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Developmental Biology 277 (2005) 316–331 www.elsevier.com/locate/ydbio

Dickkopf-1 (DKK1) reveals that fibronectin is a major target of Wnt signaling in branching morphogenesis of the mouse embryonic lung

Stijn P. De Langhea, Fre´de´ric G. Salaa, Pierre-Marie Del Morala, Timothy J. Fairbanksa, Kenneth M. Yamadab, David Warburtona, Robert C. Burnsa, Saverio Belluscia,*

aDevelopmental Biology Program, Department of Surgery, USC Keck School of Medicine and the Saban Research Institute of Children’s Hospital Los Angeles, Los Angeles, CA 90027, USA bCraniofacial Developmental Biology and Regeneration Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD 20892, USA

Received for publication 6 April 2004, revised 16 September 2004, accepted 20 September 2004 Available online 22 October 2004

Abstract

Members of the (Dkk) family of secreted are potent inhibitors of Wnt/h-catenin signaling. In this study we show that Dkk1, -2, and -3 are expressed distally in the epithelium, while Kremen1, the needed co-receptor, is expressed throughout the epithelium of the developing lung. Using TOPGAL mice [DasGupta, R., Fuchs, E., 1999. Multiple roles for activated LEF/TCF transcription complexes during hair follicle development and differentiation. Development 126, 4557–4568] to monitor the Wnt pathway, we show that canonical Wnt signaling is dynamic in the developing lung and is active throughout the epithelium and in the proximal smooth muscle cells (SMC) until E12.5. However, from E13.5 onwards, TOPGAL activity is absent in the SMC and is markedly reduced in the distal epithelium coinciding with the onset of Dkk-1 expression in the distal epithelium. To determine the role of Wnt signaling in early lung development, E11.5 organ cultures were treated with recombinant DKK1. Treated lungs display impaired branching, characterized by failed cleft formation and enlarged terminal buds, and show decreased a-smooth muscle actin (a-SMA) expression as well as defects in the formation of the pulmonary vasculature. These defects coincide with a pattern of decreased fibronectin (FN) deposition. DKK1-induced morphogenetic defects can be mimicked by inhibition of FN and overcome by addition of exogenous FN, suggesting an involvement of FN in Wnt-regulated morphogenetic processes. D 2004 Elsevier Inc. All rights reserved.

Keywords: Lung; Dickkopf-1; Wnt signaling; Fibronectin; Branching morphogenesis; Smooth muscle; Vascular development

Introduction and branch laterally and distally in a highly reproducible pattern. They develop asymmetrically, so that in the mouse Early embryonic lung development is characterized by a the right bud gives rise to four lobes, whereas the left bud succession of cycles of bud initiation, bud outgrowth, and gives rise to only one. bud arrest. Lung formation starts at E9.5, with an out- The differentiation process of the epithelium and pocketing of the endoderm from the ventral foregut. mesenchyme is tightly coordinated with the branching Initially, the two primary buds undergo repetitive outgrowth process. The most distal epithelial and mesenchymal cells are essentially undifferentiated and as they leave the distal domain, they differentiate progressively (Hogan, 1999). The * Corresponding author. Developmental Biology Program, Department differentiation process is dependent upon the combination of of Surgery, USC Keck School of Medicine and the Saban Research Institute of Children’s Hospital Los Angeles, 4650 W Sunset Boulevard, 804 SRT, signals received. Experimental evidence is rapidly emerging MS 35, Los Angeles, CA 90027. Fax: +1 323 906 8096. that the balance between activating and inhibitory signals is E-mail address: [email protected] (S. Bellusci). a mechanism of fine regulation in development. For

0012-1606/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.ydbio.2004.09.023 S.P. De Langhe et al. / Developmental Biology 277 (2005) 316–331 317 example, Sonic hedgehog activity is modulated by Hedge- expression of pro-SP-C and Vascular endothelial hog Interacting Protein (HIP), which binds to SHH and growth factor-A (Vegf-A), the latter correlating with a prevents its binding to its receptor, Patched (PTC) (Chuang reduction in alveolar capillaries. So far, inactivation of only et al., 2003). Similar inhibitory mechanisms exist between two WNT ligands has resulted in lung defects. Wnt7b,a Bone Morphogenetic Protein 4 and extracellular modulators canonical Wnt, is expressed in epithelial cells of the like Noggin, Gremlin, or Chordin (reviewed by Balemans peripheral lung. Wnt7bÀ/À mice exhibit perinatal death and Van Hul, 2002). In the Wnt pathway, secreted Frizzled- due to respiratory failure. Defects were observed in related proteins (sFRP) and more recently Dickkopf (DKK) proliferation of the lung mesenchyme resulting in lung are secreted molecules shown to inhibit Wnt signaling hypoplasia. In addition, Wnt7bÀ/À embryos and newborn (reviewed by Kawano and Kypta, 2003). mice exhibit severe defects in the smooth muscle compo- This work focuses on the role of DKK1 during lung nent of the major pulmonary vessels, with increased development. Through our study of DKK1, our goal is also to apoptosis of the vascular smooth muscle cells (VSMCs), investigate the role of the Wnt pathway during this process. resulting in rupture of the major blood vessels and The Wnt growth factor family in the mouse is comprised of hemorrhages in the lungs after birth (Shu et al., 2002). 19 different secreted ligands that interact with 10 known Wnt5a, a noncanonical Wnt, which is able to inhibit seven-transmembrane receptors of the Frizzled (Fz) canonical Wnt signaling (Topol et al., 2003; Westfall et family and either one of two single-span transmembrane al., 2003), is expressed at high levels in the distal lung proteins, low-density-lipoprotein-receptor-related proteins mesenchyme. Wnt5aÀ/À mice die perinatally from lung (LRP-5 and LRP-6) (Pinson et al., 2000; Tamai et al., 2000; defects including truncation of the trachea, overexpansion of Wehrli et al., 2000). Historically, Wnt proteins have been the peripheral airways, and delayed lung maturation. grouped into two classes, canonical and noncanonical. Absence of Wnt5a activity in mutant lungs leads to Canonical Wnts bind to frizzled receptors, inhibiting increased cell proliferation and up-regulation of the expres- glycogen-synthase kinase-3h (GSK-3h)-mediated phosphor- sion of Fgf10, Bmp4, Shh, and Ptc (Li et al., 2002). ylation of h-catenin. Hypophosphorylated h-catenin accu- To determine the role of canonical Wnt signaling in early mulates in the cytoplasm, after which it translocates to the lung development, we treated E11.5 lung explants with nucleus, where it heterodimerizes with members of the TCF/ recombinant Dickkopf-1 (DKK1), a potent and specific LEF transcription factor family to activate the transcription of diffusible inhibitor of Wnt action that is also endogenously TCF/LEF target (reviewed by Seto and Bellen, 2004). secreted by the distal lung epithelium. We show that organ Noncanonical Wnts activate other Wnt signaling pathways, cultures treated with DKK1 display impaired branching, such as the planar-cell-polarity (PCP)-like pathway that characterized by failed cleft formation and enlarged terminal guides cell movements during gastrulation (Heisenberg et buds. DKK1-treated lung explants further show reduced a- al., 2000). Activation of the PCP pathway can antagonize the smooth muscle actin (a-SMA) expression and defects in the canonical pathway intracellularly (Ishitani et al., 2003; Kuhl formation of the pulmonary vascular network. These defects et al., 2001; Torres et al., 1996). Secreted Wnt antagonists can coincide with a pattern of decreased fibronectin (FN) be divided into two functional classes, the secreted Frizzled- deposition and Platelet-derived growth factor-A (Pdgf-a) related protein (sFRP) class and the Dickkopf class. Members expression. DKK1-induced morphogenetic defects can be of the sFRP class bind directly to Wnts, thereby altering their mimicked by inhibition of FN and overcome by addition of ability to bind to the Wnt receptor complex, while members exogenous FN. Our findings suggest an intersection of the Dickkopf class inhibit Wnt signaling by binding to the between the Wnt and FN-integrin pathways and an involve- LRP5/LRP6 component of the Wnt receptor complex. In ment of FN in Wnt-regulated morphogenetic processes. addition to LRP5/6, DKK1 interacts with the high-affinity DKK1 co-receptors Kremen1 (Krm1) or Kremen2 (Krm2), which functionally cooperate with Dkk1 to block Wnt/beta- Materials and methods catenin signaling (Mao et al., 2002 and reviewed by Kawano and Kypta, 2003). DKK1, DKK3, and DKK4 act as inhibitors Transgenic embryos of canonical Wnt signaling, while DKK2 can function both as an inhibitor in the presence of Kremen2 or as an activator in TOPGAL and Sp-C-GFP mice were a generous gift of its absence (Hoang et al., 2004; Mao and Niehrs, 2003). Dr. Elaine Fuchs and Dr. Jonathan Heath, respectively. Wnt signaling plays an important role in lung develop- Flk1LacZ/+ mice were obtained from Jackson Laboratories. ment. Conditional inactivation of the b-catenin gene in the All mice were bred in the C57BL6 background. Lungs were epithelium of the developing mouse lung leads to neonatal isolated from embryonic day 11.5 (E11.5) embryos. death resulting from severe lung defects (Mucenski et al., 2003); branching of secondary bronchi is altered and the b-galactosidase staining number of small peripheral alveolar ducts and terminal saccules is markedly reduced. In addition, the epithelium TOPGAL or Flk1LacZ/+ lungs at different developmental fails to undergo proper distal differentiation, lacking the stages (E11.5–E14.5) were dissected and fixed in 4% 318 S.P. De Langhe et al. / Developmental Biology 277 (2005) 316–331 paraformaldehyde in PBS at 48C for 10 min with rocking, No differences were observed between 200 and 400 ng/ml. washed twice for 10 min in PBS at 48C, transferred into The biological effect of an initial dose of DKK1 (added at freshly prepared X-gal solution, and stained at 378C until a t = 0 h) on lung goes down with time. A peak in the clear precipitate formed (Hogan et al., 1994). After rinsing phenotype (essentially decreased cleft formation) is with PBS, lungs were post-fixed in 4% paraformaldehyde obtained at 48 h and then the treated lung resumes a in PBS. For vibratome sections, samples were embedded development that is similar to the nontreated lung. After 96 in an albumin (300 mg/ml)–gelatin (5 mg/ml) mix, cross- h of culture, the treated and untreated lungs have the same linked with glutaraldehyde (0.6%), and sectioned at 30 Am. phenotype. However, a clear phenotype can be seen when For microtome sections lungs were fixed in 4% parafor- the medium gets refreshed with DKK1 (200 ng/ml) every 24 maldehyde, washed in PBS, dehydrated, and paraffin h. This suggests that recombinant DKK1 protein is very embedded. Sections were stained with eosin for 2 s. unstable. Fibronectin was inhibited by adding anti-FN antibody R745 diluted 1:50 to cultured E11.5 lung explants. In situ hybridization Rescue experiments were performed by adding human plasma FN (2 mg/ml) dialyzed against DMEM/F12 to Microdissected and cultured lungs were fixed in 4% DKK1 (200 ng/ml) treated cultures. Explants were cultured paraformaldehyde in phosphate-buffered saline (PBS) for 2 for 3 days. Due to the limited stability of DKK1, fresh h. They were washed twice with PBS, dehydrated over- DKK1 was added every day. night in 70% ethanol at 48C, and stored in absolute ethanol at À208C. The whole-mount in situ hybridization Immunohistochemistry protocol used was based on a previously described method (Winnier et al., 1995). The following mouse cDNAs were Cultured lungs (72 h) were fixed in 4% paraformalde- used as templates for the synthesis of digoxigenin-labeled hyde for 20 min, washed in PBS, dehydrated, and paraffin riboprobes: a 1.5-kb full-length mouse Bmp-4 (Winnier et embedded. Sections were treated with a monoclonal anti al., 1995), a 818-bp full-length Dkk1 and 1.4-kb Kremen1 a-smooth muscle actin antibody, clone 1A4, Cy3 con- and -2 probes (kindly provided by Dr. Christopher jugated (from SigmaR) at 1:200 and/or polyclonal anti h- Niehrs), a 991-bp Dkk2 fragment (cloned by RT-PCR galactosidase antibody (from US Biological) at 1:200. Anti using primers Dkk2-F agttcttaagagtcccgagt and Dkk2-R Sp-C polyclonal antibody was used at dilution 1:1000 tacacaaattcacagtctgc), a 301-bp Dkk3 fragment (cloned by (a kind gift of Dr. Jeffrey Whitsett). All slides were RT-PCR using primers Dkk3-F cgtcctctgaggtgaacctggc and mounted with DAPI containing VectashieldR and photo- Dkk3-R gtctcgggtgcatagcatctgc), a 800-bp Fz7, a 1-kb Fz8 micrographs were taken. (kindly provided by Dr. Jeremy Nathan), a 360-bp Lef1 probe (kindly provided by Dr. Rudolf Grosschedl), a 642- RT-PCR bp Shh, a 630-bp Wnt-2A, a 360-bp Wnt5a, a 300-bp Wnt7b (kindly provided by Dr. Andrew McMahon), a RNA was isolated from individual lung cultures grown 900-bp Pdgf-a fragment (kindly provided by Dr. Linda in the presence or absence of DKK1, using Trizol reagent Karlsson), a 584-bp fragment of Fgf10 (Bellusci et al., (Invitrogen) according to the manufacturer’s instructions. 1997), and a 2-kb LacZ probe (kindly provided by Dr. PCR reactions were performed with a minimum number of Robert Kelly). cycles. RNA concentration was determined by spectro- photometry. cDNA was generated using Superscript first- Organ culture strand synthesis system for RT-PCR (Invitrogen) according to the manufacturer’s instructions. Competitive RT-PCR Isolated E11.5 lung explants removed on day 11.5 was performed for b-actin, Vegf-A ,andBmp4 as (E11.5) of gestation were placed on Nuclepore filters, previously described (Zhao et al., 1998) to determine the which were then laid on the surface of 800 Al DMEM/F12 effects of treatment on mRNA levels in the lungs. The medium containing 50 units/ml penicillin and 50 Ag/ml same primers were used to amplify both cDNA and streptomycin and 10% heat-inactivated fetal calf serum in competitor for each gene of interest. A known amount of Nunclon dishes (technique adapted from Lebeche et al., competitor was added to products of reverse transcription. 1999). For some experiments illustrated in Figs. 9–11, Competitive PCR, using b-actin, was performed with all organ cultures were carried out in the absence of serum. No samples to equalize the concentration of cDNA in differ- difference was seen in overall growth, budding, or differ- ent samples. entiation of the lungs compared to lungs grown with serum. Semiquantitative RT-PCR was used for the other genes We investigated the effects of DKK1 on these cultures by of interest. The number of cycles for the different PCR adding 200 ng/ml of exogenous recombinant human DKK1 reactions was chosen to be in the linear amplification (R&D Systems). Dose response experiments (25, 50, 100, phase. Oligonucleotides used were Shh-F gtcttctacgtgatc- 200, and 400 ng/ml) were carried out. No response could gagac, Shh-R ctgcgagtaccagtggat (32 cycles); Fgf10-F be seen at concentrations of DKK1 lower that 200 ng/ml. tgttttttgtcctctcctgggag, Fgf10-R ggatactgacacattgtgcctcag S.P. De Langhe et al. / Developmental Biology 277 (2005) 316–331 319

(32 cycles); Wnt2a-F atccaaagaagaaaggaagt, Wnt2a-R Results tttacactcacacttggtca (32 cycles); Bmp4-F tccatcacgaagaa- catc, Bmp4-R tagtcgtgtgatgaggtg (30 cycles); Vegf -F Wnt signaling in early lung development gtgatcaagttcatggacgtct, Vegf -R atctgcatggtgatgttgctct (30 cycles); b-actin-F, b-actin-R (28 cycles); Pdgf-a-F tcttgga- Whole-mount RNA in situ hybridizations for Wnt gatagactccgta, Pdgf-a-R acctcacatctgtctcctc (30 cycles); b- ligands, receptors, and the Tcf/Lef1 transcription factors actin-F gctctagacttcgagcaggaga, b-actin-R gatcttcatggtgc- were performed to determine the expression of some of the tagg (28 cycles); LacZ-F gcatcgagctgggtaataagcgttggcaat mediators of Wnt signaling during lung development. In the and LacZ-R gacaccgacacaactggtaatggtagcgac (30 cycles). early mouse lung (at E12.5), Wnt7b is expressed in the lung epithelium with a more intense expression distally (Fig. 1A Proliferation study and Shu et al., 2002), Wnt2a is highly expressed in the distal mesenchyme (Fig. 1B) and Wnt5a shows low expression in Organ cultures were carried out in the presence of the mesenchyme and epithelium and is highest around the bromodeoxyuridine (BrdU, Amersham Biosciences UK trachea and pharynx (Fig. 1C and Bellusci et al., 1996). Lef1 Limited) (1:500) for 3 h. Cultured lungs were fixed in (Lymphoid enhancer factor 1) is highly expressed in the 4% paraformaldehyde for 20 min, dehydrated, and distal mesenchyme and at lower levels throughout the paraffin embedded. The sections were then treated with mesenchyme adjacent to the lung epithelium (Fig. 1D and monoclonal anti-bromodeoxyuridine (Clone BU-1) RPN Tebar et al., 2001). Fz7 is expressed in the mesenchyme 202 as recommended by the manufacturer (Amersham with higher expression distally and shows weaker expres- Biosciences). FITC-labeled anti-mouse secondary anti- sion in the epithelium (Fig. 1E and data not shown). Fz8 is bodies were used. Vectashield with DAPI was used as highly expressed throughout the epithelium (Fig. 1F). Fz2, a mounting medium. The sections were then photomicro- 3,and6 are expressed both in the epithelium and graphed. The labeled epithelial cells as well as the total mesenchyme (data not shown). These expression patterns number of cells of the lungs per section, photomicro- suggest that Wnt signaling can originate from the epithelium graphed at 20Â, were counted. Five fields of distal and mesenchyme and can target both tissues in an autocrine epithelium were counted and between 50 and 80 cells and/or paracrine fashion. were counted per field. The percent of BrdU-labeled cells TOPGAL mice, which carry a b-galactosidase gene is reported. Statistical analysis was performed using the under the control of a LEF/TCF and h-catenin inducible paired t test. promoter (DasGupta and Fuchs, 1999), were used to investigate where LEF/TCF transcription factor complexes Cell death analysis were activated during early lung development. From E10.5 until E12.5, TOPGAL activation occurred throughout the Cultured lungs (72 h) were fixed in 4% paraformalde- epithelium and in the mesenchyme adjacent to the proximal hyde for 20 min, washed in PBS, dehydrated, and paraffin airways where the bronchial smooth muscle cells (SMC) embedded. The bIn situ cell death detection kit, fluo- arise (Figs. 1G–J and Maretto et al., 2003). At E13.5 (data resceinQ for detection of apoptosis at single cell level, not shown) and E14.5 (Figs. 1K,L), TOPGAL activity was based on labeling of DNA strand breaks (TUNEL no longer detected in the mesenchyme (data not shown) and technology), from Roche Applied Science, was used per the activity in the epithelium was now significantly reduced standard instructions. All slides were mounted with DAPI distally concomitant with the onset of expression of Dkk1 in containing VectashieldR and photomicrographs were the distal epithelium (Figs. 2A,B). Dkk1 (Figs. 2A,B) and taken. Statistical analysis was performed using the paired Dkk3 (Fig. 2E) expression in the distal epithelium starts at t test. E13.5 and E12.5, respectively, while Dkk2 (Figs. 2C,D) expression in the epithelium starts as early as E11.5 and is Counting of peripheral lung buds and quantitation of up-regulated distally at later stages. The expression of high- vasculogenesis affinity DKK1 co-receptors Kremen1 (Fig. 2F) and Kre- men2 (expressed at lower level, data not shown), which Lung explants were cultured for 72 h in the presence or functionally cooperate with Dkk1 to block Wnt/h-catenin absence of either DKK1, an anti-FN antibody, or DKK1 + signaling, is detected between E11.5 and E14.5 throughout FN. Peripheral lung buds were counted and statistical the epithelium. analysis was performed using the paired t test. Meta- morphk was used to quantify the surface corresponding to DKK1 treatment inhibits canonical WNT signaling the h-galactosidase staining (reported in % of the total lung surface) in Flk1LacZ/+ lungs treated with DKK1, antibodies TOPGAL transgenic mice were also used to test whether against FN or DKK1 plus FN. Statistical analysis was exogenously supplied recombinant DKK1 protein in lung performed using a single factor ANOVA followed by the explant cultures of E11.5 lungs would result in the inhibition Dunnett’s multiple comparison post-test. of canonical Wnt signaling. TOPGAL activity as shown by 320 S.P. De Langhe et al. / Developmental Biology 277 (2005) 316–331

Fig. 1. Wnt signaling and lung development. Whole-mount in situ hybridizations on E11.5 lungs (A–F) and TOPGAL activity in E10.5–14.5 lungs (G–L). (A) Wnt7b is expressed in the lung epithelium with higher expression distally. (B) Wnt2a is highly expressed in the distal mesenchyme. (C) Wnt5a shows low expression in the mesenchyme and epithelium and is highest around the pharynx and proximal trachea. (D) Lef1 is highly expressed in the distal mesenchyme and in the mesenchyme adjacent to the pulmonary epithelium. (E) Fz7 is expressed in the mesenchyme with higher expression distally. (F) Fz8 is highly expressed throughout the epithelium. (G, H) TOPGAL expression at E10.5 and E12.5 show activity of the Wnt pathway throughout the lung. (I) Vibratome sections of the lung shown in H exhibit LacZ expression in the distal epithelium. (J) Microtome section through one of the first two bronchi shows additional expression in the mesenchyme adjacent to the epithelium. (K–L) E14.5, TOPGAL expression is markedly reduced from the distal epithelial branches; this loss of TOPGAL expression coincides with the onset and domain of expression of DKK1 (see Fig. 2). h-gal staining after 72 h of culture was notably reduced in DKK1 treatment of lung explant cultures disrupts branching the distal epithelium of DKK1 treated organ cultures (Figs. 3B,D; n = 5) compared to controls (Figs. 3A,C). The Transgenic mice expressing green fluorescent protein detection of remaining TOPGAL activity in the more (GFP) under the control of the Sp-C (surfactant protein-C) proximal epithelium of DKK1 treated organ cultures (Fig. promoter (which at early developmental stages is uniformly 3B) is likely the result of the high stability of the h- expressed in the distal epithelium) were used to study the galactosidase protein. In support of this conclusion, whole- effect of exogenously supplied recombinant DKK1 protein mount in situ hybridization for LacZ was performed on lung on branching morphogenesis in explant cultures of E11.5 explants cultured for 72 h in the presence or absence of lungs. The expression of GFP in the epithelium allowed us to DKK1. The expression of LacZ was not detected after 72 h follow the growth and branching of the lung during culture of culture in lung explants treated with DKK1 (Fig. 3F) (Fig. 4). E11.5 Sp-C-GFP lung explants were cultured for 72 while control explants still showed expression throughout h in the presence or absence of 200 ng/ml of recombinant the epithelium and in the proximal mesenchyme (Fig. 3E). DKK1. DKK1-treated lungs were not significantly different This data was confirmed by semiquantitative RT-PCR for in overall size compared to controls (compare Figs. 4A,E,I,M LacZ on RNA isolated from DKK1 treated and untreated with B,F,J,N). DKK1 treatment resulted in decreased lung explants grown for 72 h (Fig. 7M). branching characterized by a defect in cleft formation and S.P. De Langhe et al. / Developmental Biology 277 (2005) 316–331 321

Fig. 2. Dkk expression in the lung epithelium. was detected by whole-mount in situ hybridization on lungs at different gestational stages. (A–B) Dkk1 is expressed from E13.5 on in the distal epithelium. (C) Dkk2 is expressed in the lung epithelium at E11.5 and is (D) up-regulated distally at later stages (shown here at E15.5). At E12.5, Dkk3 is expressed in the distal epithelium (E). At E11.5, Kremen1 is expressed throughout the epithelium (F). enlarged terminal buds compared to controls. DKK1-treated FN, a protein previously described to be downstream of lungs had an average of 24.4 F 3.2 peripheral buds compared Wnt signaling in Xenopus (Gradl et al., 1999) and the to control lungs which had 34.4 F 3.1 ( P V 0.0001, n = 5), perturbation of which leads to defects in branching in resulting in a decrease in branching of 29%. Defects in embryonic salivary glands, lung, and kidney (Sakai et al., branching started to appear after 48 h (compare Figs. 4I,K 2003). The deposition of FN was therefore investigated by with J,L) in culture and were very obvious after 72 h of immunohistochemistry on DKK1-treated and control lung culture in the presence of DKK1 (Figs. 4N,P) as compared to explants and was found to be markedly decreased in both control lungs (Figs. 4M,O). Interestingly, these defects in the epithelial basement membrane and the mesenchyme branching morphogenesis are not associated with defects in of the DKK1-treated lung explants after 72 h of culture proliferation. Proliferation rates, assessed by BrdU incorpo- (Fig. 5, n = 4). ration, in the epithelium of DKK1-treated lungs vs. control lungs, were not significantly different (36 F 9% and 40 F DKK1 treatment perturbs differentiation of the mesenchyme 9%, respectively, n =5,P = 0.2). At the same time, no significant difference in apoptosis could be seen between At E12.5, TOPGAL activity is present in the mesen- lung explants treated with DKK1 vs. control lungs (6.9 F 1% chyme around the bronchi (Fig. 6A). These cells are also and 5.3 F 1%, respectively, n =7,P = 0.05). co-expressing a-SMA (Fig. 6B) and therefore represent proximal bronchial SMC. These observations led us to Fibronectin deposition is decreased in DKK1-treated lung investigate whether DKK1 treatment of E11.5 lung explants explants would result in aberrant smooth muscle cell differentiation. E11.5 lung explants were cultured up to 72 In order to better define the observed defects in h in the presence or absence of DKK1. In control lungs, branching morphogenesis, we analyzed the deposition of a-SMA is detected at high levels around the bronchi and 322 S.P. De Langhe et al. / Developmental Biology 277 (2005) 316–331

Fig. 3. DKK1 treatment inhibits canonical WNT signaling. Lungs isolated from E11.5 TOPGAL transgenic mice were grown in culture for 72 h in the presence (B, D) or absence (A, C) of recombinant DKK1 (200 ng/ml). TOPGAL activity was analyzed by h-gal staining. TOPGAL activity is reduced in DKK1-treated lungs distally (B, D) compared to control (A, C). LacZ expression was detected by whole-mount in situ hybridization on TOPGAL lung explants isolated from E11.5 WT mice and grown in culture for 72 h in the presence (F) or absence (E) of recombinant DKK1 (200 ng/ml). Under our conditions, LacZ expression is not detected in DKK1-treated lung explants (F) vs. control (E). is excluded from the tip of the buds (Figs. 6C,E). In the expression of key regulatory molecules in lung morpho- DKK1-treated lung explants, further distal expression of genesis. The expression pattern of Pdgf-a was studied be- a-SMA was severely impaired (Figs. 6D,F; n = 10). a- cause of its known role in myofibroblast progenitor SMA expression was mostly detected proximally in proliferation and differentiation (Lindahl et al., 1997). DKK1-treated lung explants as early as 24 h after Pdgf-a expression was markedly reduced in lung explants treatment (data not shown). Interestingly, no differences grown in the presence of DKK1 (Fig. 7B) compared to could be found in Sp-C expression in the epithelium in control lungs (Fig. 7A, n = 3). Further, to test whether untreated vs. DKK1-treated lung explants (data not shown, DKK1 could be involved in a feedback loop with canonical n = 4). This result suggests that in our experimental Wnts expressed distally in the lung, the expression pattern conditions, the proximal–distal differentiation of the lung of Wnt2a was studied by whole-mount in situ hybridization. epithelium is not dramatically affected. Wnt2a was markedly reduced in the distal mesenchyme in lung explants grown in the presence of DKK1 (Fig. 7D) DKK1 treatment results in a decrease in Pdgf-a and Wnt2a compared to control lungs (Fig. 7C; n = 3). No differences expression could be detected in the expression of Fgf10 (Figs. 7F,E), Bmp4 (Figs. 7H,G), or Shh (Figs. 7J,I). This data was Because of the observed defects in branching, whole- confirmed by competitive and/or semiquantitative RT-PCR mount in situ hybridization after 72 h of culturing in the on RNA isolated from DKK1-treated and untreated lung presence or absence of DKK1 was carried out to determine explants grown for 72 h (Fig. 7M). S.P. De Langhe et al. / Developmental Biology 277 (2005) 316–331 323

Fig. 4. DKK1 treatment disrupts branching morphogenesis. Lungs isolated from E11.5 Sp-C-GFP transgenic mice were grown in culture for 72 h in the presence (B, D, F, H, J, L, N, P) or absence (A, C, E, G, I, K, M, O) of recombinant DKK1 (200 ng/ml), and branching morphogenesis was examined at 24-h time intervals. E11.5 Sp-C GFP lung explants with similar numbers of buds were selected (A–D). After 24 h, no obvious differences in branching could be detected (E–H). After 48 h, branching was clearly decreased (arrows) in the DKK1-treated cultures (J, L) compared to controls (I, K). By 72 h, DKK1-treated organ cultures (N, P) showed markedly enlarged terminal buds vs. controls (M, O).

DKK1 perturbs vascular development difference between control and DKK1-treated lung explants (3.3 F 1.1% vs. 1.5 F 0.7%, respectively, P b 0.05). This To study the effect of DKK1 on vasculogenesis in the defect occurs without a decrease in Vegf-a expression based lung, lungs isolated from E11.5 Flk1LacZ/+ mice were on competitive RT-PCR results (Fig. 7M). cultured in the presence or absence of exogenous recombi- nant DKK1. Flk1LacZ/+ mice exhibit b-galactosidase Inhibition of FN activity recapitulates DKK1 defects in the expression in endothelial cells under the control of the lung Flk1 promoter (Shalaby et al., 1995). h-gal staining illustrates abnormal vessel formation in DKK1 treated A polyclonal anti-FN antibody was used to inhibit cleft explants: Blood vessels are thinner, show reduced sprouting formation and branching in the lung as described by Sakai from preexisting vessels (angiogenesis), and impaired et al. (2003). Treatment with an anti-FN antibody formation of large vessels and establishment of intercon- significantly inhibited branching (Figs. 9A,B) of the lung nections (Figs. 8C,D), compared to untreated lung explants explants. Lung explants treated with an anti-FN antibody (Figs. 8A,B; n = 3). Quantification of the surface had an average of 15.6 F 4.6 peripheral buds compared to corresponding to the h-galactosidase staining, reported in control lungs which had an average of 35.4 F 2.4 ( P V percentage of the total lung surface, shows a significant 0.0001, n = 7) peripheral lung buds, resulting in a 324 S.P. De Langhe et al. / Developmental Biology 277 (2005) 316–331

Fig. 5. Fibronectin expression is decreased in DKK1-treated lungs. Lungs isolated from E11.5 wild-type mice where grown in culture for 72 h in the presence (B, D) or absence (A, C) of recombinant DKK1 (200 ng/ml). Fibronectin expression was analyzed by immunohistochemistry on sections. (A, C) Fibronectinis present in the basement membrane around the epithelium (arrowheads) and in the mesenchyme at sites of cleft formation (arrows). (B, D) Fibronectin deposition by the epithelium (arrowheads) into the basement membrane and at sites of cleft formation (arrows) is reduced upon DKK1 treatment. Experimental conditions and photography (exposure time) were identical between control and DKK1-treated lungs to allow for direct comparison of FN expression. The exposure time was chosen so that the maximum signal (i.e., for CTRL) was below the saturation point of the camera.

Fig. 6. DKK1 reduces SMA expression in the mesenchyme. (A) h-gal (green) immunohistochemistry on sections of E12.5 TOPGAL lungs show TOPGAL activity in the epithelium and the proximal SMC (arrows). (B) Co-immunostaining of h-gal (green) and SMA (red) (arrows) (on sections) in the proximal SMC around the upper respiratory airways. Lungs isolated from E11.5 WT mice were grown in culture for 72 h in the presence (D, F) or absence (C, E) of recombinant DKK1 (200 ng/ml). a-Smooth muscle actin (SMA) expression was analyzed by immunohistochemistry (red); nuclei were stained with DAPI (blue). (C, E) In the untreated lung explants, the expression of SMA is found in the bronchial SMC around the airways. (D, F) In DKK1-treated lung explants, SMA expression did not expand in spite of lung growth, and very little SMA expression can be detected more distally around the airways. Experimental conditions and photography (exposure time) were identical between control and DKK1 treated lungs to allow for direct comparison of a-SMA expression. The exposure time was chosen so that the maximum signal (i.e., for CTRL) was below the saturation point of the camera. S.P. De Langhe et al. / Developmental Biology 277 (2005) 316–331 325 decrease in branching of 56%. Treatment with an anti-FN epithelium but was reduced distally, concomitant with the antibody further reduced smooth muscle differentiation onset of expression of Dkk1 in the distal epithelium. To around the airways (Figs. 9C,D) and perturbed proper lung determine the role of canonical Wnt signaling in early lung vascular network formation (Figs. 8E,F), recapitulating the development, we treated E11.5 lung explants in vitro with defects seen after treatment with DKK1. Quantification of recombinant DKK1, a potent and specific diffusible the surface corresponding to the h-galactosidase staining inhibitor of Wnt action that is also endogenously secreted shows a significant difference between control and lung by the lung epithelium. We used TOPGAL, Sp-C-GFP and explants treated with antibodies against FN (3.3 F 1.1% Flk1LacZ/+ mice, respectively, to show that DKK1 treatment vs. 1.0 F 0.2%, respectively, P b 0.05). However, Pdgf-a of lung explants inhibited canonical Wnt signaling, impaired expression was unchanged in lung explants grown in the branching and resulted in defects in the formation of the presence of an anti-FN antibody (Fig. 7L) compared to pulmonary vascular network. The DKK1-treated lungs control lungs (Fig. 7K, n = 3). further displayed a reduction in a-SMA expression around the bronchi. These defects coincided with a pattern of Exogenously supplied FN rescues the DKK1 phenotype decreased FN deposition. We further observed a reduction in Wnt2a and Pdgf-a expression. However, no difference in Exogenous FN (2 mg/ml) (Sakai et al., 2003) was added expression could be detected for the major lung morphogens to DKK1-treated organ cultures to replace the FN depleted Fgf10, Shh,andBmp4,norVegf , and no significant by DKK1 treatment. Experimental supplementation of FN differences in proliferation of the epithelium could be successfully rescued branching morphogenesis (Figs. 10I,J) found. Inhibition of FN activity with an anti-FN antibody compared to organ cultures treated with DKK1 alone (Figs. recapitulated the DKK1 imposed morphogenetic defects in 10E–H) and control lungs (Figs. 10A–D). While DKK1- the lung whereas exogenously supplied FN rescued the treated lungs responded as previously described (Fig. 4), no DKK1 phenotype. significant difference in number of peripheral buds could be found between control lung explants (29 F 2.4) and lung Wnt signaling in early lung development explants treated with DKK1+FN (29.4 F 2.5) ( P = 0.3786, n = 5). Addition of exogenous FN to DKK1-treated lung In the embryonic lung, Wnt ligands, Wnt receptors, and explants did not rescue the loss of TOPGAL (Fig. 10L) associated Wnt transcription factors are detected from very activity compared to control lungs (Fig. 10K), indicating early stages of development in the epithelium and mesen- that the inhibitory effect of DKK1 on canonical Wnt chyme, suggesting that Wnt signaling can occur in both tissue signaling was not affected and that FN is acting downstream compartments. However, our results using TOPGAL mice of DKK1. Further, exogenously supplied FN not only show that Wnt signaling occurs mostly in the epithelium and rescued but enhanced a-SMA expression distally in DKK1- additionally in the mesenchymal cells directly apposed to the treated lung explants (Figs. 11E,F, n = 3) compared to, proximal bronchi (Fig. 1 and Maretto et al., 2003). The respectively, lung explants treated with DKK1 alone (Figs. mesenchymal TOPGAL-positive cells in the lung also 11C,D), or untreated control explants (Figs. 11A,B). Close express a-SMA, indicating that differentiated SMC around examination of the distal portion of the lungs showed that, the proximal airways are responding to Wnt signaling (Fig. while in control lungs, the expression of a-SMA is found in 6). TOPGAL mice have previously been shown to be the SMC around the bronchi but excluded from the tip (Fig. excellent reporters for Wnt signaling (DasGupta and Fuchs, 11B); FN treatment (in presence of DKK1) resulted in an 1999) and in vivo overexpression in the distal lung epithelium expansion of the a-SMA expression domain more distally of the noncanonical Wnt5a, which blocks canonical Wnt (Fig. 11F). Application of exogenous FN to DKK1-treated signaling, leads to a decrease in TOPGAL activity (Dr. C. Li, lung explants also partially rescued vascular network defects personal communication). (Figs. 8G,H; n = 3). Quantification of the surface Wnt signaling is modulated by secreted antagonists, like corresponding to the h-galactosidase staining shows no secreted Frizzled-related proteins (sFrp) and members of the significant difference between control and lung explants Dickkopf family. We show herein that the expression of treated simultaneously with DKK1 and FN (3.3 F 1.1% vs. Dickkopf-1, -2, and -3 in the distal epithelium of the lung 3.3 F 0.6%, respectively, P b 0.05). starts from E13.5, E11.5, and E12.5, respectively (Fig. 2). The onset of Dkk1 expression correlates with the reduction of TOPGAL activity in the distal epithelium from E13.5 Discussion onwards. Interestingly, Dkk1À/À mice have no lung pheno- type (Mukhopadhyay et al., 2001 and Westphal personal Careful analysis of TOPGAL mice showed that canonical communication). This suggests that the expression of Dkk1, Wnt signaling in the lung is dynamic. From E10.5 until Dkk2,andDkk3 (Fig. 2) in the distal epithelium is E12.5, TOPGAL activation occurred throughout the epi- redundant. thelium and in the proximal SMC. However, from E13.5 Transgenic experiments in mouse have shown that onwards,TOPGALactivitywasonlydetectedinthe Dkk1 overexpression inhibits canonical Wnt signaling. 326 S.P. De Langhe et al. / Developmental Biology 277 (2005) 316–331

Ectopically expressed Dkk1 under the control of a keratin- mammary gland development before the bud stage (Andl 14 promoter, which directs transgene expression to the et al., 2002). Transgenic mice ectopically expressing Dkk1 surface epithelium and to the basal cells of the epidermis in the gut epithelium showed a loss of intestinal crypts, during embryogenesis, resulted in a complete failure of and disrupted intestinal homeostasis (Pinto et al., 2003). hair follicle placode formation and blocked tooth and Adenoviral expression of Dkk1 in both the small intestine S.P. De Langhe et al. / Developmental Biology 277 (2005) 316–331 327

Fig. 8. DKK1 reduces vascular development. Lungs isolated from E11.5 Flk1LacZ/+ mice were grown in culture for 72 h in the presence of DKK1 (C, D), FN Ab (E, F), DKK1 + FN (G, H) or in absence of all those (A, B). Vasculogenesis was analyzed by h-gal staining. Note the abnormal vessel formation, thinner blood vessels, reduced sprouting from pre-existing vessels (angiogenesis), and impaired formation of large vessels and establishment of interconnections in lungs treated with DKK1 (C, D) or FN Ab compared to control (A, B). Application of exogenous FN to DKK1-treated lung explants (G, H) partially rescued the vascular network defects seen when treated with DKK1 alone (C, D) compared to control lungs (A, B). and colon of adult mice markedly inhibited proliferation, netic genes such as Fgf10, Shh, and Bmp4 could be detected accompanied by progressive architectural degeneration (Fig. 7). Moreover, the branching defects induced by DKK1 with the loss of crypts, villi, and glandular structure could be recapitulated by treating lung explants with a (Kuhnert et al., 2004). Here, we treated lung explants from blocking anti-FN antibody (Fig. 9) and could be rescued by TOPGAL embryos with recombinant DKK1 protein and co-treatment with exogenous FN (Fig. 10). our results show a marked decrease in TOPGAL activity, We also demonstrated that DKK1 treatment perturbed indicating that we could inhibit canonical Wnt signaling in expression of a-SMA, a terminal marker of smooth muscle the lung (Fig. 3). differentiation. This result also was associated with the decrease in FN expression, as treatment of lung explants Decreased FN deposition perturbs branching, smooth with a blocking anti-FN antibody reduced a-SMA expres- muscle differentiation, and formation of the lung sion around the bronchi (Fig. 9). Previously, it has been vasculature shown that the differentiation of lung embryonic mesen- chymal cells into SMC is triggered by their spreading along The extracellular matrix (ECM) protein FN has been the airway basement membrane. Blocking of basement shown to be essential for cleft formation during the initiation membrane assembly prevented mesenchymal cell spreading of epithelial branching in several organ systems including and smooth muscle differentiation (Yang et al., 1998). the lung (Gill et al., 2003; Sakai et al., 2003). Fibronectin is Further evidence for this was found when we studied a- secreted by the lung epithelium as part of the basement SMA expression in DKK1 treated lung explants that were membrane and has been shown to be a Wnt target gene in co-treated with exogenous FN. Treatment with exogenous Xenopus (Gradl et al., 1999). Our results suggest that the FN led to an increase in a-SMA expression around the decrease in FN deposition by the lung epithelium in bronchi, which was even further expanded distally com- response to DKK1 treatment (Fig. 5) is the primary cause pared to control lung explants (Fig. 11), suggesting for decreased branching of the epithelium (Fig. 4). Interest- premature differentiation of the SMC. ingly, the observed defect in branching seemed mainly to be Our results indicate that Wnt signaling is not likely to associated with the ECM defect as no differences in play a direct role in the differentiation of the distal epithelial proliferation could be found (data not shown), mesenchyme into bronchial SMC, because TOPGAL as well as no changes in the expression of key morphoge- activity could not be found in the mesenchyme adjacent

Fig. 7. Gene expression analysis in DKK1-treated lungs. Gene expression was detected by whole-mount in situ hybridization on lung explants isolated from E11.5 WT mice and grown in culture for 72 h in the presence (B, D, F, H, J) or absence (A, C, E, G, I) of recombinant DKK1 (200 ng/ml). (A, B) Pdgf-a expression is strongly reduced in DKK1 treated lung explants (B) vs. control (A). (C, D) Wnt2a expression is severely reduced in DKK1 treated lung explants (D) vs. control (C). No differences could be detected in the expression of Fgf10 (F, E), Bmp4 (H, G), or Shh (J, I). No difference could be detected in the expression of Pdgf-a in lung explants treated with an anti-FN antibody (L) compared to controls (K). Competitive RT-PCR (for Vegf, Bmp4, and h-actin) and semiquantitative RT-PCR (for LacZ, Wnt2a, Pdgf-A, Shh, and Fgf10) on RNA isolated from DKK1-treated and untreated lung explants grown for 72 h (M) shows a reduction in LacZ, Pdgf-a, and Wnt2a expression in DKK1-treated lungs vs. control, while the expression of Fgf10, Shh, Bmp4, and Vegf-a expression was unchanged. Competitive PCR, using b-actin, was performed on all samples to equalize the concentration of cDNA in different samples. Competitor fragments are 100 bp larger in size than product of interest. 328 S.P. De Langhe et al. / Developmental Biology 277 (2005) 316–331

Fig. 9. Inhibition of FN recapitulates DKK1 defects in the lung. (A, B) Phase contrast images of living E11.5 (inset 0 h) lung explants cultured for 72 h with (B) or without (A) anti-FN R745 antibody diluted 1:50. Treated lung explants showed a marked decrease in branching (B) compared to nontreated (A). (C, D) a-Smooth muscle actin (SMA) expression was analyzed by immunohistochemistry (red); nuclei were stained with DAPI (blue). (C) In the untreated lung explants, the expression of SMA is found in the bronchial SMC around the airways. (D) In anti-FN-treated lung explants, SMA expression was reduced more distally around the airways. Experimental conditions and photography (exposure time) were identical between control and DKK-1- treated lungs to allow for direct comparison of a-SMA expression. The exposure time was chosen so that the maximum signal (i.e., for CTRL) was below the saturation point of the camera. to the distal epithelium from E11.5 to E14.5, which is the epithelium results in a decrease of FN deposition by the developmental time frame of our organ cultures. Rather, epithelium. This would lead to a defective epithelial matrix we hypothesize that blocking Wnt signaling in the distal and basement membrane, which is necessary for the

Fig. 10. Exogenously supplied FN rescues branching in DKK1-treated lung explants. (A–J) Phase contrast images of E11.5 lung explants cultured for 72 h after treatment with PBS control in the absence (A, B) or presence (C, D) of 10% fetal bovine serum; DKK1 (200 ng/ml) in the absence (E, F) or presence of 10 fetal bovine serum (G, H); or DKK1 (200 ng/ml) plus FN (2 mg/ml) (I, J). FN treatment rescued branching defects in DKK1 treated lung explants (I, J). Lungs isolated from E11.5 TOPGAL transgenic mice were grown in culture for 72 h in the presence (L) or absence (K) of DKK1 (200 ng/ml) plus FN (2 mg/ml). TOPGAL activity was analyzed by LacZ in situ hybridization and was reduced in DKK1-treated lungs in the presence of FN (L) compared to control (K). S.P. De Langhe et al. / Developmental Biology 277 (2005) 316–331 329

Fig. 11. Exogenously supplied FN rescues and enhances a-SMA expression in DKK1-treated lung explants. Lungs isolated from E11.5 WT mice were grown in culture for 72 h in the presence of DKK1 (200 ng/ml) (C, D) or DKK1 (200 ng/ml) and FN (2 mg/ml) (E, F) or in absence of those (A, B). a-SMA expression was analyzed by immunohistochemistry (red); nuclei were stained with DAPI (blue). (A, B) In the untreated lung explants, the expression of a- SMA is found in the bronchial SMC around the bronchi. (C, D) In DKK1-treated lung explants, a-SMA expression did not expand in spite of clear lung growth and very little a-SMA expression can be detected more distally around the bronchi. (E, F) In lungs treated with DKK1 (200 ng/ml) and FN (2 mg/ml), enhanced a-SMA staining was detected and a-SMA expression was clearly expanded distally around the bronchi. Experimental conditions and photography (exposure time) were identical between control and treated lungs to allow for direct comparison of a-SMA expression. The exposure time was chosen so that the maximum signal (i.e. for DKK1 + FN) was below the saturation point of the camera. spreading of the smooth muscle progenitors and their of blood vessels (angiogenesis) is mediated by a5h1 differentiation. Interestingly, increased deposition of FN integrins through their binding to FN (Kim et al., 2000a,b, into the subepithelial space of the airways is observed in 2002). Indeed, similar defects in the lung vasculature were all forms of asthma and occurs early in the progression of obtained after treatment of lung explants with a blocking the disease (Hocking, 2002). Asthma is also characterized anti-FN antibody (Fig. 8), and defects induced by treatment by an increase in the number of bronchial SMC. FN is with DKK1 could partially be rescued by co-treating lung capable of inducing a-smooth muscle actin expression and explants with exogenous FN (Fig. 8). We cannot exclude promotes the differentiation of SMC in vitro (Thannickal that there could also be a direct effect of Wnt signaling on et al., 2003). the formation of the lung vasculature, since Wnt2a is needed Interestingly, no significant defect could be found in for proper placental vascularization, although Wnt2aÀ/À differentiation of the proximal SMC (evaluated by main- mice did not have an abnormal lung phenotype (Monkley et tenance of a-SMA expression) that had already differ- al., 1996). entiated in E11.5 lungs before the start of DKK1 treatment. TOPGAL activity could be found in the proximal SMC until Possible functions for Wnt signaling in the epithelium E12.5 in vivo (Figs. 1 and 6). This raises a question about the role of Wnt signaling in these proximal SMC. One The exact functions of Wnt signaling are still contro- possibility is that Wnt signaling is already switched off at versial. The present consensus about Wnt signaling during E12.5 and that the presence of TOPGAL activity is due to development is that the Wnt pathway is critical for the high stability of the h-galactosidase protein. This would progenitor cell proliferation and/or lineage differentiation indicate that Wnt signaling is directly involved in the and is involved in maintaining progenitor cells in an differentiation of the proximal bronchial SMC. A second, undifferentiated state and possibly maintaining the differ- less likely, possibility would be that the Wnt signaling in the entiation of fully matured cells (Batlle et al., 2002; Huelsken proximal SMC is necessary for maintenance of their et al., 2001; Merrill et al., 2001; Pinto et al., 2003; Reya et differentiation. However, we were not able to address this al., 2003, reviewed in Alonso and Fuchs, 2003). possibility because lungs isolated before E11.5 do not b-catenin inactivation in the distal epithelium leads to a develop well in vitro. Deleting b-catenin specifically in the smaller proximalized lung. This indicates that Wnt signaling SMC in vivo should therefore be very informative. is probably not critical for the development of the proximal Inhibition of canonical Wnt signaling further perturbed airway, but rather is necessary to prevent premature differ- the proper formation of the lung vasculature, probably entiation of the distal epithelium. Supporting this idea is that through the down regulation of FN deposition, as sprouting inactivating b-catenin in the proximal conducting airways 330 S.P. 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