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. Author manuscript; available in PMC: 2015 May 14.
Published in final edited form as: Science. 2014 Nov 14;346(6211):1258810. doi: 10.1126/science.1258810

Figure 4. Tissue grafts and lineage tracing show ASM progenitors are located in tip mesenchyme and form anew with new domain branches.

Figure 4

A, Graft strategy. Mesenchyme excised from stalk or tip of ZsGreen-expressing donor E11.5 lung were grafted into tip of an unlabeled littermate host lung, and grafted cells (green) were traced in culture. B, Quantification of progenitor cell migration from the graft in tip-to-tip (T>T) or stalk-to-tip (S>T) mesenchyme grafts, measuring the distance from the graft margin to the furthest invading graft cell, along the shortest inferred migration path. n, number of grafts. Note robust invasion of tip-derived cells (all grafts showed > 100 μm migration) relative to stalk-derived cells (11% showed > 100 μm migration). C, C’, Stereomicroscope image showing recipient lung four days after tip-to-tip micrograft (C), and single confocal optical slice of grafted region (C’). Note in C’ that tip cells have migrated away from lung margin (dotted line) along the airways (dashed lines) and some graft cells (arrowheads) are expressing ASM differentiation marker smooth muscle actin (red). D, Stalk-to-tip mesenchyme graft. Grafted cells fail to leave graft site, and do not migrate along airways or express smooth muscle actin (D’). E, Strategy of lineage trace of smooth muscle of parent branch of L.M1, a late-forming domain branch. Budding L.M1 (highlighted in insets) is first medial domain branch formed along left primary airway; it buds through existing ASM layer of parent branch and forms its own ASM layer. Saturating tamoxifen (3 mg) was administered at E11.5 to SM-MHC (smooth muscle myosin heavy chain)-CreER; mTmG to label differentiated ASM of parent branch and early forming lateral branches (L.L1 – L.L3) with heritable GFP expression (green bar on timeline, with tamoxifen assumed active for 24 hours) before emergence of L.M1 bud at E12.5. Lungs were analyzed at E14.5 after L.M1 has budded and formed its ASM (“new” ASM, black bar in inset). F, Confocal z-stack projections (ventral view) showing close-up of boxed region (F’) of SMMHC-CreER;mTmG E14.5 embryo treated as above and immunostained for lineage trace of smooth muscle of parent branch (GFP, green) and all airway smooth muscle (SMA, red). Note ASM of L.M1 daughter branch is not lineage labeled (border highlighted with dashed line in F’ merge) indicating a new progenitor pool formed ahead of new branch that is lineally independent of existing ASM. There is no cell mixing across smooth muscle lineage boundary (dashed line), with exception of two lineage-labeled cells (arrowheads in F') in ASM of L.M1. Dots, lineage labeled non-ASM cells. Bars, 50 μm.