Kristin K. Biris

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 (beta-catenin), we show that the canonical Wnt3a/beta-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 beta-catenin and cycling clock gene expression. Conversely, Notch pathway genes continue to oscillate in the presence of stabilized beta-catenin but boundary formation is delayed and anteriorized. Together, these results suggest that the Wnt3a/beta-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/beta-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/beta-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.

The alignment of the left-right (LR) body axis relative to the anteroposterior (AP) and dorsoventral (DV) axes is central to the organization of the vertebrate body plan and is controlled by the node/organizer. Somitogenesis plays a key role in embryo morphogenesis as a principal component of AP elongation. How morphogenesis is coupled to axis specification is not well understood. We demonstrate that Wnt3a is required for LR asymmetry. Wnt3a activates the Delta/Notch pathway to regulate perinodal expression of the left determinant Nodal, while simultaneously controlling the segmentation clock and the molecular oscillations of the Wnt/beta-catenin and Notch pathways. We provide evidence that Wnt3a, expressed in the primitive streak and dorsal posterior node, acts as a long-range signaling molecule, directly regulating target gene expression throughout the node and presomitic mesoderm. Wnt3a may also modulate the symmetry-breaking activity of mechanosensory cilia in the node. Thus, Wnt3a links the segmentation clock and AP axis elongation with key left-determining events, suggesting that Wnt3a is an integral component of the trunk organizer.

Lrp5/6 are crucial coreceptors for Wnt/beta-catenin signaling, a pathway biochemically distinct from noncanonical Wnt signaling pathways. Here, we examined the possible participation of Lrp5/6 in noncanonical Wnt signaling. We found that Lrp6 physically interacts with Wnt5a, but that this does not lead to phosphorylation of Lrp6 or activation of the Wnt/beta-catenin pathway. Overexpression of Lrp6 blocks activation of the Wnt5a downstream target Rac1, and this effect is dependent on intact Lrp6 extracellular domains. These results suggested that the extracellular domain of Lrp6 inhibits noncanonical Wnt signaling in vitro. In vivo, Lrp6-/- mice exhibited exencephaly and a heart phenotype. Surprisingly, these defects were rescued by deletion of Wnt5a, indicating that the phenotypes resulted from noncanonical Wnt gain-of-function. Similarly, Lrp5 and Lrp6 antisense morpholino-treated Xenopus embryos exhibited convergent extension and heart phenotypes that were rescued by knockdown of noncanonical XWnt5a and XWnt11. Thus, we provide evidence that the extracellular domains of Lrp5/6 behave as physiologically relevant inhibitors of noncanonical Wnt signaling during Xenopus and mouse development in vivo.