By Peter Weiss
Chicken eggs are such a common sight in refrigerators and supermarkets that they hardly seem like objects of scientific curiosity. Yet spend a few minutes talking to LBL cell biologist Rick Schwarz about the eggs warming in his laboratory incubators, and you will discover what a rich potential for scientific discovery--and for improvements in human medicine--lies within their shells.
Each embryo in those eggs makes a remarkable, developmental journey, transforming from a single cell to a hatchling that can walk, peck, and see in just three weeks. In that extraordinary transformation lie unsolved puzzles of how early embryos change from balls of identical cells into the different cell types of the body. As the embryo grows, the mysteries deepen. Biologists wonder, for instance, how newly differentiated cells are directed to build tissues and organs of just the right form and size.
A strong clue to what controls that early tissue building-- at least for one class of cells--has emerged from experiments Schwarz performs on tendon cells from his chicken embryos. The clue is a substance secreted by the cells, which Schwarz has isolated and named Cell Density Signal 1, or CDS-1. His discovery promises to shed light on a field where scientists are largely groping in the dark. "We don't understand development very well in any system," he says. As for tendon development, he adds, "we can't even answer the questions of how a tendon knows that it must connect a specific muscle to a given bone, or that it should grow to just a certain size."
Besides offering a handle on what governs tendon growth, Schwarz's discovery may also become the basis for a new drug to promote healing of human tendons and ligaments. A biotechnology company has signed a million-dollar, three-year Cooperative Research and Development Agreement (CRADA) with LBL to study CDS-1 and possibly develop it as a drug. To date, no other drug specifically helps heal injuries to those tissues. "It's a fact of life that tendons and ligaments don't heal very well," Schwarz says. "We're trying now to give it a scientific explanation."
While CDS-1 is being explored as a potential drug, Schwarz will continue to pursue basic research on the compound--and the cellular responses to it--with the goal of answering certain fundamental questions of biological development, such as whether parts of the process may be reversible. Schwarz also hopes to better understand how the external environment of cells--in particular, the distance to nearest neighbors, or what is known as cell density--affects internal functions such as protein production.
"This is a pretty fundamental process in cell and developmental biology--that a cell has to know what's around it for both growth and differentiation. The hope is that by figuring this one out, we will also have a much better idea of how other connective tissue cells might work," Schwarz says. Ligaments and bone are other examples of connective tissue. Tendons and ligaments, in particular, are very similar, but with different functions: tendons connect bones to muscles, ligaments connect bones to other bones.
Schwarz performs his experiments on tendon cells taken from 16-day-old fertilized eggs--about five days before a chick hatches. He grows them in a layer in a flask containing a warm bath of cell nutrients. To create varying cell densities in the flask, he initially confines the growing cells within a ring placed at the center of the flask bottom. When he removes the ring, the edges of the circle spread only a short distance, making an island in the middle of the flask. "In this island of cells, we have all different densities," he says.
Schwarz has found that for tendon cells, how close their neighbors are wields a strong influence on how much of a certain protein they will produce. His experiments show that the zone in which cells sense their neighbors extends approximately one millimeter from the cell. "The cell can know within a one millimeter radius how many cells are there. But if its neighbors are more than a millimeter away, the cell couldn't care less," he says. A circle with a one millimeter radius can fit more than 3000 embryonic tendon cells without overlap.
The protein whose production is affected by cell density is procollagen. Procollagen, which is secreted from embryonic tendon cells, is the precursor to another protein, called collagen--the major material from which connective tissues are made. In adult tendons and ligaments, the same type of collagen accounts for approximately 90 percent of their tissue. When newly formed procollagen leaves a cell, enzymes also secreted by the cells snip it into collagen. The collagen molecules then twist up into rope-like strands. In bones, the strands are then hardened by other processes.
In embryonic tendons, cells are abundant and procollagen production is high. An adult tendon, however, contains relatively few cells scattered here and there in a sea of tough, stretchy collagen fibers. When cells are so sparse, procollagen production drops nearly to zero. Schwarz says that the rate at which tendon cells divide also seems to depend on cell density, but in a more complex way. At both low and high cell densities, tendon cells won't grow. But at moderate densities, they grow rapidly.
Cell Density Signal-1, as its name suggests, helps cells recognize how crowded their neighborhood is. It appears to be part of a larger, more complex, chemical message that cells both send and receive, Schwarz says. As they grow, embryonic tendon cells lay down a kind of organic mortar, called the extracellular matrix. By secreting CDS-1 and certain other molecules in that mortar, the cells inform each other about how tightly they are packed.
Schwarz has found that shaking flasks of cells causes the procollagen production of some cells at the high-density centers of the islands to drop. From this and other experiments, he hypothesizes that CDS-1 becomes attached to other molecules in the extracellular matrix. In that "bound" state, CDS-1 broadcasts a message saying "boost procollagen; stop growth." But, when shaking breaks loose some CDS-1 molecules from the matrix, nearby cells get an opposite message: "now grow, but shut the procollagen machinery down." Schwarz has found in further experiments that even if cells are at low or high densities--at which they ordinarily will not grow, "if you add unbound CDS-1 to them, they think they are at moderate density and grow. It fools them."
For nearly 20 years, Schwarz has studied tendon cells in culture, trying to determine what environmental conditions they need to flourish. When he came to LBL as a postdoc in 1976, he worked with Mina Bissell, now LBL's Life Sciences Division director, who was studying those questions in chicken tendon cells and mouse mammary cells. Besides cell density, Schwarz found other external factors that affect procollagen production and tendon cell growth, such as the amount of Vitamin C in the nutrient bath. But cell density appears to be the major external control: "I think it's the real key," he says.
If he's right, fathoming cell density effects may give biologists the ability to explain growth rates of tendon tissue. "If you knew the number of cells and how much procollagen they were producing, you could make a pretty good estimate of how fast the tendon would grow. It's like building a brick house: if you knew the number of bricklayers and how many bricks each could lay per hour, you would have a good idea of how fast the house would be built." Understanding growth rates in this one type of tissue might, in turn, help developmental biologists achieve a more general understanding of what makes organs and tissues grow rapidly early in life and then shut down when proper sizes are reached.
When adult tendon cells shut down, they no longer grow or produce procollagen. Even if part of the tendon tears, an adult's over-the-hill cells won't lay down new collagen to repair the damage. Instead, fiber-secreting cells, called fibroblasts, come from outside the tissue to do the job. When they reach the injury, they secrete collagen threads that knit together the damaged area, but often not very well. Instead of an orderly, rope-like arrangement of collagen strands, "you get a patchwork pattern as if you mended something," Schwarz says.
Hence, injured tendons heal notoriously slowly, and often end up being weaker than they were originally. Yet, if adult tendon and ligament cells could be stimulated to make procollagen again, tendon and ligament repair might dramatically improve. Therein lies the potential role of CDS-1 as a drug: since, when bound to the extracellular matrix, it can signal embryonic cells to increase procollagen output, it might also revive production in mature cells. On the other hand, just why adult tendon cells become quiescent remains a puzzle. It could be that they don't make CDS-1. It could also be that, although they still make CDS- 1, they no longer respond to it. Schwarz thinks not. "CDS-1 is the critical regulator of cell growth and procollagen production in the embryo. In the adult, both functions are way down. That leads me to the assumption that CDS-1 is not present."
By revitalizing adult tendon cells, CDS-1 might not only open a new avenue for treating connective-tissue injuries, it might also create a milestone in developmental biology, Schwarz says. "I'm trying to get adult cells to be more embryonic." If he succeeds, it will be the first time a scientist has, by design, reversed cell development, he says.
Such a reversal could have important implications for another medical arena: cancer. For instance, CDS-1 could potentially be used to cure connective-tissue cancers, Schwarz says. Such cancers are rare. Osteosarcomas, or bone cancers, which are slightly more common than those of tendons or ligaments, most often strike teenagers. Amputation is frequently necessary. If instead, bound CDS-1's simultaneous growth-inhibiting and procollagen-stimulating effects were marshalled against a connective-tissue tumor, the tumor's willy- nilly growth might be transformed into rapid procollagen production. Then healthy, new connective tissue would replace the cancer.
But rejuvenating adult tendon cells would also raise even broader anti-cancer possibilities. If undesirable development proves reversible in tendon cells, the same principle might work in other tissues as well. "There are classic examples of cells in the wrong environment giving rise to tumors and then, placed in the right environment, becoming normal," Schwarz says. "That means that cells having the wrong [external] signals can cause them to grow abnormally. If we can get cells to respond to the signals they're supposed to respond to, there's a potential of their going from cancer cells to normal cells."
Cell density, in particular, Schwarz notes, is a "signal" that cancer cells are well known to miss. Whereas normal cells stop growing when they reach a certain density, cancer cells often don't. Acting as if they don't recognize that other cells are nearby, tumor cells in culture typically pile on top of each other to form vile-looking clumps.
Before CDS-1 can fulfill any of this potential as a therapy, it has many hurdles to leap--the compound has not yet even been fully identified. In the meantime, teasing developmental secrets out of chicken tendon cells is its own formidable challenge. "The combination of trying to figure out how CDS-1 works and its implications in other tissues is well beyond my five-year plan," says Schwarz. Indeed, the scientific challenge within an ordinary, simple-looking chicken egg seems enough to last several lifetimes. The rewards to biology and medicine may prove to be equally far-reaching.