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1 Supporting Information Song et al /pnas SI Materials and Methods Mice. To produce the GRP reer mouse line, the translational start TG in the mouse GRP genomic locus was replaced with a reer-frt-pgk-neo-frt cassette through gene targeting in E14 ES cells. The FRT-PGK-Neo-FRT cassette was subsequently removed by crosses with FLPe mice. p53 floxed [FVB.129- Trp53 tm1brn ] and Rb floxed [FVB;129-Rb1 tm2brn ] mice were obtained from the National ancer Institute Mouse Repository. Shh-re [B6.g-Shh tm1(egfp/cre)jt /J], Nkx2.1-re [57BL/6J-Tg (Nkx2-1-cre)2Sand/J], Pten floxed [;129S4-Pten tm1hwu /J], RO- S26 mtmg [STOK Gt(ROS)26Sor tm4(tb-tdtomato-egfp)luo /J], R26R [B6;129S4-Gt(ROS)26Sor tm1sor /J], and ROS26 egfp-t (abbreviated as ROS26 T in this study) [STOK Gt(ROS) 26Sor tm1(t)jpmb /J] mice were obtained from Jackson Laboratory. The Institutional nimal are and Use ommittee at the University of alifornia, San Francisco, approved all experiments performed in this study. Tamoxifen and Naphthalene dministration. Tamoxifen (TM; Sigma) was dissolved in Mazola corn oil to make a stock solution of 50 mg/ ml. For embryonic lineage tracing, pregnant female mice were injected intraperitoneally (i.p.) with a single dose of TM (0.25 mg/g body weight). For lineage tracing in adults, TM was injected into adult mice daily for 4 d. Lungs were collected for analysis at indicated time points. For naphthalene-induced lung injury, naphthalene (Sigma) was dissolved in corn oil at 50 mg/ml. dult mice were first injected with TM every other day for four times. The next day following the last dose of TM, mice were injected i.p. with naphthalene (275 mg/kg body weight). Lungs were collected for analysis at indicated time points. t least three animals were analyzed for each genotype. Histology, Immunohistochemistry, and β-gal Staining. Mouse lungs were inflated with 1 ml of 2% paraformaldehyde (PF) and fixed for 4 h at 4 in 2% PF. fter dehydration and processing, lungs were embedded in paraffin for sectioning and immunohistochemistry. ll of the tissues collected were sectioned at 6 μm thickness for histological analysis. Paraffin sections were stained with the following primary antibodies: chick anti-gfp (1:200; bcam), rabbit anti-calcitonin gene-related peptide (GRP, 1:400; Sigma), goat anti-lara cell 10-ka protein (, 1:200; Santa ruz), rabbit anti-prosurfactant protein (SP-, 1:400; Millipore), rabbit anti- synaptophysin (, prediluted; Life Technologies), mouse anti-scl1 (Mash1) (1:200; B), mouse anti-ki67 (1:200; B), mouse anti-acetylated (c)-tubulin (1:1,000; Sigma), and hamster anti-t1α (1:200; evelopmental Studies Hybridoma Bank). Secondary antibodies and conjugates used include donkey anti-chick ylight 488 (1:1,000; Jackson ImmunoResearch Laboratories), donkey anti-rabbit lexa Fluor 594 (1:1,000; Life Technologies), donkey anti-goat lexa Fluor 594 or 647 (1:1,000; Life Technologies), donkey anti-mouse lexa Fluor 594 or 647 (1:1,000; Life Technologies), and PI (1:10,000; Sigma). For biotinylated secondary antibodies (goat anti-rabbit, 1:1,000; donkey anti-goat, 1:1,000; and horse antimouse, 1:1,000; Jackson ImmunoResearch Laboratories), the signal was detected using HRP-conjugated streptavidin (1:1,000; Vector Laboratories) in combination with either the chromogenic substrate B (Vector Laboratories) or fluorogenic substrate lexa Fluor 594 tyramide (1:200, TS kit; Perkin-Elmer). ntibodies against scl1 (Mash1), Ki67, and may require the biotin-streptavidin amplification step for optimal signal detection. Fluorescent images were acquired using a SPOT 2.3 camera connected to a Nikon E1000 epifluorescence microscope. djustment of red/green/blue histograms and channel merges were performed using SPOT dvanced software or ImageJ. onfocal images were captured on a Leica laser-scanning confocal microscope. Multiple optical sections were obtained to distinguish cell boundaries. For X-gal staining, lungs were inflated with 1 ml of 1% PF and 0.2% gluteraldehyde and fixed 1 h at 4 in 1% PF and 0.2% gluteraldehyde. To detect β-gal activity, whole lungs or frozen sections (10 μm) were stained in X-gal staining solution following standard procedures. nalysis of ell Proliferation. In Fig. 2H, in which proliferating GRP + cells were quantified after naphthalene-induced lung injury, three mice were analyzed at each time point. total of 326 GRP + cells at day 0, 182 GRP + cells at day 3, and 245 GRP + cells at day 6 were characterized. The data (ratio of Ki67 + GRP + /GRP + ) were presented as mean ± SEM. P < by two-tailed t test. In Fig. 4L, in which proliferating GRP + cells were quantified after removal of tumor suppressors, three mice were analyzed at each time point for a given genotype. GRP + cells characterized in this study included 200 double-knockout (KO) and 200 triple-knockout (TKO) cells at 1 wk; 312 KO and 534 TKO cells at 1 mo; and 356 KO and 1,023 TKO cells at 2 mo. The data (ratio of Ki67 + GRP + /GRP + ) were presented as mean ± SEM. P < by two-tailed t test. Model of Pulmonary Neuroendocrine ell Specification and Function uring Lung Homeostasis and Injury. Lineage tracing during embryogenesis suggests that pulmonary neuroendocrine cells (PNEs) share a common lineage with alveolar cell types (type I and type II pneumocytes) from either common or separate progenitors (Fig. 1 P and Q). lonal analysis will provide further insight into this process. uring lung injury, PNEs proliferate and can give rise to lara cells and ciliated cells in addition to new PNEs (Fig. 3P),whereaslossofPNEshasnoimpactonlaracellregeneration (Fig. 3P). ccumulation of mutations in PNEs leads to progressive acquisition of carcinoid or small-cell lung cancer properties (Fig. S6). In a recent study, Li and Linnoila (1) reported that scl1 + cells can contribute to airway and alveolar cells during embryogenesis and produce lara, ciliated, and alveolar cells following naphthalene-induced injury. Further study will shed light on the relationship between the GRP + and scl1 + populations. 1. Li Y, Linnoila I (2012) Multi-directional differentiation of scl1-defined progenitors in lung development and injury repair. m J Respir ell Mol Biol, /rcmb O. 1of5

2 iliated cell c-tubulin B E SP- F T1 lara cell TII cell TI cell Fig. S1. Major epithelial cell types in the mammalian lung. ( F) Immunostaining of lung sections from wild-type (WT) adult mice to label distinct epithelial cell types, including ciliated cells (c- tubulin), PNEs in neuroepithelial bodies (s; ), lara cells (), type II cells (SP-), and type I cells (T1α). rrows in each panel points to the indicated cell type, which displays characteristic properties. (Scale bar: 75 μm.) ommon exons alcitonin-specific exon GRP-specific exons TG Wt mouse GRP locus TG 1 reer T2 tp Targeted GRP reer locus GRP reer/+ ; ROS26 mtmg/+ B +tamoxifen egfp (mtmg) anti-grp merged E +tamoxifen F G H tamoxifen I J Fig. S2. GRP reer mice confer spatial and temporal control of gene activities in PNEs. () Schematic diagram depicting gene targeting of a reert2tp cassette at the translational start (TG) of the mouse alcitonin/grp genomic locus. The resulting allele is designated GRP reer in this study. GRP reer insertion disrupts the production of alcitonin/grp mrn, but homozygous GRP reer mice are viable and exhibit no obvious phenotypes similar to previous reports of alcitonin/grp knockout mice (1). (B J) Immunostaining of lung sections from adult GRP reer/+ ;ROS26 mtmg/+ mice. re activation upon TM administration resulted in egfp expression (detected by anti-gfp antibodies) from the ROS26 mtmg allele by removing sequences that block its expression. Efficient activation of reer from the GRP locus was observed as judged by the exclusive presence of egfp signal (green) in PNEs after TM administration to adult mice. Nearly every PNE in the was labeled by egfp (B and E). PNEs were identified by anti-grp (blue;, F, and I) as well as other antibodies (not shown). No egfp signal was detected in mice without TM injection (H J). (Scale bar: 35 μm.) 1. Hoff O, et al. (2002) Increased bone mass is an unexpected phenotype associated with deletion of the calcitonin gene. J lin Invest 110(12): of5

3 egfp B anti-sm PI merged P1 E F G H P1 I J K L P30 Fig. S3. Some muscle cells (likely cardiomyocytes) in the pulmonary veins were labeled in GRP reer mice. ( L) Immunostaining of lung sections from GRP reer/+ ;ROS26 mtmg/+ mice at postnatal day 1 (P1) ( H) or P30 (I L). TM was injected at 13.5 days postcoitum (dpc). Some muscle cells [smooth muscle actin (SM + )] ( H) labeled by egfp at P1 were subsequently found in the pulmonary veins (I L). Similar results were obtained when tamoxifen was administered before 15.5 dpc. These egfp-labeled cells (I) in adults no longer express SM; instead, they display characteristic histological features, including striations reminiscent of cardiomyocytes. ardiomyocytes are well documented to be present in pulmonary veins between heart and lung (but not in intrapulmonary veins). (Scale bar: 75 μm.) egfp B egfp Brain Brain Thyroid E F G Intestine H Skin Mammary gland Kidney drenal medulla Fig. S4. etection of GRP reer activity in various mouse tissues. ( H) Immunostaining of sections from adult GRP reer/+ ;ROS26 mtmg/+ mice injected with TM postnatally. egfp signals can be detected in several mouse tissues other than the lung. For instance, several neurons and fibers in the brain were labeled. Neurons and fibers in and B are located near the cerebral cortex, as indicated by the boxed area in Inset. In addition, parafollicular cells in the thyroid, and scattered cells in the intestine, skin, mammary gland, kidney, and adrenal medulla, also displayed GRP reer activity. scl1 reer is more broadly expressed, at least in the nervous system, than GRP reer. It would be interesting to compare results of lineage and functional studies on PNEs using scl1 reer and GRP reer. (Scale bar: 25 μm.) 3of5

4 GRP reer/+ ; ROS26 T/+ B ROS26 T/+ 2.5 mon post-t activation GRP reer/+ ; ROS26 T/+ ROS26 T/+ GRP GRP E GRP reer/+ ; ROS26 T/+ F ROS26 T/+ c-tubulin c-tubulin Fig. S5. Efficient and selective ablation of PNEs by diphtheria toxin (T) expression. ( F) Immunostaining of lung sections from adult GRP reer/+ ; ROS26 T/+ mice. ROS26 T/+ mice serve as controls. ctivation of reer by TM in adult lungs induced T expression, resulting in efficient cell killing. PNEs or s could not be detected in these animals (), whereas other cell types were unaffected ( and E). We examined over 100 sections of each lung to cover different regions, and it was very difficult to find any GRP + or + cells. We did find occasional solitary PNEs in GRP reer/+ ;ROS26 T/+ lungs, likely due to incomplete ablation and not a result of PNE regeneration because the occurrence of residual PNEs is similar (1 PNE per 20 sections) at any given time point after ablation. We failed to detect any PNE clusters 1 y after PNEs ablation. istribution of cell types in ROS26 T/+ mice (B,, and F) is similar to that in WT lungs. The epithelial cells were identified by a battery of cell type-specific markers, such as (PNE), GRP (PNE), (lara cell), and c-tubulin (ciliated cell) shown here. (Scale bar: 75 μm.) Wild-type thyroid p53 ;Rb 2.5 mon post-tm p53 ;Rb ; Pten / 2 mon post-tm E B F G PNE Hyperplasia Transformation PNE (carcinoid or SL) Parafollicular cells Hyperplasia Transformation Medullary thyroid tumor Fig. S6. evelopment of thyroid tumors from GRP + cells in the thyroid. ( F) Histology of thyroid sections from WT, GRP reer/+ ;p53 f/f ;Rb f/f, and GRP reer/+ ; p53 f/f ;Rb f/f ;Pten f/f adult mice injected with TM postnatally. total of 32 GRP reer/+ ;p53 f/f ;Rb f/f adult mice and 17 GRP reer/+ ;p53 f/f ;Rb f/f ;Pten f/f adult mice were used in this analysis. ll mice developed thyroid tumors. Normal thyroid architecture is destroyed, which could lead to thyroid dysfunction. TM-injected GRP reer ;p53 f/f ;Rb f/f and GRP reer ;p53 f/f ;Rb f/f ;Pten f/f mice developed lung and thyroid tumors but not other tumors at the time of analysis. No tumors were found in the lymph nodes adjacent to the thyroid or tissues adjacent to the thyroid. These observations suggest that lung and thyroid tumors represent primary tumors, and there is no distant metastasis from thyroid or lung tumors at the time of our analysis. These mice died most likely from a combination of respiratory failure and thyroid dysfunction, including lung and thyroid tissue destruction, obstruction of the trachea by the thyroid tumor, and adverse effects from secreted amines and peptides from the tumors. (Scale bars: //E and B//F, 100 μm.) (G) model of tumor development by disruption of tumor suppressors in GRP + cells in different tissues. 4of5

5 Table S1. Time of TM administration Percentage of lineage-labeled lung epithelial cell types after TM injections at indicated time during embryonic development Labeled neuroendocrine cells, GRP + Labeled lara cells, + Labeled ciliated cells, c-tubulin + Labeled type II cells, SP- + Labeled type I cells, T1α + E11.5 0% 0% 0% 0.2 ± 0.1% (7 ± 3%) 1.2 ± 0.4% (92 ± 3%) E ± 10% (15 ± 6%) 0% 0% 0.2 ± 0.07% (6 ± 4%) 1.1 ± 0.3% (77 ± 4%) E ± 11% (21 ± 3%) 0% 0% 0.12 ± 0.3% (7 ± 2%) 0.5 ± 0.1% (70 ± 4%) E ± 7% (33 ± 4%) 0% 0% 0.09 ± 0.04% (5 ± 2%) 0.34 ± 0.2% (61 ± 5%) E ± 9% (100%) 0% 0% 0% 0% P0 66 ± 13% (100%) 0% 0% 0% 0% Lungs were collected at postnatal day 30 for analysis. The numbers in parentheses indicate the percent of egfp-labeled cells that generated a given cell type. Table S2. Post-TM administration Percentage of lineage-labeled lung epithelial cell types at different time points after four doses of TM injection in adult mice Labeled neuroendocrine cells, GRP + Labeled lara cells, + Labeled ciliated cells, c-tubulin + Labeled type II cells, SP- + Labeled type I cells, T1α + 3d 75± 7% 0% 0% 0% 0% 6mo 70± 6% 0% 0% 0% 0% 1y 61± 9% 0% 0% 0% 0% 5of5