The Role of Developmental Genes in Controlling Butterfly Eyespot Patterns

[0316283]蒙蒂罗进化-发展这一新兴领域试图通过研究基因的可变性及其控制的发育途径，来理解形成基因型如何转化为表型的进化过程。蝴蝶翅膀上的彩色和功能性图案为进化研究提供了理想的材料，因为1)它们在12000个被描述的物种内部和物种之间都是可变的，2)因为它们代表了视觉上引人注目的选择产物，通常具有已知的适应价值，3)因为图案形成的二维翅膀表皮表面非常适合发育、分子和表型特征。在蛹中最常见的图案元素是眼点：不同颜色鳞片的明显同心圆。眼点被认为是模仿脊椎动物眼睛的外观，既可以惊吓攻击者，也可以将攻击转移到翅膀边缘。最近研究表明，三个转录因子Spalt， Sal；， engiled，恩；和Distal-less， Dll在双环蝇蛹翅期的眼斑区表达。Sal和Dll在眼点的中心圆盘中表达，在那里黑色鳞片分化，而En存在于中心圆盘周围的细胞环中，与成年翅膀的金色鳞片模式相关。此外，在一种突变的B. anynana“Goldeneye”中，Sal在细胞的中央圆盘中缺失，而En在整个眼点区域中表达，并且成虫模式由一个完全金色的眼点组成。本提案中概述的实验将测试一方面Sal和Dll，另一方面En在区分眼点的黑色和金色鳞片方面是否存在因果关系。为了测试这些基因是否已经在功能上被增选以指定进化中的新模式，将开发白刺虫的种系转化技术。还将尝试开发一种热休克启动子系统，在发育过程中的特定时间激活这些转基因。候选基因的功能将通过在眼斑场中异位表达并测试其功能和是否足以指定彩色鳞片的命运来确定。如果如预期的那样，转录因子与彩色鳞片分化之间建立了因果关系，那么最终的目标将是解剖这些基因的调控区域在任意白刺内选定品系之间的进化，在具有不同眼斑形态的密切相关物种之间的进化，以及最终在具有和不具有眼斑的物种之间的进化，以了解DNA序列变异与形态进化之间的关系。优点标准1：关于进化的许多工作都是由不同物种（通常来自不同门）发育的遗传途径的特征所主导的。虽然比较研究能够而且已经产生了丰硕的成果，但在种内水平和密切相关物种之间的变异研究不应被忽视。事实上，适应性进化主要涉及通过自然选择对同一种群中个体的表型变异进行分类，而不是在不同物种的个体中进行分类。通过比较鳞翅目中一组发育基因的进化，了解这种种内变异的产生，以及可能更具“saltalal”的基因合作选择机制是很重要的。这些目标可以通过剖析调节基因的进化来实现，这些基因在区分适应性状中发挥了关键作用。这个项目旨在测试一些候选基因是否发挥了关键作用。优点标准二：蒙泰罗博士是一名新教员，目前正在培养2名本科生和2名女研究生，其中一名是俄克拉何马州乔克托族印第安人部落的成员。这个优秀的和非常有前途的研究生已经开始在这里描述的项目工作。蒙泰罗博士将继续为研究生和本科生提供群体遗传学、系统遗传学和发育生物学方面的培训，不分性别和种族，以便为他们提供广泛的多方面的进化和发展方法。此外，这个项目有一个跨学科的组成部分，研究生将与物理系的成员一起工作，并在该系学习适当的研究生课程。

0316283MonteiroThe emerging field of evo-devo seeks to understand the evolutionary processes that have shaped how genotype translates into phenotype, in part by studying variability in genes and the developmental pathways that they control. The colorful and functional patterns on the wings of butterflies provide ideal material for evo-devo research because 1) they are variable both within and across the 12,000 described species, 2) because they represent visually compelling products of selection, often with a known adaptive value, and 3) because the two-dimensional epidermal wing surfaces, where pattern formation takes place, are very amenable to developmental, molecular, and phenotypic characterization. Among the most common pattern elements within the nymphalids are the eyespots: distinct concentric rings of differently colored scales. Eyespots are thought to mimic the appearance of vertebrate eyes, and can either startle an attacking predator or deflect the attack towards the margin of the wing. Recently it was shown that three transcription factors, Spalt, Sal;, Engrailed, En; and Distal-less, Dll, are expressed in the eyespot field of Bicyclus anynana during the pupal wing stage. Sal and Dll are expressed in a central disc of the eyespot, where black scales differentiate, while En is present in a ring of cells around the central disc, correlating to the pattern of gold scales in the adult wing. Additionally, in a mutant line of B. anynana, "Goldeneye," Sal is absent from the central disc of cells, while En is expressed in the whole eyespot field, and the adult pattern consists of an entirely gold eyespot. The experiments outlined in this proposal will test whether there is a causal involvement of Sal and Dll, on the one hand, and En, on the other hand, in differentiating the black and gold colored scales of an eyespot, respectively. The technique of germ line transformation in B. anynana will be developed in order to test whether these genes have been functionally co-opted to specify novel patterns in evolution. Development of a heat-shock promoter system that can activate these transgenes at specific times during development will also be attempted. The function of the candidate genes will be determined by ectopically expressing them in the eyespot field and testing their function and sufficiency in specifying the fate of the colored scales. If, as expected, a causal relationship is established between the transcription factors and differentiation of the colored scales, the ultimate goal will be to dissect the evolution of the regulatory regions of these genes across selected lines within B. anynana, across closely related species with divergent eyespot morphologies, and ultimately across species with and without eyespots in order to understand the relationship between DNA sequence variation and morphological evolution. Merit Criteria I: Much work in evo-devo has been dominated by the characterization of the genetic pathways involved in the development of different species, often times from different phyla. Although comparative studies can, and have, generated fruitful results, studies of variation at the intra-specific level and among closely related species should not be neglected. Indeed, adaptive evolution primarily involves the sorting by natural selection of phenotypic variants present in individuals of the same population, not in individuals of different species. It is important to understand the generation of such intra-specific variation, as well as the possibly more "saltational" mechanisms of gene co-option by comparing the evolution of a set of developmental genes across the Lepidoptera. These aims can be achieved by dissecting the evolution of regulatory genes with a demonstrated key role in differentiating adaptive traits. This project aims to test whether a few candidate genes display that key role. Merit Criteria II: Dr. Monteiro is a new faculty member currently training 2 undergraduate students and two female graduate students, one of which a member of the Native American tribe Choctaw Nation of Oklahoma. This excellent and very promising graduate student has already begun working on the project described here. Dr. Monteiro will continue to train graduate and undergraduates students alike in Population Genetics, Phylogenetics, and Developmental Biology, irrespective of gender and ethnicity, in order to provide them with a broad multifaceted approach to Evolution and Development. In addition, this project has a cross-disciplinary component where graduate students will be working with members of the Physics department and taking appropriate graduate courses in that Department as well.