The Non-vertebrate Chordate Oikopleura and Evolution of Vertebrate Developmental Innovations

非脊椎动物脊索动物胸膜动物与脊椎动物发育创新的进化发育创新将古老的滤食性脊索动物转变为贪婪的捕食者，即脊椎动物的祖先。这些创新包括脑、神经嵴和表皮基板的三部分，并与广泛的基因组扩增相关。一个中心问题是对分子遗传变化的理解，这些变化使新的发育机制得以进化，从而导致脊椎动物的身体发育。除了脊椎动物（或头盖骨动物）外，脊索动物门还包括两个亚门，头脊索动物（脊椎动物的姐妹群）和尾脊索动物。尾脊索动物幼虫具有典型的脊索动物形态，包括脊索、背神经索、咽缝原基和肌肉发达的后肛尾。对海鞘纲的尾脊索动物进行了经典的、分子的和基因组的研究，但它们的幼虫通常缺乏一个完整的单向消化系统，当身体计划重组为一个无根的、滤食性的成虫时，蜕变破坏了大部分的中枢神经系统。相比之下，Larvacea（或Appendicularia）纲的尾脊索动物，包括Oikopleura dioica，作为自由生活的成虫，保持了完整的脊索体计划，这可能是研究脊索动物包计划的一个优势。此外，与头脊索动物不同的是，Oikopleura胚胎全年都可以获得，并且可以在实验室中维持许多代。本提案的总体目标是帮助理解脊椎动物发育创新的进化起源，利用双胸膜来补充对海鞘动物和头脊索动物的研究。智力优势目标：目标1。确定油松胚胎的命运图。了解细胞命运图对于研究细胞命运决定至关重要。方法包括在不同年龄的卵裂球中注射荧光染料，并在多光子共聚焦显微镜下进行四维重建。目标2。在胸膜动物中测试脊椎动物神经嵴和基板发育所需的基因同源物的发育作用。Aim - 2的检测方法包括利用morpholino反义寡核苷酸下调基因功能，以及通过瞬时转基因检测基因功能。更广泛的影响目标：目标3。培训两名少数民族本科生在为期十周的暑期研究项目中研究胸叶树分子发育遗传学，并培训一名少数民族本科生进行学年研究。目标4。增加对全球碳封存主要参与者的基本生物学知识。幼虫绕过大多数微生物食物网，将浮游植物转化为可直接供掠食性鱼类食用的生物量，并将被捕获在其废弃的房屋中的快速下沉的碳输送到海洋深处。面对全球变暖，至少部分原因是大气中二氧化碳的增加，更好地了解碳循环中这一关键全球环节的生物学是至关重要的。

NSF0345203 The Non-vertebrate Chordate Oikopleura and Evolution of Vertebrate Developmental InnovationsDevelopmental innovations transformed ancient filter-feeding chordates into voracious predators, the ancestors of the vertebrates. These innovations are proposed to include a three-part brain, neural crest, and epidermal placodes, and to be associated with extensive genome amplification. A central problem is an understanding of the molecular genetic changes that permitted the evolution of new developmental mechanisms responsible for the vertebrate bauplan. In addition to the Vertebrata (or Craniata), the phylum Chordata contains two other sub-phyla, Cephalochordata (the sister group to the Vertebrata), and the basally diverging Urochordata. Urochordate larvae have a typical chordate morphology including notochord, dorsal nerve cord, pharyngeal slit primordia, and a muscular post-anal tail. Urochordates of the class Ascidiacea are well studied classically, molecularly, and genomically, but their larvae usually lack a complete one-way digestive system, and metamorphosis destroys much of the central nervous system as the body plan reorganizes into a sessile, filter-feeding adult. In contrast, urochordates of the class Larvacea (or Appendicularia), including Oikopleura dioica, maintain a complete chordate body plan as free-living adults, which may be an advantage for the investigation of the chordate bauplan. Furthermore, in contrast to cephalochordates, Oikopleura embryos are available year round and can be maintained many generations in the lab. The overall objective of this proposal is to help understand the evolutionary origin of vertebrate developmental innovations utilizing Oikopleura dioica to complement investigations on ascidians and cephalochordates. Intellectual Merit Objectives: Aim 1. To determine the fate map of Oikopleura embryos. Understanding the fate map is essential for investigations of cell fate determination. Methods include injection of fluorescent dyes into blastomeres at various ages, and four-dimensional reconstructions in multi-photon confocal microscopy. Aim 2. To test in Oikopleura the developmental role of orthologs of genes shown to be required for the development of vertebrate neural crest and placodes. Methods for Aim 2 include down-regulating gene function with morpholino antisense oligonucleotides, and testing gene function by transient transgenesis. Broader Impact Objectives: Aim 3. To train two minority undergraduates studying Oikopleura molecular developmental genetics in a ten-week summer research program, and one minority undergraduate for academic year research. Aim 4. To increase knowledge of the basic biology of a major player in global carbon sequestration. Larvaceans bypass most of the microbial food web by converting picophytoplankton into biomass directly available to predatory fish and into fast-sinking carbon trapped in their discarded houses that are transported to ocean depths. In the face of global warming, likely due at least in part to rising atmospheric carbon dioxide, it is crucial to understand better the biology of this key global link in the carbon cycle.