DEV01726 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure 1 -

  Fig. S1. Effects of high RA doses on eye DV polarity, as detected on transverse sections of eyes after whole-mount in situ hybridization. Embryos were treated with 10 mM RA from stage 12.5/13 and analyzed at stage 33 for Vax2, ET and Pax6 expression. RA treatments upregulate Vax2 throughout the eye, completely repress ET and have no effect on Pax6. In RA-treated embryos, ET is expressed in the epidermis, but not in the eye.

• Supplemental Figure 2 -

  Fig. S2. Amino acid alignment of human, mouse, chick and Xenopus Raldh. Identical amino acids are shaded gray. The active sites and putative NAD-binding domain are indicated with arrowheads and underlines, respectively.

• Supplemental Figure 3 -

  Fig. S3. AGN194310 blocks the effects of exogenous ATR on eye DV polarity. Embryos were treated with 2 mM ATR (A) or 10 mM ATR (B) from stage 12.5/13, with or without an equimolar amount of AGN194310, and analyzed at stage 33 for the expression of Pax2 (A) or Vax2 (B). A dose of 2 mM ATR upregulates Pax2 expression in the ventral eye (A), whereas 10 mM ATR activates Vax2 in the DR (B). Both effects are inhibited by AGN194310.

• Supplemental Figure 4 -

  Fig. S4. Hh signaling can rescue expression of OS genes in embryos treated with high RA doses. Embryos were injected with 25 pg bhh mRNA, and treated with 10 mM RA from stage 12.5/13, followed by molecular marker analysis at stage 33. Expression of Pax2, Vax1b and Raldh3 in the ventral eye is detectable in bhh-injected, but not in uninjected, RA-treated embryos.

• Supplemental Figure 5 -

  Fig. S5. Inhibition of RA, Hh and FGFR signaling by low molecular weight antagonists. (A) Embryos were treated from stage 10.5 with 10 mM AGN194310 (AGN), and analyzed at stage 15/16 for the expression of HoxA1, Ptc2 and Gli1 by RT-PCR, and for the levels of phosphorylated ERK (dp-ERK) by immunoblotting. HoxA1 expression was strongly downregulated, but not Ptc2, Gli1 and dp-ERK. (B) Embryos were treated from stage 10.5 with 100 mM cyclopamine (CPM), and stage 30/31 dissected heads were analyzed as in A. Both Ptc2 and Gli1, but not HoxA1 and dp-ERK, were strongly downregulated. (C) Embryos were treated from stage 10.5 with 25 mM SU5402 (SU) and analysed at stage 15/16 as in A. A clear decrease was detected in the levels of dp-ERK, but not Ptc2, Gli1 and HoxA1 expression. RT-PCR and immunoblotting were carried out as previously described (Lupo et al., 2002; Pownall et al., 2003).

• Supplemental Figure 6 -

  Fig. S6. Expression of Shh, FGF8, Raldh2 and Raldh3 in embryos treated with antagonists of RA, Hh and FGFR signaling. (A) Embryos were treated from stage 10.5 with 10 mM AGN194310 (AGN) or 25 mM SU5402 (SU) and analyzed at stage 15/16 or 30/31 by in situ hybridization for the expression of Shh. (B) Embryos were treated from stage 10.5 with 10 mM AGN194310 or 100 mM cyclopamine (CPM) analyzed at stage 15/16 or 30/31 for the expression of FGF8. (C) Embryos were treated from stage 10.5 with 100 mM cyclopamine or 25 mM SU5402 analyzed at stage 15/16 or 30/31 for the expression of Raldh2. (D) Embryos were treated from stage 10.5 with 100 mM cyclopamine or 25 mM SU5402 analyzed at stage 30/31 for the expression of Raldh3. Raldh3 expression in the ventral eye was reduced in CPM-treated embryos, whereas no significant differences between treated and control embryos were detected in the expression of Shh, FGF8 and Raldh2 in the eye or forebrain region.