The Role of Developmental Genes in Controlling Butterfly Eyespot Patterns II

进化研究的新兴领域，在某种程度上，是基因型如何与表现型联系在一起的。本项目旨在测试几种候选转录因子在蝴蝶眼斑发育中的功能。一些已知参与果蝇翅膀和腿发育的转录因子和基因回路被假设参与了蝴蝶眼点的发育。特别是，当远端-less （Dll）在果蝇中被异位激活时，它会导致完全的腿重复，从而表明它具有主要的调节功能。在双环草中，Dll在眼斑发育的最早阶段表达于眼斑中心的信号细胞中，并且Dll的变异与眼斑大小的变化有关。蝴蝶在形成眼点的过程中是否采用了同样的“腿”发育回路？一种被激活的配体受体，磷酸化的抗十肢截瘫母亲，pMad，在蛹发育早期的眼斑区表达。形态原decapentapletic （Dpp）是否在眼斑中心产生，扩散到周围细胞并分化成完整的眼斑模式？另一种转录因子Engrailed （En）在表皮细胞环中表达，这种细胞环与双翼成虫眼斑上的金色鳞片图案有关。恩会导致眼斑中金色鳞片的分化吗？本项目概述的实验通过在发育翼上异位表达Dll、Dpp和En来验证这些假设，并观察在前两种情况下，完整的眼斑模式是否在表达位点周围分化，或者在第二种情况下，金鳞是否在表达位点发育。进一步的实验将测试在眼斑中心过表达Dll和Dpp是否会导致眼斑大小的差异。为了诱导异位表达，实验室已经开发了一种激光辅助热休克技术，该技术将允许在转基因蝴蝶发育中的翅膀上以可控的空间和时间方式激活这些基因。热休克技术已成功地用荧光标记基因进行了测试，但现在该标记将取代Dll， Dpp和En的编码序列。建立这些基因与眼斑或彩色鳞片分化之间的因果关系将允许稍后检查这些基因的进化与表型进化之间的关系。智力价值：许多关于进化的工作主要是基于基因的新表达域来推断它们的新功能。该项目打算超越相关证据，真正测试属于已知基因回路的“旧”发育基因是否在功能上被选定为进化中形态新颖的蝴蝶眼斑。一旦这种功能选择被确立，未来的工作将试图追踪眼点的进化，从它们的产生到它们的各种定量和定性修改。这项长期的研究将剖析调控基因的进化，并在这些适应性性状的分化中发挥关键作用。这个项目旨在测试一些候选基因是否发挥了关键作用。此外，该项目将提供一系列广泛适用于生物的转化结构，并为非模式生物的功能遗传学开发重要工具。更广泛的影响：蒙泰罗博士目前参与了该项目的一名博士后和三名研究生。其中一位是女性，她是俄克拉何马州印第安部落乔克托族的成员。另一位是布法罗大学电气工程系的少数族裔研究生。这两名学生都是NSF IGERT生物光子学培训补助金的获得者，并且通过与布法罗电气工程系建立的合作，真正在生物学和光子学的界面上工作。她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与发展方法。

The emerging field of evo-devo studies, in part, how genotype connects to phenotype. This project seeks to test the function of a few candidate transcription factors in the development of butterfly eyespots. Several transcription factors and gene circuits that are known to be involved in wing and leg development in Drosophila are hypothesized to be involved in the development of eyespots in butterflies. In particular, when Distal-less (Dll) is ectopically activated in Drosophila it causes complete leg duplications, thus suggesting a master regulatory function. In Bicyclus, Dll is expressed in signaling cells in the eyespot's center at the earliest stages of eyespot development, and moreover, variation in Dll is linked with variation in eyespot size. Have butterflies co-opted the same "leg" developmental circuit in the formation of eyespots? An activated ligand receptor, phosphorylated Mothers Against Decapentaplegic, pMad, is expressed in the eyespot field during early pupal development. Is the morphogen Decapentaplegic (Dpp) being produced at the eyespot's center, diffusing to surrounding cells and differentiating a complete eyespot pattern? Another transcription factor, Engrailed (En), is expressed in a ring of epidermal cells that correlates with the pattern of gold colored scales on the eyespot of the adult Bicyclus wing. Does En cause the differentiation of gold colored scales in eyespots? The experiments outlined in this project test these hypotheses by ectopically expressing Dll, Dpp, and En on the developing wing and observing whether, in the first two cases, a complete eyespot pattern differentiates around the site of expression, or whether, in the second case, gold scales develop at the site of expression. Additional experiments will test whether over-expressing Dll and Dpp at the eyespot's center will lead to differences in eyespot size. To induce ectopic expression the lab has developed a laser-assisted heat-shocking technique that will allow the activation of these genes in a controlled spatial and temporal fashion on the developing wing of transgenic butterflies. The heat-shocking technique has been successfully tested with a fluorescent marker gene but now this marker will be substituted for the coding sequences of Dll, Dpp, and En. Establishing a causal relationship between these genes and differentiation of eyespots or colored scales will allow the later examination of the relationship between the evolution of these genes and the evolution of the phenotype. Intellectual merit: Much work in evo-devo has been dominated by inferring novel functions for genes based solely on their novel expression domains. This project intends to go beyond the correlational evidence and to really test whether "old" developmental genes belonging to known gene circuits have been functionally co-opted to specify a morphological novelty in evolution, butterfly eyespots. Once this functional co-option is established future work will attempt to track the evolution of eyespots, from their creation throughout their various levels of quantitative and qualitative modifications. This long-term study will dissect the evolution of regulatory genes with a demonstrated key role in differentiating these adaptive traits. This project aims to test whether a few candidate genes display that key role. In addition, this project will make available a series of transformation constructs of wide applicability across organisms, as well as develop important tools for functional genetics in non-model organisms. Broader Impacts: Dr. Monteiro has currently involved one postdoc and 3 graduate students in this project. One is a woman and a member of the Native American tribe Choctaw Nation of Oklahoma. Another is a minority graduate student in the Department of Electrical Engeneering at Buffalo. These two students are both recipients of NSF IGERT training grants in Biophotonics, and are truly working at the interface of Biology and Photonics via an established collaboration with the Department of Electrical Engineering at Buffalo. She 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.