RESUMEN
The Notch-signalling pathway plays an important role in pattern formation in Hydra. Using pharmacological Notch inhibitors (DAPT and SAHM1), it has been demonstrated that HvNotch is required for head regeneration and tentacle patterning in Hydra. HvNotch is also involved in establishing the parent-bud boundary and instructing buds to develop feet and detach from the parent. To further investigate the functions of HvNotch, we successfully constructed NICD (HvNotch intracellular domain)-overexpressing and HvNotch-knockdown transgenic Hydra strains. NICD-overexpressing transgenic Hydra showed a pronounced inhibition on the expression of predicted HvNotch-target genes, suggesting a dominant negative effect of ectopic NICD. This resulted in a "Y-shaped" phenotype, which arises from the parent-bud boundary defect seen in polyps treated with DAPT. Additionally, "multiple heads", "two-headed" and "ectopic tentacles" phenotypes were observed. The HvNotch-knockdown transgenic Hydra with reduced expression of HvNotch exhibited similar, but not identical phenotypes, with the addition of a "two feet" phenotype. Furthermore, we observed regeneration defects in both, overexpression and knockdown strains. We integrated these findings into a mathematical model based on long-range gradients of signalling molecules underlying sharply defined positions of HvNotch-signalling cells at the Hydra tentacle and bud boundaries.
Asunto(s)
Hydra , Animales , Hydra/genética , Inhibidores de Agregación Plaquetaria , Transducción de Señal , Animales Modificados Genéticamente , PieRESUMEN
Although recent studies indicate the impact of microbes on the central nervous systems and behavior, it remains unclear how the relationship between the functionality of the nervous system, behavior, and the microbiota evolved. In this work, we analyzed the eating behavior of Hydra, a host that has a simple nervous system and a low-complexity microbiota. To identify the neuronal subpopulations involved, we used a subpopulation-specific cell ablation system and calcium imaging. The role of the microbiota was uncovered by manipulating the diversity of the natural microbiota. We show that different neuronal subpopulations are functioning together to control eating behavior. Animals with a drastically reduced microbiome had severe difficulties in mouth opening due to a significantly increased level of glutamate. This could be reversed by adding a full complement of the microbiota. In summary, we provide a mechanistic explanation of how Hydra's nervous system controls eating behavior and what role microbes play in this.
Asunto(s)
Hydra , Microbiota , Animales , Hydra/fisiología , Sistema Nervioso , Conducta AlimentariaRESUMEN
The freshwater polyp Hydra is a cnidarian used as a model organism in a number of fields, including the study of the origin and evolution of developmental mechanisms, aging, symbiosis and host-microbe interactions. Here, we describe a procedure for the establishment of stable transgenic Hydra lines by embryo microinjection. The three-stage protocol comprises (i) the design and preparation of a transgenic construct, (ii) the microinjection of the vector into early embryos of Hydra vulgaris, and (iii) the selection and enrichment of mosaic animals in order to develop uniformly transgenic clonal lines. The preparation of a transgenic construct requires ~2 weeks, and transgenic lines can be obtained within 3 months. The method allows constitutive or inducible gain- and loss-of-function approaches, as well as in vivo tracing of individual cells. Hydra polyps carrying transgenic cells reveal functional properties of the ancestral circuitry controlling animal development.
Asunto(s)
Técnicas de Transferencia de Gen , Hydra/citología , Hydra/genética , Animales , Animales Modificados Genéticamente , Blastómeros , Embrión no Mamífero , Expresión Génica , Proteínas Fluorescentes Verdes/genética , Hydra/embriología , Microinyecciones , ARN Interferente PequeñoRESUMEN
Mechanisms of programmed cell death differ between animals, plants and fungi. In animals, apoptotic cell death depends on caspases and Bcl-2 family proteins. These protein families are only found in multicellular animals, including cnidarians, insects and mammals. In contrast, members of the TMBIM-family of transmembrane proteins are conserved across all eukaryotes. Sequence comparisons of cell death related proteins between phyla indicate strong conservation of the genes involved. However, often it is not known whether this is paralleled by conservation of function. Here we present the first study to support an anti-apoptotic function of Bcl-2 like proteins in the cnidarian Hydra within a physiological context. We used transgenic Hydra expressing GFP-tagged HyBcl-2-like 4 protein in epithelial cells. The protein was localised to mitochondria and able to protect Hydra epithelial cells from apoptosis induced by either the PI(3) kinase inhibitor wortmannin or by starvation. Moreover, we identified members of the TMBIM-family in Hydra including HyBax-Inhibitor-1, HyLifeguard-1a and -1b and HyLifeguard 4. Expressing these TMBIM-family members in Hydra and human HEK cells, we found HyBax-inhibitor-1 protein localised to ER-membranes and HyLifeguard-family members localised to the plasma membrane and Golgi-vesicles. Moreover, HyBax-inhibitor-1 protected human cells from camptothecin induced apoptosis. This work illustrates that the investigated Bcl-2- and TMBIM-family members represent evolutionarily conserved mitochondrial, ER, Golgi and plasma membrane proteins with anti-apoptotic functions. The participation of ER and Golgi proteins in the regulation of programmed cell death might be a very ancient feature.
Asunto(s)
Animales Modificados Genéticamente/metabolismo , Apoptosis , Regulación de la Expresión Génica , Hydra/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Proteína X Asociada a bcl-2/metabolismo , Secuencia de Aminoácidos , Animales , Animales Modificados Genéticamente/genética , Animales Modificados Genéticamente/crecimiento & desarrollo , Células HEK293 , Humanos , Hydra/efectos de los fármacos , Hydra/genética , Inmunosupresores/farmacología , Proteínas Proto-Oncogénicas c-bcl-2/genética , Homología de Secuencia , Inanición , Wortmanina/farmacología , Proteína X Asociada a bcl-2/genéticaRESUMEN
Understanding the dynamic cellular behaviours driving morphogenesis and regeneration is a long-standing challenge in biology. Live imaging, together with genetically encoded reporters, may provide the necessary tool to address this issue, permitting the in vivo monitoring of the spatial and temporal expression dynamics of a gene of interest during a variety of developmental processes. Canonical Wnt/ß-catenin signalling controls a plethora of cellular activities during development, regeneration and adulthood throughout the animal kingdom. Several reporters have been produced in animal models to reveal sites of active Wnt signalling. In order to monitor in vivo Wnt/ß-catenin signalling activity in the freshwater polyp Hydra vulgaris, we generated a ß-cat-eGFP transgenic Hydra, in which eGFP is driven by the Hydra ß-catenin promoter. We characterized the expression dynamics during budding, regeneration and chemical activation of the Wnt/ß-cat signalling pathway using light sheet fluorescence microscopy. Live imaging of the ß-cat-eGFP lines recapitulated the previously reported endogenous expression pattern of ß-catenin and revealed the dynamic appearance of novel sites of Wnt/ß-catenin signalling, that earlier evaded detection by mean of in situ hybridization. By combining the Wnt activity read-out efficiency of the ß-catenin promoter with advanced imaging, we have created a novel model system to monitor in real time the activity of Hydra ß-cat regulatory sequences in vivo, and open the path to reveal ß-catenin modulation in many other physiological contexts.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Hydra/embriología , Regeneración/fisiología , Vía de Señalización Wnt/fisiología , beta Catenina/metabolismo , Animales , Animales Modificados Genéticamente , Tipificación del Cuerpo/fisiología , Hydra/genética , Hydra/metabolismo , Microscopía Fluorescente , Proteínas Wnt/metabolismo , beta Catenina/genéticaRESUMEN
The cnidarian Hydra is known for its unlimited lifespan and non-senescence, due to the indefinite self-renewal capacity of its stem cells. While proteins of the Lamin family are recognized as critical factors affecting senescence and longevity in human and mice, their putative role in the extreme longevity and non-senescence in long-living animals remains unknown. Here we analyze the role of a single lamin protein in non-senescence of Hydra. We demonstrate that proliferation of stem cells in Hydra is robust against the disturbance of Lamin expression and localization. While Lamin is indispensable for Hydra, the stem cells tolerate overexpression, downregulation and mislocalization of Lamin, and disturbances in the nuclear envelope structure. This extraordinary robustness may underlie the indefinite self-renewal capacity of stem cells and the non-senescence of Hydra. A relatively low complexity of the nuclear envelope architecture in basal Metazoa might allow for their extreme lifespans, while an increasing complexity of the nuclear architecture in bilaterians resulted in restricted lifespans.
Asunto(s)
Senescencia Celular/fisiología , Hydra/fisiología , Laminas/metabolismo , Lámina Nuclear/metabolismo , Células Madre/metabolismo , Envejecimiento/metabolismo , Animales , Longevidad/fisiologíaRESUMEN
Colonization of body epithelial surfaces with a highly specific microbial community is a fundamental feature of all animals, yet the underlying mechanisms by which these communities are selected and maintained are not well understood. Here, we show that sensory and ganglion neurons in the ectodermal epithelium of the model organism hydra (a member of the animal phylum Cnidaria) secrete neuropeptides with antibacterial activity that may shape the microbiome on the body surface. In particular, a specific neuropeptide, which we call NDA-1, contributes to the reduction of Gram-positive bacteria during early development and thus to a spatial distribution of the main colonizer, the Gram-negative Curvibacter sp., along the body axis. Our findings warrant further research to test whether neuropeptides secreted by nerve cells contribute to the spatial structure of microbial communities in other organisms.Certain neuropeptides, in addition to their neuromodulatory functions, display antibacterial activities of unclear significance. Here, the authors show that a secreted neuropeptide modulates the distribution of bacterial communities on the body surface during development of the model organism Hydra.
Asunto(s)
Antibacterianos/metabolismo , Hydra/microbiología , Microbiota , Neuronas/metabolismo , Neuropéptidos/metabolismo , Animales , Comamonadaceae , Ectodermo/citología , Ectodermo/metabolismo , Epitelio/metabolismo , Bacterias Grampositivas , Hydra/crecimiento & desarrollo , Hydra/metabolismoRESUMEN
Hydra's unlimited life span has long attracted attention from natural scientists. The reason for that phenomenon is the indefinite self-renewal capacity of its stem cells. The underlying molecular mechanisms have yet to be explored. Here, by comparing the transcriptomes of Hydra's stem cells followed by functional analysis using transgenic polyps, we identified the transcription factor forkhead box O (FoxO) as one of the critical drivers of this continuous self-renewal. foxO overexpression increased interstitial stem cell and progenitor cell proliferation and activated stem cell genes in terminally differentiated somatic cells. foxO down-regulation led to an increase in the number of terminally differentiated cells, resulting in a drastically reduced population growth rate. In addition, it caused down-regulation of stem cell genes and antimicrobial peptide (AMP) expression. These findings contribute to a molecular understanding of Hydra's immortality, indicate an evolutionarily conserved role of FoxO in controlling longevity from Hydra to humans, and have implications for understanding cellular aging.
Asunto(s)
Factores de Transcripción Forkhead/fisiología , Hydra/citología , Células Madre/metabolismo , Animales , Animales Modificados Genéticamente , Linaje de la Célula , Factores de Transcripción Forkhead/genética , Silenciador del Gen , Hydra/inmunología , Hydra/metabolismo , Inmunidad Innata , Datos de Secuencia MolecularRESUMEN
How distinct stem cell populations originate and whether there is a clear stem cell "genetic signature" remain poorly understood. Understanding the evolution of stem cells requires molecular profiling of stem cells in an animal at a basal phylogenetic position. In this study, using transgenic Hydra polyps, we reveal for each of the three stem cell populations a specific signature set of transcriptions factors and of genes playing key roles in cell type-specific function and interlineage communication. Our data show that principal functions of stem cell genes, such as maintenance of stemness and control of stem cell self-renewal and differentiation, arose very early in metazoan evolution. They are corroborating the view that stem cell types shared common, multifunctional ancestors, which achieved complexity through a stepwise segregation of function in daughter cells.
Asunto(s)
Linaje de la Célula/genética , Perfilación de la Expresión Génica , Hydra/citología , Hydra/genética , Células Madre/citología , Células Madre/metabolismo , Animales , Evolución Biológica , Separación Celular , Regulación de la Expresión Génica , Prueba de Complementación Genética , Ratones , Filogenia , Coloración y Etiquetado , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transcriptoma/genéticaRESUMEN
Early embryos of many organisms develop outside the mother and are immediately confronted with myriads of potential colonizers. How these naive developmental stages control and shape the bacterial colonization is largely unknown. Here we show that early embryonic stages of the basal metazoan Hydra are able to control bacterial colonization by using maternal antimicrobial peptides. Antimicrobial peptides of the periculin family selecting for a specific bacterial colonization during embryogenesis are produced in the oocyte and in early embryos. If overexpressed in hydra ectodermal epithelial cells, periculin1a drastically reduces the bacterial load, indicating potent antimicrobial activity. Unexpectedly, transgenic polyps also revealed that periculin, in addition to bactericidal activity, changes the structure of the bacterial community. These findings delineate a role for antimicrobial peptides both in selecting particular bacterial partners during development and as important components of a "be prepared" strategy providing transgenerational protection.
Asunto(s)
Bacterias/crecimiento & desarrollo , Embrión no Mamífero/microbiología , Hydra/embriología , Péptidos/fisiología , Animales , Animales Modificados Genéticamente , Datos de Secuencia MolecularRESUMEN
In an adult hydra the head organizer, located in the hypostome, is constantly active in maintaining the structure of the animal in the context of its steady state tissue dynamics. Several Wnt genes, TCF, and elevated levels of beta-catenin are expressed in the hypostome as well as during the formation of a new organizer region in developing buds suggesting they play a role in the organizer. Transgenic hydra were generated in which a modified hydra beta-catenin gene driven by an actin promoter is continuously expressed at a high level throughout the animal. These animals formed heads and secondary axes in multiple locations along the body column. Transplantation experiments indicate they have a high and stable level of head organizer activity throughout the body columns. However, none of the Wnt genes are expressed in the body columns of these transgenic animals. Further, in alsterpaullone-treated animals, which results in a transient rise in head organizer activity throughout the body column, the time of expression of the Wnt genes is much shorter than the time of the elevated level of head inducing activity. These results for the first time provide direct functional evidence that beta-catenin plays a crucial role in the maintenance and activity of the head organizer and suggest that Wnt ligands may be required only for the initiation but not in maintenance of the organizer in Hydra.
Asunto(s)
Tipificación del Cuerpo/fisiología , Embrión no Mamífero/metabolismo , Hydra/embriología , beta Catenina/metabolismo , Animales , Animales Modificados Genéticamente , Proteínas Wnt/genética , Proteínas Wnt/metabolismo , beta Catenina/genéticaRESUMEN
Multicellular organisms consist of a variety of cells of distinctive morphology, with the cell shapes often reproduced with astonishing accuracy between individuals and across species. The morphology of cells varies with tissues, and cell shape changes are of profound importance in many occasions of morphogenesis. To elucidate the mechanisms of cell shape determination and regulation is therefore an important issue. One of the simplest multicellular organisms is the freshwater polyp Hydra. Although much is known about patterning in this early branching metazoan, there is currently little understanding of how cells in Hydra regulate their shape in response to upstream signals. We previously reported generation of transgenic Hydra to trace cells and to study cell behavior in vivo in an animal at the basis of animal evolution. Here, we use a novel transgenic line which expresses enhanced green fluorescent protein (eGFP) specifically in the ectodermal epithelial cells to analyze the structure and shape of epithelial cells as they are recruited into specific regions along the body column and respond to upstream signals such as components of the canonical Wnt signaling pathway. As a general theme, in contrast to epithelial cells in more complex animals, ectodermal epithelial cells in Hydra are capable of drastic changes in structure, shape, and cell contact along the body column. The remarkable phenotypic plasticity of epithelial cells in response to positional signals allows Hydra to build its body with only a limited number of different cell types.
Asunto(s)
Células Epiteliales/citología , Hydra/citología , Hydra/genética , Adaptación Fisiológica/fisiología , Animales , Forma de la Célula/fisiología , Citoesqueleto/metabolismo , Regulación de la Expresión Génica , Organismos Modificados Genéticamente , Transducción de Señal , Proteínas Wnt/metabolismoRESUMEN
BACKGROUND: Despite decades of research, the molecular mechanisms responsible for the evolution of morphological diversity remain poorly understood. While current models assume that species-specific morphologies are governed by differential use of conserved genetic regulatory circuits, it is debated whether non-conserved taxonomically restricted genes are also involved in making taxonomically relevant structures. The genomic resources available in Hydra, a member of the early branching animal phylum Cnidaria, provide a unique opportunity to study the molecular evolution of morphological novelties such as the nematocyte, a cell type characteristic of, and unique to, Cnidaria. RESULTS: We have identified nematocyte-specific genes by suppression subtractive hybridization and find that a considerable portion has no homologues to any sequences in animals outside Hydra. By analyzing the transcripts of these taxonomically restricted genes and mining of the Hydra magnipapillata genome, we find unexpected complexity in gene structure and transcript processing. Transgenic Hydra expressing the green fluorescent protein reporter under control of one of the taxonomically restricted gene promoters recapitulate faithfully the described expression pattern, indicating that promoters of taxonomically restricted genes contain all elements essential for spatial and temporal control mechanisms. Surprisingly, phylogenetic footprinting of this promoter did not reveal any conserved cis-regulatory elements. CONCLUSIONS: Our findings suggest that taxonomically restricted genes are involved in the evolution of morphological novelties such as the cnidarian nematocyte. The transcriptional regulatory network controlling taxonomically restricted gene expression may contain not yet characterized transcription factors or cis-regulatory elements.
Asunto(s)
Genoma/genética , Genómica/métodos , Hydra/genética , Animales , Clasificación , Regulación de la Expresión Génica , Redes Reguladoras de Genes , Genes , ARN Mensajero/análisisRESUMEN
Understanding the molecular events that underlie the evolution of morphological diversity is a major challenge in biology. Here, to identify genes whose expression correlates with species-specific morphologies, we compared transcriptomes of two closely related Hydra species. We find that species-specific differences in tentacle formation correlate with expression of a taxonomically restricted gene encoding a small secreted protein. We show that gain of function induces changes in morphology that mirror the phenotypic differences observed between species. These results suggest that "novel" genes may be involved in the generation of species-specific morphological traits.
Asunto(s)
Evolución Molecular , Regulación del Desarrollo de la Expresión Génica , Hydra/genética , Factores de Transcripción/genética , Secuencia de Aminoácidos , Animales , Expresión Génica , Silenciador del Gen , Hydra/anatomía & histología , Hydra/clasificación , Hibridación in Situ , Datos de Secuencia Molecular , Fenotipo , Filogenia , Alineación de Secuencia , Especificidad de la EspecieRESUMEN
Little is known about stem cells in organisms at the beginning of evolution. To characterize the regulatory events that control stem cells in the basal metazoan Hydra, we have generated transgenics which express eGFP selectively in the interstitial stem cell lineage. Using them we visualized stem cell and precursor migration in real-time in the context of the native environment. We demonstrate that interstitial cells respond to signals from the cellular environment, and that Wnt and Notch pathways are key players in this process. Furthermore, by analyzing polyps which overexpress the Polycomb protein HyEED in their interstitial cells, we provide in vivo evidence for a role of chromatin modification in terminal differentiation. These findings for the first time uncover insights into signalling pathways involved in stem cell differentiation in the Bilaterian ancestor; they demonstrate that mechanisms controlling stem cell behaviour are based on components which are conserved throughout the animal kingdom.
Asunto(s)
Evolución Biológica , Hydra/metabolismo , Receptores Notch/metabolismo , Células Madre/citología , Proteínas Wnt/metabolismo , Animales , Animales Modificados Genéticamente , Diferenciación Celular/fisiología , Líquido Extracelular/metabolismo , Hydra/genética , Transducción de SeñalRESUMEN
Understanding the evolution of development in large part relies on the study of phylogenetically old organisms. Cnidarians, such as Hydra, have become attractive model organisms for these studies. However, despite long-term efforts, stably transgenic animals could not be generated, severely limiting the functional analysis of genes. Here we report the efficient generation of transgenic Hydra lines by embryo microinjection. One of these transgenic lines expressing EGFP revealed remarkably high motility of individual endodermal epithelial cells during morphogenesis. We expect that transgenic Hydra will become important tools to dissect the molecular mechanisms of development at the base of the Metazoan tree.