DEV008458 Supplementary Material

Files in this Data Supplement:

• Supplemental Table S1 - Adobe PDF

• Supplemental Figure S1 -

  Fig. S1. Confirmation of selected array clones by RT-PCR analysis. Twenty-five percent of the array clones that resulted upregulated by Gata5 was validated by RT-PCR analysis, reasoning that this was a representative sample size. RT-PCR was performed on cDNA synthesized from ectodermal explants expressing Gata5. ODC was used as loading control and Pdx1 as positive control for Gata5 induction. The primers used for RT-PCR analysis are available at the http://xenopus.rockefeller.edu/.

• Supplemental Figure S2 -

  Fig. S2. Establishment of the temporal hierarchy in the activation of Gata5 targets. (A-C) A hormone-inducible version of Gata5 (Gata5-GR) was injected into the animal pole of two-cell stage embryos and the animal caps were dissected at blastula stage. Half of the Gata5-injected and uninjected explants were exposed to dexamethasone for 2 hours at three developmental stages (stage 11/gastrula; stage 18/neurula; stage 30/tailbud) before being processed for RT-PCR analysis. ODC was used as loading control, Sox17α and Pdx1 as positive controls for Gata5 induction at gastrula and tailbud stages, respectively.

• Supplemental Figure S3 -

  Fig. S3. Morpholinos targeting two distinct xTGIF2 pseudoalleles. (A) Alignment of Xenopus tropicalis TGIF2 and the two Xenopus laevis TGIF2 sequences targeted by the antisense morpholino oligonucleotides used in this study (yellow). The ATG is indicated in bold. Morpholino antisense oligonucleotides were directed against the 5′UTR of the two Xenopus laevis TGIF2 pseudoalleles: MO-1 (5′-CAGGGGGAGATTCAAGGAAGATGAA-3′) and MO-2 (5′-CCTAGTGATAGACCACGAGATGGAC-3′). Standard control morpholino corresponds to 5′-CCTCTTACCTCAGTTACAATTTATA-3′. In green is the Xenopus tropicalis TGIF2 morpholino sequence that was used in a recent systematic MO-based functional screen carried out in Xenopus tropicalis. In the X. tropicalis morphants, general phenotypic defects, including shortening of the A-P axis and defects in anterior and dorsal tissues, were described (Rana et al., 2006). Similar defects were also visible in TGIF2-Mo-injected embryos (see Fig. 4B). (B) Xenopus TGIF2 translation from the mRNA encoding xTGIF2 allele 1 is specifically inhibited by TGIF2-MO1, but not by control MO. 10 µM morpholino was added to the in vitro translation reactions.

  Rana, A. A., Collart, C., Gilchrist, M. J. and Smith, J. C. (2006). Defining synphenotype groups in Xenopus tropicalis by use of antisense morpholino oligonucleotides. PLoS Genet. 2, e193.

• Supplemental Figure S4 -

  Fig. S4. Immunoprecipitation of flag-xTGIF2 with both Smad1 and Smad2. Flag-tagged TGIF2 or Myc-tagged FAST1 were injected into vegetal blastomeres (dorsally for Smad2 IP; ventrally for Smad1 IP) of four-cell stage embryos. Embryonic lysates were immunoprecipitated with anti-Smad1 or anti-Smad2 and analyzed by immunoblot with anti-flag and anti-myc antibodies. Myc-Fast1 was used as control for the specificity of the immunoprecipitation reactions, for its ability to interact exclusively with Smad2 (Chen et al., 1997). Flag-xTGIF2 immunoprecipitated complexes are indicated with **, and the myc-Fast1 complex with *. A portion of the lysates was blotted with anti-flag and anti-myc directly (without immunoprecipitation) to assess the expression of flag-xTGIF2 and myc-Fast1, respectively. In addition, equal expression of Smad1 and Smad2 was tested by immunoblotting on the crude extracts using anti-Smad1 and anti-Smad1 antibodies. TrueBlot IP beads and HRP-conjugated secondary antibodies were used to minimize interference by the heavy and light chains of the immunoprecipitating antibody in the IP/IB experiments.

  Chen, X., Weisberg, E., Fridmacher, V., Watanabe, M., Naco, G. and Whitman, M. (1997). Smad4 and FAST-1 in the assembly of activin-responsive factor. Nature 389, 85-89.