Transcriptomics Profiling of Vertebral Fusion in Evolution and Development

在脊柱的正常成熟过程中，椎体融合在一起形成骨盆带的骶骨。在鸟类中，脊柱融合更为广泛，大约有一半的椎骨融合成复合骨结构。鸟类融合的增加是在进化过程中为了适应飞行而出现的。正常椎体融合的生物学机制在很大程度上是未知的。然而，利用鸡作为模型生物，PI令人惊讶地发现，无菌炎症推动了尾巴的融合。炎症通常是对疾病或创伤的反应，但在这里，PI揭示了无菌炎症在正常骨骼发育中的新作用。PI用皮质类固醇抑制炎症，并发现这种治疗抑制尾部融合，证明皮质类固醇对脊柱成熟的作用是以前未知的。这些发现的意义是巨大的，不仅对鸟类的进化，而且对脊椎动物的轴向发育，以及对人类皮质类固醇治疗可能产生的副作用。在这个项目中，PI将确定炎症是否是正常椎体融合的共同驱动因素，或者是否有多种机制在这一过程中进化。此外，作为长期合作的一部分，PI将把蒙大拿州立大学和克莱姆森大学的基因组学能力联系起来。这项研究基础设施改善轨道4 EPSCoR研究人员（RII轨道4:NSF）项目将为蒙大拿州立大学（MSU）的助理研究教授提供奖学金。这项工作将与克莱姆森大学的研究人员合作进行。这项提议的目标是接受转录组学训练，测试无菌炎症是椎体融合的普遍驱动因素的假设，并向蒙大拿州立大学的研究人员推广克莱姆森大学的基因组学设施。PI将通过全转录组RNAseq比较融合鸡骶椎间盘组织与尾侧非融合组织的转录谱。具体来说，PI将研究鸡不同轴向区域的髓核椎间盘结构和炎症反应对融合的潜在贡献。这些数据将评估强直是通过共同机制还是独立机制发生的，并将深入了解鸟类轴向进化。克莱姆森大学是进行这些研究的理想场所，不仅因为它的基因组学设施，而且因为它的家禽养殖场，这是学术界为数不多的研究家禽中心之一。培训将包括RNA提取方法和生物信息学分析，包括GO和KEGG分析。从这次合作中收集到的专业知识将应用于RNAseq教学模块，该模块将为密歇根州立大学的研究人员建立，以利用克莱姆森的基因组学设施。MSU目前提供微阵列，但不提供RNAseq服务。这些拟议的研究将使PI能够解决鸟类的椎体融合机制，并最终将PI的研究扩展到各种脊椎动物的正常和病理融合，包括哺乳动物疾病模型。本科生指导和公众科学教育深深植根于这个项目。PI将继续培训现有学生，招收更多本科生，并开展多种多样的外联工作。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

During normal maturation of the spine, vertebrae fuse together to form the sacrum in the pelvic girdle. In birds, spine fusion is more extensive, with approximately half of the vertebrae are fused into compound bony structures. Increased fusion in birds arose during evolution as an adaptation for flight. The biological mechanisms responsible for normal vertebral fusion are largely unknown. However, using the chicken as a model organism, the PI made the surprising discovery that sterile inflammation drives fusion in the tail. Inflammation is generally a response to disease or trauma, but here the PI uncovered a novel role for sterile inflammation in normal bone development. The PI inhibited inflammation with corticosteroids, and found that this treatment inhibits tail fusion, demonstrating a previously unknown effect of corticosteroids on spine maturation. The implications for these findings are vast, not just for avian evolution but for axial development across vertebrates, and for the possible side effects of corticosteroid treatment in humans. For this project, the PI will determine whether inflammation is a common driver of normal vertebral fusion, or whether multiple mechanisms have evolved for this process. Additionally, as part of a long-standing collaboration, the PI will link the genomics capabilities of Montana State University and Clemson University.This Research Infrastructure Improvement Track-4 EPSCoR Research Fellows (RII Track-4:NSF) project would provide a fellowship to an Assistant Research Professor at Montana State University (MSU). This work would be conducted in collaboration with researchers at Clemson University. The goals of this proposal are to receive transcriptomics training, to test the hypothesis that sterile inflammation is a universal driver of vertebral fusion, and to promote Clemson University’s genomics facility to Montana State University researchers. The PI will compare the transcriptional profiles of fusing chicken synsacrum intervertebral disc tissues to caudal non-fusing tissue by whole transcriptome RNAseq. Specifically, the PI will investigate the potential contributions of nucleus pulposus disc structures and inflammatory response to fusion in different axial regions of the chicken. These data will assess if ankylosis occurs via common or independent mechanisms, and will lend insight into avian axial evolution. Clemson University is ideal for these studies not only for its genomics facility, but also for its poultry farm, one of the few research poultry centers in academia. Training will include RNA extraction methods and bioinformatics analysis, including GO and KEGG profiling. The expertise gleaned from this collaboration will be applied to an RNAseq instructional module that will be built for MSU researchers to utilize Clemson’s genomics facility. MSU currently offers microarray, but not RNAseq services. These proposed studies will allow the PI to resolve vertebral fusion mechanism(s) in birds and ultimately expand the PI’s investigations to normal and pathological fusion in a variety of vertebrates, including mammalian disease models. Undergraduate mentorship and public education of science are deeply rooted in this project. The PI will continue to train existing students, recruit additional undergraduates, and pursue multiple and varied outreach efforts.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.