Fig. 8

Correction of Nodal signaling challenges requires Lefty feedback.

(A–F) Wild type embryos with intact feedback circuits (A–C) and feedback-decoupled drug-treated lft1^-/-;lft2^-/- mutants (D–F) were challenged by decreasing Nodal signaling with injection of 0.5 pg lft1 mRNA (B,E) or by increasing Nodal signaling with injection of 7.5 pg CA-smad2 (C,F). lft1^-/-;lft2^-/- mutant embryos were exposed to 2 µM SB-505124 (Nodal inhibitor drug) at the 8 cell stage and phenotypes were assessed at 1 dpf. 0.5 pg lft1 causes mild Nodal loss-of-function phenotypes in wild type embryos (B), but strong Nodal loss-of-function phenotypes such as cyclopia and loss of head and trunk mesendoderm in feedback-decoupled embryos (E). CA-smad2 is well-tolerated in wild type embryos (C), but causes reduction of eyes, axis curvature, and accumulation of blood precursors in feedback-decoupled embryos (F). (G) Percentages of embryos with indicated phenotypes. Total embryos analyzed, lft challenge experiment: wild type + 0.5 pg gfp mRNA = 10, wild type +0.5 pg lft1 mRNA = 16, drug-treated lft1^-/-;lft2^-/- mutants + 0.5 pg gfp mRNA = 16, drug-treated lft1^-/-;lft2^-/- mutants + 0.5 pg lft1 mRNA = 12. Total embryos analyzed, CA-smad2 challenge experiment: wild type +7.5 pg gfp mRNA = 18, wild type +7.5 pg CA-smad2 mRNA = 20, drug-treated lft1^-/-;lft2^-/- mutants + 7.5 pg gfp mRNA = 19, drug-treated lft1^-/-;lft2^-/- mutants + 7.5 pg CA-smad2 mRNA = 20. (H–M’) Wild type (H–I’) and lft1^-/-;lft2^-/- mutant embryos (J–M’) were injected with 0.5 pg gfp or lft1 mRNA at the one-cell stage. Mutants were exposed to 2 µM Nodal inhibitor drug SB-505124 starting at the 8 cell stage, and expression of the endoderm marker sox32 (H, H’, J, J’, L, L’) and the mesoderm marker noto (I, I’, K, K’, M, M’) were assessed at 50% epiboly and shield stage as indicated via in situ hybridization. Note the decreased expression of noto in drug-treated double mutants injected with lft1 mRNA (K, K’) compared to wild type embryos injected with lft1 mRNA (I,I’). (N–S’) Wild type (N–O’) and lft1^-/-;lft2^-/- mutant embryos (P–S’) were injected with 7.5 pg gfp or CA-smad2 mRNA at the one-cell stage. Mutants were exposed to 2 µM Nodal inhibitor drug SB-505124 starting at the 8 cell stage, and expression of sox32 (N,N’, P, P’, R, R’) and noto (O, O’, Q, Q’,S, S’) were assessed at 50% epiboly and shield stage as indicated via in situ hybridization.