Ex Vivo Culture of Lung Buds on the Chorioallantoic Membrane of the Avian Embryo

Abstract

Rationale: Development of the vertebrate lung involves a complex series of interactions between the foregut endoderm and the adjacent mesenchyme, resulting in formation of the paired lung buds. The lung buds subsequently give rise to mature lungs through processes which include branching morphogenesis, cell commitment and differentiation. At present it remains unclear what other cell types may play a role in patterning of the developing airways and the generation of diverse cell lineages. To address this problem, we have developed an ex vivo lung culture system which allows us to experimentally manipulate the developing lung buds. The chick embryo lends itself particularly well to such manipulations and It has long served as an important model system for developmental studies. In addition, development of the respiratory system in birds has been the subject of numerous descriptive studies which can provide a useful background to more experimental approaches. Methods: We have explanted the lung buds of developing day 4 chick or quail embryos onto the chorioallantoic membranes of host embryos at a similar developmental stage. In some cases, donor king buds were taken from transgenic quail, in which all or a subset of cells were labeled constitutively through expression of fluorescent chimeric protein. The explants were cultured further before fixation and analysis by conventional and confocal laser scanning microscopy. Results: We have demonstrated that developing lung buds explanted to chorioallantoic membranes continue to develop further, in some cases surviving for a further 6 days of development. In addition, the simple airway of the explanted lung buds undergoes branching morphogenesis to form a complex airway comparable to that found in lungs of similarly-staged un-operated embryos. Moreover, the explanted lung buds develop a capillary network that appears to establish a circulation with that of the chorioallantoic membrane. Conclusions: The chorioallantoic membrane culture system can be used to sustain the growth and development of isolated chick and quail lung buds, thus permitting their experimental manipulation to determine the role of different cell populations in lung development.

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