Fig. 1-S2

lefty double mutants and morphants have distinct phenotypes.

About 97% of double lft morphants die by 24 hr post-fertilization (hpf) and undergo strongly disrupted gastrulation (Agathon et al., 2001; Feldman et al., 2002). In contrast, most lft1^-/-;lft2^-/- mutants underwent gastrulation and survived past 24 hpf. Axis bifurcation occurred with variable penetrance in lft1^-/-;lft2^-/- mutants, but has not been reported in double lft morphants. Double morphants exhibited expansion of sox32 and tb at 30% epiboly (Agathon et al., 2001; Chen and Schier, 2002), whereas expansion was only evident in double mutants starting at gastrulation (Figure 3, Figure 3—figure supplement 1). In addition, lft transcripts are present at low or undetectable levels prior to sphere stage (Pauli et al., 2012), and lft1^-/-;lft2^-/- adult females crossed to wild type males produce viable offspring (data not shown), suggesting that differences in morphant and mutant phenotypes are unlikely to be due to inhibition of maternally contributed lft. We also note that loss of sqt, but not cyc, reduced mesendodermal expansion and gastrulation defects in double morphants (Chen and Schier, 2002; Feldman et al., 2002), but loss of either sqt or cyc partially rescued morphology in double mutants (Figure 4). These differences suggest that, as other work has shown (Kok et al., 2015; Schulte-Merker and Stainier, 2014), off target morpholino effects may confound studies of gene function. (A–N) Wild type (A–D, I–K) and lft1^-/-;lft2^-/- mutant (E–H, L–N) embryos were injected with 7 ng lft1 morpholino (B,F,J,M), 7 ng lft2 morpholino (C,G,K,N), or 7 ng of each morpholino together (D,H). At 12 hpf (A–H), wild type and mutant embryos injected with both morpholinos exhibited severe gastrulation defects not observed in uninjected embryos. At 1 day post-fertilization (dpf) (I–N), all embryos injected with both morpholinos were dead. (O) Percentage of embryos alive at 1 dpf. Number of living/total embryos at 1 dpf: uninjected wild type = 38/39, wild type +lft1 MO = 25/26, wild type + lft2 MO = 18/19, wild type + lft1/2 MOs = 0/22, uninjected lft1^-/-;lft2^-/-= 47/52, lft1^-/-;lft2^-/- + lft1 MO: 7/24, lft1^-/-;lft2^-/- + lft2 MO = 1/27, lft1^-/-;lft2^-/- + lft1/2 MOs = 0/22. lft1 MO sequence: 5’-cgcggactgaagtcatctttcaag-3'. lft2 MO sequence: 5’-agctggatgaacagagccatgct-3'.