Despite the fascinating structure of the lung and its central position in health, the genetic and molecular programs that build, maintain, and regenerate the lung, and that control the seemingly simple process of breathing, have only recently begun to be elucidated. With new and emerging genetic and genomic tools, it is now possible to probe these processes and how they go awry at single-cell resolution and to follow each gene's activity during the normal and disease processes. This ability has opened a new level of understanding of lung biology and disease, with the promise of rationally designed therapies and regenerated lung tissue and, ultimately, entire lungs from stem cells.
Lung Development and Stem Cells
One of the most fundamental biological and biomedical questions is, How is the complex three-dimensional structure of our organs genetically encoded and maintained? To elucidate the molecular program that builds and maintains the lung, we use the same powerful approaches we used to dissect respiratory system development in Drosophila. We began with a systematic analysis of the mouse bronchial tree and discovered that branching is remarkably stereotyped and mathematically elegant (Fig. 1), consisting of three local branching geometries (subroutines) coupled in three different sequences. A major goal is to define the molecular basis of the subroutines and the master routine that controls their deployment, starting with identification of the key genes.
We are using the ascertained lineage of the 5,000 bronchial branches, the most complete developmental anatomic description of any mammalian organ, to map the development of the major cell types in the lung at single-cell resolution. We do this by state-of-the-art multicolor cell labeling and clonal analysis, following proliferation, movement, and differentiation of individual labeled cells in vivo. This allows us to identify lung progenitor/stem cells and their niches, both during development and in adult tissue renewal and repair. So far we have identified progenitor/stem cells of all the major mesenchymal and stromal cell types, including airway and vascular smooth muscle. We have also identified progenitor/stem cells of the endothelial cells that form pulmonary vessels, of lung macrophage lineages, and of alveoli, the exquisitely thin epithelial sacs where gas exchange occurs. Adult alveolar stem cells are unconventional stem cells: they are a small subset of the differentiated, surfactant-secreting cells, whose stem cell function is activated by dying alveolar cells (Fig. 2).
In collaborative work with Stephen Quake (HHMI, Stanford University), we reconstructed the full gene expression program of alveolar development by single-cell RNA sequencing of dozens of developmental intermediates, the first complete transcriptional program elucidated at single-cell resolution. This has identified appealing candidates for the long-sought signals and master regulators of alveolar development. We want to determine how niche signals control and pattern proliferation, migration, and morphogenesis of alveolar stem cells and other lung stem cells, and use this molecular information to build lungs in vitro and design regenerative therapies.
Lung Cancer and Other Diseases
Lung diseases are the third leading cause of death in the United States, at more than 350,000 deaths per year. For few of these diseases do we understand how, at the cellular and molecular level, the lung program goes awry, and for disappointingly few are there highly effective or curative therapies. We are using the approaches described above to elucidate at single-cell resolution the pathological programs of lung diseases. These approaches identified the alveolar stem cell as the cell of origin of the major form of lung cancer and showed that cancer-causing mutations in Egfr and K-ras oncogenes transform the stem cells into cancer by permanently activating their self-renewal program (Fig. 2). We hope to use such cellular and molecular information to discover new ways of detecting, classifying, and ultimately curing lung cancer and other deadly lung diseases.
Neural Circuit of Breathing
Breathing is a simple but essential behavior we perform 20,000 times a day. It is instantaneously adjusted in response to changes in oxygen or carbon dioxide levels, but it is also regulated by emotions (sighing, yawning, laughing, and crying) and coordinated with speech and singing, and it can be controlled voluntarily. Although the key breathing control centers have been defined anatomically over the past century (Fig. 3), and the cell physiology of the constituent neurons has been intensively investigated, we still do not understand the cellular or molecular basis of the breathing pacemaker or the identity of the oxygen and carbon dioxide sensors. We also do not know how the rate and pattern of the pacemaker – which is located in the medulla and can "beat" autonomously in culture – is modulated by physiological and higher order brain input. We are constructing a comprehensive gene expression map of the major breathing control centers at single-cell resolution, and we are using it to generate precision tools to probe the mechanism of the pacemaker and the oxygen-sensing cells and to map their connections with each other, with motor neurons of the breathing muscles, and with higher brain centers. Our goal is to elucidate the cellular and molecular basis of the breathing circuit, how it is regulated by physiology and emotions and coordinated with speech, and how it goes awry in breathing arrhythmias such as sleep apnea, anxiety disorders, and sudden infant death syndrome, a devastating disease and leading cause of death in infants.
New Genetic Model Organism
Although the mouse is the supermodel of biomedical research, there are many aspects of primate biology, behavior, and disease that are absent or poorly modeled in mice. We surveyed the animal kingdom to find other animals of similar size and genetic advantages that might better exemplify primate biology. We have established an international consortium and laboratory in Madagascar, which is partnering with Malagasy students and scientists to identify mutant mouse lemurs in the country's vanishing rain forests and follow them longitudinally in the field to determine the effects of the mutations on mouse lemur ecology, biology, behavior, physiology, and disease. We hope this spawns a new genetic model organism and new field of genetics bridging physiological, behavioral, and ecological research that at the same time will provide a model for hands-on biomedical education and training in developing countries that develops their special talents and preserves and enhances their unique natural resources.
Grants from the National Institutes of Health and support from the Vera Moulton Wall Center for Pulmonary Vascular Disease provided partial support for these projects.
As of March 23, 2016