Unraveling the essential gene regulatory network underlying notochord development in Ciona

揭示海鞘脊索发育的重要基因调控网络

• 批准号: 9884798
• 负责人: ANNA DI GREGORIO
• 金额: $ 7.93万
• 依托单位: NEW YORK UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-04-01 至 2023-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9884798
• 关键词: Address; Adult; Biological Models; Body Patterning; Brachyury protein; CRISPR/Cas technology; Cell Shape; Chordata; Chordoma; Complex; Comprehension; Congenital Abnormality; Defect; Deformity; Development; Embryo; Embryonic Development; Embryonic Structures; Ensure; Essential Genes; Event; Evolution; Gene Expression; Gene Transfer Techniques; Generations; Genes; Genetic; Genome; Genomics; Goals; Human; Intervertebral disc structure; Invertebrates; Knowledge; Laboratories; Lead; Life; Light; Link; Maintenance; Malignant - descriptor; Malignant Bone Neoplasm; Mediating; Morphogenesis; Mus; Mutation; Nature; Neural Tube Defects; Nucleic Acid Regulatory Sequences; Orthologous Gene; Pain; Pattern; Phylogenetic Analysis; Play; Public Health; Regulator Genes; Research; Resources; Role; Spinal Dysraphism; Structure; System; Techniques; Testing; Transgenic Organisms; Urochordata; Vertebral column; Vertebrates; Work; XBP1 gene; Zebrafish; ascidian; convergent extension; experimental study; genetic makeup; genome editing; insight; interest; knock-down; malformation; mutant; notochord; notochord development; postnatal development; spine bone structure; transcription factor; transcriptome; tumor; tumorigenesis

PROJECT SUMMARY/ABSTRACT In humans and all chordates the notochord plays an indispensable role in supporting and patterning the developing body plan. During embryogenesis, defects in notochord formation are the cause of birth defects such as spina bifida and vertebral deformities, while during post-natal development notochord remnants can cause painful discopathy and, more rarely, chordomas, malignant and potentially life-threatening tumors. Despite the considerable biomedical interest in the role of the notochord in spine development and in tumorigenesis, both the comprehension of the specific functions of evolutionarily conserved transcription factors in notochord development and the knowledge of the cis-regulatory mechanisms controlling notochord gene expression are still fragmentary. These gaps in knowledge greatly limit our ability to pinpoint the genes and regulatory sequences responsible for notochord-derived defects and to potentially rectify their expression. We use the invertebrate chordate Ciona (tunicate, or sea squirt) to investigate the role of evolutionarily conserved transcription factors in notochord formation and to uncover the regulatory mechanisms that ensure their proper deployment during the development of this crucial structure. Ciona was selected for these studies because it provides a fast-developing model system with an experimentally accessible notochord and a compact, fully sequenced genome, and because several genes expressed in the Ciona notochord are evolutionarily conserved across all chordates. The objective of this project is to establish in our laboratory the CRISPR/Cas9-mediated genome editing technique for systematically knocking down notochord transcription factors of interest and for editing their regulatory regions and determine the effects on gene expression. These resources will advance our research on the evolutionarily conserved genetic toolkit that is employed by widely different chordates to properly form the notochord and guide the correct formation of the body plan. The objective of this proposal will be obtained through the following approaches: the identification of the cis- regulatory modules (CRMs) that control notochord gene expression of 10 evolutionarily conserved transcription factors (Aim 1); the elucidation of the function of these transcription factors in specific steps of notochord morphogenesis (Aim 2). Completion of these experiments will shed light on the components of the notochord GRN and their reciprocal regulatory interactions and will inform and accelerate studies of notochord-derived birth defects in other chordates.

项目概要/摘要 在人类和所有脊索动物中，脊索在支撑和塑造脊索方面发挥着不可或缺的作用。 制定身体计划。在胚胎发生过程中，脊索形成缺陷是出生缺陷的原因 例如脊柱裂和脊椎畸形，而在产后发育期间，脊索残余物可以 引起疼痛性椎间盘病，更罕见的是，引起脊索瘤、恶性和可能危及生命的肿瘤。 尽管生物医学界对脊索在脊柱发育和脊柱发育中的作用产生了相当大的兴趣 肿瘤发生，既是对进化保守转录的特定功能的理解 脊索发育的因素和控制脊索的顺式调节机制的知识 基因表达仍然是碎片化的。这些知识差距极大地限制了我们查明基因的能力 以及负责脊索衍生缺陷并可能纠正其表达的调节序列。 我们使用无脊椎动物脊索动物 Ciona（被囊动物或海鞘）来研究进化的作用 脊索形成中的保守转录因子，并揭示确保 在这一关键结构的开发过程中对其进行了适当的部署。 Ciona 被选中进行这些研究 因为它提供了一个快速开发的模型系统，具有可通过实验访问的脊索和 紧凑、完全测序的基因组，并且因为玻璃海鞘脊索中表达的几个基因是 在所有脊索动物中进化上都是保守的。该项目的目标是在我们的实验室建立 CRISPR/Cas9介导的基因组编辑技术系统性地抑制脊索转录 感兴趣的因子和编辑其调控区域并确定对基因表达的影响。这些 资源将推进我们对进化保守遗传工具包的研究，该工具包被广泛使用 不同的脊索动物正确形成脊索并指导身体计划的正确形成。 该提案的目标将通过以下方法实现： 控制 10 个进化保守转录的脊索基因表达的调控模块 (CRM) 因素（目标 1）；阐明这些转录因子在脊索特定步骤中的功能 形态发生（目标 2）。这些实验的完成将揭示脊索的组成部分 GRN 及其相互调节相互作用，将为脊索衍生的研究提供信息并加速 其他脊索动物的出生缺陷。