Temporal Analysis of Combinatorial Gene Function during Vertebrate Body Elongation

项目摘要 脊柱由神经和中胚层组织组成，以及出生缺陷，如脊柱侧弯， 影响脊柱的疾病可能源于任一种组织的异常发育。有许多形式的 发育过程中可能发生的脊柱畸形，包括脊柱侧弯或脊柱内骨骼畸形 纵队。基因研究，包括我们对斑马鱼的分析表明，扰乱体细胞发生的突变会导致 斑马鱼、小鼠和人类的脊柱侧弯表型。许多关于分子基础的问题仍然存在 先天性脊柱畸形。 胚胎发育需要许多被激活和抑制的基因调控网络(GRN) 这是形态发生的动态控制的基础。这些GRN在整个过程中重复使用 发展，但会产生不同的发展结果。这些差异对于实现 整个生物体发育所需的广泛的细胞表型，同时使用相对较小的 基因的数量。脊椎动物体轴的延长过程需要组织图案化，细胞 多个组织同时分化和细胞迁移。为了让所有这些复杂的过程 为了在胚胎中发生，它们必须随着时间的推移受到多个GRN的动态控制的严格调控。这 Project使用失活的Cas9技术的创新应用来研究两个转录因子如何 调节脊柱发育早期的中胚层分化和细胞迁移。转基因 将生成线路以创建热冲击控制的双向CRISPRi和CRISPRa系统，以向下和 上调脊柱发育所需的转录因子的表达。表型将是 以形态和RT-qPCR为特征。核糖核酸测序将被用来识别 这些转录因子。在AIM 2中，CRISPRi/CRISPRa系统将用于进行有针对性的遗传 筛选这些靶基因在人体伸长过程中调节细胞迁移的作用。 这项工作将阐明这些转录因子的作用，使我们能够更好地理解组合 基因在发育中的作用以及脊椎动物身体伸长的遗传和细胞动力学。

Project Summary The spinal column is composed of neural and mesodermal tissues, and birth defects, such as scoliosis, that affect the spinal column can originate from abnormal development of either tissue. There are many forms of spinal malformation that can occur in development, including scoliosis, or misshapen bone within the spinal column. Genetic studies, including our zebrafish analyses, show that mutations that perturb somitogenesis lead to scoliosis phenotypes in zebrafish, mice and humans. Many questions remain about the molecular basis for congenital spinal malformations. Embryonic development requires many gene regulatory networks (GRN) that are activated and repressed overtime which underlie the dynamic control of morphogenesis. These GRNs are used repeatedly throughout development but give rise to different developmental outcomes. These differences are critical to achieving the wide array of cellular phenotypes required for development of an entire organism while using a relatively small number of genes. The process of elongation of the vertebrate body axis requires tissue patterning, cellular differentiation and cell migration of multiple tissues simultaneously. In order for all of these complex processes to occur in the embryo they must be tightly regulated by the dynamic control of multiple GRNs over time. This project uses an innovative application of deactivated Cas9 technology to investigate how two transcription factors regulate mesodermal differentiation and cell migration of during early spinal column development. Transgenic lines will be generated to create heat shock controlled bi-partite CRISPRi and CRISPRa systems to down- and up-regulate expression of transcription factors required for spinal column development. Phenotypes will be characterized by morphology and RT-qPCR. RNA sequencing will be used to identify the genes downstream of these transcription factors. In Aim 2, the CRISPRi/CRISPRa systems will be used to perform a targeted genetic screen to interrogate the roles of these target genes in the regulation of cell migration during body elongation. This work will elucidate the roles of these transcription factors, allowing a better understanding of combinatorial gene function in development as well as the genetic and cellular dynamics of vertebrate body elongation.