ABR: Evolution of Left-Right Asymmetry in Chordates using Cephalochordates as a Proxy for the Ancestral Chordate

Linda Z. Holland， P.I.提案编号：1353688提案标题：ABR：脊索动物中左右不对称的进化，使用头脊索动物作为祖先脊索动物的代表。非技术描述/更广泛的影响头脊索动物（文文鱼和棘鱼）与脊椎动物密切相关，但在结构和基因水平上都更简单，因为它们的基因组没有经历发生在祖先脊椎动物中的两轮全基因组复制。虽然这些复制对人类是有用的，因为一个基因的缺陷通常可以通过复制来弥补，文昌鱼缺乏基因复制使它成为确定单个基因如何协同工作以产生胚胎和成体的理想选择。因为文昌鱼与脊椎动物非常相似，所以它们的遗传机制，比如肾脏或大脑，在根本上是相同的。不同之处在于，脊椎动物通过对这些基本机制的细化，创造了额外的复杂性。一个主要的突出问题是左/右（LR）不对称是如何进化的。脊椎动物表面上是对称的，但内部器官不对称地分布在左/右（LR）轴上。本研究旨在利用两个远缘文昌鱼属（Branchiostoma和Asymmetron）来阐明LR不对称的遗传机制。重要的是，两属杂交产生的幼虫中间不对称。这项研究对理解基因网络及其进化具有深远的意义。与生物信息学家的合作将有助于识别在特定发育阶段活跃的基因。它将给本科生研究经验。2013-2014年期间，包括6名少数族裔在内的13名本科生参加了荷兰实验室的研究。培养1名博士后。研究结果将在会议上展示，并发表在科学期刊上。斯克里普斯的伯奇水族馆和圣地亚哥联合学校的“通过技术加强科学教育”项目将为中学生提供通过SKYPE直接互动的荷兰实验室虚拟之旅。这项工作将通过PI和合作者之间的学生/博士后交换来促进跨学科教育，让生物信息学的学生学习生物学，让生物学的学生学习生物信息学。长期以来，人们一直认为基岩脊索类文昌鱼掌握着脊椎动物起源的线索。这一建议解决了一个主要的未解决的问题：脊索动物中LR不对称的遗传基础是什么？它在进化过程中是如何变化的？具体目的是利用两个遥远的文昌鱼属（Branchiostoma和Asymmetron）作为祖先脊索动物的代用物，阐明脊索动物LR不对称的进化。除了脊索动物和文文鱼胚胎中保守的三联位Nodal、Lefty和Pitx的左侧表达外，脊索动物LR模式的基本遗传基础尚不清楚。我们提出，LR不对称的特征始于母体节点偏斜，并通过右侧的BMP抑制节点进行细化。文昌鱼胚胎的简单性允许解剖从卵开始到脊椎动物胚胎结束的基因网络。Branchiostoma和asymmetra提供了“金发姑娘原理”——相似到足以确定两个结构是同源的，不同到足以阐明LR不对称的遗传差异。两属之间的杂种具有中等的不对称性。荷兰实验室是佛罗里达双歧杆菌研究的先驱，是第一个研究不对称发育的实验室，并在实验室中建立了两种物种的全年繁殖。lucayanum基因组测序，目前正在对lucayanum基因组进行测序。这两个属相似，但LR不对称在几个方面有所不同。重要的是，鳃裂瘤两侧都有性腺，而非对称瘤只有右侧有性腺。该方法结合了原位杂交、抗体标记、操纵基因功能、RNA-Seq和生物信息学。比较这两个物种的LR模式和生殖细胞迁移的分子基础将阐明脊索动物LR不对称的基本基础，并验证脊椎动物是从文昌鱼类祖先进化而来的整体假设。这些结果不仅适用于头脊索动物，也适用于脊椎动物，并可能改变对脊椎动物的无数变异的进化观念，这些变异增加了一个小的非蛋黄胚胎通过内翻进行原肠受精的基本主题。

Linda Z. Holland, P.I. PROPOSAL NUMBER: 1353688 PROPOSAL TITLE: ABR: Evolution of Left-Right Asymmetry in Chordates using Cephalochordates as a Proxy for the Ancestral ChordateNontechnical description/broader impacts Cephalochordates (amphioxus and lancelets) are closely related to vertebrates but simpler both structurally and at the gene level as their genomes have not undergone the two rounds of whole-genome duplication that occurred in ancestral vertebrates. While these duplications are useful for humans as a defect in one gene can often be compensated for by a duplicate, the lack of gene duplication in amphioxus makes it ideal for determining how individual genes work together to create an embryo and adult. Because amphioxus is so vertebrate-like, the genetic mechanisms that pattern, for example, the kidney or brain are fundamentally the same for both groups. The difference is that vertebrates have created additional complexity by elaborating upon these basic mechanisms. A major outstanding question is how left/right (LR) asymmetry evolved. Vertebrates are superficially symmetric, but internal organs are asymmetrically arranged across the left/right (LR) axis. This study aims to elucidate the genetic mechanisms of LR asymmetry using two distant amphioxus genera (Branchiostoma and Asymmetron). Importantly, hybrids between the two genera yield larvae intermediate in asymmetry. This study has far reaching implications for understanding gene networks and how they evolve. A collaboration with bioinformaticians will help identify genes that are active at particular developmental stages. It will give undergraduates research experience. During 2013-2014, 13 undergraduates including 6 under-represented minorities participated in research in the Holland laboratory. One postdoctoral fellow will be trained. Results will be presented at meetings and published in scientific journals. Outreach with the Birch Aquarium at Scripps and the San Diego Unified Schools' Enhancing Science Education through Technology program will give middle school students virtual tours of the Holland laboratory with direct interactions via SKYPE. The work will foster cross-disciplinary education via exchanges of students/postdocs between the PI and collaborators, allowing bioinformatics students to learn biology and biology students to learn bioinformatics. Technical descriptionThe basal chordate amphioxus has long been thought to hold clues to vertebrate origins. This proposal addresses a major unresolved question: What is the genetic basis of LR asymmetry in chordates and how has it changed during evolution? The specific aim is to elucidate the evolution of LR asymmetry in chordates using two distant amphioxus genera (Branchiostoma and Asymmetron) as proxies for ancestral chordates. Except for left-sided expression of the conserved triad of Nodal, Lefty and Pitx in embryos of both vertebrates and amphioxus, the fundamental genetic basis of LR patterning in chordates is elusive. It is proposed that specification of LR asymmetry begins with skewed maternal Nodal and is refined by BMP suppressing Nodal on the right. The simplicity of amphioxus embryos allows dissection of gene networks starting with the egg and ending with a vertebrate-like embryo. Branchiostoma and Asymmetron offer the "Goldilocks Principle"- alike enough to be sure that two structures are homologous and unlike enough to elucidate genetic differences in LR asymmetry. Hybrids between the two genera have intermediate asymmetry. The Holland lab pioneered research on B. floridae, was the first to study Asymmetron development and establish year-round breeding of both species in the laboratory. sequencing the A. lucayanum genome and is currently sequencing the A. lucayanum genome. The two genera are similar, but LR asymmetries differ in several respects. Importantly, Branchiostoma has gonads on both sides, but Asymmetron has them only on the right. The approach combines in situ hybridization, antibody labeling, manipulating gene function, RNA-Seq and bioinformatics. Comparisons of the molecular basis of LR patterning and germ cell migration in both species will elucidate the fundamental basis of LR asymmetry in chordates and test the overall hypothesis that vertebrates evolved from an amphioxus-like ancestor. The results promise to be applicable not just to cephalochordates but also to vertebrates and may transform ideas on evolution of the myriad variations vertebrates added to the basic theme of a small non-yolky embryo gastrulating by invagination.