Evolving visual pigments: hints from the opsin-based proteins in a phylogenetically old "eyeless" invertebrate

Visual pigments are photosensitive receptor proteins that trigger the transduction process producing the visual excitation once they have absorbed photons. In spite of the molecular and morpho-functional complexity that has characterized the development of animal eyes and eyeless photoreceptive systems, opsin-based protein family appears ubiquous along metazoan visual systems. Moreover, in addition to classic rhodopsin photoreceptors, all Metazoa have supplementary non-visual photosensitive structures, mainly located in the central nervous system, that sense light without forming an image and that rather regulate the organism's temporal physiology. The investigation of novel non-visual photopigments exerting extraretinal photoreception is a challenging field in vision research. Here we propose the cnidarian Hydra as a useful tool of investigation for molecular and functional differences between these pigment families. Hydra is the first metazoan owning a nervous system and it is an eyeless invertebrate showing only an extraocular photoreception, as it has no recognized visual or photosensitive structures. In this paper we provide an overview of the molecular and functional features of the opsin-based protein subfamilies and preliminary evidences in a phylogenetically old species of both image-forming and non-visual opsins. Then we give new insights on the molecular biology of Hydra photoreception and on the evolutionary pathways of visual pigments.     

Rhodopsin: A Photopigment for Phototaxis in Euglena gracilis

For over 100 years, a major focus of photobiological studies has been the unicellular flagellate, Euglena gracilis, an organism well suited for such investigations by its special complement of organelles that may be considered an ancient, yet complete “visual” system. The possible photoreceptive roles of the cytoplasmic stigma and the photoreceptor (paraflagellar swelling) of E. gracilis are still under debate, because of conflicting interpretations of the results produced so far by the different research groups working on this microorganism. This article deals with our hypothesis, first put forward in the late 1980s, that rhodopsin-like proteins are responsible for photo-detection and that the paraxial rod is involved in the control of flagellar movements. This hypothesis uses oriented dipole and electroconformational coupling mechanisms as the physical phenomena that produce signal transduction. A model for phototaxis is presented.     

Germline stem cells and sex determination in Hydra

The sex of germline stem cells (GSCs) in Hydra is determined in a cell-autonomous manner. In gonochoristic species like Hydra magnipapillata or H. oligactis, where the sexes are separate, male polyps have sperm-restricted stem cells (SpSCs), while females have egg-restricted stem cells (EgSCs). These GSCs self-renew in a polyp, and are usually transmitted to a new bud from a parental polyp during asexual reproduction. But if these GSCs are lost during subsequent budding or regeneration events, new ones are generated from multipotent stem cells (MPSCs). MPSCs are the somatic stem cells in Hydra that ordinarily differentiate into nerve cells, nematocytes (stinging cells in cnidarians), and gland cells. By means of such a backup system, sexual reproduction is guaranteed for every polyp. Interestingly, Hydra polyps occasionally undergo sex-reversal. This implies that each polyp can produce either type of GSCs, i.e. Hydra are genetically hermaphroditic. Nevertheless a polyp possesses only one type of GSCs at a time. We propose a plausible model for sex-reversal in Hydra. We also discuss so-called germline specific genes, which are expressed in both GSCs and MPSCs, and some future plans to investigate Hydra GSCs.     

Role of epithelial cells and programmed cell death in Hydra spermatogenesis

Spermatogenesis in higher animals is a tightly regulated process, in which survival and death of sperm precursor cells depends on the presence of somatic cells in gonads. In the basal metazoan Hydra spermatogenesis takes place in anatomically simple testes and in the absence of accessory structures. Hydra sperm precursors are derived from interstitial stem cells. Here we show that large numbers of sperm precursors in testes of Hydra vulgaris undergo programmed cell death (apoptosis) and that ectodermal epithelial cells phagocytose the apoptotic sperm precursors. This is surprising since so far no evidence has been reported that epithelial cells are directly involved in germ cell differentiation in Hydra. We propose that, similar to Sertoli cells in mammals, in Hydra epithelial cells support and perhaps even control spermatogenesis.     

Diversity of opsin immunoreactivities in the extraretinal tissues of four anuran amphibians

The pineal complex, deep brain, and skin have been known to function as extraretinal photoreceptors in non-mammalian vertebrates. To see the diversity of localization of extraretinal photoreceptors in lower vertebrates having different habitats, we analyzed the opsin-like immunoreactivities in anuran amphibians, Xenopus laevis, Rana catesbeiana, Rana nigromaculata, and Bufo japonicus. An antiserum (toad Rh-AS) was raised against rhodopsin purified from the retinas of Japanese toad, B. japonicus. In the retina of all the anurans examined, the outer segments of rods were immunopositive to toad Rh-AS. The outer segments of most pinealocytes were immunopositive in R. catesbeiana, R. nigromaculata, and B. japonicus. The outer segments of photoreceptor-like cells within the frontal organ of R. nigromaculata were immunostained. Interestingly, toad Rh-AS immunostained many secretory cells of mucous glands in the head skin of B. japonicus, implying the presence of a novel photoreceptive molecule. Within the hypothalamus, toad Rh-AS immunostained many cells in the magnocellular preoptic nucleus of R. catesbeiana and B. japonicus. Toad Rh-AS also labeled cerebrospinal fluid (CSF)-contacting cells in the anterior preoptic nucleus of R. nigromaculata and those adjacent to the lateral ventricle within the septum of R. catesbeiana. Thus the distribution patterns of the rhodopsin-like immunoreactivities among the anurans were highly diverged, and there was no relationship between the distribution patterns and their habitats. J. Exp. Zool. 286:136-142, 2000.     

Hydra Peptide Project 1993–2007

A systematic screening of peptide signaling molecules (<5000 da) in Hydra magnipapillata (the Hydra Peptide Project) was launched in 1993 and at least the first phase of the project ended in 2007. From the project a number of interesting suggestions and results have been obtained. First, a simple metazoan-like Hydra appears to contain a few hundred peptide signaling molecules: half of them neuropeptides and the rest epitheliopeptides that are produced by epithelial cells. Second, epitheliopeptides were identified for the first time in Hydra . Some exhibit morphogen-like activities, which accord with the notion that epithelial cells are primarily responsible for patterning in Hydra . A family of epitheliopeptides was involved in regulating neuron differentiation possibly through neuron–epithelial cell interaction. Third, many novel neuropeptides were identified. Most of them act directly on muscle cells inducing contraction or relaxation. Some were involved in cell differentiation and morphogenesis. During the course of this study, a number of important technical innovations (e.g. genetic manipulations in transgenic Hydra , high-throughput purification techniques, etc.) and expressed sequence tag (EST) and genome databases were introduced in Hydra research. They have already helped to identify and characterize novel peptides and will contribute even more to the Hydra Peptide Project in the near future.     

GFP expression in Hydra: lessons from the particle gun

The cnidarian Hydra is an important model organism to study pattern formation and tem cell differentiation. In the past, however, it has been difficult to study gene function in Hydra because the animals have hot been accessible to gene transfection studies, we have now developed a method to transiently express GFP-tagged proteins in Hydra using a green fluorescent protein (GFP) expression plasmid under the control of the Hydra actin promoter and a particle gun to introduce it into Hydra cell nuclei. We achieve strong transient GFP expression in a small but reproducible number of epithelial and interstitial cells. Implications for the use of this method to carry out single cell assays with GFP-tagged Hydra proteins are discussed.     

Value of the Hydra model system for studying symbiosis

Green Hydra is used as a classical example for explaining symbiosis in schools as well as an excellent research model. Indeed the cosmopolitan green Hydra (Hydra viridissima) provides a potent experimental framework to investigate the symbiotic relationships between a complex eumetazoan organism and a unicellular photoautotrophic green algae named Chlorella. Chlorella populates a single somatic cell type, the gastrodermal myoepithelial cells (also named digestive cells) and the oocyte at the time of sexual reproduction. This symbiotic relationship is stable, well-determined and provides biological advantages to the algal symbionts, but also to green Hydra over the related non-symbiotic Hydra i.e. brown hydra. These advantages likely result from the bidirectional flow of metabolites between the host and the symbiont. Moreover genetic flow through horizontal gene transfer might also participate in the establishment of these selective advantages. However, these relationships between the host and the symbionts may be more complex. Thus, Jolley and Smith showed that the reproductive rate of the algae increases dramatically outside of Hydra cells, although this endosymbiont isolation is debated. Recently it became possible to keep different species of endosymbionts isolated from green Hydra in stable and permanent cultures and compare them to free-living Chlorella species. Future studies testing metabolic relationships and genetic flow should help elucidate the mechanisms that support the maintenance of symbiosis in a eumetazoan species.     

Transplantation stimulates interstitial cell migration in hydra

Migration of interstitial cells and nerve cell precursors was analyzed in Hydra magnipapillata and Hydra vulgaris (formerly Hydra attenuata). Axial grafts were made between [3H]thymidine-labeled donor and unlabeled host tissue. Migration of labeled cells into the unlabeled half was followed for 4 days. The results indicate that the rate of migration was initially high and then slowed on Days 2-4. Regrafting fresh donor tissue on Days 2-4 maintained high levels of migration. Thus, migration appears to be stimulated by the grafting procedure itself.     

Hydra and the evolution of stem cells

Hydra are remarkable because they are immortal. Much of immortality can be ascribed to the asexual mode of reproduction by budding, which requires a tissue consisting of stem cells with continuous self‐renewal capacity. Emerging novel technologies and the availability of genomic resources enable for the first time to analyse these cells in vivo. Stem cell differentiation in Hydra is governed through the coordinated actions of conserved signaling pathways. Studies of stem cells in Hydra, therefore, promise critical insights of general relevance into stem cell biology including cellular senescence, lineage programming and reprogramming, the role of extrinsic signals in fate determination and tissue homeostasis, and the evolutionary origin of these cells. With these new facts as a backdrop, this review traces the history of studying stem cells in Hydra and offers a view of what the future may hold.     

Hymyc1 downregulation promotes stem cell proliferation in Hydra vulgaris

Hydra is a unique model for studying the mechanisms underlying stem cell biology. The activity of the three stem cell lineages structuring its body constantly replenishes mature cells lost due to normal tissue turnover. By a poorly understood mechanism, stem cells are maintained through self-renewal while concomitantly producing differentiated progeny. In vertebrates, one of many genes that participate in regulating stem cell homeostasis is the protooncogene c-myc, which has been recently identified also in Hydra, and found expressed in the interstitial stem cell lineage. In the present paper, by developing a novel strategy of RNA interference-mediated gene silencing (RNAi) based on an enhanced uptake of small interfering RNAi (siRNA), we provide molecular and biological evidence for an unexpected function of the Hydra myc gene (Hymyc1) in the homeostasis of the interstitial stem cell lineage. We found that Hymyc1 inhibition impairs the balance between stem cell self renewal/differentiation, as shown by the accumulation of stem cell intermediate and terminal differentiation products in genetically interfered animals. The identical phenotype induced by the 10058-F4 inhibitor, a disruptor of c-Myc/Max dimerization, demonstrates the specificity of the RNAi approach. We show the kinetic and the reversible feature of Hymyc1 RNAi, together with the effects displayed on regenerating animals. Our results show the involvement of Hymyc1 in the control of interstitial stem cell dynamics, provide new clues to decipher the molecular control of the cell and tissue plasticity in Hydra, and also provide further insights into the complex myc network in higher organisms. The ability of Hydra cells to uptake double stranded RNA and to trigger a RNAi response lays the foundations of a comprehensive analysis of the RNAi response in Hydra allowing us to track back in the evolution and the origin of this process.     

A histological and ultrastructural study of germinal differentiation of interstitial cells arising from gland cells in Hydra viridis

Gland cells of the gastrodermis of Hydra when isolated from the epidermis are capable of dedifferentiating into interstitial cells. Under proper environmental conditions these interstitial cells are capable of undergoing meiotic divisions and forming normal gametes. This dedifferentiation and redifferentiation sequence has been studied at the level of the light and electron microscope. It is concluded that in Hydra there is no specific germinal cell line determined during embryogeny, and that a somatic cell under proper environmental conditions can be induced to undergo meiosis.     

Hydra, a model system for environmental studies

Hydra have been extensively used for studying the teratogenic and toxic potential of numerous toxins throughout the years and are more recently growing in popularity to assess the impacts of environmental pollutants. Hydra are an appropriate bioindicator species for use in environmental assessment owing to their easily measurable physical (morphology), biochemical (xenobiotic biotransformation; oxidative stress), behavioural (feeding) and reproductive (sexual and asexual) endpoints. Hydra also possess an unparalleled ability to regenerate, allowing the assessment of teratogenic compounds and the impact of contaminants on stem cells. Importantly, Hydra are ubiquitous throughout freshwater environments and relatively easy to culture making them appropriate for use in small scale bioassay systems. Hydra have been used to assess the environmental impacts of numerous environmental pollutants including metals, organic toxicants (including pharmaceuticals and endocrine disrupting compounds), nanomaterials and industrial and municipal effluents. They have been found to be among the most sensitive animals tested for metals and certain effluents, comparing favourably with more standardised toxicity tests. Despite their lack of use in formalised monitoring programmes, Hydra have been extensively used and are regarded as a model organism in aquatic toxicology.     

Molecular cloning and cellular distribution of two 14-3-3 isoforms from Hydra: 14-3-3 proteins respond to starvation and bind to phosphorylated targets

In the simple metazoan Hydra a clear link between food supply and cell survival has been established. Whilst in plants 14-3-3 proteins are found to be involved in signalling cascades that regulate metabolism, in animals they have been shown to participate in cell survival pathways. In order to explore the possibility that 14-3-3 proteins in Hydra could be involved in regulating metabolism under different conditions of food supply, we have cloned two isoforms of 14-3-3 proteins. We show here that 14-3-3 proteins bind to phosphorylated targets in Hydra and form homo- and heterodimers in vitro. 14-3-3 proteins are localised in the cytoplasm of all cells and also in the nuclei of some epithelial cells. This nuclear localisation becomes more prominent during starvation. Moreover, 14-3-3 protein is present in large amounts in food granules and from this we conclude that it performs functions which are associated with metabolism and food storage in Hydra.     

The Hydra Model System

The freshwater Hydra polyp emerged as a model system in 1741 when Abraham Trembley not only discovered its amazing regenerative potential, but also demonstrated that experimental manipulations pave the way to research in biology. Since then, Hydra flourished as a potent and fruitful model system to help answer questions linked to cell and developmental biology, as such as the setting up of an organizer to regenerate a complex missing structure, the establishment and maintainance of polarity in a multicellular organism, the development of mathematical models to explain the robust developmental rules observed in this animal, the maintainance of stemness and multipotency in a highly dynamic environment, the plasticity of differentiated cells, to name but a few. However the Hydra model system is not restricted to cell and developmental biology; during the past 270 years it has also been heavily used to investigate the relationships between Hydra and its environment, opening new horizons concerning neurophysiology, innate immunity, ecosystems, ecotoxicology, symbiosis...     

Self/nonself recognition in Cnidaria: contact to allogeneic tissue does not result in elimination of nonself cells in Hydra vulgaris

Although Cnidaria have no specialised immune cells, some colonial forms possess a genetic system to discriminate between self and nonself. Allorecognition is thought to protect them from fusion with genetically different individuals and to prevent germ line parasitism. Surprisingly, when grafting tissue of two species of the solitary freshwater polyp Hydra, we found within the contact zone phagocytozing epithelial cells which selectively eliminated cells from the other species (Bosch and David, 1986). This led us to speculate that Hydra, which never undergoes "natural transplantation", can differentiate between self and nonself (Bosch and David, 1986). In a previous paper (Kuznetsov et al., 2002) we described that cells which accumulate in the contact region of these interspecies grafts are apoptotic and that apoptosis is induced by impaired cell matrix contact. Thus, observations in such interspecies grafts did not give hints concerning the presence of a discriminative allorecognition system. To clarify whether this fundamental aspect of immunity is present in these phylogenetically old animals, we examined epithelial interactions between different strains of Hydra vulgaris. Here, we show that contact to allogeneic tissue does not evoke any response in terms of phagocytosis and elimination of allogeneic cells. We, therefore, question Hydra's ability to discriminate between self and nonself and propose that, in contrast to colonial cnidarians, the solitary polyp Hydra has either lost or substantially reduced this ability.