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Mechanical Forces Speed Up Growth of the Lung

Eighteenth century biologists were fond of pointing to the eye to illustrate how exquisitely organs are shaped to suit their purpose. The lungs would have made a fine substitute. Though drab in outward appearance, these twin shields house a labyrinth of branching ducts and sacs that appear to be perfectly designed to carry out the lungs’ main function of distributing oxygen to the body. What has not been so obvious is how the myriad branches and buds take shape in the developing embryo.

Donald Ingber and colleagues accelerated the development of embryonic mouse lungs by altering cytoskeletal tension. “I had the theory out there for years, and this is basic experimental confirmation,” said Ingber. (Photo by Graham Ramsay)

Twenty years ago, Donald Ingber worked out a theory about the way the lungs and other organs might arise. Their development was too complex to leave to the turning on and off of individual genes, he argued. Instead, he predicted that mechanical forces generated within cells of developing organs—and exerted by those cells on their surrounding matrix—play a key role in shaping tissues. But the theory would remain just that, in large part because of the difficulty in manipulating those mechanical forces in developing organs in a way that could be measured and studied. Now, Ingber, Kimberly Moore, and colleagues report in the February Developmental Dynamics that they have found a way to experimentally control these forces. Using this approach, they were able to speed up and slow down budding and branching in embryonic mouse lungs—a discovery that could lead to new approaches toward treating a host of human lung diseases, most notably those affecting newborns.

“Almost all prematurely born babies who have problems do so because they are born before their lungs develop,” said Ingber, the Judah Folkman professor of vascular biology in the Department of Pathology at HMS and Children’s Hospital Boston. “Currently they are treated with surfactants. Our study suggests that one might be able to accelerate lung development by giving drugs that could modulate these forces and that could be delivered by aerosol, as is currently done with asthma patients.”

Run in the Stocking
To understand how exactly Ingber and his colleagues ramped up lung development requires a closer examination of his original theory. In 1985, he argued that for a layer of epithelial tissue to bud, the surrounding matrix, or basement membrane, must undergo a distributed but confined thinning, much like a run in a stocking. Individual epithelial cells attached to the unraveling mesh would be pulled and stretched, which would cause their internal skeleton to become tauter. The increase in cytoskeletal tension would make the cells more responsive to growth factors and therefore more likely to proliferate, pile up, and protrude in the form of buds. Though Ingber was able to show this happening in cells grown in culture dishes, what eluded him was a way to demonstrate it in whole growing organs.

In the developing lung, bud formation begins when the basement membrane—the matrix that surrounds the epithelial cells—thins and pulls apart as shown above. The cells spread and proliferate, clustering into a bud that pokes through the membrane’s mesh. This process repeats itself to form the characteristic branching of the lungs. (Image courtesy of Donald Ingber)

In the late 1990s, researchers discovered that cytoskeleton tension was regulated by the molecule Rho through its downstream effector ROCK. Inspired by that finding, Moore, until recently an HMS clinical fellow in surgery, exposed whole embryonic mouse lungs to a Rho inhibitor, as well as to other tension-inhibiting agents, and allowed the lung rudiments to grow in culture. In each case, budding was inhibited in a reversible manner. Next, she exposed lung rudiments to a bacterial toxin that activates Rho. The opposite happened: lung budding accelerated.

The researchers believed that by altering cytoskeletal tension, they would influence basement membrane thinning. In fact, in his original theory, Ingber had specified that for thinning to occur, the matrix must be pulled taut by adjacent cells that were themselves tensed. “If you do not have tension on the stocking, there is no run,” he said. They were delighted to find that the basement membrane exhibited local thinning in the Rho-stimulated lung rudiments, but not in the Rho-inhibited tissues.

The Amazing Matrix
That the extracellular matrix controls tissue development would have been considered unlikely until quite recently. Though it is composed of vital macromolecules such as glycoproteins and collagen, biologists have tended to view the extracellular matrix merely as the glue that holds cells together. The basement membrane, which is a type of extracellular matrix, was thought to be a barrier to growth rather than a dynamic force in development. Thanks to the work of Ingber and others over the past decade, the extracellular matrix is coming out from under nearly a century’s weight of misconception. On Feb. 17, Ingber discussed his recent findings in a featured panel on “Extracellular matrix in tissue engineering” at this year’s annual meeting of the American Association for the Advancement of Science.

“What force does is spatially constrict growth. This is what creates pattern in developing tissues.”

Even so, Ingber gives the impression that he might have felt more at home with his 18th- and 19th-century predecessors than with many of his contemporaries. As a student in the late 1970s, he immersed himself in scientific writings of the 1700s and 1800s, but it was the early 20th-century biologists such as Joseph Needham and D’Arcy Wentworth Thompson who most influenced him. “The people that I resonated with were turn-of-the-century biologists,” he said. “They explained everything in terms of mechanical forces because they did not have molecules.”

It would take a few late 20th-century discoveries to refine his theory on tissue development. The first was the recognition in the 1970s that all cells have a cytoskeleton that generates tensile force. (See Focus, Sept. 3, 1999, for more on Ingber’s views on the cell as a tensile structure.) Another was the 1978 discovery by Judah Folkman, the Julia Dyckman Andrus professor of pediatric surgery at Children’s, that cells proliferate when they spread, but not when they crowd. (Ingber, working with Folkman, would later show that spreading enhances their sensitivity to growth factors.)

Ingber had a third revelation the following year when he attended an overcrowded talk by the late Merton Bernfield. Bernfield, who was the Clement A. Smith professor of pediatrics and professor of anatomy and cellular biology at HMS, told the packed room about the dynamic role played by the degradation of the extracellular matrix in the formation of buds and branches by epithelial cells, something few other scientists had appreciated. “He said the reason you get buds when you degrade matrix is because cells compress and push out through the thinning basement membrane,” Ingber said. “I raised my hand and said maybe it is a change in tension. Rather than having a weak spot that you explode through, it is possible that the basement membrane is always under tension and now it stretches open like a run in a stocking and pulls on the cells.”

Ingber’s latest finding, in particular the discovery that the basement membrane thins only when nearby cells are exerting tension, appears to confirm his decades-old theory. It also raises a question: what triggers the basement membrane to thin at particular times, in particular locations, and in this manner form buds and branches? Does tensing of the cytoskeleton of nearby epithelial cells come first? Or does the basement membrane degrade as a result of messages received from elsewhere, perhaps from the mesenchymal tissue of the embryo? It could be both. “It is possible that if you degrade and do not pull, it won’t thin. Or it may be that feedback from cells that are pulled changes matrix remodeling,” said Ingber. “So what force does is spatially constrict growth. This is what creates pattern in developing tissues.”

—Misia Landau

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