Regulation of Hydra Regeneration by Hippo Pathway effector YAP and Biomechanical forces

Abstract

Hydra is a freshwater polyp belonging to the phylum Cnidaria. These polyps are known to exhibit tremendous regenerating potential. It is still unclear how the regenerating tissue is reorganized, how the complex interplay of signaling cascades required for generating positional information in regenerating tissue is produced and regulated. Specifically, the question of how biophysical forces govern the regeneration process by integrating early injury response with positional cues has been a subject of intense research in recent times in regenerative model organisms, including Zebrafish and Axolotl. Owing to the simple tissue organization and the well-characterized morphallactic regenerative biochemical signaling pathways, Hydra provides an ideal system for developing a model system to study biomechanical regulation. In this study, we employed the paradigm of head regeneration in Hydra to understand how tissue damage invokes changes in tissue mechanics and how the mechanical forces can affect the regenerative response. Hippo signalling is a well-known pathway for mechanotransduction in cells. In this study, we report for the first time the existence of a complete repertoire of the Hippo pathway core components in Hydra. By staining Hippo effector YAP during head regeneration, we report that mechanosensitive (YAP positive) cells migrate to the site of injury early during regeneration. We show that by disrupting the Hippo pathway by the perturbation of the interaction of YAP (a transcription co-activator) and its cognate transcription factor TEAD, we can accelerate the regeneration in Hydra. Further, scanning electron microscopy (SEM) based evaluation of the ultrastructure demonstrated an extensive fibrosis-like condition of the extracellular matrix (ECM) in regenerating tips of Hydra upon YAPTEAD disruption. We then characterized the role of ECM structure and tissue stiffness in regulating regeneration in Hydra. Towards this, using Atomic Force Microscopy (AFM) to measure the Young’s modulus of Hydra body column, we show that tissue stiffness is tightly regulated during head regeneration. We also demonstrate that the fibrotic condition upon YAP-TEAD disruption causes a drastic increase in tissue stiffness. Using a combination of biochemical inhibition as well as ultrastructural studies using SEM, we show that tissue stiffness is a function of fibrillar collagen deposition and 5 cross-linking frequency in the ECM (referred to as mesoglea). Observations from this study argue in favor of the role of a hypothetical tissue stiffness threshold in the regulation of Hydra head regeneration. In support of this argument, perturbations leading to lowering the tissue stiffness during regeneration inhibit regeneration in Hydra while perturbation leading to an increase in tissue stiffness above the threshold lead to an enhanced regenerative response in Hydra. Next, we focussed on understanding the alterations in gene expression patterns during regeneration and the role of YAP-TEAD interaction towards the same. Transcriptomic analysis of regenerating Hydra under inhibition of YAP-TEAD interaction revealed upregulation of pro-fibrotic genes and early activation of crucial developmental signalling pathways. More importantly, we find that β-catenin transcriptional targets such as Brachyury (bra) are upregulated earlier than the wnt(s). This study convincingly demonstrates that YAP is an important player in the regulation of regeneration in Hydra, having the capability of modulating fibrosis induced stiffness and integrating the resulting mechanical changes to the cross-talk with β-catenin to activate its potential as a head organizer for initiating the downstream regenerative programme needed for oral fate determination. Taken together, this study not only provides a therapeutic window to enhance the regenerative response in higher model systems without genetic modifications but also predicts an interesting prospect of β- catenin. Rather than the currently implicated WNT being the head organizer molecule, our study suggests that β -catenin acts as the head organizing factor which can be activated by a simultaneous WNT-independent mechanical stimulus and WNTdependent signalling.