DEV02516 Supplementary Material

Files in this Data Supplement:

• Supplemental Tables - Adobe PDF

• Supplemental Figure 1 - Fig. S1. Hex represses axial markers in Xenopus embryos. (A) In situ hybridisation of Hex-depleted embryos at stage 10.5 for Xlim1 (upper panel) and Xenopus Brachyury (Xbra) (lower panel). Embryos were injected with a total of 40 ng Hex MO or control MO at the two-cell stage. 500 pg mouse Hex were used to rescue the phenotypes. (B) In situ hybridisation for Derrière at stage 10.5. Embryos were injected at the two-cell stage in both blastomeres with 500 pg Hex and/or 500 pg β-catenin.

• Supplemental Figure 2 -

  Fig. S2. Characterisation of Rosa26 ES cell lines (R26). (A) Western blot showing induction of Hex and HexERT2-λVP2 in response to induction of recombination by Cre. All inserts were HA-tagged and the western blot uses a α-HA antibody and therefore showed no evidence of endogenous Hex. (B) Activity of HexERT2-λVP2 in Rosa26 cell lines. Reporter assays with the Goosecoid promoter were used to optimise induction with 4-hydroxytamoxifen (4-OHT). The Goosecoid luciferase reporter is induced in response to increasing concentrations of 4-OHT in HexERT2-λVP2 Rosa26 ES cells. After we completed Affymetrix profile analysis of these cell lines, we began working with serum-free ES cell cultures. Both luciferase activity and target gene induction were enhanced in serum-free culture. We presume that this was due to the inhibitory effects of cytokines in serum and required to suppress ES cell differentiation such as BMP4.

• Supplemental Figure 3 -

  Fig. S3. Potential Hex-binding sites in the genomic regions of Nodal, Xnr1/2 and Tle4. (A) Hex-binding sites in the Nodal locus. The vertical lines in black above the locus indicate the presence of putative binding sites. Thickened lines indicate the presence of adjacent sites. The grey lines and numbering indicate genomic position. Conservation to different genomes is indicated by the plot in blue and then highlighted for each individual genome in green. Four binding sites are located upstream of Nodal. Three cluster around a conserved region, although the conservation in the binding sites is not strong. A fourth upstream site is located in a region that appears to have little conservation, but the site appears conserved in both human and dog. Two intronic sites exist in regions of high non-coding conservation. (B) Hex-binding sites in the Xnr1 and Xnr2 loci. Xnr1 and Xnr2 are located in a complex, transcribed in the opposite orientation to the Scaffold numbering. Genes are labelled in green and the arrow indicates the direction of transcription. In this case, conservation is indicated in greyscale against the zebrafish and chicken genomes. Both genes contain a cluster of three to four Hex sites approximately 1 kb upstream from the start site of transcription and a second binding site close to the start site. Both genes also contain binding sites in intron 1, in regions conserved with respect to zebrafish. (C) Sample regulatory region from mouse Tle4. Plot is labelled as in A. Two conserved clusters of Hex binding sites located within a highly conserved region of non-coding intronic sequence (intron 16). Similar regions were identified in introns 6, 12 and 14. Hex binding sites were identified by searching the regions around the genomic loci indicated with the sequence YHATTAA, allowing for no substitutions. As we were interested in conserved motifs, we applied three further criteria to generate the maps. The sites indicated were either clustered, located in the vicinity of the promoter or located in regions of conserved, non-coding genomic sequence. The resulting maps are not exhaustive, but give a strong indication as to where Hex-mediated silencer sequences may be located.