title: LRP6 exerts non-canonical effects on Wnt signaling during neural tube closure.
abstract: Low-density lipoprotein receptor related protein 6 (Lrp6) mutational effects on neurulation were examined using gain (Crooked tail, Lrp6(Cd)) 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 Lrp6(Cd/Cd) neural tube. Lrp6(-/-) embryos showed reduced proliferation and subtle patterning changes in the neural folds. Cell polarity defects in both Lrp6(Cd/Cd) and Lrp6(-/-) cranial folds were indicated by cell shape, centrosome displacement and failure of F-actin and GTP-RhoA accumulation at the apical surface. Mouse embryonic fibroblasts (MEFs) derived from Lrp6(Cd/Cd) or Lrp6(-/-) embryos exhibited elevated and decreased RhoA basal 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 Lrp6(Cd/Cd) embryos from NTDs. Lrp6 co-immunoprecipitated with Disheveled-associated activator of morphogenesis 1 (DAAM1), a formin promoting GEF activity in Wnt signaling. Biochemical and cell biological data revealed intracellular accumulation of Lrp6(Cd) 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.
authors.affiliation: Brain and Mind Research Institute and Department of Neurology.
authors.forename: Jason D
authors.initials: JD
authors.lastname: Gray
authors.forename: Stanislav
authors.initials: S
authors.lastname: Kholmanskikh
authors.forename: Bozena S
authors.initials: BS
authors.lastname: Castaldo
authors.forename: Alex
authors.initials: A
authors.lastname: Hansler
authors.forename: Heekyung
authors.initials: H
authors.lastname: Chung
authors.forename: Brian
authors.initials: B
authors.lastname: Klotz
authors.forename: Shawn
authors.initials: S
authors.lastname: Singh
authors.forename: Anthony M C
authors.initials: AM
authors.lastname: Brown
authors.forename: M Elizabeth
authors.initials: ME
authors.lastname: Ross
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000378:metabolism; D000483:Alleles; D000818:Animals; D016764:Cell Polarity; D004622:Embryo, Mammalian; D005260:Female; D018507:Gene Expression Regulation, Developmental; D057809:HEK293 Cells; D006801:Humans; D060488:Low Density Lipoprotein Receptor-Related Protein-6 / Q000235:genetics / Q000378:metabolism* / Q000502:physiology*; D051379:Mice; D008822:Mice, Transgenic; D008840:Microfilament Proteins / Q000235:genetics / Q000378:metabolism; D041681:NIH 3T3 Cells; D009432:Neural Crest / Q000378:metabolism; D054259:Neural Tube / Q000196:embryology* / Q000502:physiology; D054261:Neurulation / Q000235:genetics; D011247:Pregnancy; D051153:Wnt Proteins / Q000235:genetics / Q000378:metabolism*; D060449:Wnt Signaling Pathway; D020741:rho GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism; D020742:rhoA GTP-Binding Protein / Q000235:genetics / Q000378:metabolism
chemical_list: D048868:Adaptor Proteins, Signal Transducing; C499094:DAAM1 protein, human; C555475:LRP6 protein, human; D060488:Low Density Lipoprotein Receptor-Related Protein-6; C555476:Lrp6 protein, mouse; D008840:Microfilament Proteins; D051153:Wnt Proteins; C500365:Daam1 protein, mouse; D020741:rho GTP-Binding Proteins; D020742:rhoA GTP-Binding Protein