Guo Long, Yamashita Hiroshi, Kou Ikuyo, Takimoto Aki, Meguro-Horike Makiko, Horike Shin-ichi, Sakuma Tetsushi, Miura Shigenori, Adachi Taiji, Yamamoto Takashi, Ikegawa Shiro, Hiraki Yuji, Shukunami Chisa
Department of Cellular Differentiation, Institute for Frontier Medical Sciences, Kyoto University, Kyoto, Japan.
Department of Molecular Biology and Biochemistry, Division of Basic Life Sciences, Institute of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan.
PLoS Genet. 2016 Jan 28;12(1):e1005802. doi: 10.1371/journal.pgen.1005802. eCollection 2016 Jan.
Previously, we identified an adolescent idiopathic scoliosis susceptibility locus near human ladybird homeobox 1 (LBX1) and FLJ41350 by a genome-wide association study. Here, we characterized the associated non-coding variant and investigated the function of these genes. A chromosome conformation capture assay revealed that the genome region with the most significantly associated single nucleotide polymorphism (rs11190870) physically interacted with the promoter region of LBX1-FLJ41350. The promoter in the direction of LBX1, combined with a 590-bp region including rs11190870, had higher transcriptional activity with the risk allele than that with the non-risk allele in HEK 293T cells. The ubiquitous overexpression of human LBX1 or either of the zebrafish lbx genes (lbx1a, lbx1b, and lbx2), but not FLJ41350, in zebrafish embryos caused body curvature followed by death prior to vertebral column formation. Such body axis deformation was not observed in transcription activator-like effector nucleases mediated knockout zebrafish of lbx1b or lbx2. Mosaic expression of lbx1b driven by the GATA2 minimal promoter and the lbx1b enhancer in zebrafish significantly alleviated the embryonic lethal phenotype to allow observation of the later onset of the spinal curvature with or without vertebral malformation. Deformation of the embryonic body axis by lbx1b overexpression was associated with defects in convergent extension, which is a component of the main axis-elongation machinery in gastrulating embryos. In embryos overexpressing lbx1b, wnt5b, a ligand of the non-canonical Wnt/planar cell polarity (PCP) pathway, was significantly downregulated. Injection of mRNA for wnt5b or RhoA, a key downstream effector of Wnt/PCP signaling, rescued the defective convergent extension phenotype and attenuated the lbx1b-induced curvature of the body axis. Thus, our study presents a novel pathological feature of LBX1 and its zebrafish homologs in body axis deformation at various stages of embryonic and subsequent growth in zebrafish.
此前，我们通过全基因组关联研究在人类瓢虫同源盒1（LBX1）和FLJ41350附近鉴定出一个青少年特发性脊柱侧凸易感基因座。在此，我们对相关的非编码变异进行了表征，并研究了这些基因的功能。染色体构象捕获分析表明，与最显著相关的单核苷酸多态性（rs11190870）所在的基因组区域与LBX1 - FLJ41350的启动子区域发生物理相互作用。在HEK 293T细胞中，LBX1方向的启动子与包含rs11190870的590 bp区域结合，携带风险等位基因时的转录活性高于携带非风险等位基因时。在斑马鱼胚胎中普遍过表达人类LBX1或任何一种斑马鱼lbx基因（lbx1a、lbx1b和lbx2），而非FLJ41350，会导致身体弯曲，随后在脊柱形成之前死亡。在转录激活样效应核酸酶介导的lbx1b或lbx2基因敲除斑马鱼中未观察到这种身体轴变形。由GATA2最小启动子和lbx1b增强子驱动的lbx1b镶嵌表达在斑马鱼中显著减轻了胚胎致死表型，从而能够观察到脊柱弯曲的后期发作，无论是否伴有椎体畸形。lbx1b过表达引起的胚胎身体轴变形与原肠胚形成期胚胎主轴伸长机制的一个组成部分——汇聚延伸缺陷有关。在过表达lbx1b的胚胎中，非经典Wnt/平面细胞极性（PCP）途径的配体wnt5b显著下调。注射wnt5b或RhoA（Wnt/PCP信号的关键下游效应物）的mRNA可挽救有缺陷的汇聚延伸表型，并减轻lbx1b诱导的身体轴弯曲。因此，我们的研究揭示了LBX1及其斑马鱼同源物在斑马鱼胚胎及后续生长各阶段身体轴变形中的一种新的病理特征。
