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Fig. 5
A Burst of TGF-β Signaling Precedes tie1+ PAA Angioblast Differentiation
(A) Cartoon of zebrafish embryo highlighting the left-side pharyngeal arches 3–6 (boxed) analyzed at higher magnifications in (B)–(N). The spatial relationships between PAA progenitors (magenta), neural-crest-derived cells (green), and endodermal pouches (gray) are shown.
(B and C) Left-side dorsal views of 32 hr post-fertilization (hpf) Tg(sox10:GFP), Tg(nkx2.5:ZsYellow), or Tg(sox17:GFP),Tg(nkx2.5:ZsYellow) embryos immunostained for GFP and ZsYellow and imaged in the green and far red channels to visualize neural crest cells (pseudocolored green), endodermal pouches (pseudocolored gray), and PAA progenitor clusters (pseudocolored magenta). n = 5 for both groups.
(D–I) Spatiotemporal dynamics of TGF-β signaling during PAA angioblast differentiation. Dorsal views of Tg(nkx2.5:GFP) embryos at the indicated developmental stages immunostained for GFP and pSMAD3 imaged in the green and far red channels to visualize PAA progenitors (pseudocolored magenta) and cells experiencing active TGF-β signaling (pseudocolored yellow). 28 hpf, n = 3; 32 hpf, n = 4; 36 hpf, n = 6.
(J) Left-side dorsal views of Tg(sox17:GFP) embryos at 36 hpf immunostained for GFP and pSMAD3 imaged in the green and far red channels to visualize endodermal pouches (pseudocolored gray) and cells active for TGF-β signaling (pseudocolored yellow), n = 2.
(K–N) Left-side dorsal views of Tg(nkx2.5:GFP) embryos at 32 hpf immunostained for GFP (pseudocolored magenta) and pSMAD3 (pseudocolored yellow) following treatment from 20 hpf with DMSO (K and L; n = 3) or 50 μM LY-364947 (M and N; n = 8). Scale bars, 25 μm.