ARTICLES

A Gata6-Wnt pathway required for epithelial stem cell development and airway regeneration

Yuzhen Zhang1, Ashley M Goss2, Ethan David Cohen1, Rachel Kadzik2, John J Lepore1, Karthika Muthukumaraswamy1, Jifu Yang1, Francesco J DeMayo3, Jeffrey A Whitsett4, Michael S Parmacek1,5,6 & Edward E Morrisey1,2,5,6

Epithelial organs, including the lung, are known to possess regenerative abilities through activation of endogenous stem cell populations, but the molecular pathways regulating stem cell expansion and regeneration are not well understood. Here we show that Gata6 regulates the temporal appearance and number of bronchioalveolar stem cells (BASCs) in the lung, its absence in Gata6-null lung epithelium leading to the precocious appearance of BASCs and concurrent loss in epithelial differentiation. This http://www.nature.com/naturegenetics expansion of BASCs was the result of a pronounced increase in canonical Wnt signaling in lung epithelium upon loss of Gata6. Expression of the noncanonical Wnt Fzd2 was downregulated in Gata6 mutants and increased Fzd2 or decreased b-catenin expression rescued, in part, the lung epithelial defects in Gata6 mutants. During lung epithelial regeneration, canonical Wnt signaling was activated in the niche containing BASCs and forced activation of Wnt signaling led to a large increase in BASC numbers. Moreover, Gata6 was required for proper lung epithelial regeneration, and postnatal loss of Gata6 led to increased BASC expansion and decreased differentiation. Together, these data demonstrate that Gata6-regulated Wnt signaling controls the balance between progenitor expansion and epithelial differentiation required for both lung development and regeneration.

Organ regeneration requires the proper balance between differentia- In adult mammals, lung epithelium undergoes slow homeostatic

Nature Publishing Group Group Publishing Nature tion and self-renewal of tissue-specific progenitor cells. The transcrip- turnover, resulting in the replacement of much of the epithelium after 8 tional and signaling pathways required to direct this balance are largely approximately 4 months in rats6. Both homeostatic turnover and 200 unknown, but it is thought that many of the pathways involved in regeneration after injury are thought to involve endogenous lung © regulating embryonic development are recapitulated in progenitor progenitor cells. The progenitor cell niches within the lung are poorly expansion and tissue regeneration. characterized, but recent studies have identified several stem cell The lung is a complex organ consisting of several distinct epithelial niches located in distinct positions along the proximal–distal axis of and mesodermal cell lineages patterned in a proximal–distal manner, the airways7,8. BASCs represent one of these regional progenitor cell including specialized cell types that produce surfactant populations and are located in the bronchioalveolar duct junction and lipids as well as ones that form the thin gas exchange interface (BADJ) at the terminal ends of distal bronchioles7,9. BASCs are required for postnatal respiration (reviewed in refs. 1,2). Developmen- thought to regenerate both bronchiolar and alveolar epithelium tal defects in this patterning lead to defective differentiation and during homeostatic turnover and in response to injury9. The tran- postnatal respiratory distress, a hallmark of congenital lung disease. scriptional and signaling pathways required for the differentiation and Asmallcoreoftranscriptionfactors,includingGata6,Nkx2.1,Foxa1 expansion in BASCs are largely unknown. Given the proposed and Foxa2, are known to be important for lung airway epithelial requirement for BASCs and other progenitor cells in normal homeo- differentiation and development3–5. Altered expression or activity of static turnover in the lung as well as in regeneration and repair after any of these factors leads to severe defects in airway epithelial injury, characterization of the regulatory mechanisms controlling the differentiation and development. However, it is unclear whether any expansion and differentiation these cells could have a profound of these factors are critical for regulating the balance between differ- impact on our understanding and treatment of lung disease. entiation and progenitor cell development and regeneration in the In this study, we have examined the role of the zinc finger lung. Moreover, the molecular pathways acting downstream of these Gata6 in regulating the balance between factors are largely unknown. lung epithelial differentiation and progenitor cell expansion and

1Department of Medicine and 2Department of Cell and Developmental Biology, University of Pennsylvania, 956 BRB II/III, 421 Curie Boulevard, Philadelphia, Pennsylvania 19104, USA. 3Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas 77030, USA. 4Division of Pulmonary Biology, Cincinnati’s Children’s Hospital and Medical Center, Cincinnati, Ohio 45229, USA. 5Institute for Regenerative Medicine and 6Cardiovascular Institute, University of Pennsylvania, 956 BRB II/III, 421 Curie Boulevard, Philadelphia, Pennsylvania 19104, USA. Correspondence should be addressed to E.E.M. ([email protected]).

Received 18 December 2007; accepted 16 April 2008; published online 8 June 2008; doi:10.1038/ng.157

862 VOLUME 40 [ NUMBER 7 [ JULY 2008 NATURE GENETICS ARTICLES

WT G6 flox:Sftpc-cre WT G6 flox:Sftpc-cre Figure 1 Loss of Gata6 leads to lung epithelial differentiation defects. a g (a–n) Wild-type (WT) and Gata6 flox/flox:Sftpc-cre mutants (G6 flox:Sftpc-cre) d k with hematoxylin and eosin staining (a–f) or immunohistochemical staining for lung epithelial marker proteins (g–n). Gata6 flox/flox:Sftpc-cre mutants SP-C E12.5 showed dilated airways beginning as early as E12.5 (d,e; arrowheads). Gata6 flox/flox:Sftpc-cre mutants showed decreased or absent expression of b e hl SP-C (g,k), CC10 (h,l), SP-B (i,m), and T1a (j,n). However, ectopic * expression of the Clara cell marker CC10 was observed in distal E14.5 CC10 regions of the airways of Gata6 flox/flox:Sftpc-cre mutants (l, arrowheads). Asterisk, a large bronchiolar airway that has reduced CC10 in the mutant. c f i m Scale bars: a,b,d,e,g–n,500mm; c,f,800mm. SP-B E18.5 Gata6 flox/flox:Sftpc-cre mutants that lack Gata6 in airway epithelium11. n The Sftpc-rtTA system has been shown to inducibly express transgenes j both in distal airway epithelium and in bronchiolar epithelium12–14. α

T1 Treatment of pregnant dams from E0.5 to birth with doxycycline resulted in lethality in all Gata6 flox/flox:Sftpc-cre mutants within minutes after birth due to respiratory failure. Examination of Gata6 flox/flox:Sftpc-cre mutants earlier in gestation showed that loss regeneration. Specific ablation of Gata6 in lung epithelium led to a of Gata6 in lung epithelium led to increased airway dilation, which marked loss in airway epithelial differentiation and to neonatal death. could be observed as early as E12.5 (Fig. 1a–f). However, lung size and Unexpectedly, this was associated with the ectopic and premature lobation was normal in Gata6 flox/flox:Sftpc-cre mutants (Fig. 1a–f and appearance of BASCs. Gata6 acts through direct regulation of Fzd2, data not shown). Normal lobe patterning suggests that Gata6 is not which inhibits the canonical Wnt pathway in lung epithelial cells, and required for some of the earliest stages of lung branching. http://www.nature.com/naturegenetics loss of Gata6 led to activation of Wnt–b-catenin signaling in the lung. To determine the extent of epithelial differentiation in Gata6 flox/flox: Postnatal deletion of Gata6 resulted in compromised airway regenera- Sftpc-cre mutants, we performed immunohistochemistry using anti- tion associated with defective BASC expansion and differentiation, bodies recognizing markers of distinct epithelial cell lineages within which also occurred upon postnatal activation of canonical Wnt the lung airways, including (SP-C), a marker of signaling. These studies define a new pathway mediated by Gata6- alveolar epithelial type 2 cells (AEC-2); Clara cell 10-kDa protein Wnt that controls the balance between progenitor or stem cell (CC10), a marker of nonciliated Clara cells of the bronchiolar airways; expansion and epithelial differentiation and regeneration in the lung. (SP-B), which is expressed by both AEC-2 and Clara cells; and T1a, a marker of type 1 alveolar epithelial cells RESULTS (AEC-1). Whereas wild-type mice at E18.5 expressed typical spatial Loss of Gata6 results in precocious appearance of BASCs patterns and amounts of all of these proteins, Gata6 flox/flox:Sftpc-cre

Nature Publishing Group Group Publishing Nature Gata6 was expressed robustly in both the developing distal airway mutants showed little or no expression of these proteins (Fig. 1g–n). 8 epithelium and in the more proximal bronchiolar epithelium (Sup- Notably, sporadic expression of CC10 was observed in cells within the 200 plementary Fig. 1 online and ref. 10). To examine the role of Gata6 in distal airways of Gata6 flox/flox:Sftpc-cre mutants, a region of the lung © lung epithelial development, we crossed mice with a conditional null that normally does not contain Clara cells (Fig. 1l). Thus, loss of loxP-flanked (flox) allele of Gata6 (Gata6 flox/flox) to the Sftpc-rtTA: Gata6 expression led to defective lung epithelial differentiation and tetO-cre bigenic system (hereafter referred to as Sftpc-cre)togenerate ectopic expression of CC10 in distal airway epithelium.

flox Figure 2 Loss of Gata6 in lung epithelium WT G6 :Sftpc-cre g + 70 results in the precocious appearance of BASCs. a b 60 (a–g)ExpressionofSP-CandCC10(a,b)andSP- 50 40 C, CC10 and Sca1 (c–f) in wild-type (WT; a)and 30 Gata6 flox/flox:Sftpc-cre mutants (G6 flox:Sftpc-cre; 20 10

b–f) using double and triple immunofluorescence, other cell markers Percentage CC10 Percentage 0 cells also expressing with quantification of cells positive for various SP-C/CC10 SP-C/CC10 + + + + + + flox/flox combinations (g). Gata6 :Sftpc-cre mutants Sca1 SP-C Sca1 SP-C c + /Sca-1 + /Sca-1 contained many SP-C+CC10+ double-positive d BASCs in airways (c,d, arrows; g), whereas wild- SP-C SP-C flox type lungs did not (a,g). Many of these double- WT G6 :Sftpc-cre positive cells also expressed Sca1 (e,f, arrows). hiWT G6 flox:Sftpc-cre + + + 104 104 In addition to the SP-C CC10 Sca1 triple- SP-C CC10 0.029 2.42 0.012 13.8 + + positive population, a CC10 Sca1 population 103 103 was also upregulated in Gata6 flox/flox:Sftpc-cre e f 102 102

mutants (d–f, arrowheads; g). (h,i)FACSanalysis CC10 using SP-C and CC10 antibodies on both wild- 101 101 flox/flox type (h)andGata6 :Sftpc-cre mutant (i) 90.4 7.14 40.7 45.5 100 100 permeabilized lung epithelial cells at E18.5 Sca1 Overlay 100 101 102 103 104 100 101 102 103 104 showed a substantial increase in BASC numbers in the mutant lungs (2.4% versus 13.8%). Scale SP-C bars: a,b,500mm; c,d,50mm. Error bars, s.e.m. based on a minimum of four embryos.

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WT G6 flox:Sftpc-cre a b e WT h ij WT Medium G6 flox:Sftpc-cre f CC10 Ki-67 CC10/Ki-67 cd kl m Sftpc-cre : Small 45 g 40 flox

35 G6 30 CC10 Ki-67 CC10/Ki-67 cells 25 + 20 flox 15 n 60 WT G6 :Sftpc-cre Ki-67 Percentage 10 + 50 5 op Figure 3 Increased proliferation in bronchiolar 0 40 flox/flox flox Ki-67 epithelium of Gata6 :Sftpc-cre mutants. WT G6 : + 30 Sftpc-cre (a–g) Increased staining for Ki-67 in 20 Percentage Gata6 flox/flox:Sftpc-cre mutant (G6 flox:Sftpc-cre) CC10 10 0 Ki-67/alveolar versus wild-type (WT) lungs in bronchiolar epithelium of both medium- and WT G6 flox: small-caliber airways (a–d; enclosed by dotted lines). This corresponded to a more Sftpc-cre

disorganized appearance in this epithelium, with layers of cells growing on top of q + 7 6 one another (e,f). Quantitation showed an approximately fourfold increase in proliferation in bronchiolar epithelium (g). 5 (h–n) Double immunofluorescence microscopy showed that most of the increased cell proliferation was observed in cells weakly 4 positive for CC10 in Gata6 flox/flox:Sftpc-cre mutants (arrowheads; green, CC10; red, Ki-67). CC10 immunofluorescence in the 3 2 mutants was very low, and the data represent a longer exposure than used in wild-type samples. (o–q) No increase in distal 1 airway epithelial proliferation was observed in Gata6 flox/flox:Sftpc-cre mutants by Ki-67 immunostaining. Scale bars: Percentage Ki-67 0 http://www.nature.com/naturegenetics WT G6 flox: a–d,40mm; e,f,25mm; h–p,30mm. Error bars, s.e.m. based on a minimum of four embryos. Sftpc-cre

CC10-positive Clara cells are thought to include several progenitor immunostaining for the proliferation marker Ki-67 and CC10, which cell populations in the lung airway, including BASCs, which express was expressed at very low levels in the bronchiolar epithelium of both SP-C and CC10 proteins, a trait not shared by other epithelial cell mutants (Fig. 3h–n). Notably, cellular proliferation was relatively types within the lung9. To assess whether the cells ectopically expres- unaffected in the distal airways of Gata6 flox/flox:Sftpc-cre mutants sing CC10 in Gata6 flox/flox:Sftpc-cre mutants also expressed SP-C, we (Fig. 3o–q). These data are consistent with a model wherein Gata6 performed double immunofluorescence staining at E18.5. A high regulates differentiation and proliferation of the bronchiolar epithelial percentage of cells expressing CC10 within the distal airways also niche, expanding the BASC population.

Nature Publishing Group Group Publishing Nature expressed SP-C, indicating that these represented BASCs (Fig. 2a–d). 8 Moreover, a high percentage of the BASCs double-positive for Fzd2 antagonizes Wnt–b-catenin signaling in lung epithelium 200 SP-C and CC10 in Gata6 flox/flox:Sftpc-cre mutants also expressed The precocious appearance of BASCs and increased proliferation in © Sca1, another marker of BASCs (Fig. 2e,f and ref. 9). There seemed CC10+ cells suggested that Gata6 regulates molecular pathway(s) to be at least two populations of Sca1-expressing cells: CC10+Sca1+ required for progenitor cell development in the lung. We performed and SP-C+CC10+Sca1+. Whereas approximately 8–10% of CC10+ cells microarray analysis to identify Gata6-dependent pathways in lung expressed Sca1 in wild-type embryos at E18.5, Gata6 flox/flox:Sftpc-cre epithelium (Fig. 4a). One of the identified from these analyses mutants showed a fivefold increase in CC10+Sca1+ cells (Fig. 2d–g). was Fzd2, encoding a Wnt receptor previously reported to activate Notably, at this stage of development, we did not identify substantial the noncanonical Wnt pathway15,16. Fzd2 expression was specifically numbers of SP-C+CC10+ BASCs in wild-type littermates by immu- downregulated in airway epithelium of Gata6 flox/flox:Sftpc-cre nostaining (Fig. 2g). However, using FACS analysis for SP-C and mutants, as determined by RT-PCR and in situ hybridization analyses CC10 expression on permeabilized lung epithelial cells, we did observe (Fig. 4b–f). In contrast, expression of Wnt7b and Wnt2,whichencode a small percentage of SP-C+CC10+ BASCs in wild-type mice at E18.5, two ligands in the Wnt family expressed in the lung, was not affected which was markedly increased in Gata6 flox/flox:Sftpc-cre mutants (Fig. 4g–k). Although in vitro studies have indicated that non- (Fig. 2h,i). These data demonstrate that a lung epithelium–specific canonical Wnt signaling antagonizes the b-catenin–dependent loss of Gata6 led to premature appearance and increased numbers canonical Wnt pathway, less is known about what function this has of BASCs. in vivo16–18. To assess whether loss of Fzd2 led to altered canonical Wnt signaling in lung epithelia, we used short interfering RNA Gata6 regulates bronchiolar epithelial proliferation (siRNA)-mediated knockdown to inhibit Fzd2 expression in the To further elucidate the cellular defects arising from loss of Gata6 lung epithelial cell line MLE-15, which expresses Fzd2. Fzd2 expres- expression in lung epithelium, we assessed cellular proliferation. In sion was reduced by approximately 75% as compared to expression wild-type lungs, cellular proliferation is normally very low in bronch- in control siRNA–transfected cells, based on quantitative PCR iolar epithelium (Fig. 3 and ref. 6). By contrast, cellular prolif- (Q-PCR; data not shown). Unexpectedly, cotransfection of the eration was substantially increased in bronchiolar epithelium of TOPFLASH Wnt reporter revealed that inhibition of Fzd2 expression Gata6 flox/flox:Sftpc-cre mutants (Fig. 3a–g). Moreover, the bronchiolar led to a substantial increase in TOPFLASH activity (Fig. 4l). Gata6 epithelium had a more disorganized appearance, with cells growing on was also able to transactivate the –1.5-kb proximal mouse Fzd2 top of one another (Fig. 3e,f). This phenotype is probably the result promoter in NIH-3T3 cells (Fig. 4m). Moreover, chromatin immuno- of increased proliferation of Clara cells, as shown by double precipitation (ChIP) assays showed a direct association of Gata6 with

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WT1 WT2 WT3 KO2 KO2 KO3 Figure 4 Fzd2 is a target of Gata6 in lung a Galnt13 Rpl17 epithelium and negatively regulates canonical Dsc2 MGI:1930803 Wnt signaling. (a) Microarray studies comparing Pof1b Fut9 WT Arsk b g flox expression profiles in wild-type (WT) versus Aox3 1.2 1.4 G6 :Sftpc-cre flox/flox Aqp4 Gata6 :Sftpc-cre mutants (KO). Heat map Maob 1.0 1.2 Aqp4 represents a portion of the genes upregulated or Emb 0.8 1.0 Mpzl2 flox/flox Gsto1 0.8 downregulated in Gata6 :Sftpc-cre mutants, Cd36 0.6

Mettl7a1 Fzd2 0.6 Svt14 0.4 highlighting Fzd2. Red, increased expression Cab391 0.4 Fabp4 0.2 versus wild-type samples; green, decreased Fabp4 0.2 Relative expression Relative Soat1 expression Relative expression. (b) Q-PCR showing that Fzd2 Col4a4 0 0 Srebf2 flox Slc35e3 WT G6 :Sftpc-cre Wnt7b Wnt2 expression was decreased approximately 80% in Rab27a flox/flox flox 2810003C17Rik Gata6 :Sftpc-cre (G6 :Sftpc-cre)mutants. Slc7a7 Muc1 Insig1 (c–f) In situ hybridization showing that Fzd2 Sdpr WTG6 flox:Sftpc-cre WT G6 flox:Sftpc-cre Chst11: Phactr1 expression was lost in airway epithelium (arrows) Thbs3 Eif5a2 Pcbp3 cd hi but was still observed in mesenchyme of the lung Alad flox/flox Adcy7 in Gata6 :Sftpc-cre mutants. (g)Q-PCR Mvd ai BB376947 E12.5 Slco3a1 Wnt7b showing that Wnt7b and Wnt2 expression Dusp7 flox/flox Trpm6 ai remained unchanged in Gata6 :Sftpc-cre Kcnj15 Tmem16a mutants. (h–k) In situ hybridization showing that Rerg ef jk Dcbld1 Dcbld1 ai spatial expression of these Wnt ligands also Fzd2 Fzd2

Slco4c1 ai Wnt2 remained unchanged. (l) Knockdown of Fzd2 Dhcr7 E14.5 Sc4mol Fzd2 Fzd2 expression by siRNA in MLE-15 cells resulted in Sema3a 9230110J10 increased TOPFLASH activity. (m) The proximal Cvp51 Galnt14 Ccbp2 –1.5-kb Fzd2 promoter was transactivated by Ppid; Lamp3 –1.5-kb mouse Fzd2 promoter Idi1 lmGata6 in a dose-dependent manner in NIH-3T3 Tead4 4 Gpr172b Gipc2 (Semcap2) cells. (n,o)ChIPassaysshowedthatGata6was Bspry 3 http://www.nature.com/naturegenetics Ctsb associated with the mouse Fzd2 promoter, Slco3a1 GATA DNA binding site 9430028L06Rik 2 7 using Q-PCR (n) and shown by agarose gel Slco4c1 Txnrd3 activity 6 Lama3 1 electrophoresis (o). ai, airways. Scale bars, Fdps 5 Tekt5 in MLE-15 cells 100 mm. Error bars, s.d. from the representative Tspan8 0 4 Parp3 TOPFLASH Relative BB047533 Control siRNA Fzd2 siRNA 3 assays performed in triplicate. Fabp5 Tmem108 2 Egfr Rhof 1 Relative activation Relative Dusp7 0 Cvp51 Adcv7 pCMV 0.25 µg 0.5 µg Hmha1 n 12 Tuba8 reported .Thispronouncedincreasein 6530401D17Rik 4 Tmod1 canonical Wnt signaling is also the likely Slco3a1 Col4a3 3 Ldlr cause of the increased proliferation observed Egfr o Fabp5 2 flox/flox Akap5 in Clara cells of Gata6 :Sftpc-cre Spock2 lgG Gata6 lgG Input

Nature Publishing Group Group Publishing Nature Tspan11 Cpm 1 mutants (Fig. 3). Previously described targets 8

Matn4 activity Relative

Cpm normal versus lgG Akap5 0 of canonical Wnt signaling such as Fgfr2, Abca3 200 Abca3 Normal lgG Gata6 lgG Cldn18 Mycn (N-)andBmp4 were also upregu- © Cldn18 flox/flox Camk2b lated in Gata6 :Sftpc-cre mutants (Supplementary Fig. 2 online). However, the mouse Fzd2 promoter in the lung (Fig. 4n,o). Thus, mouse Fzd2 is intestinal genes previously shown to be upregulated upon forced a direct target of Gata6. activation of b-catenin signaling in the lung were relatively unaffected, On the basis of these data, we predicted that Gata6 flox/flox:Sftpc-cre suggesting distinct differences between this model of hyperactivated mutants would show increased canonical Wnt signaling in airway canonical Wnt signaling and the upregulation of canonical Wnt epithelia in vivo. Given that canonical Wnt signaling has been signaling observed in the Gata6 flox/flox:Sftpc-cre mutants (Supplemen- demonstrated to promote expansion of progenitor cells in other tary Fig. 2). Together, these data demonstrate that canonical Wnt tissues19–22, increased activity in this pathway could help explain the signaling is markedly upregulated by loss of Gata6 and Fzd2, which increased proliferation of CC10+ cells and expansion of BASCs that we may lead to the imbalance in BASC proliferation and differentiation. observed. Therefore, we determined canonical Wnt signaling activity in Gata6 flox/flox:Sftpc-cre mutants. Increased expression of activated b- Rescue of epithelial defects in Gata6 flox/flox:Sftpc-cre mutants catenin protein was readily observed in Gata6 flox/flox:Sftpc-cre mutants To determine whether loss of Fzd2 was responsible for the lung at E18.5 (Fig. 5a,b). Next, we crossed the transgenic Wnt–b-catenin epithelial differentiation defects observed in Gata6 flox/flox:Sftpc-cre reporter line BAT-lacZ (BAT-gal)intotheGata6 flox/flox:Sftpc-cre mutants, we tested whether reexpression of Fzd2 could rescue the mutants and stained embryos for b-galactosidase expression at block in epithelial differentiation, as assayed by loss of SP-C expression E13.5 and E16.5. We have previously reported that b-galactosidase in Gata6 flox/flox:Sftpc-cre mutants (Fig. 5i–m). As expected, electro- expression from this Wnt reporter declines rapidly in airway poration of a control plasmid into lung explants did not rescue the epithelium after E11.5 (ref. 12). Notably, there was a pronounced loss of SP-C expression observed in Gata6 flox/flox:Sftpc-cre mutants increase in b-galactosidase expression in the airway epithelium of (Fig. 5l). By contrast, reexpression of Fzd2 in Gata6 flox/flox:Sftpc-cre Gata6 flox/flox:Sftpc-cre mutants, indicating that Wnt–b-catenin signal- mutant lungs by electroporation of a Fzd2 expression plasmid led to a ing was upregulated in airway epithelium upon loss of Gata6 expres- substantial increase in SP-C expression (Fig. 5m). Expression of sion (Fig. 5c–h). In wild-type mice, b-galactosidase expression was FGFR2, N-myc and BMP4 was also upregulated upon loss of Gata6 observed in scattered lung mesenchymal cells at E13.5 (Fig. 5d) expression, and this increase was reversed upon rescue with Fzd2 and in a few epithelial cells at E16.5 (Fig. 5g), as previously reexpression in lung explants (Supplementary Fig. 3 online).

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Figure 5 Increased canonical Wnt signaling in lung epithelium upon loss of Gata6 expression ab i and rescue of these defects by reexpression of

-catenin Electroporate Fzd2 or decreased b-catenin expression. β flox E11.5 lung bud (a,b) Activated b-catenin protein expression was WT G6 :Sftpc-cre Analyze for flox increased in Gata6 flox/flox:Sftpc-cre (G6 flox:Sftpc- c WT G6 : G6 flox: expression Sftpc-cre after 48 h cre) mutant airway epithelium (b, arrowheads) Sftpc-cre relative to wild-type (WT; a). (c–h) BAT-lacZ Wnt G6 flox:Sftpc-cre G6 flox:Sftpc-cre reporter mice showed increased canonical Wnt WT G6 flox:Sftpc-cre + control + Fzd2 activity in the lungs of Gata6 flox/flox:Sftpc-cre

E13.5 jk lm mutants at both E13.5 (c–e)andE16.5(f–h). This activity was strong enough to be observed through the chest wall of the mutants at E13.5 (c, arrows). It was confined to airway epithelium Ctnnb1-lacZ de (e,h, asterisks). Residual b-galactosidase expression was observed in wild-type littermates * * WT G6 flox:Sftpc-cre G6 flox:Sftpc-cre in scattered mesenchymal or epithelial cells flox (d,g, arrowheads). f, lung wholemounts. WT G6 :Sftpc-cre n op (i–m) SP-C expression after electroporation (i)of f

Fzd2 construct. SP-C expression was observed in SP-C the airway epithelium of the wild-type explants (j). Gata6 flox/flox:Sftpc-cre mutants lacked SP-C E16.5 expression (k). Electroporation of the control q rs flox vector did not result in increased SP-C expression WT G6 :Sftpc-cre * * (l), whereas the Fzd2 expression plasmid resulted gh

CC10 *

in reexpression of SP-C (m). (n–s) Expression Ctnnb1-lacZ * http://www.nature.com/naturegenetics of SP-C and CC10 in wild-type mice (n,q), * flox/flox Gata6 :Sftpc-cre mutants (o,r)and WT G6 flox:Sftpc-cre Gata6 flox/flox:Ctnnb1flox/+:Sftpc-cre (G6 flox:Ctnnb1flox/+:Sftpc-cre) mutants (p,s). Gata6 flox/flox:Sftpc-cre mutants had substantially decreased SP-C and CC10 expression, with ectopic CC10 expression in distal airways (o,r, arrowheads). However, loss of one allele of b-catenin in Gata6 flox/flox:Ctnnb1flox/+:Sftpc-cre mutants led to a marked increase in SP-C and CC10 expression and a decrease in ectopic CC10 expression (p,s). Asterisks, airway epithelium. Scale bars: a,b,d,e,n–s,50mm; g,h,75mm.

Because an increase in Wnt–b-catenin signaling in Gata6 flox/flox: Sftpc-cre:Ctnnb1flox/+ mutants to reduce b-catenin expression and Sftpc-cre mutants could contribute to the increase in BASC expansion signaling by 50% in a Gata6 null background. Loss of one copy of and epithelial differentiation defects, we generated Gata6 flox/flox: the b-catenin gene led to increased SP-C expression, reestablishment

Nature Publishing Group Group Publishing Nature SP-C CC10 Overlay 8 abBADJ efg k 200 BADJ 5 © WT 4

Uninjured 2 d after injury 3

cd : hij 2 BADJ region

ex3flox 1

BADJ of BASCs per No. BADJ 0 ex3flox

Scgb1a1-cre WT Ctnnb1 : Ctnnb1 4 d after injury 7 d after injury Scgb1a1-cre

SP-C CC10 Overlay SP-C CC10 Overlay lmn rst x 70 60 + 50

SP-C 40 + Scgb1a1-cre : 30 WT

Percentage 20 opq uvw CC10 ex3flox 10 0 ex3flox Ctnnb1 WT Ctnnb1 : Scgb1a1-cre

Figure 6 Canonical Wnt signaling is activated upon lung regeneration and forced activation of Wnt–b-catenin signaling leads to expansion of BASCs. Lung airways of BAT-lacZ mice were injured by naphthalene to induce airway regeneration. In uninjured mice, only rare b-galactosidase+ cells were observed in the BADJ region of the airways (a, arrowhead). (b–d) After naphthalene injury and during the epithelial regeneration process, a marked increase in b-galactosidase staining, indicating increased canonical Wnt signaling, was observed in cells within the BADJ niche. (e–k)StainingwithSP-CandCC10 showed that BASC numbers were increased in the BADJ region of Ctnnb1ex3flox:Scgb1a1-cre mice (e–j, arrows; quantified in k). Seven days after naphthalene induced lung injury and regeneration, there was a substantial increase in BASC number in Ctnnb1ex3flox:Scgb1a1-cre mice (l–w;compare arrowheads to brackets). (x) Quantitation showed that approximately 50% of the cells regenerating in the bronchiolar airways were SP-C+CC10+ double- positive BASCs. Scale bars, 50 mm. Errors bars, s.e.m. based on four embryos.

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flox flox Figure 7 Gata6 regulates BASC expansion and WT G6 :Scgb1a1-cre WT G6 :Scgb1a1-cre e 20

+ differentiation in airway epithelial regeneration. a b cd 15 (a,b) Adults of both wild-type (WT) mice and

Ki-67 flox/flox flox + 10 Gata6 :Scgb1a1-cre (G6 :Scgb1a1-cre)

No injury 5 mutants showed normal expression of CC10 Percentage CC10 flox/flox CC10/Ki67 CC10/Ki67 0 (green). (c–e) However, Gata6 :Scgb1a1-cre 100 WT G6 flox: mutants showed increased proliferation (Ki-67 g n 80 WT Scgb1a1-cre expression). (f–k) Naphthalene injury of f flox 60 G6 :Scgb1a1-cre Gata6 flox/flox:Scgb1a1-cre mutants resulted in 40 + Day 2 Day 20 decreased regeneration of Clara cells (CC10 0 +

Percentage cells Percentage cells, green) at 14 d after injury. (l–n)CC10 expression CC10 expression Negative Low High cells that did regenerate were divided into two h i o p q categories: low-expressing cells (m, arrowheads) and high-expressing cells (m, arrows). Few if any

Day 4 Day CC10 high-expressing cells regenerated in Gata6 flox/flox:Scgb1a1-cre mutants (n). (o–u)This CC10 SP-C Overlay loss in Clara cell regeneration was accompanied

by an increase in BASCs (o–r) and increased jkr + 40 30 proliferation in the CC10- and SP-C/CC10– SP-C

+ 20 expressing cells in the mutants (s–u). (v) Model: Day 14 Day 10 Gata6 regulates Fzd2 expression, which in turn Percentage CC10 0 antagonizes canonical Wnt signaling in lung WT G6 flox:Scgb1a1-cre epithelia and allows proper epithelial WT G6 flox:Scgb1a1-cre 100 u 80 differentiation and development of BASCs, which

m + l s t 60 is required for lung regeneration. Scale bars: 40

Ki-67 a,b,f–k,500mm; c,d,o–q,40mm; l,m,s,t 50 mm. Day 14 Day 20 http://www.nature.com/naturegenetics Percentage Percentage Error bars, s.e.m. based on four embryos.

bronchiolar cells 0 CC10/DAPI CC10/DAPI CC10/Ki-67/DAPI CC10/Ki-67/DAPI 10

WT-CC10Mut-CC10 v Gata6 Gata6 Progenitor cell Mut-SP-C/CC To determine whether increased activity of Progenitor cell (BASC) (BASC) proliferation Wnt–b-catenin signaling would lead to Fzd2 proliferation and differentiation Fzd2 Progenitor cell increased numbers of BASCs, we crossed a (BASC) differentiation Scgb1a1-cre mouse line, which targets Cre expression specifically to Clara cells using the promoter of the CC10 gene (Scgb1a1), to a of proper CC10 expression in bronchiolar epithelium and reduced line carrying the Ctnnb1ex3flox allele, which produces a constitutively

Nature Publishing Group Group Publishing Nature numbers of SP-C+CC10+ double-positive BASCs as compared to activated form of b-catenin that activates canonical Wnt signaling to 8 Gata6 flox/flox:Sftpc-cre mutants (Fig. 5n–s and data not shown). high levels25. Cre expression from this mouse line is initiated at 200 Although this degree of rescue was substantial, these mice still approximately E18.5 and spares alveolar epithelium, thus allowing © succumbed to respiratory failure at birth, indicating that rescue was us to target a more specific pool of cells containing BASCs and not complete (data not shown). circumventing the perinatal lethality due to respiratory failure observed in Gata6 flox/flox:Sftpc-cre mutants26. Ctnnb1ex3flox: Wnt–b-catenin signaling regulates BASC expansion and lung Scgb1a1-cre adult mice were viable and expressed CC10 normally regeneration (data not shown). However, they had increased numbers of BASCs The above data suggest that canonical Wnt–b-catenin signaling located in the BADJ region (Fig. 6e–k). Using naphthalene injury promotes expansion of BASCs. However, it is unknown whether to induce epithelial regeneration, we compared wild-type and Wnt–b-catenin signaling is activated during lung epithelial regenera- Ctnnb1ex3flox:Scgb1a1-cre mice for differences in the number of tion in the adult as well as whether forced activation of this pathway BASCs during airway regeneration. Notably, in Ctnnb1ex3flox: leads to BASC expansion. To determine whether canonical Wnt–b- Scgb1a1-cre mice, most cells found in the regenerating airways after catenin signaling is activated upon lung epithelial regeneration, injury were SP-C+CC10+ double-positive BASCs (Fig. 6l–x). This was we injured adult BAT-lacZ Wnt–b-catenin reporter mice with supported by a pronounced increase in double-positive BASCs using naphthalene and examined the activity of Wnt signaling using FACS analysis (Supplementary Fig. 4 online). These data show that b-galactosidase histochemical staining23. Bronchiolar Clara cells, Wnt signaling is activated in the niche containing BASCs and that except for BASCs, express cytochrome P450-2F2, making them forced activation of Wnt–b-catenin signaling results in a marked susceptible to naphthalene toxicity24. Depletion of Clara cells with increase in BASCs. naphthalene induces expansion and differentiation of BASCs as they regenerate the bronchiolar epithelium, and this injury is normally Gata6 is essential for lung epithelial regeneration resolved after 10–14 d (refs. 7,9). In uninjured mice and 2 d after On the basis of the data showing that there is increased Wnt injury, we observed small clusters of one to three b-galactosidase- signaling after loss of Gata6 in lung epithelium and that activated positive cells within the BADJ region (Fig. 6a,b). By 4 d after injury, Wnt signaling leads to increased BASC numbers, we predicted that we saw a substantial increase in the number of cells staining for postnatal loss of Gata6 would lead to an increase in BASC number and b-galactosidase expression, and by 7 d after injury, we saw a marked decreased airway regeneration. As the Gata6 flox/flox:Sftpc-cre mutants increase in b-galactosidase staining in the BADJ niche, where BASCs die perinatally, we crossed the Gata6 flox/flox mice to the Scgb1a1-cre are located (Fig. 6c,d). mice to delete Gata6 in Clara cells. Gata6 flox/flox:Scgb1a1-cre mutants

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had viability similar to that of wild-type littermates (data not shown). transcription, and the regulatory regions of most lung epithelium- However, the Gata6 flox/flox:Scgb1a1-cre mutants showed increased specific genes contain evolutionarily conserved GATA DNA binding proliferation in CC10-positive cells similar to that observed in sites5,10,27–30. Gata6 regulation of Fzd2 reveals an unexpected role for Gata6 flox/flox:Sftpc-cre mutants at E18.5 (Fig. 7a–e). noncanonical Wnt antagonism of canonical Wnt signaling in con- To determine whether BASC differentiation and airway regenera- trolling progenitor cell expansion and differentiation. The role for tion were affected in Gata6 flox/flox:Scgb1a1-cre mutants, we studied Fzd2 in Wnt signaling has been somewhat controversial. Most studies naphthalene-induced acute lung injury. The initial response in both have shown that Fzd2 regulates effectors of noncanonical Wnt signal- wild-type and Gata6 flox/flox:Scgb1a1-cre mutants was similar at 2 d and ing, including G-protein signaling and activation of calcium/calmo- 4 d after naphthalene treatment, with both controls and mutants dulin-dependent protein kinase II (CamKII) (refs. 31–33). Because showing severe loss of CC10-expressing cells (Fig. 7f–i). However, by increased canonical Wnt signaling leads to defective epithelial differ- 14 d after injury, the Gata6 flox/flox:Scgb1a1-cre mutants showed a entiation and to tumor formation in the lung34,35,itisimperativethat substantial impairment in Clara cell regeneration in the bronchiolar this pathway be controlled by multiple signals. Gata6 regulation of airways (Fig. 7j,k). The CC10-positive cells within the bronchi of Fzd2 may be key in this, through fine-tuning the activity of canonical Gata6 flox/flox:Scgb1a1-cre mutants were also qualitatively different: Wnt signaling in airway epithelium. instead of uniform CC10 expression, as observed in wild-type mice, Gata factors and Wnt signaling have each been implicated in CC10 expression in the mutants fell into two categories: high expres- progenitor cell expansion and differentiation. Gata6 seems to oppose sion, as observed in wild-type lungs, or low expression (Fig. 7l,m). Nanog in regulating primitive endoderm development, with loss of More than 50% of bronchiolar epithelial cells were negative for CC10 Gata6 in embryonic stem cells leading to increased proliferation and expression and less than 10% expressed high levels of CC10 in decreased primitive endoderm differentiation36–38.Wntsignalinghas Gata6 flox/flox:Scgb1a1-cre mutants (Fig. 7n). The different levels of also recently been implicated in progenitor self-renewal and tissue CC10 expression observed in these mice may reflect abnormal devel- regeneration. In zebrafish, tail regeneration requires canonical Wnt opment of Clara cells repopulating the airways because of the disrup- signaling, and inhibition of this pathway inhibits regeneration21. tion in BASC expansion and differentiation. Most of the remaining Zebrafish cardiac regeneration and expansion of anterior heart field http://www.nature.com/naturegenetics CC10-negative cells in the bronchiolar airways expressed b-tubulin-IV, progenitors in mammals also seems to require canonical Wnt indicating that they were ciliated epithelium (data not shown). signaling19,21,39. Although hyperactive canonical Wnt signaling has To determine whether the loss of Clara cell regeneration was been shown to disrupt lung development, leading to a loss of epithelial associated with altered BASC development, we immunostained for differentiation and ectopic expression of intestinal marker genes, these SP-C and CC10. Consistent with previous reports, a small expansion studies have not addressed the role of this pathway in regulating of BASCs was observed at 4 d after injury in both wild-type and progenitor development in the lung34,35.Weshowthatincreased Gata6 flox/flox:Scgb1a1-cre mutants (data not shown and ref. 9). How- canonical Wnt signaling in lung epithelium not only results in ever, at 14 d after injury, a marked increase in BASCs was observed in defective epithelial differentiation but also increased progenitor cell Gata6 flox/flox:Scgb1a1-cre mutants that was not observed in wild-type development, including precocious appearance of BASCs. Our studies mice (Fig. 7o–r). These cells were associated with an increase in also highlight regulatory cross-talk between canonical and noncano-

Nature Publishing Group Group Publishing Nature proliferation in both CC10+ and SP-C+CC10+ cells in the terminal nical Wnt signaling pathways in progenitor cell development by 8 bronchioles (Fig. 7s–u and data not shown). Together, these data revealing that Fzd2 inhibits canonical Wnt signaling in lung epithelia, 200 indicate that Gata6 is essential in airway epithelial regeneration which helps to balance epithelial differentiation with progenitor © through regulation of BASC expansion and differentiation in the cell expansion. adult lung by means of modulation of Wnt signaling (Fig. 7v). BASCs were originally defined by their expression of two lung epithelium-specific marker proteins, SP-C and CC10, in addition to DISCUSSION expression of other general stem cell markers such as Sca1 (ref. 9). The The molecular programs regulating endogenous tissue-specific stem involvement of BASCs in lung cancer biology is supported by the cell expansion and differentiation are of obvious importance in observation that BASC numbers were substantially increased in a developing techniques to harness the ability of these cells to regenerate K-ras tumor model of lung adenocarcinoma9. The data on BASC damaged and diseased organs. We show that the transcription factor development and differentiation have remained scant, with recent Gata6, acting through Fzd2-mediated Wnt signaling, is required for studies focusing on their expansion upon disruption of the cell cycle/ proper development of BASCs. Loss of Gata6 leads to unrestrained proliferation genes p27kip1, MAPK p38 and PTEN (refs. 40–43). Our expansion of BASCs at the expense of lung epithelial differentiation data implicate BASC function in normal regeneration after lung injury during both lung development and airway regeneration after injury, and show that Gata6 regulates a Wnt-mediated pathway required for similar to what occurs upon forced activation of canonical Wnt this regeneration. We show a pronounced increase in bronchiolar signaling. This finding underscores an important aspect of stem cell epithelial proliferation in response to increased canonical Wnt signal- biology: the recapitulation of critical developmental pathways in the ing in airway epithelium. Such an increase is likely to contribute to the regulation of progenitor cell expansion is required for tissue regenera- precocious appearance and expansion of BASCs upon loss of Gata6 in tion in the adult. the lung. The role of most tissue restricted transcription factors in progenitor Our current study has revealed that Gata6 is required for the cell expansion and differentiation remains less well understood than balance between BASC expansion and epithelial differentiation in their role in normal development. Although it is commonly thought the lung. Gata6 regulates Fzd2 gene expression, and Fzd2 acts as a that these transcription factors and pathways play key roles in tissue negative regulator of canonical Wnt signaling in lung epithelium. Our repair by directing self-renewal and differentiation of endogenous data show that Wnt signaling is activated upon lung epithelial progenitor cells, this hypothesis has not been tested in many tissues, regeneration, and increased Wnt signaling expands the numbers of including the lung. Gata6 is expressed throughout the developing BASCs in the lung. Given this ability of canonical Wnt signaling to foregut endoderm and is important for regulating lung epithelial gene expand progenitor cell populations in the lung, it may be possible in

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the future to use agonists of this pathway to increase lung injury repair was minced and fixed with 1% formaldehyde, and chromatin was sheared by and regeneration. However, given the role of Wnts as oncogenes and sonication to an average length of 500–600 bp, then immunoprecipitated with a the fact that BASCs are thought to be progenitors of lung adeno- GATA6-specific antibody (Santa Cruz Biotechnology, C-10). Reverse cross- carcinomas, careful consideration will have to be given before linked immunoprecipitated chromatin was subjected to PCR using the primers modulating the Wnt pathway for this purpose. listed in Supplementary Table 1. Naphthalene injury. Mice were injected intraperitoneally with 300 mg/kg of naphthalene dissolved in corn oil; control mice were injected with the same METHODS flox/flox flox/flox ex3flox volume of corn oil. For immunohistochemistry, lungs were inflation-fixed at Mice. Gata6 , Sftpc-rtTA, tetO-cre, Ctnnb1 , Ctnnb1 , Scgb1a1- 25 cm water pressure with 4% paraformaldehyde (PFA) at the indicated time cre and BAT-lacZ mice were previously generated and were genotyped as points after naphthalene injection and further fixed by submersion in 4% PFA described11,13,14,23,26,44,45. Sftpc-rtTA:tetO-cre mice were treated with doxycycline for 24 h. For b-galactosidase staining, lungs were fixed with 2% PFA and (1 mg per kilogram body weight in food and 1 mg/ml in water) starting at processed as previously described47. E0.5 for developmental deletion. All animal experiments were performed with the approval of the University of Pennsylvania Institutional Animal Care and Lung explant studies. Lungs from E11.5 wild-type or Gata6 flox/flox:Sftpc-cre Use Committee. mutants were isolated and the airways were microinjected with 1 mg/ml of either a Fzd2 expression plasmid or a control expression plasmid, rinsed briefly 46 Histology. In situ hybridization was performed as previously described . in PBS to remove extraneous external DNA and electroporated in a BTX ECM Immunohistochemistry and immunofluorescence staining was performed 2001 electroporator using 3 pulses at 50 V, 25 ms. Explants were then cultured using the following antibodies at the indicated dilutions: SP-C (Chemicon, as previously described for 48 h, after which they were collected and fixed 1:500), CC10 (Santa Cruz T-18, 1:50), SP-B (Chemicon, 1:250), rat antibody to for histology47. Ki-67 (Dako, clone TEC-3), Gata6 (R&D Systems AF1700, 1:50), Sca1 (R&D Systems AF1226, 1:50), T1a (Developmental Studies Hybridoma Bank mono- URLs. SAM, http://www-stat.stanford.edu/~tibs/SAM/; Treeview, http:// clonal antibody 8.1.1, University of Iowa, 1:100), b-catenin (BD Biosciences, rana.lbl.gov. 1:50), activated b-catenin (Millipore clone 8E7, 1:100) and b-tubulin-IV (BioGenex MU178-UC, 1:100). b-Galactosidase histochemical staining was Accession codes. Gene Expression Omnibus microarray data: GSE11165. http://www.nature.com/naturegenetics performed as previously described47. Note: Supplementary information is available on the Nature Genetics website. Quantitative assessment of BASCs. Immunostained cells were counted ACKNOWLEDGMENTS 200 mm proximal and distal to the BADJ in relation to the branching airways, The authors thank J. Epstein and C. Simon for helpful suggestions and critical using a protocol similar to that previously described9. We used a minimum of reading of the manuscript. We thank S. Piccolo (University of Padua) for five sections from each sample for quantitative measurements and analyzed at providing the BAT-lacZ mice, M. Taketo (Kyoto University) for providing least four mice of each described genotype in all experiments. FACS analysis the Ctnnb1ex3flox mice and M.M. Lu for excellent technical assistance with the was performed using lung epithelial cells isolated from E18.5 or adult lungs, as histological studies. These studies were supported by funding from the US indicated, using a previously described protocol9.Lungepithelialcellswere National Institutes of Health to E.E.M. (HL064632, HL075215, and HL087825) permeabilized using the Fix and Perm Cell kit (Caltag Labs) and then stained and to M.S.P. (HL075215). for SP-C and CC10 expression using the antibodies described above followed by Alexa Fluor 647 and 488 secondary antibodies, respectively40,42. AUTHOR CONTRIBUTIONS Nature Publishing Group Group Publishing Nature

8 Y.Z. and E.E.M. developed the project; Y.Z., A.M.G., E.D.C., J.J.L. and R.K. Microarray studies. RNA was isolated from E18.5 lungs from wild-type and performed mouse genetics experiments; K.M. and Y.Z. performed cell culture 200 Gata6 flox/flox:Sftpc-cre mutant littermates (three samples of each genotype). experiments; J.Y. performed histology experiments; F.J.D., J.A.W. and M.S.P. © Total RNA was transcribed to generate biotinylated cRNA to use as a probe for contributed materials; and Y.Z. and E.E.M. wrote the paper. Affymetrix mouse 230A GeneChips. Three chips were hybridized for each genotype. Data from these arrays were normalized using Microarray Suite 5.0 Published online at http://www.nature.com/naturegenetics/ (MAS5, Affymetrix) and Significance Analysis of Microarrays (SAM). Twofold Reprints and permissions information is available online at http://npg.nature.com/ reprintsandpermissions/ and greater changes in gene expression were considered significant. Treeview software was used to generate the heatmap. Total RNA was isolated with Trizol and Q-PCR was performed using the oligonucleotides listed in Supplementary 1. Cardoso, W.V. Molecular regulation of lung development. Annu. Rev. Physiol. 63, 471–494 (2001). Table 1 online and an Applied Biosystems 7900HT system with Sybr Green 2. Cardoso, W.V. & Lu, J. Regulation of early lung morphogenesis: questions, facts and reaction mixture as previously described48. controversies. Development 133,1611–1624(2006). 3. Minoo, P., Su, G., Drum, H., Bringas, P. & Kimura, S. Defects in tracheoesophageal Cell culture transfection assays. MLE-15 cells were transfected with the and lung morphogenesis in Nkx2.1( / )mouseembryos.Dev. Biol. 209,60–71 À À TOPFLASH luciferase reporter plasmid along with either Fzd2 siRNA Smart- (1999). 4. Wan, H. et al. Compensatory roles of Foxa1 and Foxa2 during lung morphogenesis. pool or control siRNA (Dharmacon) using Lipofectamine 2000 (Invitrogen). A J. Biol. Chem. 280,13809–13816(2005). Renilla luciferase control expression plasmid was included in the transfections 5. Yang, H., Lu, M.M., Zhang, L., Whitsett, J.A. & Morrisey, E.E. GATA6 regulates to control for transfection efficiency. Cells were collected 48 h after transfection, differentiation of distal lung epithelium. Development 129,2233–2246(2002). and luciferase assays were performed as previously described49. 6. Blenkinsopp, W.K. Proliferation of respiratory tract epithelium in the rat. Exp. Cell Res. 46,144–154(1967). The proximal 1.5 kb promoter region of mouse Fzd2 was amplified and 7. Giangreco, A., Reynolds, S.D. & Stripp, B.R. Terminal bronchioles harbor a unique cloned into the pGL3basic vector to generate pGL3Fzd2pro.luc. The oligo- airway stem cell population that localizes to the bronchoalveolar duct junction. Am. J. nucleotide sequences used to generate the Fzd2 promoter plasmid are listed in Pathol. 161,173–182(2002). Supplementary Table 1. This plasmid was transfected along with a Gata6 8. Rawlins, E.L. & Hogan, B.L. Epithelial stem cells of the lung: privileged few or opportunities for many? Development 133,2455–2465(2006). expression plasmid into NIH-3T3 cells and a control Renilla luciferase expres- 9. Kim, C.F. et al. Identification of bronchioalveolar stem cells in normal lung and lung sion plasmid. Cells were collected 48 h after transfection and luciferase assays cancer. Cell 121,823–835(2005). were performed as previously described49. 10. Zhang, Y. et al. GATA and Nkx factors synergistically regulate tissue-specific gene expression and development in vivo. Development 134,189–198(2007). Quantitative PCR and chromatin immunoprecipitation (ChIP) assays. Total 11. Lepore, J.J. et al. GATA-6 regulates semaphorin 3C and is required in cardiac neural crest for cardiovascular morphogenesis. J. Clin. Invest. 116,929–939(2006). RNA was isolated with Trizol, and Q-PCR was performed using the primers 12. Shu, W. et al. Wnt/b-catenin signaling acts upstream of N-myc, BMP4, and FGF listed in Supplementary Table 1. Chromatin was made from E18.5 mouse lung signaling to regulate proximal-distal patterning in the lung. Dev. Biol. 283,226–239 tissue using a commercially available kit (Upstate Biotechnology). Lung tissue (2005).

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