Fig. 6

Artificial, uniform Nodal inhibition rescues Lefty loss.

Wild type and lft1^-/-;lft2^-/- mutant embryos were exposed to 0, 1.25, 2.5, 3.75, 5, or 6.25 ?M SB-505124 (Nodal inhibitor drug) starting at the 8 cell stage, sphere stage, or shield stage. (Exposure to 30?50 ?M fully inhibits Nodal signaling (Fan et al., 2007; Hagos et al., 2007; Hagos and Dougan, 2007).) Drug was removed at 24 hr post-fertilization (hpf). (A) Inhibition is decoupled from signaling in drug-treated lft1^-/-;lft2^-/- mutants. (B?F) Exposure to 2.5 ?M SB-505124 at the 8 cell (D) or sphere (E) stage or 5 ?M at shield stage (F) rescues gross morphological defects in lft1^-/-;lft2^-/- mutants (C); compare to wild type embryo (B). Embryos were imaged at 1 day post-fertilization (dpf). (G,H) Drug exposure rescues lft1^-/-;lft2^-/- mutants to adulthood. (G) 10 month old wild type TE adult male. (H) 5 month old lft1^-/-;lft2^-/- mutant male treated with 3.5 ?M SB-505124 from sphere stage to 24 hpf. (I) Percent of wild type (black bars) or double mutant (gray bars) embryos with normal gross morphology at 1 dpf after drug exposure is plotted. See Figure 6?figure supplements 1 and 2 for further details. (J?U??) Endodermal (sox32/casanova) and mesodermal (noto/floating head) gene expression in embryos treated as indicated was assessed using in situ hybridization. lft1^-/-;lft2^-/- mutants treated starting at the 8 cell stage (2.5 ?M) exhibit decreases in endoderm compared to wild type embryos (J?L??), but have mesodermal gene expression similar to wild type embryos (M?O??). lft1^-/-;lft2^-/- mutants treated at shield stage (5 ?M) exhibit upregulation of mesendoderm until ~5 hr post-exposure (P?U??). See Figure 6?figure supplement 3 for in situ hybridization of embryos treated with drug at sphere and shield stage, as well as additional time points and treatments.