Balance is often difficult to achieve. The painstakingly long process of evolution has produced a miraculous level of balance both in organisms and in ecosystems. But as mankind has been made aware, with disease and ecological disasters, this state of equilibrium rests on precarious ground. Biologists have long struggled to understand the basis of cellular, tissue and organ construction and function. During development, for example, cells of the early embryo divide at a controlled pace, move with determined precision and differentiate in specified manners to produce tissues, organs and organ systems that work in harmony. From the gross level of an organ to the micro level of the cell, size, shape and function are fairly uniform between individuals in a species. Such uniformity speaks to the exquisite biologic regulation that exists at the genetic level. The genes and the products that are made from them, in specific time, space and quantity, dictate how frequently a cell divides, how the cell will differentiate and what type of structural and biochemical repertoire the cell will possess. For every gene product that serves to stimulate cell division and maturation there is a counteracting gene product to ensure that things do not speed out of control. It is the balanced regulation of growth that biologists seek to understand no matter what their topic of interest may be.
Pulmonary neonatologist, Dr. David Warburton and his group of collaborators, post-doctoral associates, graduate students and technicians take special interest in studying the lung as a model to understand how developmental processes are balanced. As members of CHLA/USC's Human Developmental Biology Program, they have a special appreciation for the genetic regulation of lung development because many of them have seen, first hand, what happens to premature infants whose lungs have not developed properly. As physician researchers and basic scientists, they rationalize that an understanding of the normal processes of development will give them a handle on how these processes can go astray. The narratives that follow are devoted to the work of three such scientists from the Human Developmental Biology Program, Dr. Jing-Song Zhao, Dr. Matthew Lee, and graduate student, Denise Tefft.
Sorting out tangled paths- Dr. Jing-Song Zhao
The goal of development is to control growth and maturation of biologic structures by regulating how genes are expressed. It makes sense then that numerous genetic pathways would exist to accomplish such a complicated task. For years the challenge to scientists has been to tease out individual genetic paths and see how each contributed to gene expression. In a simplified scenario, a protein growth factor binds the surface of a cell and sparks the propagation of a signal to the nucleus. Depending on the nature of this signal, gene expression can be turned on or off. The simple scenario paints a clear image. But of late, scientists have faced the reality that numerous genetic paths can contribute to whether or not a gene is activated. Researchers now have a redefined mandate. Rather than just untangle genetic paths into a series of individual routes they must find the points where these paths intersect.
Dr. Jing-Song Zhao, Assistant Research Professor and active member of David Warburton's Developmental Biology Program, focuses his research on deciphering how one such transcription pathway affects the development of the lung. This pathway is activated by a family of peptide growth factors called Transforming Growth Factor ß (TGF-ß). TGF-ß signaling controls diverse cellular activities such as cell division, differentiation and apoptosis. In some developing tissues TGF-ß increases cell proliferation and in others retards it. In some cells, TGF- ß stimulates cell maturation but in still others prevent it. In the early lung, TGF-ß helps control what lung biologists call "branching morphogenesis" of the early respiratory structures.
The lungs start out as a bud of tissue that out-pockets from the primitive pharynx. This single bud splits into two new buds. Each of these ends divides again and again until a final structure with a densely organized system of branches is produced. The terminal divisions of the lung, the alveolar sacs, provide a large surface area for the efficient exchange of gases to and from the blood. When Zhao first started his post-doctoral work with Warburton, the group was generating the first evidence that TGF-ß stimulation seemed to throw the brakes on this process. Excess TGF-ß activation demonstrably reduced how often the embryonic lungs would branch.
Two serious respiratory conditions afflicting premature infants, respiratory distress syndrome (RDS) and lung fibrosis, consistently feature heightened levels of TGF-ß. Much of Zhao and Warburton's research efforts have originated from these early observations and emphasize the idea that abnormal TGF-ß signaling somehow plays a role in abnormal lung development.
As signaling with growth factors is initiated at the cell surface and propagated through the cytoplasm to the nucleus, there are various points along the TGF-ß signaling route that could alter lung branching morphogenesis. The group started first with the TGF-ß ligand itself, then moved to its receptor and now focuses on the next step in the pathway of gene control, the molecules involved in transferring the TGF- ß signal to the cell's nucleus. This systematic approach to defining the roles of individual molecules in the TGF- ß pathway was made with the use of an elegant, yet simple experimental approach called the embryonic lung organ explant system.
Zhao removes embryonic lungs from fetal mice and puts the tiny organs into a specialized culture dish that contains only basic nutritional elements. One at a time, molecules, be they soluble proteins or transcription blocking DNA fragments, can be added to or removed from the embryonic lungs. Adenoviral vectors can even be microinjected directly into the lumen of the embryonic lung trachea. Each treatment can then be assessed for its contribution to the continued branching morphogenesis and maturation of the lung. Zhao and Warburton utilize the explant system because it is well defined. "The ex vivo system can be used without any maternal or systemic factors complicating things," explains Zhao. The lung explant system has allowed Zhao and others to demonstrate that interference with any one of the TGF-ß signaling players, be it the peptide ligand, the receptor or a cytosolic signaling protein, usually leads to abnormal lung development of the lung explants.
The point is not however, to prevent lung formation but to allow it to proceed in a controlled manner so that a modeled, functional organ is produced. The negative regulation of lung development is thus counteracted and balanced by positive regulators. Over the past few years, lung biologists, including Zhao, have shed light on some of the forward mediators of lung development. Zhao has demonstrated that even the growth factor path generally associated with slowing of lung morphogenesis, TGF-ß, can be tipped toward positive regulation. This counterbalancing effect on lung development is made possible by a series of cytoplasmic signaling proteins called Smads. The Smads work inside the cytosol to transfer the signal from the TGF- ß receptor to the nucleus where gene transcription can be activated or repressed. The different Smad isoforms compete with one another in the cytoplasm to effect such change. When the Smad3 isoform predominates then the TGF- ß signaling circuit is complete and results in decreased branching. If Smad3's competitor, Smad7, is abundant, it binds to the TGF-ß receptor complex and reduces the TGF- ß signal that gets to the nucleus. In this scenario, total branch number increases.
Smad7, it turns out, may be a spoiler to the negative effects of TGF-ß. Its capacity to dampen TGF-ß's effects, explains Zhao, are made possible by the powerful antagonistic properties of another growth factor system, the tumor necrosis factor a (TNF-a) pathway. Currently, Zhao is examining the evolving relationship between TGF-ß and TNF-a. In isolation, TNF-a acts as a positive regulator of lung branching morphogenesis. The transmission of the TNF-a signal to the nucleus occurs through an intermediate protein called NF-kB. NF-kB, in turn, binds Smad7 and stimulates its production. Zhao and his small group have recently demonstrated that the addition of TNF-a to lung cultures results in a stimulation of Smad7 gene expression and enhanced branching. "The enhanced branching could be a direct effect of the TNF-a pathway or it could be an effect of suppression of the TGF-ß pathway," said Zhao. He speculates that the heightened production of Smad7 serves to out compete Smads, such as Smad3, that propagate the TGF- ß signal. "Smad7 appears to be a very important molecule that can interplay between these antagonistic pathways," said Zhao.
Balancing Shc- Dr. Matthew Lee
Finding balance means different things to different people. To neonatologist, Matthew Lee, the search for balance comes in his scientific as well as personal endeavors. Lee who received his medical education at the University of Chicago and continued with specialty training at Stanford and the University of Washington, came full-time to the University of Southern California/CHLA in the early 90s. He arrived there as a neonatologist who wanted to spend time in the laboratory to understand the science behind the problem of lung immaturity. At the time, the field of molecular lung embryology was in its own infancy. There were, it seemed many questions and few answers. In the ten years that followed significant numbers of genes were identified as being important to lung development. Many questions about basic embryology and persistent clinical conditions linger to this day. Lee explains a particularly troublesome clinical condition, "The primary problem for premature babies is the development of chronic lung disease." Premature infants, he describes, are at a high risk for developing pulmonary fibroproliferation, a thick, stiff, scar tissue in the lungs that compromises blood flow and breathing. Increased prematurity only heightens an infant's risk for developing this lung fibrosis. The lungs of an infant who has died from this disorder possess a mesenchymal compartment, the space between the alveolar sacs and the vasculature, that has proliferated uncontrollably. "What we want to understand is why these mesenchymal cells grow like crazy in pre-term babies but are much less likely to do so in full-term babies," says Lee.
When he first started as a research fellow in Warburton's lab, Lee focused his efforts on how the growth factors, Epidermal Growth Factor (EGF) and Transforming Growth Factor b (TGF-b), exerted effects over lung proliferation and maturation. As an independent investigator, Lee trained his sights one step closer to the nucleus, on the intermediate proteins that transfer the signal to the nucleus and help stimulate gene activity. "Being rather simplistic, we started looking at the signaling pathways that control cell proliferation and what components in these pathways were different in pre-term as opposed to full-term lungs," explained Lee. One signal transduction protein, Shc66, sparked particular interest. Lee's first finding on the Shc protein was that its expression, while abundant in fetal lungs, was nearly absent in lungs that had reached full development. This observation has held true in experimental animals such as rodents and primates and appears to be the case in developing human lungs as well. Shc protein localizes primarily to the mesenchymal cells that are the problematic cell population in lung fibrosis. "That's the fascinating thing about this protein. It's found in the cells that grow uncontrollably and cause the problems. Shc is also a putative component of a major growth pathway that could be telling these cells to grow like crazy," said Lee.
The Shc protein could impact the development of pulmonary fibrosis by affecting cellular mechanisms other than just cell division. As a fetus develops, its lungs undergo a type of remodeling. Early on, the mesenchymal population is in the majority and the epithelial population in the minority. As the fetus approaches term, the mesenchymal cells decrease in number or become blood vessels. Blood vessels and epithelial cells that mature into alveoli predominate in the mature lung. As Lee explains, "It very well could be that it's not the proliferation but the maturation or apoptosis (death) of these cells that is regulated by Shc". An Italian group that removed the function of Shc66 from the mouse genome indicated that Shc66 plays at least a partial role in promoting programmed cell death in the mesenchymal cells of the fetal lung. When Shc66 was removed the mice lived longer and their lungs were resistant to the effects of the apoptosis-inducing lung toxin, periquot. "I hesitate to use their paper as a gold standard because some of their conclusions were preliminary but there are at least some people on the planet that believe the Shc66 is modulating apoptosis," explained Lee.
Mediating cellular activities as diverse as cell division, maturation and death may appear to be a complicated endeavor. Generally, it is. Shc66 and two highly related Shc isoforms can be activated by more than one signal transduction pathway making these activities entirely within the realm of possibility. As Lee describes, "Shc proteins were originally described as being in the pathway of the TGF-b activated, tyrosine-kinase receptor cascade. Subsequently, they were also found to be activated by integrin receptors, cytosolic tyrosine kinases and even by calcium modulated kinases. It seems the Shc proteins act as signal integrators in numerous, responsive signaling pathways."
Shc66's role is further balanced by the existence of the two Shc isoforms, Shc42 and Shc56, each of which is proposed to perform different functions. "If one Shc isoform is highly expressed early on and it's expression crashes when the baby is born, the expression of that particular isoform may be modulating contributions to different signaling pathways. The relative expression of each isoform can govern the relative contribution to these separate signaling pathways.
Lee calls himself fortunate to be both an active clinician and basic scientist. "If I had been working exclusively in the clinic I think I would have burned out a long time ago. Doing science means that I do not have to do neonatology full time. Conversely, doing neonatology takes some of the pressure off of doing science," says Lee. It is a personal balance Lee has managed to strike that thus far, is very much to his liking.
Denise Tefft on Branching, Breathless and Sprouty
The lowly fruit fly, known in scientific circles as Drosophila malanogaster, has reliably provided useful genetic crossover information to scientists who study mammalian systems. Humorously coined genes like "hedgehog" and "cheap date" may speak to the quirky nature of Drosophila scientists but the names also describe the basic physical characteristics that result when a gene has been mutated. Interference with the hedgehog gene results in stunted Drosophila larvae that resemble the compressed body of a wobbly little hedgehog. Compromised function of "cheap date" hinders alcohol metabolism and produces precocious drunkenness in adult flies. The clever naming strategy also applies to a gene called sprouty, which in the Drosophila controls bifurcation of the insect trachea.
Working in a group that focuses its efforts on the growth and branching of the mammalian lung, graduate student Denise Tefft took a natural interest in the Drosophila sprouty gene. "I was working with Jing-Song Zhao on a TGF-b receptor that was involved in lung development, but I was also trying to find something of my own for a Ph.D. project," recalled Tefft. When Tefft first read the papers on Drosophila sprouty and its counteracting gene branchless, she had a feeling she had found a project she could call her own. But to work with sprouty in the mouse mammalian system employed by her mentor David Warburton, she needed to come up with the homologous genetic sequence for mouse. At first, people were mildly reluctant to support her efforts. "Everybody told me not to work on it because it would be too hard to clone the sequence from fly to mouse. But they did have some of the human homologue already sequenced so I decided to try it anyway," she said.
Tefft was able to clone the mouse sequence for sprouty-2 and from there quickly went to determine the gene's role in the mouse lung. Sprouty, she found, was localized to the tips of the epithelial, knob-like lung buds at the points where the embryonic structures would branch. To study the function of sprouty in the murine lung Tefft turned first to the lung organ culture system frequently utilized by Warburton's group. By removing newly formed lung from mouse embryos and growing them in culture, in the presence or absence of various factors, scientists can assess the general function a gene has on early aspects of lung development. Tefft interfered with the ability of embryonic lung buds to use sprouty by flooding the culture system with short, complementary pieces of the sprouty DNA sequence. These sequences, called anti-sense oligonucleotides (AS-ODN), effectively destroy the sprouty message needed to make sprouty protein. When Tefft removed sprouty function from the lung culture system with the sprouty AS-ODNs, the embryonic lungs branched robustly, much more so than the normal lungs, and contained increased amounts of surfactant protein-C associated with lung maturity.
In effect, Tefft demonstrated that sprouty-2 exerts a pressure on murine lungs not to branch. The counterbalance to the "do not split" signal in Drosophila is the branchless gene. Its normal function is to promote branching. "FGF-10 is thought to be the mouse homologue to the fly's branchless and since Drosophila sprouty is downstream in the pathway, I'm now looking at the interaction between FGF-10 (fibroblast growth factor-10) and sprouty-2 in embryonic lung development," explained Tefft. To this end, Tefft designed an adenoviral vector to overproduce murine sprouty and microinjected the vector into her embryonic mouse lungs. As she anticipated, the excess sprouty served to diminish branching. It also substantially reduced the presence of FGF-10 in the lung tissues. These and other experiments point to the fact that sprouty and FGF-10 seem to keep one another in check, increasing one serves to decrease the other. Sprouty, as Tefft believes, may help limit the diffusion of FGF-10 signaling and thus stop new buds from occurring at the points where it is expressed.
In the lungs, FGF-10 is produced in the mesenchyme overlying the branching epithelial buds that express sprouty-2, Tefft explains. The FGF peptide works by binding cell surface receptors on adjoining cells, and activates gene expression via signaling pathways such as MAP kinase. Activation of the MAP kinase pathway, in turn, is associated with cell proliferation and migration.
In Drosophila, the sprouty homologue intracellularly binds proteins found in the MAP kinase pathway. Tefft wants to know if murine sprouty binds similar proteins in the mammalian MAP kinase path. "If sprouty does bind," asks Tefft, "does this decrease MAP kinase activation?" FGF signaling is necessary to promote cell proliferation, migration and, in the big picture of lung development, branching. Perhaps, Tefft suggests, FGF stimulates the cells in the lung buds to continue growing. In places where sprouty expression is high, the FGF signal is diminished, and the cells do not continue their expansion.
Tefft and Warburton's sprouty collaborator in France has made a transgenic mouse that overexpresses sprouty. The phenotype of the transgenic mouse mirrors that elaborated by Tefft with the lung organ culture system. "I haven't seen the figures yet but there's a significant decrease in epithelial proliferation, which is no surprise. There's no lobular formation, or branch formation. I understand that the phenotype is quite striking," said Tefft.
At present, Tefft is expanding her studies beyond the confines of the lung explant system because as she says, "You can show that adding FGF-10 expands the lungs and changes sprouty levels and vice versa when you add sprouty. But that's it. You really don't know what happens after that." During her last years as a graduate student with Warburton, Tefft intends to use basic cell culture techniques to explore exactly how sprouty earned its name.
A selection of publications
Lee MK, Zhao J, Smith SM, Tefft JD, Bringas P, Hwang C, Warburton D. The Shc 66 and 46 kD isoforms are differentially downregulated at parturition in the fetal mouse lung. Pediatr Res. 1998 Dec; 44(6): 850-9.
Tefft JD, Lee M, Smith S, Leinwand M, Zhao J, Bringas P Jr, Crowe DL, Warburton D. Conserved function of mSpry-2, a murine homolog of Drosophila sprouty, which negatively modulates respiratory organogenesis. Curr Biol. 1999 Feb 25; 9(4): 219-22.
Warburton D, Lee MK. Current concepts on lung development. Curr Opin Pediatr. 1999 Jun; 11(3): 188-92.
Warburton D, Zhao J, Berberich MA, Bernfield M. Molecular embryology of the lung: then, now, and in the future. Am J Physiol. 1999 May; 276(5 Pt 1):L697-704. Review.
Zhao J, Shi W, Chen H, Warburton D. Smad7 and Smad6 Differentially Modulate TGFb-Induced Inhibition of Embryonic Lung Morphogenesis. J Biol Chem. 2000 Aug 4; 275(31): 23992-7.
Zhao J, Crowe DL, Castillo C, Wuenschell C, Chai Y, Warburton D. Smad7 is a TGF-ß-inducible attenuator of Smad2/3-mediated inhibition of embryonic lung morphogenesis. Mech Dev. 2000 May; 93(1-2): 71-81.
Zhao J, Sime PJ, Bringas P Jr, Tefft JD, Buckley S, Bu D, Gauldie J, Warburton D. Spatial-specific TGF-beta1 adenoviral expression determines morphogenetic phenotypes in embryonic mouse lung. Eur J Cell Biol. 1999 Oct; 78(10): 715-25.
Zhao J, Sime PJ, Bringas P Jr, Gauldie J, Warburton D. Adenovirus-mediated decorin gene transfer prevents TGF-beta-induced inhibition of lung morphogenesis. Am J Physiol. 1999 Aug; 277(2 Pt 1): L412-22.
Zhao J, Tefft JD, Lee M, Smith S, Warburton D. Abrogation of betaglycan attenuates TGF-beta-mediated inhibition of embryonic murine lung branching morphogenesis in culture. Mech Dev. 1998 Jul; 75(1-2): 67-79.
Zhao J, Sime PJ, Bringas P Jr, Gauldie J, Warburton D. Epithelium-specific adenoviral transfer of a dominant-negative mutant TGF-beta type II receptor stimulates embryonic lung branching morphogenesis in culture and potentiates EGF and PDGF-AA. Mech Dev. 1998 Mar; 72(1-2): 89-100.
Zhao J, Lee M, Smith S, Warburton D. Abrogation of Smad3 and Smad2 or of Smad4 gene expression positively regulates murine embryonic lung branching morphogenesis in culture. Dev Biol. 1998 Feb 15; 194(2): 182-95.
Zhao J, Warburton D. Matrix Gla protein gene expression is induced by transforming growth factor-beta in embryonic lung culture. Am J Physiol. 1997 Jul; 273(1 Pt 1): L282-7.
Zhao J, Bu D, Lee M, Slavkin HC, Hall FL, Warburton D. Abrogation of transforming growth factor-beta type II receptor stimulates embryonic mouse lung branching morphogenesis in culture. Dev Biol. 1996 Nov 25; 180(1): 242-57.
