Brain and Mind Research Institute and Department of Neurology.
Hum Mol Genet. 2013 Nov 1;22(21):4267-81. doi: 10.1093/hmg/ddt277. Epub 2013 Jun 16.
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.
低密度脂蛋白受体相关蛋白 6 (Lrp6) 突变对神经胚形成的影响,使用功能增益(卷曲尾,Lrp6(Cd)) 和功能缺失(Lrp6(-))) 小鼠品系进行了研究。在 Lrp6(Cd/Cd) 神经管中,与经典 Wnt 信号通路相关的两个特征,背腹模式和增殖,与野生型(WT)没有不同。Lrp6(-/-) 胚胎表现出增殖减少和神经褶细微的模式变化。Lrp6(Cd/Cd) 和 Lrp6(-/-) 颅褶中的细胞极性缺陷,表现为细胞形状、中心体位移以及顶端表面 F-肌动蛋白和 GTP-RhoA 积累的失败。从 Lrp6(Cd/Cd) 或 Lrp6(-/-) 胚胎衍生的小鼠胚胎成纤维细胞(MEFs)分别表现出升高和降低的 RhoA 基础活性水平。虽然配体非依赖性的经典 Wnt 信号转导激活,绕过 Lrp-Frizzled 受体,不能激活 RhoA,但 Lrp6(-/-) MEFs 中非经典 Wnt5a 刺激 RhoA 活性受损。与 WT 同窝仔相比,在培养的 Lrp6 敲除胚胎中 RhoA 抑制加剧了 NTDs。相反,ROCK 抑制剂从 NTDs 中拯救了 Lrp6(Cd/Cd) 胚胎。Lrp6 与 Disheveled 相关形态发生激活物 1 (DAAM1) 共免疫沉淀,DAAM1 是一种促进 Wnt 信号转导中 GEF 活性的形成蛋白。生化和细胞生物学数据显示,Lrp6(Cd) 蛋白在细胞内积累,其与 DAAM1 的相互作用可以解释观察到的 RhoA 活性升高。相反,消除 Lrp6 与 DAAM1 相互作用的无效突变导致 Lrp6(-/-) 胚胎中基础 RhoA 活性降低。这些结果表明,Lrp6 不仅介导经典 Wnt 信号转导,还可以调节涉及 RhoA 依赖性机制的非经典途径,从而影响神经胚形成,可能通过与 DAAM1 的细胞内复合物。
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