© 2019. Published by The Company of Biologists Ltd | Disease Models & Mechanisms (2019) 12, dmm040139. doi:10.1242/dmm.040139

RESEARCH ARTICLE Lack of whey acidic protein (WAP) four-disulfide core domain protease inhibitor 2 (WFDC2) causes neonatal death from respiratory failure in mice Kuniko Nakajima1, Michio Ono1,UrošRadović1, Selma Dizdarević1, Shin-ichi Tomizawa1, Kazushige Kuroha1, Go Nagamatsu2, Ikue Hoshi1, Risa Matsunaga1, Takayuki Shirakawa1,*, Takeyuki Kurosawa3, Yasunari Miyazaki4, Masahide Seki5, Yutaka Suzuki5, Haruhiko Koseki6, Masataka Nakamura7, Toshio Suda8,9 and Kazuyuki Ohbo1,‡

ABSTRACT INTRODUCTION Respiratory failure is a life-threatening problem for pre-term and term During the transition from fetal to neonatal life, the cardio-respiratory infants, yet many causes remain unknown. Here, we present evidence system undergoes a drastic and sudden change: placental circulation that whey acidic protein (WAP) four-disulfide core domain protease switches to a pulmonary circulation and the respiratory system goes inhibitor 2 (Wfdc2), a protease inhibitor previously unrecognized in from being fluid filled to air filled. Malfunctions of this switch can respiratory disease, may be a causal factor in infant respiratory failure. cause respiratory failure (Reuter et al., 2014), in particular in premature Wfdc2 transcripts are detected in the embryonic lung and analysis of a infants in whom the immature lung tissues have produced inadequate Wfdc2-GFP knock-in mouse line shows that both basal and club cells, mucosal fluid (Reuter et al., 2014). The components of this fluid are and type II alveolar epithelial cells (AECIIs), express Wfdc2 neonatally. produced by respiratory epithelial cells. There are at least eight different Wfdc2-null-mutant mice display progressive atelectasis after birth with types of these cells, which form the primary barrier between air and a lethal phenotype. Mutant lungs have multiple defects, including lung tissue (Hogan et al., 2014; Montoro et al., 2018; Plasschaert et al., impaired cilia and the absence of mature club cells from the tracheo- 2018; Rock and Hogan, 2011; Treutlein et al., 2014). In the airways, bronchial airways, and malformed lamellar bodies in AECIIs. RNA the epithelium consists of basal stem cells, and ciliated, club, goblet sequencing shows significant activation of a pro-inflammatory and neuroendocrine cells, as well as other rare cell types, including pathway, but with low-quantity infiltration of mononuclear cells in the newly described ionocytes (Montoro et al., 2018; Plasschaert et al., lung. These data demonstrate that Wfdc2 function is vitally important 2018). The goblet and club cells secrete mucins and glycoproteins that for lung aeration at birth and that gene deficiency likely causes failure of have bactericidal and anti-inflammatory functions; the ciliated cells the lung mucosal barrier. move the mucus and entrap particles out of the lungs; and the ionocytes, which highly express cystic fibrosis transmembrane KEY WORDS: Cilia, Surfactant, Atelectasis, Respiratory disease conductance regulator (CFTR), likely play a role in regulating fluid secretion (Montoro et al., 2018; Plasschaert et al., 2018). In the alveolar region, the type II epithelial cells (AECIIs) produce important surfactants that consist of lipids [e.g. phosphatidylcholine (PC), 1Department of Histology and Cell Biology, Yokohama City University, School of phosphatidylglycerol (PG)] and proteins (e.g. SP-A, -B, -C, -D), which Medicine, 3-9 Fukuura, Kanazawa-ku, Yokohama, 236-0004, Japan. 2Department are stored in specialized organelles known as lamellar bodies of Stem Cell Biology, Kyushu University, Faculty of Medical Sciences, 3-1-1, Maidashi, Higashi-ku, Fukuoka City, 812-8582, Japan. 3Department of Respiratory (Chakraborty and Kotecha, 2013; Lopez-Rodriguez and Pérez-Gil, Medicine, Toho University, School of Medicine, 5-21-16, Ohmorinishi, Ohta-ku, 2014; Olmeda et al., 2017; Pérez-Gil, 2008; Rooney et al., 1994; Tokyo, Japan. 4Department of Respiratory Medicine, Tokyo Medical and Dental University, 1-5-45, Yushima, Bunkyo-ku, Tokyo, 113-8510, Japan. 5Department of Whitsett and Alenghat, 2015). Surfactants form thin films that reduce Computational Biology and Medical Sciences, Graduate School of Frontier surface tension, open up the alveolar space, and are critical for both Sciences, The University of Tokyo, Chiba, 277-8562, Japan. 6Laboratory for preventing collapse of the airways (atelectasis) and for contributing to Developmental Genetics, RIKEN Center for Integrative Medical Sciences, 1-7-22, Tsurumi-ku, Yokohama, 230-0045, Japan. 7Human Gene Sciences Center, Tokyo the host defense strategy (Chakraborty and Kotecha, 2013; Rooney Medical and Dental University, 1-5-45, Yushima, Bunkyo-ku, Tokyo, 113-8510, et al., 1994; Whitsett and Alenghat, 2015; Whitsett et al., 2010). Japan. 8Cancer Science Institute of Singapore, National Singapore University In addition to mucins and surfactants, the epithelial cells of the Centre for Translational Medicine, 14 Medical Drive, #12-01, Singapore 117599. 9International Research Center for Medical Sciences, Kumamoto University, 2-2-1 lung secrete numerous proteases and protease inhibitors – such as Honjo, Chuo-ku, Kumamoto, 860-0811, Japan. serine proteases, matrix metalloproteinases (MMPs), serine protease *Present address: Mitsubishi Tanabe Pharma, Yokohama office, 1000, Kamoshida- – cho, Aoba-ku, Yokohama, 227-0033, Japan. inhibitors and tissue inhibitor of metalloproteinases (TIMPs) that together form the protease/protease-inhibitor web (Löffek et al., ‡Author for correspondence ([email protected]) 2011; Abboud, 2012; Meyer and Jaspers, 2015). These proteases and

M.O., 0000-0001-9054-5298; S.T., 0000-0002-8291-6623; G.N., 0000-0002- protease inhibitors are deeply involved in the pathophysiology of 4901-8514; T.K., 0000-0002-8640-8853; Y.M., 0000-0002-9073-9815; Y.S., 0000- neonatal respiratory distress syndrome (RDS) as well as other lung 0002-7559-5139; H.K., 0000-0001-8424-5854; M.N., 0000-0002-9797-0673; T.S., diseases such as emphysema, cystic fibrosis, infection and pulmonary 0000-0001-7540-1771; K.O., 0000-0001-5319-7319 fibrosis (Meyer and Jaspers, 2015; Taggart et al., 2017). RDS is This is an Open Access article distributed under the terms of the Creative Commons Attribution mainly caused by pulmonary surfactant deficiency in premature License (https://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed. infants, and the lungs of RDS patients are diffusely atelectatic (Bhandari, 2014; Reuter et al., 2014). A subpopulation of RDS

Received 12 April 2019; Accepted 11 September 2019 infants who show limited responses to surfactant may go on to Disease Models & Mechanisms

1 RESEARCH ARTICLE Disease Models & Mechanisms (2019) 12, dmm040139. doi:10.1242/dmm.040139 develop chronic neonatal lung injury known as bronchopulmonary (P)1.5. Transcripts were already expressed at E11.5 and were strongly dysplasia (BPD), which involves failure of alveogenesis and upregulated at P1.5 (Fig. 1A). Analysis of lungs 6-8 h after caesarean vasculogenesis, and the risk of developing long-term morbidities section at E18.5 revealed that mRNA expression rose significantly (Bhandari, 2014; Dumpa and Bhandari, 2018). Inflammation is a after respiration began (Fig. 1A). To track the lung epithelial cells that cornerstone in the transition from RDS to BPD (Bhandari, 2014); its produce WFDC2, we generated knock-in mouse lines driving either causes include hyperoxia and mechanical stress from ventilation, but GFP or lacZ from the Wfdc2 locus (Fig. S1A-D). In agreement with proteases such as neutrophil elastase (NE) have also been shown to the mRNA expression data, Wfdc2-GFP embryos showed signal from play crucial roles in the induction of IL-1α and TNFβ (Bhandari, E14.5 (the pseudoglandular stage) in the proximal region of the 2014). However, the failure of α-antitrypsin therapy trials in human bronchial tubes. The GFP-positive cells were located in the mesial infants implies that other proteases and/or protease inhibitors may part of the Sox2-positive proximal region (Fig. 1B), and few, if any, participate in the pathogenesis of both RDS and BPD, although a were seen in the distal, Sox9-positive, region (Fig. 1C). possibility remains that drug delivery efficiency to the affected areas Mice heterozygous for the GFP knock-in allele (Wfdc2GFP/+) were might need to be improved (Beam et al., 2014). indistinguishable in lifespan and phenotype from wild-type One of the serine protease inhibitor families predicted to function littermates (Table 1). By contrast, the frequency of Wfdc2GFP/GFP in the innate immune system in the lung is the evolutionarily mice followed Mendel’s laws until birth, but thereafter declined, and conserved group of whey acidic protein (WAP) four-disulfide core none survived post-weaning (Table 1). Wfdc2GFP/GFP neonatal mice (FDC) proteins (WFDC protein family; Bingle and Vyakarnam, became cyanotic immediately after delivery (Movie 1), and all died 2008). The WFDC signature – eight conserved cysteine residues within 10 days (Table 2), suggesting the cause of death was either linked by four disulfide bonds – is often found in antimicrobial and heart failure or respiratory failure. Hearts from Wfdc2GFP/GFP mice did antifungal molecules (Jia et al., 2017; Scaletta et al., 2017), and not show any abnormality in volume or structure (Fig. S1E). inhibits protease activity (Bingle and Vyakarnam, 2008). Among the Wfdc2GFP/GFP lungs also did not show any abnormalities until birth 14 WFDC mammalian genes (at least), many map to a cluster on (Fig. S1F). However, at P1.5, apparently collapsed regions could be chromosome 20q12-13 in humans (e.g. SLPI, EPPIN and WFDC2) detected (Fig. 1D), and this was confirmed by examining histological or chromosome 2 in mice (Clauss et al., 2005; Bingle and sections (Fig. S1G). The atelectasis was progressive (Fig. S1H). Vyakarnam, 2008). This genomic region is amplified in many Among the 29 Wfdc2-deficient mice that died between P0.5 and P1.5, epithelial cancers, including breast, colon, lung, ovarian, pancreatic 93.1% had macroscopic atelectasis (Fig. 1E). By contrast, of the 31 and stomach (Liu et al., 2013; O’Neal et al., 2013). In patients with surviving Wfdc2-deficient mice, only 48.3% had atelectasis (Fig. 1E). serous and endometrioid epithelial ovarian carcinomas, a high serum These results suggest that Wfdc2 is indispensable for functioning of WFDC2 level has been reported, and affords a good diagnostic and the respiratory system after birth. prognostic marker (Drapkin et al., 2005; Jia et al., 2017; Scaletta et al., 2017). Supporting the idea that the protein not only marks Differentiation of Scgb1a1-positive club cells in the cancer cells but promotes cancerous growth, overexpression of tracheobronchial region is impaired in Wfdc2-deficient mice WFDC2 reportedly induces the proliferation and invasion of human To examine Wfdc2 expression at the cellular level, we tracked GFP ovarian cancer cells (Moore et al., 2014; Zhu et al., 2013). Similarly, expression in Wfdc2GFP/+ mice. This showed that a subset of basal in the respiratory system, the majority of adenocarcinomas and a [positive for keratin 5 (KRT5pos)] and club (Scgb1a1pos) cells percentage of squamous, small cell, and large cell carcinomas express express GFP (Fig. 2A,C). To adjust the GFP gene dosage between high levels of WFDC2 protein, a marker again linked to a poor knock-out and heterozygous mice, Wfdc2GFP/LacZ mice were prognosis (Yamashita et al., 2012; Zhong et al., 2017). In a kidney established and used instead of Wfdc2GFP/GFP mice. In both fibrosis model, Wfdc2 reportedly suppresses the activity of serine Wfdc2GFP/+ and Wfdc2GFP/LacZ mice, a subpopulation of basal proteases and metalloproteases (LeBleu et al., 2013). However, the cells positive for KRT5 were positive for GFP (Fig. 2A,B), and physiological roles of Wfdc2 are just beginning to be revealed. GFPpos/KRT5pos basal cells in Wfdc2GFP/LacZ mice were fewer than We show here that deleting Wfdc2 in mice causes perinatal death those in Wfdc2GFP/+ mice (Fig. 2B). due to respiratory failure soon after birth. Wfdc2-deficient neonatal At P1.5, Scgb1a1 expression levels in Wfdc2GFP/+ mice varied mice have lung atelectasis of variable magnitude and at various significantly, ranging from low to high (Fig. S2A). Histological locations. Our findings suggest that this phenotype is likely caused analysis of Wfdc2GFP/LacZ lungs showed far fewer Scgb1a1high cells by damage to cilia, elimination of mature club cells in the than wild type (Fig. 2C, Fig. S2B). When we graded Scgb1a1- tracheobronchial region and impairment of the processing of positive cells into three categories – low, middle and high (Fig. S2C) – surfactants in AECIIs. Although histological analyses show Wfdc2GFP/LacZ mice contained fewer Scgb1a1high and Scgb1a1middle low-quantity infiltration of mononuclear cells, RNA sequencing cells (Fig. 2D), suggesting that club cells in the homozygous mutants (RNA-seq) of lung samples showed significantly upregulated fail to terminally differentiate. Interestingly, the phenotype was only inflammatory networks, in postnatal but not in embryonic stages. evident at the large airways of the trachea and primary bronchi, Taken together, these results suggest that, in vivo, Wfdc2 plays critical whereas, in the intralobular airways, Scgb1a1 expression was roles in multiple aspects of lung function: it not only promotes normal (Fig. S2D). mucociliary clearance but also confers anti-inflammatory activity and reduces surface tension. Therefore, Wfdc2 dysfunction may be a key Cilia formation is impaired in Wfdc2-deficient mice factor in driving infant respiratory failure. To explore the cause of the atelectasis, ciliated epithelial cells were visualized by staining for acetylated tubulin (Ac-tub) in RESULTS combination with either Foxj1 or neuronal calcium sensor-1 Expression of Wfdc2 during development, and lung atelectasis (NCS-1), which is specifically expressed in ciliated cells (Fig. 3A, and perinatal death in Wfdc2 homozygous-null mutants Fig. S3A) (Treutlein et al., 2014). The number of NCS-1pos ciliated We initially measured Wfdc2 RNA levels in the developing mouse cells in the tracheobronchial area of Wfdc2-deficient mice was lung at embryonic day (E)11.5, E14.5, E18.5 and postnatal day similar to control (Fig. 3B). However, the pattern of Ac-tub staining Disease Models & Mechanisms

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Fig. 1. Wfdc2 expression is detectable in the mouse proximal lung epithelium before birth and expression is upregulated after birth. (A, left) Relative mRNA expression of Wfdc2 during development. Data are shown as mean±s.e.m. (stages E11.5, n=4; E14.5-P1.5, n=2; *P<0.05; **P<0.01). (A, right) Induction of Wfdc2 mRNA expression after cesarean section (CS), 1 day before due delivery. E18.5 embryos were obtained from pregnant mice by CS, resuscitated and processed for experimental samples 6-8 h after the CS (w/ res). As a control, other pregnant mice were sacrificed at the same point in time as the resuscitated fetus collection (w/o res). Data are shown as mean±s.e.m. (n=7 mice; **P<0.05). (B) Confocal images of the proximal region of a Wfdc2GFP/GFP E14.5 lung. Yellow dotted boxes are enlarged in the right panel. Note that only the mesial parts (arrowheads) of the Sox2-positive region (red) express GFP (green). Nuclear DNA (DAPI) is shown in white. Scale bars: 100 µm. (C) Confocal images of the distal region of a Wfdc2GFP/GFP E14.5 lung. The areas boxed by yellow dotted lines are enlarged in the right panels. There was no GFP (green) expression in Sox9-positive (red) regions (arrowheads). Nuclear DNA (DAPI) is shown in white. Scale bars: 100 µm. (D) Fresh lung specimens. (a,b,d,e) Dorsal view. (c,f) Frontal view. Both black and white arrows in e and f indicate areas affected by atelectasis. Boxed areas in panels a and d are enlarged in b and e, respectively. Wfdc2+/+ mice: a-c. Wfdc2GFP/GFP mice: d-f. L, left lobe. Scale bars: 1 mm. (E) Wfdc2GFP/GFP mice with atelectasis reveal high mortality. Out of 60 Wfdc2-deficient mice, 31 pups were alive and 29 pups died at between P0.5 and P1.5. Fifteen out of 31 live knockout mice showed atelectasis. However, 27 out of 29 dead pups displayed macroscopic atelectasis. Disease Models & Mechanisms

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Table 1. Genotyping of embryos, pups and adults Table 2. Fewer Wfdc2GFP/GFP mice than heterozygotes survive after delivery Age WT Ht KO Measurement Wfdc2GFP/+ Wfdc2GFP/GFP E14.5 10 20 11 E18.5 44 72 33 No. pups (P1.5) 25 8 Adult* 229 429 0 Age at death Wfdc2GFP/+ Wfdc2GFP/GFP Status of P1.5 pups WT Ht KO P0.5∼P2.5 0 3 Total 161 285 87 P3.5∼P5.5 0 4 Alive 146 265 35 P6.5∼P8.5 0 1 Dead 15 20 52 Status Wfdc2GFP/+ Wfdc2GFP/GFP Percent of death (%) 16.9 22.5 58.4 WT, wild type; Ht, heterozygous; KO, knock out. *4-10 weeks. Dead 0 8 Alive 25 0 Pups were given back to their mother at P0.5. was abnormal (Fig. 3A, Fig. S3A), suggesting that the structure of the cilia was severely altered. This was confirmed by scanning electron microscopy (SEM) at P1.5 (Fig. 3C). In Wfdc2GFP/GFP Abnormal lamellar body formation and surfactant mice, the cilia were consistently shorter than in Wfdc2GFP/+ mice processing in Wfdc2-deficient lungs and their morphology was abnormal (Fig. 3C). This difference was IHC analysis showed low levels of Wfdc2-GFP expression in the not seen before birth (i.e. at E18.5) (Fig. 3D). Immunohistochemical alveolar region of the lung (Fig. S4A) in a subfraction of cells (IHC) analysis showed that the clustered CGRPpos neuroendocrine positive for ProSP-C and ABCA3, markers of AECIIs (Fig. 4A, cells were clearly GFP negative (Fig. 3E, Fig. S3B), and the Fig. S4B). Wfdc2-deficient mice showed a higher frequency of quantitative analysis showed no difference in the frequency of these GFP-positive AECIIs (Fig. S4B). By contrast, podoplanin- cells between Wfdc2GFP/+ and Wfdc2GFP/GFP mice (Fig. S3B). positive type I alveolar epithelial cells (AECIs) did not express

Fig. 2. Wfdc2 deficiency impairs differentiation of secretory cells. (A) Representative confocal images of GFPpos (green) and KRT5pos (red) cells in the P1.5 bronchus. White dotted boxes in panels a and f are enlarged in b and g, respectively. Yellow dotted boxes in panels b and g are enlarged in c-e and h-j, respectively. Arrowheads indicate KRT5pos/GFPneg basal cells. Asterisks indicate KRT5pos/GFPpos basal cells. Nuclear DNA (DAPI) is shown in white. Es, esophagus; Br, main bronchus. Scale bars: 100 µm (a,b,f,g); 10 µm (c-e,h-j). (B) Quantification of GFPpos/KRT5pos double-positive cells among total KRT5pos cells in littermates of Wfdc2GFP/+ (G/+) and Wfdc2GFP/LacZ (G/L). Data are shown as mean±s.e.m. (n=3 mice; ***P<0.005). P-values were calculated as compared with controls. (C) Representative confocal images of GFPpos (green) and Scgb1a1pos (red) cells in the P1.5 bronchus. Nuclear DNA (DAPI) is shown in white. Scale bars: 100 µm (a,d); 10 µm (b,c,e,f). (D) Quantification of Scgb1a1high, Scgb1a1middle and Scgb1a1low cells among total (DAPIpos) cells in control Wfdc2GFP/+ (G/+) and Wfdc2GFP/LacZ (G/L) mice. Representative examples of Scgb1a1high, Scgb1a1middle and Scgb1a1low cells are shown in Fig. S2C. Fewer secretory cells expressed high to middle levels of Scgb1a1 in Wfdc2GFP/LacZ mice. Data are shown as mean±s.e.m. (n=3 mice; ***P<0.005, ****P<0.001). P-values were calculated as compared with littermate controls. Disease Models & Mechanisms

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Fig. 3. Wfdc2 deficiency causes cilia abnormalities in proximal conducting airways. (A) NCS-1pos ciliated cells lacking Wfdc2 show poor cilia formation. Yellow dotted areas in panels a and c are enlarged in b and d, respectively. Enlargements of the white boxed areas in b and d are shown at the bottom right corner of each panel. Note that clusters of acetylated tubulin (Ac-tub)- positive cells are common in Wfdc2GFP/+ but are rarely seen in Wfdc2GFP/LacZ mice. Green, GFP; cyan, NCS-1; red, Ac-tub; white, DAPI; Es, esophagus; Br, main bronchus. Scale bars: 50 µm. (B) The frequency of NCS-1-positive ciliated cells is similar among Wfdc2GFP/+ (G/+) and Wfdc2GFP/LacZ (G/L) mice. (C) SEM analysis reveals shorter and less abundant cilia in the P1.5 main bronchus of Wfdc2GFP/GFP mice. Scale bars: 10 µm. (D) SEM analysis reveals that cilia in the main bronchus of Wfdc2GFP/GFP mice are comparable to those in Wfdc2GFP/+ mice at E18.5. Scale bars: 20 µm. (E) CGRP-positive neuroendocrine cells are largely negative for GFP. Green, GFP; red, CGRP; white, DAPI. Scale bars: 20 µm.

GFP (Fig. 4B). To quantify GFP expression in AECIs, we used Secondly, RNA-seq analyses showed that mRNA levels of HopxandconfirmedthelackofGFPexpressioninHopx-positive/ hydrophilic surfactant peptides (SP-A and SP-D) were ProSP-C-negative AECIs (Fig. S4D) (Yin et al., 2006; Barkauskas significantly upregulated in Wfdc2GFP/GFP mice (Fig. 4D). In et al., 2013). Given that Wfdc2 is expressed in AECIIs, we agreement with the mRNA expression data, western blotting speculated that the lung collapse and respiratory distress in null analyses showed increased levels of SP-A and SP-D proteins in mutants is caused by dysfunction of these cells and that this Wfdc2GFP/GFP mice (4.07 and 2.06 times, respectively), as protease inhibitor normally regulates surfactant composition compared with those of littermate controls (Fig. 4E, Table S1), through synthesis and/or recycling of surfactant components. and matSP-B showed a little increase (Fig. 4E, Table S1). Lastly, This hypothesis is supported by the following observations. when the levels of two major phospholipids, PC and PG, were Firstly, transmission electron microscopy (TEM) of Wfdc2GFP/GFP quantified, levels of PC but not PG were significantly lower in mice showed that mutant AECIIs were significantly impaired in the lung of Wfdc2GFP/GFP mice than in littermate controls the ability to form lamellar bodies at inflated regions (Fig. 4C). (Fig. 4F). Disease Models & Mechanisms

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Fig. 4. AECIIs express proper markers but produce abnormal surfactants. (A) P1.5 lung specimens were stained with GFP (green), ProSP-C (red) and DAPI (white); (a,c,e) Wfdc2GFP/+ mice; (b,d,f) Wfdc2GFP/GFP mice. Yellow dotted areas in panels a and b are enlarged in c, e and d,f, respectively. Yellow asterisks indicate GFPpos/ProSP-Cpos (double- positive) cells. Yellow arrowheads indicate GFPneg/ProSP-Cpos cells. Scale bars: 30 µm. (B) P1.5 lung specimens were stained for GFP (green), podoplanin (Pod; red) and DAPI (white); (a,c,e) Wfdc2GFP/+ mice; (b,d,f) Wfdc2GFP/GFP mice. Yellow dotted areas in panels a and b are enlarged in c,e and d,f, respectively. Scale bars: 20 µm. (C) TEM analysis of lamellar bodies in AECIIs. Yellow dotted areas in panels a-c are enlarged in d-f, respectively. Scale bars: 500 nm. (D) RNA-seq analyses of hydrosoluble surfactant proteins. mRNA expression changes of SP-A, SP-B, SP- C and SP-D are shown as log2 fold change between Wfdc2+/+ and Wfdc2GFP/GFP lung tissues at P1.5. The dotted line at 1.0 indicates twofold upregulation in Wfdc2GFP/GFP mice. Error bars show mean±s.e.m. (n=2). (E) Representative western blotting of liposoluble surfactant proteins. (Left) SP-A and SP-D are upregulated in Wfdc2GFP/GFP mice. (Middle and right) matSP-B shows a little increase in Wfdc2GFP/GFP mice. +/+, Wfdc2+/+ mice; G/G, Wfdc2GFP/GFP mice; β-cat, β-catenin; α-tub, α-tubulin. (F) Wfdc2GFP/GFP mice have low levels of phosphatidylcholine (PC) compared with Wfdc2+/+ mice, but comparable levels of phosphatidylglycerol (PG). Data are shown as mean±s.e.m. (n=3 mice; *P<0.05). P-values were determined compared to controls.

Wfdc2-deficient mice upregulate pro-inflammatory and before birth, at E18.5 (Fig. 5C,D). Among the upregulated genes, defense/immune-response mRNAs IL-1α and TNFβ were notable, with approximately 5.4- and 4.9-fold To uncover how Wfdc2 deficiency affects lung gene expression, we overexpression in Wfdc2GFP/GFP mice. The upregulation of used RNA-seq to profile and compare the lung transcriptome of Ccl4/MIP1β and Cxcl2/MIP-2 was also prominent, with 11.5- and Wfdc2GFP/GFP and littermate control mice at P1.5. This revealed 16.0-fold overexpression in Wfdc2GFP/GFP mice. upregulation of a set of genes involved in the immune response Since inflammation is controlled at both the transcriptional and (P=2.3e–17; n=23 genes) and its related functions, which include a translational levels, we used ELISA to measure levels of four number of pro-inflammatory and anti-inflammatory cytokines as proteins: two cytokines (IL-1α and TNFβ) and two chemokines well as various CC chemokines and CXC chemokines (Fig. 5A,B, [CCL4/MIP1β (CC class) and CXCL2/MIP-2 (CXC class)].

Fig. S5A, Table 3). Significantly, this upregulation was not observed Analysis of lungs from Wfdc2-deficient mice confirmed increases Disease Models & Mechanisms

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Fig. 5. See next page for legend. in protein products of all four molecules (Fig. 5E). mesenchymal regions of the alveolus; in addition, mononuclear Immunohistochemical analysis showed strong IL-1α and CXCL2 cells appeared in alveolar spaces more often in Wfdc2GFP/GFP mice signals in some of the epithelial cells in the bronchus, and in than in littermate controls (Fig. 5F,G). Disease Models & Mechanisms

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Fig. 5. Wfdc2 deficiency causes inflammation in the neonatal lung. Table 3. Classification of upregulated immune response genes in (A) RNA-seq scatter plot showing mRNA expression levels of all genes (gray Wfdc2-deficient lung dots) expressed in Wfdc2+/+ and Wfdc2GFP/GFP lungs at P1.5. Values are indicated as log2-normalized read count per million mapped reads. Wfdc2 Immune response genes listed by type mRNA is reduced, and mRNA for inflammatory response genes (red dots) is CC chemokines CXC upregulated in Wfdc2GFP/GFP mice. (B) GO term enrichment analysis of the Cytokines and receptors chemokines Others upregulated genes (>fourfold) in Wfdc2GFP/GFP mice. (C) Multidimensional β scaling plot showing distances between RNA-seq datasets of different IL-1 * Ccl3 Cxcl1 Prg2 conditions. The plot was generated using the plotMDS function of EdgeR. IL-1 family member 5 (IL35RN)* Ccl4 Cxcl2 Slpi α White and black circles indicate Wfdc2+/+ (+/+) littermate control samples from TNF * Ccl7 Cxcl3 H2-Ab2 E18.5 and P1.5, respectively; white and black triangles indicate Wfdc2- TNF receptor superfamily 9 Ccl12 Cxcl5 GFP/GFP IL-6* Ccl17 Cxcl9 knockout (G/G; Wfdc2 ) samples from E18.5 and P1.5, respectively. † R, replicate #1; R2, replicate #2. (D) The gene expression profile of E18.5 IL-10 Ccl20 Cxcl13 Wfdc2GFP/GFP fetuses is similar to that of Wfdc2+/+ fetuses. Only the Wfdc2 gene CSF-3 Ccl22 expression is significantly downregulated (black arrow). Red dots indicate Ccr7 inflammatory response genes that are upregulated at P1.5 in Wfdc2GFP/GFP Each column lists a group of upregulated immune response genes (entries in each mice. (E) Induction of acute inflammatory response proteins in Wfdc2GFP/GFP row are unrelated). *Pro-inflammatory signaling; †anti-inflammatory signaling. mice at P2.5. Data are shown as mean±s.e.m. (n=4, *P<0.05). (F) Immunohistochemical analysis of IL-1α in neonatal lung at P1.5. Black dotted boxes are enlarged in the right panels. IL-1α-positive cells (brown) are epithelium from damage, which include lamellar body formation in detected in some epithelial cells in bronchioles and mesenchymal areas, and AECIIs and mucociliary clearance machinery in bronchi (Fig. 6E). mononuclear cells appeared in alveolar spaces in Wfdc2GFP/GFP mice. Scale We have shown here that the mice develop atelectasis as a bars: 1 mm (a,c); 100 µm (b,d). (G) Immunohistochemical analysis of CXCL2 consequence of Wfdc2 deficiency, leading to neonatal death. in neonatal lung at P1.5. Black dotted boxes are enlarged in the right panels. Ninety percent of pulmonary natural surfactant is composed of CXCL2-positive cells (brown) are also detected in some epithelial cells in lipids, and PC makes up approximately 80% of the lipid. PC bronchioles and mesenchymal areas, and mononuclear cells appeared in alveolar spaces in Wfdc2GFP/GFP mice. Scale bars: 1 mm (a,c); 100 µm (b,d). functions to reduce the surface tension, which prevents collapse of alveoli (Echaide et al., 2017; Parra and Pérez-Gil, 2015). Our study has shown that PC levels significantly decreased in response to Changes in protein expression and localization in Wfdc2 deficiency, likely due to an impairment of synthesis and/or Wfdc2-deficient mice recycling of PC (Chakraborty and Kotecha, 2013). Given that the In a kidney fibrosis model, Wfdc2 overexpression reportedly reduction of PC concentration increases the surface tension causes up- and down-regulation of proteases at the protein and (Hallman et al., 1991), a decrease in PC may be linked to mRNA levels, as well as at the enzymatic activity level (LeBleu progressive atelectasis and respiratory failure in Wfdc2-deficient et al., 2013). Therefore, the observed disruption in lung mice. This is also indirectly supported by clinical reports showing homeostasis might be caused by an imbalance of the protease/ that patients of acute respiratory distress syndrome (ARDS) protease-inhibitor system. In the kidney fibrosis model, protein accompanied by severe atelectasis exhibit decreased levels of PC levels of Prss35 and MMP9 were upregulated in parallel with the (Cogo et al., 2007; Dushianthan et al., 2014; Gregory et al., 1991). upregulation of Wfdc2, and Wfdc2 inhibits enzymatic activity of Meanwhile, we also found that SP-A and SP-D levels were Prss35 and MMP9 (LeBleu et al., 2013). In our lung knockout increased in Wfdc2-deficient mice. Given that an increase of the SP- model of Wfdc2, only PRSS35 revealed a little downregulation A/PC ratio experimentally causes an increase of surface tension (Fig. 6A, Table S1). We also checked two other lung-disease- (Hallman et al., 1991), the upregulation of SP-A and the related proteases, MMP12 and ADAM10. Of those, ADAM10 downregulation of PC in Wfdc2-deficient mice may accelerate showed marginal changes in Wfdc2-deficient lungs at P1.5 lung atelectasis. Abnormal processing of surfactants may also cause (Fig. 6A, Table S1). secondary changes in lung physiology at birth: mice lacking other As protease levels increase, target proteins likely are digested, surfactant components – SP-B and ABCA3 – have been shown to perturbing homeostasis and self-defense mechanisms. As a die of RDS (Clark et al., 1995; Cheong et al., 2007; Fitzgerald et al., consequence, the structure of the lung tissue may not mature and 2007; Hammel et al., 2007). may collapse. Wfdc2 target molecules might mediate cell Club cells produce SP-A, SP-B and SP-D as well as an inhibitor adherence and tight junctions, or be a structural part of the of phospholipase A2 (CC10) (Han and Mallampalli, 2015), and alveolar epithelial barrier, an idea supported by recent studies in have crucial secretory functions contributing to the mucus pool. The the mouse colon (Parikh et al., 2019). To track the integrity of mucus, together with surfactants, interacts with and subsequently intercellular junctions, expression levels of the adherence-junction kills pathogens to prevent their dissemination. The mucus is then protein E-cadherin and the tight-junction protein ezrin were cleared by a constant upward flow generated by cilia. Therefore, the measured and their localization was analyzed (Fig. 6B-D, decreased number of mature club cells in Wfdc2-deficient mice may Table S1). Although the expression levels of E-cadherin and lead to a breakdown of the self-defense barrier function. Even ezrin were not different among the mice (Fig. 6B, Table S1), in worse, the impairment of cilia formation in Wfdc2-deficient mice Wfdc2-deficient mice, E-cadherin was often expressed in an may fail to clear pathogens in the mucus. unusual pattern, at the basal side of the epithelial cells of the Various types of lung disorders, such as the chronic obstructive intralobular conducting airway, compared with at the lateral wall in lung disease, are known to be associated with protease/antiprotease wild-type controls (Fig. 6C,D). imbalance (Abboud, 2012; Goopts et al., 2009). At present, the target proteases of Wfdc2 are unclear. Until now, multiple examples DISCUSSION carrying dysfunctions in other protease systems have been presented Our study in the lung has presented, for the first time, evidence that to be associated with lung diseases: neutrophil elastase/α1- the protease inhibitor Wfdc2 plays an important role in a wide range antitrypsin imbalance causing human emphysema (Goopts et al., of aspects of the barrier mechanisms that protect respiratory airway 2009; Meyer and Jaspers, 2015); mouse α1-antitrypsin (serpina1) Disease Models & Mechanisms

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Fig. 6. See next page for legend. knockout leading to spontaneous emphysema (Borel et al., 2018); located on the same chromosome, possibly as a consequence of and chymotrypsin-like elastase 1 responsible for emphysema, rapid evolutionary changes (Hurle et al., 2007), our single-knockout proven by an antisense oligo model for the α1-antitrypsin model indicates that Wfdc2 exhibits non-redundant yet diverse deficiency (Joshi et al., 2018). However, the phenotype of the functions in the lung. Wfdc2-deficient mice is different from those of the above-described During the preparation of the manuscript, a new report was mice. Moreover, although many Wfdc family genes are closely published showing that human WFDC2 is downregulated in goblet Disease Models & Mechanisms

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Fig. 6. Disturbance of homeostasis in the Wfdc2-deficient lung. glucose DMEM, L-glutamine, sodium pyruvate (Thermo Fisher Scientific) (A) Western blotting analysis of proteases. +/+, Wfdc2+/+ mice; G/G, containing 15% FBS (Hyclone, Thermo Fisher Scientific), 0.1% non- Wfdc2GFP/GFP mice. The asterisk and the arrowhead in the ADAM10 blot essential amino acids, 50 U/ml penicillin (Sigma-Aldrich) and streptomycin indicate the precursor and the active form, respectively. The arrowhead in the (Nacalai Tesque), 0.1 mM 2-mercaptoethanol (Millipore) and 1000 U/ml PRSS35 blot indicates PRSS35. (B) Western blotting analysis of E-cadherin LIF supplement (ESGRO, Millipore). IRES-EGFP-rabbit β-globin-polyA +/+ GFP/GFP (E-cad) and ezrin. +/+, Wfdc2 mice; G/G, Wfdc2 mice. cDNA was integrated into a pKSTKNeoLoxP vector carrying PGK-neo- GFP/GFP (C) Mislocalization of E-cadherin in lateral walls of Wfdc2 airways. Red, polyA for positive selection and the HSV-tk gene for negative selection E-cadherin; blue, laminin; green, GFP; white, DAPI. Yellow dotted boxes in (Fig. S1A) (Nagamatsu et al., 2006). The targeting vector contains a 6.3-kb panels a and f are enlarged in b-e and g-j, respectively. E-cadherin localizes at long arm, carrying a part of exon 1, and a 2 kb short arm, encompassing the lateral walls of conducting airways in Wfdc2GFP/+ mice (b-e). In Wfdc2GFP/GFP exon 3 and intron 3, from 129-derived genomic DNA. The vector lacks a mice, E-cadherin (red in h) shows mislocalization to the basement membrane GFP/GFP part of exon 1 and the entire exon 2 (Fig. S1A). A targeted vector carrying (visualized by laminin, blue in h). Note that only the Wfdc2 samples EGFP Not show white signals at the basement membrane, as a result of overlapping cDNA was linearized with I and electroporated into TT2 ES cells, E-cadherin and laminin signals (arrowheads in h). Asterisks in g-i show the cell which were then selected in 250 mg/ml (active weight) neomycin (G418; shown in D. Scale bars: 100 µm (a,f); 10 µm (b-e,g-j). (D) Super-resolution Invitrogen) for 10 days and ganciclovir for 7 days (Sigma-Aldrich). After microscopy analysis of E-cadherin and laminin signals. A high-magnification confirming recombination of neomycin-resistant colonies, the ES cells were image of an epithelial cell in the conducting airway shown in C (Wfdc2GFP/GFP aggregated with ICR mouse morulae to generate chimeric mice. After mice, indicated by asterisks in Cg-Cj). The HyVolution system is used for confirming germline transmission from chimeric mice, heterozygous mice super-resolution imaging. Single-channel images of E-cadherin and laminin were crossed with CAG-Cre mice to delete the drug-resistant marker, PGK- are shown in the middle and the bottom panels, respectively. Scale bars: 1 µm. neo-polyA (Wfdc2GFP/+), and then homozygous mice (Wfdc2GFP/GFP) were (E) Quantification of colocalization signals of E-cadherin and laminin at the generated. Southern blotting and genomic PCR analyses verified that the basal side of intralobular bronchioles. Data are shown as mean±s.e.m. (n=3, Wfdc2 gene was deleted (Fig. S1B and S1C); RNA-seq did not detect any ****P<0.001). (F) Summary of the Wfdc2-deficiency status in the lung. Wfdc2 is Wfdc2 mRNA in the mutant mouse lung (Fig. S1D). A second mutant line, expressed in a subpopulation of basal cells, ciliated cells and club cells at the with the lacZ gene knocked in at the Wfdc2 locus, was generated and proximal airway, but not in neuroendocrine cells. At the distal airway, a intercrossed with Wfdc2-knockout mice, generating mutant mice carrying subpopulation of AECIIs but not in AECIs express Wfdc2. Wfdc2 deficiency both GFP and lacZ (Wfdc2GFP/lacZ mice) (Fig. S1A). leads to damaged cilia, a reduction of terminally differentiated club cells, which have secretory functions (pink and red cytosol), and abnormal distribution of E- cadherin (red line). At the distal airway, proper lamellar body formation in Mice AECIIs is also impaired. Altogether, many abnormalities arising from Wfdc2 Mouse husbandry and experiments were carried out under the guidelines of deficiency give rise to a breakdown of the multi-layered barrier system. Yokohama City University, Japan, and all animal experiments were approved by the Committee for Animal Care and Use at Yokohama City University. CAG-Cre mice were reported previously (Glaser et al., 2009). cells in inflammatory bowel disease (IBD) patients and participates The lacZ knock-in mice were generated from ES cells obtained from in the pathogenesis of IBD (Parikh et al., 2019). Analysis in the EUCOMM. colon has shown that WFDC2 has bactericidal activity, preserves the integrity of tight junctions and participates in forming a mucus Southern blotting layer to block bacteria penetration into epithelial cells. This finding Ten µg of genomic DNA was prepared from E18.5 fetuses, as described raises another possibility: that mislocalization of the adherens- previously with slight modifications (Ohbo et al., 1996), and digested with junction molecule E-cadherin in Wfdc2-deficient lung epithelia may the indicated restriction enzymes (Fig. S1B), separated by electrophoresis on reflect the loss of epithelial barrier integrity. Indeed, some major 0.7% agarose gels in TBE buffer, transferred to Hybond-N (RPN303B, proteases, such as ADAM15, kallikrein 6 and MMPs, reportedly Amersham) and hybridized with the probes indicated in Fig. S1A. mediate E-cadherin shedding and affect cell-cell adhesion (Biswas et al., 2010; Klucky et al., 2007; Kwon et al., 2014; Najy et al., Genomic PCR 2008; Symowicz et al., 2007; Zuo et al., 2011). Since the protein Primer sequences for genotyping PCR are listed in Table S2. levels of the proposed Wfdc2-target proteases in the kidney fibrosis model did not change significantly in our knockout lung study, the Antibodies Antibodies for IHC analysis are listed in Table S3. target proteases of Wfdc2 may be tissue specific. Therefore, future studies in the lung system will address which proteases are targeted Histology and immunostaining by Wfdc2 and whether the effect of their interactions extends to Fixation and immunostaining of lung were done as described previously epithelial integrity in multiple tissues. (Shirakawa et al., 2013). Briefly, neonatal lung was fixed by perfusion with Collectively, our results suggest that Wfdc2 deficiency causes 4% paraformaldehyde (PFA) for 4 h (P0.5 and older). Embryonic and fetus impairments in a series of barrier mechanisms required to protect lung was immersed in 4% PFA for 1 h (E11.5) and 4 h (E14.5, E18.5), respiratory airway epithelium. The deficiency not only induces respectively. After mounting in O.C.T. compound (Tissue-Tek), blocks disruption of surfactants, which leads to a collapsed lung, but also were sliced and subjected to staining as follows. The sections were initially causes insufficient mucociliary clearance due to damaged cilia, incubated for 30 min with PBS supplemented with 2% BSA (BSA/PBS), which impedes a constant upward flow of respiratory secretion to and then incubated for either 2 h at room temperature or overnight at 4°C the mouth (Fig. 6E). In the lung, since Wfdc2 is induced after birth with an appropriate primary antibody, followed by incubation with a and plays crucial roles in preventing atelectasis and possibly secondary antibody for 1 h at room temperature (Table S3). The sections were mounted with ProLong Gold (Thermo Fisher Scientific) and observed maintaining the barrier function, the decrease in WFDC2 expression by confocal laser microscopy (FV-1000; Olympus). For hematoxylin and might be a factor in human respiratory failure, an issue that should eosin (H&E) staining, lung and heart were embedded in paraffin, sectioned be addressed in future studies. and stained with H&E. For 3,3′-diaminobenzidine (DAB) staining, the sectioned specimens were incubated with a primary antibody overnight at MATERIALS AND METHODS 4°C, and then specimens were incubated with a secondary antibody Mouse ES cell culture and generation of GFP knock-in mice conjugated with horseradish peroxidase (HRP) and reacted with 0.05% Mouse TT2 embryonic stem cells (ES cells) were used to generate GFP DAB and 0.015% hydrogen peroxide for 8 min at room temperature. GFP/+ GFP/LacZ knock-in mice. ES cells were cultured on 0.1% gelatin-coated plates in high- Wfdc2 and Wfdc2 mice were used for all the quantification Disease Models & Mechanisms

10 RESEARCH ARTICLE Disease Models & Mechanisms (2019) 12, dmm040139. doi:10.1242/dmm.040139 experiments for the frequency of GFP-positive cells in epithelial cells. The Data analysis measurements of the GFP frequency in neuroendocrine cells, AECIs and RNA-seq reads were quality- and adapter-trimmed with Trim Galore AECIIs, as well as the measurements of the overlapped region between E- (version 0.4.0) (http://www.bioinformatics.babraham.ac.uk/projects/trim_ cadherin and laminin, were performed blinded. The measurements of the galore/) after 3′ ends of reads longer than 36 nucleotides were removed using GFP frequency in the basal cells, the ciliated cells and the club cells were an NGS QC Toolkit (version 2.3.1). The trimmed reads were mapped onto performed non-blinded. Super-resolution fluorescence signals were mm10 using TopHat (version 2.1.1) (Trapnell et al., 2009) with default acquired with a microscope system Leica TCS SP8 (Leica Biosystems) parameters with a guide by a gtf file containing reference mRNA equipped with HyD detectors and a hybrid super-resolution (HyVolution) coordinates. Total number of sequencing reads and mapping efficiency system. Z-section images were captured using the HyD detectors, and the are shown in Table S4. Read counts and quantification were done using raw data were deconvolved using Huygens (Scientific Volume Imaging) in SeqMonk (version 1.44.0) (http://www.bioinformatics.babraham.ac.uk/ the HyVolution package with default parameters. projects/seqmonk/) and the values were normalized as log2 reads per kilobase per million mapped reads (RPKM) for downstream analyses. Gene Transmission and scanning electron microscopy Ontology (GO) analysis was performed using DAVID (version 6.8) using all Mice were fixed by perfusion using 0.1 M phosphate buffer (pH 7.4) mouse genes as background (Huang et al., 2009). containing 2% glutaraldehyde and 2% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). Fixed specimens were cut into small blocks and immersed in ELISA and colorimetric/fluorometric assay the same fixative at room temperature for 2 h. Subsequently, after washing ELISA was performed as per the manufacturer’s protocol (Quantikine® with 0.1 M phosphate buffer, the blocks were post-fixed with 1% OsO4 at 4°C ELISA; R&D systems). Briefly, lung tissues were homogenized with Cell for 1 h. After washing with distilled water, the blocks were stained with 4% Lysis Buffer 2 provided by R&D Systems. Proteins of standard, control and solution of uranyl acetate at room temperature for 1 h, then dehydrated in samples were incubated with monoclonal antibodies specific for IL-1α, graded concentrations of ethanol solutions. For TEM observation, the lung TNFβ, CCL4 and CX2 individually pre-coated onto microplates for 2 h at blocks were replaced by propylene oxide, embedded in Epon812 resin room temperature. After washing five times, HRP-conjugated antibodies (TAAB Laboratories Equipment) and polymerized at 60°C for 48 h. The against individual antigens were incubated for 2 h at room temperature. After ultra-thin sections of lung were made using Reichert Ultracut N again washing five times, a substrate solution was added to the wells. After the Ultramicrotome (Leica Microsystems) and stained with 2% uranyl acetate developing reaction, the stop solution was added and the intensity of color was in 70% ethanol and 0.4% lead citrate. The sections were analyzed by an measured by Sunrise™ (Tecan Life Sciences). A PG/cardiolipin assay kit H-7500 transmission electron microscope (Hitachi) operated at 80 kV. For (MET-5024, CELL BIOLABS) and PC assay kit (ab83377, abcam) were SEM observation, the dehydrated blocks of trachea and bronchus were put in used to measure PG and PC, respectively. To measure PG, lung tissues were t-butyl alcohol, and then freeze-dried with VFD-21S (vacuum device). After homogenized and glycerol in the tissues was extracted by MeOH/chloroform/ the dried blocks were coated with platinum-palladium using an E102 ion 1 M NaCl and subsequently treated with lipase to hydrolyze PG. The yielded coater (Hitachi), we imaged the lumen of the trachea and bronchus with an glycerol was then phosphorylated and oxidized to produce hydrogen S-4800 scanning electron microscope (Hitachi) operated at 10 kV. peroxide, which subsequently reacted with the fluorometric probe. To measure PC after homogenization of lung specimens, the extracts were RNA purification, cDNA construction and qRT-PCR incubated with the OxiRed Probe supplemented with hydrolysis enzyme. The Total RNA was extracted from lung using Isogen (Nippon Gene) according intensity of color was measured by Wallac 1420 ARVOMX (Perkin Elmer). to the manufacturer’s instructions and treated with RQ1 DNase (Promega) at 37°C for 30 min. The total RNA was used for first-strand cDNA synthesis Statistical analyses with Superscript III (Life Technologies). Expression levels were determined Statistical data are presented as means±standard error of mean (s.e.m.). by Applied Biosystems 7900HT Fast Real Time PCR System using Significance is indicated with asterisk(s): *P<0.05, **P<0.01, ***P<0.005, FastStart SYBR Green Master (Roche). Primers specific for each gene are ****P<0.001. provided in Table S2. Acknowledgements Western blotting We are grateful to Dr Brigid L. M. Hogan from Duke University for critical reading of the manuscript. We also thank Dr Atsuyasu Sato from Kyoto University, Western blotting was performed as described (Shirakawa et al., 2013). Briefly, Dr Kazutoshi Cho from Hokkaido University and Dr Yoshinori Sato from Yokohama whole-cell extracts from lung were resolved by sodium-dodecyl-sulfate City University for discussion and suggestions. PAGE (SDS-PAGE) with a 7.5% (SP-D and ADAM10), 12.5% (SP-A), 10-20% (proSP-B, matSP-B, proSP-C, matSP-C, MMP9) gradient gel Competing interests (ePAGEL, E-R1020L, Atto), and 5-20% (MMP12, PRSS35, E-cadherin, The authors declare no competing or financial interests. ezrin) gradient gel (ePAGEL, E-R520L, Atto). The proteins were then transferred to polyvinylidene difluoride (PVDF) membranes (Immobilon-P; Author contributions Merck Millipore). Membranes were incubated with the indicated antibodies Conceptualization: K.O.; Methodology: K.N., M.O., G.N.; Validation: M.O., S.T., overnight at 4°C, followed by incubation with an appropriate HRP-conjugated K.K.; Investigation: K.N., M.O., U.R., S.D., S.T., K.K., G.N., I.H., R.M., T. Shirakawa, secondary antibody for 1 h at room temperature (Table S3). Positive signals T.K., M.S., Y.S.; Resources: H.K.; Data curation: K.N., M.O., S.T., Y.S., K.O.; were detected and visualized with Chemi-Lumi One L or Super reagents Writing - original draft: K.O.; Writing - review & editing: M.N.; Supervision: Y.M., H.K., (Nacalai Tesque). Antibodies for western blotting analysis are listed in M.N., T. Suda; Project administration: K.O.; Funding acquisition: S.T., K.O. Table S3. The band intensities were quantified using the LAS3000 mini system and Multi-Gauge version 2.3 (Fuji Film). The results of western blot Funding quantification are summarized in Table S1. This work was partly supported by Grant-in-Aid for Scientific Research on Innovative Areas on “Regulatory Mechanism of Gamete Stem Cells” from the Ministry of RNA-seq Education, Culture, Sports, Science, and Technology (MEXT) to K.O. (25114004), Total lung RNA was purified using Isogen (Nippon Gene) according to the Grant-in-Aid for Scientific Research (C) from Japan Society for the Promotion of ’ Science (JSPS) KAKENHI to K.O. (15K08156) and to K.O., M.S. and Y.S. manufacturer s instructions. Quality control assessment of RNA was done (16H06279), and Grant-in-Aid for Young Scientists (B) from JSPS KAKENHI to S.T. using Bioanalyzer (Agilent) with RNA 6000 Nano Kit (Agilent). Genomic (26860137). DNA was digested using RQ1 DNase (Promega) at 37°C for 30 min, and the resulting RNA was used for library preparation using NEBNext Ultra Data availability Directional RNA Library Prep Kit for Illumina (NEB). The libraries were All RNA-seq datasets have been deposited at NCBI Gene Expression Omnibus sequenced using either Illumina HiSeq2500 or GAIIx. under accession number GSE129006. Disease Models & Mechanisms

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