LRP6 exerts non-canonical effects on Wnt signaling during neural tube closure

Abstract

Low-density lipoprotein receptor related protein 6 (Lrp6) mutational effects on neurulationwere examined using gain (Crooked tail, Lrp6Cd) and loss (Lrp6-) of function mouse lines. Two features often associated with canonical Wnt signaling, dorsal-ventral patterning and proliferation, were no different from wild-type (WT) in the Lrp6Cd/Cd neural tube. Lrp6-/- embryos showed reduced proliferation and subtle patterning changes in the neural folds. Cell polarity defects in both Lrp6Cd/CdandLrp6-/- cranial foldswereindicatedbycell shape, centrosome displacement and failure of F-actin and GTP-RhoA accumulation at the apical surface. Mouse embryonic fibroblasts (MEFs) derived from Lrp6Cd/Cd or Lrp6-/- embryos exhibited elevated and decreasedRhoAbasal activity levels, respectively. While ligand-independent activation of canonical Wnt signaling, bypassing Lrp- Frizzled receptors, did not activate RhoA, non-canonical Wnt5a stimulation of RhoA activity was impaired in Lrp6-/- MEFs. RhoA inhibition exacerbated NTDs in cultured Lrp6 knockout embryos compared with WT littermates. In contrast, a ROCK inhibitor rescued Lrp6Cd/Cd embryos from NTDs. Lrp6 co-immunoprecipitated with Disheveled-associated activator of morphogenesis 1 (DAAM1), a formin promoting GEF activity inWnt signaling. Biochemical and cell biological data revealed intracellular accumulation of Lrp6Cd protein where interaction with DAAM1 could account for observed elevated RhoA activity. Conversely, null mutation that eliminates Lrp6 interaction with DAAM1 led to lower basal RhoA activity in Lrp6-/- embryos. These results indicate that Lrp6 mediates not only canonical Wnt signaling, but can also modulate non-canonical pathways involving RhoA-dependent mechanisms to impact neurulation, possibly through intracellular complexes with DAAM1. © The Author 2013. Published by Oxford University Press. All rights reserved.