HydRA: Deep-learning models for predicting RNA-binding capacity from protein interaction association context and protein sequence.

RNA-binding proteins (RBPs) control RNA metabolism to orchestrate gene expression and, when dysfunctional, underlie human diseases. Proteome-wide discovery efforts predict thousands of RBP candidates, many of which lack canonical RNA-binding domains (RBDs). Here, we present a hybrid ensemble RBP classifier (HydRA), which leverages information from both intermolecular protein interactions and internal protein sequence patterns to predict RNA-binding capacity with unparalleled specificity and sensitivity using support vector machines (SVMs), convolutional neural networks (CNNs), and Transformer-based protein language models. Occlusion mapping by HydRA robustly detects known RBDs and predicts hundreds of uncharacterized RNA-binding associated domains. Enhanced CLIP (eCLIP) for HydRA-predicted RBP candidates reveals transcriptome-wide RNA targets and confirms RNA-binding activity for HydRA-predicted RNA-binding associated domains. HydRA accelerates construction of a comprehensive RBP catalog and expands the diversity of RNA-binding associated domains.

Regulation of Genomic Output and (Pluri)potency in Regeneration.

Regeneration is a remarkable phenomenon that has been the subject of awe and bafflement for hundreds of years. Although regeneration competence is found in highly divergent organisms throughout the animal kingdom, recent advances in tools used for molecular and genomic characterization have uncovered common genes, molecular mechanisms, and genomic features in regenerating animals. In this review we focus on what is known about how genome regulation modulates cellular potency during regeneration. We discuss this regulation in the context of complex tissue regeneration in animals, from Hydra to humans, with reference to ex vivo-cultured cell models of pluripotency when appropriate. We emphasize the importance of a detailed molecular understanding of both the mechanisms that regulate genomic output and the functional assays that assess the biological relevance of such molecular characterizations.

A chromosome-scale epigenetic map of the Hydra genome reveals conserved regulators of cell state.

The epithelial and interstitial stem cells of the freshwater polyp Hydra are the best-characterized stem cell systems in any cnidarian, providing valuable insight into cell type evolution and the origin of stemness in animals. However, little is known about the transcriptional regulatory mechanisms that determine how these stem cells are maintained and how they give rise to their diverse differentiated progeny. To address such questions, a thorough understanding of transcriptional regulation in Hydra is needed. To this end, we generated extensive new resources for characterizing transcriptional regulation in Hydra, including new genome assemblies for Hydra oligactis and the AEP strain of Hydra vulgaris, an updated whole-animal single-cell RNA-seq atlas, and genome-wide maps of chromatin interactions, chromatin accessibility, sequence conservation, and histone modifications. These data revealed the existence of large kilobase-scale chromatin interaction domains in the Hydra genome that contain transcriptionally coregulated genes. We also uncovered the transcriptomic profiles of two previously molecularly uncharacterized cell types: isorhiza-type nematocytes and somatic gonad ectoderm. Finally, we identified novel candidate regulators of cell type-specific transcription, several of which have likely been conserved at least since the divergence of Hydra and the jellyfish Clytia hemisphaerica more than 400 million years ago.

A complete biomechanical model of Hydra contractile behaviors, from neural drive to muscle to movement.

How does neural activity drive muscles to produce behavior? The recent development of genetic lines in Hydra that allow complete calcium imaging of both neuronal and muscle activity, as well as systematic machine learning quantification of behaviors, makes this small cnidarian an ideal model system to understand and model the complete transformation from neural firing to body movements. To achieve this, we have built a neuromechanical model of Hydra's fluid-filled hydrostatic skeleton, showing how drive by neuronal activity activates distinct patterns of muscle activity and body column biomechanics. Our model is based on experimental measurements of neuronal and muscle activity and assumes gap junctional coupling among muscle cells and calcium-dependent force generation by muscles. With these assumptions, we can robustly reproduce a basic set of Hydra's behaviors. We can further explain puzzling experimental observations, including the dual timescale kinetics observed in muscle activation and the engagement of ectodermal and endodermal muscles in different behaviors. This work delineates the spatiotemporal control space of Hydra movement and can serve as a template for future efforts to systematically decipher the transformations in the neural basis of behavior.

Patterning of morphogenetic anisotropy fields.

Orientational order, encoded in anisotropic fields, plays an important role during the development of an organism. A striking example of this is the freshwater polyp Hydra, where topological defects in the muscle fiber orientation have been shown to localize to key features of the body plan. This body plan is organized by morphogen concentration gradients, raising the question how muscle fiber orientation, morphogen gradients and body shape interact. Here, we introduce a minimal model that couples nematic orientational order to the gradient of a morphogen field. We show that on a planar surface, alignment to a radial concentration gradient can induce unbinding of topological defects, as observed during budding and tentacle formation in Hydra, and stabilize aster/vortex-like defects, as observed at a Hydra's mouth. On curved surfaces mimicking the morphologies of Hydra in various stages of development-from spheroid to adult-our model reproduces the experimentally observed reorganization of orientational order. Our results suggest how gradient alignment and curvature effects may work together to control orientational order during development and lay the foundations for future modeling efforts that will include the tissue mechanics that drive shape deformations.

The Hippo pathway regulates axis formation and morphogenesis in Hydra.

How did cells of early metazoan organisms first organize themselves to form a body axis? The canonical Wnt pathway has been shown to be sufficient for induction of axis in Cnidaria, a sister group to Bilateria, and is important in bilaterian axis formation. Here, we provide experimental evidence that in cnidarian Hydra the Hippo pathway regulates the formation of a new axis during budding upstream of the Wnt pathway. The transcriptional target of the Hippo pathway, the transcriptional coactivator YAP, inhibits the initiation of budding in Hydra and is regulated by Hydra LATS. In addition, we show functions of the Hippo pathway in regulation of actin organization and cell proliferation in Hydra. We hypothesize that the Hippo pathway served as a link between continuous cell division, cell density, and axis formation early in metazoan evolution.

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 ß-catenin signaling or the application of recombinant Wnts. We propose a model in which a ß-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.

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.

De novo evolution of transmissible tumours in hydra.

While most cancers are not transmissible, there are rare cases where cancer cells can spread between individuals and even across species, leading to epidemics. Despite their significance, the origins of such cancers remain elusive due to late detection in host populations. Using Hydra oligactis, which exhibits spontaneous tumour development that in some strains became vertically transmitted, this study presents the first experimental observation of the evolution of a transmissible tumour. Specifically, we assessed the initial vertical transmission rate of spontaneous tumours and explored the potential for optimizing this rate through artificial selection. One of the hydra strains, which evolved transmissible tumours over five generations, was characterized by analysis of cell type and bacteriome, and assessment of life-history traits. Our findings indicate that tumour transmission can be immediate for some strains and can be enhanced by selection. The resulting tumours are characterized by overproliferation of large interstitial stem cells and are not associated with a specific bacteriome. Furthermore, despite only five generations of transmission, these tumours induced notable alterations in host life-history traits, hinting at a compensatory response. This work, therefore, makes the first contribution to understanding the conditions of transmissible cancer emergence and their short-term consequences for the host.

Nonlinear elasticity and short-range mechanical coupling govern the rate and symmetry of mouth opening in Hydra.

Hydra has a tubular bilayered epithelial body column with a dome-shaped head on one end and a foot on the other. Hydra lacks a permanent mouth: its head epithelium is sealed. Upon neuronal activation, a mouth opens at the apex of the head which can exceed the body column diameter in seconds, allowing Hydra to ingest prey larger than itself. While the kinematics of mouth opening are well characterized, the underlying mechanism is unknown. We show that Hydra mouth opening is generated by independent local contractions that require tissue-level coordination. We model the head epithelium as an active viscoelastic nonlinear spring network. The model reproduces the size, timescale and symmetry of mouth opening. It shows that radial contractions, travelling inwards from the outer boundary of the head, pull the mouth open. Nonlinear elasticity makes mouth opening larger and faster, contrary to expectations. The model correctly predicts changes in mouth shape in response to external forces. By generating innervated : nerve-free chimera in experiments and simulations, we show that nearest-neighbour mechanical signalling suffices to coordinate mouth opening. Hydra mouth opening shows that in the absence of long-range chemical or neuronal signals, short-range mechanical coupling is sufficient to produce long-range order in tissue deformations.

Phototaxis is a satiety-dependent behavioral sequence in Hydra vulgaris.

Understanding how internal states such as satiety are connected to animal behavior is a fundamental question in neuroscience. Hydra vulgaris, a freshwater cnidarian with only 12 neuronal cell types, serves as a tractable model system for studying state-dependent behaviors. We found that starved hydras consistently move towards light, while fed hydras do not. By modeling this behavior as a set of three sequences of head orientation, jump distance and jump rate, we demonstrate that the satiety state only affects the rate of the animal jumping to a new position, while the orientation and jump distance are unaffected. These findings yield insights into how internal states in a simple organism, Hydra, affect specific elements of a behavior, and offer general principles for studying the relationship between state-dependent behaviors and their underlying molecular mechanisms.

Hydra and the hair follicle - An unconventional comparative biology approach to exploring the human holobiont.

The microbiome of human hair follicles (HFs) has emerged as an important player in different HF and skin pathologies, yet awaits in-depth exploration. This raises questions regarding the tightly linked interactions between host environment, nutrient dependency of host-associated microbes, microbial metabolism, microbe-microbe interactions and host immunity. The use of simple model systems facilitates addressing generally important questions and testing overarching, therapeutically relevant principles that likely transcend obvious interspecies differences. Here, we evaluate the potential of the freshwater polyp Hydra, to dissect fundamental principles of microbiome regulation by the host, that is the human HF. In particular, we focus on therapeutically targetable host-microbiome interactions, such as nutrient dependency, microbial interactions and host defence. Offering a new lens into the study of HF - microbiota interactions, we argue that general principles of how Hydra manages its microbiota can inform the development of novel, microbiome-targeting therapeutic interventions in human skin disease.

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.

Does the Symbiotic Relationship Between Hydra Viridissima and Photoautotrophic Alga Provide an Evolutionary Advantage in Protecting DNA against Damage by the Cytotoxic or Genotoxic Mode of Action of Environmental Stressors?

The aim of this paper was to evaluate whether symbiotic cooperation between green hydra (Hydra viridissima) and photoautotrophic alga gives higher resistance of the preservation of DNA integrity compared to brown hydra (Hydra oligactis). Norflurazon concentrations were 0.061 or 0.61 mg/L and UV-B light 254 nm, 0.023mWcm- 2 applied separately or simultaneously. By alkaline comet assay primary DNA damage was assessed and cytotoxicity by fluorescent staining. Norflurazon at 0.61 mg L- 1 significantly increased DNA damage in brown hydras compared to the control (6.17 ± 0.6 µm, 5.2 ± 1.7% vs. 2.9 ± 0.2 µm, 1.2 ± 0.2%). Cytotoxicity was significantly elevated, being higher in brown hydras (25.7 ± 3.5% vs. 8.2 ± 0.2%). UV-B irradiation induced significant DNA damage in brown hydras (13.5 ± 1.0 µm, 4.1 ± 1.0%). Simultaneous exposure to UV-B and norflurazon led to a synergistic DNA damaging. The frequency of cytotoxicity and hedgehog nucleoids was more pronounced in brown (78.3 ± 9.4%; 56.4 ± 6.0%) than in green hydras (34.7 ± 2.5%; 24.2 ± 0.6%). Evolutionary established symbiotic cooperation proved to provide resistance against cyto/genotoxicity.

Traffic light Hydra allows for simultaneous in vivo imaging of all three cell lineages.

We present a new transgenic Hydra vulgaris line expressing a distinct fluorescent protein in each of the three cell lineages of the adult polyp. Plasmid microinjection was used to generate a novel transgenic Hydra line expressing the yellow fluorescent protein YPet in the ectodermal epithelial cell lineage. Tissue grafting was then used to combine a YPet animal with a line that expresses DsRed2 in the endodermal epithelial lineage and eGFP in the interstitial cell (i-cell) lineage. The resulting triple-labeled ("tricolored") transgenic line provides, for the first time, a Hydra in which all three cell lineages can be imaged simultaneously in vivo. We show example confocal images of whole animals and individual cells to illustrate the imaging capabilities that this new line makes possible. We also used this line to carry out new studies of cell fate in the tentacles. Specifically, we evaluated the well-accepted notion that all tentacle cells are terminally differentiated and are displaced or migrate exclusively towards the distal end of the tentacle. We found that ectodermal and endodermal epithelial cells are displaced distally, as expected. In contrast, members of the i-cell lineage, which resembled neuronal precursors, could migrate out of a tentacle into the body column. This example illustrates how this tricolored transgenic line enables new in vivo studies of cell behaviors in Hydra.

Ancestral role of TNF-R pathway in cell differentiation in the basal metazoan Hydra.

Tumour necrosis factor receptors (TNF-Rs) and their ligands, tumour necrosis factors, are highly conserved proteins described in all metazoan phyla. They function as inducers of extrinsic apoptotic signalling and facilitate inflammation, differentiation and cell survival. TNF-Rs use distinct adaptor molecules to activate signalling cascades. Fas-associated protein with death domain (FADD) family adaptors often mediate apoptosis, and TNF-R-associated factor (TRAF) family adaptors mediate cell differentiation and inflammation. Most of these pathway components are conserved in cnidarians, and, here, we investigated the Hydra TNF-R. We report that it is related to the ectodysplasin receptor, which is involved in epithelial cell differentiation in mammals. In Hydra, it is localised in epithelial cells with incorporated nematocytes in tentacles and body column, indicating a similar function. Further experiments suggest that it interacts with the Hydra homologue of a TRAF adaptor, but not with FADD proteins. Hydra FADD proteins colocalised with Hydra caspases in death effector filaments and recruited caspases, suggesting that they are part of an apoptotic signalling pathway. Regulating epithelial cell differentiation via TRAF adaptors therefore seems to be an ancient function of TNF-Rs, whereas FADD-caspase interactions may be part of a separate apoptotic pathway.

Differential gene regulation in DAPT-treated Hydra reveals candidate direct Notch signalling targets.

In Hydra, Notch inhibition causes defects in head patterning and prevents differentiation of proliferating nematocyte progenitor cells into mature nematocytes. To understand the molecular mechanisms by which the Notch pathway regulates these processes, we performed RNA-seq and identified genes that are differentially regulated in response to 48 h of treating the animals with the Notch inhibitor DAPT. To identify candidate direct regulators of Notch signalling, we profiled gene expression changes that occur during subsequent restoration of Notch activity and performed promoter analyses to identify RBPJ transcription factor-binding sites in the regulatory regions of Notch-responsive genes. Interrogating the available single-cell sequencing data set revealed the gene expression patterns of Notch-regulated Hydra genes. Through these analyses, a comprehensive picture of the molecular pathways regulated by Notch signalling in head patterning and in interstitial cell differentiation in Hydra emerged. As prime candidates for direct Notch target genes, in addition to Hydra (Hy)Hes, we suggest Sp5 and HyAlx. They rapidly recovered their expression levels after DAPT removal and possess Notch-responsive RBPJ transcription factor-binding sites in their regulatory regions.

Cancer: slaying the nine-headed Hydra.

Modern medicine continues to evolve, and the treatment armamentarium for various diseases grows more individualized across a breadth of medical disciplines. Cure rates for infectious diseases that were previously pan-fatal approach 100% because of the identification of the specific pathogen(s) involved and the use of appropriate combinations of drugs, where needed, to completely extinguish infection and hence prevent emergence of resistant strains. Similarly, with the assistance of technologies such as next-generation sequencing and immunomic analysis as part of the contemporary oncology armory, therapies can be tailored to each tumor. Importantly, molecular interrogation has revealed that metastatic cancers are distinct from each other and complex. Therefore, it is conceivable that rational personalized drug combinations will be needed to eradicate cancers, and eradication will be necessary to mitigate clonal evolution and resistance.

An interview with Celina Juliano.

Celina Juliano is an Assistant Professor at UC Davis, where she uses Hydra as a model system to understand development and regeneration. She is co-founder of the Cnidarian Model Systems Meeting (Cnidofest) biennial conference and the OpenHydra Hydra resource platform. This year, she was awarded the Elizabeth D. Hay New Investigator award for outstanding developmental biology research during the early stages of her independent career by the Society for Developmental Biology (SDB). Following the virtual SDB 2020 meeting, we met with Celina over Zoom to hear more about her life and career.

Deficient autophagy in epithelial stem cells drives aging in the freshwater cnidarian Hydra.

Hydra possesses three distinct stem cell populations that continuously self-renew and prevent aging in Hydra vulgaris However, sexual animals from the H. oligactis cold-sensitive strain Ho_CS develop an aging phenotype upon gametogenesis induction, initiated by the loss of interstitial stem cells. Animals stop regenerating, lose their active behaviors and die within 3 months. This phenotype is not observed in the cold-resistant strain Ho_CR To dissect the mechanisms of Hydra aging, we compared the self-renewal of epithelial stem cells in these two strains and found it to be irreversibly reduced in aging Ho_CS but sustained in non-aging Ho_CR We also identified a deficient autophagy in Ho_CS epithelial cells, with a constitutive deficiency in autophagosome formation as detected with the mCherry-eGFP-LC3A/B autophagy sensor, an inefficient response to starvation as evidenced by the accumulation of the autophagosome cargo protein p62/SQSTM1, and a poorly inducible autophagy flux upon proteasome inhibition. In the non-aging H. vulgaris animals, the blockade of autophagy by knocking down WIPI2 suffices to induce aging. This study highlights the essential role of a dynamic autophagy flux to maintain epithelial stem cell renewal and prevent aging.