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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 μM SB-505124 (Nodal inhibitor drug) starting at the 8 cell stage, sphere stage, or shield stage. (Exposure to 30–50 μM 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 μM SB-505124 at the 8 cell (D) or sphere (E) stage or 5 μM 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 μM 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 μM) 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 μM) 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.