Spaw and Vg1/Gdf3 form heterodimers. (A) Anti-GFP reducing immunoblot of wild-type embryos injected with 25 pg of spaw-sfGFP, vg1-sfGFP or vg1-sfGFP and spaw mRNA. Black arrowhead indicates the position of full-length protein; open arrowhead represents cleaved protein. Lower panel: anti-α-Tubulin loading control. (B) Animal cap of sphere-stage live embryos injected at the one-cell stage with 50 pg of spaw-sfGFP, 50 pg of vg1-sfGFP mRNA or 50 pg of vg1-sfGFP and 50 pg of spaw mRNA. Scale bar: 20 µm. (C) Animal cap of a sphere-stage live embryo injected with 50 pg of vg1-sfGFP mRNA at the one-cell stage (left) or 50 pg of vg1-sfGFP mRNA at the one-cell stage and 25 pg of a spaw mRNA/mCherry-CAAX mRNA mix at the four-cell stage (right). Scale bar: 40 µm. (D) Anti-Flag reducing immunoblot (IB) of anti-HA immunoprecipitates (IPs) from lysates of wild-type embryos injected with 50 pg of squint-HA (sqt-HA), 50 pg of spaw-HA and/or 50 pg of vg1-Flag mRNA. Black arrowheads indicate the position of full-length protein; open arrowheads represent cleaved protein. The input and IP blots were exposed for different lengths of time. (E) Expression of lefty1 in wild-type and maternal vg1 (Mvg1) mutant embryos injected with 0.25-50 pg of spaw mRNA. Scale bar: 150 µm.

Dand5 and Lefty1 regulate the timing and speed of spaw propagation. (A) spaw and myod1 expression in six-somite stage (6SS) to 14SS wild-type and mutant embryos, staged by myod1 somite expression. Black arrowheads represent the earliest stage spaw expression appears in the lateral plate mesoderm. The diagram indicates myod1 expression (dark purple) and spaw expression (light purple). N, notochord, S, somite, LPM, lateral plate mesoderm. Scale bar: 150 µm.(B) Scatterplot of distance spaw has traveled through the LPM in wild-type and mutant embryos. The spaw, dand5;spaw and lefty1;spaw graphs overlap at zero. n=160 (wild type),183 (dand5), 148 (lefty1), 149 (spaw), 115 (dand5;lefty1), 126 (dand5;spaw) and 90 (lefty1;spaw). These values represent the sum of three to six independent experiments. To calculate the rate of spaw propagation, a linear regression was fitted to the data after spaw had initiated in the LPM, see Fig. S2.

Uniform Nodal inhibition rescues lefty1 mutants. (A) Quantification of heart jogging in wild-type, lefty1 embryos and lefty1 embryos soaked in 1 µM of the Nodal inhibitor SB-505124. (B) Examples of left, bilateral and no spaw expression in wild-type, lefty1 and SB-505124-treated lefty1 mutant embryos. Scale bar: 150 µm.(C) Quantification of spaw expression in wild-type, lefty1 and SB-505124-treated lefty1 mutant embryos. (D) Quantification of spaw expression, lft2 expression and heart jogging in wild-type, lefty1, and 0.8 µM and 1 μM SB-505124-treated lefty1 embryos.