Volume 18 Number 2 Fall 2001


Totally tubular
by Susan Dieterle

Discovering how simple tubes transform to become our lungs

Consider the basic building blocks of most organisms – molecules, genes, cells. And tubes. Though tubes don’t spring to mind as readily as the others, they are one of the most fundamental structures found in internal organs. The lungs, kidneys, vascular system, liver, pancreas and mammary glands are all made of branched tubular structures that transport gases and liquids.

But relatively little is known about how these biological tubes form. For instance, how do cells make tubes, and what regulates the size and shape of the tubes? And how do they branch into complex patterns that ensure an adequate flow of gases and liquids to various parts of the body?

The answers to these questions have profound implications for understanding both their normal formation during development and what happens when development goes awry, resulting in disease or the growth of a tumor in these structures. And with precise knowledge of how such diseases and tumors form, scientists could design molecular cures to pinpoint and repair the trouble spots.

But first they need a solid understanding of the tube formation process. Enter Mark Krasnow, MD, PhD, a Stanford professor of biochemistry and an investigator for the Howard Hughes Medical Institute.

For the past 10 years, Krasnow and a tenacious team of researchers have studied the tracheal system of the fruit fly, properly known as Drosophila. Their efforts have resulted in a cellular-level understanding of how the simple tubular structure forms. The team plans to spend the next few years refining that knowledge while also using it to guide similar research into the formation of a vastly more complex system — the mouse lung.

To do that, the team is using the latest technologies and “old-school” science — patiently researching literature, following leads that sometimes turn into blind alleys and developing simple descriptions of the processes.

“What we’re doing is rather unusual. There are people who are studying mouse lung development and there are people who are studying Drosophila respiratory system development, but as far as I know we’re the only ones trying to do both,” Krasnow says.

And already the unique approach has provided some surprising results. The team has discovered that the genetic process controlling the sprouting of branches in Drosophila’s simple tracheal system is remarkably similar to that of the highly complex mammalian lung.

“The findings are surprising because, although the respiratory system in Drosophila and the lung have similar physiological functions, no evolutionary biologist had thought these structures would be under similar genetic control,” Krasnow says. “It raises the possibility that the tracheal system in Drosophila and the lungs in mammals might not only be functionally similar but they might have some evolutionary relationship as well.”

Krasnow succumbed to the siren song of Drosophila more than 15 years ago. He had come to Stanford in the 1980s as a postdoctoral fellow working in the lab of David Hogness, the emeritus Rudy J. and Daphne Donohue Munzer Professor in the School of Medicine, at a time when Hogness and others were pioneering techniques that enabled them to understand how genes controlled the developmental processes of the fruit fly.

“The biggest successes during that time came with understanding some of the very first events during development — the events that happen right after fertilization that establish the major body axes and that make the head different from the tail and the back different from the front,” Krasnow says. “The results were so powerful in providing an initial molecular and genetic understanding of those developmental processes that it encouraged people like me to say, ‘If this can be done with the early developmental events, why not look at some of the later developmental events, like the ones that occur during organ formation?’”

Using techniques that had shed light on the early developmental processes, Krasnow and others made great leaps in understanding how Drosophila’s organs form. Like most researchers, Krasnow chose to concentrate on a single organ. He selected the tracheal system — the intricate, branched structure of threadlike tubes that carry oxygen to the cells of the fruit fly’s body.

“Branched tubular structures are one of the most fundamental structures of organ design,” Krasnow says. Drosophila’s tracheal system made an excellent model for studying the formation of branched structures because of its simplicity — each tube is made of a single layer of cells without any surrounding support structure. “You can’t make a simpler cellular tube,” Krasnow notes. And Drosophila’s tracheal system consists of a mere 10,000 branches, compared with the millions of branches in the mammalian lung.

Mark Krasnow

“There are 80 cells that make up the couple hundred branches that form in each segment, so you can get down to a cellular level of understanding this problem. In fact, we’ve named every cell in the larval tracheal system,” he says.

Krasnow’s Drosophila team includes postdoctoral fellows Amin Ghabrial and Mark Metzstein, graduate students Farhad Imam and Stephanie Toering, and until recently, former postdoc Eric Johnson, who in September moved to the University of Oregon as an assistant professor of biology. The researchers use genetic, cellular and molecular methods to identify and characterize the genes involved in the development process.

Johnson, who works primarily on understanding the branch patterning of the tracheal system, says he was initially drawn to the work for aesthetic reasons. “I thought the Drosophila tracheal system was very beautiful,” he says. “And then I figured that there must be some really interesting biology behind it.”

The team discovered that the cells in the branch segments form a sac from which small groups of cells begin to migrate in different directions as they form the primary branches of the tracheal system. The primary branches sprout secondary branches and then the fine terminal branches.

The team has documented how the branches sprout and what the cells are doing during the branching events. “This gives us a cellular-resolution view of these complex organ formations,” Krasnow says. Their work has involved identifying, cloning and characterizing each gene to determine how it contributes to the overall process of sprouting branches and forming tubes.

Krasnow’s team named the genes — bestowing such monikers as “branchless,” “sprouty,” “pruned,” “stumps” and “trimmed” — and worked to characterize the molecules they encode. As the scientists studied the branchless gene, they discovered it produced a fibroblast growth factor similar to one produced in mammals. Fibroblast growth factors, or FGFs, are powerful substances implicated in the development of branched structures, such as blood vessel networks and lungs.

“We began to understand how the Drosophila branchless FGF controlled branching,” Krasnow recalls. “It was very exciting because David Sutherland [a graduate student at that time] found that the gene turns on just before branching begins, but it’s not expressed in the tracheal cells themselves. Rather, it’s expressed in clusters of cells that surround the tracheal system at every position where a new branch will soon sprout. The tracheal cells sprout new branches by growing out toward these FGF signals that are basically a chemo-attractant — a come-hither signal — that tells the branches where they should grow.”

Further research indicated that the influence of the branchless FGF extends beyond the formation of the primary tracheal branches. As the primary branches approach the FGF signal centers, genes involved in the secondary branching event are activated. “And then we found more recently that the gene turns back on again a couple of hours later, now in a completely different pattern, and controls the sprouting of the very fine terminal branches,” Krasnow says. “That was a surprise because we didn’t expect to find a signal that would control so many important aspects of the complex branching structure.”

The first two stages of branching — the primary and secondary branching — are controlled genetically so that the process always turns on in the same places. “It’s highly reproducible, highly stereotyped; it’s part of the hard-wired developmental program,” Krasnow says. But a key discovery by Johnson and postdoctoral fellow Jill Jarecki showed that when the terminal branches start to form at the end of embryogenesis, the process switches to physiological control, meaning that the oxygen needs of individual cells in the body dictate where the terminal branches form.

“This makes a lot of sense because the initial hard-wired program gets the main branches out near the target tissues,” Krasnow says. “But to get the very fine branches out to the cells in the tissues, the process is controlled by oxygen. Any cell that starts to become starved for oxygen — either because it has used up its available supply or because it happens to be in a region that hasn’t received any tracheal branches — somehow knows that it’s in an oxygen crisis and responds by turning up expression of this FGF gene.

“So you have this intricate feedback system, which ensures that the branches, although they look very random in their distribution, are actually very precisely guided to the individual cells in each tissue that need oxygen the most.”

It didn’t take long for mammalian parallels to crop up. Within the last two years, other researchers exploring mammalian lung development reported that sprouting of new bronchial branches is substantially controlled by an FGF and an FGF receptor expressed in the cells that line the pulmonary passages. “In addition, we discovered a gene called sprouty in Drosophila that was an antagonist — an inhibitor — of branching,” Krasnow says. “It was a new gene, but we soon found mammalian homologs for sprouty in mice and also in humans.”

By the end of the 1990s, Krasnow knew his research team had made considerable advances in understanding the formation process of the tracheal system in Drosophila. “We’ve made enough progress that at least the questions we have to answer are now clear. That means that the project is maturing,” he says.

But as the researchers sought to compare mammalian lung development with the findings about the Drosophila tracheal system, they realized that much regarding mammalian lung development was murky. For one thing, most of the initial descriptions of lung development were at least 40 years old and were written before the availability of the powerful microscopes and reagents commonly used in today’s laboratories.

And yet, few scientists seemed interested in revisiting those descriptions. “In today’s science, if you were to write a grant to the NIH and say you want to describe the gross branching pattern of the mouse lung, the reviewers would say, ‘Wasn’t that done a half-century ago? I think you should find another source of funds,’” Krasnow says.

He decided to assemble a second team in his lab to launch a parallel investigation into the development of the mouse lung. Current team members include MD/PhD student Ross Metzger and postdoctoral fellow Hernan Espinoza. For the past few years, Metzger has been mapping out the sprouting process in the mouse lung and looking at how the blood vessels, smooth muscle cells and cartilage develop in concert with the bronchial tree. Metzger characterizes his work as “old-school science with new techniques.”

Mouse lungs: Day 11. Embryonic mouse lungs. Primary bronchi have formed. Day 12: Primary brnochi elongate and additional bronchi bud. Day 13: The newly formed bronchi are smaller than those formedearlier. Day 14: The lungs' five lobes take shape - one in the left lung and four in the right. Day 15: At this stage, the lungs measure about 1/4 inch across.

“What I’ve done is not glamorous,” Metzger says with a laugh. But he notes that he was able to pioneer a method that allows him to stain and microscopically examine the entire mouse lung. Previously, researchers were only able to stain and examine slices of the developing lung. The beauty of Metzger’s technique is that it allows researchers to see the formation of the airways in the developing lung in three dimensions.

“The first time I saw this gorgeous, elaborate network of mesh, it was unbelievable,” Metzger says.

Additionally, Espinoza has been examining the molecules and their expression patterns during these developmental branching events. Espinoza has also been leading the effort in the first large-scale gene expression screen in the lung, which will provide an overall look at the major gene expression patterns during mouse lung development. “Because the sequences of all those genes are known, when we find genes that are expressed in an intriguing pattern we can often make pretty reasonable predictions as to how they may be functioning in lung development,” Krasnow says. “The genomics is going to lead the genetics in mouse lung development, rather than the way it worked in Drosophila where the genetics has led the genomics.”

Though it has been slow going during the past few years for the lab’s mouse lung research, Krasnow believes it’s laying an essential foundation. “We’ve taken an approach that others in the field of lung development hadn’t seen as critical,” he says. “This type of foundation is really necessary to get to the kind of genetic and molecular detail achieved in the Drosophila tracheal system. I think our approach is beginning to be appreciated now that we’ve started talking about it at meetings. It’s opening up a new understanding, and I think it heralds a renaissance in understanding lung development.

“I hope that these careful groundwork studies for an organ like the lung will encourage other laboratories around the world to spend a similar amount of time in setting up the framework for understanding the development of other important organs.”

The information from the gene expression screen will give Krasnow’s team both genetic and developmental overviews of the mouse lung development process. If the research follows the pattern established by the Drosophila work, the overviews will then guide the team in relating specific genes to discrete developmental events and eventually to identifying all of the genes involved in lung development. “It’s fun to do it in stages because you get a lot of intellectual gratification from providing the initial outline,” Krasnow says. “Then in the second phase there’s more intellectual gratification in seeing the genetic program come into view.”

And having the Drosophila research serve as a conceptual guide should help speed up the progress in the mouse lung work in the years ahead. “We have to figure out ways to make the genetic and molecular approaches move faster than they traditionally have for the mouse lung research, but that’s one of the challenges we think can be partially overcome by the genome projects that are going on,” Krasnow says. “When we started with Drosophila, just finding individual genes was a big deal. But now with the mouse and with humans, you’re starting with literally the whole catalog of genes. We just have to figure out the ones that are relevant for lung development.”

Though the challenges of the mouse lung project are great, Krasnow points out that the goal of the research is the same as for the Drosophila project: To understand the complete developmental program of a key organ. And once researchers have obtained a genetic understanding of how lungs form in mice – and, eventually, in humans – they can begin to understand how the normal developmental program can go wrong, resulting in lung cancer and other diseases. “It will suggest ways of treating those diseases,” Krasnow says.

“Researchers will begin to make reasonable predictions as to how to fix them in a way that’s never before been possible. Ultimately, it might even be possible to trigger the lung development program in an adult so that if, for instance, a portion of a lung is resected because of a tumor, we could reactivate the development program in the residual lung to generate a new lobe and restore functionality.”

Although that scenario may be decades away, Krasnow has seen enough advances during his career to believe that it’s within mankind’s grasp.

“A few years ago, I wouldn’t have thought it was possible to do a project like mouse lung development or to even think about the possibility of regenerating a lung. It was too complicated, and finding the genes and understanding the process seemed like it was so far in the future that it wasn’t even worth spending time on,” he says.

“But I’ve been so encouraged by how much progress the lab has made in the 10 years of studying the Drosophila tracheal system that, with all the new technologies, I think we’re really in reach of knowing the complete developmental program for the Drosophila tracheal system.”

Perhaps advances in understanding mammalian organs are also close at hand. Says Krasnow: “We’re also at least in the position to be thinking that it’s possible — in our lifetime — to gain a similarly high level of understanding of lung development, blood vessel development and the development of other organs.”


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Copyright © 2001, Stanford University School of Medicine. All rights reserved.