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First published online 27 April 2005
doi: 10.1242/dev.01842

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Dissecting Wnt/ß-catenin signaling during gastrulation using RNA interference in mouse embryos

Heiko Lickert^1,*, Brian Cox^1,4, Christian Wehrle², Makoto M. Taketo³, Rolf Kemler² and Janet Rossant^1,4,

¹ Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto M5G 1X5, Canada
² Max-Planck Institute of Immunobiology, Department of Molecular Embryology, Freiburg 79108, Germany
³ Kyoto University Graduate School of Medicine, Department of Pharmacology, Yoshida-Konoe-cho, Sakyo-ku, Kyoto 606-8501, Japan
⁴ University of Toronto, Department of Medical Genetics and Microbiology, Toronto M5S 1A8, Canada

View larger version (37K): [in a new window]   Fig. 1. Gene expression profiling of conditional ß-catenin mutant embryos. (A) Hierarchical clustering of differentially regulated genes (see Materials and methods). The number of upregulated (shades of red) and downregulated (shades of green) genes per GeneChip is indicated. Different shades indicate the signal log ratios between the individual comparisons, and a color scale bar represents these values. (B) Enrichment of genes encoding for developmental regulatory factors among the potential ß-catenin target genes. Approximately 6000 genes on the U74Av2 GeneChip are annotated and have a gene ontology (GOID) term. The left pie chart represents the percentage of genes with the indicated GO terms among all genes with a GO term. The right pie chart represents the percentage of genes with the indicated GO terms among the 49 downregulated genes of the U74Av2 GeneChip.

View larger version (101K): [in a new window]   Fig. 2. Confirmation of the GeneChip results by whole-mount in situ hybridization. (A-I) Late-gastrula stage wild-type (wt) and conditional ß-catenin mutant (CKO) embryos hybridized with the indicated probes. All embryos are depicted in a lateral view, anterior to the left. (A-H) Axin2, Wnt3a, Wnt8, Frzb1, Notch1, Dll1, Gbx2 and Hoxb1 are all expressed in the primitive streak of wild-type embryos, but are downregulated in conditional ß-catenin mutants to varying extents. (I) Sox2 is normally expressed in the extraembryonic chorion and anterior epiblast in wild-type embryos at this stage. The expression in the extraembryonic region is unaffected in mutant embryos, but the whole epiblast expresses Sox2.

View larger version (69K): [in a new window]   Fig. 3. Whole-mount expression screening to identify potential ß-catenin target genes. (A-H) Whole-mount in situ hybridization with the indicated probes on wild-type embryos at different developmental stages. All embryos are depicted in a lateral view, anterior to the left, except for the 6- to 7-somite stage embryo in H, which is depicted in a ventral view. (A) Grsf1 is strongly expressed in the primitive streak (ps) at neural-plate stage (NP). At 9-somite stage, Grsf1 is restricted to the posterior neurectoderm in the tailbud (tb), and the midbrain (mb; arrow) and hindbrain region (hb; arrow). (B) Scap2 is expressed in the primitive streak at head-fold stage. At the 9-somite stage, mRNA expression is detected in the tailbud region and foregut pocket (arrow). (C) EST6 is expressed (arrows) in the extraembryonic ectoderm at early-streak (ES) and mid-streak (MS) stage. At head-fold stage, EST6 shows expression in a row of cells anterior to the node (arrows). (D) Punc is expressed in the primitive streak at head-fold stage. At the 11-somite stage, expression is seen in the midbrain (mb) and hindbrain (hb), as well as in the somites (so), inflow tract (ift) and posterior neurectoderm. (E) Zic3 is expressed in the primitive streak at head-fold stage. At 11-somite stage, expression is detected in anterior neurectoderm (ne), heart (h), inflow tract, gut (g), somites, midbrain (mb) and hindbrain (hb), and posterior paraxial mesoderm (pm). (F) EST10 is strongly expressed throughout the definitive endoderm at mid-streak, late-streak (LS), late-bud (LB) and head-fold stage. (G) Fragilis2 is first detected in the region where the allantois will form at mid-streak stage (arrow). At head-fold stage, expression is prominent in the primitive streak region and in the lateral-plate mesoderm (arrows). At 10-somite stage, expression is confined to the lateral-plate mesoderm (lpm), paraxial mesoderm and first forming somite (arrow) in the tailbud region. (H) EST16 shows expression in the primitive streak at late head-fold stage. At 6- to 7-somite stage, expression is confined to the posterior neurectoderm.

View larger version (50K): [in a new window]   Fig. 4. The response of Grsf1 and Fragilis2 to Wnt signaling and shRNA silencing. (A) Co-culture of ES cells on lacZ and Wnt1-expressing fibroblasts induces Grsf1 endogenous mRNA expression in Wnt1-treated ES cells, but not Fragilis2 expression. Gene-specific expression was normalized to Gapdh and the fold change was calculated. Results of three independent co-culture experiments are shown. Whole-mount in situ hybridization of wild-type and ß-catenin mutants (B,C), and of wild-type and shRNA-silenced embryos (E,F) with Grsf1 and Fragilis2 probes at late-gastrulation stage. All embryos are depicted in a lateral view, anterior to the left. Grsf1 and Fragilis2 are strongly downregulated in the primitive streak of ß-catenin mutant embryos (B,C) and knock-down embryos (E,F). Arrow in C indicates remaining Fragilis2 expression at the base of the allantois. (D) Northern blot analysis for Grsf1 and Fragilis2 for eight different shRNA ES-cell lines (1-8) and wild-type ES cells. 28S, 18S and 5.8S ribosomal RNAs are indicated as molecular length markers, and the ethidium bromide (EtBr)-stained agarose gels served as loading control.

View larger version (74K): [in a new window]   Fig. 5. Grsf1-silenced embryos mimic mid/hindbrain and posterior truncation phenotypes of Wnt mutants. (A) Posterior truncations are more pronounced at E9.0 (the first forming somite is indicated by an asterisk in A and B). Additionally, Grsf1-silenced embryos show a clear thickening in the midbrain (mb) region (red arrows), a large alantois (al) and abnormal anterior hindbrain development (white arrow; red arrowhead indicates mid/hindbrain boundary). (B) At E8.5, Grsf1-silenced embryos show a reduced tailbud (tb) region (indicated by dotted area). (C,D) Comparison of Hematoxylin and Eosin-stained transverse sections of wild-type and Grsf1-silenced embryos at E9.0. fb, forebrain; hb, hindbrain; nt, neural tube. (E-K) Whole-mount in situ analysis of wild-type and Grsf1-silenced embryos with indicated probes. (E,F) Comparison of Fgf8 expression in 7-8 somite wild-type and Grsf1-silenced embryos. (E) Lateral view, anterior up. (F) Dorsal view on the mid/hindbrain boundary. (G,H) Comparison of hindbrain marker gene expression in wild-type and Grsf1-silenced embryos at 7-somite stage; dorsal view, anterior up. (I-L) Expression analysis of potential (Gbx2) and known Wnt/ß-catenin target genes (Cdx1 and T) in wt and knock-down embryos at gastrulation stage. L, lateral view, anterior to the left; A, anterior view, anterior is up; P, posterior view, posterior is up. (I,J) Gbx2 and Cdx1 are normally expressed in Grsf1-silenced embryos at head-fold stage. Note, that the posterior axis is slightly shortened in knock-down mutants. (K,L) Comparison of T expression in wild-type and Grsf1-silenced embryos at E7.5 (K) and head-fold stage (L). Arrow indicates the anterior expression border. A, anterior view; P, posterior view; L, lateral view.

View larger version (88K): [in a new window]   Fig. 6. Fragilis2-silenced embryos mimic the somite phenotype of the ß-catenin mutants. (A,B,D) Embryos are depicted in a dorsal view; anterior is up. Comparison of wild-type and Fragilis2-silenced embryos at E8.75 (A) and E8.5 (B) demonstrates abnormal development in the posterior region, formation of a large allantois (al), a kinked neural tube and abnormal somite formation (magnified in b' and b''). (C) Comparison of Hematoxylin and Eosin-stained frontal sections of wild-type and Fragilis2-silenced embryos at E8.5. (D) Tissue non-specific alkaline phosphatase (AP) staining to detect primordial germ-cell (PGC) formation at head-fold stage. Embryos are depicted in a posterior view, focusing on the embryonic-extraembryonic border. (E) Whole-mount in situ analysis of wild-type and Fragilis2-silenced embryos with the indicated probes at the 2- to 3-somite stage (upper panels; posterior view, posterior is up) and the 9- to 10-somite stage (lower panel; dorsal view, posterior is down). T and Tbx6 are normally expressed in Fragilis2-silenced embryos at 2-3 somite stage, but expression is slightly reduced at the 9- to 10-somite stage in the tailbud region. In wild-type embryos, PAPC is expressed in two presomitic stripes (s0 and s-1). At the 2- to 3-somite stage, only one presomitic stripe is clearly visible in knock-down mutants, and PAPC expression further diminishes at the 9- to 10-somite stage.