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Fig. S5 Validation of grem2-MO. (A-I) Unilateral flat-mount dissections of 5 dpf facial skeletons. The moderately affected grem2-MO #1 (600 μM) example (B) displayed a mild Hm defect (black arrow) and Op shape change (black arrowhead), whereas the more severe example (C) showed a severely reduced/transformed Hm (blue arrow) and missing Sy and joint (blue asterisk). grem2-MO #1 (300 μM) (D) and grem2-MO #2 (400 μM) (E) caused no defects on their own, but together (F) caused synergistic skeletal defects, including transformed Hm (green arrow), missing Sy and joint, and altered Pq (green arrowhead). Two examples of hsp70I:Gal4; UAS:Grem2 larvae subjected to a 16-17 hpf heat-shock (G,H) show increasing reduction/transformation of Ch, Sy and joints. The Op was expanded in less severely affected UAS:Grem2 animals (G, red arrow), yet lost in more severely affected animals (H, red arrowhead). (J) Quantification of dorsal Hm defects, scored as: 0, not affected; 1, mildly defective (e.g. B); and 2, transformed/reduced (e.g. C,F). Standard errors of the mean are shown, and asterisks show statistically significant comparisons. (K-M) Ventral views of dissected facial skeletons in wild-type (K), edn1-/- mutant (L) and edn1-/- embryos injected with grem2-MO #2 (400 μM) (L). Grey arrowheads depict rescue of ventral Ch. Scale bars: 50 μm.