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Exosomes are small extracellular vesicles (EVs) secreted by many cell types in both normal and pathogenic circumstances. Because EVs, particularly exosomes, are known to transfer biologically active proteins, RNAs and lipids between cells, they have recently become the focus of intense interest as potential mediators of cell-cell communication, particularly in long-range and juxtacrine signaling events associated with adaptive immune function and progression of cancer. Among the EVs, exosomes appear particularly adapted for long-range delivery of cargoes between cells. Because of their association with disease states, the exciting potential for exosomes to serve as diagnostic biomarkers and as target-specific biomolecule delivery vehicles has stimulated a broad range of biomedical investigations to learn how exosomes are generated, what their cargoes are, and how they might be tailored for uptake by remote targets. Addressing these questions requires experimental models in which biochemically useful amounts of material can be harvested, gene expression easily manipulated, and interpretable biological assays developed. The early Xenopus embryo fulfills these model-system ideals in an in vivo context: during morphogenesis the embryo develops several large, fluid-filled extracellular compartments across which numerous tissue-specifying signals must cross, and which are abundantly endowed with exosomes and other EVs. Importantly, certain surface-facing tissues avidly ingest EVs during gastrulation. Recent work has demonstrated that EVs can be isolated from these interstitial spaces in amounts suitable for proteomic and transcriptomic analysis. With its large numbers, great cell size, well-understood fate map, and tolerance of a variety of experimental approaches, the Xenopus embryo provides a unique opportunity to both understand and manipulate the basic cell biology of exosomal trafficking in the context of an intact organism.
Figure 1.
EVs present numerous possibilities for cell-cell communication. (a) Exosome biogenesis via ILV formation in MVBs; (b) ectosome biogenesis via blebbing from cell surface; (c) regulated exosome release into interstitial space via MVB exocytosis; (d) transfer of cargo by exosome fusion directly to cell surface or following phagocytosis; (e) exosome signaling via ligand-receptor interaction, either at cell surface or following phagocytosis; (f) retrograde trafficking (âsurfingâ) of exosomes along filopodia to site of endocytosis at filopodium's proximal base; (g) transcytosis of phagocytosed EVs from one cell to another
Figure 2.
EVs are highly abundant in Xenopus embryonic extracellular spaces. (a) Viewed from the side, a cleaving embryo displays substantial volume of PV fluid between the vitelline envelope and the pigmented animal pole. (b) EVs in this PV fluid fluoresce (green in this figure) upon exposure to styryl dye FM1-43. (c) Electron microscopy of particles pelleted via u006Ctracentrifugation reveals exosome-sized membranous vesicles. (d) Nanosight particle size profile of PV fluid. (e) Blastocoel surface is carpeted with EVs. (f) TEMreveals unusual ultrastructure of blastocoelic EVs. EVs associate with short, motile (g) and thin, blastocoelspanning (h) filopodia. Bars: 1 μm in (c); 100 μm in (e); 500 nm in (f); 10 μm in (g); 5 μm in (h)
Figure 3.
Extensive morphogenetic movements of gastrulating Xenopus embryo redeploy extracellular fluids from PV space and blastocoel into the archenteron. (a) Four frames from a time-lapse recording of an embryo undergoing gastrulation. Epibolic movements of superficial ectoderm, pressing against the vitelline envelope displace perivitelline EVs, fluorescing green following exposure to FM1-43, toward blastopore lip and into the expanding archenteron. (b) Higher-mag time-lapse frames of blastopore closing around the yolk plug during last phase of gastrulation. (c) Summary of fluid exchange between extracellular compartments during gastrulation. Greenâ=âPV fluid; blackâ=âblastocoelic fluid
Figure 4.
Epithelial cells of the prospective Xenopus endoderm ingest fluorescent EVs from the PV space after involution. (a) Stage 12.5 neurula, displaying cut-lines used to expose the postinvolution endodermal cells lining the roof of the archenteron. (b, c) Bright fluorescence of cells around blastopore indicates active incorporation of FM1-43-labeled EVs at the time of involution. (d, e) closer details of the circumblastoporal endodermal cells, revealing incorporated EVs in vesicular compartment in apical surfaces of some, but not all exposed archenteron surface cells
Figure 5.
Harvesting extracellular fluids from Xenopus embryos. (a) Embryos in dish awaiting (lower portion of panel) or having just undergone (upper portion) manual devitellination. Released PV fluid is easily obtained without rupturing a single embryo. (b) Manual devitellination of a cleavage-stage embryo. (c) Late-stage blastulae or gastrulae have a large blastocoel and thin blastocoel roof; blastocoel contents can beharvested directly via aspiration using a blunt-tip micropipette. (d) Proteomes of perivitelline EVs from stage 1 and stage 10 differ. Five perivitelline EV samples, each from 400 embryos at stages 1 and 10, were processed and analyzed via an Orbitrap Fusion mass spectrometer. Peptides and proteins were identified using Discoverer 1.4 software (Thermo Scientific) and the recently available X. laevis protein data base derived from egg and embryo mRNA sequences (Wühr et al., 2014). Total protein intensities for unique peptides identifying 34 fertilization-associated (red) and 128 exosome-associated (blue) were plotted from stage 1 (x-axis) and stage 10 (y-axis) samples, from a total of 1102 peptide identifications
