Figure S2.

Rapid loss of mGFP fluorescent lineage marker in flattening epithelial cells during AT1 differentiation in injury and embryonic development. (A) Strategy for pan-alveolar epithelial lineage marking with mGFP prior to pulsed epithelial ablation. (B and C) Direct fluorescence (top row) and co-staining for pan-epithelial lineage marker (mGFP) and AT1 cells (Pdpn; bottom row) in control adult lung (B) and 24 h after DT administration (C) shows the absence of the pan-epithelial mGFP in the majority of AT1 cells by direct fluorescence and antibody staining. (D) Low- (left column) and high- (right column) magnification micrographs of co-staining for pan-epithelial lineage marker (mGFP), AT1 cells (Pdpn), and AT2 cell injury marker (Krt8) show loss of mGFP protein in AT1 cells, including ones co-expressing the AT2 cell injury marker (arrows; n = 5 control and 5 DT-treated mice). (E–G) Co-staining for pan-epithelial lineage marker (mGFP), AT1 cells (Pdpn), and AT2 cells (pro-SpC) in sacculating embryonic day 18 lung (E) with separated channels of boxed regions (F and G) shows mGFP in nascent AT2 cells (asterisks) but patchy absence in newly forming AT1 cells at varying stage of flattening (arrows; n = 5 mice). Scale bars, 100 µm (B top, C top, D left), 50 µm (E), 25 µm (B bottom, C bottom, D right), 10 µm (F and G).