Figure 3. Alk5 restricts EC proliferation in the cardiac OFT and is required for ventral aorta formation.

(A) Schematics of OFT ECs at 36 and 72 hpf. (B) Quantification of EC number (darker cells shown in A) in 36 and 72 hpf alk5+/+ and alk5-/- OFTs (36 hpf: n = 11; 72 hpf: n = 6). (C–H) Confocal images (C, D, F, G) and quantification (E, H) of the percentage of EdU⁺ ECs in Tg(kdrl:EGFP) alk5+/+ and alk5-/- OFTs. Dotted lines outline the OFT; white dots mark EdU⁺ ECs. All the other EdU⁺ cells in the OFT region are kdrl:EGFP^-. (I) Protocol used for photoconversion experiment. (J–K’) Confocal images of the OFT in 74 hpf Tg(fli1a:Kaede) larvae treated with DMSO or Alk5 inhibitor. Magenta, photoconverted ECs; dotted lines outline the OFT. (L) Quantification of the distance covered by photoconverted ECs between 54 and 72 hpf in DMSO and Alk5 inhibitor-treated larvae. (C, D, J–K’) Maximum intensity projections. (F, G) Single confocal planes. (B, E, H, L) Plot values represent means ± SD; p values from t-tests (E, H) or Mann Whitney (B, L). Scale bars: (C-G) 10 μm; (J–K’) 20 μm. See also Figure 3—figure supplement 1.

Figure 3—figure supplement 1. Phenocopy of alk5 mutants by Alk5 inhibitor treatment.

(A) Protocol used for Alk5 inhibitor treatments. (B–E) Confocal images of Tg(kdrl:EGFP) animals treated with DMSO or Alk5 inhibitor starting at 36 hpf and analyzed at 54 (B, n = 11; C, n = 11) or 78 (D, n = 13; E, n = 17) hpf. Asterisk, absence of the VA in Alk5 inhibitor-treated larvae. (F) Percentage of OFT expansion in 78 hpf control and Alk5 inhibitor-treated larvae; means ± SD; p value from t-test. For details about the quantifications, see Materials and methods ‘Defining the landmarks of the OFT’. (G) Schematics of the area photoconverted in H. (H, I) Confocal images of untreated photoconverted Tg(fli1a:Kaede) embryos at 54 (H, n = 5) and 74 (I, n = 5) hpf. (J) Schematics of the area photoconverted in K and L. (K, L) Confocal images of 54 hpf photoconverted Tg(fli1a:Kaede) DMSO (n = 9) or Alk5 inhibitor treated (n = 7) embryos. (H–L) Magenta, photoconverted ECs in the OFT (yellow arrowheads). (B–L) Dotted lines outline the OFT. (H, K, L) Single confocal planes. (B–E, I) Maximum intensity projections. AA1- 1° aortic arch, VA- ventral artery, BV- bulbo-ventricular canal. Scale bars: (B-E) 20 μm; (I–L) 10 μm; (H) 5 μm.

Figure 3—video 1. Time lapse-imaging of a Tg(fli1a:Kaede) fish treated with DMSO starting at 36 hpf and imaged from 56 to 74 hpf.

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Magenta, photoconverted cell.

Figure 3—video 2. Time lapse-imaging of a Tg(fli1a:Kaede) fish treated with Alk5 inhibitor starting at 36 hpf and imaged from 56 to 74 hpf.

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Magenta, photoconverted cell.