Two years ago, a team of researchers led by Carmen Birchmeier, PhD, head of the developmental biology/signal transduction lab at the Berlin-based Max Delbrück Center for Molecular Medicine in the Helmholtz Association (MDC), showed that the development of stem cells into muscle cells is regulated with the help of two proteins, Hes1 and MyoD. These proteins are produced in progenitor cells in an oscillatory manner. Both proteins are involved in the Notch signaling pathway, a widespread mechanism by which cells respond to external stimuli and communicate with other cells. The signaling pathway is named after its receptor “Notch,” onto which the ligand “Delta,” a cell surface protein, latches.

Now, working with researchers from Japan and France, Birchmeier and four other scientists at the MDC say they also uncovered the role of a third protein that, along with Hes1 and MyoD, forms a dynamic network within the cells. As the team reports “Oscillations of Delta-like1 regulate the balance between differentiation and maintenance of muscle stem cells” in Nature Communications, this protein is the Notch ligand Delta-like1, or Dll1 for short.

“It is produced in activated muscle stem cells in a periodically fluctuating manner, with the oscillation period lasting two to three hours,” Birchmeier explains. “Whenever a portion of the stem cells expresses more Dll1, the amount in the other cells is correspondingly lower. This rhythmic signaling determines whether a stem cell becomes a new stem cell or develops into a muscle cell.”

She points out that in the current study, the researchers provided “unequivocal evidence” that oscillation in muscle tissue is not just some strange phenomenon of the cells involved. The rhythmic fluctuations in gene expression are actually crucial for transforming stem cells into muscle cells in a balanced and controlled manner, according to Birchmeier.

“Cell-cell interactions mediated by Notch are critical for the maintenance of skeletal muscle stem cells. However, dynamics, cellular source and identity of functional Notch ligands during expansion of the stem cell pool in muscle growth and regeneration remain poorly characterized. Here we demonstrate that oscillating Delta-like 1 (Dll1) produced by myogenic cells is an indispensable Notch ligand for self-renewal of muscle stem cells in mice,” write the investigators.

“Dll1 expression is controlled by the Notch target Hes1 and the muscle regulatory factor MyoD. Consistent with our mathematical model, our experimental analyses show that Hes1 acts as the oscillatory pacemaker, whereas MyoD regulates robust Dll1 expression. Interfering with Dll1 oscillations without changing its overall expression level impairs self-renewal, resulting in premature differentiation of muscle stem cells during muscle growth and regeneration.

“We conclude that the oscillatory Dll1 input into Notch signaling ensures the equilibrium between self-renewal and differentiation in myogenic cell communities.”

Team carried out further investigations

In their experiments with isolated stem cells, individual muscle fibers and mice, Birchmeier and her team further investigated how the Hes1 and MyoD proteins are involved in muscle growth. “Put simply, Hes1 acts as the oscillatory pacemaker, while MyoD increases Dll1 expression,” says Ines Lahmann, PhD, a scientist in Birchmeier’s lab and a lead author of the study along with Yao Zhang from the same team. “These findings were demonstrated not only in our experimental analyses, but also in the mathematical models created by Professor Jana Wolf and Dr. Katharina Baum at the MDC,” Birchmeier says.

Using gene-modified mice, the researchers obtained evidence that Dll1 oscillation plays a critical role in regulating the transformation of stem cells into muscle cells. “In these animals, a specific mutation in the Dll1 gene causes production of the protein to occur with a time delay of a few minutes,” Birchmeier explains. “This disrupts the oscillatory production of Dll1 in cell communities, but does not alter the overall amount of the ligand.”

“Nevertheless, the mutation has severe consequences on the stem cells, propelling them to prematurely differentiate into muscle cells and fibers,” reports Zhang, who performed a large portion of the experiments. As a result, he says, the stem cells were depleted quickly, which resulted, among other things, in an injured muscle in the mice’s hind legs regenerating poorly and remaining smaller than it had been before the injury.

“Quite obviously, this minimal genetic change manages to disrupt the successful communication, in the form of oscillation, between stem cells,” Zhang adds.

“Only when Dll1 binds to the Notch receptor in an oscillatory manner and thus periodically initiates the signaling cascade in the stem cells is there a good equilibrium between self-renewal and differentiation in the cells,” Birchmeier says.

The MDC researcher hopes that a better understanding of muscle regeneration and growth may one day help create more effective treatments for muscle injuries and diseases.