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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., (© 2024. The Author(s).)

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.

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.

Tissue morphogenesis comprises the self-organized creation of various patterns and shapes. Although detailed underlying mechanisms are still elusive in many cases, an increasing amount of experimental data suggests that chemical morphogen and mechanical processes are strongly coupled. Here, we develop and test a minimal model of the axis-defining step (i.e., symmetry breaking) in aggregates of the Hydra polyp. Based on previous findings, we combine osmotically driven shape oscillations with tissue mechanics and morphogen dynamics. We show that the model incorporating a simple feedback loop between morphogen patterning and tissue stretch reproduces a wide range of experimental data. Finally, we compare different hypothetical morphogen patterning mechanisms (Turing, tissue-curvature, and self-organized criticality). Our results suggest the experimental investigation of bigger (i.e., multiple head) aggregates as a key step for a deeper understanding of mechanochemical symmetry breaking in Hydra., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)