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First published online November 17, 2003
doi: 10.1242/10.1242/dev.00859

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ß-Catenin regulates Cripto- and Wnt3-dependent gene expression programs in mouse axis and mesoderm formation

¹ Max Delbrueck Center for Molecular Medicine, Robert-Roessle-Strasse 10, 13125 Berlin, Germany
² Department of Molecular Genetics, The University of Texas, M. D. Anderson Cancer Center, Houston, TX 77030, USA
³ Center for Advanced Biotechnology and Medicine and Dept. of Pediatrics, UMDNJ-Robert Wood Johnson Medical School, 679 Hoes Lane, Piscataway, NJ 08854, USA
⁴ Department of Immunology, University Hospital Utrecht, NL-3584 CX Utrecht, The Netherlands
⁵ Department of Pharmacology, Kyoto University Graduate School of Medicine, Yoshida-Konoe-cho, Sakyo-ku, Kyoto 606-8501, Japan

View larger version (29K): [in a new window]   Fig. 1. Expression of genes that are deregulated in ß-catenin-/- embryos, as determined by microarray analysis. (A) Transcriptional changes that occur between E6.0 and E6.5 in wild-type embryos, and comparison with ß-catenin mutants. The expression of 130 genes is downregulated, and expression of 673 genes is upregulated in wild-type embryos between these two stages. In ß-catenin-/- embryos, most genes are similarly modulated (green; 127 are also downregulated and 591 are also upregulated). A minority of genes are oppositely regulated (red; three genes are downregulated and 82 genes upregulated). (B) Upper panel: dissection scheme for separation of embryonic tissues. X, extra-embryonic tissues (green); VE, visceral endoderm (yellow); EE, embryonic ectoderm including primitive streak region (blue); M, mesoderm (red). Lower panel: assessment of the expression of genes that are known to be expressed in a tissue-specific manner. Pou5f1 is expressed in EE, brachyury (T) in EE and M, Cer1 in VE, and Bmp4 in X (Wilkinson et al., 1990; Rosner et al., 1990; Belo et al., 1997; Lawson et al., 1999). Red, high relative expression; blue, low relative expression; bright area, genes with consistent measurements; dark area, genes with variable or low measurements in certain tissues. (C) Assessment of the tissue specificity of expression of genes that are deregulated in ß-catenin-/- embryos, as determined by expression profiling. Genes that are deregulated at E6.0 or E6.5 greater than twofold (Change p-Value<0.05) are displayed. Names of selected genes are indicated on the right.

View larger version (129K): [in a new window]   Fig. 2. Expression patterns of selected genes, as determined by in situ hybridisation in wild-type and ß-catenin mutant embryos. Whole-mount in situ hybridisation (A,B,I-L,N), in situ hybridisation of sections of embryos in utero (C-H,O-R), and transverse section through primitive streak region (M) are shown. Arrows in M indicate cells that leave the primitive streak to become mesoderm. Arrow in Q indicates expression in visceral endoderm.

View larger version (47K): [in a new window]   Fig. 3. Comparison of gene expression profiles of ß-catenin and Cripto or Wnt3 mutant embryos. Fold changes of expression of selected genes in mutants compared with wild-type embryos, and the tissue specific expression of these genes in wild-type embryos are shown. Genes were selected from all genes that are deregulated in the respective mutants (see http://www.mdcberlin.de/~zelldiff). (A) Genes commonly deregulated in both ß-catenin and Cripto mutants at E6.0. Upper panel, commonly downregulated genes with putative signalling or developmental functions; lower panel, commonly upregulated genes of which ESTs have been preferentially isolated from neural tissues. (B) Genes commonly downregulated in both ß-catenin and Wnt3 mutants at E6.5. Upper panel, genes downregulated specifically at E6.5 (late genes); lower panel, genes deregulated at both E6.5 and E6.0 (early genes); in brackets, fold change at E6.0. nd, not determined. Red, high relative expression; blue, low relative expression; bright area, genes with consistent measurements; dark area, genes with variable or low measurements in certain tissues.

View larger version (139K): [in a new window]   Fig. 4. Expression patterns of genes in wild-type embryos (left panels) and ß-catenin gain-of-function mutants (right panels) that are important in embryonic patterning, as determined by immunohistochemistry (A,B) and in situ hybridisation (C-N). Transverse sections of the epiblast region at E6.5 are shown. Posterior is towards the right in wild type, as indicated above A.

View larger version (41K): [in a new window]   Fig. 5. Cripto is regulated by ß-catenin/Tcf in colon cancer cells and tumours. (A) Northern blot analysis of Cripto expression in Ls174T colon adenocarcinoma cells. Expression of Cripto in control Ls174T cells and in cells that express dominant-negative Tcf4 in a tetracycline-inducible manner is shown. Cripto expression was assessed before (-) and 12 hours after (+) induction with doxycyclin. RNA-loading control, 28s rRNA. (B) In situ hybridisation on consecutive sections of intestinal epithelium of Min (APC mutant) mice that contain an adenoma. The ß-catenin target gene conductin marks the crypts and the adenoma. Cripto and Tssc3 (see Table 1) are also upregulated in the adenoma. (C) Identity plot of murine and human genomic sequences in the region of the Cripto locus. The mouse sequence is indicated on the horizontal axis. The transcribed region of Cripto is indicated by a horizontal arrow, the position of Cripto exons by black boxes. The Cripto enhancer (8 kb upstream) and immediate 5' flanking regions are indicated by red and green boxes, respectively. Conserved sequence stretches between mouse and human sequences are indicated by the short horizontal lines (50% to 100% identity). Consensus sequences for Lef/Tcf binding sites (WWCAAAG) in the murine genome are indicated by vertical red lines. Sequences were aligned using the Pipmaker program (Schwartz et al., 2000). (D,E) Identification of ß-catenin/Tcf responsive elements in the Cripto enhancer by luciferase reporter gene assay, using a Tcf4-ß-catenin hybrid effector plasmid. ß-Catenin-responsive TOPflash and inactive FOPflash have been described previously (Molenaar et al., 1996). Mutations in the three Lef/Tcf-binding sites (3xMUT) are described in the Materials and methods.

View larger version (17K): [in a new window]   Fig. 6. Schematic representation of developmental steps in mouse embryos that depend on ß-catenin. (A) Scheme of wildtype, Cripto, Wnt3 and ß-catenin mutant phenotypes. Blue, embryonic ectoderm; green, extra-embryonic ectoderm; yellow, visceral endoderm; orange, signalling centre in visceral endoderm; red, primitive streak; A, anterior; P, posterior. (B) Schematic representation of putative signalling events. At E6.0, ß-catenin regulates expression of Cripto, which is required for anteroposterior axis positioning. Components upstream of ß-catenin are unknown in this pathway. At E6.5, Wnt3 controls ß-catenin, which directly or indirectly regulates the expression of downstream genes (such as brachyury, Nanog and others) and leads to primitive streak and mesoderm formation. Red arrows indicate the order of signal transduction events, the broken arrow indicates crosstalk between the ß-catenin and Nodal signalling pathways via Cripto. Continuous and broken horizontal lines separate the extracellular, cytoplasmic and nuclear compartments.

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