Exosomal Trafficking in Xenopus Development

胚胎体计划的发育是一个复杂的过程，需要自主细胞活动与局部和远程细胞间信号的整合，以指定各种组织和器官。本项目研究早期胚胎是否使用细胞外微泡（外泌体）作为组织指定信号剂。外泌体是最近发现的一类由细胞分泌的膜结合的小颗粒。现在已经知道，外泌体在其表面携带参与细胞-细胞识别和信号传导的蛋白质，并且它们含有具有改变接受它们的细胞的基因表达模式的潜力的microrna和mrna。从一种细胞类型分泌的外泌体在远距离“重编程”其他细胞的能力已在组织培养中得到证实，但这种令人兴奋的特性是否在正常胚胎发育中被利用尚不清楚。目前的项目通过观察血液形成需要外泌体来调查这种可能性。本研究的目的是研究青蛙胚胎发生过程中外泌体交换的细胞生物学。该项目将研究外泌体蛋白的定性或定量变化是否发生在早期发育过程中？哪些组织分泌外泌体？外泌体对早期青蛙胚胎中的特定组织有针对性吗？如果是这样，这些组织是如何反应的？改变外泌体摄取的分泌或特异性能改变胚胎血液发育吗？回答这些问题将推进新兴的、快速发展的外泌体细胞生物学领域，并扩大我们对胚胎细胞在正常发育早期使用的信号机制的理解。本研究的许多方面为各级学生和学员提供了极好的实践机会，因此可以作为发展生物学或生物医学事业的关键起点或进步步骤。该项目探索了一种新的假设，即早期非洲爪蟾胚胎使用细胞外微泡，特别是外泌体，进行胚胎组织图案。外泌体最近被认为是膜相关信号配体和受体、功能性microrna和mrna的有效载体，但大多数此类证明都是在有限的组织培养情况下进行的。最近的研究表明，完整的早期非洲爪蟾胚胎的细胞外腔室富含外泌体。质谱蛋白质组学分析证实了外泌体标记物的丰度，电子显微镜证实了外泌体典型大小范围内颗粒的丰度。缺乏微囊泡的胚胎发育具有一组特征性的形态缺陷，特别是造血功能的严重减少，这表明外泌体依赖的信号传导在早期发育的组织规范步骤中起着重要作用。这种重要的细胞生物学活性以前还没有在脊椎动物胚胎中被探索过，所以这个项目的目标是表征外泌体，包括它们出现的时间过程，它们的货物和表面蛋白，这些蛋白可能定义它们的组织靶特异性。这些信息将用于开发探针和试剂来研究它们的组织起源和潜在的组织目标。

Development of the embryonic body plan is a complex process, requiring integration of autonomous cellular activities with both local and long-range intercellular signaling to specify various tissues and organs. This project investigates whether the early embryo uses extracellular microvesicles (exosomes), as tissue-specifying signaling agents. Exosomes are a recently discovered class of small, membrane-bound particles secreted from cells. It is now known that exosomes carry proteins involved with cell-cell recognition and signaling on their surfaces, and also that they contain microRNAs and mRNAs with the potential to alter gene expression patterns of cells receiving them. The ability of exosomes secreted from one cell type to "reprogram" other cells at a distance has been demonstrated in tissue culture, but whether this exciting property is utilized during normal embryonic development is not known. The current project investigates this possibility by pursuing the observation that blood formation requires exosomes. The goals of this research are to investigate the cell biology of exosomal exchange in during embryogenesis in frogs. The project will address whether qualitative or quantitative changes in the exosomal proteins occur during early development? Which tissues secrete exosomes? Are specific tissues in the early frog embryo targeted by exosomes; if so, how do those tissues respond? Can altering the secretion or specificity of exosome uptake alter embryonic blood development? Answering these questions will advance the nascent, rapidly growing field of exosomal cell biology, and expand our understanding of the repertoire of signaling mechanisms used by embryonic cells early in normal development. Many aspects of this research present excellent hands-on opportunities for students and trainees at all levels, and thus can serve as a critical starting point or advancement step for developing careers in biology or biomedicine.This project explores the novel hypothesis that the early Xenopus embryo uses extracellular microvesicles, specifically exosomes, for embryonic tissue patterning. Exosomes have recently been recognized as potent conveyors of membrane-associated signaling ligands and receptors, functional microRNAs and mRNAs, but most such demonstrations have been in limited tissue-culture situations. Recent work demonstrated that an extracellular compartment in the intact early Xenopus embryo is highly enriched with exosomes. Mass spectroscopic proteomic analysis confirms an abundance of exosomal markers, and electron microscopy confirms an abundance of particles in the size range typical of exosomes. Embryos reared deprived of microvesicles develop with a characteristic set of morphological defects, particularly a profound reduction in hematopoiesis, suggesting a fundamental role for exosome-dependent signaling during tissue specification steps of early development. This significant cell-biological activity has not previously been explored in a vertebrate embryo, so the goals of this project are to characterize exosomes, including the time course of their appearance, their cargo and the surface proteins that potentially define their tissue target specificity. This information will be used to develop probes and reagents to investigate their tissue origins and potential tissue targets.