Morpho-functional characterization of the microenvironment in muscle cell development of Hirudo verbana

Muscle regeneration is a process of great interest to the scientific community because is involved in a big number of disorders that are still without a cure. To study the mechanisms behind this process, the most used animal models have always been rodents, for their closeness to humans. Nonetheless, the rising ethical awareness has encouraged a reduction in the use of vertebrates in the research, but at the same time the complex puzzle in which the different factors interact for muscle regeneration has not been clarified, so the contribution of the in vivo experiments is still fundamental. The leech is an inexpensive and easily manageable animal model and represents an invaluable and necessary alternative for these studies. In fact, in a simple body organization it is able to elicit complex processes (wound-healing, angiogenesis, immune response, and muscle regeneration) characterized by the same phases described for vertebrates. For all these reasons, taking also in consideration that regeneration recapitulates some of the processes employed during development and that no satellite cells were identified in leech, we aimed to study the structure and composition of the microenvironment where myogenic precursors mature during the body wall growth of the juvenile leech. In this regard, we performed morphological analyses of the adult and of the juvenile of Hirudo verbana, focusing on the extracellular matrix (ECM) organization and on the cell’s level of differentiation. Moreover, we also reconstructed one of the main pathways by which mechanical stimuli are integrated and transduced into transcriptional activity: the Hippo signaling pathway. Our data suggest that ECM has a pivotal role in controlling myocytes’ proliferation, migration and differentiation thanks to the control exerted by Yap1. Moreover, many cellular types, which concur in this development as stem-like cells of different lineages, were identified in these processes. These preliminary data confirmed that juvenile leech can be a good model for understanding how muscle fibres can growth and differentiate in relation to the 21 stiffness of ECM and to its conformations, to other mechanical, and chemical stimuli and to the interactions with different cell types.