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.

项目摘要/摘要 在人类和所有和弦中，脊索在支持和构图中起着必不可少的作用 制定身体计划。在胚胎发生过程中，脊索形成的缺陷是出生缺陷的原因 例如脊柱裂和椎骨畸形，而在产后发育期间，诺诺遗迹可以 引起痛苦的乳腺病，更少是脊柱，恶性和潜在的威胁生命的肿瘤。 尽管对脊索在脊柱发育中的作用以及在 肿瘤发生，既是对进化保守转录的特定功能的理解 脊索发展的因素以及控制脊索的顺式调节机制的知识 基因表达仍然是碎片的。这些知识的差距极大地限制了我们查明基因的能力 以及负责脊索衍生的缺陷的调节序列，并有可能纠正其表达。 我们使用无脊椎动物脊椎动物ciona（皮肤膜或海喷）来研究进化的作用 脊索形成中保守的转录因子，并发现确保的调节机制 它们在这种关键结构的发展过程中的适当部署。这些研究选择了ciona 因为它提供了一个具有实验可访问的曲线和一个快速开发的模型系统和 紧凑的，完全测序的基因组，并且因为在ciona notochord中表达的几个基因是 在所有和弦上的进化保守。该项目的目的是在我们的实验室中建立 CRISPR/CAS9介导的基因组编辑技术，用于系统地击倒脊索转录 感兴趣的因素和编辑其调节区域并确定对基因表达的影响。这些 资源将推进我们对广泛使用的进化保守基因工具包的研究 不同的和弦正确形成脊索并指导身体计划的正确形成。 该提案的目的将通过以下方法获得：识别顺式 控制10个进化转录的脊索基因表达的调节模块（CRMS） 因素（目标1）；在脊索的特定步骤中阐明这些转录因子的功能 形态发生（AIM 2）。这些实验的完成将阐明脊索的组成部分 GRN及其相互监管的相互作用，并将为诺修（Notochord）衍生的研究提供信息和加速研究 其他和弦中的出生缺陷。