Molecular Cell Biology Laboratory, Research Institute of Pharmaceutical Sciences, Faculty of Pharmacy, Musashino University, Tokyo, 202-8585, Japan; Department of Physiological Chemistry, Graduate School of Pharmaceutical Sciences, University of Tokyo, Tokyo, 113-0033, Japan; RIKEN Center for Brain Science, Saitama, 351-0198, Japan.
Molecular Cell Biology Laboratory, Research Institute of Pharmaceutical Sciences, Faculty of Pharmacy, Musashino University, Tokyo, 202-8585, Japan; Department of Physiological Chemistry, Graduate School of Pharmaceutical Sciences, University of Tokyo, Tokyo, 113-0033, Japan.
Biochem Biophys Res Commun. 2018 Jun 27;501(3):786-790. doi: 10.1016/j.bbrc.2018.05.074. Epub 2018 May 18.
Ras related (R-Ras), a small GTPase, is involved in the maintenance of apico-basal polarity in neuroepithelial cells of the zebrafish hindbrain, axonal collapse in cultured murine hippocampal neurons, and maturation of blood vessels in adult mice. However, the role of R-Ras in neural tube formation remains unknown. Using antisense morpholino oligonucleotides (AMOs), we found that in the spinal cord of zebrafish embryos, the lumen was formed bilaterally in rras morphants, whereas it was formed at the midline in control embryos. As AMO can cause off-target effects, we generated rras mutant zebrafish lines using CRISPR/Cas9 technology. Although these rras mutant embryos did not have a bilateral lumen in the spinal cord, the following findings suggest that the phenotype is unlikely due to an off-target effect of rras AMO: 1) The rras morphant phenotype was rescued by an injection of AMO-resistant rras mRNA, and 2) a bilaterally segregated spinal cord was not observed in rras mutant embryos injected with rras AMO. The results suggest that the function of other ras family genes may be redundant in rras mutants. Previous research reported a bilaterally formed lumen in the spinal cord of zebrafish embryos with a mutation in a planar cell polarity (PCP) gene, van gogh-like 2 (vangl2). In the present study, in cultured cells, R-Ras was co-immunoprecipitated with Vangl2 but not with another PCP regulator, Pricke1. Interestingly, the interaction between R-Ras and Vangl2 was stronger in guanine-nucleotide free point mutants of R-Ras than in wild-type or constitutively active (GTP-bound) forms of R-Ras. R-Ras may regulate neural tube formation in cooperation with Vangl2 in the developing zebrafish spinal cord.
Ras 相关（R-Ras），一种小分子 GTPase，参与斑马鱼后脑神经上皮细胞的顶底极性维持、培养的鼠海马神经元轴突崩溃以及成年小鼠血管成熟。然而，R-Ras 在神经管形成中的作用尚不清楚。使用反义形态发生素寡核苷酸（AMOs），我们发现斑马鱼胚胎脊髓中，rras 突变体的管腔在两侧形成，而在对照胚胎中则在中线形成。由于 AMO 可能引起脱靶效应，我们使用 CRISPR/Cas9 技术生成了 rras 突变斑马鱼系。尽管这些 rras 突变体胚胎的脊髓中没有双侧管腔，但以下发现表明该表型不太可能是由于 rras AMO 的脱靶效应：1）rras 突变体的表型被 AMO 抗性 rras mRNA 的注射挽救，并且 2）rras AMO 注射的 rras 突变体胚胎中未观察到双侧分离的脊髓。结果表明，其他 ras 家族基因的功能在 rras 突变体中可能是冗余的。先前的研究报道，平面细胞极性（PCP）基因 vangl2 突变的斑马鱼胚胎脊髓中形成双侧管腔。在本研究中，在培养的细胞中，R-Ras 与 Vangl2 共免疫沉淀，但与另一个 PCP 调节剂 Pricke1 不沉淀。有趣的是，与野生型或组成性激活（GTP 结合）形式的 R-Ras 相比，R-Ras 的无核苷酸点突变体与 Vangl2 的相互作用更强。R-Ras 可能与 Vangl2 合作调节斑马鱼脊髓中神经管的形成。
