Lung development and repair: Contributionof the ciliated lineageEmma L. Rawlins*, Lawrence E. Ostrowski†, Scott H. Randell†, and Brigid L. M. Hogan*‡

*Department of Cell Biology, Duke University Medical Center, Durham, NC 27710; and †Cystic Fibrosis/Pulmonary Research and Treatment Center,University of North Carolina, Chapel Hill, NC 27599

This contribution is part of the special series of Inaugural Articles by members of the National Academy of Sciences elected on May 3, 2005.

Contributed by Brigid L. M. Hogan, December 5, 2006 (sent for review November 15, 2006)

The identity of the endogenous epithelial cells in the adult lungthat are responsible for normal turnover and repair after injury isstill controversial. In part, this is due to a paucity of highly specificgenetic lineage tools to follow efficiently the fate of the majorepithelial cell populations: the basal, secretory, ciliated, neuroen-docrine, and alveolar cells. As part of a program to address thisproblem we have used a 1-kb FOXJ1 promoter to drive CreER in theciliated cells of the embryonic and adult lung. Analysis of FOXJ1-GFP transgenic lungs shows that labeled cells appear in a proximal-distal pattern during embryogenesis and that the promoter drivesexpression in all ciliated cells. Using FOXJ1CreER adult mice, wehave followed the fate of ciliated cells after epithelial injury bynaphthalene or sulfur dioxide. From quantitative analysis andconfocal microscopy we conclude that ciliated cells transientlychange their morphology in response to lung injury but do notproliferate or transdifferentiate as part of the repair process.

ciliated cell � injury � lung stem cells

W ith each breath we take, air passes through the highlybranched epithelial tubes of the respiratory tree into the

terminal alveoli, where gas exchange takes place. Under normalconditions, cell turnover in the lung is relatively low, at leastcompared with tissues such as the intestine and skin. Neverthe-less, if the epithelium is extensively damaged, the lung caneffectively replace lost cells. Identifying the stem or progenitorcell populations responsible for this repair is an active area ofresearch not lacking in controversy (reviewed in refs. 1 and 2).In addition, compared with organs such as the pancreas andnervous system, we have a rather elementary view about how themajor epithelial cell types of the lung are generated in theembryo (3). Because the repair of adult tissues may involve, atleast in part, the recapitulation of developmental mechanisms,discoveries in one area are likely to inform the other.

The lung develops during embryogenesis from two primary budsthat arise in the ventral foregut. These buds undergo extensivebranching morphogenesis, directed by reciprocal signaling betweenthe epithelial endoderm and the surrounding mesoderm. Duringthis branching stage it is thought that the terminal buds contain apopulation of multipotent epithelial progenitors (4). As the tubesextend, descendants of these cells give rise to the progenitors of themajor cell types of the conducting airways—certainly to the ciliatedand secretory (Clara) cells (5). The appearance of morphologicallydifferentiated epithelial cells begins proximally and proceeds dis-tally (6, 7). We need to know a great deal more about the stepsinvolved in generating these differentiated cells: the identity ofintermediate cell types, whether these intermediates self renew astransit amplifying populations, their lineage relationships, and theprecise mechanism of their commitment to different fates. Oncemorphogenesis is complete, the lung continues to increase in size.During this phase, new epithelial cells are generated in the intralob-ular airways, alveoli, and trachea. There is evidence that during thisphase, cells expressing Clara cell markers can give rise to ciliatedcells (8, 9). Moreover, in the proximal airways, columnar epithelial

cells give rise to basal cells (10). Once formed, basal cells canself-renew and contribute to other lineages, but differentiatedciliated cells do not appear to divide (10, 11).

A number of experimental systems are used for studying repairprocesses in the adult lung. In all cases, the response of theepithelium varies with the region being studied (proximal versusdistal airways) and mouse strain and sex. One widely used injurymodel is naphthalene exposure, which destroys most of the secre-tory Clara cells of both the proximal and distal conducting airways(12, 13). Studies have identified two cell populations that proliferatein response to this injury. These are the basal cells in the trachea andprimary bronchi, and naphthalene-resistant Clara cells in the moredistal bronchi, bronchioles and the bronchoalveolar duct junction(BADJ) (14–18). The evidence that the naphthalene-resistantClara cells give rise to other lineages after proliferation is indirect.First, repair does not occur if all Scgb1a1� cells are ablated,including both the naphthalene-sensitive Clara cells and the naph-thalene-resistant Clara (putative stem) cells (16). Second, cellsisolated from the BADJ can give rise to several differentiated celltypes when cultured in vitro (18). By contrast, the evidence thatbasal cells in the upper airways can both proliferate and function asstem cells after naphthalene injury is based on in vivo lineage-labeling (14, 15). In addition, indirect evidence that the populationof basal cells in the upper airway and submucosal gland (SMG)ducts includes label-retaining, putative stem cells comes from aninjury model using high doses of sulfur dioxide (SO2) gas thatdestroys most of the epithelial cell layer (19).

Recently, based on morphological and immunohistochemicaldata, it has been suggested that ciliated cells can proliferate andtransdifferentiate into Clara cells in response to naphthaleneinjury or partial pneumonectomy (20). This result is surprisingbecause ciliated cells are generally considered to be terminallydifferentiated and nonproliferative (for example, see refs. 21–23). However, there is morphological evidence for transdiffer-entiation of ciliated cells into mucus secreting cells in murinemodels of asthma and viral infection (24, 25).

One way to follow cell fate during lung development, ho-meostasis, and repair is to use genetic lineage-labeling tech-niques to indelibly mark the descendants of specific cell types. Inthis method, the daughter cells express the lineage markerwhatever phenotype they acquire. Moreover, the genetic lineage-labeling tools can be designed to have other advantages; they canbe used to isolate progenitor cells and their daughters for geneexpression analysis to investigate the mechanisms of cell fate

Author contributions: E.L.R., S.H.R., and B.L.M.H. designed research; E.L.R. performedresearch; L.E.O. contributed new reagents/analytic tools; E.L.R. analyzed data; and E.L.R.and B.L.M.H. wrote the paper.

The authors declare no conflict of interest.

Freely available online through the PNAS open access option.

Abbreviations: BADJ, bronchoalveolar duct junction; CGRP, calcitonin gene-related pep-tide; En, embryonic day n; SMG, submucosal gland.

‡To whom correspondence should be addressed. E-mail: b.hogan@cellbio.duke.edu.

© 2006 by The National Academy of Sciences of the USA

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specification and differentiation, and they can be exploited forfunctional studies based on gene inactivation and overexpres-sion. Such tools have so far only been applied in a very limitedway to the lung. Moreover, they have sometimes been used in theabsence of a completely cell type-specific promoter, makinginterpretation difficult (5, 8, 14, 15, 26). A long-term goal in thefield is thus to engineer mice for efficient lineage-labeling ofeach of the different airway epithelial cell types.

Here, we use two transgenic mouse strains to investigate ciliatedcells and their precursors during lung development and repair afterinjury. Both strains use a promoter region of FOXJ1, a gene thatencodes a forkhead domain transcription factor required for latestages of ciliogenesis (27, 28). Our analysis of FOXJ1-GFP embry-onic lungs provides a unique overview of the appearance of ciliatedcells during development. Moreover, the FOXJ1-GFP transgene isultimately expressed in virtually all of the ciliated cells of the adultlung. We have also made a new transgenic mouse strain in which thesame promoter directs expression of a tamoxifen-inducible allele ofthe Cre DNA recombinase (CreER2T). This allele allows us toindelibly mark both ciliated cells and any of their descendents in vivoafter two different kinds of injury: exposure to naphthalene andsulfur dioxide.

ResultsCiliated Cells in Lung Development. Previous studies have shownthat a 1-kb fragment of the human FOXJ1 upstream promoterregion is sufficient to direct expression of GFP in ciliated cellsof the adult mouse, including the trachea and lungs (29). Weused 100-�m vibratome sections and confocal microscopy ofthese FOXJ1-GFP transgenic mice to qualitatively describe theappearance of ciliated cells throughout the trachea and intralob-ular airways during development. At embryonic day (E) 13.5,there was no detectable GFP expression from the transgene (Fig.1A). By E14.0, a small number of scattered GFP� cells werevisible in the trachea and primary bronchi (data not shown). Theappearance of these GFP� cells clearly proceeded in a proximal-distal sequence. Thus, at E14.5, the primary and most proximalintralobular bronchi had many GFP� cells (Fig. 1B), but only afew GFP� cells were seen at lower airway generations and noneat all were seen in the most distal tubes undergoing branching(Fig. 1C). This gap between GFP� proximal regions and theterminal tubules was still seen at E15.0 and E15.5 (Fig. 1 D andE). E15.5 was the first time at which we were able to detect theClara cell marker, Scgb1a1 (secretoglobin 1a1, also known asCC10 and CCSP) by immunostaining. The Scgb1a1� cells wereinterspersed with the GFP� cells. At E16.5, the lung was stillincreasing in size, but GFP� cells now extended to the BADJ(Fig. 1F). E16.5 was the first time that we detected, by immu-nohistochemistry, the neuroendocrine cell marker CGRP (cal-citonin gene-related peptide). It was also the first time, inagreement with other studies, when surface cilia were detected,using antibodies to �-tubulin (6, 30). It was now possible toobserve, as previously described in human embryonic lungsamples, that some of the CGRP� neuroendocrine bodies aresurrounded by Scgb1a1� Clara precursors, whereas others areringed by a mixture of ciliated and Clara precursor cells (31). AtE16.5, in contrast to the earlier stages, there was no significantpopulation of epithelial cells that did not express one of the threemarkers: GFP, Scgb1a1, and CGRP. Taken together, theseresults confirm that morphological differentiation of the airwayepithelium occurs in a proximal-distal sequence. It is importantto note that by E17.5 and later, FOXJ1-GFP is apparentlyexpressed in all of the ciliated cells in the lung and trachea (Fig.1 G and J). Moreover, the presence of GFP� cells before theappearance of surface cilia suggests that the 1-kb promoterregion is able to drive transgene expression in precursors ofciliated cells that are not yet fully differentiated, as judged by thepresence of surface cilia.

To assess whether the FOXJ1� ciliated cell precursors candivide, we labeled cells in S-phase with BrdU at E15.0 whenFOXJ1-GFP is expressed but surface cilia are not yet detectable.We used immunofluorescence of paraffin sections to detect GFPor FoxJ1 protein and BrdU, and then analyzed the lungs byconfocal microscopy to accurately determine cell boundaries.After a 1-h BrdU pulse, we scored 1,991 BrdU� cells and 499FoxJ1� cells and found only 1 (BrdU�, FoxJ1�) cell (n � 8embryos) (Fig. 1H). Moreover, we were unable to detect FoxJ1�

cells that also express the G2/M-phase marker phosphorylatedhistone H3 (data not shown). We repeated the BrdU analysis atpostnatal stages 2–3 weeks, when the rate of proliferation of lungcells is dramatically decreased. We counted 108 BrdU� cells and1,142 FoxJ1� cells and found 0 (BrdU�, FoxJ1�) cells (n � 5lungs) (Fig. 1I). Taken together, these findings suggest that onceciliated cell precursors express detectable levels of FoxJ1, theyeither do not proliferate or have a significantly longer cell cycletime than other dividing lung epithelial cells.

FOXJ1CreER2T Mouse Strain. We made transgenic mice expressingCreER2T under the control of the same FOXJ1 promoter fragment(Fig. 2A). Crossing the FOXJ1CreER2T mouse line to the Rosa26R(Gt(ROSA)26Sortm1Sor) reporter strain and exposing the adultoffspring to tamoxifen gave widespread activation of the heritablereporter gene in epithelial cells throughout the airways (Fig. 2 B andC). Sections were stained for �-tubulin, which strongly labels cilia,to confirm that recombination of the reporter gene was restrictedto ciliated cells (Fig. 2 D and E). For each experiment, theproportion of ciliated cells that were lineage-labeled varied from50% to 70%, depending on the dose of tamoxifen. This level ofrecombination compares favorably with that seen in similar studiesusing other CreER transgenes (see Discussion). Injection of theFOXJ1CreER2T; Rosa26R mice with corn oil vehicle did not resultin detectable expression of the reporter gene (Fig. 7 B and G),demonstrating that Cre activation is not leaky.

Behavior of Ciliated Cells in Response to Naphthalene Injury. Previousreports have demonstrated that the severity of naphthaleneinjury depends on the strain and sex of the mice used (32, 33).As expected, a 250 mg/kg dose of naphthalene was sufficient tokill the majority, but not all, of the Clara cells in the bronchioles(Fig. 3 A–D). Analysis of cell proliferation showed that in malemice, DNA replication begins throughout the airways at 52 hafter the injury and continues for several more days (Fig. 3 E andF and data not shown). In female mice, the injury was moresevere, and proliferation began slightly later (data not shown).We used both male and female mice in our experiments to elicita range of repair responses and so increase the scope of ouranalysis. However, no significant difference was seen in thebehavior of ciliated cells between the sexes. Importantly, at thisdose of naphthalene, Clara cells were destroyed at all airwaylevels including the tracheobronchial region and the bronchiolar/BADJ level (data not shown). Our analysis included ciliated cellsin all of these different airway regions. However, the lineage-labeled ciliated cells in different regions of the airways did notbehave differently and are therefore described together.

To assess the fate of the ciliated cells after naphthalene injury,we exposed male and female FOXJ1CreER2T; Rosa26R mice totamoxifen and then, at least 1 week later, to naphthalene. Theoverall extent of labeling of ciliated cells throughout the airwaysbefore injury in these experiments was 50% [4,047 of 8,103ciliated cells (n � 5 mice in two independent experiments)]. Ciliacould not be detected on 0.8% of lineage-labeled cells; these maybe Foxj1-expressing precursors of ciliated cells generated duringsteady-state turnover. In addition, 1% of lineage-labeled cellscoexpressed the Clara cell marker Scgb1a1. Morphologically,these cells did not have cilia [11 of 1,599 X-gal� cells (n � 3 mice

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in two independent experiments); in contrast, 2,943 (X-gal�,Scgb1a1�) cells were also scored].

Lineage-labeled ciliated cells survived the naphthalene expo-sure but became flattened (Fig. 4 A and B). Squamation ofciliated cells has previously been observed in response to naph-thalene injury (12, 32).

We assessed proliferation at 52, 60, and 72 h after the injury whenlevels of BrdU incorporation are highest. Lineage-labeled cells weredetected by X-gal staining or with anti-�-galactosidase, and anti-body staining was performed to score proliferating (BrdU�) cells.Images were captured on a laser scanning confocal microscope,analyzed in more than one z-section plane to clearly determine cellboundaries, and the number of proliferating (BrdU�) lineage-

labeled (�-gal�) cells was scored. Overall, we counted 11,270 cellsfrom 14 animals in three independent experiments. We scored2,081 BrdU� cells and 9,188 �-gal� cells and found only 1 cell thatwas both (BrdU�, �-gal�). Moreover, in experiments where sur-viving Scgb1a1� Clara cells were detected, 93% of the BrdU� cellswere also Scgb1a1� [85 of 91 BrdU� cells (n � 7 mice); Fig. 4C] inthe intralobular airways and terminal bronchioles. This is consistentwith previous reports that identified the dividing cells in distalregions of naphthalene-injured lungs as naphthalene-resistantClara cells and neuroendocrine cells (16, 21, 34). In the trachea,Scgb1a1 was not detectable on the proliferating cells, which insteadhad the morphology of basal cells (Fig. 4D and data not shown).This is consistent with previous reports which demonstrated that

Fig. 1. Ciliated cell patterning during development. (A–G) One hundred-micrometer vibratome sections of FOXJ1-GFP transgenic lungs. (A–E) Green,FOXJ1-GFP; red, anti-Scgb1a1; blue, anti-E-cadherin. (A) E13.5 mainstem-bronchi. FOXJ1-GFP is not yet expressed (green staining in the mesenchyme isnonspecific). (B) E14.5 mainstem-bronchus. FOXJ1-GFP is expressed in many cells. (C) E14.5 distal bronchiole from the same lung. FOXJ1-GFP-expressing cells aremore scattered and are absent from distal tubules (*). (D) E15.0 lung lobe. FOXJ1-GFP is expressed in scattered cells in the bronchioles but does not extend tothe branching tips (brackets). (E) E15.5. FOXJ1-GFP is expressed in more cells but still does not extend to the branching tips (brackets). Scgb1a1 is first detectableat this stage by immunostaining. (F) E16.5. Green, FOXJ1-GFP; red, anti-Scgb1a1; blue, anti-CGRP. FOXJ1-GFP expression extends to the terminal bronchioles(arrows). Almost every epithelial cell expresses one of the three markers, but no cell expressing more than one marker was detected (the small amount of yellowlabeling in the image is a result of boosting Scgb1a1 detection to visualize the more weakly expressing cells). Note that some CGRP-expressing cells are surroundedby clusters of Scgb1a1� cells (*). (G) E17.5. FOXJ1-GFP (green) colocalizes with anti-�-tubulin, a marker of surface cilia (red). (H) E15.0. Seven-micrometer paraffinsection through an intralobular airway after 1 h of BrdU exposure. Green, anti-BrdU; red, anti-FoxJ1. The BrdU label does not colocalize with FoxJ1. (I) FOXJ1-GFPpostnatal stage 3 weeks; 7-�m paraffin section through a bronchiole after 1 h of BrdU exposure. Green, anti-GFP; red, anti-BrdU. Proliferating cells (arrows) donot colocalize with FoxJ1. (J and J�) Whole-mount view of adult FOXJ1-GFP (green in J) bronchiole stained with tomato lectin (grayscale in J�) to visualize cellsurfaces including cilia. The merged image (J�) shows that FOXJ1-GFP is expressed in all of the ciliated cells. (Scale bars: A–C, 100 �m; D–G, 200 �m; H, 20 �m;I and J, 40 �m.)

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basal cells can act as multipotent progenitors after naphthaleneinjury (14, 15). Our comprehensive survey of BrdU� cells duringthe peak period of proliferation leads us to conclude that although

ciliated cells change their shape in response to naphthalene injury,they do not divide. Therefore, they are neither self-renewing norfunctioning as progenitor cells in this injury model.

To test the hypothesis that ciliated cells can transdifferentiateto another epithelial cell type without undergoing cell division,we killed animals 2–3 weeks after the naphthalene injury. At thistime, repair was essentially complete, based on normal epithelialmorphology and the absence of BrdU labeling (Fig. 5 B and C).

Fig. 2. FOXJ1CreER2T transgene efficiently lineage-labels ciliated cells aftertamoxifen injection. (A) FOXJ1CreER2T transgene construct. (B–E) X-gal-stained (blue) FOXJ1-CreER2T; Rosa26R adult airways after tamoxifen injec-tion. (B) Whole-mount ventral trachea. (C) Intralobular airway. (D and E)Sections of anti-�-tubulin-stained trachea (brown) (D) and distal bronchiole(E). Most ciliated cells but no Clara cells (arrows) are lineage-labeled. (Scalebars: B and D, 200 �m; C and E, 20 �m.)

Fig. 3. Characterization of the extent of the naphthalene injury andtiming of proliferation. (A–F) Paraffin sections of bronchiole epitheliumfrom male mice. (A and B) Hematoxylin and eosin staining. (A) Thirty-sixhours after control corn oil injection. (B) Thirty-six hours after naphthalene.Arrowheads mark cells with no attachment to the basal lamina. (C–F)Anti-Scgb1a1 (green), anti-BrdU (red), and DAPI (blue). (C) Fifty-two hoursafter control injection. Clara cells predominate the airway epithelium andno dividing cells are present. (D) Thirty-six hours after naphthalene. Claracells have detached from the basal lamina (arrowheads), but small numbersare retained at the BADJ (arrows) and at intervals throughout the bron-chioles (data not shown). Cells have not yet started to divide. (E) Fifty-twohours after naphthalene injection. Dividing epithelial cells are observed;they are either Scgb1a1� (arrows) or Scgb1a1� (arrowheads). (F) Seventy-two hours after naphthalene injection. Dividing Scgb1a1� (arrows) andScgb1a1� (arrowhead) cells are still observed, and many more Clara cellsare also present. (Scale bars: A and B, 20 �m; C–F, 200 �m.)

Fig. 4. Response of ciliated cells to naphthalene injury. (A–D) FOXJ1-CreER2T;Rosa26R 7-�m paraffin sections. (A and B) X-gal-stained (blue) and eosin-stained (pink) bronchioles. (A) Fifty-two hours after control corn oil injection.(B) Fifty-two hours after naphthalene. Domed Clara cells are absent, andX-gal� cells are flattened. (C and C�) Fifty-two hours after naphthalene. Twoconfocal z-sections of the same BADJ. Green, anti-�-gal; red, anti-BrdU; blue,anti-Scgb1a1. Lineage-labeled cells (arrows) have not incorporated BrdU.Proliferating cells are Scgb1a1� (arrowheads). (D) Proximal tracheal section52 h after naphthalene injury. Black cytoplasm, X-gal; red cilia, anti-�-tubulin;green nuclei, anti-BrdU. Lineage-labeled cells have not incorporated BrdU.(Scale bars: 20 �m.)

Fig. 5. Ciliated cells do not transdifferentiate after naphthalene injury.FOXJ1-CreER2T; Rosa26R airways. (A) One hundred days after naphthaleneexposure. Whole-mount image of X-gal-stained (blue), microdissected bron-chiole. The X-gal� cells are tightly clustered and separated by regions ofunlabeled cells. Compare with Fig. 2C. (B–E) Seven-micrometer paraffin sec-tions of bronchioles 3 weeks after naphthalene injury. (B) X-gal (blue) andanti-�-tubulin (brown). Ninety-eight percent of lineage-labeled cells areclearly �-tubulin� (arrows). Domed Clara cells are not lineage-labeled (arrow-heads). Two percent of lineage-labeled cells do not have visible cilia (redarrowhead). (C–E) Single confocal z-sections of lineage-labeled X-gal� cells(black) and Scgb1a1� cells (red). (C and D) Typical bronchioles. Lineage-labeled cells are Scgb1a1� (arrows). (E) One percent of lineage-labeled cellscolabel with Scgb1a1 (arrowheads). (Scale bars: A, 200 �m; B, 40 �m; C, 60 �m;D and E, 20 �m.)

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The lineage-labeled cells were visualized by using X-gal stainingand colabeled either with anti-�-tubulin (cilia) or Scgb1a1 (Claracells) (Fig. 5 B–E). Throughout the airways, 98% of the lineage-labeled cells had visible cilia [4,548 of 4,642 X-gal� cells (n � 7mice in three independent experiments)]. By contrast, of 4,111Scgb1a1� cells scored, none were X-gal�, showing that there wasnot a large-scale transdifferentiation of lineage-labeled ciliatedcells into Clara cells. Only 1% of the lineage-labeled cellsstrongly expressed Scgb1a1 [19 of 1,575 X-gal� cells (n � 8 micein two independent experiments)]. This corresponds very wellwith the 1% of Scgb1a1-expressing lineage-labeled cells ob-served in control animals (see above). We do not know whetherthe (Scgb1a1�, X-gal�) cells also express FoxJ1. However, in theuninjured FoxJ1-GFP transgenic animal, a few GFP� cells arealso Scgb1a1� [4 of 1,162 GFP� cells (n � 1 mouse)]. Afternaphthalene injury, the absence of dividing lineage-labeled cellsor increased numbers of (Scgb1a1�, X-gal�) cells suggests thatthe dual-positive (Scgb1a1�, X-gal�) cells survived the injury butdid not contribute to the repair process. Other studies havesuggested that cells expressing Scgb1a1 and a marker of anotherdifferentiated lineage (type II alveolar or neuroendocrine cells)function as stem cells after naphthalene injury (18, 21). Ourevidence does not support this possibility for the ciliated cells.

Whole-mount analysis of the patterns of X-gal staining in thesmaller bronchi and bronchioles at 2–3 weeks (data not shown) and100 days (compare Figs. 5A to 2C) after naphthalene injuryrevealed regions of largely X-gal� or largely X-gal� epithelium.This indirect evidence is consistent with idea that an X-gal�progenitor cell population proliferates to repair the injury, andin the process the nondividing lineage-labeled cells are pushedtogether.

Behavior of Ciliated Cells in Response to SO2 Exposure. Inhalation ofhigh doses of SO2 has been used to induce cell proliferation inthe trachea to identify label retaining cells (putative stem cells)(19). We exposed adult male mice to 500 ppm SO2 for 3 h. Thiscaused sloughing of large sheets of tracheal epithelial cells (Fig.6 A–C). Repair was rapid, and the epithelium was virtuallyindistinguishable from wild type 2 weeks after the injury. Thisconclusion is based on normal morphology and the absence ofBrdU incorporation (Fig. 8 A–D).

To follow the fate of the lineage-labeled ciliated cells after SO2injury to the trachea, we injected FOXJ1CreER2T; Rosa26R adultmales with tamoxifen and then exposed the animals to SO2 gas. Theoverall extent of lineage-labeling of ciliated cells in these experi-ments was 65% [3,884 of 6,012 ciliated cells (n � 6 mice in threeindependent experiments); Fig. 7A]. Cilia were not visible on 0.4%of lineage-labeled cells. Twenty-four hours after the injury, very fewlineage-labeled cells remained in the proximal trachea (Fig. 7C),and those cells that did survive were located in the ducts of theSMGs (Fig. 7H). The number surviving in the distal half of thetrachea was more variable; either there were no lineage-labeledcells in this region or labeled cells were scattered throughout thesurface epithelium (Fig. 7E). In both cases, there was no apparentincrease in the number of lineage-labeled cells when mice werekilled 2 weeks after the injury (Fig. 7D and data not shown).Moreover, the lineage-labeled cells remained in the SMG ducts(Fig. 7 H and I) or scattered throughout the luminal epithelium(data not shown). They were not found in patches as might beexpected if they had divided.

To confirm this lack of proliferation, we scored BrdU� cellsat 24 h after the injury, when proliferation is at its peak (Fig. 6D).We could not identify a lineage-labeled ciliated cell that hadincorporated BrdU in either the SMG ducts or the trachealepithelium [0 of 3,667 BrdU� cells; 223 X-gal� cells scored (n �6 mice in two independent experiments); Fig. 8 A and B]. Thisanalysis included animals with many surviving lineage-labeledcells in the distal trachea as well as animals where almost all of

the lineage-labeled cells were killed. The repaired tracheas allcontained the normal complement of ciliated epithelial cells.These were not lineage-labeled (Fig. 8C) and must thereforehave been derived from a different unlabeled epithelial cell type.These data strongly support the idea that after the SO2 injury,the ciliated cells do not function as either a self-renewing or astem cell population.

To test the hypothesis that the ciliated cells transdifferentiatein response to SO2 injury, we assessed which epithelial cell typescarried the lineage-label 2 weeks after the SO2 exposure whenthe epithelium was largely repaired. Ninety-nine percent of thelineage-labeled cells had cilia [548 of 551 X-gal� cells (n � 9 micein three independent experiments); Fig. 8D]. We counted 3,609(Scgb1a1�, X-gal�) cells after repair. Only 0.1% of the lineage-labeled cells expressed the Clara cell marker Scgb1a1 [1 of 870X-gal� cells (n � 7 mice in three independent experiments)].This analysis does not provide any support for the hypothesis thatciliated cells can transdifferentiate into another cell lineage inresponse to SO2 injury.

DiscussionOur results confirm the usefulness of transgenic reporter andlineage tracing mouse strains for investigating basic questions

Fig. 6. Characterization of the extent of SO2 injury and timing of prolifer-ation. (A–C) Transverse tracheal sections. (A) Uninjured control. (B) Six hoursafter SO2 inhalation. Epithelial cells are sloughing off. (C) Twenty-four hoursafter SO2 inhalation. The luminal epithelium consists of a monolayer of lowcuboidal cells. (D) Percentage of epithelial cells (in the most proximal one-third of the trachea) that incorporated BrdU in a 1-h pulse at different timesafter the injury. Bars are the standard error of the mean. Epithelial cellproliferation peaks 24 h after the injury. (Scale bars: 200 �m.)

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related to lung epithelial cell differentiation and repair afterinjury. They also illustrate some of the limitations that have tobe taken into consideration when interpreting the data.

Ciliated Cell Specification and Differentiation During Development.We first detected FOXJ1-GFP expression at E14.0, which agreeswith published data that Foxj1 protein expression begins in theairways around this time (35). Evidence suggests that Foxj1functions in airway cells during late stages of ciliogenesis and isnot sufficient to drive cilia formation in nonciliated adult epi-thelial cells (27, 28). Therefore, FOXJ1-GFP expression in cellswithout cilia most likely marks embryonic cells already commit-ted to the ciliated lineage. Similarly, we find that Scgb1a1 is only

reliably detected by immunohistochemistry in secretory cells asthey begin to differentiate. Our results show that the appearanceof epithelial differentiation markers clearly proceeds in aproximal-distal temporal and spatial sequence, as reported byothers (6, 36). However, using 100-�m vibratome sections tovisualize distal intralobular airways in three dimensions, we wereable to see that morphogenesis does not involve a wave ofdifferentiation that simultaneously enjoins all of the cells at aparticular airway level. Rather, GFP� cells initially appear in ascattered or mosaic pattern, and additional cells are specified asdevelopment progresses. This observation is consistent withpotential models in which Notch signaling is involved in thespecification of the ciliated lineage in the lung (37, 38). However,the field urgently needs earlier markers of Clara and ciliated cellfate to test this hypothesis and to determine exactly how thepattern is set up.

Ciliated Cells in Injury and Repair. We were unable to detectproliferating FoxJ1-positive ciliated cell precursors during devel-opment. Similarly, we have found no evidence that lineage-labeledciliated cells can self-renew, transdifferentiate, or function as stemcells in response to airway injury. This analysis included examina-tion of the effects of naphthalene-mediated destruction of the Claracells in all regions of the conducting airways from the trachea to theBADJ, and of SO2-mediated injury to all epithelial cells in thetracheobronchial epithelium. These results contradict the recentclaims that ciliated cells can transdifferentiate into Clara cells inresponse to naphthalene injury in the bronchiolar regions (22). Weobserved the same cell shape changes in the ciliated cells inresponse to naphthalene injury as Park and colleagues (20). Thissuggests that the extent of the injury did not differ significantlybetween the two sets of experiments. One explanation for thedifferent conclusions is that the method used by Park et al. (20) totrace the fate of ciliated cells after injury (immunohistochemistryfor tubulin) does not provide a precise longitudinal analysis ofheritably marked cells. One potential drawback of our approach isthat we only label 50–70% of the ciliated cells in the adult lung,

Fig. 7. Ciliated cells after SO2 inhalation. Shown are FOXJ1-CreER2T; Rosa26R ventral trachea. Blue, X-gal. (A–E) Whole-mount images of ventral trachealepithelial surface. (F–I) Seven-micrometer paraffin sections taken from the regions between the dotted lines. (A and F) Tamoxifen-treated positive control. X-gal�

cells (arrows) are in the luminal epithelium and SMG ducts (*). (B and G) Negative control (no tamoxifen injections). Low level of endogenous X-gal staining inthe SMG mucus cells (arrowhead). (C and H) Tamoxifen-treated, 24 h after SO2 inhalation. X-gal� cells (arrows) are restricted to the SMG ducts. (D and I)Tamoxifen-treated, 2 weeks after SO2 inhalation. X-gal� cells (arrows) are still restricted to the SMG ducts. (E) Tamoxifen-treated, 24 h after SO2 inhalation. Insome animals, the surviving X-gal� cells are scattered throughout the luminal epithelium, particularly in the distal region. (Scale bars: A–E, 0.5 mm; G–I, 10 �m.)

Fig. 8. Ciliated cells do not divide or transdifferentiate after SO2 exposure.Shown are FOXJ1-CreER2T; Rosa26R X-gal-stained 7-�m paraffin sections ofthe trachea. (A and B) Twenty-four hours after SO2 inhalation. (A) Proximaltrachea with SMG duct. Anti-BrdU (black) and X-gal (blue; arrows) do notcolocalize. (B) Single confocal z-section of distal trachea. Anti-BrdU (red) andX-gal (black) do not colocalize. (C and D) Two weeks after SO2 inhalation.Black, anti-�-tubulin; blue, X-gal. (C) Typical trachea section; ciliated cells havedifferentiated in the repaired epithelium but are not lineage-labeled. (D)Lineage-labeled cells have cilia (arrows). Domed Clara cells have not inheritedthe lineage label (arrowheads). (Scale bars: 20 �m.)

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depending on the dose of tamoxifen. This failure to label all cellsis an inherent limitation of using tamoxifen to stochastically inducetranslocation of Cre into the nucleus and has been seen in manyother lineage tracing studies with adult mice (for example, thosestudies described in refs. 39–41). For this limitation to affect theconclusions of our study it would be necessary to argue thatrecombination only occurs, either by chance or for mechanisticreasons, in a specific subpopulation of ciliated cells that is unable toundergo transdifferentiation. We consider this argument unlikelyfor two reasons. First, we examined a total of 42 mice, which makesit very unlikely that failure to activate recombination occurred in aspecific subpopulation by chance. Second, we show that the FOXJ1promoter is able to drive transgene expression in all ciliated cells,as well as in precursors that do not have surface cilia and maytherefore be more likely to transdifferentiate.

The most parsimonious explanation of our results is thereforethat ciliated cells do not function as progenitors during develop-ment or in two commonly used models of airway injury and repair.However, our findings do not exclude the possibility that ciliatedcells can function as progenitors in response to different injuries orin models of diseases such as acute asthma or viral infection wherethere is morphological evidence that ciliated cells transdifferentiateinto mucous-secreting cells (24, 25). In the future, these possibilitiescan be tested by using our FOXJ1CreER2T mouse line.

Materials and MethodsTransgenic Mice. A CreER2T cassette (39) was inserted intoStuI/MluI restriction sites in the 1,008-bp human FOXJ1 pro-moter vector (29), just 3� of the mouse transthyretin (TTR)intron (Fig. 2 A). Transgenic mice were created by pronuclearinjection into (C57BL/6 � DBA) fertilized eggs. Five indepen-dent FOXJ1CreER2T founder lines were established and tested bymating to Rosa26R (Gt(ROSA)26Sortm1Sor) reporter mice (42)and injecting the adult progeny with tamoxifen (Sigma, St. Louis,MO). Three of the lines gave indistinguishable and efficientrecombination specifically in the ciliated cells of the adult lung.

Mouse Strains. The FOXJ1-GFP mice were maintained by crossingto C57BL/6. Timed matings for embryonic studies were generatedby crossing with the outbred ICR strain (Harlan Sprague Dawley,Indianapolis, IN). For injury/repair experiments, we used C57BL/6(Charles River Laboratories, Wilmington, MA) as wild type andFOXJ1CreER2T and Rosa26R strains at the N2 and N3 C57BL/6backcross generations. FOXJ1CreER2T; Rosa26R animals (8–12weeks) were injected i.p. (intraperitoneally) up to five times (every2–3 days) with 4 mg of tamoxifen in Mazola (Cordova, TN) corn oil.Control mice were injected with corn oil only. At least 1 weekseparated the final tamoxifen injection and airway injury.

Airway Injury and BrdU Exposure. Adult (10–14 weeks) mice wereinjected i.p. with 250 mg/kg naphthalene (Sigma) dissolved inMazola corn oil between 8 and 10 a.m. Adult males were placedin individual compartments of a chamber and exposed to 500ppm SO2 in air for 3 h. To detect cell proliferation, mice were

injected i.p. with 10 �l per g of body weight of a solutioncontaining 3 mg/ml BrdU (Amersham Biosciences, Piscataway,NJ) 2 h (naphthalene-injured) or 1 h (SO2-injured and devel-opmental stages) before sacrifice.

Fixation and X-gal/Lectin Staining. Adult lungs were inflated with1 ml of fixative. Trachea and lungs were placed into 4%paraformaldehyde or 10% formalin for anti-�-galactosidase(anti-�-gal) staining and fixed for 4–6 h at 4°C. The X-gal colorreaction was as described (43). To label the surface of the airwayepithelial cells, we incubated lungs for 20 min with biotin-labeledtomato lectin (Lycopersicon esculentum) (Vector Laboratories,Burlingame, CA) and detected the biotin label using a TSA kitand Cy3-tyramide (PerkinElmer, Wellesley, MA) (6). Embry-onic lungs were fixed 1–2 h at 4°C in 4% paraformaldehyde. Forvibratome sectioning, they were embedded in 4% agarose, and100-�m sections were cut.

Immunohistochemistry. Vibratome sections were stained by using awhole-mount protocol. Paraffin sections were immunostained byusing standard protocols, including controls lacking the primaryantibody. Mouse anti-�-tubulin (1:1,500; BioGenex, San Ramon,CA), rat anti-E-cadherin (1:2,000; Zymed, South San Francisco,CA), and rabbit anti-CGRP (1:1,000; Peninsula Laboratories) wereused without antigen retrieval. Other antibodies required antigenretrieval: 0.05% trypsin digestion for 5 min for goat anti-secretoglobin 1a1 (Scgb1a1) (1:10,000) and rabbit anti-Scgb1a1(1:5,000; both kindly provided by Barry Stripp, University ofPittsburgh, Pittsburgh, PA), 30 min at 37°C in 2 M HCl followed bytrypsin digestion for mouse anti-BrdU (1:200; Sigma) and ratanti-BrdU (1:200; Accurate Chemical, Westbury, NY), boiling for5 min in 100 mM sodium citrate buffer (pH 6) followed by trypsindigestion for rabbit anti-�-gal (1:5,000; Cappel), and 4 h heating at60°C in 100 mM Tris (pH 10) for mouse anti-FoxJ1 (1:500; kindlyprovided by Steve Brody, Washington University, St. Louis, MO).Biotinylated secondary antibodies (1:500; Jackson ImmunoRe-search, West Grove, PA) were amplified with the VECTASTAINElite ABC kit and detected with DAB (Vector Laboratories).Alexa Fluor-coupled secondary antibodies (Invitrogen, Carlsbad,CA) were used at 1:500 dilution.

Confocal Microscopy and Cell Counting. Cells were counted manu-ally. Approximately equal numbers of cells were counted foreach experimental lung. Cells whose apical surface was notvisible because of the plane of the section were not counted. Allimmunofluorescence images used for scoring cells consisted ofa z-series of optical sections captured on a Zeiss LSM 510 Metalaser scanning confocal microscope. Multiple optical sectionswere examined to accurately determine cell boundaries.

We thank W. Michael Foster and Barry Stripp for advice on injury modelsand Rodney Gilmore, Lisa Jones, Wanda O’Neal, and Lindsey Becker fortechnical assistance. Work in the B.L.M.H. laboratory is supported byNational Institutes of Health Grant NIH-080517, and SO2 exposures aresupported by National Institutes of Health Grant ES011961.

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