(A) Experimental design. Deletion of Dll4 in ECs was induced by injecting tamoxifen (Tx) at 0, 2, and 4 dpf. Fractured bone samples were evaluated on day 4, 7, and 14 by histology and day 21 by microCT and torsion testing. (B) Dll4 deletion led to a decreased callus size at 4 dpf (Cre^– n = 6, Cre⁺ n = 9), 7 dpf (Cre^– n = 9, Cre⁺ n = 8), and 14 dpf (Cre^– n = 8, Cre⁺ n = 10). (C) Dll4 deletion also resulted in decreased proliferation at 4 dpf (Cre^– n = 5, Cre⁺ n = 9), which increased by 7 dpf (Cre^– n = 9, Cre⁺ n = 8) in Cre⁺ compared with Cre^– animals. (D and F) Cre⁺ mice had significantly less cartilage. Sample numbers at 4 dpf (Cre^– n = 5, Cre⁺ n = 9), 7 dpf (Cre^– n = 9, Cre⁺ n = 8), and 14 dpf (Cre^– n = 8, Cre⁺ n = 10) were analyzed by evaluating Safranin O–stained sections, as observed on representative sections. (E and G) Mineralized area was analyzed by von Kossa staining (Cre^– n = 8, Cre⁺ n = 10), as shown on Cre^- and Cre⁺ representative sections. (H) MicroCT analysis showed decreased callus bone mass on day 14 and no difference in callus volume, with representative 3D reconstructions of Cre^– and Cre⁺ fractures on the right. At 7 dpf, Cre^– n = 8, Cre⁺ n = 6; and at 21 dpf Cre^– n = 7, Cre⁺ n = 9. (I) Biomechanical properties were evaluated by torsion testing and are presented as bone strength (maximum torque), stiffness as a measure of torsional rigidity, and toughness as a work to fracture measure, with no change between Cre^– and Cre⁺ fractured bones at 21 dpf. Cre^– n = 9, Cre⁺ n = 6. (J) Bglap gene expression analysis at 7 dpf (Cre^– n = 6, Cre⁺ n = 6). (K) Histological analysis of osteocalcin-stained samples at 14 dpf (Cre^– n = 8, Cre⁺ n = 10). (L) Evaluation of CD31⁺ cells within the periosteal callus at 7 dpf (Cre^– n = 9, Cre⁺ n = 12) and 14 dpf (Cre^– n = 7, Cre⁺ n = 9). Deletion of Dll4 in ECs with (M) representative magnified images of CD31 staining within the mineralized callus at 14 dpf. Scale bar: 200 μm. (N) Proportion of osterix-stained cells within the periosteal callus (Cre^– n = 5, Cre⁺ n = 6). (O) Representative images of osterix-stained fractured femurs with magnified areas of cartilaginous callus (a and b), mineralized callus (a’ and b’), and cortical bone with the area next to the pin insertion (a” and b”). The analyzed periosteal callus is shown by the yellow line. Scale bars: 1 mm (left) and 100 μm (right). PT, pin trace; CB, cortical bone; P, periosteum. Unpaired, 2-tailed Student’s t test. *P < 0.05, **P < 0.01.