85 RESEARCH ARTICLE INTRODUCTION Gastrulation is a major early event in the development of the mammalian embryo. The primitive streak (PS) plays a crucial role in gastrulation because it is the site where mesoderm and endoderm progenitors are specified. The paraxial presomitic mesoderm (PSM) cells that will give rise to the musculoskeletal system of the trunk and tail, arise in the anterior PS (Tam and Beddington, 1987). The morphogenetic process of gastrulation continuously drives the movement of PSM progenitors from the streak to the posterior end of the PSM. Cells in the posterior PSM remain in an undetermined and immature state, and become anteriorly displaced as new cells are added to the posterior PSM. When PSM cells reach a prescribed position in the anterior PSM, they undergo a dramatic transition in gene expression, initiating a segmentation program that determines where and when a morphological segment boundary will form. The rhythmic formation of a new boundary in the anterior PSM, every 2 hours, leads to the formation of somites. Thus the coordinated addition of new mesodermal cells to the posterior PSM, coupled with the cleaving of new somites from the anterior PSM, is critical for the maintenance of the PSM and, ultimately, for the rapid growth and posterior extension of the body axis that occurs during vertebrate embryogenesis (Aulehla and Herrmann, 2004; Dubrulle and Pourquie, 2004; Pourquie, 2001; Saga and Takeda, 2001). The bHLH transcription factor Mesp2, under the control of the Notch signaling pathway, plays an important role in the segmentation program. Mesp2 is expressed in a segmental prepattern in the anterior PSM prior to the formation of overt boundaries, and is required for segment polarity and boundary formation (Saga et al., 1997). The prevailing ‘clock and wavefront’, or ‘clock and gradient’ models postulate that segment boundaries are positioned along the anterior-posterior (AP) axis by gradients of fibroblast growth factor 8 (Fgf8) and/or Wnt3a and an opposing gradient of retinoic acid (RA), which together define a boundary determination front in the anterior PSM. The periodicity of boundary formation is thought to be controlled by an oscillating segmentation clock driven by the Wnt and Notch signaling pathways (Aulehla and Herrmann, 2004; Pourquie, 2003; Rida et al., 2004). The molecular mechanisms linking these signaling pathways to the clock and to boundary formation, are not well understood. Feedback suppressor loops in the Wnt and Notch pathways are considered central molecular components of the segmentation clock. Notch activity oscillates in the PSM, driving periodic expression of its target genes lunatic fringe (Lfng) and Hes7 (Bessho et al., 2001; Morimoto et al., 2005). The glycosyltransferase Lfng, and the transcriptional repressor Hes7, function as negative regulators of Notch signaling and are required for proper segmentation (Bessho et al., 2003; Evrard et al., 1998). Similarly, the Wnt target genes Axin2 and Nkd1 encode negative regulators of Wnt signaling, oscillate in the PSM, and are thought to function as integral components of the clock to periodically suppress Wnt signaling (Aulehla et al., 2003; Ishikawa et al., 2004). Oscillating genes in both the Wnt and Notch pathways depend upon Wnt3a (Aulehla et al., 2003; Nakaya et al., 2005), however the significance of Wnt- centered feedback loops for the clock remains unclear because mutations in Axin2 or Nkd1 do not lead to somite or mesodermal phenotypes (Li et al., 2005; Yu et al., 2005). Wnt3a/-catenin signaling controls posterior body development by coordinating mesoderm formation and segmentation William C. Dunty, Jr 1 , Kristin K. Biris 1 , Ravindra B. Chalamalasetty 1 , Makoto M. Taketo 2 , Mark Lewandoski 1 and Terry P. Yamaguchi 1, * Somitogenesis is thought to be controlled by a segmentation clock, which consists of molecular oscillators in the Wnt3a, Fgf8 and Notch pathways. Using conditional alleles of Ctnnb1 (-catenin), we show that the canonical Wnt3a/-catenin pathway is necessary for molecular oscillations in all three signaling pathways but does not function as an integral component of the oscillator. Small, irregular somites persist in abnormally posterior locations in the absence of -catenin and cycling clock gene expression. Conversely, Notch pathway genes continue to oscillate in the presence of stabilized -catenin but boundary formation is delayed and anteriorized. Together, these results suggest that the Wnt3a/-catenin pathway is permissive but not instructive for oscillating clock genes and that it controls the anterior-posterior positioning of boundary formation in the presomitic mesoderm (PSM). The Wnt3a/-catenin pathway does so by regulating the activation of the segment boundary determination genes Mesp2 and Ripply2 in the PSM through the activation of the Notch ligand Dll1 and the mesodermal transcription factors T and Tbx6. Spatial restriction of Ripply2 to the anterior PSM is ensured by the Wnt3a/-catenin-mediated repression of Ripply2 in posterior PSM. Thus, Wnt3a regulates somitogenesis by activating a network of interacting target genes that promote mesodermal fates, activate the segmentation clock, and position boundary determination genes in the anterior PSM. KEY WORDS: Wnt3a, -catenin, Gastrulation, Mesoderm, Segmentation, Somitogenesis Development 135, 85-94 (2008) doi:10.1242/dev.009266 1 Cancer and Developmental Biology Laboratory, Center for Cancer Research, National Cancer Institute-Frederick, NIH. Frederick, MD 21702, USA. 2 Department of Pharmacology, Graduate School of Medicine, Kyoto University, Sakyo, Kyoto, 606- 8501, Japan. *Author for correspondence (e-mail: tyamaguchi@ncifcrf.gov) Accepted 16 September 2007 DEVELOPMENT