Resolution of defective dorsal aortae patterning in Sema3E-deficient mice occurs via angiogenic remodeling

Abstract

Background: Neuronal guidance cues influence endothelial cell (EC) behavior to shape the embryonic vascular system. The repulsive neuronal guidance cue, Semaphorin 3E (Sema3E), is critical for creating avascular zones that instruct and subsequently pattern the first embryonic vessels, the paired dorsal aortae (DA). Sema3E(-) (/) (-) embryos develop highly branched plexus-like vessels during vasculogenesis, instead of smooth paired vessels. Unexpectedly, despite these severe DA patterning defects, mutant mice are viable throughout adulthood.

Results: Examination of Sema3E(-) (/) (-) mice reveals that the plexus-like DA resolve into single, unbranched vessels between embryonic day (E) E8.25 and E8.75. Although fusion of Sema3E(-) (/) (-) DA occurs slightly earlier than in heterozygotes, the DA are otherwise indistinguishable, suggesting a complete "rescue" in their development. Resolution of the DA null plexuses occurs by remodeling rather than by means of changes in cell proliferation or death.

Conclusions: Normalization of Sema3E(-) (/) (-) DA patterning defects demonstrates resilience of embryonic vascular patterning programs. Additional repulsive guidance cues within the lateral plate mesoderm likely re-establish avascular zones lost in Sema3E(-) (/) (-) embryos and guide resolution of mutant plexus into branchless, parallel aortae. Our observations explain how Sema3E(-) (/) (-) mice survive throughout development and into adulthood, despite severe initial vascular defects.

Figure 1. Dorsal aortae defects in Sema3E deficient embryos resolve over developmental time

Sema3E^+/− (A,B,E,F,I) and Sema3E^−/− (C,D,G,H,J) embryos (A–H) and adults (I,J) in whole mount views stained for PECAM (A,C) and sections (B,D,E–J) stained with hematoxylin-eosin (H&E) (E,G) or for indicated antigens (B,D,F,H-J). Note that initial blood vessels in Sema3E^−/− form aberrant plexus-like vascular structures (C,D), instead of smooth parallel vessels (A,B). Aortic vessels in Sema3E^−/− are located much closer to midline notochord compared to Sema3E^+/− (arrows). Dashed white lines indicate notochord in (A,C) and outline notochord and lateral plate mesoderm in (B,D). a, aorta; da, dorsal aortae; ev, extraembryonic vasculature; lpm, lateral plate mesoderm; nc, notochord; s, somite. Dorsal aorta diameter and morphology is indistinguishable between Sema3E^+/− and Sema3E^−/− 23S embryos (E–H) and adults (I,J). Note that ECs of normal appearance line the aortic lumen and layers of smooth muscle are of similar thickness in both genotypes. Scale bars: 100 μM (A,C), 12.5 μM (B,D) and 25 μM (lower right panels I and J)

Figure 2. Sema3E^−/− aortic plexus-like vessels remodel into unbranched dorsal aortae by 11S

(A) PECAM-DAB staining of Sema3E^+/− (left hand column) and Sema3E^−/− (right hand column) aortae. Note that Sema3E^+/− DA are unbranched and initially wide set, while Sema3E^−/− plexus vessels are initially highly branched, of smaller diameter, and closer to each other across the midline. These vessels rapidly converge between 7S and 11S to form two large unbranched vessels. Arrows highlight ectopic lateral vessels that connect aortic vessels to extraembryonic vessels. Black dashed lines mark the embryonic midline. Scale bar: 100 μM (A). (B–D) Quantification of parameters from (A), including average (ave.) number of total, or lateral branch points, as well as average vessel thickness. ev, extraembryonic vasculature; ns, not statistically significant; S, somite.

Figure 3. Resolution of dorsal aortae in Sema3E^−/− embryos is not due to changes in cell proliferation or death

Sections of Sema3E^+/− (A,D,G,G′) and Sema3E^−/− (B,E,H,H′) embryos assessing cell proliferation with PHH3 (A,B), cell death with staining for cleaved Caspase-3 (D,E) and total EC number with PECAM and Endomucin (G,H,). A,B,D,E: 9 somites (S) and G,G′,H,H′: 7S. Arrows mark positive ECs for indicated stainings and arrowheads mark ECs. Midline notochord and lateral plate mesoderm (lpm) are outlined by dashed white lines. (C,F,I) Quantification of PHH3 (C) and cleaved Caspase-3 (F) stained ECs, and the average (ave.) total number of ECs (I) reveals no significant changes in cell proliferation, programmed death and number. ns, not statistically significant. Scale bars: 25 μM (A,B,D,E) and 25 μM: 25 (G,G′,H,H′).

Figure 4. Sema3E null embryos form an interconnected aortic plexus, however select vessels carry blood flow

(A–C) PECAM-DAB staining of a 6 somite (6S) Sema3E^−/− embryo shown in anterior (A), ventral (B) and posterior views (C). White lines mark aortic vessels that connect to form a continuous circulatory loop. Black arrows indicate blunt ended vessels. (D–F) Anterior views of a 9S Sema3E^−/− embryo injected with Isolectin IB4 and stained for PECAM and Endomucin. White arrowheads indicate interconnected vessels devoid of Isolectin IB4. Black and white dashed lines demarcate the embryonic midline. ev, extraembryonic vasculature. Scale bars: 100 μM (A–C) and 100 μM (D–F).

Figure 5. Sema3E^−/− dorsal aortae precociously fuse at the embryonic midline

Representative images of Sema3E^+/− (A,C,E) and Sema3E^−/− (B,D,F) sectioned embryos stained for hematoxylin and eosin. Note early contact between the DA of Sema3E^−/− embryos at 14 somite (14S) and precocious fusion of the DA at 15S, compared to Sema3E^+/− embryos. Scale bar: 100 μM. (G) Graph of Sema3E^+/− and Sema3E^−/− DA categorized as separate, touching or fusing, as a total percentage of the embryos analyzed.

Figure 6. PlexinD1^−/− embryos display similar dorsal aortae defects to Sema3E^−/− embryos

Ventral views of PlexinD1^+/− (A,C) and PlexinD1^−/− (B,D) embryos stained for PECAM and Endomucin. Note the branched, disorganized and plexus-like appearance of PlexinD1 mutant DA. Dashed white lines indicate the midline. da, dorsal aortae; S, somite. Scale bar: 100 μM.

Figure 7. Schematic representation of morphogenetic consequences of Sema3E ablation on dorsal aortae formation

A) Sema3E from the midline notochord and lateral plate mesoderm, as well as a multiple signals from the notochord (including chordin, noggin, slit and netrin - blue), repel migrating angioblasts (precursor ECs) from 0S to 6S, constraining them to parallel locations where they coalesce and form two smooth aortic vessels that run along the anterioposterior axis. B) By 10S, the paired, parallel aortic tubes enlarge in diameter, remaining free of sprouting branches. C) In the absence of Sema3E, angioblasts migrate freely, except for a narrow domain near the notochord, and form a plexus on both sides of the embryo, that connects with extraembryonic vessels. D) Aortic vessels are rescued over time in Sema3E^−/− embryos. Repulsion from the lateral plate mesoderm occurs between 8S and 11S, suggesting onset of expression of an as yet unknown cue. Aberrantly branched aortic plexus structures resolve as branches fuse into large smooth DA by 10-11S. All panels show ventral views, anterior is up; posterior is down. Blood vessels, red; da, dorsal aortae; ev, extraembryonic vasculature; Sema3E protein, green; notochord (nc) cues, blue; lateral plate mesoderm (lpm) unknown cue, yellow. Dashed black brackets indicate the midline avascular space between aortic vessels.