Temporal Analysis of Combinatorial Gene Function during Vertebrate Body Elongation

脊椎动物身体伸长过程中组合基因功能的时间分析

• 批准号: 10606014
• 负责人: Abigail Alexandra Kindberg
• 金额: $ 6.72万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-03-06 至 2026-03-05
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10606014
• 关键词: Address; Affect; Anterior; Biological Assay; CRISPR interference; CRISPR-mediated transcriptional activation; Candidate Disease Gene; Cells; Cellular Morphology; Complex; Computer Assisted; Congenital Abnormality; Data; Data Analyses; Development; Developmental Process; Down-Regulation; Embryo; Embryonic Development; Exhibits; Family; Gene Expression; Gene Expression Profiling; Genes; Genetic; Genetic Screening; Genetic study; Heat-Shock Response; Human; Image; Individual; Lead; Mesoderm; Messenger RNA; Molecular; Morphogenesis; Morphology; Motion; Mus; Mutation; Neural tube; Organism; Outcome; Pattern; Phenotype; Play; Process; Regulation; Repression; Role; Somites; Spinal; System; Tail; Technology; Time; Tissues; Transcription Coactivator; Transcription Repressor; Transgenes; Transgenic Organisms; Up-Regulation; Vertebral column; Visual; Work; Zebrafish; bone; cell motility; combinatorial; deactivated CRISPR-Cas9; gain of function; gastrulation; gene function; gene regulatory network; genetic analysis; innovation; interest; loss of function; malformation; member; migration; mutant; neural; novel strategies; pleiotropism; progenitor; recruit; reverse genetics; scoliosis; screening; somitogenesis; transcription factor; transcriptome sequencing

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.

项目摘要 脊柱由神经和中胚层组织组成，以及诸如脊柱侧弯的先天缺陷， 影响脊柱的可能起源于任何两组的异常发育。有多种形式 发育中可能发生的脊柱畸形，包括脊柱侧弯或脊柱中的骨骼畸形 柱子。包括我们的斑马鱼分析在内的遗传研究表明，扰动体重生成的突变 在斑马鱼，小鼠和人类中进行脊柱侧弯表型。关于分子基础的许多问题仍然存在 先天性脊柱畸形。 胚胎开发需要许多受激活和抑制的基因调节网络（GRN） 加时赛是形态发生动态控制的基础。这些GRN在整个过程中反复使用 发展但会带来不同的发展结果。这些差异对于实现 使用相对较小的整个生物所需的大量细胞表型 基因数量。脊椎动物体轴伸长的过程需要组织图案，细胞 多个组织的分化和细胞迁移同时。为了所有这些复杂的过程 要在胚胎中发生，它们必须通过随着时间的推移的多个GRN的动态控制来严格调节。这 项目使用停用CAS9技术的创新应用来研究两个转录因素 调节早期脊柱发育过程中的中胚层分化和细胞迁移。转基因 将生成线路，以创建热量控制的双方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。 RNA测序将用于识别下游的基因 这些转录因子。在AIM 2中，CRISPRI/CRISPRA系统将用于执行目标遗传 筛选以询问这些靶基因在身体伸长过程中细胞迁移调节中的作用。 这项工作将阐明这些转录因子的作用，从而更好地了解组合 基因在发育以及脊椎动物体伸长的遗传和细胞动力学中的功能。