Evolution of segmentation in chordates: dissecting the genetic mechanism of somitogenesis in the basal chordate, amphioxus

非技术。了解组成生物体基因组（基因型）的数千个基因如何相互作用，创造出共同组成生物体（表现型）的组织和器官，是国家研究委员会确定的“21世纪生物学的五大挑战”之一。剖析这种基因型-表型关系具有重要的现实意义，是大量研究工作的重点。重要的是，它是理解生物体如何进化的关键，对健康科学、农业、生物技术和生态学有着广泛的影响。为了解决基因型-表型的关系，这项研究使用了文昌鱼，一种简单的鱼状动物，其祖先在5亿年前从脊椎动物的进化路线中分离出来。具体的问题是文文鱼的基因组是如何产生分段的身体肌肉的（类似于脊椎动物，如鱼，允许左右运动的每侧重复的肌肉）。技术。该研究将阐明介导肌肉分割的基因网络，并在单细胞水平上显示该网络如何控制细胞分割成离散的片段（somites）。与脊椎动物的比较将表明，随着脊椎动物变得更大、更复杂，这种遗传机制是如何变化的。文昌鱼的肌肉节延伸全身，但脊椎动物的头部没有肌肉节。究竟是脊椎动物失去了头节，还是文昌鱼是一种新奇的物种，这是有争议的。在文文鱼和脊椎动物之间，肌肉分割的大部分基因网络似乎都是保守的——在两者中，一个“时钟”指定肌肉片段何时形成，Notch/Delta信号介导肌肉分割。然而，在脊椎动物中，分泌蛋白的梯度（主要是Fgfs）决定了节段边界的形成位置，但在文文鱼中，只有头部节段需要Fgfs，而躯干或尾部的节段不需要Fgfs。为了验证这一理论，即随着脊椎动物变大并失去头节，Fgf梯度从头部向后节被增调，文文鱼发育过程中的关键信号通路（Wnt/β-catenin; Fgf, Notch）将在特定的发育时期受到化学抑制剂和激活剂的干扰。基因敲低和过表达会显示出基因层次。通过连续块面扫描电子显微镜（SBSEM）在三维中重建对分割形态的影响，当间隔进行扫描时，可以显示单细胞水平上的形态发生步骤。研究人员将让本科生和高中生直接参与该项目，并与斯克里普斯海洋学研究所的伯奇水族馆和圣地亚哥县教育办公室合作，为高中教师举办讲习班。这些讲习班将利用当地的海洋无脊椎动物向教师展示如何指导学生进行实验设计和数据分析，这些技能是培养下一代科学家的基础。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

Non-technical. Understanding how the thousands of genes that make up an organism’s genome (the genotype) interact to create the tissues and organs that together make up that organism (the phenotype) is one of the five ‘grand challenges of 21st century biology’ identified by the National Research Council. Dissecting this genotype-phenotype relationship is of great practical importance and is the focus of enormous research efforts. Importantly, it is key to understanding how organisms evolve and has wide-ranging implications for the health sciences, agriculture, biotechnology, and ecology. To address the genotype-phenotype relationship, this research uses amphioxus, a simple fish-like animal whose ancestors separated from the evolutionary line leading to vertebrates half a billion years ago. The specific question asked is how the amphioxus genome produces the segmented body muscles (similar to the repeating muscles along each side of a vertebrate such as a fish that allow side to side movement). Technical. The research will elucidate the gene network mediating muscle segmentation, and show at the single-cell level how this network controls the partitioning of cells into discrete segments (somites). Comparisons with vertebrates will show how this genetic mechanism has changed as vertebrates became larger and more complex. In amphioxus, muscle segments extend the full length of the body, but in vertebrates, there are none in the head. It is controversial whether vertebrates lost head segments or they are an amphioxus novelty. Much of the gene network for muscle segmentation appears to be conserved between amphioxus and vertebrates—in both, a “clock” specifies when segments will form, and Notch/Delta signaling mediates segmentation. However, in vertebrates, gradients of secreted proteins, chiefly Fgfs, specify where segment boundaries will form, but in amphioxus only the head segments, not those in the trunk or tail, require Fgfs. To test the theory that as vertebrates grew larger and lost head segments, the Fgf gradient was co-opted from the head to the posterior segments, key signaling pathways (Wnt/β-catenin; Fgf, Notch) in developing amphioxus will be perturbed with chemical inhibitors and activators at specific developmental times. Gene knockdown and overexpression will show gene hierarchy. Effects on morphology of segmentation will be reconstructed in 3-D by serial blockface scanning electron microscopy (SBSEM), which when done at intervals can show the steps in morphogenesis at the single cell level. Researchers will involve undergraduates and high school students directly in the project and also partner with the Birch Aquarium at Scripps Institution of Oceanography and the San Diego County Office of Education to lead workshops for high school teachers. These workshops will use locally-available marine invertebrates to show teachers how to instruct students in experimental design and data analysis—skills that are fundamental to training the next generation of scientists.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.