Figure 1

Secondary Axis Inducing Nodal and BMP Double Clones Produce a Localized Region of pSmad2 Activity Overlapping with a Broad Domain of pSmad5 Activity

(A) Nodal and BMP form orthogonal overlapping gradients in zebrafish embryos. Transplanting ectopic sources of Nodal and BMP induces the formation of a secondary axis, which contains both anterior and posterior structures such as the hindbrain, otic vesicles, notochord, and tail.

(B) Double clones of Bmp2b/7-sfGFP and Squint-mVenus imaged 30 min and 180 min post-transplantation. The first row depicts confocal microscopy images of Bmp2b/7-sfGFP (red) and Squint-mVenus (green). The second row shows light-sheet microscopy images of embryos immunostained with anti-pSmad2 (green) or anti-pSmad5 (red) antibodies as well as a cross-reactive anti-GFP antibody to detect Bmp2b/7-sfGFP and Squint-mVenus (blue). The third row shows comparable wild-type embryos. Nodal clones are traced in cyan and BMP clones are traced in white. Scale bar, 150 μm.

(C) Higher magnification of images shown in (B) with separate fluorescent channels. Scale bar, 150 μm.

(D) Images showing Nodal/BMP double clones with different spacings of transplanted cells taken immediately after transplantation. Scale bar, 150 μm.

(E) Nodal/BMP double clones were transplanted with different spacings into blastula-stage zebrafish embryos: narrow (~0 μm between clones, n = 60), moderate (40–50 μm between clones, n = 44), wide (120–150 μm between clones, n = 29), and very wide (>170 μm between clones, n = 20). Narrow to wide spacings support the formation of secondary axes, whereas secondary axis formation fails with extremely wide spacing between Nodal and BMP clones. Quantification was performed at 24 h post-transplantation.