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.

Blood flow is critical for normal cardiac development. Hemodynamic stimuli outside of normal ranges can lead to overt cardiac defects, but how early heart tissue remodels in response to altered hemodynamics is poorly understood. This study investigated changes in tissue collagen in response to hemodynamic overload in the chicken embryonic heart outflow tract (OFT) during tubular heart stages (HH18 to HH24, ~24 h). A suture tied around the OFT at HH18 was tightened to constrict the lumen for ~24 h (constriction range at HH24: 15–60%). Expression of fibril collagens I and III and fibril organizing collagens VI and XIV were quantified at the gene and protein levels via qPCR and quantitative immunofluorescence. Collagen I was slightly elevated upstream of the band and in the cushions in banded versus control OFTs. Changes in collagen III were not observed. Collagen VI deposition was elevated downstream of the band, but not overall. Collagen XIV deposition increased throughout the OFT, and strongly correlated to lumen constriction. Interestingly, organization of collagen I fibrils was observed for the tighter banded embryos in regions that also showed increase in collagen XIV deposition, suggesting a potentially key role for collagens I and XIV in the structural adaptation of embryonic heart tissue to hemodynamic overload.

Subdivision-based image registration has previously been applied to co-localize digital information extracted from rigid structures in biological specimens, such as the brain. Here, we describe and demonstrate the creation and application of a two-dimensional subdivision-based atlas representing a dynamic structure: the outflow tract of the developing chicken heart. The atlas is designed to segment three different anatomical layers of the outflow tract, and is demonstrated on the characterization of collagen XIV in both control and induced abnormal flow specimens. Abnormal blood flow in the embryonic developing heart can lead to congenital heart disease. Comparing local cellular and sub-cellular changes that are caused by abnormal flow can assist in understanding the molecular pathways involved in maladaptations of the heart and congenital defects. This study demonstrates the approach and potential for more extensive applications of the subdivision-based atlas for the embryonic chicken heart.

The pattern of the earliest cell divisions in a vertebrate embryo lays the groundwork for later developmental events such as gastrulation, organogenesis, and overall body plan establishment. Understanding these early cleavage patterns and the mechanisms that create them is thus crucial for the study of vertebrate development. This chapter describes the early cleavage stages for species representing ray-finned fish, amphibians, birds, reptiles, mammals, and proto-vertebrate ascidians and summarizes current understanding of the mechanisms that govern these patterns. The nearly universal influence of cell shape on orientation and positioning of spindles and cleavage furrows and the mechanisms that mediate this influence are discussed. We discuss in particular models of aster and spindle centering and orientation in large embryonic blastomeres that rely on asymmetric internal pulling forces generated by the cleavage furrow for the previous cell cycle. Also explored are mechanisms that integrate cell division given the limited supply of cellular building blocks in the egg and several-fold changes of cell size during early development, as well as cytoskeletal specializations specific to early blastomeres including processes leading to blastomere cohesion. Finally, we discuss evolutionary conclusions beginning to emerge from the contemporary analysis of the phylogenetic distributions of cleavage patterns. In sum, this chapter seeks to summarize our current understanding of vertebrate early embryonic cleavage patterns and their control and evolution.

Ras homolog enriched in brain (RHEB1) is a member within the superfamily of GTP-binding proteins encoded by the RAS oncogenes. RHEB1 is located at the crossroad of several important pathways including the insulin-signaling pathways and thus plays an important role in different physiological processes. To understand better the physiological relevance of RHEB1 protein, the expression pattern of RHEB1 was analyzed in both embryonic (at E3.5–E16.5) and adult (1-month old) mice. RHEB1 immunostaining and X-gal staining were used for wild-type and Rheb1 gene trap mutant mice, respectively. These independent methods revealed similar RHEB1 expression patterns during both embryonic and postnatal developments. Ubiquitous uniform RHEB1/β-gal and/or RHEB1 expression was seen in preimplantation embryos at E3.5 and postimplantation embryos up to E12.5. Between stages E13.5 and E16.5, RHEB1 expression levels became complex: In particular, strong expression was identified in neural tissues, including the neuroepithelial layer of the mesencephalon, telencephalon, and neural tube of CNS and dorsal root ganglia. In addition, strong expression was seen in certain peripheral tissues including heart, intestine, muscle, and urinary bladder. Postnatal mice have broad spatial RHEB1 expression in different regions of the cerebral cortex, subcortical regions (including hippocampus), olfactory bulb, medulla oblongata, and cerebellum (particularly in Purkinje cells). Significant RHEB1 expression was also viewed in internal organs including the heart, intestine, urinary bladder, and muscle. Moreover, adult animals have complex tissue- and organ-specific RHEB1 expression patterns with different intensities observed throughout postnatal development. Its expression level is in general comparable in CNS and other organs of mouse. Thus, the expression pattern of RHEB1 suggests that it likely plays a ubiquitous role in the development of the early embryo with more tissue-specific roles in later development.

Asymmetric development of the vertebrate embryo has fascinated embryologists for over a century. Much has been learned since the asymmetric Nodal signaling cascade in the left lateral plate mesoderm was detected, and began to be unraveled over the past decade or two. When and how symmetry is initially broken, however, has remained a matter of debate. Two essentially mutually exclusive models prevail. Cilia-driven leftward flow of extracellular fluids occurs in mammalian, fish and amphibian embryos. A great deal of experimental evidence indicates that this flow is indeed required for symmetry breaking. An alternative model has argued, however, that flow simply acts as an amplification step for early asymmetric cues generated by ion flux during the first cleavage divisions. In this review we critically evaluate the experimental basis of both models. Although a number of open questions persist, the available evidence is best compatible with flow-based symmetry breakage as the archetypical mode of symmetry breakage.

Alterations in blood flow at early embryonic stages can lead to detrimental remodeling and heart defects, but these structural adaptations are not well understood. We hypothesize that deposition of collagens will be increased as shear stress is increased — leading to a stiffer wall. To test this hypothesis a suture (OTB) was tightened around the outflow tract (OFT) of stage HH18 chick embryos for 24 hours to reduce cross sectional area of the lumen. Sham and OTB embryos were immunostained for collagen I, III, VI and XIV, imaged with confocal microscopy, and staining was quantified by grayscale analysis. Changes in fibril collagens I and III were not observed, however deposition of collagens VI and XIV increased in a degree-of-constriction dependent manner. The observed increase in collagen VI and XIV deposition suggests they play a key role in structural adaptation to increased hemodynamic pressure.

In the frog Xenopus laevis, dorsal–ventral axis specification involves cytoskeleton-dependent transport of localized transcripts and proteins during the first cell cycle, and activation of the canonical Wnt pathway to locally stabilize translated beta-catenin which, by as early as the 32-cell stage, commits nuclei in prospective dorsal lineages to the subsequent expression of dorsal target genes. Maternal ligands important for activating this dorsal-specific signaling pathway are thought to interact with secreted glypicans and coreceptors in the blastocoel. While diffusion between cells is generally thought of as sufficient to accomplish the distribution of secreted maternal ligands to their appropriate targets, signaling may also involve other potential mechanisms, including direct transfer of morphogens via membrane-bounded entities, such as argosomes, exosomes, or even filopodia. In Xenopus, the blastocoel-facing, basolateral surfaces where signaling interactions ostensibly take place have not been previously examined in detail. Here, we report that the cleavage-stage blastocoel is traversed by hundreds of extremely long cellular protrusions that maintain long-term contacts between nonadjacent blastomeres during expansion of the interstitial space in early embryogenesis. The involvement of these protrusions in early embryonic patterning is suggested by the discoveries that (a) they fragment into microvesicles, whose resorption facilitates considerable exchange of cytoplasm and membrane between blastomeres; and (b) they are active in caveolar endocytosis, a prerequisite for ligand-receptor signaling.

In fish and mammalian neurula embryos midline epithelia (Kupffer's vesicle, posterior notochord/node) harbor polarized monocilia, which rotate in a clockwise manner to produce a leftward extracellular fluid flow. Flow induces asymmetric gene expression on the left side, resulting in asymmetric organ morphogenesis and placement. In frog, much earlier asymmetries were described for the monoamine serotonin and the ion pump gastric H+/K+-ATPase (ATP4). The 'ion-flux' model proposed that an ATP4-generated voltage gradient drives serotonin through gap junctions to asymmetrically enrich on one side of the embryo to break symmetry upstream of flow. Here we show that both serotonin and ATP4 were symmetrically expressed in Xenopus and required for Wnt-mediated setup of leftward flow. Flow and asymmetry were lost in embryos in which serotonin signaling was down-regulated. Serotonin was required as competence factor of Wnt signaling, which provides the instructive signal to specify the superficial mesoderm (SM), i.e. the structure which gives rise to the ciliated gastrocoel roof plate (GRP) where flow in frog occurs. Gene knock-down or pharmacological inhibition of ATP4 compromised organ situs, asymmetric gene expression and leftward flow. The GRP revealed fewer, shortened and misaligned cilia. FoxJ1, a master control gene of motile cilia, was down-regulated in the SM. Specifically, ATP4 was required for Wnt/s-catenin regulated Foxj1 induction and Wnt/PCP dependent cilia polarization. Our work confirms flow as primary mechanism of symmetry breakage in frog and argues for evolutionary conservation of symmetry breakage in vertebrates.

In vertebrates, most inner organs are asymmetrically arranged with respect to the main body axis [1]. Symmetry breakage in fish, amphibian, and mammalian embryos depends on cilia-driven leftward flow of extracellular fluid during neurulation [2-5]. Flow induces the asymmetric nodal cascade that governs asymmetric organ morphogenesis and placement [1, 6, 7]. In the frog Xenopus, an alternative laterality-generating mechanism involving asymmetric localization of serotonin at the 32-cell stage has been proposed [8]. However, no functional linkage between this early localization and flow at neurula stage has emerged. Here, we report that serotonin signaling is required for specification of the superficial mesoderm (SM), which gives rise to the ciliated gastrocoel roof plate (GRP) where flow occurs [5, 9]. Flow and asymmetry were lost in embryos in which serotonin signaling was downregulated. Serotonin, which we found uniformly distributed along the main body axes in the early embryo, was required for Wnt signaling, which provides the instructive signal to specify the GRP. Importantly, serotonin was required for Wnt-induced double-axis formation as well. Our data confirm flow as primary mechanism of symmetry breakage and suggest a general role of serotonin as competence factor for Wnt signaling during axis formation in Xenopus.

Over the past half century, the Xenopus laevis embryo has become a popular model system for studying vertebrate early development at molecular, cellular, and multicellular levels. The year-round availability of easily fertilized eggs, the embryo's large size and rapid development, and the hardiness of both adults and offspring against a wide range of laboratory conditions provide unmatched advantages for a variety of approaches, particularly "cutting and pasting" experiments, to explore embryogenesis. There is, however, a common perception that the Xenopus embryo is intractable for microscope work, due to its store of large, refractile yolk platelets and abundant cortical pigmentation. This chapter presents easily adapted protocols to surmount, and in some cases take advantage of, these optical properties to facilitate live-cell microscopic analysis of commonly used experimental manipulations of early Xenopus embryos.

2003). In contrast to thismodel,whenFGFsignaling is inhibited, cyclopsand lefty1 are bilaterallyexpressed in thebrain even in the absenceof southpaw. Two transcription factors, six3b and six7, are required for repression of asymmetric lefty1 expression in the brain (Inbal et al., Neuron 2007). We have found that FGF signaling regulates expression of these transcription factors. Fromour results, we propose amodel for brain laterality,where FGF signaling activates six3b and six7,which in turn inhibits lefty1 expression in the brain. Here, southpaw, rather than initiating lefty1 and cyclops, inhibits the repressive activity of six3b and six7 in the left-side of the brain, acting as a permissive factor for normal lefty1 and cyclops expression.

Epithelial membrane polarity develops early in Xenopus development, with membrane inserted along the earliest cleavage furrows by means of localized exocytosis. The added surface constitutes a new basolateral domain important for early morphogenesis. This basolateral surface becomes isolated from the outside by furrow closure, a zippering of adjacent apical-basolateral margins. Time-lapse microscopy of membrane-labeled embryos revealed two distinct kinds of protrusive activity in furrow closure. Early in furrowing, protrusive activity was associated with purse-string contractility along the apical-basolateral margins. Later in furrow progression, a basolateral protrusive zone developed entirely within the new membrane domain, with long motile filopodia extending in contractile bands from the exposed surfaces. Filopodia interacting with opposing cell surfaces across the cleavage furrow appeared to mediate blastomere-blastomere adhesion, contact spreading and lamellipodial protrusion. Interference with these dynamic activities prevented furrow closure, indicating a basic role for both marginal and basolateral protrusive activities in early embryogenesis.

Vertebrate embryos define an anatomic plane of bilateral symmetry by establishing rudimentary anteroposterior and dorsoventral (DV) axes. A left-right (LR) axis also emerges, presaging eventual morphological asymmetries of the heart and other viscera. In the radially symmetric egg of Xenopus laevis, the earliest steps in DV axis determination are driven by microtubule-dependent localization of maternal components toward the prospective dorsal side. LR axis determination is linked in time to this DV-determining process, but the earliest steps are unclear. Significantly, no cytoskeletal polarization has been identified in early embryos capable of lateral displacement of maternal components. Cleaving Xenopus embryos and parthenogenetically activated eggs treated with 2,3-butanedione monoxime (BDM) undergo a dramatic large-scale torsion, with the cortex of the animal hemisphere shearing in an exclusively counterclockwise direction past the vegetal cortex. Long actin fibers develop in a shear zone paralleling the equator. Drug experiments indicate that the actin is not organized by microtubules, and depends on the reorganization of preexisting f-actin fibers rather than new actin polymerization. The invariant chirality of this drug response suggests a maternally inherited, microfilament-dependent organization within the egg cortex that could play an early role in LR axis determination during the first cell cycle. Consistent with this hypothesis, brief disruption of cortical actin during the first cell cycle randomizes the LR orientation of tadpole heart and gut.

In dividing Xenopus eggs, furrowing is accompanied by expansion of a new domain of plasma membrane in the cleavage plane. The source of the new membrane is known to include a store of oogenetically produced exocytotic vesicles, but the site where their exocytosis occurs has not been described. Previous work revealed a V-shaped array of microtubule bundles at the base of advancing furrows. Cold shock or exposure to nocodazole halted expansion of the new membrane domain, which suggests that these microtubules are involved in the localized exocytosis. In the present report, scanning electron microscopy revealed collections of pits or craters, up to ∼1.5 μm in diameter. These pits are evidently fusion pores at sites of recent exocytosis, clustered in the immediate vicinity of the deepening furrow base and therefore near the furrow microtubules. Confocal microscopy near the furrow base of live embryos labeled with the membrane dye FM1-43 captured time-lapse sequences of individual exocytotic events in which irregular patches of ∼20 μm2 of unlabeled membrane abruptly displaced pre-existing FM1-43-labeled surface. In some cases, stable fusion pores, approximately 2 μm in diameter, were seen at the surface for up to several minutes before suddenly delivering patches of unlabeled membrane. To test whether the presence of furrow microtubule bundles near the surface plays a role in directing or concentrating this localized exocytosis, membrane expansion was examined in embryos exposed to D2O to induce formation of microtubule monasters randomly under the surface. D2O treatment resulted in a rapid, uniform expansion of the egg surface via random, ectopic exocytosis of vesicles. This D2O-induced membrane expansion was completely blocked with nocodazole, indicating that the ectopic exocytosis was microtubule-dependent. Results indicate that exocytotic vesicles are present throughout the egg subcortex, and that the presence of microtubules near the surface is sufficient to mobilize them for exocytosis at the end of the cell cycle.