Explant culture of mouse embryonic whole lung, isolated epithelium, or mesenchyme under chemically defined conditions as a system to evaluate the molecular mechanism of branching morphogenesis and cellular differentiation

Abstract

Lung primordial specification as well as branching morphogenesis, and the formation of various pulmonary cell lineages, requires a specific interaction of the lung endoderm with its surrounding mesenchyme and mesothelium. Lung mesenchyme has been shown to be the source of inductive signals for lung branching morphogenesis. Epithelial-mesenchymal-mesothelial interactions are also critical to embryonic lung morphogenesis. Early embryonic lung organ culture is a very useful system to study epithelial-mesenchymal interactions. Both epithelial and mesenchymal morphogenesis proceed under specific conditions that can be readily manipulated in this system (in the absence of maternal influence and blood flow). More importantly this technique can be readily done in a serumless, chemically defined culture media. Gain and loss of function can be achieved using expressed proteins, recombinant viral vectors, and/or analysis of transgenic mouse strains, antisense RNA, as well as RNA interference gene knockdown. Additionally, to further study epithelial-mesenchymal interactions, the relative roles of epithelium versus mesenchyme signaling can also be determined using tissue recombination (e.g., epithelial and mesenchymal separation) and microbead studies.

Fig. 5.1

Dissection of E12 embryonic lungs. (a) E12.5 whole embryo viewed from the right side. (b) Right forelimb has been removed from the embryo. (c) Forceps held by the left hand holding the embryo steady in the dish. Arrows indicate plan of dissection. (d) Embryonic lung lies posterior to the heart (removed) and anterior to the spine. This figure shows the lungs after skin and heart removal. (e) Pharynx removal allows separation of the intact lung from the embryo. (f) Extraneous pharyngeal tissue and esophagus have been trimmed away. Dissected E12.5 embryonic lung with intact trachea and larynx is shown. (Cr) Cranial lobe, (Med) Medial lobe, (Ca) Caudal lobe, (Acc) Accessory lobe, (Le) Left lobe.

Fig. 5.2

FGF9 induces expansion of the mesenchyme and the dilation of the epithelium in lung grown in vitro (6). (a–c) E12.5 lung grown for 48 h in the absence of FGF9. Note the increase in branching over time. (d–f) E12.5 lung grown for 48 h in the presence of FGF9. Note the dilation of the epithelium and mesenchyme as early as after 24 h of culture (e). After 48 h (f), the effect on the epithelium is even more pronounced. Scale bar: (a–f) 400 μm.

Fig. 5.3

Effect of FGF9 on isolated distal lung epithelium and mesenchyme (6). (a, b) Isolated distal mesenchyme grown for 48 h in the absence of FGF9 undergoes necrosis (b). (c, d) In the presence of FGF9, the mesenchymal explant grows and many mesenchymal cells invading the Matrigel are observed in the periphery. (e, f) Isolated distal epithelium grown for 48 h in the absence of FGF9 undergoes necrosis (f). (g, h) In the presence of FGF9, the epithelial explant grows considerably to form a cyst-like structure. Scale bar: (a–h) 80 μm.