Genetic interference with HvNotch provides new insights into the role of the Notch-signalling pathway for developmental pattern formation in Hydra.

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.

Novel technologies uncover novel 'anti'-microbial peptides in Hydra shaping the species-specific microbiome.

The freshwater polyp Hydra uses an elaborate innate immune machinery to maintain its specific microbiome. Major components of this toolkit are conserved Toll-like receptor (TLR)-mediated immune pathways and species-specific antimicrobial peptides (AMPs). Our study harnesses advanced technologies, such as high-throughput sequencing and machine learning, to uncover a high complexity of the Hydra's AMPs repertoire. Functional analysis reveals that these AMPs are specific against diverse members of the Hydra microbiome and expressed in a spatially controlled pattern. Notably, in the outer epithelial layer, AMPs are produced mainly in the neurons. The neuron-derived AMPs are secreted directly into the glycocalyx, the habitat for symbiotic bacteria, and display high selectivity and spatial restriction of expression. In the endodermal layer, in contrast, endodermal epithelial cells produce an abundance of different AMPs including members of the arminin and hydramacin families, while gland cells secrete kazal-type protease inhibitors. Since the endodermal layer lines the gastric cavity devoid of symbiotic bacteria, we assume that endodermally secreted AMPs protect the gastric cavity from intruding pathogens. In conclusion, Hydra employs a complex set of AMPs expressed in distinct tissue layers and cell types to combat pathogens and to maintain a stable spatially organized microbiome. This article is part of the theme issue 'Sculpting the microbiome: how host factors determine and respond to microbial colonization'.

Multiple neuronal populations control the eating behavior in Hydra and are responsive to microbial signals.

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.

Spontaneous body wall contractions stabilize the fluid microenvironment that shapes host-microbe associations.

The freshwater polyp Hydra is a popular biological model system; however, we still do not understand one of its most salient behaviors, the generation of spontaneous body wall contractions. Here, by applying experimental fluid dynamics analysis and mathematical modeling, we provide functional evidence that spontaneous contractions of body walls enhance the transport of chemical compounds from and to the tissue surface where symbiotic bacteria reside. Experimentally, a reduction in the frequency of spontaneous body wall contractions is associated with a changed composition of the colonizing microbiota. Together, our findings suggest that spontaneous body wall contractions create an important fluid transport mechanism that (1) may shape and stabilize specific host-microbe associations and (2) create fluid microhabitats that may modulate the spatial distribution of the colonizing microbes. This mechanism may be more broadly applicable to animal-microbe interactions since research has shown that rhythmic spontaneous contractions in the gastrointestinal tracts are essential for maintaining normal microbiota.

Spontaneously occurring tumors in different wild-derived strains of hydra.

Hydras are freshwater cnidarians widely used as a biological model to study different questions such as senescence or phenotypic plasticity but also tumoral development. The spontaneous tumors found in these organisms have been so far described in two female lab strains domesticated years ago (Hydra oligactis and Pelmatohydra robusta) and the extent to which these tumors can be representative of tumors within the diversity of wild hydras is completely unknown. In this study, we examined individuals isolated from recently sampled wild strains of different sex and geographical origin, which have developed outgrowths looking like tumors. These tumefactions have common features with the tumors previously described in lab strains: are composed of an accumulation of abnormal cells, resulting in a similar enlargement of the tissue layers. However, we also found diversity within these new types of tumors. Indeed, not only females, but also males seem prone to form these tumors. Finally, the microbiota associated to these tumors is different from the one involved in the previous lineages exhibiting tumors. We found that tumorous individuals hosted yet undescribed Chlamydiales vacuoles. This study brings new insights into the understanding of tumor susceptibility and diversity in brown hydras from different origins.

The Role of DNA Methylation in Genome Defense in Cnidaria and Other Invertebrates.

Considerable attention has recently been focused on the potential involvement of DNA methylation in regulating gene expression in cnidarians. Much of this work has been centered on corals, in the context of changes in methylation perhaps facilitating adaptation to higher seawater temperatures and other stressful conditions. Although first proposed more than 30 years ago, the possibility that DNA methylation systems function in protecting animal genomes against the harmful effects of transposon activity has largely been ignored since that time. Here, we show that transposons are specifically targeted by the DNA methylation system in cnidarians, and that the youngest transposons (i.e., those most likely to be active) are most highly methylated. Transposons in longer and highly active genes were preferentially methylated and, as transposons aged, methylation levels declined, reducing the potentially harmful side effects of CpG methylation. In Cnidaria and a range of other invertebrates, correlation between the overall extent of methylation and transposon content was strongly supported. Present transposon burden is the dominant factor in determining overall level of genomic methylation in a range of animals that diverged in or before the early Cambrian, suggesting that genome defense represents the ancestral role of CpG methylation.

Tumors (re)shape biotic interactions within ecosystems: Experimental evidence from the freshwater cnidarian Hydra.

While it is often assumed that oncogenic processes in metazoans can influence species interactions, empirical evidence is lacking. Here, we use the cnidarian Hydra oligactis to experimentally explore the consequences of tumor associated phenotypic alterations for its predation ability, relationship with commensal ciliates and vulnerability to predators. Unexpectedly, hydra's predation ability was higher in tumorous polyps compared to non-tumorous ones. Commensal ciliates colonized preferentially tumorous hydras than non-tumorous ones, and had a higher replication rate on the former. Finally, in a choice experiment, tumorous hydras were preferentially eaten by a fish predator. This study, for the first time, provides evidence that neoplastic growth has the potential, through effect(s) on host phenotype, to alter biotic interactions within ecosystems and should thus be taken into account by ecologists.

Prototypical pacemaker neurons interact with the resident microbiota.

Pacemaker neurons exert control over neuronal circuit function by their intrinsic ability to generate rhythmic bursts of action potential. Recent work has identified rhythmic gut contractions in human, mice, and hydra to be dependent on both neurons and the resident microbiota. However, little is known about the evolutionary origin of these neurons and their interaction with microbes. In this study, we identified and functionally characterized prototypical ANO/SCN/TRPM ion channel-expressing pacemaker cells in the basal metazoan Hydra by using a combination of single-cell transcriptomics, immunochemistry, and functional experiments. Unexpectedly, these prototypical pacemaker neurons express a rich set of immune-related genes mediating their interaction with the microbial environment. Furthermore, functional experiments gave a strong support to a model of the evolutionary emergence of pacemaker cells as neurons using components of innate immunity to interact with the microbial environment and ion channels to generate rhythmic contractions.

Ancestral roles of atypical cadherins in planar cell polarity.

Fat, Fat-like, and Dachsous family cadherins are giant proteins that regulate planar cell polarity (PCP) and cell adhesion in bilaterians. Their evolutionary origin can be traced back to prebilaterian species, but their ancestral function(s) are unknown. We identified Fat-like and Dachsous cadherins in Hydra, a member of phylum Cnidaria a sister group of bilaterian. We found Hydra does not possess a true Fat homolog, but has homologs of Fat-like (HyFatl) and Dachsous (HyDs) that localize at the apical membrane of ectodermal epithelial cells and are planar polarized perpendicular to the oral-aboral axis of the animal. Using a knockdown approach we found that HyFatl is involved in local cell alignment and cell-cell adhesion, and that reduction of HyFatl leads to defects in tissue organization in the body column. Overexpression and knockdown experiments indicate that the intracellular domain (ICD) of HyFatl affects actin organization through proline-rich repeats. Thus, planar polarization of Fat-like and Dachsous cadherins has ancient, prebilaterian origins, and Fat-like cadherins have ancient roles in cell adhesion, spindle orientation, and tissue organization.

Dynamic interactions within the host-associated microbiota cause tumor formation in the basal metazoan Hydra.

The extent to which disturbances in the resident microbiota can compromise an animal's health is poorly understood. Hydra is one of the evolutionary oldest animals with naturally occurring tumors. Here, we found a causal relationship between an environmental spirochete (Turneriella spec.) and tumorigenesis in Hydra. Unexpectedly, virulence of this pathogen requires the presence of Pseudomonas spec., a member of Hydra´s beneficial microbiome indicating that dynamic interactions between a resident bacterium and a pathogen cause tumor formation. The observation points to the crucial role of commensal bacteria in maintaining tissue homeostasis and adds support to the view that microbial community interactions are essential for disease. These findings in an organism that shares deep evolutionary connections with all animals have implications for our understanding of cancer.

Temperature and insulin signaling regulate body size in Hydra by the Wnt and TGF-beta pathways.

How multicellular organisms assess and control their size is a fundamental question in biology, yet the molecular and genetic mechanisms that control organ or organism size remain largely unsolved. The freshwater polyp Hydra demonstrates a high capacity to adapt its body size to different temperatures. Here we identify the molecular mechanisms controlling this phenotypic plasticity and show that temperature-induced cell number changes are controlled by Wnt- and TGF-ß signaling. Further we show that insulin-like peptide receptor (INSR) and forkhead box protein O (FoxO) are important genetic drivers of size determination controlling the same developmental regulators. Thus, environmental and genetic factors directly affect developmental mechanisms in which cell number is the strongest determinant of body size. These findings identify the basic mechanisms as to how size is regulated on an organismic level and how phenotypic plasticity is integrated into conserved developmental pathways in an evolutionary informative model organism.

Transgenesis in Hydra to characterize gene function and visualize cell behavior.

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.

Apoptosis in Hydra: function of HyBcl-2 like 4 and proteins of the transmembrane BAX inhibitor motif (TMBIM) containing family.

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.

Rethinking the Role of the Nervous System: Lessons From the Hydra Holobiont.

Here we evaluate our current understanding of the function of the nervous system in Hydra, a non-bilaterian animal which is among the first metazoans that contain neurons. We highlight growing evidence that the nervous system, with its rich repertoire of neuropeptides, is involved in controlling resident beneficial microbes. We also review observations that indicate that microbes affect the animal's behavior by directly interfering with neuronal receptors. These findings provide new insight into the original role of the nervous system, and suggest that it emerged to orchestrate multiple functions including host-microbiome interactions. The excitement of future research in the Hydra model now relies on uncovering the common rules and principles that govern the interaction between neurons and microbes and the extent to which such laws might apply to other and more complex organisms.

Real time dynamics of ß-catenin expression during Hydra development, regeneration and Wnt signalling activation.

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.

Non-senescent Hydra tolerates severe disturbances in the nuclear lamina.

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.

Stem Cell Transcription Factor FoxO Controls Microbiome Resilience in Hydra.

The aging process is considered to be the result of accumulating cellular deterioration in an individual organism over time. It can be affected by the combined influence of genetic, epigenetic, and environmental factors including life-style-associated events. In the non-senescent freshwater polyp Hydra, one of the classical model systems for evolutionary developmental biology and regeneration, transcription factor FoxO modulates both stem cell proliferation and innate immunity. This provides strong support for the role of FoxO as a critical rate-of-aging regulator. However, how environmental factors interact with FoxO remains unknown. Here, we find that deficiency in FoxO signaling in Hydra leads to dysregulation of antimicrobial peptide expression and that FoxO loss-of-function polyps are impaired in selection for bacteria resembling the native microbiome and more susceptible to colonization of foreign bacteria. These findings reveal a key role of FoxO signaling in the communication between host and microbiota and embed the evolutionary conserved longevity factor FoxO into the holobiont concept.

Spontaneous body contractions are modulated by the microbiome of Hydra.

Spontaneous contractile activity, such as gut peristalsis, is ubiquitous in animals and is driven by pacemaker cells. In humans, disruption of the contraction pattern leads to gastrointestinal conditions, which are also associated with gut microbiota dysbiosis. Spontaneous contractile activity is also present in animals lacking gastrointestinal tract. Here we show that spontaneous body contractions in Hydra are modulated by symbiotic bacteria. Germ-free animals display strongly reduced and less regular contraction frequencies. These effects are partially restored by reconstituting the natural microbiota. Moreover, soluble molecule(s) produced by symbiotic bacteria may be involved in contraction frequency modulation. As the absence of bacteria does not impair the contractile ability itself, a microbial effect on the pacemakers seems plausible. Our findings indicate that the influence of bacteria on spontaneous contractile activity is present in the early-branching cnidarian hydra as well as in Bilateria, and thus suggest an evolutionary ancient origin of interaction between bacteria and metazoans, opening a window into investigating the roots of human motility disorders.

Back to the Basics: Cnidarians Start to Fire.

The nervous systems of cnidarians, pre-bilaterian animals that diverged close to the base of the metazoan radiation, are structurally simple and thus have great potential to reveal fundamental principles of neural circuits. Unfortunately, cnidarians have thus far been relatively intractable to electrophysiological and genetic techniques and consequently have been largely passed over by neurobiologists. However, recent advances in molecular and imaging methods are fueling a renaissance of interest in and research into cnidarians nervous systems. Here, we review current knowledge on the nervous systems of cnidarian species and propose that researchers should seize this opportunity and undertake the study of members of this phylum as strategic experimental systems with great basic and translational relevance for neuroscience.

How do environmental factors influence life cycles and development? An experimental framework for early-diverging metazoans.

Ecological developmental biology (eco-devo) explores the mechanistic relationships between the processes of individual development and environmental factors. Recent studies imply that some of these relationships have deep evolutionary origins, and may even pre-date the divergences of the simplest extant animals, including cnidarians and sponges. Development of these early diverging metazoans is often sensitive to environmental factors, and these interactions occur in the context of conserved signaling pathways and mechanisms of tissue homeostasis whose detailed molecular logic remain elusive. Efficient methods for transgenesis in cnidarians together with the ease of experimental manipulation in cnidarians and sponges make them ideal models for understanding causal relationships between environmental factors and developmental mechanisms. Here, we identify major questions at the interface between animal evolution and development and outline a road map for research aimed at identifying the mechanisms that link environmental factors to developmental mechanisms in early diverging metazoans. Also watch the Video Abstract.