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藻类进入脊椎动物宿主细胞的例子。该协会的寄主是北美东部常见的斑点火蜥蜴(Ambystoma Maculata)。在蜥蜴胚胎在临时的春季池塘中开始发育后，绿藻共生体很快就进入了组织和细胞。该项目将使用几种实验方法，包括细胞共培养、信使核糖核酸测序和基于激光的细胞分选，以确定藻细胞进入火蜥蜴宿主的机制以及这种独特联系的生理意义。该项目的成功完成还将使在藻类共生体和火蜥蜴宿主中引入外来基因(转基因)的新工具可用，这将使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培训，内容包括多种蜥蜴物种的野外采样以及细胞培养、细胞分选和下一代测序方面的先进技术。该项目还将通过美国自然历史博物馆(AMNH)支持博物馆的视频展览，并资助博物馆的一名博士后研究员。这个项目概述了几个高风险、高回报的实验，这些实验将极大地促进我们对这种密切联系的分子机制的理解，并为未来的研究提供一套研究工具。为实现这些目标将采用的方法包括：收集纽约州、宾夕法尼亚州和华盛顿州的火蜥蜴胚胎；培养两个火蜥蜴物种的胚胎和藻类，并在宿主之间切换共生体；对含有藻类内共生体的宿主细胞进行分离和荧光激活细胞分选；利用转录组学和伴随的生物信息学方法，确定宿主+内共生体细胞群中的差异表达基因；建立宿主内胚层细胞培养体系，以便与藻类共生体进行体外共培养；利用在密切相关模式生物体中建立的协议和载体，对宿主和共生体细胞培养进行转基因转化试验。这项工作的结果将揭示宿主S对有益共生体的反应与已知脊椎动物对进入宿主细胞的有害病原体的反应有何不同。

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.