Fig. 1

Formin Activity Is Required for Vessel Lumen Formation and Maintenance

(A–D) Tg(fli1ep:Lifeact-EGFP);Tg(kdr-l:ras-Cherry)^s916 embryos were injected with control or fmnl3 morpholino or treated with DMSO or 5 µM SMIFH2 from 31 to 46 hpf and examined at 50 hpf. The asterisk shows lumenized ISV, and arrowheads show junctional F-actin cables. Scale bars represent 10µm.

(E–G) Mosaic expression of EGFP or fmnl3ΔC-EGFP in ISVs of Tg(kdr-l:ras-Cherry)^s916 embryos at 52 hpf. ISVs with EGFP or fmnl3ΔC-EGFP expression were phenotyped for lumen defects (G). EGFP, n = 41 ISVs, n = 16 embryos; fmnl3ΔC-EGFP, n = 52 ISVs, n = 27 embryos. Scale bars represent 10 µm.

(H–J) Tg(kdr-l:ras-Cherry)^s916 embryos were injected with quantum dots (Qdot) at 54–57 hpf. The arrow shows discontinuous perfusion. Control MO, n = 134 ISVs, n = 28 embryos; fmnl3 MO, n = 186 ISVs, n = 36 embryos. Scale bars represent 50 µm.

(K) Quantification of blood flow through ISVs. Control MO, n = 79 embryos; Fmnl3 MO, n = 87 embryos; Fmnl3 MO + 100pg fmnl3 mRNA, n = 64 embryos. Data represent mean ± SD.

(L–O) Uninjected or Qdot-injected Tg(fli1ep:Lifeact-EGFP);Tg(kdr-l:ras-Cherry)^s916 embryos were treated with DMSO or 10 µM SMIFH2 at 49–50 hpf and imaged 4–5 hr later. (L and M) Arrowheads show junctional F-actin cables. The arrow shows apical membrane, and the asterisk shows lumen. (O) Arrows show fragments of Qdot-filled vessels, and the arrowhead shows vessel disconnection. Scale bars represent 20 µm.

(P–S) Mosaic endothelial EGFP or fmnl3ΔC-EGFP expression (serrated lines) was induced in Tg(kdr-l:ras-Cherry)^s916 embryos at 2 dpf and examined at 71–77 hpf for lumen defects. Arrows show unlumenized vessel. EGFP, n = 42 ISVs, n = 16 embryos; fmnl3ΔC-EGFP, n = 57 ISVs, n = 33 embryos. Scale bars represent 20 µm.

See also Figure S1.