Abstract
Early mouse lung development, including specification of primordia, patterning of early endoderm and determination of regional progenitor cell fates, is tightly regulated. The ability to culture explanted embryonic lung tissue provides a tractable model to study cellular interactions and paracrine factors that regulate these processes. We provide up-to-date protocols for the establishment of this culture model and its application to investigate hedgehog signaling in the developing lung.
Keywords: Mouse embryonic lung, In vitro, Explant culture
Background
Mouse lung development initiates as an endodermal diverticulum of the anterior foregut endoderm at day 9.5 postcoitum (E9.5), with subsequent closure of a proximal tracheoesophageal septum for the formation of distinct tracheal and esophageal tubes (Minoo and King, 1994). Subsequent branching of primitive endodermal tubes yields a planar lung structure by E12.5, with subsequent orthogonal branches yielding three-dimensional structure characteristic of the mature lung (Metzger et al., 2008). The planar structure of lung rudiments isolated prior to E12.5 is suitable for in vitro culture at an air liquid interface (Carraro et al., 2010; Del Moral and Warburton, 2010). Embryonic lung is isolated by dissection using a stereo microscope either under bright field illumination or by fluorescence illumination when coupled with lineage tracing and fluorescent reporters. Herein we describe the use of a ShhCre/RosamTmG reporter mouse allowing Cre-mediated activation of membrane-localized GFP within anterior foregut endoderm from approximately E8.75 (Montgomery et al., 2007; Goss et al., 2009; Yao et al., 2017). Accordingly lung endoderm is visualized by green fluorescence and surrounding tissue by red fluorescence, allowing clear identification and microdissection of developing endodermal structures, including the lung, and imaging during in vitro culture.
Materials and Reagents
Equipment
Software
Procedure
Data analysis
Images were taken and processed using Zen blue software, statistics analysis was performed using PRISM software version 7.
Notes
Recipes
Acknowledgments
This work was funded by the National Institutes of Health 1T32HL134637-01, R01HL135163-01; California Institution of Regenerative Medicine CIRM LA1-06915. The protocol described here is adapted from Yao et al. (2017). All authors declared that no competing interest exists.
References
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