The intricate network of branched airways of the lung originates as a simple epithelial tube. A sequence of recursive branching events

INTRODUCTION The intricate network of branched airways of the lung originates as a simple epithelial tube. A sequence of recursive branching events then transforms this simple tubular geometry into a complex system of airways that conduct air and promote gas exchange postnatally. In the early chicken embryo, this process is monopodial; that is, daughter branches emerge laterally along the length of their parent branch (Locy and Larsell, 1916; Hogan, 1999). This branching strategy generates a structure distinct from that in the mammalian lung, which is formed instead by a combination of lateral and dichotomous branching modes (Metzger et al., 2008). Moreover, because airflow is unidirectional in the lungs of birds, as opposed to tidal in mammals, some of these monopodial branches anastomose to form circulatory loops called air capillaries. Other branches dilate at their distal tips to form large air sacs, which then function like bellows to maintain a constant unidirectional flow. These dramatically different architectures suggest distinct underlying developmental mechanisms. Yet, remarkably, several signaling pathways, including those downstream of fibroblast growth factor (FGF), bone morphogenetic protein (BMP) and sonic hedgehog (SHH), are conserved across different species and different organ systems (Metzger and Krasnow, 1999; Affolter et al., 2003; Morrisey and Hogan, 2010). This molecular homology underlies the hypothesis that the physical mechanisms that sculpt these branching structures are likewise conserved (Hogan, 1999; Davies, 2002). Branching of many organs is thought to result from local increases in proliferation along the epithelium (Goldin and Wessells, 1979; Goldin and Opperman, 1980; Moore et al., 2002; Moore et al., 2005; Morrisey and Hogan, 2010), but unambiguous experimental evidence for this mechanism is lacking (Wessells, 1970; Nogawa et al., 1998). Whether patterned proliferation is sufficient to generate the observed deformations remains unclear. Recent studies, however, have suggested a possible regulatory role for mechanical forces and cytoskeletal contractility during branching (Michael et al., 2005; Moore et al., 2005; Nelson and Gleghorn, 2012). Quantitative studies focused on the mechanics of branching (Caussinus et al., 2008; Gjorevski and Nelson, 2010) are therefore required to define the physical forces that shape the developing airways. Here, we examined the relative roles of actomyosin contraction and cell proliferation in the initiation and outgrowth of buds during monopodial branching of the embryonic chicken lung. Combining quantitative image analysis with computational modeling, we found that apical constriction of the airway epithelium is sufficient to drive the changes in cell and tissue shape required for initial budding during monopodial branching. Moreover, we also found a complex pattern of proliferation along the primary bronchus, enriched along the dorsal and ventral aspects of the tube and highest in regions prior to branching. Nonetheless, both experiments and simulations revealed that proliferation, coupled with the forces generated by apical constriction, was not sufficient to fully shape the emerging lung bud. These results challenge the generally accepted view that patterned proliferation drives airway branching morphogenesis, and suggest instead that active shape changes of the epithelium play a major role in the branching process.