Implications of tissue stiffness in growth control during limb regeneration in salamanders (Ambystoma mexicanum)

Project Information

ProxDistReg

Grant agreement ID: 101022810

DOI

10.3030/101022810

Project closed

EC signature date 16 April 2021

Start date 1 May 2021

End date 30 April 2023

Funded under

• EXCELLENT SCIENCE - Marie Skłodowska-Curie Actions

Total cost

€ 174 806,40

EU contribution

€ 174 806,40

Coordinated by

TECHNISCHE UNIVERSITAET DRESDEN
Germany

Periodic Reporting for period 1 - ProxDistReg (Implications of tissue stiffness in growth control during limb regeneration in salamanders (Ambystoma mexicanum))

Reporting period: 2021-05-01 to 2023-04-30

Summary of the context and overall objectives of the project

Salamanders are able to regenerate full limbs following amputation and the regenerating limb grows until it catches up with the development of the intact contralateral limb. This is a remarkable example of growth control since the regenerated limb must grow to a size that is larger than it was at the time of amputation in order to accommodate to the animal’s growing body. Even more strikingly, when a salamander is given a complete amputation on one limb and a digit amputation on the other, regeneration of both structures is completed in approximately the same period. Thus, distally amputated limbs grow slower than proximally amputated ones, resulting in an overall length of regeneration that is independent of the tissue volume to be reformed. Although this phenomenon was first observed centuries ago, the underlying mechanisms are still unknown. Furthermore, whether such differences in growth are already encoded in undamaged tissues, or if the differences only arise during regeneration, is undetermined as well.
Differential adhesion strength and extracellular matrix (ECM) were reported along the proximodistal (PD) axis during axolotl limb regeneration. Therefore, considering that cell–cell interactions and cell-ECM interactions play key roles in force transmission to and between cells, controlling signalling pathways that regulate stem cell self-renewal and differentiation, we hypothesize that tissue mechanical properties are regulated in gradient along the PD axis and are thus majorly responsible for the differential growth rates observed during regeneration between proximally and distally-amputated limbs.
Therefore, the central aim of this project is understanding how biomechanical properties of tissues affect regeneration, which may have important implications for the design of biomaterials to be used in regenerative medicine.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

Different techniques were combined to address our hypothesis studying limb regeneration in the axolotl (Ambystoma mexicanum). Main results are summarised below:
Limb growth: tissue growth during regeneration was evaluated in differently sized animals and data was used to generate a mathematical model describing growth dynamics and differences. From such model we were able to demonstrate that there is a linear correlation between the amputation site and maximum growth rate, as well as the mean cell cycle length during early regeneration phases. We also demonstrated that animal’s development is not affected by limb amputations.
Cell proliferation: we demonstrated that cell cycle is regulated differentially along the P/D axis during regeneration. Such differences were already detected during the early blastema stage.
Cell differentiation: we show that limb patterning occurs faster after distal amputations, as opposed to proliferation, suggesting a differential regulation of cell proliferation/differentiation decisions along the PD axis.
Tissue mechanics: we optimized 2 different techniques for the measurement of mechanical properties in the regenerating axolotl limbs: Brillouin confocal and atomic force microscopy. Our results indicate that distally amputated limbs present stiffer tissues during regeneration than proximally amputated ones.
Mechanical dependence: we proved that cultured blastema cells derived from proximal and distal blastemas don’t present any differences in proliferation, revealing the importance of the in vivo context for the regulation of growth. Furthermore, we developed a technique to culture axolotl cells in three-dimensional gels of differing degrees of stiffness, and proved a mechanical dependence in which cells decrease their proliferation when cultured in stiffer extracellular contexts.
In summary, our results go in line with the hypothesis proposed in the application, i.e. faster growth rate correlates with softer tissue during early phases of regeneration, whereas slower growth rate with stiffer tissues. We are currently characterizing the molecular mechanism behind our observations.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

Mechanical forces have emerged as a key player in the regulation of size during development and regeneration. A better understanding of these forces and their underlying mechanisms could lead to new therapeutic strategies for promoting tissue regeneration and repair in the clinic.
Thus far, the state-of-the-art was lacking studies associating growth regulation with mechanical properties during regeneration. Therefore, our results will importantly contribute to the field of regenerative biology.

When a salamander limb is amputated at different levels along the proximo-distal axis, regeneration