EAGER: Collaborative Research: Mechanisms of Establishing and Maintaining an Algal Endosymbiont in a Vertebrate Host

研究人员最近发现了第一个有益的共生体进入脊椎动物宿主细胞的例子。该协会的宿主是来自北美东部的常见斑点蝾螈（Ambystoma maculatum）。在蝾螈胚胎开始发育后不久，绿色藻类共生体进入蝾螈胚胎的组织和细胞。该项目将使用几种实验方法，包括细胞共培养，mRNA测序和基于激光的细胞分选，以确定藻类细胞进入蝾螈宿主的机制以及这种独特关联的生理意义。 该项目的成功完成还将为在藻类共生体和蝾螈宿主中引入外源基因（转基因）提供新的工具，这将允许进一步研究实现这种密切联系的细胞和分子机制。该项目将包括对葛底斯堡学院的本科生进行多个蝾螈物种的实地采样以及细胞培养、细胞分选和下一代测序方面的先进技术培训。该项目还将通过美国自然历史博物馆（AMNH）支持博物馆视频展览，并资助博物馆的博士后研究人员。该项目概述了几个高风险高回报的实验，这些实验将大大促进我们对这种亲密关系的分子机制的理解，并为未来的研究提供一套研究工具。本研究将采用的方法包括：从纽约、宾夕法尼亚和华盛顿州采集蝾螈胚胎，培养两种蝾螈的胚胎和藻类，并在宿主之间转换共生体，解离和荧光激活细胞分选（FACS）含有藻类内共生体的宿主细胞，并在宿主细胞中进行转化。转录组学和伴随的生物信息学以确定宿主+内共生体细胞群体中差异表达的基因;建立用于与藻类共生体体外共培养的宿主内胚层细胞培养物;使用在密切相关的模式生物中建立的方案和载体对宿主和共生体细胞培养物进行先导转基因转化。这项工作的结果将揭示如何主机？脊椎动物对有益共生体的反应可能不同于已知的脊椎动物对进入宿主细胞的有害病原体的反应。

The investigators have recently discovered the first example of a beneficial, symbiotic alga entering cells of a vertebrate host. The host in this association is the common spotted salamander (Ambystoma maculatum) from Eastern North America. The green algal symbiont enters tissues and cells of the salamander embryos soon after they begin development in temporary vernal pools. The project will use several experimental approaches, including cell co-culturing, mRNA sequencing, and laser-based cell sorting, to determine the mechanisms of algal cell entry into the salamander host and the physiological implications of this unique association. The successful completion of this project will also make new tools available for introducing foreign genes (transgenics) in both the algal symbiont and salamander host, which will allow further investigation into the cellular and molecular mechanisms that enable this intimate association. The project will include training of undergraduate students from Gettysburg College in both field sampling of multiple salamander species as well as advanced techniques in cell culture, cell sorting, and next generation sequencing. The project will also support a museum video exhibit through the American Museum of Natural History (AMNH), and fund a post-doctoral researcher at the museum.This project outlines several high-risk high-reward experiments that will significantly advance our understanding of the molecular mechanisms of this intimate association and provide a suite of research tools for future investigations. The methods that will be employed to accomplish these goals include: Collecting salamander embryos from New York, Pennsylvania and Washington State; Culturing embryos and their algae from two salamander species and switching symbionts between hosts; Dissociation and fluorescence-activated cell sorting (FACS) of host cells containing algal endosymbionts; Transcriptomics and accompanying bioinformatics to determine differentially expressed genes in the host+endosymbiont cell population; Establishing host endoderm cell culture for in vitro co-culturing with algal symbionts; Pilot transgene transformation of both host and symbiont cell cultures using protocols and vectors established in closely related model organisms. The results from this work will reveal how a host?s response to a beneficial symbiont can differ from known vertebrate reactions to harmful pathogens that enter host cells.