Figure 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.

Figure 8—figure supplement 1. Response of wild type and lefty1^-/-;lefty2^-/- mutant embryos to squint injection.

Wild type and lft1^-/-;lft2^-/- mutant embryos were injected with 0.2 or 0.4 pg sqt mRNA at the one-cell stage. Embryos were then exposed to 2.5 μM SB-505124 (Nodal inhibitor drug) starting at the 8 cell stage (see Figure 6, Figure 6—figure supplements 1, 2 and 3, and Figure 2—figure supplement 1). (A–D’’) Phenotypes at 1 day post-fertilization (dpf). Embryos injected with sqt mRNA exhibit dramatic morphological defects and frequently die by 1 dpf, whereas embryos injected with sqt mRNA and treated with Nodal inhibitor drug have more normal morphology and higher survival rates. (E) Percentages of embryos with the indicated phenotypes. Injected, drug-treated embryos have higher survival rates and more normal morphology than injected embryos that were not treated with drug. Total embryos analyzed: wild type = 24, wild type + 0.2 pg sqt = 24, wild type + 0.4 pg sqt = 24, wild type + drug = 15, wild type + 0.2 pg sqt + drug = 19, wild type + 0.4 pg sqt +drug = 20, lft1^-/-;lft2^-/- mutants = 24, lft1^-/-;lft2^-/- mutants + 0.2 pg sqt = 24, lft1^-/-;lft2^-/- mutants + 0.4 pg sqt = 24, lft1^-/-;lft2^-/- mutants + drug = 27, lft1^-/-;lft2^-/- mutants + 0.2 pg sqt + drug = 25, lft1^-/-;lft2^-/- mutants + 0.4 pg sqt + drug = 24. (F–M’’) Embryos were treated as described above, and fixed when uninjected wild type embryos reached 50% epiboly. Injection of 0.2 pg (F’) or 0.4 pg (F’’) sqt mRNA caused upregulation of the mesoderm marker goosecoid in wild type embryos. (G–G’’) Compared to wild type embryos, lft1^-/-;lft2^-/- mutants exhibited stronger upregulation of goosecoid upon injection of 0.2 pg (G’) or 0.4 pg (G’’) sqt mRNA. (H–I’’) Exposure to Nodal inhibitor drug decreased the size of the goosecoid expression domain (H,I). Injection of 0.2 pg (H’,I’) or 0.4 pg (H’’, I’’) sqt mRNA did not affect goosecoid expression in drug-treated wild type or mutant embryos. (J–K’’) The endoderm marker sox32/casanova was not upregulated in wild type embryos injected with 0.2 pg (J’) or 0.4 pg (J’’) sqt mRNA, but was upregulated in injected lft1^-/-;lft2^-/- mutant embryos (K’,K’’). (L–M’’) Exposure to Nodal inhibitor drug dampened sox32 expression in both wild type and mutant embryos (L,M). Injection of 0.2 pg (L’,M’) or 0.4 pg (L’’,M’’) sqt mRNA did not affect sox32 expression in drug-treated wild type or mutant embryos. Wild type embryos did not have a clear advantage over drug-treated lft1^-/-;lft2^-/- mutants in this assay.