DEV037317 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Id2 and Cre protein expression in wild-type and Id2^CreERT2/+ lungs. (A) E11.5 wild-type lung section. Green, anti-Id2. Blue, DAPI (nuclei). Arrow marks Id2⁺ cells in the distal endoderm. Arrowheads mark Id2⁺ cells in the distal mesoderm. Id2 is observed in both the cytoplasm and the nucleus (endoderm), or cytoplasm alone (mesoderm). The protein localization is consistent with previous observations in other cell types (Hua et al., 2006; Russell et al., 2004; Samanta and Kessler, 2004). (B-E) E16.5 sections stained with anti-Cre (black). Cre protein is observed in the distal epithelium of the Id2^CreERT2/+ (B,C), but not the wild-type (D,E) lungs. Antibody staining was performed on paraformaldehyde-fixed frozen sections using rabbit anti-Id2 (1:200, Cal Bioreagents) or rabbit anti-Cre (1:1000, Novagen) with boiling in citrate buffer pH 6.0 antigen retrieval. Scale bar: 500 µm.

• Supplemental Figure S2 -

  Fig. S2. T1α is co-expressed with pro-SftpC in the early postnatal stages of lung development. (A) Wild-type P7 alveolar section. Green, anti-pro-SftpC. Red, anti-T1α. Arrows mark cells that co-express pro-SftpC and T1α. (A′) Green channel showing pro-SftpC alone. All the observed pro-SftpC protein is located in lamellar bodies of type 2 cells. (A′′) Red channel showing T1α staining alone. The plasma membrane-localized T1α is observed in the type 2 cells and additional (type 1) cells. (A′′′) DIC image of the same alveolar region to show cell outlines. Scale bar: 20 µm.

• Supplemental Figure S3 -

  Fig. S3. Lineage-tracing Id2⁺ cells in the adult lung. (A) Four tmx injections of 0.25 mg/gram body weight were administered every other day to Id2-CreER^T2; Rosa26R-lacZ or Rosa26R-YFP adults (8-12 weeks old). The mice were either sacrificed at intervals over a 10-month period (upper time course), or they were exposed to naphthalene 1 week after the final tmx injection to injure Clara cells (lower time course). Naphthalene-treated mice were sacrificed 3 days post-naphthalene (the beginning of the repair period) or 3 weeks post-naphthalene (when epithelial repair is largely complete). (B-D) Eosin-stained lung sections harvested (B) 1 week, (C) 4 weeks or (D) 10 months following the final tmx injection. Patches of lineage-labeled cells do not appear in the bronchioles, suggesting that the lineage-labeled bronchiolar epithelial cells did not proliferate during the experiment. The labeled cells in the alveoli are mostly endothelial cells (not shown). (E,F) One week post-final tmx injection. Single optical confocal sections showing labeled Clara and ciliated cells in the bronchioles. Green, anti-GFP (lineage-labeled cells). Red, anti-Scgb1a1 (Clara cells). Blue, anti-acetylated-tubulin (ciliated cells). Arrowhead, lineage-labeled Clara cell. Arrow, lineage-labeled ciliated cell. (G,H) Blue, X-Gal, lineage-labeled cells. Brown, anti-acetylated tubulin, ciliated cells. (G) Four weeks. (H) Ten months following the final tmx injection. A mixture of both ciliated cells (arrows) and Clara cells (arrowheads) were initially labeled. By the end of the experiment, only lineage-labeled ciliated cells were seen. The overall Clara cell population has been shown to both self-renew and produce new ciliated cells during homeostasis (Rawlins et al., 2009). These data suggest that the subpopulation of Id2⁺ Clara cells that is lineage-labeled in this experiment can give rise to ciliated cells, but does not self-renew extensively. (I-L) Lung sections from naphthalene-exposed mice. (I,K) Three days-post-naphthalene exposure. (J,L) Three weeks post-naphthalene exposure. (I,J) Eosin staining. Lineage-labeled cells (blue) could survive naphthalene treatment, but they have not proliferated to contribute to the repair process. No lineage-labeled Clara cells survived naphthalene exposure (Clara cells are the naphthalene-sensitive population). This was confirmed by antibody staining. (K,L) Blue, X-Gal, lineage-labeled cells. Brown, anti-acetylated tubulin, cilia. The lineage-labeled cells that survived naphthalene injury were all ciliated (arrows). Cilia are often very short and located only at one vertex of the cell following injury. Following repair, all lineage-labeled cells were still ciliated and there was no evidence that they had proliferated. Taken together, these data suggest that although Id2 is expressed in some Clara cells, it is not expressed in the naphthalene-resistant subpopulation of Clara cells that act as epithelial progenitors in response to naphthalene injury. Adult mice were injected intraperitoneally with 250 mg/kg naphthalene (Sigma) dissolved in Mazola corn oil and sacrificed 72 hours or 3 weeks later. The final tmx injection was administered 1 week before injury. Scale bars: 1 mm in B-D,I,J; 25 µm in E,F; 20 µm in G,H; 200 µm in K,L.

• Supplemental Figure S4 -

  Fig. S4. Current model of distal tip organization. The Id2⁺ distal tip population at the pseudoglandular stage gives rise to all epithelial cell types of the proximal airways (Clara, ciliated, NE). The pseudoglandular Id2⁺ cells also self-renew and give rise to the canalicular stage Id2⁺ distal tip epithelium, which contributes descendents to the alveolar lineages. Important questions that still need to be answered include: is the tip population heterogeneous at the single cell level? Can the canalicular stage progenitors still generate bronchiolar descendents if given the right conditions?

  References

  Hua, H., Zhang, Y. Q., Dabernat, S., Kritzik, M., Dietz, D., Sterling, L. and Sarvetnick, N. (2006). BMP4 regulates pancreatic progenitor cell expansion through Id2. J. Biol. Chem. 281, 13574-13580.

  Russell, R. G., Lasorella, A., Dettin, L. E. and Iavarone, A. (2004). Id2 drives differentiation and suppresses tumor formation in the intestinal epithelium. Cancer Res. 64, 7220-7225.

  Samanta, J. and Kessler, J. A. (2004). Interactions between ID and OLIG proteins mediate the inhibitory effects of BMP4 on oligodendroglial differentiation. Development 131, 4131-4142.