Arterial-venous segregation by selective cell sprouting: an alternative mode of blood vessel formation

Abstract

Blood vessels form de novo (vasculogenesis) or upon sprouting of capillaries from preexisting vessels (angiogenesis). With high-resolution imaging of zebrafish vascular development, we uncovered a third mode of blood vessel formation whereby the first embryonic artery and vein, two unconnected blood vessels, arise from a common precursor vessel. The first embryonic vein formed by selective sprouting of progenitor cells from the precursor vessel, followed by vessel segregation. These processes were regulated by the ligand EphrinB2 and its receptor EphB4, which are expressed in arterial-fated and venous-fated progenitors, respectively, and interact to orient the direction of progenitor migration. Thus, directional control of progenitor migration drives arterial-venous segregation and generation of separate parallel vessels from a single precursor vessel, a process essential for vascular development.

Fig. 1. The CV forms by selective angioblast sprouting

(A-E) Mid-trunk transverse sections (A, D), lateral views (B, E) or lateral time-lapse (C; movie S1) of Tg(kdrl:GFP)^s843 (A-C) or Tg(kdrl:GFP)^s843;Tg(gata1:dsRed)^sd2 (D, E) embryos. Angioblasts coalesce and remodel to form the DA by 22hpf (white/red brackets; A-C). Between 21-24hpf, venous angioblasts sprout ventrally from the DA (arrowheads and dotted lines; A-C) and contribute to the CV primordium (yellow/blue brackets; A-C). ISVs sprout dorsally from 23hpf (arrows; B). By 25hpf, venous angioblasts surround Tg(gata1:dsRed)^sd2-positive erythrocytes (asterisks, A, D; red cells, D, E). Erythrocyte displacement typically clears the CV lumen by 26hpf (A, D, E) but not in tnnt2 MO-injected embryos (E). Scale bars, 35 μm.

Fig. 2. Vegf signaling limits ventral sprouting

(A, B) Lateral views of Tg(kdrl:GFP)^s843 embryos (A) or transverse sections of Tg(kdrl:GFP)^s843;Tg(gata1:dsRed)^sd2 embryos (B) injected with control, vegfa, plc1 or hey2 MO, or exposed to DMSO, SU5416, DAPT or LY294002. vegfa or plcγ1 MO-injection blocks ISV sprouting (asterisks), promotes excessive ventral sprouting (arrowheads), leads to loss of the DA (white bracket) and generates a single venous tube (yellow bracket). SU5416, DAPT, or hey2 MO-injection, also promote loss of the DA, whereas LY294002 blocks CV morphogenesis. Scale bars in, 35 μm.

Fig. 3. Flt4/PI3K signaling mediates venous angioblast migration

(A-C) Tg(kdrl:GFP)^s843 embryos were exposed to DMSO, LY294002 or AS605240, processed for microscopy (23hpf; A) and then scored for (B) the number of ventral sprouts (as a percentage of DMSO treated embryos) or (C) the percentage of cells in the DA (white bracket; a in graph) or CV (yellow bracket; v in graph). PI3K-inhibition blocks ISV sprouting (arrows), venous sprouting (asterisks) and angioblast ventral migration (arrowheads). Lateral images (D) and quantification (E) of donor-derived Tg(kdrl:GFP)^s843 cells in wild-type hosts at 48hpf. Fewer cells from flt4 MO-injected donors contribute to the CV versus controls. Excessive ventral migration of angioblasts from plcγ1 MO-injected donors is reduced upon co-injection with flt4 MO. Scale bars, 35 μm. Error bars represent mean ± SEM. (*P < 0.05 versus control; **P < 0.05 versus plcγ1 MO; Student's t test).

Fig. 4. Ephb4a/Efnb2a-signaling determines the direction of angioblast migration

(A, B) Lateral projections of Tg(kdrl:GFP)^s843 embryos (A) or transverse sections of Tg(kdrl:GFP)^s843;Tg(gata1:dsRed)^sd2 embryos (B) injected with control, efnb2a, or ephb4a MO (A, B). Reduced Efnb2a expression promotes excessive ventral sprouting (A; arrowheads), loss of the DA (B; white bracket) and formation of a single vessel (A, B; yellow bracket) without affecting initiation of ISV sprouting (A; asterisks). Similarly, posterior arterial/venous segregation is disrupted in ephb4a MO-injected embryos. (C) Quantification of donor-derived Tg(kdrl:GFP)^s843 cells in wild-type hosts at 30hpf. More cells from efnb2a MO-injected or ΔC-efnb2a over-expressing donors contribute to the CV versus controls, whereas less cells from ephb4a MO-injected donors contribute to the CV. Over-expression of efnb2a or ephb4a enhances the number of donor cells contributing to ISVs. Scale bars, 35 μm. Error bars represent mean ± SEM. (*P < 0.05; Student's t test).