Fig 2: A single club cell can differentiate into alveolar cells in 3-D culture.(A–D) Representative DAPI (blue), EGFP (green) and PDPN (red) staining of secondary colonies derived from a single EGFP+ club cell. Higher magnification of selected area in A with combination of different channels was shown as B-D. Arrows indicate cells positive for both EGFP and PDPN in alveolar-like structures. (E–H) Adjacent section of (A) was stained for DAPI (blue), EGFP (green) and pro-SPC (red). Higher magnification of selected area in E with combination of different channels was shown as F–H. Arrows indicate cells positive for both EGFP and pro-SPC in alveolar-like structures. Scale bars: (A,E) 100 µm; (B–D, F–H) 25 µm.

Fig 3: Club cells differentiate into alveolar cells in 3-D culture.(A,B) Representative DAPI (blue), EGFP (green) and Cyp2f2 (red) staining of colonies formed by EGFP+ club cells at day 7. Channels of EGFP and Cyp2f2 are shown separately in panel (A) and (B) for clarity. Higher magnification of the same selected area in (A) and (B) are shown as indicated. (C,D) Adjacent section of (A) is stained for DAPI (blue), EGFP (green) and pro-SPC (red). Channels of EGFP and pro-SPC are shown separately in panel (C) and (D). Higher magnification of the same selected area in (C) and (D) are shown as indicated. (E) Representative DAPI (blue), EGFP (green) and PDPN (red) staining of colonies formed by EGFP+ club cells at day 14. Combinations of different channels are shown as indicated in different panels. Arrows indicate cells positive for both EGFP and PDPN in alveolar-like structures. (F) Adjacent section of (E) is stained for DAPI (blue), EGFP (green) and pro-SPC (red). Combinations of different channels are shown as indicated. Scale bars: (A–D) 500 µm; (E,F) 25 µm.

Fig 4: QPC-deficient LECs gradually up-regulate the expression of BEC markers.(A and B) At E11.5, QPCf/f;PdpnGFPCre LECs show severely reduced Vegfr3 levels, but similar to controls, Prox1+ LECs do not coexpress BEC markers such as CD34 (n = 3). (C and D) At around E12.5, Vegfr3 expression becomes undetectable in mutant LECs and Prox1+(low) LECs abnormally up-regulate the expression of CD34, an indication that they are losing LEC fate and regaining BEC fate. White arrows indicate Prox1+ CD34+ LECs. Blood cells are abnormally seen inside the mutant lymph sacs (D), most likely a consequence of defective lymphovenous valves. White box insets correspond to higher magnification of the regions in dotted boxes in each panel. CD34+ Prox1+ cells were quantified and shown as percentage of Prox1+ in each group (E). Each data point represents a biological replicate. All results are presented as means ± SEM and analyzed by two-tailed Student’s t test. Scale bar, 50 µm (n = 6).

Fig 5: (A) Fluorescence micrographs show circumferential distribution of LepR+ cells (red) around HEVs and lymphatics in Leprcre;tdTomato mice LNs. LepR (red) colocalized with PDPN and ER-TR7. Scale bar, 50µm. (B) Fluorescence micrographs of LNs from Leprcre;tdTomato mice showed no colocalization of LepR (red) with NG2 and PDGFRß. Scale bar, 100µm. (C) Fluorescence micrographs of Leprcre;tdTomato mice LNs showed no colocalization of LepR (red) with RANKL, CD35 and MAdCAM. Scale bar, 50µm. (D) LepR expression in CD45-PDPN+CD31-(FRCs) and CD45-PDPN-CD31-(DN) populations of LNs were evaluated by flow cytometry. Data are representative of three independent experiments (n=3). (E) Gating strategy to exclude hematopoietic and endothelial cells for LepR+ cells in LepRcre;tdTomato LNs. (F) The percentages of LepR+ cells of LNs in stromal marker panel Sca-1, CD29, CD90, CD44, CD73, CD105 and CD106 were evaluated based on the gating strategy in (E). Data are representative of three independent experiments (n=3). (G) Fluorescence micrographs of PND1 and PND14 LNs showed location of LepR+ cells (red) in relation to HEVs, lymphatics, PDPN and ER-TR7. Scale bar, 100µm.