Single-Cell Imaging Clarifies How Cells Tell Time

A new imaging approach offers the first real-time visual evidence of how cells know when to enter the next phase of their development.

As they develop, embryos form vertebrae in a sequential, time-controlled way. Scientists have determined previously that this process of body segmentation is controlled by a kind of molecular “clock,” but just how this timing system works has been unclear.

An international cross-disciplinary collaboration between physicists and molecular genetics researchers from Ohio State and McGill universities sheds light on the clock mechanism by fusing a yellow fluorescent protein to a protein that defines the cell’s cyclical behavior. The findings advance scientists’ understanding of the Notch signaling pathway, one of a handful of messaging systems cells use to communicate with their neighbors.

Previous scientific studies have examined the oscillation phenomenon in the tissue of mouse embryos. In the new study, however, the researchers observed and analyzed it in single cells. To do so, the researchers genetically modified zebrafish — a freshwater fish whose body is nearly transparent during early development, making its anatomy easy to observe — by introducing a fluorescent marker to monitor the concentration of a certain cyclic protein whose production rises and falls with the oscillating expression of the molecular clock genes.

In their experiment with the zebrafish, the scientists disabled the Notch signal to observe the effect on the oscillation pattern in individual cells and their neighbors. They combined the imaging tool with three mutant cell types with disabled Notch signals.

In zebrafish embryos, under normal circumstances, cells oscillate in synchrony with their neighbors as they prepare to make segments that later become muscle and vertebrae. When a color map (top left corner) is used to indicate the phase of oscillation in each cell at any fixed snapshot of time, with cool colors representing the peak of the gene activation wave and warm colors the lower levels of activation, it is evident in the top image that neighboring cells are in a similar phase, or transitioning smoothly to the next phase. However, in embryos lacking a powerful messaging system called Notch signaling, that synchrony is lost. In the bottom map, cells in mutant embryos that lack the Notch signal are oscillating, but the random assortment of colors without smooth transitions shows that Notch is required to synchronize the oscillations in neighboring cells. Courtesy of Ohio State University.

What the investigators discovered is that cells in all three mutants could oscillate individually but not in a synchronized fashion. With or without the signal, cells can activate their genes in oscillating patterns. However, to maintain synchronization with nearby cells and to form segments that will become tissue, the cells must receive the Notch signal.

“For the first time, this nails it,” said Ohio State molecular genetics professor Sharon Amacher, who conducted the research at the University of California, Berkeley, before joining the Ohio State faculty in July. “This provides the data that cells with disabled Notch signaling can oscillate just fine, but what they can’t do is synchronize with their neighbors.”

Defects in Notch signaling are associated with human congenital developmental disorders characterized by malformed ribs and vertebrae; this work could offer insight into potential therapies to prevent these diseases.

“In humans, defects in Notch signaling are associated with congenital developmental disorders called spondylocostal dysostosis, that are typified by scoliosis and trunk dwarfism caused by malformed ribs and vertebrae," Amacher said. "Studies such as ours may provide insight into potential therapies for human disease. It is likely that many cells in our bodies — stem cells, cancer cells — have similar molecular oscillators that regulate response to environmental signals. By unraveling such molecular clocks, we can understand how to modify them and thus change the number of oscillating cells that respond to differentiating signals, providing tremendous insight for studies in stem cell and cancer biology, and tissue engineering."

The team also explored whether cell division interrupted the synchrony needed for creating segments. Mitosis, occurring among 10 to 15 percent of embryonic cells at any one time, is considered a source of biological “noise” because when cells divide, they stop activating genes. If division happened randomly, as previously thought, instead of in a pattern, those divisions would disrupt the clock synchrony, creating problems that segmenting organisms would have to overcome.

During experiments, it was found that mitosis is not a random event. Instead, the team discovered that division occurs when neighboring cells are at a low point of gene activation for signal reception. The two daughter cells created from a fresh division are more tightly synchronized with each other than are any other cell neighbors in the area, the team said.

For embryos lacking Notch signaling, these daughter cells appear as a pair of tightly synchronous cells in a largely asynchronous area, showing that oscillation can resume without the signaling pathway. Without Notch, the cells gradually drift out of synchrony, becoming like their asynchronous neighbors.

These findings could be incorporated into models of developmental cell behavior to further advance cell biology research, Amacher said.

“Most of our tissues and organs are not made up of the same types of cells. They have different jobs. So you don’t want them to respond identically to every signal; you want them to have different responses,” she said. “We need to understand systems like this that help cells not only interpret the signals in their environment, but do the right thing when they get that signal.”

The study appeared in Developmental Cell (doi: 10.1016/j.devcel.2012.09.009)

For more information, visit: www.osu.edu