DEV01723 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure 1 -

  Fig. S1. pitx3 mRNA translation is blocked by pitx3 morpholinos. (A-D) pitx3 mRNA rescues pitx3 morpholino-injected embryos (8 ng morpholino/embryo). (E-G) Embryos co-injected with increasing doses of pitx3 morpholino and pitx3:eGFP mRNA (mRNA from 5¢UTR of pitx3 cDNA linked to eGFP; 300 pg/embryo). Morpholinos suppress eGFP fluorescence in a dose-dependent manner (F,G). By contrast, eGFP fluorescence is not blocked by pitx3 morpholino in embryos injected with a modified mRNA construct 5Dpitx3:egfp that contains five mismatches in the morpholino-binding site of the pitx3 5¢ UTR (I). (A-D) Frontal view, dorsal towards the top; (E-I) Side views, animal pole towards top. Scale bars: 200 mm in A-D; 100 mm in E-I.

• Supplemental Figure 2 -

  Fig. S2. Hedgehog signaling is required for pituitary cell specification and regulates olfactory placode size. Reduced Hedgehog signal transduction increases lens and olfactory placode domains, while reducing pituitary placode specification. (A,B) pitx3 expression in wild-type embryos. (C) dlx3b expression in wild-type embryos. Eighty-nine percent of smoothened mutants (n=83) have ectopic and retina-associated lenses (D,G), 7% have distorted lens (E) and 4% have fused lens (F). In smoothened mutants, we observe normal pitx3 expression in anterior neural plate border ectoderm at bud stage (not shown), and in lens tissue at prim-5 stage (D-G). By contrast, head mesenchyme and branchial arches fail to express pitx3 and expression in ventral forebrain is reduced (D,G). (C,H) In 97 smoothened mutant embryos, 5% had larger olfactory placodes (H) than wild-type embryos (C). Although the olfactory placodes never fuse across the ventral midline, this phenotype correlates with the extended and fused lens placodes in smoothened mutants. (A,D-F) Frontal view, dorsal is upwards; (B,C,G,H) side views, anterior towards the left, dorsal towards the top. Scale bars: 100 mm

• Supplemental Figure 3 -

  Fig. S3. pitx3 and dlx3b are required for primary lens fiber cell differentiation. (A-D) zl-1 immunofluorescence labeling of primary lens fiber cells (red) in (A) control, (B) pitx3 morpholino-injected, (C) pitx3 mRNA-injected, and (D) dlx3b and dlx4b morpholino-injected embryos. Inset in B shows that lens form in presence of pitx3 morpholino. (A-D) Frontal view, dorsal towards the top. MO, morpholino. Scale bar: 200 mm.