expression is normal in yot embryos (G) and in hsp70:gfpsema3a1myc transgenic embryos at 25 hpf following heat induction at 15 hpf (H). Scale bar: 20 µm.

View larger version (79K): [in a new window]   Fig. 5. Induced ubiquitous expression of sema3a1 induces constriction of the dorsal aorta and blocks flow of blood cells in the trunk vascular system. In all panels, embryos are 30 hpf. Blood cells flow through the dorsal aorta (right-pointing arrow) and axial vein (left-pointing arrow) in wild-type embryos (A) but not in hsp70:gfpsema3a1myc embryos following heat induction at 15 hpf (B,C). Asterisk in C denotes abnormal cavity filled with putative blood cells. When labeled with Berlin Blue ink, the dorsal aorta can be seen to be constricted in hsp70:gfpsema3a1myc embryos following heat induction at 15 hpf (arrow in E) compared with wild-type embryos (D). In yot embryos, the lumen of the dorsal aorta was tapered (arrow in F). High pressures that caused the cardinal vein to rupture (asterisks in F) were required to fill the dorsal aorta with ink. Scale bar: 20 µm.

View larger version (67K): [in a new window]   Fig. 6. Knockdown of Sema3a1 by antisense MO disrupts the migration of putative neuropilin 1-positive angioblasts and the formation of the dorsal aorta. (A) Heat induction of GFPSema3a1myc was significantly reduced in a hsp70:gfpsema3a1myc embryo at 25 hpf by prior injection of antisense sema3a1 antisense MO (left), but not by injection of sema3a1 control MO (right). (B,C) There are fewer neuropilin 1-expressing cells than normal between the notochord and yolk tube in a 22 hpf antisense sema3a1 MO-injected wild-type embryo seen in a lateral view (B, compare with Fig. 2E) and a transverse section (C, compare with Fig. 2J). Some neuropilin 1-positive cells can be seen just ventral to the notochord but the strips of cells extending dorsally from the yolk tube are missing. (D) Normal pressures that would label the dorsal aorta and the cardinal vein in the trunk of wild-type embryos with Berlin Blue ink only label the cardinal vein in 30 hpf embryos injected with sema3a1 antisense MO. (E) Higher pressures that lead to rupture of the cardinal vein (asterisk) sometimes labeled the dorsal aorta, but the aorta was severely constricted (arrow) in 30 hpf embryos injected with sema3a1 antisense MO. Scale bar: 20 µm.

View larger version (28K): [in a new window]   Fig. 7. The regulation of angioblast migration in the trunk by sema3a1, vegf and neuropilin 1. (Left) A side view of the trunk showing the expression of sema3a1, vegf and neuropilin 1 with the posterior-most somite showing the earliest pattern of somitic sema3a1 expression, and each successive anterior somite showing a slightly more mature pattern. (Right) Transverse section showing the expression of sema3a1, vegf and neuropilin 1 in a segment corresponding to the middle segments shown in the side view. Initially expression of vegf by medial somitic cells attracts neuropilin 1-positive angioblasts to migrate dorsally (arrows), whereas expression of sema3a1 in the posterior somitic cells channels these angioblasts to the anterior regions of each somite. As the angioblasts migrate dorsally to their position just ventral to the notochord, sema3a1 is changing so that it is no longer expressed by the horizontal myoseptal cells at the level of the notochord but is expressed by the ventral third of the somite. The new pattern of Sema3a1 is proposed to restrict the angioblasts to their final destination. Overlapping expression of Vegf and Sema3a1 may generate functional gradients of attraction by Vegf and repulsion by Sema3a1