Finding the Genes That Matter: Profiling Gene Expression in Strongylocentrotid Sea Urchins with Different Biogeographic and Temperature Distributions

本项目的目标是：(1)验证温度影响海洋生态系统物种分布模式的假设；(2)利用基因组学方法和其他分子技术评估适应不同温度和不同自然生物地理分布的同属海胆基因表达的变化。该项目将使用四种温带海胆（圆心海胆属）作为研究生物：S. purpuratus、S. droebacachiensis、S. franciscanus和S. pallidus。这些海洋无脊椎动物是本研究的理想研究生物，因为：(1)同系物关系密切，但有不同的生态温度范围，便于比较研究；(2)可以相对容易地在早期生活史阶段以及成虫和胚胎中进行研究，对温度的反应有种内差异；(3)紫色海胆，S. purpuratus，这是海胆基因组计划的成果。基因表达谱将被评估，以便在基因组水平上解决不同热适应海胆物种对生态相关温度的反应。基因组学方法的策略需要使用DNA大阵列来筛选差异表达的基因，然后使用其他基因组学技术- DNA微阵列和实时PCR进行分析。问题1：就基因表达模式和耐热性而言，强中心海胆的早期生活史阶段对温度的敏感性是否存在差异？该项目的这一方面将测试海胆同类的胚胎对温度是否有不同的敏感性。胚胎将使用标准程序饲养，并在热试验中评估不同阶段的耐热性。每个物种的不同胚胎阶段将暴露在一定的温度下，然后采样，以描述胚胎中温度依赖的基因表达。将使用基因组学策略评估幼龄海胆的基因表达模式，其中使用宏阵列筛选基因表达模式。问题2：在基因组尺度上，成年人对温度是否表现出不同的反应？每个物种的成年标本将适应相同的温度，分子技术将用于评估不同的基因表达模式。同种成虫将在5℃和15℃的水族箱中驯化4周。将分析样品（解剖管足）在适应后立即和2小时热挑战样品中的表达模式变化。利用cDNA宏阵列技术，对目标基因进行DNA微阵列实验，比较其在海胆中的表达。问题3：在自然界中分布明显不同的两种圆梭菌同系物中，温度依赖的基因表达模式在纬度尺度上是如何变化的？海胆同系物的不同分布与基因表达差异的假设有关，即不同的转录丰度和种类。将在生物地理范围内的不同地点收集标本，并利用DNA微阵列测量转录本剖面，将对其进行评估，并将其与物种的热范围相关联。更广泛的影响&教育：这项研究的最终贡献是将功能基因组学技术用于海洋系统的问题。这些结果将提供深入了解海洋生物如何在海洋环境中发挥作用，以及它们如何应对物理环境的变化。在教育和培训方面，除了对本科生和研究生进行生物研究方面的标准培训外，PI还将与“海洋活着！”这是UCSB海洋科学研究所和研究经验与教育设施（REEF）正在进行的教育推广项目。REEF是一个学习实验室，致力于为K-12学生和公众提供正式和非正式的教育活动。π。他的实验室将与REEF经理和MSI教育主任合作，为REEF讲解员开发当地潮间带生物的解释性展示以及教育和培训材料。

The goal of this project is: (1) to test the hypothesis that temperature contributes to setting species distribution patterns in marine ecosystems and (2) to employ genomic approaches and other molecular techniques to assess changes in gene expression in congeneric sea urchins that are adapted to different temperatures and have different biogeographical distributions in nature. The project will use four congeneric species of temperate sea urchin (genus Strongylocentrotus) as study organisms: S. purpuratus, S. droebachiensis, S. franciscanus, and S. pallidus. These marine invertebrates are ideal study organisms for this study because (1) the congeners are closely-related yet have different ecological temperature ranges that facilitate comparative studies, (2) it is possible to work on early life history stages as well adults with relative ease and the embryos have intraspecific differences in response to temperature, and (3) there are significant, readily-available molecular resources for the purple sea urchin, S. purpuratus, as a result of the Sea Urchin Genome Project. Gene expression profiles will be assessed in order to address how the differentially thermally adapted sea urchin species respond to ecologically relevant temperatures at the genomic level. The strategy for the genomics approach entails using DNA macroarrays to screen for differentially expressed genes that will then be analyzed using other genomics techniques -- DNA microarrays and real time PCR. The objectives of the research component are focused around the following three questions: Question 1: Are early life history stages of strongylocentrotid sea urchins differentially sensitive to temperature with respect to patterns gene expression and thermotolerance? This aspect of the project will test whether embryos of the sea urchin congener are differentially sensitive to temperature. Embryos will be raised using standard procedures and thermotolerance of different stages will be assessed in thermal trials. Different embryo stages for each species will be exposed to a range of temperatures and then sampled in order to profile temperature-dependent gene expression in the embryos. Gene expression patterns in larval stage sea urchins will be assessed using the genomics strategy where macroarrays are used to screen for gene expression patterns. Question 2: Do adults display differential responses to temperature as detected at the genomic scale? Adult specimens from each species will be acclimated to the same temperatures and molecular techniques will be used to assess differential gene expression patterns. Congeneric adults will be acclimated in aquaria at 5 and 15 C for 4 weeks. Samples (dissected tube feet) will be analyzed for expression pattern variations in the acclimated sea urchins immediately following acclimation and in the 2 h heat challenge samples. cDNA macroarray, followed by DNA microarray experiments on targeted genes, will be used to compare the expression in the sea urchins. Question 3: How do temperature-dependent patterns of gene expression vary on a latitudinal scale in two Strongylocentrotus congeners with distinctly different distributions in nature? The hypothesis that differential gene expression will be tested, i.e. variable transcript abundance and species, correlates with the different distributions of the sea urchin congeners. Specimens will be collected at different points in the biogeographic range and the transcript profile, as measured using DNA microarrays, will be assessed and correlated to thermal range of the species. Broader Impacts & Education: The ultimate contribution of this study is to bring techniques of functional genomics to bear on questions in marine systems. The results will provide insight into how marine organisms function in the marine environment, and how they respond to variation in their physical environment. Regarding education and training, in addition to the standard training of undergraduates and graduate students in biological research, the PI will collaborate with Oceans Alive!, an on-going educational outreach program at UCSB's Marine Science Institute and the Research Experience and Education Facility (REEF). REEF is a learning lab committed to formal and informal educational activities for K-12 students and the public. The PI.s lab will work with the REEF manager and the MSI educational director to develop interpretive displays of local intertidal organisms and educational and training materials for REEF docents.