新转录因子VRTN调控胸椎数的分子机制研究

The vertebral column is the defining characteristic of vertebrates. Through genetic and molecular assays, we firstly identified that a novel transcription factor, VRTN, controls the number of thoracic vertebrae in pigs and mice. The mutant pigs with higher VRTN expression have one more thoracic segment. VRTN-null mice are embryonically lethal with fewer somites than their wild-type and heterozygous littermates at the late thoracic somite stages. Among heterozygous animals, one third has one less thoracic vertebra and others exhibit abnormal phenotypes on last thoracic element. Based on these results, we planned to study the segment of thoracic vertebrae using the existed VRTN knockout mice as follows. To identify the target genes of VRTN and the involved signal pathway, we planned to measure gene expression profiles with RNA-seq and identify the target gene with ChIP-seq for whole embryos of mice with knockout, heterozygous and wild-type genotypes from mates between VRTN+/- mice. Through in situ hybridization assays, we might identify the different expression of VRTN and the above critical gene in whole embryos with three genotypes. We aim to give a hypothesis how to VRTN control the number of thoracic vertebrae.The findings would provide important insights into the existing mechisms on the segment of thoracic vertebra, and provide necessary theoretical evidences to genetic diagnosis of human congenital vertebra deformity and to add the number of thoracic vertebrae in livestock using gene manipulation technique.

脊椎骨是脊椎动物的最主要特征。新转录因子VRTN是申请人前期通过遗传和分子手段鉴别到的控制猪和小鼠胸椎数量的基因。在猪中，增加VRTN表达量的因果突变导致猪增加一根胸椎；VRTN基因敲除小鼠胚胎致死、胸部体节分节异常，杂合子小鼠缺少一根胸椎或最后一根胸椎缺少单侧肋骨。基于上述结果，项目组拟将VRTN敲除鼠作为研究脊椎动物胸部体节分节的模型。通过原位杂交技术，确定VRTN在敲除、杂合和野生型胚胎中的时空表达情况。通过ChIP-seq和RNA-seq鉴别敲除、杂合和野生型鼠胚在胸部体节发育期的差异基因，确定VRTN的靶基因及调节的信号通路。进一步通过分子手段，检测VRTN下游与分节相关基因的表达情况，最终揭示VRTN参与胸部体节分节和分化的机理。预期结果将丰富现有胸椎分节理论，同时为人类先天性脊柱畸形的遗传诊断和利用基因修饰技术增加家畜的脊椎数提供重要理论依据。

【背景】申请人前期通过遗传手段鉴别到控制哺乳动物胸椎数量的因果基因VRTN。课题组发现增加VRTN表达量的因果突变导致猪增加一根胸椎；VRTN基因敲除小鼠胚胎致死，杂合子小鼠缺少一根胸椎或最后一根胸椎缺少单侧肋骨。本课题旨在明确VRTN调控胸椎数量的分子机制，为畜牧胸椎数量选育和人类先天性脊柱畸形诊断提供理论依据。.【结果】整合猪和小鼠遗传研究结果，课题组确定VRTN的剂量决定胸椎数量，上调VRTN表达增加胸椎数量，下调VRTN表达减少胸椎数量。活体和细胞水平实验证明VRTN在胸部体节分节时高量表达，因果突变增加体节分节区域VRTN的表达。整合多组学和细胞水平研究，课题组首次明确VRTN是转录因子，调控Hes5的表达。通过对E9.0，E9.5,E10.0和E10.5时期的Vrtn-/-和Vrtn+/+胚胎的转录组测序，发现VRTN敲除下调了上述时期Hes5的表达。染色质免疫共沉淀方法发现VRTN直接调控Hes5的转录。前人研究已发现Hes5缺陷小鼠体节分节异常和胸椎数量减少，与VRTN杂合小鼠的表型一致。整合课题组现在的发现及过去的报道，我们提出一个解释VRTN剂量决定哺乳动物胸椎数量的模型。VRTN是胸部体节分节的关键因子，上调Hes5的表达，作为Notch信号的效应物Hes5通过影响胸部体节分节的速率和特化决定体节的数量和椎骨的类型。此外，VRTN敲除小鼠在心脏、神经系统也表现异常，说明VRTN参与多个器官的发育。 .【结论】总之，课题组确定VRTN是哺乳动物胸部体节发育的关键增强子，其剂量决定了胸椎的数量。VRTN上调Notch信号效应物Hes5的表达，影响着体节分节的速率，最终决定了胸椎的数量。此外，VRTN也参与了心脏和神经的发育。现有结果丰富了胸椎分节理论，并为今后开展VRTN的功能研究奠定了基础。