Light sheet theta microscopy for rapid high-resolution imaging of large biological samples.

BACKGROUND: Advances in tissue clearing and molecular labeling methods are enabling unprecedented optical access to large intact biological systems. These developments fuel the need for high-speed microscopy approaches to image large samples quantitatively and at high resolution. While light sheet microscopy (LSM), with its high planar imaging speed and low photo-bleaching, can be effective, scaling up to larger imaging volumes has been hindered by the use of orthogonal light sheet illumination. RESULTS: To address this fundamental limitation, we have developed light sheet theta microscopy (LSTM), which uniformly illuminates samples from the same side as the detection objective, thereby eliminating limits on lateral dimensions without sacrificing the imaging resolution, depth, and speed. We present a detailed characterization of LSTM, and demonstrate its complementary advantages over LSM for rapid high-resolution quantitative imaging of large intact samples with high uniform quality. CONCLUSIONS: The reported LSTM approach is a significant step for the rapid high-resolution quantitative mapping of the structure and function of very large biological systems, such as a clarified thick coronal slab of human brain and uniformly expanded tissues, and also for rapid volumetric calcium imaging of highly motile animals, such as Hydra, undergoing non-isomorphic body shape changes.

Species-specific viromes in the ancestral holobiont Hydra.

Recent evidence showing host specificity of colonizing bacteria supports the view that multicellular organisms are holobionts comprised of the macroscopic host in synergistic interdependence with a heterogeneous and host-specific microbial community. Whereas host-bacteria interactions have been extensively investigated, comparatively little is known about host-virus interactions and viral contribution to the holobiont. We sought to determine the viral communities associating with different Hydra species, whether these viral communities were altered with environmental stress, and whether these viruses affect the Hydra-associated holobiont. Here we show that each species of Hydra harbors a diverse host-associated virome. Primary viral families associated with Hydra are Myoviridae, Siphoviridae, Inoviridae, and Herpesviridae. Most Hydra-associated viruses are bacteriophages, a reflection of their involvement in the holobiont. Changes in environmental conditions alter the associated virome, increase viral diversity, and affect the metabolism of the holobiont. The specificity and dynamics of the virome point to potential viral involvement in regulating microbial associations in the Hydra holobiont. While viruses are generally regarded as pathogenic agents, our study suggests an evolutionary conserved ability of viruses to function as holobiont regulators and, therefore, constitutes an emerging paradigm shift in host-microbe interactions.

Imaging inward and outward trafficking of gold nanoparticles in whole animals.

Gold nanoparticles have emerged as novel safe and biocompatible tools for manifold applications, including biological imaging, clinical diagnostics, and therapeutics. The understanding of the mechanisms governing their interaction with living systems may help the design and development of new platforms for nanomedicine. Here we characterized the dynamics and kinetics of the events underlying the interaction of gold nanoparticles with a living organism, from the first interaction nanoparticle/cell membrane, to the intracellular trafficking and final extracellular clearance. By treating a simple water invertebrate (the cnidarian Hydra polyp) with functionalized gold nanoparticles, multiple inward and outward routes were imaged by ultrastructural analyses, including exosomes as novel undescribed carriers to shuttle the nanoparticles in and out the cells. From the time course imaging a highly dynamic picture emerged in which nanoparticles are rapidly internalized (from 30 min onward), recruited into vacuoles/endosome (24 h onward), which then fuse, compact and sort out the internalized material either to storage vacuoles or to late-endosome/lysosomes, determining almost complete clearance within 48 h from challenging. Beside classical routes, new portals of entry/exit were captured, including exosome-like structures as novel undescribed nanoparticle shuttles. The conservation of the endocytic/secretory machinery through evolution extends the value of our finding to mammalian systems providing dynamics and kinetics clues to take into account when designing nanomaterials to interface with biological entities.

Subcellular localization of the epitheliopeptide, Hym-301, in Hydra.

Peptides, as signaling molecules, play a number of roles in cell activities. An epitheliopeptide, Hym-301, has been described as a peptide involved in morphogenesis in hydra. However, little is known about the intracellular location of the peptide or its specific functions. To investigate the mechanism of morphogenesis that involves peptidic molecules, we have examined the intracellular localization of Hym-301 in hydra by using immunohistochemical and immunogold electron-microscopic analyses. We have found that the pattern of distribution of mature peptide is slightly different from that of its mRNA, and that the peptide is stored in vesicles located adjacent to the cell membrane. We have also found that the peptide is released both extracellularly and internally to the cytoplasm of the cells. Based upon these observations, we have constructed a possible model mechanism of homeostatic regulation of the distribution of the Hym-301 peptide in a dynamic tissue context.

Preparation techniques for transmission electron microscopy of Hydra.

Hydra is a classical model organism in developmental and cell biology with a simple body plan reminiscent of a gastrula with one body axis and a limited number of cell types. This rather simple organism exhibits a regeneration capacity that is unique among all eumetazoans and is largely dependent on the stem cell properties of its epithelial stem cell population. Molecular work in the past few years has revealed an unexpected genetic complexity of these simple animals, making them an interesting model for studying the generation of animal form and regeneration. In addition, Hydra has an interstitial stem cell system with a unique population of nematocytes, neuronal cells that are characterized by an explosive exocytotic discharge. Here, we compare classical and modern transmission electron microscopy (TEM) fixation protocols including protocols for TEM immunocytochemistry (post-embedding immunogold labeling). We presume that TEM studies will become an important tool to analyze cell-cell interactions as well as cell matrix interrelationships in Hydra in the future.

The dynamic genome of Hydra.

The freshwater cnidarian Hydra was first described in 1702 and has been the object of study for 300 years. Experimental studies of Hydra between 1736 and 1744 culminated in the discovery of asexual reproduction of an animal by budding, the first description of regeneration in an animal, and successful transplantation of tissue between animals. Today, Hydra is an important model for studies of axial patterning, stem cell biology and regeneration. Here we report the genome of Hydra magnipapillata and compare it to the genomes of the anthozoan Nematostella vectensis and other animals. The Hydra genome has been shaped by bursts of transposable element expansion, horizontal gene transfer, trans-splicing, and simplification of gene structure and gene content that parallel simplification of the Hydra life cycle. We also report the sequence of the genome of a novel bacterium stably associated with H. magnipapillata. Comparisons of the Hydra genome to the genomes of other animals shed light on the evolution of epithelia, contractile tissues, developmentally regulated transcription factors, the Spemann-Mangold organizer, pluripotency genes and the neuromuscular junction.

Effects of norflurazon on green and brown hydra.

For the first time effects of norflurazon on green (Hydra viridissima Pallas, 1766) and brown hydra (Hydra oligactis Pallas, 1766) in high- and low-light conditions were investigated in order to establish the extent of damage that this substance inflicts, with special emphasis on the "bleaching effect" and the effect on hydra-algae symbiosis. Green hydra is a typical example of an endosymbiotic organism. The gastrodermal myoepithelial cells of green hydra contain endosymbiotic algae. Norflurazon is a selective translocational herbicide that induces a "bleaching effect" on newly developed chloroplasts, resulting in a decrease of photosynthetic activity and viability of the organism. In the experiment, morphological (binocular), cytological and histological (Bouin fixative, dehydration, paraplast embedding, Hämalaun-eosine staining, light microscope) and conventional transmission electron microscopy (cTEM) (glutaraldehyde, dehydration, raisin, uranyl-acetate, Pb-citrate) were used. Depending on the concentration and light conditions, norflurazon caused mortality, deformations, changes in behavior, locomotion and asexual reproduction, changes in the structure of all 3 layers in hydras, changes in the position and shape of endosymbiotic algae inside the hydra body as well as ultrastructural changes of treated hydras and endosymbiotic algae. Under low concentrations of norflurazon the effects on hydra were similar to the controls, while in the highest concentrations especially manifested were antichloroplastal and antimitochondrial effects. Norflurazon caused a great extent of damage and induced deleterious effects also upon other cellular components such as cellular membranes, ER, Golgi apparatus, ribosomes. Newly developed buds in symbiotic green hydras were not bleached. After a recovery period, the green hydra individuals that had survived re-established regular endosymbiosis with algae and recovered completely, whereas brown hydras recovered only partially.

Uncovering the evolutionary history of innate immunity: the simple metazoan Hydra uses epithelial cells for host defence.

Although many properties of the innate immune system are shared among multicellular animals, the evolutionary origin remains poorly understood. Here we characterize the innate immune system in Hydra, one of the simplest multicellular animals known. In the complete absence of both protective mechanical barriers and mobile phagocytes, Hydra's epithelium is remarkably well equipped with potent antimicrobial peptides to prevent pathogen infection. Induction of antimicrobial peptide production is mediated by the interaction of a leucine-rich repeats (LRRs) domain containing protein with a TIR-domain containing protein lacking LRRs. Conventional Toll-like receptors (TLRs) are absent in the Hydra genome. Our findings support the hypothesis that the epithelium represents the ancient system of host defence.

The extracellular matrix of hydra is a porous sheet and contains type IV collagen.

Hydra, as an early diploblastic metazoan, has a well-defined extracellular matrix (ECM) called mesoglea. It is organized in a tri-laminar pattern with one centrally located interstitial matrix that contains type I collagen and two sub-epithelial zones that resemble a basal lamina containing laminin and possibly type IV collagen. This study used monoclonal antibodies to the three hydra mesoglea components (type I, type IV collagens and laminin) and immunofluorescent staining to visualize hydra mesoglea structure and the relationship between these mesoglea components. In addition, hydra mesoglea was isolated free of cells and studied with immunofluorescence and scanning electron microscopy (SEM). Our results show that type IV collagen co-localizes with laminin in the basal lamina whereas type I collagen forms a grid pattern of fibers in the interstitial matrix. The isolated mesoglea can maintain its structural stability without epithelial cell attachment. Hydra mesoglea is porous with multiple trans-mesoglea pores ranging from 0.5 to 1 microm in diameter and about six pores per 100 microm(2) in density. We think these trans-mesoglea pores provide a structural base for epithelial cells on both sides to form multiple trans-mesoglea cell-cell contacts. Based on these findings, we propose a new model of hydra mesoglea structure.

New observations on green hydra symbiosis.

New observations on green hydra symbiosis are described. Herbicide norflurazon was chosen as a "trigger" for analysis of these observations. Green hydra (Hydra viridissima Pallas, 1766) is a typical example of endosymbiosis. In its gastrodermal myoeptihelial cells it contains individuals of Chlorella vulgaris Beij. (KESSLER & HUSS 1992). Ultrastructural changes were observed by means of TEM. The newly described morphological features of green hydra symbiosis included a widening of the perialgal space, missing symbiosomes and joining of the existing perialgal spaces. Also, on the basis of the newly described mechanisms, the recovery of green hydra after a period of intoxication was explained. The final result of the disturbed symbiosis between hydra and algae was the reassembly of the endosymbiosis in surviving individuals.

Synthesis and biological assay of GSH functionalized fluorescent quantum dots for staining Hydra vulgaris.

Quantum dots (QDs) have been used extensively as fluorescent markers in several studies on living cells. Here, we report the synthesis of conjugates based on glutathione (GSH) and QDs (GSH-QDs) and we prove how these functionalized fluorescent probes can be used for staining a freshwater invertebrate called Hydra vulgaris. GSH is known to promote Hydra feeding response by inducing mouth opening. We demonstrate that GSH-QDs as well are able to elicit biological activity in such an animal, which results in the fluorescent staining of Hydra. GSH-QDs, once they reach the gastric region, are internalized by endodermal cells. The efficiency of GSH-QD internalization increases significantly when nanoparticles are coadministrated with free GSH. We also compared the behavior of bare QDs to that of GSH-QDs both in the presence and in the absence of free GSH. The conclusions from these series of experiments point to the presence of GSH binding proteins in the endodermal cell layer and uncover a novel role played by glutathione in this organism.

Structure/function analysis of spinalin, a spine protein of Hydra nematocysts.

The nematocyst capsules of the cnidarians are specialized explosive organelles that withstand high osmotic pressures of approximately 15 MPa (150 bar). A tight disulfide network involving cysteine-rich capsule wall proteins, like minicollagens and nematocyst outer wall antigen, characterizes their molecular composition. Nematocyst discharge leads to the expulsion of a long inverted tubule that was coiled inside the capsule matrix before activation. Spinalin has been characterized as a glycine-rich, histidine-rich protein associated with spine structures on the surface of everted tubules. Here, we show that full-length Hydra spinalin can be expressed recombinantly in HEK293 cells and has the property to form disulfide-linked oligomers, reflecting its state in mature capsules. Furthermore, spinalin showed a high tendency to associate into dimers in vitro and in vivo. Our data, which show incomplete disulfide connectivity in recombinant spinalin, suggest a possible mechanism by which the spine structure may be linked to the overall capsule polymer.

Endosymbiotic alga from green hydra under the influence of cinoxacin.

Cinoxacin (Cxn) showed a strong effect on the endosymbiotic alga Chlorella; it was significantly damaged. Changes in algal color, position, structure and ultrastructure were found. In some algal cells ultrastructures were completely destroyed. The antichloroplastal and antimitochondrial effect was especially expressed. Damage to the thylakoid system of chloroplasts was more pronounced with increasing Cxn concentration. Some of the mitochondria were swollen and some of them were completely destroyed. From the evolutionary point of view, the correlation between antibacterial, and antichloroplastal and antimitochondrial effect of Cxn points to the evolutionary connection of chloroplasts and mitochondria with eubacteria.

4-nonylphenol-induced toxicity and apoptosis in Hydra attenuata.

Effects of 4-nonylphenol (4-NP) on the morphology and survival of the cnidarian Hydra attenuata were studied under acute exposure conditions. The lethal concentration value inducing 50% mortality after 96 h was 97.5 +/- 20 microg/L, whereas the lethal concentration value inducing 10% mortality after 96 h was 64 +/- 25.5 microg/L. The no-observed-effect concentration based on morphological criteria was less than 25 microg/L. Hydra was one of the most sensitive freshwater invertebrate species behind the amphipod Hyalella azteca. Toxicity effects appeared rapidly and did not evolve substantially between 24 and 96 h of exposure. Induction of apoptosis was registered during the first hour of exposure to 4-NP at lethal concentrations, indicating rapid effects of the chemical. Abnormal increase of apoptosis may explain the acute toxicity of 4-NP in hydra. Results show that hydra viability is affected in the short term at 4-NP concentrations normally found in contaminated sites, but not at those concentrations reflecting lower levels of environmental contamination.

The glycoprotein NOWA and minicollagens are part of a disulfide-linked polymer that forms the cnidarian nematocyst wall.

The nematocyst is a unique extrusive organelle involved in the defense and capture of prey in cnidarians. Minicollagens and the glycoprotein NOWA are major components of the nematocyst capsule wall, which resists osmotic pressure of 15 MPa. Here we present the recombinant expression of NOWA, which spontaneously assembles to globular macromolecular particles that are sensitive to reduction as the native wall structure. Ultra-structural analysis showed that the Hydra nematocyst wall is composed of several layers of globular particles, which are interconnected via radiating rodlike protrusions. Evidence is presented that native wall particles contain NOWA and minicollagen, supposed to be linked via disulfide bonds between their homologous cysteine-rich domains. Our data suggest a continuous suprastructure of the nematocyst wall, assembled from wall proteins that share a common oligomerization motif.

Epithelial interactions in Hydra: apoptosis in interspecies grafts is induced by detachment from the extracellular matrix.

Apoptosis plays an important role in immunity and is widely used to eliminate foreign or infected cells. Cnidaria are the most basal eumetazoans and have no specialised immune cells, but some colonial cnidarians possess a genetic system to discriminate between self and non-self. By grafting epithelia of different species we have previously shown that the freshwater polyp Hydra eliminates non-self cells by phagocytosis. Here we have investigated whether apoptosis is involved in the histocompatibility reactions. We studied epithelial interactions between Hydra vulgaris and Hydra oligactis and show that a large number of apoptotic cells accumulate in the contact region of interspecies grafts. Histological analysis of the graft site revealed that displacement of the endodermal layer of Hydra vulgaris by endoderm from Hydra oligactis coincided with impaired cell-cell and cell-matrix contacts. We therefore suggest that in interspecies grafts, apoptosis is induced by the detachment of epithelial cells from the extracellular matrix (anoikis) and not by a discriminative allorecognition system.

Nowa, a novel protein with minicollagen Cys-rich domains, is involved in nematocyst formation in Hydra.

The novel protein Nowa was identified in nematocysts, explosive organelles of Hydra, jellyfish, corals and other CNIDARIA: Biogenesis of these organelles is complex and involves assembly of proteins inside a post-Golgi vesicle to form a double-layered capsule with a long tubule. Nowa is the major component of the outer wall, which is formed very early in morphogenesis. The high molecular weight glycoprotein has a modular structure with an N-terminal sperm coating glycoprotein domain, a central C-type lectin-like domain, and an eightfold repeated cysteine-rich domain at the C-terminus. Interestingly, the cysteine-rich domains are homologous to the cysteine-rich domains of minicollagens. We have previously shown that the cysteines of these minicollagen cysteine-rich domains undergo an isomerization process from intra- to intermolecular disulfide bonds, which mediates the crosslinking of minicollagens to networks in the inner wall of the capsule. The minicollagen cysteine-rich domains present in both proteins provide a potential link between Nowa in the outer wall and minicollagens in the inner wall. We propose a model for nematocyst formation that integrates cytoskeleton rearrangements around the post-Golgi vesicle and protein assembly inside the vesicle to generate a complex structure that is stabilized by intermolecular disulfide bonds.

Poly-gamma-glutamate synthesis during formation of nematocyst capsules in Hydra.

Nematocysts are explosive organelles found in all Cnidaria. Explosion of nematocyst capsules is driven by the high pressure within the capsule formed by the high concentration of poly-gamma-glutamate in the capsule matrix. Poly-gamma-glutamate is a polyanion that binds cations tightly, including the fluorescent cationic dyes acridine orange and DAPI (4',6-diamidino-2-phenylindole). We have used acridine orange and DAPI staining to localize poly-gamma-glutamate within capsules and to follow the biosynthesis of poly-gamma-glutamate during capsule formation. The results indicate that poly-gamma-glutamate biosynthesis occurs late in capsule formation after invagination of the tubule and that it is accompanied by swelling of the capsule due to increasing osmotic pressure. The matrix in all four capsule types is homogeneously filled with poly-gamma-glutamate. In vivo this poly-gamma-glutamate is complexed with monovalent cations. In addition, poly-gamma-glutamate is formed within the tubule lumen of stenoteles. We argue that this poly-gamma-glutamate is required to drive the two-step explosion process in stenotele nematocysts.

The process of cell adhesion among dissociated single cells of Hydra: morphological observations.

Ultrastructural observations were made on the initial adhesion process at the adherent region of Hydra endodermal cell pairs brought into contact (following dissociation) using a three-dimensional laser manipulator. Total contact length across the diameter of the adherent region decreased during the period 10-60 min after initial adhesion. However, the mean numbers of closest (<4 nm) and medium (5-25 nm) separation distances between membranes (thought to be important in total cell adhesion) were not significantly different. These data indicate that adherent cell pairs maintain a constant adhesiveness during the first 60 min of the adhesion process, despite membrane rearrangements. The relative length of each separation distance in adherent cell pairs approached that reported previously for intact Hydra. The sums of lengths in both the closest and medium categories (as a proportion of total contact length) increased because the length of cleavages (distances >25 nm) decreased significantly during the same time period. These results suggest that adherent cell pairs undergo rapid, active membrane changes in the adherent region, which might be associated with cell sorting. The possible significance of these changes for active rearrangement are discussed.

The novel peptide HEADY specifies apical fate in a simple radially symmetric metazoan.

One of the first steps in animal development is axis formation, during which an uneven distribution of signals and/or transcription factors results in the establishment of polarity in the embryo. Hydra, one of the simplest metazoan animals, shows characteristics of a permanent embryo. Even adult polyps have a striking capacity to regenerate, suggesting that molecular mechanisms underlying de novo pattern formation are permanently active and self regulatory. Here we show that HEADY, a short, amidated peptide, plays a central role in the specification of apical fate in this simple metazoan. The HEADY gene, whose transcripts accumulate at the apical organizing center, is required for specification of apical fate, as disruption of HEADY function by dsRNA mediated interference (RNAi) results in severe defects in head formation. Conversely, an instructive role of HEADY in head specification is demonstrated by the application of synthetic HEADY peptide, which induces formation of secondary axes with head morphology. Thus, the HEADY peptide acts as developmental switch to pattern the apical-basal axis of Hydra, providing a first insight into how initial asymmetry is specified in lower metazoan animals.