Journal: Development (Cambridge, England)
Article Title: Lung epithelial tip progenitors integrate glucocorticoid- and STAT3-mediated signals to control progeny fate
Figure Lengend Snippet: Extrinsic factors are the major determinant of progenitor cell identity in the developing mouse lung. (A) Experimental outline. Epithelial progenitors (tip or stalk) were microdissected from donor E12.5 or E16.5 Tomato + ( Rosa26R mT−mG/+ ) lungs and grafted into the mesenchyme of unlabelled E12.5 hosts. Hosts and grafts were cultured for 8 days without Dx, or with addition of 50 nM Dx at culture day 4 or 5, followed by serial sectioning and staining to determine graft fate. (A′,A″) Grafts integrate into the host lung, grow and form a lumen. (B-G) Sections of grafted lungs with alternate slides stained for: green, LPCAT1 (alveolar fate); red, Tomato (graft); white, PDPN (basal and AT1 cells), or: green, SOX2 (bronchiolar fate); red, RFP (Tomato + graft); white, acetylated tubulin (ACT; cilia) to determine graft fate. Examples of tip grafts with alveolar, bronchiolar and mixed broncho-alveolar fate are shown. Note D/D′ and G/G′ are different sections of the same graft. (H,I) Quantitation of graft fate as a percentage of numbers of grafts analysed. Each type of graft was analysed in at least three independent experiments. Scale bars: 25 μm in A″, 100 μm in B-G′.
Article Snippet: Primary antibodies: acetylated tubulin (mouse, 1:3000, Sigma, T7451), CEBPA (rabbit, 1:500, Santa Cruz, sc-61), cleaved caspase 3 (rabbit, 1:100, Abcam, ab2302), E-CAD (rat, 1:1000, Invitrogen, 13-1900; or mouse, 1:1000, BD Biosciences, 610182), GFP (chick, 1:1000, Abcam, AB13970), GR (rabbit, 1:100, Santa Cruz, sc-1004), HOPX (rabbit, 1:50, Santa Cruz, sc-30216, clone FL-73), KI67 (mouse, 1:200, BD, 550609), LAMP3 (rat, 1:100, Dendritics, DDX0192, clone 1006F7.05), LIF (goat, 1:100, R & D Systems, AB-449-NA), LPCAT1 (rabbit, 1:500, Proteintech), PDPN (hamster, 1:1000, DSHB, 8.1.1), RFP (rabbit, 1:250, Rockland, 600-401-379), pro-SFTPC (rabbit, 1:500, Millipore, AB3786), SOX2 (goat, 1:250, Santa Cruz, sc-17320, clone Y-17), SOX9 (goat, 1:200, R & D Systems, AF3075), pSTAT3-Tyr705 (rabbit, 1:200, Cell Signaling, 9145), STAT5a (rabbit, 1:20, Abcam, ab7968).
Techniques: Cell Culture, Staining, Activated Clotting Time Assay, Quantitation Assay