Mechanical control of tissue growth during limb regeneration

The axolotl is a highly regenerative species, capable of restoring full limbs, regardless of the amputation site. However, the regeneration rate is adjusted with the plane of amputation along the proximo-distal (PD) axis, leading to equivalent regeneration times regardless of the extent of tissue removal. We hypothesized that this phenomenon could be partly explained by differences in tissue mechanical properties. In this work, we describe tissue growth mathematically and evaluate cell cycle parameters of regenerating limbs amputated at different levels along the PD axis, demonstrating a linear correlation between the cell cycle length and the amputation site during early regeneration phases. We show as well, that blastema cells require their endogenous context to retain such proliferation differences. We measured mechanical properties in regenerating limbs with in vivo optical and standard indentation-based techniques and demonstrated that distal blastema cells are stiffer than proximal ones. Accordingly, we demonstrated that axolotl cells decrease their proliferation with increased extracellular matrix stiffness in vitro. Next, we evaluated the activity of the mechanotransducers YAP/TAZ in vivo by using a GTIIC-based reporter line combined with target gene expression data, which indicated that their activity peaks during the blastema stage, with higher activity after proximal amputations. Hence, our findings strongly suggest a mechanical dependence for the position-dependent regulation of cell proliferation during axolotl limb regeneration, where YAP/TAZ likely plays a role in the mechanotransduction mechanism.