DEV000901 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Effect of SB-431542 on cCer expression. (A-D) Chick embryos were grafted on the left side with beads (arrowheads) soaked in DMSO (control; A and C) or 10 mM SB-432542 (B and D) at HH5 and fixed for whole-mount in situ hybridization at HH8 (A and B) or HH9 (C and D). cCer left-side expression was normal in control embryos (A,C; white arrows), whereas it was downregulated in SB-432542-treated embryos. (B,D; black arrows).

• Supplemental Figure S2 -

  Fig. S2. Lefty expression is not altered in cCer morphant embryos. (A-B′) Chick embryos were electroporated with fluorescein-tagged control (CoMO; A and A′) or cCer (MO; B and B′) morpholinos at HH5 and fixed at HH9. (A,B) Nodal and Lefty transcripts detected by whole-mount in situ hybridization. (A′,B′) Merge of bright-field and fluorescein green fluorescence images. Whereas Nodal expression was ectopically induced on the right side of cCer morphants (B; white arrow), Lefty expression was similar in control (A; white arrowhead) and cCer morphant (B; white arrowhead) embryos.

• Supplemental Figure S3 -

  Fig. S3. Efficacy of cCer morpholinos to inhibit Cer-Luc translation. In vitro transcription and translation of luciferase reporter plasmids containing Cer0.36 or PCR5 regulatory sequences (see Fig. 2) was performed using the TNT Quick Coupled Reticulocyte Lysate system (Promega) and luciferase activity was measured using a luminometer (see Materials and methods). 200 ng of each reporter plasmid were transcribed either alone (Cer0.36 and PCR5) or in the presence of 4 nmol of cCer morpholinos (Cer0.36+CoMO; Cer0.36+MO and PCR5+MO). Data are expressed as means of triplicates from two independent experiments and are relative to the highest luciferase activity values (Cer0.36+CoMO and PCR5). In vitro translation of Cer0.36-Luc was blocked by cCer MO (lane 2) but not by the control morpholino (CoMO) (lane 1). By contrast, cCer MO did not block the translation of PCR5-Luc, a plasmid that does not contain the cCer MO complementary sequence upstream the ATG.