Elucidating the Mechanism of Precision in Vertebral Segmentation

摘要 尽管基因表达不可避免的波动，胚胎发育是稳健和可重复的， 这需要几种缓冲随机基因表达的机制。一个有趣的例子， 时空模式是体节的节律性分割，体节是脊椎动物的前体。 柱体节的周期性分裂受Hes/Her基因的振荡表达控制 家族;被称为脊椎动物分段时钟。为了测量振荡的幅度及其细胞间的相互作用， 细胞变异性（噪音），我们计数RNA分子转录的两个主分割时钟基因（her 1 和her 7）使用单分子荧光原位杂交（smFISH）。我们发现低振幅， 野生型胚胎中HER 1和HER 7转录的噪音和转录爆发。在Notch信号中 突变体，由于转录爆发减少，振荡幅度降低，变异性增加 这是由于基因外部噪声增加。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。损失涉及多个因素 在基本的转录机制造成分割缺陷和减少转录的时钟 基因.这些蛋白质，包括Rtf 1，Ctr 9和Spt 6，释放近端启动子暂停的Pol-II。底层 机制仍然难以捉摸，但我们初步的酵母双杂交数据显示，Spt 6与Her 7相互作用， 主分段时钟调节器。在本提案中，我们将测试以下假设，这些假设建立在我们的 广泛的初步数据和文献：1）聚合酶在时钟基因的近端启动子处暂停 导致转录爆发; Pol II暂停的频率由Her 1/7阻遏物和Notch控制 2）Fgf、Wnt和RA信号传导活性的后-前梯度控制所观察到的 转录噪声的空间分布，3）基因表达噪声被时钟中的冗余缓冲 机制，以及短距离和长距离细胞间信号传导：目的1。确定的来源 分段时钟基因表达的随机波动。目标2.调查信号是如何 梯度缓冲分段时钟中的表达噪声。目标3：了解噪声传播是如何 在分段时钟的下游被抑制。Hes/Her蛋白的振荡控制着从 在各种组织中从增殖到分化。在某些癌症中检测到它们的表达， 它们的抑制恢复分化。阐明指导其表达的分子机制， 体节发生对于理解和潜在地预防脊椎畸形是重要的， 增强干细胞增殖和开发抗癌疗法。因此，该应用程序具有 与国家卫生研究所的使命密切相关。

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.