The Branching Program of Mouse Lung Development

Mammalian lungs are branched networks containing thousands to millions of airways arrayed in intricate patterns that are crucial for respiration. How such trees are generated during development, and how the developmental patterning information is encoded, have long fascinated biologists and mathematicians. However, models have been limited by a lack of information on the normal sequence and pattern of branching events. Here we present the complete three-dimensional branching pattern and lineage of the mouse bronchial tree, reconstructed from an analysis of hundreds of developmental intermediates. The branching process is remarkably stereotyped and elegant: the tree is generated by three geometrically simple local modes of branching used in three different orders throughout the lung. We propose that each mode of branching is controlled by a genetically-encoded subroutine, a series of local patterning and morphogenesis operations, which are themselves controlled by a more global master routine. We show that this hierarchical and modular program is genetically tractable, and it is ideally suited to encoding and evolving the complex networks of the lung and other branched organs. Many organs are composed of highly ramified tubular networks, each with a distinct architecture tailored to its physiological function. The bronchial tree of the human lung has over 105 conducting and 107 respiratory airways arrayed in an intricate pattern crucial for oxygen flow1–4. Classical studies of lung structure5–8 raise the question of how the information required to generate a tree of such complexity is biologically encoded9. Individually configuring thousands or millions of branches would require a tremendous amount of patterning information, far more than is biologically plausible, to specify when and where each branch forms during development, and the size, shape, and direction of outgrowth of each branch. One possibility is that the process is not precisely controlled, for example if branching occurs randomly to fill available space. Another is that control is precise but coding is simplified by repeated use of a branching mechanism, as in Mandelbrot’s fractal model and other elegant algorithms10–17. 4 Corresponding author: Dr. Mark Krasnow, Department of Biochemistry and HHMI, Stanford University School of Medicine, Stanford, CA 94305-5307; [email protected]; Phone 650-723-7191; FAX 650-723-6783. 3Present addresses: R.J.M.: Department of Anatomy, School of Medicine, University of California at San Francisco, CA 94158-2517. O.D.K: Departments of Orofacial Sciences and Pediatrics, and Institute of Human Genetics, Schools of Dentistry and Medicine, University of California at San Francisco, San Francisco, CA 94143-0442 Full Methods and any associated references are available in the online version of the paper at www.nature.com/nature. Author Contributions R.J.M. and M.A.K. conceived the experiments. R.J.M. designed and performed experiments and collected data. O.K. and G.R.M. contributed to conception and design of Spry2 experiments and provided genotyped Spry2 embryos. R.J.M. and M.A.K. analyzed the data and wrote the manuscript. All authors discussed results and edited the manuscript. Author Information Correspondence and requests for materials should be addressed to R.J.M. ([email protected]) or M.A.K. ([email protected]). NIH Public Access Author Manuscript Nature. Author manuscript; available in PMC 2010 June 28. Published in final edited form as: Nature. 2008 June 5; 453(7196): 745–750. doi:10.1038/nature07005. N IH -P A A uhor M anscript N IH -P A A uhor M anscript N IH -P A A uhor M anscript Even with these attractive models and recent progress in identifying lung development genes18, understanding of the program that directs branching remains rudimentary. This is largely due to the complexity of the bronchial tree, which makes it difficult to follow branching dynamics beyond the earliest events19–21. Although branching of the lung and other organs can occur in culture22–25, it is unlikely these recapitulate the full pattern. Here, we have determined the complete in vivo pattern of branching and branch lineage of the mouse bronchial tree, and show that it is generated using three geometrically distinct local modes of branching coupled in three different sequences. The branch lineage of the mouse bronchial tree The bronchial tree develops by branching of airway epithelium into surrounding mesenchyme. Although the process cannot be visualized in living embryos with current techniques, we reasoned we could reconstruct the branching sequence from fixed specimens, provided the process is stereotyped. An immunostaining procedure was developed to visualize the full threedimensional structure of the bronchial tree in fixed lungs (Fig. 1a). Examination of hundreds of wild-type CD1 specimens collected between embryonic day (E) 11 and E15 revealed that the branching pattern is remarkably stereotyped. This allowed us to reconstruct the sequence of events — where, when, and in what order, branches form — from finely staged specimens (Fig. 1b). This information was used to construct a lineage diagram representing the developmental history of the ~5000 branches of the bronchial tree (Fig. 1c, d; Supplementary Fig. 1). We found that there are three branching modes used repeatedly throughout the lung, which we call domain branching, planar bifurcation and orthogonal bifurcation.