2599 Introduction Signaling molecules of the Wnt family regulate many cellular behaviors, including differentiation, proliferation and morphogenesis, and are involved in gastrulation and axial development (reviewed by Sokol, 1999; Yamaguchi, 2001). β- Catenin, besides acting as a central component of the cadherin cell-adhesion complex, plays an essential role in the canonical Wnt/β-catenin signaling pathway (reviewed by Huber et al., 1996). Upon Wnt stimulation, β-catenin enters the nucleus and acts in a complex with members of the Tcf/Lef (T-cell factor/Lymphoid enhancer factor) family of transcription factors to activate target genes (Hecht and Kemler, 2000). Mutations in a number of Wnt genes and Wnt signaling components highlight the crucial role of the Wnt/β-catenin signaling pathway in the initiation of primitive streak formation, as well as in the patterning and morphogenesis of the gastrulation-stage embryo (Beddington and Robertson, 1999; Lu et al., 2001). Mutant mouse embryos that lack functional Wnt3 or β-catenin fail to establish an anterior- posterior (A-P) axis and do not form a primitive streak, thus they fail to generate endoderm and mesoderm, resulting in an arrest of development before gastrulation (Liu et al., 1999; Haegel et al., 1995; Huelsken et al., 2000). Both Wnt3a null mutants and Lef1;Tcf1 compound homozygous null mutants fail to differentiate paraxial mesoderm, do not form somites caudal to the forelimb buds and exhibit severe posterior truncations (Takada et al., 1994; Galceran et al., 1999). In addition, Wnt3a controls directly the expression of Axin2 and Dll1 in the paraxial mesoderm, and thereby, links the Notch and Wnt signaling pathways in the processes of somitogenesis (Aulehla et al., 2003; Galceran et al., 2004; Hofmann et al., 2004). By the end of gastrulation and the beginning of neurulation, secreted Fgf8 and Wnt1 molecules from the isthmic organizer play an important role in patterning the mid/hindbrain region along the A-P axis (reviewed by Liu and Joyner, 2001; Wurst and Bally-Cuif, 2001). Recently, using the Cre/loxP system, we have conditionally inactivated β-catenin in the visceral endoderm (VE) and the anterior primitive streak (APS) by using a Cytokeration 19 (K19)-driven Cre (Lickert et al., 2002). Similar to in Wnt3 and β-catenin null mutants, A-P axis formation was affected; however, the conditional β-catenin mutants proceeded through gastrulation. This revealed a crucial function for β-catenin during later developmental processes, such as posterior axis Differential gene regulation integrated in time and space drives developmental programs during embryogenesis. To understand how the program of gastrulation is regulated by Wnt/β-catenin signaling, we have used genome-wide expression profiling of conditional β-catenin mutant embryos. Known Wnt/β-catenin target genes, known components of other signaling pathways, as well as a number of uncharacterized genes were downregulated in these mutants. To further narrow down the set of differentially expressed genes, we used whole-mount in situ screening to associate gene expression with putative domains of Wnt activity. Several potential novel target genes were identified by this means and two, Grsf1 and Fragilis2, were functionally analyzed by RNA interference (RNAi) in completely embryonic stem (ES) cell-derived embryos. We show that the gene encoding the RNA-binding factor Grsf1 is important for axial elongation, mid/hindbrain development and axial mesoderm specification, and that Fragilis2, encoding a transmembrane protein, regulates epithelialization of the somites and paraxial mesoderm formation. Intriguingly, the knock-down phenotypes recapitulate several aspects of Wnt pathway mutants, suggesting that these genes are components of the downstream Wnt response. This functional genomic approach allows the rapid identification of functionally important components of embryonic development from large datasets of putative targets. Key words: Wnt/β-catenin signaling, Gastrulation, RNA interference (RNAi), Target genes, Expression profiling, Grsf1, Fragilis2, Functional genomics Summary Dissecting Wnt/β-catenin signaling during gastrulation using RNA interference in mouse embryos Heiko Lickert 1, *, Brian Cox 1,4 , Christian Wehrle 2 , Makoto M. Taketo 3 , Rolf Kemler 2 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 *Present address: GSF – National Research Center for Environment and Health, Institute of Stem Cell Research, Neuherberg 85764, Germany † Author for correspondence (e-mail: rossant@mshri.on.ca) Accepted 30 March 2005 Development 132, 2599-2609 Published by The Company of Biologists 2005 doi:10.1242/dev.01842 Research article Development