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，并且出生缺陷，如脊柱侧凸， 影响脊柱的疾病可能源于任何一种组织的异常发育。有许多形式的 在发育过程中可能发生的脊柱畸形，包括脊柱侧凸或脊柱内的畸形骨 柱遗传研究，包括我们对斑马鱼的分析，表明干扰体节发生的突变导致 斑马鱼、小鼠和人类的脊柱侧凸表型。许多问题仍然存在的分子基础， 先天性脊柱畸形 胚胎发育需要许多基因调控网络（GRN），这些网络被激活和抑制 这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，同时使用相对小的 基因数量。脊椎动物体轴的伸长过程需要组织图案化、细胞图案化、细胞图案化和细胞图案化。 细胞分化和细胞迁移。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GRNs的动态控制的严格调节。这 一个项目使用灭活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 将产生线，以创建热冲击控制的双向CRISPRi和CRISPRa系统， 上调脊柱发育所需的转录因子的表达。表型将是 通过形态学和RT-qPCR表征。RNA测序将用于识别基因的下游， 这些转录因子。在目标2中，CRISPRi/CRISPRa系统将用于进行靶向遗传修饰。 筛选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，使人们更好地了解组合的 基因在发育中的功能以及脊椎动物身体伸长的遗传和细胞动力学。