Conservation and Non-Conservation of Engrailed Gene Regulation in Metazoan Evolution: Amphioxus as a Model for the Ancestral Vertebrate

后生动物进化中基因调控的保守与非保守：文昌鱼作为祖先脊椎动物的模型

• 批准号: 0236171
• 负责人: Linda Holland
• 金额: $ 36万
• 依托单位: University of California-San Diego Scripps Inst of Oceanography
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-02-01 至 2007-01-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0236171&HistoricalAwards=false
• 关键词: Conservation; Non; Engrailed; Gene; Regulation

0236171HollandThe long term goal of this research is to elucidate the evolutionary origins of the patterning mechanisms that led to the vertebrate body plan. This proposal focuses on the evolution of cis-regulatory elements of the engrailed gene in relation to the evolution of body plans using amphioxus (Branchiostoma) as a proxy for the ancestral vertebrate. Amphioxus is vertebrate-like but lacks the extensive gene duplications characteristic of vertebrates. Thus, developmental gene cascades in amphioxus are proving to be similar to those in vertebrates, but less complex and easier to study. Evolutionary changes in developmental gene regulation are one important way to effect large-scale changes in animal body plans. An understanding of the evolution of cis-regulation of the engrailed gene can give insights into the ancestral roles of engrailed in development, in particular, and the evolutionary conservation of the roles of developmental genes versus their co-option for new roles in newly evolved structures, in general. Theories regarding the ancestral role(s) of engrailed include functions in (1) boundary formation, (2) specification of particular nerve cells, (3) specification of subsets of muscle cells and (4) segmentation. The last is particularly controversial. To test these ypotheses, expression of lacZ reporter constructs of cis-regulatory DNA from amphioxus engrailed will be compared in amphioxus, the zebrafish, a tunicate (Ciona), Drosophila and the lamprey. In preliminary work, a construct including 8.5 kb of amphioxus engrailed regulatory DNA, which shares many transcription factor binding sites with the mouse engrailed-2 region that directs expression to jaw muscle precursors, was expressed in the first 4 of these organisms. Initial results suggest that (1) engrailed regulation may be conserved during evolution of the vertebrate jaw muscles from the anterior somites of an amphioxus-like ancestor and (2) a role for engrailed in specification of muscle cell identity and/or properties may be conserved in Drosophila, amphioxus and the vertebrates. In the present research, smaller constructs will be expressed in the 5 organisms to determine if the same constellation of transcription factor binding sites directs expression to muscle and the nerve cord in all organisms. Comparative experiments will be done with the mouse engrailed-2 regulatory regions known to direct expression to the vertebrate jaw muscles. Finally, by overexpressing and blocking expression of amphioxus engrailed in amphioxus embryos, it will be determined if engrailed really does function in segmentation of the amphioxus mesoderm as its expression suggests. The combined results from these experiments will give insights into the evolution of regulation of the engrailed gene in particular, and the evolution of cis-regulation of genes in general. An understanding of the evolution of gene regulation is central to an understanding of how life evolved on earth. This research will continue a history of training undergraduate students, including woman, under-represented minorities and those with physical disabilities, in basic techniques of molecular biology and embryology. It will also provide advanced training for a postdoctoral fellow in experimental embryology and molecular biology. The results will be published in peer-reviewed journals and presented at scientific meetings.

0236171 Holland这项研究的长期目标是阐明导致脊椎动物身体计划的模式机制的进化起源。该提案的重点是使用Amphioxus（Branchiostoma）作为祖先脊椎动物的替代物的身体计划的演变，从而介绍了界面基因的顺式调节元件的演变。 Amphioxus is vertebrate-like but lacks the extensive gene duplications characteristic of vertebrates.因此，事实证明，两栖动物中的发育基因级联反应与脊椎动物中的级联相似，但较不复杂和易于研究。发育基因调控的进化变化是影响动物体计划大规模变化的一种重要方法。对界面基因的顺式调节的演变的理解可以使人们对发展中的祖先作用，尤其是对发育基因的作用的进化保护，而不是其在新进化结构中的新作用的合作。关于属于祖先作用的理论包括在（1）边界形成中的功能，（2）特定神经细胞的规范，（3）肌肉细胞子集的规范和（4）分割。最后一个特别有争议。为了测试这些YPOTHES，将在两栖二氧化碳，斑马鱼，柔毛（ciona），果蝇和lamprey中比较来自闪石焦虑的顺式调节DNA的LACZ报告构建体的表达。在初步工作中，包括8.5 kb的两栖动物融合的调节性DNA，该结构在这些生物体的前4个中表达了许多转录因子结合位点，该结构与小鼠grounded-2区域共享，该结合位点将其引导至下颌肌肉前体。最初的结果表明，（1）在脊椎动物下颌肌肉的演变中可以保留的（1）构成的调节，（2）在肌肉细胞身份和/或特性中的规定中，可以在果蝇，Amphioxus和perteperate中固定构成肌肉细胞身份和/或特性的作用。在本研究中，将在5个生物体中表达较小的构建体，以确定转录因子结合位点的相同星座是否将表达引导到所有生物体中的肌肉和神经线。比较实验将使用已知将表达到脊椎动物下颌肌肉的狭窄2调节区域进行。 最后，通过过度表达和阻断植物胚胎中的两性轴的表达，将确定是否确实在分割中胚层的表达表达的表达中确实在分割中确实发挥作用。这些实验的综合结果将洞悉尤其是植入基因的调节演变，以及一般基因的顺式调节的演变。对基因调节的演变的理解对于理解生命在地球上的发展至关重要。这项研究将继续培训本科生，包括女性，代表性不足的少数民族以及身体残疾的人，在分子生物学和胚胎学基本技术中。它还将为实验性胚胎学和分子生物学的博士后研究员提供高级培训。结果将在同行评审的期刊上发表，并在科学会议上介绍。