DEV010595 Supplementary Material

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• Supplemental Figure S1 (Adobe PDF) -

  Fig. S1. Oligonucleotide sequences used for RT-PCR and electrophoretic mobility shift assay, and locations of potential Cdx1-binding motifs on foxa2 and gata5 genes. (A) Degenerate primers used to amplify the 435-bp cdx1b DNA fragment in the PCR reaction. The CDXNC and CDXCA primer pair was used in the first PCR and the CDXNC and CDXCD primer pair was used in the subsequent nested PCR. I represents inosine. (B) Oligonucleotides used for the electrophoretic mobility shift assay. Double-stranded oligonucleotide spanning bp 644 to 673 of foxa2 intron 1 with the wild-type sequence (Cdx1b-binding motifs are in boldface) is shown. Double-stranded oligonucleotide spanning bp 644 to 673 of foxa2 intron 1 with mutations from bp 657 to 661 (mutated nucleotides are underlined) is shown. (C) Paircomp, FamilyRelationsII and Cartwheel were used for interspecific sequence comparisons to search for Cdx1-binding motifs. One conserved Cdx1-binding motif was located in intron 1 of foxa2 genes from zebrafish, human and mouse. (D) Two conserved Cdx1-binding motifs were detected in the respective 5′ upstream region and 3′ untranslated region (UTR) of gata5 genes from zebrafish and stickleback. The corresponding position on the respective chromosome is shown. Identical nucleic acid sequences are shown in red, and Cdx1-binding motifs are boxed.

• Supplemental Figure S2 (Adobe PDF) -

  Fig. S2. Amino acid sequence comparison and phylogenetic tree analysis of zebrafish cdx1b and homologues from Xenopus, mouse and human. (A) Identical amino acid sequences &γτ;5 are shown in boldface. Amino acid sequences in the homeodomain are italicized. (B) Phylogenetic analyses were performed using PHYLIP3.6 (Felsenstein, 2000) and a neighbor-joining tree is presented. Bootstrap values of >700 are shown. Sequences used were human CDX1, CDX2 Hcdx1 and Hcdx2 (Mallo et al., 1997) and CDX4 Hcdx4 (Horn and Ashworth, 1995); mouse Cdx1 Mcdx1 (Duprey et al., 1988), Cdx2 Mcdx2 (Suh et al., 1994) and Cdx4 Mcdx4 (Gamer and Wright, 1993); Xenopus tropicalis cdx2, cdx1 and cdx4 Xtcad1, Xtcad2 and Xtcad3 (Reece-Hoyes et al., 2002); and zebrafish cdx1a (Zcdx1a; NM_212836), cdx1b (Zcdx1b; this study) and cdx4 Zcdx4 (Joly et al., 1992). The bar represents a distance of 100.

  Duprey, P., Chowdhury, K., Dressler, G. R., Balling, R., Simon, D., Guenet, J.-L. and Gruss, P. (1988). A mouse gene homologous to the Drosophila gene caudal is expressed in epithelial cells from the embryonic intestine. Genes Dev. 2, 1647-1654.

  Gamer, L. W. and Wright, C. V. (1993). Murine cdx4 bears striking similarities to the Drosophila gene in its homeodomain sequence and early expression pattern. Mech. Dev. 43, 71-81.

  Horn, J. M. and Ashworth, A. (1995). A member of the caudal family of homeobox genes maps to the X-inactivation centre region of the mouse and human X chromosomes. Hum. Mol. Genet. 4, 1041-1047.

  Joly, J. S., Maury, M., Joly, C., Duprey, P., Boulekbache, H. and Condamine, H. (1992). Expression of a zebrafish caudal homeobox gene correlates establishment of posterior cell lineages at gastrulation. Differentiation 50, 75-87.

  Mallo, G. V., Rechreche, H., Frigerio, J.-M., Rocha, D., Zweibaum, A., Lacasa, M. Jordan, B. R., Dusetti, N. J., Dagorn, J. C. and Iovanna, J. L. (1997). Molecular cloning, sequencing and expression of the mRNA encoding human cdx1 and cdx2 homeobox. Down-regulation of cdx1 and cdx2 mRNA expression during colorectal carcinogenesis. Int. J. Cancer 74, 35-44.

  Reece-Hoyes, J. S., Keenan, I. D. and Isaacs, H. V. (2002). Cloning and expression of the cdx family from the frog Xenopus tropicalis. Dev. Dyn. 223, 134-140.

  Suh, E., Chen, L., Taylor, J. and Traber, P. G. (1994). A homeodomain protein related to caudal regulates intestine-specific gene transcription. Mol. Cell. Biol. 14, 7340-7351.

• Supplemental Figure S3 (Adobe PDF) -

  Fig. S3. Maps showing the gene order with conserved syntenic groups between human chromosome 5, mouse chromosome 18, and zebrafish linkage group 7. Genes sharing &γτ;51% amino acid sequence identity and their respective start positions (×10⁶ bp) on human chromosome 5 (Has 5), mouse chromosome 18 (Mch 18), and zebrafish linkage group 7 (LG 7) are shown. The scales differ for the different chromosomes. The external gene ID is shown next to each chromosome.

• Supplemental Figure S4 -

  Fig. S4. Analyses of relationships between cdx1b and components of Nodal signaling. A slightly increased expression area of cdx1b in the intestines was detected in a 48-hpf squint^cz35 mutant embryo (n=9) (B) when compared with a sibling wild-type embryo (A). However, a subsequent semiquantitative RT-PCR revealed no significant differences when comparing cdx1b expression levels in 48-hpf squint^cz35 homozygous mutants with their sibling wild-type or AB wild-type embryos. No differences in cdx1b expression was observed in 48-hpf cyc^b16 (n=7) (D) and 4-6s oep^m134 mutants (n=26) (F) compared with sibling wild-type embryos (C,E). Respective expression levels of cyclops (n=54) and oep (n=54) in 85% epiboly cdx1b morphants (H,J) were not altered compared with wild-type embryos (G,I). Similar expression levels of sqt (n=172) and bon (n=91) were detected in 30% (L) and 40% (N) epiboly morphants when compared with respective wild-type embryos (K,M). Scale bars: 100 µm.

• Supplemental Figure S5 -

  Fig. S5. Inhibition of cdx1b function affects expression domains of respective gata5, gata6 and hnf4α in the liver, pancreas and intestines of cdx1b morphants. Expression of gata5 in 54-hpf wild-type embryos (A) and respective morphants (B,C); gata6 expression in wild-type embryos (D) and respective morphants (E,F); hnf4α expression in wild-type embryos (G) and respective morphants (H,I). Ep, exocrine pancreas; i, intestine; l, liver. Scale bars: 100 µm.