The cnidarian freshwater polyp Hydra sp. exhibits an unparalleled regeneration capacity in the animal kingdom. Using an integrative transcriptomic and stable isotope labeling by amino acids in cell culture proteomic/phosphoproteomic approach, we studied stem cell-based regeneration in Hydra polyps. As major contributors to head regeneration, we identified diverse signaling pathways adopted for the regeneration response as well as enriched novel genes. Our global analysis reveals two distinct molecular cascades: an early injury response and a subsequent, signaling driven patterning of the regenerating tissue. A key factor of the initial injury response is a general stabilization of proteins and a net upregulation of transcripts, which is followed by a subsequent activation cascade of signaling molecules including Wnts and transforming growth factor (TGF) beta-related factors. We observed moderate overlap between the factors contributing to proteomic and transcriptomic responses suggesting a decoupled regulation between the transcriptional and translational levels. Our data also indicate that interstitial stem cells and their derivatives (e.g., neurons) have no major role in Hydra head regeneration. Remarkably, we found an enrichment of evolutionarily more recent genes in the early regeneration response, whereas conserved genes are more enriched in the late phase. In addition, genes specific to the early injury response were enriched in transposon insertions. Genetic dynamicity and taxon-specific factors might therefore play a hitherto underestimated role in Hydra regeneration.

Although mounting in vitro studies suggest a critical impact of structural and mechanical properties of the surrounding microenvironment on stem cell maintenance and differentiation, little is known about how extracellular matrix (ECM) remodeling co-ordinates stem cell behavior in vivo. In this study we used the native ECM (mesoglea) of the freshwater polyp Hydra and determined the mesoscopic ECM structure by grazing-incidence small-angle X-ray scattering using a nano-focused beam. We found that the packing of Hydra collagen fibrils is comparable to that in vertebrates. It is also highly anisotropic with respect to the main (oral-aboral) body axis explaining the very dynamic contraction and extension behavior of a Hydra polyp. We monitored the spatio-temporal evolution of ECM mechanics ex vivo during Hydra maturation by mapping the elastic modulus of Hydra mesoglea along the main body axis before and after the budding with aid of nano-indentation. The extracted characteristic elasticity patterns implied that freshly detached polyps bear a mesoglea of uniform elasticity. By time, the mesoglea becomes softer in the upper gastric region but stiffer in the middle and lower region, where new buds emerge. The complementary proteome analysis demonstrated that the observed changes in mechanical patterns are correlated with the activity of proteases. Our data are consistent with a model according to which regions of high ECM stiffness facilitate stem cell activity in a given tissue.

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