Additional Supporting Information may be found in the online version of this article.

DVDY_22558_sm_suppinfofig1.tif3388KSupporting Information Figure 1. Developmental profile of pSmad1/5/8-mediated transcriptional activity in Tg(bre:egfp)pt509 embryos. In all images, green indicates bre-driven EGFP expression. a–d: Macro images of Tg(bre:egfp)pt509 (a, c) and Tg(bre:egfp)pt510 (b, d) at 1 dpf (a, b) and 2 dpf (c, d). e–o: 2D projections of confocal Z-series, Tg(bre:egfp)pt509. Magnification, 400×. e, e′: Pineal gland, 1 dpf. f, f′: Hypothalamus, 1 dpf. g, g′: Stomodeum, 2 dpf. h, h′: Trigeminal ganglia, 1 dpf. i, i′: Pharyngeal arches, 1 dpf. j, j′: Somites, 1 dpf. k, k′: Spinal cord neurons, 2 dpf. Brackets denote spinal cord. l, l′: Mesenchymal cells of the median finfold, 2 dpf. m, m′: Cloaca, 1 dpf. n, n′: Heart, 1dpf. o, o′: Pectoral fin, 2 dpf. a–d, h–l, n, o: Lateral view, anterior left. e, f: Frontal view, left to the right. g: Ventral view, anterior up. m: Ventral view, anterior left.
DVDY_22558_sm_suppinfofig2.tif2836KSupporting Information Figure 2. Temporal relationship between pSmad1/5/8 activity and EGFP fluorescence in Tg(bre:egfp) embryos. Embryos were collected every 2 hr between 10–18 hpf and 28–38 hpf and assayed for pSmad1/5/8 via immunofluorescence (a–k), 100×; egfp mRNA via in situ hybridization (a′–k′), 80×; and EGFP fluorescence (a′′–k′′), 80×. Results demonstrate a 4-hr delay between pSmad1/5/8 expression and EGFP fluorescence, and an 8–10-hr perdurance of EGFP after pSmad1/5/8 is lost. Green indicates bre-driven EGFP expression, 80× magnification. White arrows, dorsal retina; yellow arrows, trigeminal ganglia. All images lateral view, anterior left.
DVDY_22558_sm_suppinfofig3.tif6964KSupporting Information Figure 3. smad5 knockdown down-regulates bre:egfp transgene expression primarily in somites. Embryos were injected with 2 ng standard control morpholino (a, d, f), 2 ng smad5 morpholino (b, e, g), or 4 ng smad5 morpholino (c) at the one- to two-cell stage and imaged at 1 dpf. In all images, green indicates bre-driven EGFP expression. Brightfield (a, b, c) and fluorescent (d, e) images, lateral view, anterior left, 80× magnification. f, g: 2D projections of confocal Z-series showing somites, EGFP, and transmission overlays, 400× magnification. Lateral views, anterior left.
DVDY_22558_sm_suppinfofig4.tif7448KSupporting Information Figure 4. smad1 knockdown downregulates expression of the bre:egfp transgene expression in additional domains. Embryos were injected with 2 ng standard control morpholino (a, c, e) or 2 ng smad1 morpholino (b, d, f) at the one- to two-cell stage. Images are 2D projections of confocal Z-series showing EGFP and transmission overlays, 400× magnification; focused on cloaca,1 dpf (a, b), somites, 2 dpf (c,d), and median finfold, 2 dpf (e, f). a, b: Ventral view, anterior left. c–f: Lateral view, anterior left.

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