Hydra Regeneration Research Articles

Notch signaling mediates between two pattern-forming processes during head regeneration in Hydra.

Hydra head regeneration consists of hypostome/organizer and tentacle development, and involves Notch and Wnt/β-catenin signaling. Notch inhibition blocks hypostome/organizer regeneration, but not the appearance of the tentacle tissue. β-Catenin inhibition blocks tentacle, but not hypostome/organizer regeneration. Gene expression analyses during head regeneration revealed the Notch-promoting expression of HyWnt3, HyBMP2/4, and the transcriptional repressor genes CnGsc, Sp5, and HyHes, while blocking HyBMP5/8b and the c-fos-related gene HyKayak β-Catenin promotes the expression of the tentacle specification factor HyAlx, but not of HyWnt3 This suggests HyWnt3 and HyBMP4 as parts of a hypostome/organizer gene module, and BMP5/8, HyAlx, and β-catenin as parts of a tentacle gene module. Notch then functions as an inhibitor of tentacle production to allow regeneration of a hypostome/head organizer. HyKayak is a candidate target gene for HvNotch-induced repressor genes. Inhibiting HyKayak attenuated the expression of HyWnt3 Polyps of Craspedacusta do not have tentacles and thus after head removal only regenerate a hypostome structure. Notch signaling was not needed for head regeneration in Craspedacusta, corroborating the idea of its requirement during Hydra head regeneration to harmonize two co-operating pattern-forming processes.

Mechanical characterization of regenerating Hydra tissue spheres

Hydra vulgaris, long known for its remarkable regenerative capabilities, is also a long-standing source of inspiration for models of spontaneous patterning. Recently it became clear that early patterning during Hydra regeneration is an integrated mechanochemical process whereby morphogen dynamics is influenced by tissue mechanics. One roadblock to understanding Hydra self-organization is our lack of knowledge about the mechanical properties of these organisms. In this study, we combined microfluidic developments to perform parallelized microaspiration rheological experiments and numerical simulations to characterize these mechanical properties. We found three different behaviors depending on the applied stresses: an elastic response, a viscoelastic response, and tissue rupture. Using models of deformable shells, we quantify their Young’s modulus, shear viscosity, and the critical stresses required to switch between behaviors. Based on these experimental results, we propose a description of the tissue mechanics during normal regeneration. Our results provide a first step toward the development of original mechanochemical models of patterning grounded in quantitative experimental data.

Assessing teratogenic risks of gadolinium in freshwater environments: Implications for environmental health

Gadolinium (Gd) is among the rare earth elements extensively utilized in both industrial and medical applications. The latter application appears to contribute to the rise in Gd levels in aquatic ecosystems, as it is excreted via urine from patients undergoing MRI scans and often not captured by wastewater treatment systems. The potential environmental and biological hazards posed by gadolinium exposure are still under investigation. This study aimed to assess the teratogenic risk posed by a gadolinium chelate on the freshwater cnidarian Hydra vulgaris. The experimental design evaluated the impact of pure Gadodiamide (25 μg/l, 50 μg/l, 100 μg/l, 500 μg/l) and its commercial counterpart compound (Omniscan®; 100 μg/l, 500 μg/l, 782.7 mg/l) at varying concentrations using the Teratogenic Risk Index (TRI). Here we showed a moderate risk (Class III of TRI) following exposure to both tested formulations at concentrations ≥ 100 μg/l. Given the potential for similar concentrations in aquatic environments, particularly near wastewater discharge points, a teratogenic risk assessment using the Hydra regeneration assay was conducted on environmental samples collected from three rivers (Tiber, Almone, and Sacco) in Central Italy. Additionally, chemical analysis of field samples was performed using ICP-MS. Analysis of freshwater samples revealed low Gd concentrations (≤ 0.1 μg/l), despite localized increases near domestic and/or industrial wastewater discharge sites. Although teratogenic risk in environmental samples ranged from high (Class IV of TRI) to negligible (Class I of TRI), the low Gd concentrations, particularly when compared to higher levels of other contaminants like arsenic and heavy metals, preclude establishing a direct cause-effect relationship between Gd and observed teratogenic risks in environmental samples. Nevertheless, the teratogenic risks observed in laboratory tests warrant further investigation.

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.

Universal calcium fluctuations in Hydra morphogenesis

Understanding the collective physical processes that drive robust morphological transitions in animal development necessitates the characterization of the relevant fields involved in morphogenesis. Calcium (Ca2+) is recognized as one such field. In this study, we demonstrate that the spatial fluctuations of Ca2+ during Hydra regeneration exhibit universal characteristics. To investigate this phenomenon, we employ two distinct controls, an external electric field and heptanol, a gap junction-blocking drug. Both lead to the modulation of the Ca2+ activity and a reversible halting of the regeneration process. The application of an electric field enhances Ca2+ activity in the Hydra’s tissue and increases its spatial correlations, while the administration of heptanol inhibits its activity and diminishes the spatial correlations. Remarkably, the statistical characteristics of Ca2+ spatial fluctuations, including the coefficient of variation and skewness, manifest universal shape distributions across tissue samples and conditions. We introduce a field-theoretic model, describing fluctuations in a tilted double-well potential, which successfully captures these universal properties. Moreover, our analysis reveals that the Ca2+ activity is spatially localized, and the Hydra’s tissue operates near the onset of bistability, where the local Ca2+ activity fluctuates between low and high excited states in distinct regions. These findings highlight the prominent role of the Ca2+ field in Hydra morphogenesis and provide insights into the underlying mechanisms governing robust morphological transitions.

Hydra gasdermin-gated pyroptosis signalling regulates tissue regeneration

Pyroptosis, an inflammatory form of programmed cell death, is directly executed by gasdermin (GSDM) depending on its N-terminal pore-forming fragment-mediated membrane-disrupting, triggering intracellular contents release, which plays important roles in mammalian anti-infection and anti-tumor immune responses. However, whether pyroptosis engages in the regulation of tissue regeneration remains largely unknown. Here, utilizing Hydra vulgaris as the research model, we found that an HyCARD2-HyGSDME-mediated pyroptosis signalling is activated in both head and foot regenerated tips after amputation. Impeding pyroptosis by knocking down the expression of either HyGSDME or HyCARD2 significantly hampered both head and foot regeneration in Hydra. Mechanistically, the activation of HyCARD2-HyGSDME axis at wound sites is dependent of intracellular mitochondrial reactive oxygen species (mtROS), the removing of which hindered Hydra head regeneration. Moreover, the HyCARD2-HyGSDME axis-gated pyroptosis was found to enhance the initial secretion and upregulated expression of Wnt3. Collectively, these findings indicate that gasdermin-gated pyroptosis is critical for the evoking of Wnt signalling to facilitate Hydra tissue regeneration, which provides insights into functional diversification within the gasdermin family in the animal kingdom.

Teratogenic effects of environmental concentration of plastic particles on freshwater organisms

Given the widespread presence of plastics, especially in micro- and nanoscale sizes, in freshwater systems, it is crucial to identify a suitable model organism for assessing the potential toxic and teratogenic effects of exposure to plastic particles. Until now, the early life stage of freshwater organisms and the regeneration capacity in relation to plastic particles exposure is a still poorly investigated topic. In this study, we examine the teratogenic effect on diatom Cocconeis placentula and cnidarian Hydra vulgaris under controlled exposure conditions of poly(styrene-co-methyl methacrylate) (P(S-co-MMA)) particles. Significant effects were observed at the lowest concentrations (0.1 μg/L). A significant increase in the teratological frequency in C. placentula and a significant decrease in the regeneration rate in H. vulgaris were found at the lowest concentration. The delay in hydra regeneration impaired the feeding capacity and tentacles reactivity at 96 h of exposure. No effects on diatom growth were observed upon exposure to P(S-co-MMA) particles (0.1, 1, 100, 10,000 μg/L) for 28 days and these findings agree with other studies investigating algal growth. The application of the Teratogenic Risk Index, modified for diatoms, highlighted a moderate risk for the lowest concentration evaluating C. placentula and low risk at the lowest and the highest concentrations considering H. vulgaris. This study suggests the importance of testing organisms belonging to different trophic levels as diverse teratogenic effects can be found and the need to evaluate environmentally relevant concentrations of plastic particles.

Hydra morphogenesis as phase transition dynamics

Abstract We utilize whole-body Hydra regeneration from a small tissue segment to develop a physics framework for animal morphogenesis. Introducing experimental controls over this process, an external electric field and a drug that blocks gap junctions, allows us to characterize the essential step in the morphological transition —from a spherical shape to an elongated spheroid. We find that spatial fluctuations of the Ca2+ distribution in the Hydra's tissue drive this transition and construct a field-theoretic model that explains the morphological transition as a first-order–like phase transition resulting from the coupling of the Ca2+ field and the tissue's local curvature. Various predictions of this model are verified experimentally.

H4K20me1 plays a dual role in transcriptional regulation of regeneration and axis patterning in Hydra.

The evolution of the first body axis in the animal kingdom and its extensive ability to regenerate makes Hydra, a Cnidarian, an excellent model system for understanding the underlying epigenetic mechanisms. We identify that monomethyltransferase SETD8 is critical for regeneration in Hydra because of its conserved interaction with β-catenin to fine-tune the associated gene regulatory network. Inhibition of SETD8 activity abolishes head and foot regeneration in Hydra Furthermore, we show that H4K20me1, the histone mark imparted by SETD8, colocalizes with the transcriptional activation machinery locally at the β-catenin-bound TCF/LEF-binding sites on the promoters of head-associated genes, marking an epigenetic activation mode. In contrast, genome-wide analysis of the H4K20me1 occupancy revealed a negative correlation with transcriptional activation. We propose that H4K20me1 acts as a general repressive histone mark in Cnidaria and describe its dichotomous role in transcriptional regulation in Hydra.

Understanding Hydra Regeneration

Understanding Hydra Regeneration Charisios Tsiairis from the Friedrich Miescher Institute for Biomedical Research explores Hydra Regeneration through genetic programs. Hydras inhabit pristine springs throughout the world and are shaped as hollow tubes constructed by two epithelial layers. These Hydras have a natural ability to regrow a whole organism from a small piece of tissue, and the animal is named after a mythical serpent monster that regrew two heads for every head chopped off. Hydra research can help identify genetic programs that may be present in our own cells, and help people to understand the mechanism of common diseases, such as multiple cancer. This link seems to be present in some of the earliest multicellular species, testifying to the importance of mechanical signals for the proper behaviour of cells.

A novel thioredoxin glutathione reductase from evolutionary ancient metazoan Hydra

Thioredoxin (Trx) and glutathione disulfide (GSSG), are regenerated in reduced state by thioredoxin reductase (TrxR) and glutathione reductase (GR) respectively. A novel protein thioredoxin glutathione reductase (TGR) capable of reducing Trx as well as GSSG, linking two redox systems, has only been reported so far from parasitic flat worms and mammals. For the first time, we report a multifunctional antioxidant enzyme TGR from the nonparasitic, nonmammalian cnidarian Hydra vulgaris (HvTGR) which is a selenoprotein with unusual fusion of a TrxR domain with glutaredoxin (Grx) domain. We have cloned and sequenced HvTGR which encodes a polypeptide of 73 kDa. It contains conserved sequence CPYC of Grx domain, and CVNVGC and GCUG domains of thioredoxin reductase. Phylogenetic analysis revealed HvTGR to be closer to TGR from mammals rather than to TGR from parasitic helminths. We then subcloned HvTGR in plasmid pSelExpress-1 and expressed it in HEK293T cells to ensure selenocysteine incorporation. Purified HvTGR showed Grx, glutathione reductase and TrxR activities. Both thioredoxin and GSSG disulfide reductase activities were inhibited by 1-Chloro-2,4-dinitrobenzene (DNCB) supporting the existence of an essential selenocysteine residue. HvTGR expression was induced in response to H2O2 in Hydra. Interestingly, inhibition of HvTGR by DNCB, inhibited regeneration in Hydra indicating its involvement in other cellular processes.

Injury-induced MAPK activation triggers body axis formation in Hydra by default Wnt signaling

Hydra's almost unlimited regenerative potential is based on Wnt signaling, but so far it is unknown how the injury stimulus is transmitted to discrete patterning fates in head and foot regenerates. We previously identified mitogen-activated protein kinases (MAPKs) among the earliest injury response molecules in Hydra head regeneration. Here, we show that three MAPKs-p38, c-Jun N-terminal kinases (JNKs), and extracellular signal-regulated kinases (ERKs)-are essential to initiate regeneration in Hydra, independent of the wound position. Their activation occurs in response to any injury and requires calcium and reactive oxygen species (ROS) signaling. Phosphorylated MAPKs hereby exhibit cross talk with mutual antagonism between the ERK pathway and stress-induced MAPKs, orchestrating a balance between cell survival and apoptosis. Importantly, Wnt3 and Wnt9/10c, which are induced by MAPK signaling, can partially rescue regeneration in tissues treated with MAPK inhibitors. Also, foot regenerates can be reverted to form head tissue by a pharmacological increase of β-catenin signaling or the application of recombinant Wnts. We propose a model in which a β-catenin-based stable gradient of head-forming capacity along the primary body axis, by differentially integrating an indiscriminate injury response, determines the fate of the regenerating tissue. Hereby, Wnt signaling acquires sustained activation in the head regenerate, while it is transient in the presumptive foot tissue. Given the high level of evolutionary conservation of MAPKs and Wnts, we assume that this mechanism is deeply embedded in our genome.

Plasticity of body axis polarity in Hydra regeneration under constraints

One of the major events in animal morphogenesis is the emergence of a polar body axis. Here, we combine classic grafting techniques with live imaging to explore the plasticity of polarity determination during whole body regeneration in Hydra. Composite tissues are made by fusing two rings, excised from separate animals, in different configurations that vary in the polarity and original positions of the rings along the body axes of the parent animals. Under frustrating initial configurations, body axis polarity that is otherwise stably inherited from the parent animal, can become labile and even be reversed. Importantly, the site of head regeneration exhibits a strong bias toward the edges of the tissue, even when this involves polarity reversal. In particular, we observe head formation at an originally aboral tissue edge, which is not compatible with models of Hydra regeneration based only on preexisting morphogen gradients or an injury response. The site of the new head invariably contains an aster-like defect in the organization of the supra-cellular ectodermal actin fibers. While a defect is neither required nor sufficient for head formation, we show that the defect at the new head site can arise via different routes, either appearing directly following excision as the tissue seals at its edge or through de novo defect formation at the fusion site. Altogether, our results show that the emergence of a polar body axis depends on the original polarity and position of the excised tissues as well as structural factors, suggesting that axis determination is an integrated process that arises from the dynamic interplay of multiple biochemical and mechanical processes.

Optical Control of Tissue Regeneration through Photostimulation of Organic Semiconducting Nanoparticles.

Next generation bioengineering strives to identify crucial cues that trigger regeneration of damaged tissues, and to control the cells that execute these programs with biomaterials and devices. Molecular and biophysical mechanisms driving embryogenesis may inspire novel tools to reactivate developmental programs in situ. Here nanoparticles based on conjugated polymers are employed for optical control of regenerating tissues by using an animal with unlimited regenerative potential, the polyp Hydra, as in vivo model, and human keratinocytes as an in vitro model to investigate skin repair. By integrating animal, cellular, molecular, and biochemical approaches, nanoparticles based on poly-3-hexylthiophene (P3HT) are shown able to enhance regeneration kinetics, stem cell proliferation, and biomolecule oxidation levels. Opposite outputs are obtained with PCPDTBT-NPs (Poly[2,6-(4,4-bis-(2-ethylhexyl)-4H-cyclopenta [2,1-b;3,4-b'] dithiophene)-alt-4,7(2,1,3-benzothiadiazole)], causing a beneficial effect on Hydra regeneration but not on the migratory capability of keratinocytes. These results suggest that the artificial modulation of the redox potential in injured tissues may represent a powerful modality to control their regenerative potential. Importantly, the possibility to fine-tuning materials' photocatalytic efficiency may enable a biphasic modulation over a wide dynamic range, which can be exploited to augment the tissue regenerative capacity or inhibit the unlimited potential of cancerous cells in pathological contexts.

Canalized Morphogenesis Driven by Inherited Tissue Asymmetries in Hydra Regeneration.

The emergence and stabilization of a body axis is a major step in animal morphogenesis, determining the symmetry of the body plan as well as its polarity. To advance our understanding of the emergence of body axis polarity, we study regenerating Hydra. Axis polarity is strongly memorized in Hydra regeneration even in small tissue segments. What type of processes confer this memory? To gain insight into the emerging polarity, we utilize frustrating initial conditions by studying regenerating tissue strips which fold into hollow spheroids by adhering their distal ends of opposite original polarities. Despite the convoluted folding process and the tissue rearrangements during regeneration, these tissue strips develop in a reproducible manner, preserving the original polarity and yielding an ordered body plan. These observations suggest that the integration of mechanical and biochemical processes supported by their mutual feedback attracts the tissue dynamics towards a well-defined developmental trajectory biased by weak inherited cues from the parent animal. Hydra thus provide an example of dynamic canalization in which the dynamic rules are instilled, but, in contrast to the classical picture, the detailed developmental trajectory does not unfold in a programmatic manner.

Studying Mechanical Oscillations During Whole-Body Regeneration in Hydra.

Cells of the freshwater cnidarian Hydra possess an exceptional regeneration ability. In small groups of these cells, organizer centers emerge spontaneously and instruct the patterning of the surrounding population into a new animal. This property makes them an excellent model system to study the general rules of self-organization. A small tissue fragment or a clump of randomly aggregated cells can form a hollow spheroid that is able to establish a body axis de novo. Interestingly, mechanical oscillations (inflation/deflation cycles of the spheroid) driven by osmosis accompany the successful establishment of axial polarity. Here we describe different approaches for generating Hydra tissue spheroids, along with imaging and image analysis techniques to investigate their mechanical behavior.

Fluid–solid coupling dynamic model for oscillatory growth of multicellular lumens

The development of multicellular lumens involves the interplay of cell proliferation, oscillation, and fluid transport. In this paper, a fluid–solid coupling dynamic model is proposed to investigate the physical mechanisms underlying the oscillatory growth of lumens. On the basis of experimental observations, the periodic oscillation of a lumen is interpreted by the fracturing–healing mechanism of cell–cell contacts, which induces a hydraulic-controlled outward flow switch. This model reproduces the oscillations of lumen sizes, in agreement with the experimental results of Hydra regeneration. It is found that the overall change trend of the lumen volume is determined by the tissue development induced by cell proliferation and the fluid transport induced by the osmotic pressure, while the outward flow due to the fracturing of cell–cell contacts regulates the oscillatory volume and the stress level in an appropriate scope. This work not only deepens our understanding of biomechanical mechanisms under the development of fluid-containing lumens, but also provides a theoretical framework to rationalize the dynamics of lumen-like tissues.

Cloning and characterization of Thioredoxin 1 from the Cnidarian Hydra.

Thioredoxins, small disulphide-containing redox proteins, play an important role in the regulation of cellular thiol redox balance through their disulfide reductase activity. In this study, we have identified, cloned, purified and characterized thioredoxin 1 (HvTrx1) from the Cnidarian Hydra vulgaris Ind-Pune. Bioinformatics analysis revealed that HvTrx1 contains an evolutionarily conserved catalytic active site Cys-Gly-Pro-Cys and shows a closer phylogenetic relationship with vertebrate Trx1. Optimum pH and temperature for enzyme activity of purified HvTrx1 was found to be pH7.0 and 25°C, respectively. Enzyme activity decreased significantly at acidic or alkaline pH as well as at higher temperatures. HvTrx1 was found to be expressed ubiquitously in whole mount in situ hybridization. Treatment of Hydra with hydrogen peroxide (H2O2), a highly reactive oxidizing agent, led to a significant increase in gene expression and enzyme activity of Trx1. Further experiments using PX12, an inhibitor of Trx1, indicated that Trx1 plays an important role in regeneration in Hydra. Finally, by using growth assay in Escherichia coli and wound healing assay in human colon cancer cells, we demonstrate that HvTrx1 is functionally active in both prokaryotic and eukaryotic heterologous systems.

Cellular, Metabolic, and Developmental Dimensions of Whole-Body Regeneration in Hydra.

Here we discuss the developmental and homeostatic conditions necessary for Hydra regeneration. Hydra is characterized by populations of adult stem cells paused in the G2 phase of the cell cycle, ready to respond to injury signals. The body column can be compared to a blastema-like structure, populated with multifunctional epithelial stem cells that show low sensitivity to proapoptotic signals, and high inducibility of autophagy that promotes resistance to stress and starvation. Intact Hydra polyps also exhibit a dynamic patterning along the oral-aboral axis under the control of homeostatic organizers whose activity results from regulatory loops between activators and inhibitors. As in bilaterians, injury triggers the immediate production of reactive oxygen species (ROS) signals that promote wound healing and contribute to the reactivation of developmental programs via cell death and the de novo formation of new organizing centers from somatic tissues. In aging Hydra, regeneration is rapidly lost as homeostatic conditions are no longer pro-regenerative.

A screen of kinase inhibitors reveals a potential role of Chk1 in regulating Hydra head regeneration and maintenance

The cnidarian Hydra possesses remarkable regenerative capabilities which allow it to regrow lost or damaged body parts in a matter of days. Given that many key regulators of regeneration and development are evolutionarily conserved, Hydra is a valuable model system for studying the fundamental molecular mechanisms underlying these processes. In the past, kinase inhibitors have been useful tools for determining the role of conserved signaling pathways in Hydra regeneration and patterning. Here, we present a systematic screen of a commercially available panel of kinase inhibitors for their effects on Hydra regeneration. Isolated Hydra gastric segments were exposed to 5 µM of each kinase inhibitor and regeneration of the head and foot regions were scored over a period of 96 hours. Of the 80 kinase inhibitors tested, 28 compounds resulted in abnormal regeneration. We directed our focus to the checkpoint kinase 1 (Chk1) inhibitor, SB 218078, considering the role of Chk1 in G2 checkpoint regulation and the importance of G2-paused cells in Hydra regeneration. We found that Hydra exposed to SB 218078 were unable to regenerate the head and maintain head-specific structures. Furthermore, SB 218078-treated Hydra displayed a reduction in the relative proportion of epithelial cells; however, no differences were seen for interstitial stem cells or their derivatives. Lastly, exposure to SB 218078 appeared to have no impact on the level of mitosis or apoptosis. Overall, our study demonstrates the feasibility of kinase inhibitor screens for studying Hydra regeneration processes and highlights the possible role for Hydra Chk1 in head regeneration and maintenance.