Integration of repulsive guidance cues generates avascular zones that shape mammalian blood vessels

Abstract

Rationale: Positive signals, such as vascular endothelial growth factor, direct endothelial cells (ECs) to specific locations during blood vessel formation. Less is known about repulsive signal contribution to shaping vessels. Recently, "neuronal guidance cues" have been shown to influence EC behavior, particularly in directing sprouting angiogenesis by repelling ECs. However, their role during de novo blood vessel formation remains unexplored.

Objective: To identify signals that guide and pattern the first mammalian blood vessels.

Methods and results: Using genetic mouse models, we show that blood vessels are sculpted through the generation of stereotyped avascular zones by EC-repulsive cues. We demonstrate that Semaphorin3E (Sema3E) is a key factor that shapes the paired dorsal aortae in mouse, as sema3E(-/-) embryos develop an abnormally branched aortic plexus with a markedly narrowed avascular midline. In vitro cultures and avian grafting experiments show strong repulsion of ECs by Sema3E-expressing cells. We further identify the mouse notochord as a rich source of multiple redundant neuronal guidance cues. Mouse embryos that lack notochords fail to form cohesive aortic vessels because of loss of the avascular midline, yet maintain lateral avascular zones. We demonstrate that lateral avascular zones are directly generated by the lateral plate mesoderm, a critical source of Sema3E.

Conclusions: These findings demonstrate that Sema3E-generated avascular zones are critical regulators of mammalian cardiovascular patterning and are the first to identify a repulsive role for the lateral plate mesoderm. Integration of multiple, and in some cases redundant, repulsive cues from various tissues is critical to patterning the first embryonic blood vessels.

Figure 1. Formation of the dorsal aortae and avascular zones during mouse vasculogenesis

(A-E) Flk1-EGFP highlights ECs (green) in 1-6S stage embryos (anterior views). (A) Free angioblasts (white arrowheads) align in two bilateral rows, at 1-2S. Note absence of angioblasts at the midline and lateral regions. (B) At 3S, angioblasts coalesce into cord-like structures (white arrowhead). Few angioblasts are located lateral to aortic cords. (C) Avascular zones (outlined in white) surround the DA at 4S. By 5S (D), angioblasts differentiate into ECs and begin forming lumens. (E) DA are lumenized by 6S, and avascular zones are clearly demarcated. (A’-E’) Ventral views of 1-6S embryos. Lateral avascular zones are forming from 1-4S (A’-C’), with angioblasts (arrowheads) still present. By 5S(D’), lateral regions are virtually EC-free. Scale bar: 200 μm. da, dorsal aorta; S, somite; ys, yolk sac.

Figure 2. The notochord expresses multiple repulsive guidance cues during embryonic vasculogenesis

(A,B) Cartoon depiction of an E8.25 embryo; (A) anterior and (B) cross-section views showing DA (red), notochord (blue) and surrounding tissues. (C,D) E8-8.25 Flk1-LacZ embryos stained for β-galactosidase (light blue) and eosin (D, red); (C) anterior and (D) cross-section views. (E-N) In situ hybridization for chordin, noggin, netrin1, slit2 and sema3E at E8-8.25. (E,G,I,K, M) Anterior and (F,H,J,L,N) cross-section views. Arrows mark the notochord and DA are outlined in black. Note expression of chordin, noggin, netrin1, slit2 and sema3E in the notochord. The scale bars represent 200 μm (C,E,G,I,K,M) and 25 μm (D,F,H,J,L,N). (D’,F’,H’,J’,L’,N’) Cartoon schematics of D,F,H,J,L,N respectively. Stained tissues shown in red. da, dorsal aorta; ect, ectoderm; end, endoderm; h, heart; lpm, lateral plate mesoderm; nc, notochord; nt, neural tube; s, somites;

Figure 3. Dorsal aortae are severely disrupted in notochordless Foxh1 and Foxa2 null embryos

(A, B) Expression of shh in Foxh1^+/- and Foxh1^-/- E8.25 embryos (anterior views). Shh marks the notochord (A) but is absent in Foxh1^-/- mutants (B). (C-J) β-galactosidase staining (light blue) and eosin staining (G,H, red) of E8.25 Foxh1^+/-;Flk1-LacZ, Foxh1^-/-;Flk1-LacZ, Foxa2^+/-;Flk1-LacZ and Foxa2^-/-;Flk1-LacZ embryos; anterior (C,D), posterior (I, J) and cross-section (E-H) views. Note presence of midline ECs (arrowheads) in Foxh1^-/- and Foxa2^-/- embryos (D,F,H,J). Scale bars: 200 μm (A-D,I,J) and 50 μm (E-H). (K) The percent of Foxh1 (het, n = 23; mut, n = 14) and Foxa2 (het, n = 11; mut, n = 8) embryos with ECs located at the midline. (L) The percent of Foxh1 embryos with lumenized DA (het, n = 7; mut, n = 7). Arrows, DA. lpm, lateral plate mesoderm; nc, notochord; np, neural plate; nt, neural tube; s, somite.

Figure 4. Midline repulsive guidance cues are lost in notochordless embryos

(A-H) In situ hybridization for noggin and sema3E transcripts in E8.0 Foxh1^+/- and Foxh1^-/- embryos: (A,C,E,G) anterior and (B,D,F,H) cross-section views. Arrows mark the notochord and DA are outlined in black. (Note F and H show posterior sections where Sema3E is expressed throughout the neural tube). Scale bars: 200 μm (A,C,E,F) and 25 μm (B,D,F,H). (B’,D’,F’,H’) Cartoon depiction of stained tissues (red) in B,D,F,H respectively. da, dorsal aorta; ect, ectoderm; end, endoderm; lpm, lateral plate mesoderm; nc, notochord; np, neural plate; nt, neural tube: s, somite.

Figure 5. Sema3E expression corresponds to the lateral avascular zones in wild-type and Foxh1^-/- embryos

(A-H) Expression of plexinD1 and sema3E in E8.25 Foxh1^+/-, Foxh1^-/- and Flk1-LacZ embryos; (A,C,G,H) anterior and (E,F) cross-section views. Lateral avascular zones (arrows), sema3E expression in the lpm (arrowhead) and DA (outlined in black) are indicated. Anterior sema3E expression is lost at the midline (D) but maintained in the posterior neural plate (F) of Foxh1 mutants. (G) Cartoon of plexinD1 expression (light blue) in the DA of embryo (A) superimposed onto (C). (H) Sema3E expression (purple) and β-galactosidase staining (light blue) in a Flk1-LacZ embryo. Scale bars: 200 μm (A-D,G), 100 μm (H) and 25 μm (E,F). da, dorsal aorta; lpm, lateral plate mesoderm; nc, notochord; nt, neural tube.

Figure 6. Sema3E is both sufficient for lateral avascular zones and required for dorsal aortae patterning

Sema3E^+/-;Flk1-LacZ and sema3E^-/-;Flk1-LacZ embryos stained for (A-D) PECAMor β-galactosidase (E,F). (A,B) Anterior view of 5S, (C,D) lateral view of 6S and (E,F) cross-section view of 8S embryos. E and F stained with eosin (red). (B,D) Blood vessels in sema3E mutants form a plexus-like network across lateral avascular regions (arrowheads). Note vessels (arrowheads) in closer proximity to the notochord (outlined in black) in a sema3E mutant (F) than in a heterozygote (arrows, DA) (E). Brackets indicate width of avascular zone around the notochord. Quantification of (G) total aortic branch points and (H) ectopic branchpoints within lateral avascular regions, in sema3E^+/- and sema3E^-/- embryos. Branchpoints within 100 sq μm areas, in both left and right lateral regions (anterior, representative fields of view), were counted in 5-9S embryos (n = 3-4 embryos per somite stage). (I) Quantification of midline avascular areas (sq μm/height) in sema3E^+/- and sema3E^-/- embryos, in anterior regions of 5-9S embryos (n = 3-4 embryos per somite stage). (J-U) Cultured ECs are repelled by HEK293-Sema3E cells (K, green), but not by control HEK293 cells (J, green). (L-Q) ‘Wound-healing’ assays with mouse MS1 (L-P) or bend.3 EC lines (Q). MS1 cells at 0 hours (hrs) (L, M) and after 18 hrs cultured (N, O) in media conditioned by control or HEK293-Sema3E cells. (P, Q) Quantification of MS1 and bend.3 cells migration (arbitrary units) at 12 and 18 hrs post ‘scratch’ (n=3). (R-U) In vivo response of ECs in control and Sema3E-HEK293 cells in quail embryos at 11S. ECs contact implanted control cells (R), but not HEK293-Sema3E cells (S). Asterisks denote auto-fluorescing cell implants. Quantification of embryos with disrupted DA (T) and of cell implants contacting ECs (U) (control n=3, and Sema3E n=8). da, dorsal aorta. Scale bars: 200 ⌈m (A-D), 25 ⌈m (E,F) and (R,S) 100 μm.

Figure 7. Model: Dorsal aortae formation is regulated by repulsive signals from the notochord and lateral plate mesoderm

Redundant EC-repulsive cues are expressed by the midline notochord (blue/green), while Sema3E is the primary cue in the lateral plate mesoderm (green). da, dorsal aortae; ect, ectoderm; end, endoderm; lpm, lateral plate mesoderm; nc, notochord; nt, notochord; s, somite.