A synthetic biology approach to investigate the role of ultradian oscillations in myoblast differentiation

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Simona Ventre, Chiara Fracassi, Luca Cardone, Diego di Bernardo

Telethon institute of genetics and medicine (TIGEM), Italy

Cyclic expression of genes (i.e. oscillations) is essential for multicellular life and it is involved in basic processes such as the cell-cycle and the circadian clock. Ultradian oscillations, i.e. with periods much shorter than 24 hours, have been observed also in the major signalling pathways, but their relevance is still unclear. Specifically in myoblasts, ultradian oscillations of the Notch-effector gene Hes1 have been observed but their role is unknown. Differentiated myoblasts, however, do not exhibit any ultradian oscillation. The Notch family of cell surface receptors is involved in contact-mediated cell-to-cell signaling (Artavanis-Tsakonas et al.,1995; Weinmaster,1997). The Notch pathway regulates downstream responses, such as cell-fate specification, progenitor cell maintenance, boundary formation, cell proliferation and apoptosis. Notch signaling also mediates the synchronization of cyclic gene expression of effector genes among cells, an essential process during the somite segmentation of vertebrate embryos. The aim of the project is to understand the physiological significance of Hes1 cyclic gene expression in myoblasts. We will use a synthetic biology approach to investigate the role of Notch-Delta signaling in myotube differentiation, to verify the role of cyclic gene expression in cell proliferation, cell differentiation and in specification of compartment and boundary cells. Whether these oscillators regulate the timing of differentiation system as a clock also remains to be determined. We chose as a model the mouse C2C12 myoblast cells. We first evaluated existing gene expression profiles (Dilusha A. William, Kenro Kusum, 2007) to estimate the endogenous expression level of Notch family and its ligands. We then confirmed the results by quantitative RT-PCR. Our preliminary results show that the Notch pathway is indeed active in C2C12 cells. We will now investigate Hes1 oscillations in single cells during differentiation, and the effect of blocking Hes1 oscillations by constitutively activating or inhibiting Notch signaling.