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.

Combining BrdU-Labeling to Detection of Neuronal Markers to Monitor Adult Neurogenesis in Hydra.

The nervous system is produced and maintained in adult Hydra through the continuous production of nerve cells and mechanosensory cells (nematocytes or cnidocytes). De novo neurogenesis occurs slowly in intact animals that replace their dying nerve cells, at a faster rate in animals regenerating their head as a complete apical nervous system is built in few days. To dissect the molecular mechanisms that underlie these properties, a precise monitoring of the markers of neurogenesis and nematogenesis is required. Here we describe the conditions for an efficient BrdU-labeling coupled to an immunodetection of neuronal markers, either regulators of neurogenesis, here the homeoprotein prdl-a, or neuropeptides such as RFamide or Hym-355. This method can be performed on whole-mount animals as well as on macerated tissues when cells retain their morphology. Moreover, when antibodies are not available, BrdU-labeling can be combined with the analysis of gene expression by whole-mount in situ hybridization. This co-immunodetection procedure is well adapted to visualize and quantify the dynamics of de novo neurogenesis. Upon continuous BrdU labeling, the repeated measurements of BrdU-labeling indexes in specific cellular populations provide a precise monitoring of nematogenesis as well as neurogenesis, in homeostatic or developmental conditions.

Model systems for regeneration: Hydra.

The freshwater polyp Hydra provides a potent model system for investigating the conditions that promote wound healing, reactivation of a developmental process and, ultimately, regeneration of an amputated body part. Hydra polyps can also be dissociated to the single cell level and can regenerate a complete body axis from aggregates, behaving as natural organoids. In recent years, the ability to exploit Hydra has been expanded with the advent of new live-imaging approaches, genetic manipulations that include stable transgenesis, gene silencing and genome editing, and the accumulation of high-throughput omics data. In this Primer, we provide an overview of Hydra as a model system for studying regeneration, highlighting recent results that question the classical self-enhancement and long-range inhibition model supposed to drive Hydra regeneration. We underscore the need for integrative explanations incorporating biochemical as well as mechanical signalling.

Cellular and Molecular Mechanisms of Hydra Regeneration.

Regeneration of lost body parts is essential to regain the fitness of the organism for successful living. In the animal kingdom, organisms from different clades exhibit varied regeneration abilities. Hydra is one of the few organisms that possess tremendous regeneration potential, capable of regenerating complete organism from small tissue fragments or even from dissociated cells. This peculiar property has made this genus one of the most invaluable model organisms for understanding the process of regeneration. Multiple studies in Hydra led to the current understanding of gross morphological changes, basic cellular dynamics, and the role of molecular signalling such as the Wnt signalling pathway. However, cell-to-cell communication by cell adhesion, role of extracellular components such as extracellular matrix (ECM), and nature of cell types that contribute to the regeneration process need to be explored in depth. Additionally, roles of developmental signalling pathways need to be elucidated to enable more comprehensive understanding of regeneration in Hydra. Further research on cross communication among extracellular, cellular, and molecular signalling in Hydra will advance the field of regeneration biology. Here, we present a review of the existing literature on Hydra regeneration biology and outline the future perspectives.

Stem cell differentiation trajectories in Hydra resolved at single-cell resolution.

The adult Hydra polyp continually renews all of its cells using three separate stem cell populations, but the genetic pathways enabling this homeostatic tissue maintenance are not well understood. We sequenced 24,985 Hydra single-cell transcriptomes and identified the molecular signatures of a broad spectrum of cell states, from stem cells to terminally differentiated cells. We constructed differentiation trajectories for each cell lineage and identified gene modules and putative regulators expressed along these trajectories, thus creating a comprehensive molecular map of all developmental lineages in the adult animal. In addition, we built a gene expression map of the Hydra nervous system. Our work constitutes a resource for addressing questions regarding the evolution of metazoan developmental processes and nervous system function.

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.

Immunocytochemical localization of a putative strychnine-sensitive glycine receptor in Hydra vulgaris.

Previous biochemical studies have identified strychnine-sensitive glycine receptors in membrane preparations of Hydra vulgaris (Cnidaria: Hydrozoa). Electrophysiological and behavioral evidence has shown that these receptors play a role in modulating pacemaker activity and feeding behavior. Here, we present our genomic analysis that revealed hydra proteins having strong homology with the strychnine-binding region of the human receptor protein, GlyRα1. We further present immunocytochemical evidence for the specific labeling of cell and tissue preparations of hydra by a commercially available polyclonal anti-GlyRα1 antibody, selected through our genomic analysis. Tissue pieces and cell macerates from the upper and lower thirds of the body and ablated tentacles were double-labeled with this antibody and with an antibody specific for α-tubulin, to identify the glycine receptors and microtubules, respectively. Extensive receptor labeling was evident on the membranes, cell bodies and myonemes of endodermal and ectodermal epithelial cells, cell bodies and neurites of nerve cells, cnidocytes and interstitial cells. Labeling of the membranes of epithelial cells frequently corresponded to conspicuous varicosities (presumptive presynaptic sites) in the associated nerve net. Our findings support the idea that glycine receptors form an integral part of the nerve and effector systems that control hydra behavior.

Loss of Neurogenesis in Aging Hydra.

In Hydra the nervous system is composed of neurons and mechanosensory cells that differentiate from interstitial stem cells (ISCs), which also provide gland cells and germ cells. The adult nervous system is actively maintained through continuous de novo neurogenesis that occurs at two distinct paces, slow in intact animals and fast in regenerating ones. Surprisingly Hydra vulgaris survive the elimination of cycling interstitial cells and the subsequent loss of neurogenesis if force-fed. By contrast, H. oligactis animals exposed to cold temperature undergo gametogenesis and a concomitant progressive loss of neurogenesis. In the cold-sensitive strain Ho_CS, this loss irreversibly leads to aging and animal death. Within four weeks, Ho_CS animals lose their contractility, feeding response, and reaction to light. Meanwhile, two positive regulators of neurogenesis, the homeoprotein prdl-a and the neuropeptide Hym-355, are no longer expressed, while the "old" RFamide-expressing neurons persist. A comparative transcriptomic analysis performed in cold-sensitive and cold-resistant strains confirms the downregulation of classical neuronal markers during aging but also shows the upregulation of putative regulators of neurotransmission and neurogenesis such as AHR, FGFR, FoxJ3, Fral2, Jagged, Meis1, Notch, Otx1, and TCF15. The switch of Fral2 expression from neurons to germ cells suggests that in aging animals, the neurogenic program active in ISCs is re-routed to germ cells, preventing de novo neurogenesis and impacting animal survival.

In Vivo Toxicity Assessment of Hybrid Diatomite Nanovectors Using Hydra vulgaris as a Model System.

Drug nanocarriers based on nanostructured materials are very promising for precision and personalized medicine applications. Diatomite porous biosilica has been recently proposed as a novel and effective material in formulations of drug systems for oral and systemic delivery. In this paper, the cytotoxicity of hybrid diatomite silica functionalized nanovectors is assessed in vivo in a living model organism, the cnidarian freshwater polyp Hydra vulgaris. Hydra specimens are exposed to modified diatomite nanoparticles by prolonged incubation within their medium. Uptake and toxicological effects on Hydra are examined from viability and genetic points of view. High concentrations, up to 3.5 g L-1 for 72 h, of diatomite modified nanoparticles do not affect Hydra morphology nor do growth rate and the genetic analysis confirm the biosafety of this material, opening the way to new applications in nanomedicine.

Non-developmental dimensions of adult regeneration in Hydra.

An essential dimension of 3D regeneration in adult animals is developmental, with the formation of organizers from somatic tissues. These organizers produce signals that recruit surrounding cells and drive the restoration of the missing structures (organs, appendages, body parts). However, even in animals with a high regenerative potential, this developmental potential is not sufficient to achieve regeneration as homeostatic conditions at the time of injury need to be "pro-regenerative". In Hydra, we identified four distinct homeostatic properties that provide a pro-regenerative framework and we discuss here how these non-developmental properties impact regeneration. First, both the epithelial and the interstitial-derived cells are highly plastic along the animal body, a plasticity that offers several routes to achieve regeneration. Second, the abundant stocks of continuously self-renewing adult stem cells form a constitutive pro-blastema in the central body column, readily activated upon bisection. Third, the autophagy machinery in epithelial cells guarantees a high level of fitness and adaptation to detrimental environmental conditions, as evidenced by the loss of regeneration in animals where autophagy is dysfunctional. Fourth, the extracellular matrix, named mesoglea in Hydra, provides a dynamically-patterned environment where the molecular and mechanical signals induced by injury get translated into a regenerative process. We claim that these homeostatic pro-regenerative features contribute to define the high regenerative potential of adult Hydra.

Impact of cycling cells and cell cycle regulation on Hydra regeneration.

Hydra tissues are made from three distinct populations of stem cells that continuously cycle and pause in G2 instead of G1. To characterize the role of cell proliferation after mid-gastric bisection, we have (i) used flow cytometry and classical markers to monitor cell cycle modulations, (ii) quantified the transcriptomic regulations of 202 genes associated with cell proliferation during head and foot regeneration, and (iii) compared the impact of anti-proliferative treatments on regeneration efficiency. We confirm two previously reported events: an early mitotic wave in head-regenerating tips, when few cell cycle genes are up-regulated, and an early-late wave of proliferation on the second day, preceded by the up-regulation of 17 cell cycle genes. These regulations appear more intense after mid-gastric bisection than after decapitation, suggesting a position-dependent regulation of cell proliferation during head regeneration. Hydroxyurea, which blocks S-phase progression, delays head regeneration when applied before but not after bisection. This result is consistent with the fact that the Hydra central region is enriched in G2-paused adult stem cells, poised to divide upon injury, thus forming a necessary constitutive pro-blastema. However a prolonged exposure to hydroxyurea does not block regeneration as cells can differentiate apical structures without traversing S-phase, and also escape in few days the hydroxyurea-induced S-phase blockade. Thus Hydra head regeneration, which is a fast event, is highly plastic, relying on large stocks of adult stem cells paused in G2 at amputation time, which immediately divide to proliferate and/or differentiate apical structures even when S-phase is blocked.

Physical Mechanisms Driving Cell Sorting in Hydra.

Cell sorting, whereby a heterogeneous cell mixture organizes into distinct tissues, is a fundamental patterning process in development. Hydra is a powerful model system for carrying out studies of cell sorting in three dimensions, because of its unique ability to regenerate after complete dissociation into individual cells. The physicists Alfred Gierer and Hans Meinhardt recognized Hydra's self-organizing properties more than 40 years ago. However, what drives cell sorting during regeneration of Hydra from cell aggregates is still debated. Differential motility and differential adhesion have been proposed as driving mechanisms, but the available experimental data are insufficient to distinguish between these two. Here, we answer this longstanding question by using transgenic Hydra expressing fluorescent proteins and a multiscale experimental and numerical approach. By quantifying the kinematics of single cell and whole aggregate behaviors, we show that no differences in cell motility exist among cell types and that sorting dynamics follow a power law with an exponent of ∼0.5. Additionally, we measure the physical properties of separated tissues and quantify their viscosities and surface tensions. Based on our experimental results and numerical simulations, we conclude that tissue interfacial tensions are sufficient to explain cell sorting in aggregates of Hydra cells. Furthermore, we demonstrate that the aggregate's geometry during sorting is key to understanding the sorting dynamics and explains the exponent of the power law behavior. Our results answer the long standing question of the physical mechanisms driving cell sorting in Hydra cell aggregates. In addition, they demonstrate how powerful this organism is for biophysical studies of self-organization and pattern formation.

Evidence that polycystins are involved in Hydra cnidocyte discharge.

Like other cnidarians, the freshwater organism Hydra is characterized by the possession of cnidocytes (stinging cells). Most cnidocytes are located on hydra tentacles, where they are organized along with sensory cells and ganglion cells into battery complexes. The function of the battery complexes is to integrate multiple types of stimuli for the regulation of cnidocyte discharge. The molecular mechanisms controlling the discharge of cnidocytes are not yet fully understood, but it is known that discharge depends on extracellular Ca2+ and that mechanically induced cnidocyte discharge can be enhanced by the presence of prey extracts and other chemicals. Experiments in this paper show that a PKD2 (polycystin 2) transient receptor potential (TRP) channel is expressed in hydra tentacles and bases. PKD2 (TRPP) channels belong to the TRP channel superfamily and are non-selective Ca2+ channels involved in the transduction of both mechanical and chemical stimuli in other organisms. Non-specific PKD2 channel inhibitors Neo (neomycin) and Gd3+ (gadolinium) inhibit both prey capture and cnidocyte discharge in hydra. The PKD2 activator Trip (triptolide) enhances cnidocyte discharge in both starved and satiated hydra and reduces the inhibition of cnidocyte discharge caused by Neo. PKD1 and 2 proteins are known to act together to transduce mechanical and chemical stimuli; in situ hybridization experiments show that a PKD1 gene is expressed in hydra tentacles and bases, suggesting that polycystins play a direct or indirect role in cnidocyte discharge.

The two nerve rings of the hypostomal nervous system of Hydra vulgaris-an immunohistochemical analysis.

In Hydra vulgaris, physiological and pharmacological evidence exists for a hypostomal circumferential neuro-effector pathway that initiates ectodermal pacemaker activity at tentacular-hypostomal loci coordinating body and tentacle contractions. Here, we describe an ectodermal nerve ring that runs below and between the tentacles, and an anti-GABAB receptor antibody-labeled ring coincident with it. The location of this ring is consistent with the physiology of the hypostomal pacemaker systems of hydra. We also describe a distally located, ectodermal ring of nerve fibers that is not associated with anti-GABAB receptor antibody labeling. The neurites and cell bodies of sensory cells contribute to both rings. The location of the distal ring and its sensory cell neurites suggests an involvement in the behavior of the mouth. Between the two rings is a network of anastomosing sensory and ganglion cell bodies and their neurites. Phase contrast, darkfield, and antibody-labeled images reveal that the mouth of hydra comprises five or six epithelial folds whose endoderm extensively labels with anti-GABAB receptor antibody, suggesting that endodermal metabotrobic GABA receptors are also involved in regulating mouth behavior.

Sequential development of apical-basal and planar polarities in aggregating epitheliomuscular cells of Hydra.

Apical-basal and planar cell polarities are hallmarks of metazoan epithelia required to separate internal and external environments and to regulate trans- and intracellular transport, cytoskeletal organization, and morphogenesis. Mechanisms of cell polarization have been intensively studied in bilaterian model organisms, particularly in early embryos and cultured cells, while cell polarity in pre-bilaterian tissues is poorly understood. Here, we have studied apical-basal and planar polarization in regenerating (aggregating) clusters of epitheliomuscular cells of Hydra, a simple representative of the ancestral, pre-bilaterian phylum Cnidaria. Immediately after dissociation, single epitheliomuscular cells do not exhibit cellular polarity, but they polarize de novo during aggregation. Reestablishment of the Hydra-specific epithelial bilayer is a result of short-range cell sorting. In the early phase of aggregation, apical-basal polarization starts with an enlargement of the epithelial apical-basal diameter and by the development of belt-like apical septate junctions. Specification of the basal pole of epithelial cells occurs shortly later and is linked to synthesis of mesoglea, development of hemidesmosome-like junctions, and formation of desmosome-like junctions connecting the basal myonemes of neighbouring cells. Planar polarization starts, while apical-basal polarization is already ongoing. It is executed gradually starting with cell-autonomous formation, parallelization, and condensation of myonemes at the basal end of each epithelial cell and continuing with a final planar alignment of epitheliomuscular cells at the tissue level. Our findings reveal that epithelial polarization in Hydra aggregates occurs in defined steps well accessible by histological and ultrastructural techniques and they will provide a basis for future molecular studies.

[Dissociation-reaggregation experiments in cnidarians as a model system for study of regulative capacity of metazoan development].

Developmental processes of cnidarians, the basal metazoan representatives, possess extremely high regulative ability. It is known that any isolated fragment of the freshwater polyp hydra's body can regenerate an intact animal. Moreover, in many cnidarian species, suspension of single dissociated cells can form aggregates, which regenerate normal body plan of polyp or medusa. This process can be considered as an extreme case of regeneration. The development of cell reaggregates of Hydra is a conventional experimental system to study the physical basis of morphogenesis. Investigations of the cnidarians' reaggregate development help to clarify basic rules and mechanisms of the metazoan body plan formation and the role of self-organization in the metazoan early development. In this review, we summarize the data revealed by dissociation - reaggregation experiments performed on the representatives of different cnidarian taxa. We also analyze the data on the morphogenetic and molecular basis of the reaggregate development from randomly organized group of cells to cnidarian-specific body plan.

The ancestral gene repertoire of animal stem cells.

Stem cells are pivotal for development and tissue homeostasis of multicellular animals, and the quest for a gene toolkit associated with the emergence of stem cells in a common ancestor of all metazoans remains a major challenge for evolutionary biology. We reconstructed the conserved gene repertoire of animal stem cells by transcriptomic profiling of totipotent archeocytes in the demosponge Ephydatia fluviatilis and by tracing shared molecular signatures with flatworm and Hydra stem cells. Phylostratigraphy analyses indicated that most of these stem-cell genes predate animal origin, with only few metazoan innovations, notably including several partners of the Piwi machinery known to promote genome stability. The ancestral stem-cell transcriptome is strikingly poor in transcription factors. Instead, it is rich in RNA regulatory actors, including components of the "germ-line multipotency program" and many RNA-binding proteins known as critical regulators of mammalian embryonic stem cells.

Unraveling the non-senescence phenomenon in Hydra.

Unlike other metazoans, Hydra does not experience the distinctive rise in mortality with age known as senescence, which results from an increasing imbalance between cell damage and cell repair. We propose that the Hydra controls damage accumulation mainly through damage-dependent cell selection and cell sloughing. We examine our hypothesis with a model that combines cellular damage with stem cell renewal, differentiation, and elimination. The Hydra individual can be seen as a large single pool of three types of stem cells with some features of differentiated cells. This large stem cell community prevents "cellular damage drift," which is inevitable in complex conglomerate (differentiated) metazoans with numerous and generally isolated pools of stem cells. The process of cellular damage drift is based on changes in the distribution of damage among cells due to random events, and is thus similar to Muller's ratchet in asexual populations. Events in the model that are sources of randomness include budding, cellular death, and cellular damage and repair. Our results suggest that non-senescence is possible only in simple Hydra-like organisms which have a high proportion and number of stem cells, continuous cell divisions, an effective cell selection mechanism, and stem cells with the ability to undertake some roles of differentiated cells.

On-chip immobilization of planarians for in vivo imaging.

Planarians are an important model organism for regeneration and stem cell research. A complete understanding of stem cell and regeneration dynamics in these animals requires time-lapse imaging in vivo, which has been difficult to achieve due to a lack of tissue-specific markers and the strong negative phototaxis of planarians. We have developed the Planarian Immobilization Chip (PIC) for rapid, stable immobilization of planarians for in vivo imaging without injury or biochemical alteration. The chip is easy and inexpensive to fabricate, and worms can be mounted for and removed after imaging within minutes. We show that the PIC enables significantly higher-stability immobilization than can be achieved with standard techniques, allowing for imaging of planarians at sub-cellular resolution in vivo using brightfield and fluorescence microscopy. We validate the performance of the PIC by performing time-lapse imaging of planarian wound closure and sequential imaging over days of head regeneration. We further show that the device can be used to immobilize Hydra, another photophobic regenerative model organism. The simple fabrication, low cost, ease of use, and enhanced specimen stability of the PIC should enable its broad application to in vivo studies of stem cell and regeneration dynamics in planarians and Hydra.

Robust G2 pausing of adult stem cells in Hydra.

Hydra is a freshwater hydrozoan polyp that constantly renews its two tissue layers thanks to three distinct stem cell populations that cannot replace each other, epithelial ectodermal, epithelial endodermal, and multipotent interstitial. These adult stem cells, located in the central body column, exhibit different cycling paces, slow for the epithelial, fast for the interstitial. To monitor the changes in cell cycling in Hydra, we established a fast and efficient flow cytometry procedure, which we validated by confirming previous findings, as the Nocodazole-induced reversible arrest of cell cycling in G2/M, and the mitogenic signal provided by feeding. Then to dissect the cycling and differentiation behaviors of the interstitial stem cells, we used the AEP_cnnos1 and AEP_Icy1 transgenic lines that constitutively express GFP in this lineage. For the epithelial lineages we used the sf-1 strain that rapidly eliminates the fast cycling cells upon heat-shock and progressively becomes epithelial. This study evidences similar cycling patterns for the interstitial and epithelial stem cells, which all alternate between the G2 and S-phases traversing a minimal G1-phase. We also found interstitial progenitors with a shorter G2 that pause in G1/G0. At the animal extremities, most cells no longer cycle, the epithelial cells terminally differentiate in G2 and the interstitial progenitors in G1/G0. At the apical pole ~80% cells are post-mitotic differentiated cells, reflecting the higher density of neurons and nematocytes in this region. We discuss how the robust G2 pausing of stem cells, maintained over weeks of starvation, may contribute to regeneration.