Alk1 controls arterial endothelial cell migration in lumenized vessels

Abstract

Heterozygous loss of the arterial-specific TGFβ type I receptor, activin receptor-like kinase 1 (ALK1; ACVRL1), causes hereditary hemorrhagic telangiectasia (HHT). HHT is characterized by development of fragile, direct connections between arteries and veins, or arteriovenous malformations (AVMs). However, how decreased ALK1 signaling leads to AVMs is unknown. To understand the cellular mis-steps that cause AVMs, we assessed endothelial cell behavior in alk1-deficient zebrafish embryos, which develop cranial AVMs. Our data demonstrate that alk1 loss has no effect on arterial endothelial cell proliferation but alters arterial endothelial cell migration within lumenized vessels. In wild-type embryos, alk1-positive cranial arterial endothelial cells generally migrate towards the heart, against the direction of blood flow, with some cells incorporating into endocardium. In alk1-deficient embryos, migration against flow is dampened and migration in the direction of flow is enhanced. Altered migration results in decreased endothelial cell number in arterial segments proximal to the heart and increased endothelial cell number in arterial segments distal to the heart. We speculate that the consequent increase in distal arterial caliber and hemodynamic load precipitates the flow-dependent development of downstream AVMs.

Keywords: Alk1/Acvrl1; Angiogenesis; Arteriovenous malformation; Endocardium; Hereditary hemorrhagic telangiectasia; Zebrafish.

Fig. 1.

Schematic of 36 hpf zebrafish cranial vasculature. (A) Frontal view; (B) lateral view. Blood flows from the heart (dark red) through an ordered series of arteries including the alk1-positive first aortic arch (AA1; green), internal carotid artery (ICA; yellow) and caudal division of the ICA (CaDI; red), and the alk1-negative basal communicating artery (BCA; blue); posterior communicating segments (PCS; light gray), basilar artery (BA; light gray) and metencephalic artery (MtA; pink). Veins (dark gray) are alk1 negative and include the primordial midbrain channel (PMBC), primordial hindbrain channel (PHBC) and midcerebral vein (MCeV). The BCA drains to the PMBC through transient connections (red arrowheads); these connections are maintained in alk1 mutants.

Fig. 2.

Arterial endothelial cell numbers are altered in alk1-deficient embryos in a segment-specific manner. (A,C) 2D maximum projections of selected time points from confocal time-lapse imaging of Tg(fli1a.ep:mRFP-CAAX)^pt505 control and alk1-morphant embryos, 24-36 hpf, showing proximal cranial vessels/ventral planes (A) and distal cranial vessels/dorsal planes (C) of a single embryo. Dorsofrontal views, anterior down. Shaded regions correspond to vessel segments analyzed for cell number in B,D: green, AA1; yellow, proximal ICA; red, CaDI. Arrow in A points to transient AA1 stenosis. Scale bars: 50 µm. (B,D) Endothelial cell counts in AA1 and ICA (B) and CaDI (D) in precisely staged alk1^−/− embryos and wild-type (wt) siblings. Data are mean±s.e.m., n≥4 embryos for each data point. Data were analyzed by two-way ANOVA followed by Bonferroni's multiple comparisons test. Wild type and alk1^−/− comparisons: significance indicated above time point. Within-treatment temporal comparisons: significance indicated to right of graph. ns, not significant, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

Fig. 3.

alk1 deficiency does not affect endothelial cell proliferation. (A) Cumulative number of CaDI endothelial cell divisions in time-lapse two-photon movies of control (n=4) and alk1 morphant (n=6) Tg(fli1a:nEGFP)^y7 embryos, 24-36 hpf. All alk1 morphants analyzed developed a fully expressive alk1 mutant phenotype. Values represent mean±s.e.m. Two-tailed Student's t-test, not significant. (B) Number of mAG-positive (proliferating) endothelial cells in alk1-positive cranial arteries (CaDI; blue), alk1-negative cranial arteries (PCS, MtA; black) and cranial veins (optic vein, MCeV, PMBC; white) at 28, 30 and 32 hpf in control (solid bars) and alk1-morphant (stippled bars) embryos. Data represent mean±s.e.m., n=3-13 independent samples. Two-way ANOVA followed by Bonferroni's multiple comparisons test; control versus alk1 morphant, not significant.

Fig. 4.

Arterial endothelial cell displacement towards the heart requires alk1 and blood flow. (A) Representative two-dimensional confocal projections of control and alk1-morphant Tg(fli1a:GAL4FF)^ubs4;Tg(UAS:kaede)^rk8 embryos at 24 hpf (pre- and post-photoconversion of AA1 with 405 nm laser) and 48 hpf. Green, native Kaede; magenta, photoconverted Kaede. Arrowheads denote segment originally photoconverted. 24 hpf: merge; dorsal view, anterior down. 48 hpf: 488 nm laser (native Kaede; green), 516 nm laser (photoconverted Kaede; magenta), and merge; frontal view, dorsal up. Trio of images at right shows a single plane of a wild-type ventricle at 48 hpf, demonstrating the contribution of AA1-derived cells to the endocardium. Scale bars: 50 µm. (B) Schematic of 48 hpf heart and cranial vasculature showing color-coded segmental boundaries used for analysis of location of photoconverted cells. (C-K) Percentage of 48 hpf control (C-E), alk1-morphant (F-H) and tnnt2a-morphant (I-K) embryos exhibiting photoconverted cells in specified region of heart and cranial vessels; data are laid out proximal (left) to distal (right) with respect to the heart. Cells were photoconverted at 24 hpf in proximal AA1 (C,F,I), proximal ICA (D,G,J) or at the CaDI/ICA junction (E,H,K). Gray shading denotes site of photoconversion. n (number of embryos) noted in each panel, upper right.

Fig. 5.

Endothelial cell migration against blood flow is impaired in AA1 in alk1-deficient embryos. (A) 2D maximum projections of selected time points from time-lapse two-photon imaging of proximal cranial vessels in control and alk1-morphant Tg(fli1a:nEGFP)^y7 embryos, ∼24-34 hpf. Vessels of interest are outlined to improve clarity. AA1 nuclei are color coded according to their behavior over the course of the movie. Green, cells remain in proximal AA1; pink, cells move from AA1 into the OFT/heart; yellow, cells enter proximal AA1 from more distal segments. Scale bar: 50 μm. (B) Quantification of endothelial cells grouped by initial (Time_i, 24 hpf) and final (Time_f, 34 hpf) positions. n=3 controls, 4 alk1 morphants. Below the graph, names of alk1-positive vessels are shaded gray, and direction of cell migration with respect to blood flow is depicted by black arrows. (C) 2D maximum projections of selected time points from time-lapse confocal/two-photon imaging of the right side of AA1 in Tg(fli1a:nEGFP)^y7;Tg(fli1a.ep:mRFP-CAAX)^pt505 control and alk1-morphant embryos. See also Movies 1,2. Endothelial cell membranes, magenta; endothelial cell nuclei, gray. White arrowheads track a single cell nucleus over time in each embryo. Dorsal views, anterior down. Scale bar: 20 µm. (D-F) From these movies, we quantified summed migration track length (D), net nuclear displacement (E) and tortuosity ratio (F). Each data point represents mean values for a single embryo, 6-10 cells per embryo. Only cells that could be tracked for a minimum of four consecutive frames were included in the analysis. Graphs represent mean±s.e.m. for n=3 controls and 3 alk1 morphants. Two-tailed Student's t-test, *P<0.05, **P<0.01.

Fig. 6.

Endothelial cell migration in the direction of blood flow is enhanced in the CaDI in alk1-deficient embryos. (A) 2D maximum projections of selected time points from two-photon time-lapse imaging of distal cranial blood vessels in control and alk1-morphant Tg(fli1a:nEGFP)^y7 embryos, ∼24-36 hpf. See also Movies 3,4. Vessels of interest are outlined to improve clarity. Selected nuclei are color coded according to their position at the beginning of the time series. Red, cells originate in CaDI; blue, cells originate from PMBC; pink, cells originate from MtA; yellow, cells originate from ICA. Dorsal views, anterior down. Scale bar: 50 µm. (B) Quantification of endothelial cells grouped by initial (Time_i, 24 hpf) and final (Time_f, 36 hpf) positions. n=4 control, 6 alk1 morphants. Below the graph, names of alk1-positive vessels are shaded gray, and direction of cell migration with respect to blood flow is depicted by black arrows. Graphs represent mean±s.e.m. Two-tailed Student's t-test, *P<0.05.