Identification of Components of the Hippo Pathway in Hydra and Potential Role of YAP in Cell Division and Differentiation

The Hippo signaling pathway has been shown to be involved in regulating cellular identity, cell/tissue size maintenance and mechanotransduction. The Hippo pathway consists of a kinase cascade which determines the nucleo-cytoplasmic localization of YAP in the cell. YAP is the effector protein in the Hippo pathway, which acts as a transcriptional cofactor for TEAD. Phosphorylation of YAP upon activation of the Hippo pathway prevents it from entering the nucleus and abrogates its function in the transcription of the target genes. In Cnidaria, the information on the regulatory roles of the Hippo pathway is virtually lacking. Here, we report the existence of a complete set of Hippo pathway core components in Hydra for the first time. By studying their phylogeny and domain organization, we report evolutionary conservation of the components of the Hippo pathway. Protein modelling suggested the conservation of YAP-TEAD interaction in Hydra. Further, we characterized the expression pattern of the homologs of yap, hippo, mob and sav in Hydra using whole-mount RNA in situ hybridization and report their possible role in stem cell maintenance. Immunofluorescence assay revealed that Hvul_YAP expressing cells occur in clusters in the body column and are excluded in the terminally differentiated regions. Actively proliferating cells marked by Ki67 exhibit YAP colocalization in their nuclei. Strikingly, a subset of these colocalized cells is actively recruited to the newly developing bud. Disruption of the YAP-TEAD interaction increased the budding rate indicating a critical role of YAP in regulating cell proliferation in Hydra. Collectively, we posit that the Hippo pathway is an essential signaling system in Hydra; its components are ubiquitously expressed in the Hydra body column and play a crucial role in Hydra tissue homeostasis.

The Wnt-specific astacin proteinase HAS-7 restricts head organizer formation in Hydra

Background The Hydra head organizer acts as a signaling center that initiates and maintains the primary body axis in steady state polyps and during budding or regeneration. Wnt/beta-Catenin signaling functions as a primary cue controlling this process, but how Wnt ligand activity is locally restricted at the protein level is poorly understood. Here we report a proteomic analysis of Hydra head tissue leading to the identification of an astacin family proteinase as a Wnt processing factor. Results Hydra astacin-7 (HAS-7) is expressed from gland cells as an apical-distal gradient in the body column, peaking close beneath the tentacle zone. HAS-7 siRNA knockdown abrogates HyWnt3 proteolysis in the head tissue and induces a robust double axis phenotype, which is rescued by simultaneous HyWnt3 knockdown. Accordingly, double axes are also observed in conditions of increased Wnt activity as in transgenic actin::HyWnt3 and HyDkk1/2/4 siRNA treated animals. HyWnt3-induced double axes in Xenopus embryos could be rescued by coinjection of HAS-7 mRNA. Mathematical modelling combined with experimental promotor analysis indicate an indirect regulation of HAS-7 by beta-Catenin, expanding the classical Turing-type activator-inhibitor model. Conclusions We show the astacin family protease HAS-7 maintains a single head organizer through proteolysis of HyWnt3. Our data suggest a negative regulatory function of Wnt processing astacin proteinases in the global patterning of the oral-aboral axis in Hydra.

The Hippo pathway transcriptional co-activator YAP is involved in head regeneration and bud development in Hydra

The Hippo signaling pathway has been shown to be involved in the regulation of cellular identity, cell/tissue size maintenance and mechanotransduction. The Hippo pathway consists of a kinase cascade which determines the nucleo-cytoplasmic localization of YAP in the cell. YAP is the effector protein in the Hippo pathway which acts as a transcriptional cofactor for TEAD. Phosphorylation of YAP upon activation of the Hippo pathway prevents it from entering the nucleus and hence abrogates its function in transcription of target genes. In Cnidaria, the information on the regulatory roles of the Hippo pathway is virtually lacking. Here, we report for the first time the existence of a complete set of Hippo pathway core components in Hydra. By studying their phylogeny and domain organization, we report evolutionary conservation of the components of the Hippo pathway. Protein modelling suggested conservation of YAP-TEAD interaction in Hydra. We also characterized the expression pattern of the homologs of yap, hippo, mob and sav in Hydra using whole mount RNA in situ hybridization and report their possible role in stem cell maintenance. Immunofluorescence assay revealed that Hvul_YAP expressing cells occur in clusters in the body column and are excluded in the terminally differentiated regions. The YAP expressing cells are recruited early during head regeneration and budding implicating the Hippo pathway in early response to injury or establishment of oral fate. These cells exhibit a non-clustered existence at the site of regeneration and budding, indicating the involvement of a new population of YAP expressing cells during oral fate specification. Collectively, we posit that the Hippo pathway is an important signaling system in Hydra, its components are ubiquitously expressed in the Hydra body column, and may play crucial role in Hydra oral fate specification.

Nicotine Stimulates Peristalsis in N. Vectensis: From Behavior to Nicotinic Receptor Genes

Nicotinic acetylcholine receptors (nAChRs) play a pivotal role in the nervous system, and there is great interest in understanding their evolution. Cnidarians, an ancestral group of metazoans, display an early nervous system utilizing complex neural architecture that provides insight into the early evolution and function of nAChRs. Using the anthozoan Nematostella vectensis, we found that the rate of peristalsis, a radial constriction of the body column, is modulated by nicotinic agonists. Nicotine caused a robust, dose dependent increase in the rate of peristalsis. The peristalsis rate also increased in response to ACh in the presence of edrophonium, an acetylcholine esterase inhibitor. Thus, N. vectensis possesses functional nAChRs. We developed a phylogeny of nAChRs from 15 cnidarian and 16 bilaterian species. Our results indicate that the large number of cnidarian and bilaterian nAChR gene copies is convergent, resulting from gene expansions after the two lineages diverged. These independent expansions led to an average of 31 and 34 paralogous nAChR gene copies in sampled bilaterians and cnidarians, respectively. The N. vectensis proteome contains 49 paralogous nAChR copies. The independent expansions indicate that nAChRs receptors in cnidarians may have distinct pharmacological and biochemical characteristics in comparison to their bilaterian counterparts.

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. FADD-adaptors often mediate apoptosis and TRAF-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 homolog of a TRAF-adaptor, but not with FADDs. Hydra-FADD proteins co-localised 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 tissue stiffness of body column facilitates locomotion of Hydra on solid substrates

ABSTRACTThe bell-shaped members of the Cnidaria typically move around by swimming, whereas the Hydra polyp can perform locomotion on solid substrates in an aquatic environment. To address the biomechanics of locomotion on rigid substrates, we studied the ‘somersaulting’ locomotion in Hydra. We applied atomic force microscopy to measure the local mechanical properties of Hydra's body column and identified the existence of differential Young's modulus between the shoulder region versus rest of the body column at 3:1 ratio. We show that somersaulting primarily depends on differential tissue stiffness of the body column and is explained by computational models that accurately recapitulate the mechanics involved in this process. We demonstrate that perturbation of the observed stiffness variation in the body column by modulating the extracellular matrix polymerization impairs the ‘somersault’ movement. These results provide a mechanistic basis for the evolutionary significance of differential extracellular matrix properties and tissue stiffness.

Bacteria- and temperature-regulated peptides modulate β-catenin signaling inHydra

Animal development has traditionally been viewed as an autonomous process directed by the host genome. But, in many animals, biotic and abiotic cues, like temperature and bacterial colonizers, provide signals for multiple developmental steps.Hydraoffers unique features to encode these complex interactions of developmental processes with biotic and abiotic factors, and we used it here to investigate the impact of bacterial colonizers and temperature on the pattern formation process. InHydra, formation of the head organizer involves the canonical Wnt pathway. Treatment with alsterpaullone (ALP) results in acquiring characteristics of the head organizer in the body column. Intriguingly, germfreeHydrapolyps are significantly more sensitive to ALP compared to control polyps. In addition to microbes, β-catenin–dependent pattern formation is also affected by temperature. Gene expression analyses led to the identification of two small secreted peptides, named Eco1 and Eco2, being up-regulated in the response to bothCurvibactersp., the main bacterial colonizer ofHydra, and low temperatures. Loss-of-function experiments revealed that Eco peptides are involved in the regulation of pattern formation and have an antagonistic function to Wnt signaling inHydra.

A Wnt-specific astacin proteinase controls head formation in Hydra

The Hydra head organizer acts as a signaling center that initiates and maintains the primary body axis in steady state polyps and during budding or regeneration. Wnt/beta-Catenin signaling functions as a primary cue controlling this process, but how Wnt ligand activity is locally restricted at the protein level is poorly understood.Here we report the identification of an astacin family proteinase as a Wnt processing factor. Hydra astacin-7 (HAS-7) is expressed from gland cells as an apical-distal gradient in the body column, peaking close beneath the tentacle zone. HAS-7 siRNA knockdown abrogates HyWnt3 proteolysis in the head tissue and induces a robust double axis phenotype, which is rescued by simultaneous HyWnt3 knockdown. Accordingly, double axes are also observed in conditions of increased Wnt levels as in transgenic actin::HyWnt3 and HyDkk1/2/4 siRNA treated animals. HyWnt3-induced double axes in Xenopus embryos could be rescued by co-injection of HAS-7 mRNA. Mathematical modelling combined with experimental promotor analysis indicate an indirect regulation of HAS-7 by beta-Catenin, expanding the classical Turing-type activator-inhibitor model.Our data suggest a negative regulatory function of Wnt processing astacin proteinases in the global patterning of the oral-aboral axis in Hydra.

Differential tissue stiffness of body column facilitates locomotion of Hydra on solid substrates

The bell-shaped members of Cnidaria typically move around by swimming, whereas the Hydra polyp can perform locomotion on solid substrates in aquatic environment. To address the biomechanics of locomotion on rigid substrates, we studied the ‘somersaulting’ locomotion in Hydra. We applied atomic force microscopy to measure the local mechanical properties of Hydra’s body column and identified the existence of differential Young’s modulus between the shoulder region versus rest of the body column at 3:1 ratio. We show that somersault primarily depends on differential tissue stiffness of the body column and is explained by computational models that accurately recapitulate the mechanics involved in this process. We demonstrate that perturbation of the observed stiffness variation in the body column by modulating the extracellular matrix (ECM) polymerization impairs the ‘somersault’ movement. These results provide mechanistic basis for the evolutionary significance of differential extracellular matrix properties and tissue stiffness.

Differential expression of BMP antagonists, gremlin and noggin in hydra: antagonism between Wnt and BMP pathways

Mechanisms regulating BMP and Wnt signaling pathways have been widely studied in many organisms. One of the mechanisms by which these pathways are regulated is by binding of extracellular ligands. In the present study, we report studies with two BMP antagonists, gremlin and noggin from Hydra vulgaris Ind-Pune and demonstrate antagonistic relationship between BMP and Wnt pathways. Gremlin was ubiquitously expressed from the body column to head region except in the basal disc and hypostome. During budding, gremlin was expressed predominantly in the budding region suggesting a possible role in budding; this was confirmed in polyps with different stages of buds. Noggin, on the other hand, was predominantly expressed in the endoderm of hypostome, base of the tentacles, lower body column and at the basal disc in whole polyps. During budding, noggin was expressed at the sites of emergence of tentacles suggesting a role in tentacle formation. This was confirmed in alsterpaullone-treated polyps, which showed noggin expression as distinct spots where ectopic organizers and ectopic tentacles eventually formed. Using RT-PCR, we found that up-regulation of Wnt is accompanied with down-regulation of BMP5-8b demonstrating antagonism between the two pathways. Down-regulation of noggin and gremlin, however, occurred only after 24 h recovery. The data suggest that inhibition of BMP pathway by Wnt signaling in hydra does not directly involve noggin and gremlin. Our findings indicate that the BMP/Noggin antagonism evolved early for setting up and/or maintaining the head organizer while involvement of these BMP antagonists during vertebrate axial patterning are recent evolutionary acquisitions.Summary statementWe show that setting up of the Organizer by BMP/Noggin antagonism and role of BMP inhibitors in tissue patterning are evolutionarily ancient, probably arising for the first time in hydra