Figure 3

Nerve ablation halts lung branching . (A) Nerves were ablated to the left lung but not the right. Red and black asterisks mark left and right lung terminal buds, respectively, at the time of ablation and mark these original positions after 43 to 46 hours in culture in (B). New branches have formed beyond asterisks in the right lung but on the left only one branch has extended slightly. (C) The panel diagrams the branches present on explantation (black) and new branches formed over two days (green). (D) As a control, equivalent laser energy was delivered to ablate non-neural mesenchymal cells from the left lung of a matched explant, avoiding epithelium, neurons and endothelium. Asterisks mark the original terminal buds and (E) shows how budding has progressed beyond these original terminal locations after two days. Both lungs branch substantially, as illustrated in the schematic (F). (G-L) shows that neural ablation (G) halts right lung branching without affecting the non-intervened left lung (H,I) while control ablation (J) leaves branching unperturbed in both lungs (K,L). Scale bar = 200 μm throughout. (M) Nerve ablation significantly reduces lung branching after two days in culture, while control mesenchymal ablation has no affect. (N) Muscarinic inhibitor 4-DAMP did not alter lung budding compared to controls. Red lines indicate median values. 4-DAMP, 1,1-dimethyl-4-diphenylacetoxypiperidinium iodide.