ALK5- and TGFBR2-independent role of ALK1 in the pathogenesis of hereditary hemorrhagic telangiectasia type 2 -- Park et al. 111 (2): 633 Data Supplement - <font size="3">Supplemental material for: Park et al</font> -- Blood

Blood, Vol. 111, Issue 2, 633-642, January 15, 2008

ALK5- and TGFBR2-independent role of ALK1 in the pathogenesis of hereditary hemorrhagic telangiectasia type 2
Blood Park et al. 111: 633

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. Alk1^1loxP/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 Alk1^1loxP/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^2,4.
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.