mapk7基因突变介导斑马鱼脊索异常发育致特发性脊柱侧凸的机制研究

Idiopathic scoliosis (idiopathic scoliosis, IS) is the most common spinal deformity, the underlying causes are still unknown. We have identified a new IS-predisposing gene named MAPK7 in IS families and genotyped in multicenter populations (unpublished). However, the specific mechanism underlying MAPK7 predisposing to IS is still unknown. Zebrafish has been an important animal model to study the developmental deformities of spine. The pre-experiment showed that mapk7-morpholinos knockdown zebrafish showed structural abnormalities of notochord and body curvature deformity. It has been previously demonstrated that defects of notochord structure and function can cause varied developmental disorders in spine. We hypothesized that variants in MAPK7 would contribute to IS by causing abnormalities of notochord. This project intends to further establish mapk7-knockout zebrafish model by CRISPR/Cas9 technology (F0 showed IS phenotype), Tg (snap25a::mapk7) zebrafish by tol2 system, conditional mapk7 knockout zebrafish by vivo-morpholino, and explore the development of notochord and spine of these models by hybridization in situ, notochord staining and bone staining. The relationship between spatio-temporal expression of MAPK7 and structural and functional protein of notochord in human embryo will also be explored. This project will illuminate the mechanism how mutant mapk7 predisposes to scoliosis by effecting abnormal development of the notochord in vivo.

特发性脊柱侧凸（idiopathic scoliosis, IS）是最常见的脊柱畸形，病因不明。我们前期定位出IS一个新致病基因——MAPK7（未发表），然而MAPK7突变导致IS的机制未知。斑马鱼是研究脊柱发育的重要工具。预实验发现morpholino（MO）敲低斑马鱼mapk7出现脊索鞘结构异常和中轴侧偏畸形，而研究表明脊索结构和功能缺陷可造成不同程度的脊柱发育障碍。因此，我们提出MAPK7基因突变可能通过介导脊索异常发育导致IS发生的假设。本项目拟应用CRISPR/Cas9敲除mapk7构建IS斑马鱼侧凸模型（F0出现侧凸表型）、Tol2系统脊索特异性回补mapk7表达模型、及脊索时间特异性敲除mapk7模型，通过原位杂交、脊索与骨骼染色等研究各模型脊索、体节及脊柱发育状况，并进一步验证人胚胎MAPK7的时空表达与脊索发育关系，阐明MAPK7突变通过脊索异常发育致IS发生的分子机制。

特发性脊柱侧凸（idiopathic scoliosis, IS）是最常见的脊柱畸形，病因不明。我们前期定位出IS一个新致病基因——MAPK7，然而MAPK7突变导致IS的机制未知。本项目利用CRISPR/Cas9技术构建mapk7基因敲除的斑马鱼模型，成鱼中轴骨侧方出现不同程度偏曲畸形。Morpholino（MO）特异性敲除斑马鱼mapk7构建的突变体出现脊索发育异常，中轴骨侧方偏曲及钙化延迟，注射野生型MAPK7mRNA可纠正MO诱导的斑马鱼中轴骨侧方偏曲畸形，而突变型MAPK7mRNA则无法纠正其侧方偏曲畸形表型。为了进一步研究mapk7突变是否通过调控下游rsk2蛋白导致侧弯，我们设计打靶rps6ka3b（rsk2编码基因）剪切受体部位的突变型MO并注射斑马鱼胚胎，导致rsk2的磷酸化位点缺失，mapk7无法作用于rsk2, 结果斑马鱼出现体轴偏曲。共注射野生型rps6ka3bmRNA可以逆转纠正突变rps6ka3b导致的斑马鱼体轴偏曲，说明mapk7突变导致的脊柱侧弯部分削弱了下游rsk2的激活。这些研究结果部分解释了mapk7敲除导致小鼠脊柱畸形的可能机制。本项目为阐明mapk7基因突变致脊柱侧凸的致病机理，对于推动IS临床特异性诊疗技术的发展具有重要意义。