Laser micro-scalpel yields biological insights into tissue dynamics

Using a laser beam scalpel so fine it could inscribe words on the surface of a fly egg, researchers have snipped their way to a new understanding of a key process in a fruit fly’s embryonic development. The process, called dorsal closure, is the complex mechanism by which the embryonic skin of the fruit fly Drosophila knits itself together to protect its innards from the outside world.

Understanding this seemingly arcane process is important because dorsal closure uses molecular and cellular mechanisms very similar to those involved in wound-healing as well as those that can go awry in humans to produce the spinal malformation spina bifida.

The researchers’ achievements were reported in an online article in the February 6, 2003, Sciencexpress — and will appear in the April 4, 2003, print version of Science — by an interdisciplinary Duke research team that includes biologists, physicists and a mathematician. It was this broad collaboration, said the scientists, that enabled them to refine the laser scalpel, to perform the microsurgery to dissect the fly tissue and to model the forces involved in key developmental machinery.

“Dorsal closure is a good system for studying these processes because it’s tractable,” said lead author Shane Hutson. “We only have to deal with a few different kinds of cells that are arranged in a planar fashion.”

According to Hutson — a postdoctoral fellow in Duke’s Free Electron Laser Laboratory (FELL) — dorsal closure involves the interplay of forces among three kinds of tissues in the fly embryo, which is smaller than a grain of rice. The amnioserosa cells form an inner sheet of tissue involved in knitting the closure; the lateral epidermis is the tissue layer that ultimately forms the fly’s outer covering; and in between these two tissues is a group of “leading edge” cells that form a purse-string structure that somehow tightens to contribute to closure.

“As closure proceeds, the cells of the amnioserosa contract, the purse-string along the boundary contracts, the lateral epidermis cells are stretched, and the two sheets of lateral epidermis along those purse-strings are zipped together into a seam. And so those four processes contribute to how dorsal closure proceeds,” explained Hutson.

The mystery, said Hutson, was precisely how these different tissues, and the forces they exert, work together to effect dorsal closure.

“There are lots of ways you could build a model such that closure would occur,” said Hutson. “It could be the amnioserosa doing all the work. It could be the purse-strings doing all the work. It could be zipping. It could be the lateral epidermis actually growing and pushing itself over the amnioserosa.

“And so, we wanted to systematically investigate the forces in the system to figure out which of these processes were really contributing to closure, and which were simply following along.”

To attack the problem, the team needed to be able to selectively dissect the force-producing tissues, and to simultaneously observe the result through a high-powered microscope. Thus, they designed an optical and steering system for the laser beam scalpel that was implemented and refined by graduate students Yoichiro Tokutake and Ming-Shien Chang at the FELL. The resulting system can produce and guide a laser beam as small as a half-micron in diameter — roughly a hundredth the diameter of a human hair. Tokutake and Chang went on to become the group’s master laser surgeons.

Said FELL Director Glenn Edwards, one of the paper’s senior authors, “These four forces are working in concert, so in essence we are trying to understand the ‘symphony’ of dorsal closure — how these forces are coordinated in space and time.” The dissection of the symphony produced surprises, said Hutson. “For example, we found that the system was very resilient,” he said. “When we perturbed only one or another of the tissues, the process kept right on going.

“We were surprised by this finding because we thought we’d find that at least one of these processes was absolutely essential,” he said. “But it does make sense in the end that you’d want a system where, if something’s not quite right in one process, you can compensate and still complete dorsal closure.”

Key to the rigorous understanding of this intricate system was the physical reasoning inherent in creating a quantitative model describing the process — the result of a team effort led by Edwards, Hutson, Duke biologist Dan Kiehart and Duke mathematician Stephanos Venakides.

As laser surgery produced experimental results, the research team incorporated those results into a model describing the interplay of forces. The next steps, said the scientists, will be to use their approach to further expand their explorations.

Said Dan Kiehart, the other senior author, “One of the next goals of this collaboration will be to use the same kind of modeling to study wound-healing. We may begin with Drosophila, but then progress to studying vertebrate cell cultures, fish or mice, where genetic studies may be of more direct interest to physicians.”

However, emphasized Kiehart — an expert on the molecular machinery of such contractile processes as dorsal closure — Drosophila remains an attractive research model because the flies can be genetically manipulated so easily.

Other major questions include how the multiple forces involved in dorsal closure are synchronized, and how the system initially launches the process.

Said Venakides, “One way this system might work is like the unwinding of a clock, with the closure proceeding on its own initial energy. Another analogy might be the driving of a car, where a steady force is being guided.”

More broadly, said Kiehart, such studies “are going to provide a model not only for dorsal closure and wound-healing, but for studying any such developmental process that involve tissue migration and closure.” Essential for these scientific advances, said Edwards, is the value of such interdisciplinary collaboration.

“The FEL Lab is an interdisciplinary think tank, and in this environment my group, Dan’s group and Stephanos came together to work on this problem at the interface of traditional disciplines. And we’ve made a lot of progress towards cracking it,” he said