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Fig. S1. Notch-expressing cells do not contribute to notochord.
NICD-myc mRNA was injected with (B, C) or without (E, F) β-galactosidase mRNA as a lineage tracer into both dorsal blastomeres to target the midline. Embryos were fixed, bisected transversely, stained for lacZ (pink, A–C), and processed by ISH for AxPC expression in the notochord (purple, A–F). This injection technique efficiently targets the midline (A), with labeled cells in FP, notochord, dorsal endoderm and sometimes medial somite. Embryos with activated Notch have reduced (B, E) or absent (C, F) notochord. Co-injected lineage tracer never labels any notochord that forms (B). Staining for NICD protein (αnti-myc, brown in E, F) shows that NICD+ cells never give rise to the notochord. |
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Fig.3. Shh is not a major regulator of dorsal midline fates.
Shh signaling was blocked by injection of Shh MO or by cyclopamine treatment (“−Shh” B, E, G, J, M, P) and activated by injection of Shh mRNA (“+Shh,” C, H, K, N, Q). Netrin expression in the floor plate was decreased in 42% of − Shh embryos (B, N = 387) and slightly upregulated in 44% of + Shh embryos (C, N = 139). Floor plate expression of Shh was slightly narrower in 29% of embryos with blockade of Shh signaling (E inset, N = 194). Perturbation of Shh signaling had no effect on notochord (AxPC, F–H and Tor70, I–Q) or hypochord (O–Q) or floor plate markers F-spondin and FoxA2 in the spinal cord of tadpoles (I–N). (A–H) Dorsal views, anterior up, with transverse bisection in inset (A–E). (G–O) Transverse vibratome sections through middle of spinal cord at tailbud stages. |
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Fig. 1.
Notch signaling promotes formation of floor plate and hypochord and represses notochord.
Notch signaling was repressed by injection of mRNA encoding Su(H) DNA-binding mutant (SDBM, B, E, H, K, N) and activated by Notch intracellular domain mRNA (NICD, C, F, I, L, O). Floor plate development was assayed by expression of Netrin (A–C), F-spondin (G–I), and FoxA2 (J–L). Notochord formation was assayed by Axial protocadherin (AxPC, D–F) and Tor70 staining (brown, G–O). Tor70 staining has high variability and background, and thus should not be considered quantitative. Hypochord development was assayed by F-spondin (G–I, staining ventral to notochord) and VEGF (M–O). (A–F) Dorsal views, anterior up. (G–O) Transverse vibratome sections through middle of spinal cord at tailbud stages. |
