Fig. 8-S1

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.