Elucidating the Mechanism of Precision in Vertebral Segmentation

阐明椎骨分割的精确机制

基本信息

批准号：
9889967
负责人：
Ertugrul M Ozbudak
金额：
$ 31.2万
依托单位：
CINCINNATI CHILDRENS HOSP MED CTR
依托单位国家：
美国
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-07-01 至 2021-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9889967
关键词：
Anterior Biological Buffers Cells Congenital Abnormality DNA Polymerase II Data Defect Embryo Embryonic Development Feedback Fluorescent in Situ Hybridization Frequencies Gene Expression Gene Family Genes Genetic Genetic Transcription Genome Human In Situ Hybridization Individual Left Literature Malignant Neoplasms Measures Mission Molecular Mosaicism Mutation Noise Pathway interactions Pattern Penetrance Periodicity Pharmaceutical Preparations Phenotype Polymerase Proteins RNA Recurrence Reproducibility Role Segmentation Clock Pathway Siblings Side Signal Transduction Somites Source Testing Time Tissues Transgenic Animals Tretinoin United States National Institutes of Health Vertebral column Yeasts base dosage experimental study gene discovery gene environment interaction loss of function malformation mutant notch protein prevent progenitor promoter single molecule somitogenesis spatiotemporal spine bone structure stem cell proliferation therapy development transcription factor yeast two hybrid system

项目摘要

Abstract Despite unavoidable fluctuations in gene expression, embryonic development is robust and reproducible, which necessitates several mechanisms buffering stochastic gene expression. An intriguing example of robust spatiotemporal patterning is the rhythmic segmentation of somites, which are precursors of the vertebral column. Periodic segmentation of somites is controlled by the oscillatory expression of the Hes/Her gene family; known as the vertebrate segmentation clock. To measure the amplitude of oscillations and their cell-to- cell variability (noise), we counted RNA molecules transcribed by two master segmentation clock genes (her1 and her7) using single molecule fluorescent in situ hybridization (smFISH). We found low amplitudes, high noise and transcriptional bursts of her1 and her7 transcription in wild-type embryos. In Notch-signaling mutants, amplitudes of oscillations decreased due to reduced transcriptional bursts, and variability increased due to increased gene extrinsic noise. Furthermore, transcriptional noise increased from the posterior progenitor zone towards the anterior segmentation zone, in wild-type embryos. Loss of several factors involved in the basic machinery of transcription resulted in segmentation defects and reduced transcription of clock genes. These proteins, including Rtf1, Ctr9 and Spt6, release proximal-promoter paused Pol-II. The underlying mechanism remains elusive but our preliminary, yeast-two-hybrid data show that Spt6 interacts with Her7, one of the master segmentation clock regulators. In this proposal, we will test the following hypotheses built on our extensive preliminary data and literature: 1) polymerase pausing at the proximal promoters of clock genes causes bursts of transcription; the frequency of Pol II pausing is controlled by Her1/7 repressors and Notch activators, 2) the posterior-to-anterior gradients of Fgf, Wnt, and RA signaling activity control the observed spatial profile of transcriptional noise, 3) gene expression noise is buffered by redundancy in the clock machinery, as well as short- and long-distance cell-to-cell signaling: Aim 1. Determine the sources of stochastic fluctuations in the expression of segmentation clock genes. Aim 2. Investigate how signaling gradients buffer expression noise in the segmentation clock. Aim 3. Understand how noise propagation is suppressed downstream of the segmentation clock. Oscillations of Hes/Her proteins control the switch from proliferation to differentiation in various tissues. Their expression has been detected in certain cancers, while their inhibition restores differentiation. Elucidating the molecular mechanisms that guide their expression in somitogenesis is significant for understanding and potentially preventing vertebral malformations, but also for enhancing stem cell proliferation and developing therapies against cancer. Therefore, this application has strong relevance to the mission of the National Institute of Health.

抽象的尽管基因表达的波动不可避免，但胚胎发育是稳健且可重复的，这需要多种缓冲随机基因表达的机制。一个有趣的稳健例子时空模式是体节的节律分割，体节是椎体的前身柱子。体节的周期性分割由 Hes/Her 基因的振荡表达控制家庭;称为脊椎动物分节时钟。测量振荡的幅度及其细胞间的细胞变异性（噪声），我们计算了由两个主分段时钟基因（her1 和her7) 使用单分子荧光原位杂交(smFISH)。我们发现低振幅，高野生型胚胎中her1和her7转录的噪音和转录爆发。陷波信号突变体中，由于转录爆发减少，振荡幅度降低，变异性增加由于基因外源噪声增加。此外，转录噪音从后部增加在野生型胚胎中，祖细胞区朝向前分割区。涉及多个因素的损失转录的基本机制导致分段缺陷和时钟转录减少基因。这些蛋白质，包括 Rtf1、Ctr9 和 Spt6，释放近端启动子暂停的 Pol-II。底层的机制仍然难以捉摸，但我们的初步酵母双杂交数据显示 Spt6 与 Her7 相互作用，Her7 是其中之一主分段时钟调节器。在本提案中，我们将测试基于我们的以下假设大量的初步数据和文献：1) 聚合酶在时钟基因的近端启动子处暂停引起转录爆发； Pol II 暂停的频率由 Her1/7 阻遏物和 Notch 控制激活剂，2）Fgf、Wnt 和 RA 信号活动的后到前梯度控制观察到的转录噪声的空间分布，3) 基因表达噪声通过时钟冗余来缓冲机器，以及短距离和长距离细胞间信号传导：目标 1. 确定信号源分段时钟基因表达的随机波动。目标 2. 研究信号如何传递梯度缓冲分段时钟中的表达噪声。目标 3. 了解噪声传播方式抑制分段时钟的下游。 Hes/Her 蛋白的振荡控制着从在各种组织中增殖至分化。它们的表达已在某些癌症中检测到，而它们的抑制可恢复分化。阐明指导其表达的分子机制体节发生对于理解和潜在预防椎骨畸形具有重要意义，而且对于增强干细胞增殖并开发抗癌疗法。因此，该应用程序具有与国立卫生研究院的使命密切相关。