Journal: Development (Cambridge, England)

Article Title: Lung epithelial tip progenitors integrate glucocorticoid- and STAT3-mediated signals to control progeny fate

doi: 10.1242/dev.134023

Figure Lengend Snippet: Evolution of alveolar epithelial gene expression patterns in the developing mouse lung. Sections of E15.5, 16.5, 17.5 and 18.5 wild-type mouse lungs stained for markers of differentiation. (A) Green, SOX2 (differentiating bronchioles); red, SOX9 (tips); white, LPCAT1 (tip cells from E16.5, then AT2 cells). (B) Green, CEBPA (sub-set of tip cells from E16.5, then AT2 cells); red, pro-SFTPC (embryonic epithelium, stronger from E16.5, later specific to AT2 cells). (C) Green, pro-SFTPC (stronger from E16.5, later specific to AT2 cells); red, LAMP3 (rare tip cells; AT2 cells); magenta, PDPN (tip cells from E16.5, then AT1 cells). (D) Green, LPCAT1 (tip cells from E16.5, then AT2 cells); red, LAMP3 (rare tip cells; AT2 cells); magenta, PDPN (tip cells from E16.5, then AT1 cells). (E) Green, HOPX (stalk cells from E16.5, AT1 cells); red, SOX9 (tip cells); white, E-CAD (epithelial cells). (F) Green, SOX2 (differentiating bronchioles); red, SOX9 (tips); white, HOPX (stalk cells from E16.5, AT1 cells). (G) Green, HOPX (stalk cells from E16.5, AT1 cells); red, LPCAT1 (tip cells from E16.5, then AT2 cells). Arrows, LPCAT1 + HOPX + cells; arrowheads, LPCAT1 + HOPX − cells. Blue, DAPI (nuclei). Dashed line, edge of lung. Scale bars: 50 μm in A-F, 20 μm in G and insets.

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: Gene Expression, Staining

Journal: Development (Cambridge, England)

Article Title: Lung epithelial tip progenitors integrate glucocorticoid- and STAT3-mediated signals to control progeny fate

doi: 10.1242/dev.134023

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, Quantitation Assay

Journal: Development (Cambridge, England)

Article Title: Lung epithelial tip progenitors integrate glucocorticoid- and STAT3-mediated signals to control progeny fate

doi: 10.1242/dev.134023

Figure Lengend Snippet: Glucocorticoid signalling is sufficient, but not essential, to specify alveolar fate. (A) Experimental design: Tomato + E12.5 or 16.5 tip or stalk was grafted into E12.5 host lung and grown with 50 nM Dx throughout culture. (B) Examples of alveolar-fated tip grafts stained for: green, LPCAT1 (alveolar fate); red, Tomato (graft); white, PDPN (basal and AT1 cells). Arrowheads, PDPN + AT1 cells. (C) Split bar graph showing results from B. Each type of graft was analysed in at least three independent experiments. (D) E12.5 wild-type lungs were grown with or without Dx for up to 6 days; two independent experimental replicates. Note precocious expression of alveolar markers in the presence of Dx. Lungs cultured without Dx do express LPCAT1 from experimental day 5. Green, LPCAT1 (late tip progenitors and type 2 cells); red, SOX9 (tip progenitors). (E,F) Sections of GR −/− and GR +/+ sibling lungs at E17.5 and E18.5 stained for: green, HOPX (AT1 cells); red, SOX9 (tip progenitors); white, E-CAD (epithelium) (E), and: green, LPCAT1 (late tip progenitors and AT2 cells); red, LAMP3 (AT2 cells); magenta, PDPN (late tip progenitors and AT1 cells) (F). A total of five GR −/− and 5 GR +/+ sibling lungs from three independent litters were observed at both E17.5 and E18.5. Blue, DAPI. Dashed line, edge of lung. Scale bars: 100 μm in B; 50 μm in D-F.

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: Staining, Expressing, Cell Culture

Journal: Development (Cambridge, England)

Article Title: Lung epithelial tip progenitors integrate glucocorticoid- and STAT3-mediated signals to control progeny fate

doi: 10.1242/dev.134023

Figure Lengend Snippet: Lung epithelial specific knock-out of Stat3 results in a brief delay in lung development. (A-D) Sections of E15.5, 16.5, 17.5 and 18.5 wild-type mouse lungs stained to show phosphorylated (active) STAT3 protein. Red, pSTAT3; white, E-CAD (epithelium). Arrowheads, distal tips. (E) RT-qPCR for Stat3 in Nkx2.1-Cre; Stat3 Δ/fx and sibling Stat3 fx/+ lungs. Five lungs of each genotype were collected from three independent litters. (F-H) pSTAT3 staining in sibling control (F) and Stat3 cKO (G,H) lungs shows the highly variable extent of recombination. Red, pSTAT3; white, E-CAD (epithelium). (I-K) RT-qPCR from Nkx2.1-Cre; Stat3 Δ/fx and sibling Stat3 fx/+ lungs. Mature differentiation markers SftpD and Aqp5 at E16.5 and E18.5 (I,J) and late progenitor and/or early differentiation markers at E16.5 (K). (L-W) Sections of Stat3 cKO and sibling lungs. (L,M) E17.5 sections stained for: green, SOX9 (tip progenitors); red, pSTAT3; white, E-CAD (epithelium). Arrowheads, distal tips. (N,O) E18.5 sections stained for: red, pSTAT3; white, PDPN (type 1 cells). (P-S) Green, LPCAT1 (late tip progenitors and type 2 cells); red, pSTAT3; white, E-CAD (epithelium) at E16.5 (P,Q) and E18.5 (R,S). (T-W) Green, HOPX (type 1 cells); red, pSTAT3; white, E-CAD (epithelium) at E16.5 (T,U) and E18.5 (V,W). Blue, DAPI. Dashed line, edge of lung. Scale bars: 50 μm, except 100 μm in L,M. Error bars represent s.e.m. * P <0.05; ** P <0.01 by two-tailed t -tests with unequal variance; n =5.

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: Knock-Out, Staining, Quantitative RT-PCR, Control, Two Tailed Test

Journal: Development (Cambridge, England)

Article Title: Lung epithelial tip progenitors integrate glucocorticoid- and STAT3-mediated signals to control progeny fate

doi: 10.1242/dev.134023

Figure Lengend Snippet: Ectopic IL6 family ligands result in accelerated AT2 differentiation via STAT3 activation. (A) Schematic and section of E15.5 slice culture resulting in differentiation of mature saccules with AT1 and AT2 cells in the presence of Dx. Green, pro-SFTPC; red, PDPN. (B) Schematic of IL6 and LIF experiments. Slices from individual lungs were split between two conditions for internal controls. (C,D) Sections from control, IL6- and LIF-exposed wild-type lungs. (C) Red, pSTAT3; white, E-CAD (epithelium). (D) Red, LAMP3 (differentiating AT2 cells); green LPCAT1 (late tip and AT2 cells). (E) Sections from control ( Nkx2.1-Cre; Stat3 +/fx ) and mutant ( Nkx2.1-Cre; Stat3 Δ/fx ) lungs with and without IL6. n =9 Nkx2.1-Cre; Stat3 Δ/fx lungs analysed in three independent experiments. Top panels: green, pro-SFTPC; red, LAMP3. Lower panels: red, pSTAT3; white, E-CAD (epithelium). Blue, DAPI. Dashed line, edge of lung. Scale bars: 50 μm in A,D; 100 μm in C,E.

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: Activation Assay, Control, Mutagenesis