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

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。本研究以文昌鱼（Branchiostoma）作为脊椎动物祖先的代表，重点研究了与身体结构进化相关的基因顺式调控元件的进化。文昌鱼是脊椎动物，但缺乏脊椎动物广泛的基因复制特征。因此，文昌鱼的发育基因级联被证明与脊椎动物相似，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体型大规模变化的重要途径之一。对缠绕基因顺式调控的进化的理解可以让我们深入了解缠绕基因在发育中的祖先角色，特别是发育基因的角色的进化保护，以及它们在新进化结构中的新角色的共同选择。关于遗传作用的理论包括：(1)边界形成，(2)特定神经细胞的规范，(3)肌肉细胞亚群的规范和(4)分割的功能。最后一点尤其有争议。为了验证这些假设，我们将比较文昌鱼顺式调控DNA的lacZ报告结构在文昌鱼、斑马鱼、被囊动物（Ciona）、果蝇和七鳃鳗中的表达。在初步研究中，在文昌鱼的前4个生物体中表达了一个包含8.5 kb文昌鱼嵌合调控DNA的结构，该结构与小鼠的嵌合-2区域共享许多转录因子结合位点，后者指导下颌肌肉前体的表达。初步结果表明：(1)从文文鱼类祖先的前体开始，在脊椎动物颚肌的进化过程中，遗传调控可能是保守的；(2)在果蝇、文文鱼和脊椎动物中，遗传调控在肌肉细胞身份和/或特性的规范中可能是保守的。在本研究中，我们将在这5种生物中表达较小的结构体，以确定在所有生物中是否都有相同的转录因子结合位点引导肌肉和神经索的表达。比较实验将在已知直接表达到脊椎动物颚肌的小鼠镶嵌-2调节区域进行。最后，通过在文昌鱼胚胎中过表达和阻断文昌鱼基因的表达，我们将确定文昌鱼基因是否真的如其表达所暗示的那样在文昌鱼中胚层的分割中起作用。这些实验的综合结果将使我们深入了解嵌入基因的调控进化，以及基因顺式调控的进化。理解基因调控的进化对于理解地球上的生命是如何进化的至关重要。这项研究将继续培养本科生的历史，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的博士后提供高级培训。研究结果将发表在同行评议的期刊上，并在科学会议上发表。

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.