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ALK5- and TGFBR2-independent role of ALK1 in the pathogenesis of hereditary hemorrhagic telangiectasia type 2

Sung O. Park, Young Jae Lee, Tsugio Seki, Kwon-Ho Hong, Naime Fliess, Zhigang Jiang, Alice Park, Xiaofang Wu, Vesa Kaartinen, Beth L. Roman and S. Paul Oh

Data supplements

  • Supplemental material for: Park et al

    Files in this Data Supplement:

    • Document 1. Supplemental methods (PDF, 74 KB)
    • Document 2. Supplemental references (PDF, 12 KB)
    • Figure S1. L1cre embryos express the Cre recombinase in the developing blood vessels of the yolk sac (PDF, 140 KB) -
      L1cre(-);R26R(+) (A, C, E, G) and L1cre(+);R26R(+) (B, D, F, H) yolk sacs at E10.5 (A, B), E12.5 (C, D), E14.5 (E, F), and E16.5 (G, H) stages were stained with X-gal, and mounted in a slide with a cover glass for photographs. X-gal positive cells are restricted to the blood vessels (in the endothelial cells—not shown). X-gal staining was found homogeneously throughout the yolk sac, but it is clear that not every endothelial cell was X-gal positive.

    • Figure S2. Alk11loxP/1loxP embryos are morphologically indistinguishable from the previously characterized Alk1-/- embryos (PDF, 531 KB) -
      A, B, Dissection microscope views of E9.5 WT (A) and Alk11loxP/1loxP (B) embryos. The yolk sac blood vessels are visible in WT, but not in the mutants. C, Dissection microscope views of a WT and three mutant E9.5 embryos taken out of the yolk sac. The mutant embryos exhibit growth retardation. Often accumulation of blood was found in the head (or tail) region of the mutant (arrowheads), similar to the previously characterized Alk1-/- embryos
    • Figure S3. SB-431542 effectively inhibits Alk5 activity in zebrafish embryos (PDF, 138 KB) -
      Injection of 1 pg constitutively active alk5a or alk5b into 1 to 4-cell embryos induces gsc expression at 6 hpf (A-C), and this induction (as well as endogenous gsc expression) can be inhibited by incubation with 100 µM SB-431542 from the 8-cell stage (D-F). Exposure to 100 µM SB-431542 beginning at the 8 to 10-somite stage inhibits left-sided expression of pitx2c in the gut (white arrow, G and I) and diencephalon (black arrowhead, H and J) at 24 hpf, demonstrating the ability of this drug to penetrate the embryo at later times. To further verify efficacy of 8 to 10-somite exposure, one-cell stage wild type embryos were injected with 85 pg pgl2-basic (3TP-lux backbone), BRElux, or 3TP-lux, incubated in 1% DMSO or 100 µM SB-431542 beginning at the 8 to 10-somite stage, and assayed for luciferase activity at 32 hpf (K); drug treatment significantly decreased 3TP-lux activity. A-J, in situ hybridization: A and D, animal pole at top; B-C and E-F, random orientation to best show staining.; G-J, dorsal views, anterior to the left. In K, each bar represents mean + standard error for three independent biological replicates (20 embryos per sample), each of which was assayed in triplicate. Results are representative of two independent experiments. Significance was determined by Student�s t-test: for BRE-lux activity, p = 0.0003; for 3TP-lux activity, p = 0.0000002.

Article Figures & Data

  • Figure 1

    Multiple versions of TGF-β signaling pathways in endothelial and smooth muscle cells. During the activation phase of angiogenesis, endothelial cells degrade their vascular basement membranes, migrate into extracellular spaces, proliferate, and form vascular lumens. During the resolution phase, endothelial cells cease to migrate and to proliferate and instead reconstitute their basement membranes. The maturation and remodeling of the vessels also occur in this phase, as mesenchymal cells are recruited for endothelial tube ensheathment. (A,B) It has been reported that both ALK1 and ALK5 are TGF-β subfamily type I receptors in ECs: that is, they are both activated by TGF-β subfamily ligands binding to TGFBR2. As ALK1 and ALK5 signal through different SMAD proteins, it has been suggested that the opposing activities of these 2 type I receptors regulate angiogenesis. However, whereas some studies have suggested a role or ALK1 in resolution and ALK5 in activation,14,17 others have suggested opposite roles, with ALK5 being necessary for ALK1 function.16,20 (C) Both balance models (A,B) are called into question by an expression study in mice showing that, whereas Alk1 is endothelial-specific, Alk5 is expressed not in the endothelium but in neighboring smooth muscle cells.21 (D) Data presented do not support a role for TGF-β subfamily ligands and TGFBR2 in ALK1 function, suggesting that TGF-β superfamily ligands outside of the TGF-β subfamily may be physiologic ligands for ALK1 in endothelial cells. This hypothesis is supported by recent biochemical data demonstrating that BMP9 serves as an ALK1 ligand.12,13

  • Figure 2

    Tg(Alk1-cre)-L1 mice express the Cre recombinase predominantly in the pulmonary vascular endothelial cells. The cells in which Cre–mediated recombination has occurred were visualized by staining the Tg(Alk1-cre);R26R bigenic embryos with X-gal for the β-gal activity at E10.5 (A-D), E13.5 (E-H), and E15.5 (I-K) stages. (A,B) X-gal-positive staining is visible in the blood vessels throughout embryos in the Tg(Alk1-cre)-B (A) and -D (B) lines. (C) Transverse sections of X-gal stained Tg(Alk1-cre)-D embryo showing lacZ expressions in the vascular ECs, endocardial cells in atria and ventricles, and mesenchymal cells in the atrioventricular cushion (AVC). (D) In contrast with that in the B and D lines, almost no lacZ expression was detected in the L1cre embryos; only a spotty staining pattern in the head region (arrow). (E,F) Dorsal aorta (DA) view of the heart and lungs of L1cre:R26R embryos stained with X-gal, showing a strong lacZ expression in the lung in comparison with a patch staining in the heart (E) and body trunk (F). (G,H) Histologic sections of the X-gal stained lung was counterstained with NFR (G) or costained with anti-PECAM antibodies (H). The inset in panel H is a magnified view of the area indicated by the arrow. Note that X-gal-positive cells resided in pulmonary ECs, but only in a subpopulation of PECAM-positive cells. (I) Ventral view of the X-gal stained L1cre:R26R lung attached to the body trunk. The heart was removed for clarity of the view. Note a strong X-gal staining in the lung but not in the body trunk. (J,K) Histologic sections demonstrate that most PECAM-positive cells are positive for X-gal staining in E15.5 embryonic lungs, yet no X-gal-positive cells were detected in airway epithelial and smooth muscle cells. Insets are magnified views of the areas indicated by the arrow in each panel.

  • Figure 3

    Generation of Alk1-conditional alleles. (A) Schematic diagram of the Alk1 wild-type allele, Alk1-conditional targeting vector, and Alk13loxP, Alk12loxP, and Alk11loxP alleles. Exons and loxP sequences are indicated by boxes and arrowheads, respectively. Locations of primer pairs used for amplifying specific regions containing a loxP sequence are also indicated. (B) Genomic Southern blot analysis from EcoRI digested DNA isolated from several ES clones, showing the homologous recombination of the Alk13loxP vector into the Alk1 locus. (C) Representative PCR genotyping results from intercrosses of Alk1+/3loxP (top) and Alk1+/2loxP (bottom). The arrowheads indicate the PCR amplicon containing the loxP sequence; Alk13loxP/3loxP (lanes 2 and 5); Alk1+/3loxP (lanes 1, 4); Alk1+/+ (lane 3); Alk1+/2loxP (lanes 6 and 8); and Alk12loxP/2loxP (lanes 7, 9, and 10). (D) PCR detection of the Alk11loxP allele from genomic DNA isolated from multiple organs/tissues of E16.5 L1cre(+)Alk13loxP/3loxP (top), L1cre(+)Alk1+/3loxP (middle), and L1cre(−);Alk13loxP/3loxP (bottom) fetuses, demonstrating tissue-specific Cre activities. The arrowheads indicate the Alk11loxP-specific PCR amplicon.

  • Figure 4

    Alk1 deletion resulted in abnormal extraembryonic vasculature in E16.5 L1cre(+);Alk13loxP/3loxP fetuses. (A,B) Gross morphology of control and L1cre(+);Alk13loxP/3loxP mutant fetuses enclosed in the yolk sac attached to the placenta (PL). Note bulged arteries (A) and veins (V) in the mutant yolk sac. The inset in panel B shows magnified view of typical dilated, tortuous vitelline vessels in the mutants. (C,D) Umbilical arteries (UA) and veins (UV) are connected to the placenta, whereas vitelline arteries (VA) and veins (VV) are connected to the yolk sac. Note markedly enlarged VA and AVMs (circled; see enlarged view in Figure 5D) in the mutants. (E,F) Cross-sectional view of the extraembryonic vessels indicated by the scissors symbols in panels C,D demonstrates marked dilation and thinning of mutant VA (F), which has a similar morphology as control VV (E).

  • Figure 5

    Alk1 deletion results in multiple AVM formations. Dissection microscopic views of representative arteries (A) and veins (V) in control (A,C,E,G) and L1cre(+);Alk13loxP/3loxP (B,D,F,H) E16.5 embryonic yolk sacs. In the control yolk sac (A,C), arteries and veins are intercalating and not connected directly to one another. In the mutants (B,D), however, there are numerous regions where dilated and tortuous arteries and veins are directly connected without the connecting capillaries. (E-H) India ink was injected into the vitelline artery to visualize the yolk sac vessels. In control (E,G), arteries and veins were easily distinguishable and were connected by capillaries. In mutants (F,H), numerous AVMs (indicated by arrows) were formed between arteries and veins.

  • Figure 6

    Alk1 deletion resulted in abnormal pulmonary vasculature in E17.5 L1cre(+);Alk13loxP/3loxP fetuses. Transverse sections of the left lobe of the control (A,C,D) and mutant (B,E,F) lungs. Blood vessels are readily identifiable by the red blood cells in them. Dissection microscopic views of the left lung are shown as insets. The control lung displayed organized vascular trees (A, inset), whereas the mutant lung exhibited dilated, tortuous, and irregular blood vessels (B, inset, arrows). In the control lungs (A,C), bronchial trees and blood vessels are coordinated, and blood vessels are well defined as a circular shape. Br indicates bronchus; PA, pulmonary artery; and H&E, hematoxylin and eosin. In the mutant lungs (B,E), the bronchial lumens are not expanded as much as control lungs, and blood vessels are noticeably enlarged and irregular, presumably resulting from fusions between neighboring vessels (arrowheads in E). (D,F) Immunostaining with anti-αSMA antibodies revealed thinning of blood vessel walls with irregular thickness of smooth muscle layers (arrowheads in F).

  • Figure 7

    The Alk5 and Tgfbr2 are deleted in the lungs of L1cre(+);Alk5loxP/loxP and L1cre(+);Tgfbr2loxP/loxP mice, respectively, yet no noticeable pathologic signs were observed in the lungs of 2-month-old mutants. (A) PCR genotyping with primers β and γ (top) and Cre (bottom), showing L1cre(+);Alk5loxP/loxP (lane 1) and L1cre(+);Alk5+/+ (lane 2) mice. WT, wild-type allele (B) PCR amplification of the Alk5 null allele is specific for the lung. Genomic DNA isolated from the lung (top), liver (middle), and tail (bottom) were used as template to amplify the null allele by primers α and γ. Another primer set detecting a diploid genome (ie, Alk1 = control) was also included in the PCR reaction to demonstrate equal loading of the template. (C) PCR genotyping with primers x and y (top) and cre (bottom), showing L1cre(+);Tgfbr2loxP/loxP (lane 3), L1cre(−);Tgfbr2loxP/loxP (land 4), and L1cre(+);Tgfbr2+/+ (lane 5) mice. (D) PCR amplification of the Tgfbr2 null allele is specific for the lung. Genomic DNA isolated from the lung (top), liver (middle), and tail (bottom) was used as template to amplify the null allele by primers x and z. Another primer set detecting a diploid genome (ie, Alk1) was also included in the PCR reaction to demonstrate equal loading of the template. (E-J) Histologic sections of the lungs of 2-month-old control (ie, L1cre(+);Alk5+/+;R26R; E,H), L1cre(+);Alk5loxP/loxP;R26R (F,I), and L1cre(+);Tgfbr2loxP/loxP;R26R (G,J) mice. (E-G) Histologic sections of the X-gal stained lungs were counterstained with NFR, demonstrating that the Cre–mediated recombination has occurred in these lungs as expected. Insets are high magnification views showing that lacZ expression is restricted to pulmonary ECs, not in bronchial epithelial or smooth muscle cells. (H-J) Anti-αSMA antibody staining of the control and mutant lungs showing no specific pathologic signs. Insets show that similar thickness of the VSMC layers of distal arteries.

  • Figure 8

    Zebrafish alk5a and alk5b are not expressed in the endothelium and activities are not necessary for vessel development. alk5a is expressed in the eye, brain, pharyngeal arches, and endoderm at 24 and 48 hpf (A-D). alk5b is also expressed in these tissues, as well as in the spinal cord and ventral somites (E-H). Neither seems to be expressed in blood vessels (compare A-H with I-L, vecad expression). Exposure of phenotypically wild-type zebrafish embryos to 100 μM SB-431542 beginning at the 8- to 10-somite stage had no effect on trunk (M,N) or cranial (O,P) vascular anatomy at 24 or 48 hpf, respectively. This same exposure regimen did not exacerbate the cranial vascular phenotype in alk1−/− embryos (Q,R). Comparing panels Q and R with O and P, note enlargement of basal communicating artery (asterisk), posterior connecting segments (arrows), and primordial hindbrain channel (arrowhead). (A-L) In situ hybridization, lateral views, anterior to the left. First and third rows, head; second and fourth rows, trunk and tail. M-R, 2-dimensional reconstructions of laser scanning confocal Z-series of TG(flk1:GFP)la116 embryos. (M,N) Lateral views of the trunk, anterior to the left. (O-R) Dorsal views of the head, anterior to the left.

  • Table 1

    L1cre (+); Alk13loxp/3loxP fetuses resulted in embryonic lethality at late gestational stages

    Cross*StageProgeny, total no.Mutants [L1cre(+);Alk13loxP/3loxP], no.Embryos exhibiting phenotype* among the mutants, no.Embryos found dead among the mutants, no.
    L1cre(+); Alk1+/3loxPE14.513440
    × Alk13loxP/3loxP orE15.52410100
    Alk1+/3loxPE16.59117143
    E17.578936
    E18.522808
    • * Phenotype in L1cre(+);Alk13loxP/3loxP refers to the bulged yolk sac blood vessels similar to those in Figure 4B.

    • The mutant yolk sac vessels looked enlarged, but not as distinctive as the ones in E15.5 and later stages.

Supplementary Materials

  • Figure S1

    Supplementary PDF file available online.

  • Figure S2

    Supplementary PDF file available online.

  • Figure S3

    Supplementary PDF file available online.

  • Document S1

    Supplementary PDF file available online.