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. 2020 Sep 29;9:e57603. doi: 10.7554/eLife.57603

Figure 2. p-Smad3 is observed in OFT ECs at 24 hpf and reduced in alk5 mutants.

(A, A’) 24 hpf Tg(kdrl:EGFP) alk5+/+ hearts immunostained for p-Smad3 (white). Green, ECs; magenta, CMs. (B, C) Intensity ratio of p-Smad3 immunostaining (normalized to DAPI) between ECs and surrounding cells at 24 and 75 hpf (B) and between OFT ECs and heart ECs in 24 hpf alk5+/+ embryos (C). Every dot represents the ratio for one embryo. (D–E’) 24 hpf Tg(kdrl:EGFP) alk5+/+ (D) and alk5-/- (E) hearts immunostained for p-Smad3 (white). (F) Quantification of p-Smad3 immunostaining (normalized to DAPI) in ECs and surrounding cells comparing 24 hpf alk5+/+ and alk5-/- OFTs. (G–J) Confocal images of the OFT in 75 hpf Tg(kdrl:EGFP) alk5+/- (alk5a-/-;alk5b+/-; G, H) and alk5-/- (I, J) larvae treated with DMSO (G, I) or SIS3 (H, J) from 36 until 75 hpf. Asterisks point to the absence of the VA. (K) OFT maximum area in 75 hpf larvae treated with DMSO or SIS3. (A–J) Dotted lines outline the OFT ECs. (B, C, F, K) Plot values represent means ± SD; p values from Mann Whitney (B, K) and t-test (F). In K, p values refer to the comparisons highlighted. Scale bars: (A, A’, D–E’) 20 μm; (G–J) 15 μm. See also Figure 2—figure supplement 1 and Figure 2—source data 1 and 2.

Figure 2—source data 1. Quantification of p-Smads immunostaining in 24 and 75 hpf wild-type animals.
Related to Figure 2 and Figure 2—figure supplement 1.
Figure 2—source data 2. Quantification of p-Smads immunostaining in 24 hpf alk5+/+ and alk5-/- embryos.
Related to Figure 2 and Figure 2—figure supplement 1.

Figure 2.

Figure 2—figure supplement 1. Evidence for p-Smad3 presence and function in OFT ECs at 24 hpf, but no evidence for p-Smad1/5/8 presence.

Figure 2—figure supplement 1.

(A–B’) Confocal images showing Tg(12xSBE:EGFP) expression (white) in 24 (A) and 75 (B) hpf hearts. Green, ECs; white dotted lines outline the OFT. (C–D’) Confocal images of 75 hpf wild-type larvae immunostained for p-Smad3 (white, C) or showing Tg(12xSBE:EGFP) expression (white, D); white dotted lines outline the OFT; green dotted lines outline the ECs. (E) Schematics of a 36 hpf heart showing ECs inside (OFT ECs) and outside (heart ECs) the OFT quantified in Figure 2C. (F) Intensity ratio of p-Smad3 and p-Smad1/5/8 immunostaining (normalized to DAPI) between ECs and surrounding cells in the OFT at 24 hpf. Every dot represents the ratio of ECs vs surrounding cells in each embryo. (G–H’) 24 hpf Tg(kdrl:EGFP) alk5+/+ (G) and alk5-/- (H) hearts immunostained for p-Smad1/5/8 (white). Dotted lines outline OFT ECs. (I) Quantification of p-Smad1/5/8 immunostaining (normalized to DAPI) in ECs and surrounding cells comparing 24 hpf alk5+/+ and alk5-/- OFTs. (F, I) Plot values represent means ± SD; p values from t-tests. Scale bars: (A, A’, C–D’) 10 μm; (B, B’, G–H’) 20 μm.