Figure 2. Capillary-venous loss of ALK1 leads to AVMs.

(A) Schematic representation of the experimental strategy used to delete Alk1 in mice (P4-P6). (B-D) P6 retina flat mount images labeled with IB4 (blue) and GFP (white) from Alk1^f/f Mfsd2a Cre^ERT2 mTmG (B), Alk1^f/f Esm1 Cre^ERT2 mTmG (C) and Alk1^f/f Bmx Cre^ERT2 mTmG pups (D) injected with 100 μg Tx at P4 and dissected at P6. White arrows indicate AVMs. (E) Quantification of AVM number. n = 6–11 mice per group. Error bars: SEM. **** P-value < 0.0001, ns: nonsignificant, One-way ANOVA with Sidak’s multiple comparisons test. (F-I) Vascular labeling with latex dye (red) of retinal and brain vessels in Alk1^f/f (F and G) and Alk1^f/f Mfsd2a Cre^ERT2 (H and I) P6 pups. White arrows indicate AVMs. (J and K) GFP (white) and VE-Cad (blue) staining of mesentery and gastrointestinal (GI) tract (J) and lacteals (K) from P14 Esm1 Cre^ERT2 mTmG. 100 μg Tx was injected at P4. An arrow indicates Esm1 positive capillary ECs (J). (L and M) VE-Cad (blue) and GFP (white) staining of jejunum lacteals from P14 Alk1^f/f (L) and Alk1^f/f Esm1 Cre^ERT2 mTmG (M). (N-Q and T-W) 100 μg Tx was injected at P4 and dissected at P12. Vascular labeling with latex dye (red) of villi, GI tracts, retinas and brains in Alk1^f/f (N,P,T and V) and Alk1^f/f Esm1 Cre^ERT2 (O, Q, U and W) P12 pups. (R and S) 100 μg Tx was injected at P4 and dissected at P12 (R and S). IB4 (blue) and GFP (white) staining of retinal flat mounts from Esm1 Cre^ERT2 mTmG (R) and Alk1^f/f Esm1 Cre^ERT2 mTmG (S) P12 mice. An arrow indicates vascular malformations (Q, U and W). A: artery, V: vein, Scale bars: 500 μm (B-D), 200 μm (F and H), 2 mm (G and I). 400 μm (J), 25 μm (K-M), 200 μm (N and O), 1 mm (P-G and V-W), 500 μm (R-U), 200 μm.