Microtubule organization of vertebrate sensory neurons in vivo.

Dorsal root ganglion (DRG) neurons are the predominant cell type that innervates the vertebrate skin. They are typically described as pseudounipolar cells that have central and peripheral axons branching from a single root exiting the cell body. The peripheral axon travels within a nerve to the skin, where free sensory endings can emerge and branch into an arbor that receives and integrates information. In some immature vertebrates, DRG neurons are preceded by Rohon-Beard (RB) neurons. While the sensory endings of RB and DRG neurons function like dendrites, we use live imaging in zebrafish to show that they have axonal plus-end-out microtubule polarity at all stages of maturity. Moreover, we show both cell types have central and peripheral axons with plus-end-out polarity. Surprisingly, in DRG neurons these emerge separately from the cell body, and most cells never acquire the signature pseudounipolar morphology. Like another recently characterized cell type that has multiple plus-end-out neurites, ganglion cells in Nematostella, RB and DRG neurons maintain a somatic microtubule organizing center even when mature. In summary, we characterize key cellular and subcellular features of vertebrate sensory neurons as a foundation for understanding their function and maintenance.

Experimental Tools to Study Regeneration in the Sea Anemone Nematostella vectensis.

Animal regeneration is a biological process leading to the reformation of injured or lost tissues/body parts. One of the most fascinating regenerative phenomena is the so-called whole-body regeneration, leading to the reformation of fully functional organisms within days after bisection. The sea anemone Nematostella vectensis is currently emerging as novel whole-body regeneration model. Here we describe the methods of inducing the regenerative process in this cnidarian as well as the fixation and staining protocols for morphological, molecular, and cellular analysis.

Functional and Structural Variation among Sticholysins, Pore-Forming Proteins from the Sea Anemone Stichodactyla helianthus.

Venoms constitute complex mixtures of many different molecules arising from evolution in processes driven by continuous prey-predator interactions. One of the most common compounds in these venomous cocktails are pore-forming proteins, a family of toxins whose activity relies on the disruption of the plasmatic membranes by forming pores. The venom of sea anemones, belonging to the oldest lineage of venomous animals, contains a large amount of a characteristic group of pore-forming proteins known as actinoporins. They bind specifically to sphingomyelin-containing membranes and suffer a conformational metamorphosis that drives them to make pores. This event usually leads cells to death by osmotic shock. Sticholysins are the actinoporins produced by Stichodactyla helianthus. Three different isotoxins are known: Sticholysins I, II, and III. They share very similar amino acid sequence and three-dimensional structure but display different behavior in terms of lytic activity and ability to interact with cholesterol, an important lipid component of vertebrate membranes. In addition, sticholysins can act in synergy when exerting their toxin action. The subtle, but important, molecular nuances that explain their different behavior are described and discussed throughout the text. Improving our knowledge about sticholysins behavior is important for eventually developing them into biotechnological tools.

A bipolar role of the transcription factor ERG for cnidarian germ layer formation and apical domain patterning.

Germ layer formation and axial patterning are biological processes that are tightly linked during embryonic development of most metazoans. In addition to canonical WNT, it has been proposed that ERK-MAPK signaling is involved in specifying oral as well as aboral territories in cnidarians. However, the effector and the molecular mechanism underlying latter phenomenon is unknown. By screening for potential effectors of ERK-MAPK signaling in both domains, we identified a member of the ETS family of transcription factors, Nverg that is bi-polarily expressed prior to gastrulation. We further describe the crucial role of NvERG for gastrulation, endomesoderm as well as apical domain formation. The molecular characterization of the obtained NvERG knock-down phenotype using previously described as well as novel potential downstream targets, provides evidence that a single transcription factor, NvERG, simultaneously controls expression of two different sets of downstream targets, leading to two different embryonic gene regulatory networks (GRNs) in opposite poles of the developing embryo. We also highlight the molecular interaction of cWNT and MEK/ERK/ERG signaling that provides novel insight into the embryonic axial organization of Nematostella, and show a cWNT repressive role of MEK/ERK/ERG signaling in segregating the endomesoderm in two sub-domains, while a common input of both pathways is required for proper apical domain formation. Taking together, we build the first blueprint for a global cnidarian embryonic GRN that is the foundation for additional gene specific studies addressing the evolution of embryonic and larval development.

Morphological Variability and Distinct Protein Profiles of Cultured and Endosymbiotic Symbiodinium cells Isolated from Exaiptasia pulchella.

Symbiodinium is a dinoflagellate that plays an important role in the physiology of the symbiotic relationships of Cnidarians such as corals and sea anemones. However, it is very difficult to cultivate free-living dinoflagellates after being isolated from the host, as they are very sensitive to environmental changes. How these symbiont cells are supported by the host tissue is still unclear. This study investigated the characteristics of Symbiodinium cells, particularly with respect to the morphological variability and distinct protein profiles of both cultured and endosymbiotic Symbiodinium which were freshly isolated from Exaiptasia pulchella. The response of the cellular morphology of freshly isolated Symbiodinium cells kept under a 12 h L:12 h D cycle to different temperatures was measured. Cellular proliferation was investigated by measuring the growth pattern of Symbiodinium cells, the results of which indicated that the growth was significantly reduced in response to the extreme temperatures. Proteomic analysis of freshly isolated Symbiodinium cells revealed twelve novel proteins that putatively included transcription translation factors, photosystem proteins, and proteins associated with energy and lipid metabolism, as well as defense response. The results of this study will bring more understandings to the mechanisms governing the endosymbiotic relationship between the cnidarians and dinoflagellates.

In vivo imaging of Nematostella vectensis embryogenesis and late development using fluorescent probes.

BACKGROUND: Cnidarians are the closest living relatives to bilaterians and have been instrumental to studying the evolution of bilaterian properties. The cnidarian model, Nematostella vectensis, is a unique system in which embryology and regeneration are both studied, making it an ideal candidate to develop in vivo imaging techniques. Live imaging is the most direct way for quantitative and qualitative assessment of biological phenomena. Actin and tubulin are cytoskeletal proteins universally important for regulating many embryological processes but so far studies in Nematostella primarily focused on the localization of these proteins in fixed embryos. RESULTS: We used fluorescent probes expressed in vivo to investigate the dynamics of Nematostella development. Lifeact-mTurquoise2, a fluorescent cyan F-actin probe, can be visualized within microvilli along the cellular surface throughout embryonic development and is stable for two months after injection. Co-expression of Lifeact-mTurquoise2 with End-Binding protein1 (EB1) fused to mVenus or tdTomato-NLS allows for the visualization of cell-cycle properties in real time. Utilizing fluorescent probes in vivo helped to identify a concentrated 'flash' of Lifeact-mTurquoise2 around the nucleus, immediately prior to cytokinesis in developing embryos. Moreover, Lifeact-mTurquoise2 expression in adult animals allowed the identification of various cell types as well as cellular boundaries. CONCLUSION: The methods developed in this manuscript provide an alternative protocol to investigate Nematostella development through in vivo cellular analysis. This study is the first to utilize the highly photo-stable florescent protein mTurquoise2 as a marker for live imaging. Finally, we present a clear methodology for the visualization of minute temporal events during cnidarian development.

GABA and glutamate immunoreactivity in tentacles of the sea anemone Phymactis papillosa (LESSON 1830).

Sea anemones have a structurally simple nervous system that controls behaviors like feeding, locomotion, aggression, and defense. Specific chemical and tactile stimuli are transduced by ectodermal sensory cells and transmitted via a neural network to cnidocytes and epithelio-muscular cells, but the nature of the neurotransmitters operating in these processes is still under discussion. Previous studies demonstrated an important role of peptidergic transmission in cnidarians, but during the last decade the contribution of conventional neurotransmitters became increasingly evident. Here, we used immunohistochemistry on light and electron microscopical preparations to investigate the localization of glutamate and GABA in tentacle cross-sections of the sea anemone Phymactis papillosa. Our results demonstrate strong glutamate immunoreactivity in the nerve plexus, while GABA labeling was most prominent in the underlying epithelio-muscular layer. Immunoreactivity for both molecules was also found in glandular epithelial cells, and putative sensory cells were GABA positive. Under electron microscopy, both glutamate and GABA immunogold labeling was found in putative neural processes within the neural plexus. These data support a function of glutamate and GABA as signaling molecules in the nervous system of sea anemones.

Formation of the apical flaps in nematocysts of sea anemones (cnidaria: actiniaria).

Using scanning and transmission electron microscopy, we studied formation of the structure at the apical end of sea anemone nematocysts through which the tubule everts at discharge. In anemones of the genus Metridium, we found that each of the three solid triangular apical flaps comprises two layers that are continuous with those of the capsule wall: the electron-lucent inner layer is bound to the electron-dense outer layer. The two-layer structure is obvious in some discharged capsules in which, perhaps due to fixation, the layers part at the flap's periphery. Before the nematocyst discharges, a channel leads from a pore at the tip of the joined flaps into the lumen of the inverted tubule. The thin laminate layer that coats each flap lines the channel. The base of the nematocyst tubule adheres to the capsule wall near the capsule's apical end, and a branch of the tubule underlies part of the laminate layer that coats the flaps. Thus the tubule is not continuous with the capsule wall but structurally separate from it. This helps reconcile differences in understanding of the number of layers constituting the capsule wall, and makes clear that the tubule should be considered part of the capsule contents.

Active nematocyst isolation via nudibranchs.

Cnidarian venoms are potentially valuable tools for biomedical research and drug development. They are contained within nematocysts, the stinging organelles of cnidarians. Several methods exist for the isolation of nematocysts from cnidarian tissues; most are tedious and target nematocysts from specific tissues. We have discovered that the isolated active nematocyst complement (cnidome) of several sea anemone (Cnidaria: Anthozoa) species is readily accessible. These nematocysts are isolated, concentrated, and released to the aqueous environment as a by-product of aeolid nudibranch Spurilla neapolitana cultures. S. neapolitana feed on venomous sea anemones laden with stinging nematocysts. The ingested stinging organelles of several sea anemone species are effectively excreted in the nudibranch feces. We succeeded in purifying the active organelles and inducing their discharge. Thus, our current study presents the attractive possibility of using nudibranchs to produce nematocysts for the investigation of novel marine compounds.

Cadherin 23-like polypeptide in hair bundle mechanoreceptors of sea anemones.

We investigated hair bundle mechanoreceptors in sea anemones for a homolog of cadherin 23. A candidate sequence was identified from the database for Nematostella vectensis that has a shared lineage with vertebrate cadherin 23s. This cadherin 23-like protein comprises 6,074 residues. It is an integral protein that features three transmembrane alpha-helices and a large extracellular loop with 44 contiguous, cadherin (CAD) domains. In the second half of the polypeptide, the CAD domains occur in a quadruple repeat pattern. Members of the same repeat group (i.e., CAD 18, 22, 26, and so on) share nearly identical amino acid sequences. An affinity-purified antibody was generated to a peptide from the C-terminus of the cadherin 23-like polypeptide. The peptide is expected to lie on the exoplasmic side of the plasma membrane. In LM, the immunolabel produced punctate fluorescence in hair bundles. In TEM, immunogold particles were observed medially and distally on stereocilia of hair bundles. Dilute solutions of the antibody disrupted vibration sensitivity in anemones. We conclude that the cadherin 23-like polypeptide likely contributes to the mechanotransduction apparatus of hair bundle mechanoreceptors of anemones.

Embryonic and larval development of the host sea anemones Entacmaea quadricolor and Heteractis crispa.

Little information is available on the sexual reproductive biology of anemones that provide essential habitat for anemonefish. Here we provide the first information on the surface ultrastructural and morphological changes during development of the embryos and planula larvae of Entacmaea quadricolor and Heteractis crispa, using light and scanning electron microscopy. Newly spawned eggs of E. quadricolor and H. crispa averaged 794 microm and 589 microm diameter, respectively, and were covered by many spires of microvilli that were evenly distributed over the egg surface, except for a single bare patch. Eggs of both species contained abundant zooxanthellae when spawned, indicating vertical transmission of symbionts. Fertilization was external, and the resulting embryos displayed superficial cleavage. As development continued, individual blastomeres became readily distinguishable and a round-to-ovoid blastula was formed, which flattened with further divisions. The edges of the blastula thickened, creating a concave-convex dish-shaped gastrula. The outer margins of the gastrula appeared to roll inward, leading to the formation of an oral pore and a ciliated planula larva. Larval motility and directional movement were first observed 36 h after spawning. E. quadricolor larval survival remained high during the first 4 d after spawning, then decreased rapidly.

Gastrulation in the sea anemone Nematostella vectensis occurs by invagination and immigration: an ultrastructural study.

The sea anemone Nematostella vectensis has recently been established as a new model system for the understanding of the evolution of developmental processes. In particular, the evolutionary origin of gastrulation and its molecular regulation are the subject of intense investigation. However, while molecular data are rapidly accumulating, no detailed morphological data exist describing the process of gastrulation. Here, we carried out an ultrastructural study of different stages of gastrulation in Nematostella using transmission electron microscope and scanning electron microscopy techniques. We show that presumptive endodermal cells undergo a change in cell shape, reminiscent of the bottle cells known from vertebrates and several invertebrates. Presumptive endodermal cells organize into a field, the pre-endodermal plate, which undergoes invagination. In parallel, the endodermal cells decrease their apical cell contacts but remain loosely attached to each other. Hence, during early gastrulation they display an incomplete epithelial-mesenchymal transition (EMT). At a late stage of gastrulation, the cells eventually detach and fill the interior of the blastocoel as mesenchymal cells. This shows that gastrulation in Nematostella occurs by a combination of invagination and late immigration involving EMT. The comparison with molecular expression studies suggests that cells expressing snailA undergo EMT and become endodermal, whereas forkhead/brachyury expressing cells at the ectodermal margin of the blastopore retain their epithelial integrity throughout gastrulation.

Heat stress induces different forms of cell death in sea anemones and their endosymbiotic algae depending on temperature and duration.

Bleaching of reef building corals and other symbiotic cnidarians due to the loss of their dinoflagellate algal symbionts (=zooxanthellae), and/or their photosynthetic pigments, is a common sign of environmental stress. Mass bleaching events are becoming an increasingly important cause of mortality and reef degradation on a global scale, linked by many to global climate change. However, the cellular mechanisms of stress-induced bleaching remain largely unresolved. In this study, the frequency of apoptosis-like and necrosis-like cell death was determined in the symbiotic sea anemone Aiptasia sp. using criteria that had previously been validated for this symbiosis as indicators of programmed cell death (PCD) and necrosis. Results indicate that PCD and necrosis occur simultaneously in both host tissues and zooxanthellae subject to environmentally relevant doses of heat stress. Frequency of PCD in the anemone endoderm increased within minutes of treatment. Peak rates of apoptosis-like cell death in the host were coincident with the timing of loss of zooxanthellae during bleaching. The proportion of apoptosis-like host cells subsequently declined while cell necrosis increased. In the zooxanthellae, both apoptosis-like and necrosis-like activity increased throughout the duration of the experiment (6 days), dependent on temperature dose. A stress-mediated PCD pathway is an important part of the thermal stress response in the sea anemone symbiosis and this study suggests that PCD may play different roles in different components of the symbiosis during bleaching.

Localization of a symbiosis-related protein, Sym32, in the Anthopleura elegantissima-Symbiodinium muscatinei Association.

Cnidarian-dinoflagellate symbioses are widespread in the marine environment. Growing concern over the health of coral reef ecosystems has revealed a fundamental lack of knowledge of how cnidarian-algal associations are regulated at the cellular and molecular level. We are interested in identifying genes that mediate interactions between the partners, and we are using the temperate sea anemone Anthopleura elegantissima as a model. We previously described a host gene, sym32, encoding a fasciclin domain protein, that is differentially expressed in symbiotic and aposymbiotic A. elegantissima. Here, we describe the subcellular localization of the sym32 protein. In aposymbiotic (symbiont-free) hosts, sym32 was located in vesicles that occur along the apical edges of gastrodermal cells. In symbiotic hosts, such vesicles were absent, but sym32 was present within the symbiosome membranes. Sym32 (or a cross-reactive protein) was also present in the accumulation bodies of the symbionts. Although the anti-sym32 antiserum was not sufficiently specific to detect the target protein in cultured Symbiodinium bermudense cells, Western blots of proteins from two Symbiodinium species revealed a protein doublet of 45 and 48 kDa, suggesting that the symbionts may also produce a fasciclin domain protein. We suggest that host sym32 is relocated from gastrodermal vesicles to the symbiosome membrane when symbionts are taken into host cells by phagocytosis.

Ultrastructure of neuro-spirocyte synapses in the sea anemone Aiptasia pallida (Cnidaria, Anthozoa, Zoantharia).

Using transmission electron microscopy of serially sectioned tentacles from the sea anemone Aiptasia pallida, we located and characterized two types of neuro-spirocyte synapses. Clear vesicles were observed at 10 synapses and dense-cored vesicles at five synapses. The diameters of vesicles at each neuro-spirocyte synapse were averaged; clear vesicles ranged from 49-89 nm in diameter, whereas the dense-cored vesicles ranged from 97-120 nm in diameter. One sequential pair of synapses included a neuro-spirocyte synapse with clear vesicles (81 nm) and a neuro-neuronal synapse with dense-cored vesicles (168 nm). A second synapse on the same cell had dense-cored vesicles (103 nm). An Antho-RFamide-labeled ganglion cell and three different neurites were observed adjacent to spirocytes, but no neuro-spirocyte synapses were present. Many of the spirocytes also were immunoreactive to Antho-RFamide. The presence of sequential neuro-neuro-spirocyte synapses suggests that synaptic modulation may be involved in the neural control of spirocyst discharge. The occurrence of either dense-cored or clear vesicles at neuro-spirocyte synapses suggests that at least two types of neurotransmitter substances control the discharge of spirocysts in sea anemones.

Hair bundles of sea anemones as a model system for vertebrate hair bundles.

Sea anemones are marine invertebrates that use hair bundles to detect swimming movements of prey. Prey are captured by nematocysts (stinging capsules) that discharge into the prey. To further characterize anemone hair bundles and to compare hair bundles in anemones with hair bundles in vertebrates, we investigated fine structure and cytochemistry of anemone hair bundles. In addition, using a biological assay based on counting nematocysts discharged into vibrating test probes, we examined sensitivity of vibration detection to aminoglycoside antibiotics, Ca(2+)-free seawater, and amiloride. Like vertebrate hair bundles, anemone hair bundles are composed of stereocilia, possess lateral linkages between stereocilia whose preservation for transmission electron microscopy is enhanced by ruthenium red, and possess tip links morphologically similar to vertebrate tip links. Furthermore, vibration-dependent discharge of nematocysts is reversibly inhibited by 10(-4) M streptomycin and abolished by brief exposure to Ca(2+)-free seawater. However, unlike vertebrate hair bundles, anemone hair bundles appear to be insensitive to amiloride since vibration-dependent discharge of nematocysts is unaffected by up to mM amiloride. Thus, anemone hair bundles may serve as a useful model system for vertebrate hair bundles with the interesting feature of being insensitive to amiloride.

Ultrastructure of neurons and synapses in the tentacle epidermis of the sea anemone Calliactis parasitica.

The anatomical organization of neutrons and synaptic pathways in tentacles of sea anemones is poorly understood. Transmission electron microscopy of serial thin sections was carried out on various regions of tentacles of the sea anemone Calliactis parasitica in order to locate and characterize typical epidermal neutrons and synapses. Both surface-oriented sensory cells with ciliary cones and basally located ganglion cells lacking a cilium have Golgi-derived granular or faintly cored vesicles. Similar vesicles are present at synaptic loci on some ganglion and muscle cells. The synaptic contacts on the longitudinal muscle cells are generally en passant rather than terminal. They vary from single neuromuscular synapses to pairs of neurites innervating the same muscle cell or one neurite innervating two or more muscle cells. Both two-way and one-way interneuronal synapses with vesicles aligned at paired synaptic membranes with dense material in a 14-20-nm-wide cleft are present in the epidermal nerve plexus. The vesicles average from 50 to 80 nm in diameter and vary from electron lucent to faintly cored. The results of this study demonstrate the presence of a complex system of epidermal neuronal pathways with specific synaptic loci in this modern representative of a first-evolved nervous system.

Neurofilament-like immunoreactivity in the sea anemone Condylactis gigantea (Cnidaria: Anthozoa).

The neuronal cytoskeleton contains neurofilament proteins that serve as markers for nervous tissue in many species across phyla. Antiserum generated to mammalian neurofilaments was used for immunocytochemical staining of tissues in the sea anemone Condylactis gigantea (Cnidaria: Anthozoa). Specific staining, visible at the light and electron microscope levels, was found in the tissues of the tentacle. Proteins were extracted from the tissues and solubilized. SDS-polyacrylamide gel electrophoresis and Western blotting revealed two bands of MWr 156 kD and 74 kD that reacted with antiserum generated to neurofilaments. The protein bands also bound a monoclonal antibody shown to react with a highly conserved epitope in many classes of intermediate filaments. These data suggest that the neurons of this anthozoan contain neurofilament-like proteins with molecular properties similar to those of mammalian neurons.

Ultrastructure of nematocyst discharge in catch tentacles of the sea anemone Haliplanella luciae (Cnidaria: Anthozoa).

The mature nematocyst lies just beneath the cnidodyte plasma membrane. A microtubule array surrounds the nematocyst capsule just beneath the capsule tip. We propose that the array helps to hold the capsule at the cnidocyte cell surface until discharge. The undischarged capsule tip is sealed by three apical flaps, joined together along complex radial seams. The seams are filled with subunits that appear to bind the flaps together. Upon discharge, the flaps separate along the radial seams to permit thread eversion. The everted thread is lined on both sides by subunits that are stained by antimonate, indicating that they bind calcium. We suggest that, together, the subunits hold the uneverted thread in its folded and coiled configuration. Thread eversion would follow subunit uncoupling. The capsule and thread interiors of partially discharged nematocysts are stained by antimonate. In contrast, the capsule and thread interiors of fully discharged nematocysts are not stained by antimonate. Thus, nematocyst calcium might be injected into the target tissue where it is presumed to act in conjunction with nematocyst venom to promote cell death.