flow in Wnt11bMO and Wnt11b DNA injected specimens reached r-value of 0.5160.17 and 0.660.28, respectively. While these data clearly demonstrate the impact of Wnt11b knockdown on flow directionality, the residual flow has not lost directionality altogether. Flow velocities were calculated from the same set of time-lapse movies. Velocities in Wnt11b morphants and following Wnt11b DNA injection were found at 1.37mm/s60.32 and 1.19mm/s60.23, respectively, compared to uninjected controls which displayed a mean velocity of 2.54mm/ s60.94. Velocity thus was affected in a more pronounced manner than flow directionalty. These data pinpoint flow as a decisive step for Wnt11b function during LR development. Wnt11b-manipulated Embryos show Loss of Asymmetric Pitx2c Expression Wnt11b function in LR development 11303052 was addressed by morpholino oligonucleotide mediated knockdown of Wnt11b translation and in gain-of-function experiments using a full length Wnt11b DNA expression construct. Short of a Xenopus Wnt11b-specific antibody, knockdown efficiencies could not be addressed directly. SM and GRP were targeted by injecting 4-cell embryos into the dorsal marginal zone. Specimens were cultured until they reached stage 31 and analyzed for Pitx2c gene expression by whole mount in situ hybridization. Remarkably, both gain and loss of Wnt11b function resulted predominantly in loss of asymmetric Pitx2c expression in the left LPM. In Wnt11b morphants Pitx2c expression was mostly absent, while bilateral expression of Pitx2c represented the most frequently encountered phenotype following Wnt11b DNA injection. Ectopic expression of Wnt11b in addition resulted in severely shortened anterior-posterior axes, indicative of convergent extension defects. Next we asked whether changes in Foxj1 expression might correlate with absence of Pitx2c expression in morphants. Unexpectedly, differences from the wildtype pattern were recorded only in a minority of cases. As both organizer function and notochord formation are required for normal LR development, we analyzed Xnr3 and Not mRNA expression in Wnt11b morphants. Both were expressed in wildtype fashion, demonstrating that organizer and notochord were not affected. In order to test whether ligand-mediated Wnt signaling was indeed required for Foxj1 induction in the SM, we used a previously characterized antisense MO to interfere with translation of the canonical Wnt 9450616 receptor Frizzled 8, which was 
shown to be active on the dorsal side of the Xenopus gastrula. As shown in Wnt11b Regulates Wnt/PCP Dependent Cilia Polarization and Morphogenesis of the GRP Next we analyzed cilia polarization, a process known to depend on Wnt/PCP. GRP explants from Wnt11b manipulated embryos were stained for cilia and cell borders using an antibody against acetylated tubulin and phalloidin. In uninjected control GRPs most cells were ciliated and cilia were localized to the posterior pole. Analysis of Wnt11b morphants and embryos injected with DNA encoding either a dominant negative Wnt11b construct, which is specific for Wnt5/11-type ligands without affecting the canonical pathway, or wildtype Wnt11b revealed disturbed cilia polarization. Remarkably, clear differences were seen between loss-of-function scenarios and ectopic expression of Wnt11b. The MedChemExpress MRT-67307 ciliation rate was reduced to 50% and 66% in Wnt11b morphants and following injection of dnWnt11b DNA, respectively. Overexpression of Wnt11b did not alter the wildtype ciliation rate of about 80%, but