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.

荷兰这项研究的长期目标是阐明导致脊椎动物身体计划的图案形成机制的进化起源。这项建议侧重于利用文昌鱼(Branchiostoma)作为原始脊椎动物的替代品，与文昌鱼(Branchiostoma)体型计划的进化有关的嵌合基因顺式调控元件的进化。文昌鱼与脊椎动物相似，但缺乏脊椎动物特有的广泛的基因复制。因此，文昌鱼的发育基因级联被证明与脊椎动物的发育基因级联相似，但不那么复杂，更容易研究。发育基因调控的进化变化是影响动物身体计划大规模变化的重要途径之一。了解嵌合基因顺式调控的进化可以深入了解嵌合基因在发育中的祖先角色，特别是总体上，以及发育基因角色的进化保守与它们在新进化结构中对新角色的选择。关于进化的祖先角色(S)的理论包括(1)边界形成、(2)特定神经细胞的指定、(3)肌肉细胞亚群的指定和(4)分割的功能。最后一个问题尤其有争议。为了测试这些假说，文昌鱼的顺式调控DNA的LacZ报告结构的表达将在文昌鱼、斑马鱼、被膜鱼(Ciona)、果蝇和七鳃鳗中进行比较。在初步工作中，一个包括8.5kb文昌鱼调控DNA的构建体在前4个生物体中表达，该DNA与小鼠Engrailed-2区有许多共同的转录因子结合位点，将表达定向到颌肌前体。初步结果表明：(1)在文昌鱼类祖先的前体节的脊椎动物颌肌进化过程中，内聚化调控可能是保守的；(2)在果蝇、文昌鱼和脊椎动物中，内聚化调控在肌肉细胞特性和/或特性的指定中可能是保守的。在目前的研究中，较小的结构将在5个生物体中表达，以确定是否相同的转录因子结合位点星座在所有生物体中定向表达到肌肉和神经索。将与已知直接表达到脊椎动物颌部肌肉的小鼠Engraated-2调节区进行比较实验。最后，通过文昌鱼胚胎中文昌鱼的过度表达和阻断表达，将确定文昌鱼是否真的像其表达所显示的那样在文昌鱼中胚层的分割中发挥作用。这些实验的综合结果将使我们深入了解尤其是增强基因调控的进化，以及一般基因顺式调控的进化。对基因调控进化的理解是理解地球上生命是如何进化的核心。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和身体残疾的学生，学习分子生物学和胚胎学的基本技术。它还将为实验胚胎学和分子生物学方面的博士后研究员提供高级培训。研究结果将发表在同行评议的期刊上，并在科学会议上公布。