Small GTPase R-Ras participates in neural tube formation in zebrafish embryonic spinal cord Shinya Ohata a, b, c, * , Hideko Uga a, b , Hitoshi Okamoto c , Toshiaki Katada a, b a Molecular Cell Biology Laboratory, Research Institute of Pharmaceutical Sciences, Faculty of Pharmacy, Musashino University, Tokyo, 202-8585, Japan b Department of Physiological Chemistry, Graduate School of Pharmaceutical Sciences, University of Tokyo, Tokyo,113-0033, Japan c RIKEN Center for Brain Science, Saitama, 351-0198, Japan article info Article history: Received 8 May 2018 Accepted 12 May 2018 Available online xxx Keywords: R-Ras Neural tube defect Vangl2 Zebrafish abstract Ras related (R-Ras), a small GTPase, is involved in the maintenance of apico-basal polarity in neuro- epithelial 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. © 2018 Elsevier Inc. All rights reserved. 1. Introduction The conformation and activity of the Ras family of GTPase pro- teins are altered by binding to guanine nucleotides [guanosine triphosphate (GTP) and diphosphate (GDP)], and they depend on GTP/GDP forms to transduce intracellular signaling [1 ,2]. Ras related (R-Ras) belongs to the Ras family and functions as a mo- lecular “switch” in the intracellular signaling pathway [3]. In mice, R-Ras promotes maturation of blood vessels and induces endo- thelial lumenogenesis [4e6]. In cultured hippocampal neurons, R- Ras determines neuronal polarity and is involved in axonal collapse [7 ,8]. In the zebrafish developing hind brain, R-Ras maintains apical-basal polarity and apically restricted cell division in neuro- epithelial stem cells [9]. However, the role of R-Ras in early neural development in vivo remains unclear. Neural tube defects (NTDs) are severe neurological birth defects, affecting about 6% of all births [10]. Genes regulating planar cell polarity (PCP) have been implicated in NTDs [11]. For example, van gogh-like 2 (vangl2), a gene encoding a four-transmembrane pro- tein, is mutated in loop-tail mutant mice [12], which develop NTDs [13e15], and in patients with cranial NTDs [16]. In the zebrafish embryonic spinal cord, the neural plate invaginates at the midline, and the lumen is formed by mirror-symmetric cell division of neural stem cells [17]. In vangl2 mutant zebrafish embryos, invag- ination is delayed, but the developmental timing of mirror- symmetric cell division is not affected [17]. Consequently, the lumen is formed bilaterally, resulting in a bilaterally segregated spinal cord. The potential role of R-Ras in the regulation of neural Abbreviations: AMO, anti-sense morpholino oligonucleotide; NTD, neural tube defect; PCP, planar cell polarity; R-Ras, Ras related; Vangl2, van Gogh-like 2; WT, wild type. * Corresponding author. Molecular Cell Biology Laboratory, Research Institute of Pharmaceutical Sciences, Faculty of Pharmacy, Musashino University, Tokyo, 202- 8585, Japan. E-mail address: shiohata@musashino-u.ac.jp (S. Ohata). Contents lists available at ScienceDirect Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc https://doi.org/10.1016/j.bbrc.2018.05.074 0006-291X/© 2018 Elsevier Inc. All rights reserved. Biochemical and Biophysical Research Communications xxx (2018) 1e5 Please cite this article in press as: S. Ohata, et al., Small GTPase R-Ras participates in neural tube formation in zebrafish embryonic spinal cord, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.05.074