Genetic Control of Airway Development
The lungs and vascular system are tubular networks that transport oxygen and blood throughout the body. The walls of the tubes are sheets of cells (an epithelium) or in some cases individual cells rolled up into a tube. Each organ contains thousands or millions of tubes, and the proper size and pattern of tubes and connections between them are crucial for efficient flow through the networks. Our goal is to identify the genes and molecular pathways that control embryonic development of such complex structures. We want to answer three basic questions: (1) What specifies the complex pattern of branching -- where each branch sprouts, the direction it grows, and when it sprouts again to form the next generation of branches &endash; and how is this patterning information encoded in the genome? (2) How does an epithelium migrate and assemble into tubes of the appropriate size and shape? (3) How does oxygen influence the process? Our work focuses on the Drosophila melanogaster respiratory system and the mouse lung because the excellent genetics and cell biology in these model organisms provide powerful tools to address these questions in molecular detail. Ultimately we hope to understand how the developmental program goes awry in lung cancer and other lung diseases, and how we can trigger the program in the adult to restore diseased tissue.
Genetic dissection of airway development in Drosophila
During development of most branched organs, a small unbranched epithelial tube or sac initially forms and then new branches succesively sprout from it to form a tree-like structure of interconnected tubes. Despite the similar overall appearance of the branches, our analysis of Drosophila airway development showed that each successive generation of branches uses a different set of genes and a different cellular mechanism of tube formation.
The Drosophila airways (the tracheal system) arise from twenty unbranched sacs of ~80 cells. The six main (primary) branches begin to form when one or two cells at six positions in each sac migrate out in specific directions. A small number of cells follow the lead cells, organizing into tubes as they migrate. Several hours later, secondary branches sprout from the tips of the primary branches. These are formed by individual tracheal cells that roll up into unicellular tubes. Subsequently, secondary branches ramify into dozens of terminal branches, which are long cytoplasmic extensions that form very fine tubules that directly contact the tissues. In this way, each of the 20 sacs generates a small network of tubes. During the process, specific branches in each sac find and fuse to branches in the neighboring sacs to form an interconnected network of over 5000 tubes that transport oxygen throughout the body.
Our initial screens for mutants with defective airways identified over 50 genes that control various aspects of airway development. Different sets of genes are required for each of the three levels of branching. The three sets are organized into a regulatory hierarchy, with genes required for one level of branching also required to trigger expression of genes that control the next level of branching. Thus, although each level of branching is genetically distinct, their development is coupled by a gene regulatory hierarchy that insures that branching occurs in a defined sequence with bigger branches forming before the next generation of smaller branches. A fourth set of genes controls the fusions of tracheal tubes. Yet another set of genes is necessary for proper lumen formation and to maintain tube size and shape.
We are conducting new screens for tracheal mutants, and we are using DNA microarrays to monitor expression of all genes in the developing respiratory system. Our goal is to discover all the critical genes and to elucidate the full genetic program for respiratory development in Drosophila, and use this information to recreate a tracheal system in vitro.
An FGF pathway patterns airway branching
To understand how the identified genes control airway development, we are characterizing their protein products. This has begun to reveal at a molecular level how the pattern of tracheal branches is specified. branchless encodes a homolog of a major family of vertebrate signaling molecules called fibroblast growth factors (FGFs). The gene is expressed in a remarkably complex and dynamic pattern -- near the position where each new branch is sprouting. The secreted branchless FGF functions as a chemoattractant that guides the migrating tracheal cells to their correct destinations. It does so by activating a transmembrane receptor tyrosine kinase called Breathless expressed on the developing tracheal cells.
Some of the other gene products we have recently characterized regulate production of the FGF signal. Others function in the receiving cells to transduce the signal from the receptor to the cellular machinery responsible for cell migration and tube formation, but how they do so is a mystery we hope to solve. Another gene we discovered called sprouty is a negative regulator of the pathway. It restricts the range of the FGF signal, so in its absence too many tracheal cells receive the signal and too many branches sprout. The biochemical studies of the FGF pathway and its regulation now underway should reveal how binding of the ligand to its receptor leads to formation of new tubes at the appropriate positions in the developing airway.
Oxygen regulation of airway branching
Although the major branches that form in the embryo are stereotyped, the fine terminal branches that form later in development are variable and regulated by oxygen need of the target tissues. We discovered that branchless also plays the critical role in this physiological control of branching. Larval cells respond to oxygen starvation by turning on expression of branchless. The secreted FGF signals nearby tracheal cells to grow towards the signaling cells and supply them with more oxygen. The process is highly dynamic: other cells become hypoxic and the process repeats, generating a complex pattern of branches that precisely matches the oxygen needs of the target tissue.
We are characterizing other genes that are required for this process. Some encode components of the oxygen sensing pathway used by oxygen-starved cells to recognize and initiate the response to the crisis. Two are transcription factors that become active only under low-oxygen conditions. We are examining how low-oxygen tension activates the transcription factors and how this leads to increased expression of branchless and increased branching. One of the transcription factors relocates from the cytoplasm to the nucleus under low oxygen conditions. We hope to identify the cellular oxygen sensor that monitors local oxygen conditions and triggers this nuclear localization when oxygen is low, and all of the target genes it and the other transcription factor regulate. A grant from the National Institutes of Health provides support for this project.
Genetic dissection of lung development
We are extending our studies of respiratory system development to the mammalian lung. Lung development is considerably more complex than airway development in Drosophila, as there are more than 15 million airway branches in the human lung, plus a similar number of blood vessels. Are the same mechanisms and types of molecules that are used to pattern the Drosophila airways also used in the mammalian lung? Results from a number of labs indicate that homologs of some of the key Drosophila regulators -- FGFs, FGF receptors, and sprouty's -- also play important roles early in mouse lung development. We are investigating these and homologs of other Drosophila tracheal genes, and we have initiated a systematic search for genes that control mouse lung development. Elucidation of the genetic pathways that control mammalian lung development should lead to a molecular understanding of the process. It should also provide insights into human lung diseases, and may aid in the design of interventions that reactivate the developmental program in the adult to restore diseased tissue.
Select Publications
Manning, G. and Krasnow, M.A. (1993). Development of the Drosophila tracheal system. In The Development of Drosophila melanogaster (Eds. M. Bate and A. Martinez Arias), Cold Spring Harbor Press (Cold Spring Harbor, New York), pp. 609-685.
Samakovlis, C., Hacohen, N., Manning, G., Sutherland, D., Guillemin, K. and Krasnow, M.A. (1996). Development of the Drosophila tracheal system occurs by a series of morphologically distinct but genetically coupled branching events. Development 122, 1395-1407.
Samakovlis, C., Manning, G., Steneberg, P., Hacohen, N., Cantera, R., and Krasnow, M.A. (1996) Genetic control of epithelial tube fusion during Drosophila tracheal development. Development 122:3531-3536.
Sutherland, D., Samakovlis, C., and Krasnow, M.A. (1996) branchless encodes a Drosophila FGF homolog that controls tracheal cell migration and the pattern of branching. Cell 87, 1091-1101.
Guillemin, K. and Krasnow, M.A. (1997) The hypoxic response: Huffing and HIFing. Cell 89, 9-12.
Hacohen, N., Kramer, S. , Sutherland, D., Hiromi, Y, and Krasnow, M.A. (1998) sprouty encodes a novel antagonist of FGF signaling that patterns apical branching of the Drosophila airways. Cell 92, 253-263.
Metzger, R. and Krasnow, M.A. (1999) Genetic control of branching morphogenesis. Science 284, 1635-1639
Jarecki, J., Johnson, E. and Krasnow, M.A. (1999) Oxygen regulation of airway branching in Drosophila is mediated by Branchless FGF. Cell 99, 211-220.
Beitel, G. J. and Krasnow, M.A. (2000) Genetic control of epithelial tube size in the Drosophila tracheal system. Development 127, 3271- 3282.
Shim, K., Blake, K., Jack, J. and Krasnow, M.A. (2001) The Drosophila ribbon gene encodes a nuclear BTB domain protein that promotes epithelial migration and morphogenesis. Development 128, 4923-4933.
Cho, N.K., Keyes, L., Johnson, E., Heller, J., Ryner, L., Karim, F., and Krasnow, M.A. (2002) Developmental control of blood cell migration by the Drosophila VEGF pathway. Cell 108, 865-876.
Arbeitman, M.N., Furlong, E.E., Imam, F., Johnson, E., Null, B.H., Baker, B.S., Krasnow, M.A., Scott, M.P., Davis, R.W., and White, K.P. (2002) Gene expression during the life cycle of Drosophila melanogaster. Science 297, 2270-2275.
Lubarsky, B., and Krasnow, M.A. (2002) Tube morphogenesis: making and shaping biological tubes. Cell 112, 19-28.
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