(BMP) 4 Signaling Antagonist in Controlling Mouse Lung Development

Follistatin-like 1 (Fstl1) is a bone morphogenetic protein (BMP) 4 signaling antagonist in controlling mouse lung development

Yan Genga,1, Yingying Donga,1, Mingyan Yua,1, Long Zhangb, Xiaohua Yanb, Jingxia Suna, Long Qiaoa, Huixia Genga, Masahiro Nakajimac, Tatsuya Furuichic, Shiro Ikegawac, Xiang Gaoa, Ye-Guang Chenb,2, Dianhua Jiangd,2, and Wen Ninga,e,2

aModel Animal Research Center, Nanjing University, Nanjing 210061, China; bState Key Laboratory of Biomembrane and Membrane Biotechnology, School of Life Sciences, Tsinghua University, Beijing 100084, China; cCenter for Genomic Medicine, RIKEN, Tokyo 108-8639, Japan; dDepartment of Medicine, Division of Pulmonary, Allergy, and Critical Care Medicine, Duke University School of Medicine, Durham, NC 27710; and eCollege of Life Sciences, Nankai University, Tianjin 300071, China

Edited by Gail Martin, University of California, San Francisco, CA, and approved March 7, 2011 (received for review June 18, 2010) Lung morphogenesis is a well orchestrated, tightly regulated BMP4 gain of function in the lung results in less extensive process through several molecular pathways, including TGF-β/bone branching and decreased distal epithelial differentiation (11). morphogenetic protein (BMP) signaling. Alteration of these signal- The precise mechanism of TGF-β family members in regulating ing pathways leads to lung malformation. We investigated the role lung development is largely unclear. of Follistatin-like 1 (Fstl1), a secreted follistatin-module–containing Follistatin-like 1 (Fstl1), first identified as a TGF-β1–inducible glycoprotein, in lung development. Deletion of Fstl1 in mice led to gene (13), encodes a secreted extracellular glycoprotein belong- postnatal lethality as a result of respiratory failure. Analysis of the ing to the Fst-SPARC family, whose amino acid sequence con- mutant phenotype showed that Fstl1 is essential for tracheal carti- tains a follistatin-like domain (14, 15). Its functions and the lage formation and alveolar maturation. Deletion of the Fstl1 gene underlying mechanism are poorly understood. Studies in zebra- resulted in malformed tracheal rings manifested as discontinued fish (16, 17) suggest a developmental role of Fstl1 in early dor- rings and reduced ring number. Fstl1-deficient mice displayed sep- soventral body axis establishment. In vitro studies have shown tal hypercellularity and end-expiratory atelectasis, which were as- that Fstl1 is one of the mesenchymal factors determining ovi- sociated with impaired differentiation of distal alveolar epithelial ductal epithelial cell fate (18). In this study, we generated Fstl1- cells and insufficient production of mature surfactant proteins. deficient mice to examine the role of Fstl1 in lung development Mechanistically, Fstl1 interacted directly with BMP4, negatively and found that Fstl1 is essential for normal tracheal formation as regulated BMP4/Smad1/5/8 signaling, and inhibited BMP4-induced well as alveolar maturation. Furthermore, we demonstrated that surfactant gene expression. Reducing BMP signaling activity by Fstl1 regulates the differentiation of lung epithelial cells, in part, Noggin rescued pulmonary atelectasis of Fstl1-deficient mice. through negative regulation of BMP4 signaling. Therefore, we provide in vivo and in vitro evidence to demonstrate that Fstl1 modulates lung development and alveolar maturation, in Results part, through BMP4 signaling. Generation of Fstl1-Deficient Mice. To determine the biological − function of Fstl1 in vivo, we first generated Fstl1+/ mice by fl − − lung atelectasis | trachea formation | surfactant protein C | intercrossing EIIa-Cre;Fstl1 ox/+ mice (Fig. S1 A and B). Fstl1 / − lung epithelial differentiation mice were then generated by intercrossing Fstl1+/ mice. Western blotting confirmed the loss of Fstl1 protein expression (Fig. 1A). −/− ung development is a well orchestrated process that is tightly Fstl1 pups were born alive at the expected Mendelian ratio Lregulated by transcription factors, hormones, growth factors, (27%, 127 of 472). However, all homozygous pups breathed ir- and other factors in temporal and spatial manners (1, 2). The regularly and displayed a cyanotic skin color, then died shortly −/− mouse lung is derived from foregut endoderm in an embryonic day after birth (Fig. 1B). In addition, Fstl1 neonates displayed (E) 9.5 embryo. Trachea arises from the more proximal foregut multiple defects, including abnormal dorsal–ventral pattern of tube, whereas the rest of the lung develops from two ventral buds the neural tube, hydroureter, and overall skeletal defects. Con- that form at the distal end of the trachea, and undergoes branching sistent with the pleiotropic developmental defects caused by the morphogenesis to produce the pulmonary tree (3, 4). This branching loss of Fstl1, in situ hybridization revealed widespread Fstl1 ex- morphogenesis is accompanied by differentiation of epithelial cell pression during mouse embryonic development, including in types along a proximal–distal axis, including bronchial Clara cells the lung (19). (proximal) and alveolar type I and type II epithelial cells (AEC-I and Fstl1 fi −/− AEC-II, respectively; distal) (5). These highly specialized cell types Tracheal Malformation in -De cient Mice. Fstl1 neonates had render specific functions in the respiratory tract, such as the func- soft and unusually large tracheal tubes (Fig. 1C). Transverse tional alveolar surface area formed by AEC-I and AEC-II cells for gas exchange (6). TGF-β superfamily growth factors regulate organogenesis, Author contributions: Y.G., Y.-G.C., D.J., and W.N. designed research; Y.G., Y.D., M.Y., L.Z., X.Y., J.S., L.Q., H.G., M.N., and T.F. performed research; S.I., X.G., Y.-G.C., and D.J. con- including that of the lung (1, 7). For example, they exert an in- tributed new reagents/analytic tools; Y.G., Y.D., S.I., Y.-G.C., D.J., and W.N. analyzed data; hibitory effect on lung branching morphogenesis (8). Interference and Y.G. and W.N. wrote the paper. of TGF-β signaling with a dominant-negative TβRII (9) or anti- The authors declare no conflict of interest. Smad2/3 oligos (10) in embryonic lung organ cultures stimulates This article is a PNAS Direct Submission. branching morphogenesis. BMP4 is dynamically expressed in the 1Y.G., Y.D., and M.Y. contributed equally to this work. distal epithelium; disruption of BMP4 signaling in lungs of Sp-C- 2To whom correspondence may be addressed. E-mail: [email protected], Xnoggin or Sp-C-dnAlk6 transgenic mice abrogates the proximal– [email protected], or [email protected]. distal patterning in the lung where distal epithelial differentiation This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. is inhibited while proximal differentiation is promoted (11, 12). 1073/pnas.1007293108/-/DCSupplemental.

7058–7063 | PNAS | April 26, 2011 | vol. 108 | no. 17 www.pnas.org/cgi/doi/10.1073/pnas.1007293108 Downloaded by guest on September 26, 2021 It has been suggested that tracheal cartilage formation is a multistep process. The committed mesenchymal cells first con- dense and proliferate to form cartilage primordia that prefigure the overall shape of future cartilages (E10.5–E12.5). Expression of cartilage-specific proteins (such as type II collagen) is then − − initiated and cells differentiate into chondrocytes (E13.5–E15.5). Fig. 1. Generation of Fstl1 / mice. (A) Western blot analysis of Fstl1 proteins from E15.5 embryos (Upper) or E18.5 lung tissues (Lower). (B) Exami- After E15.5, well formed C-rings can be observed (20). To further nation of neonates after birth revealed that Fstl1−/− neonates were cyanotic. examine the function of Fstl1 in cartilaginous development, we (C) Autopsy observation showed that WT lungs were expanded by in- generated stable clones overexpressing Fstl1 using murine mes- halation of air, which can be seen as air bubbles in the distal regions, but enchymal cells, ATDC5, which can differentiate into chon- − − Fstl1 / lungs were collapsed and did not show evidence of air in the distal drocytes in the presence of insulin-transferrin-sodium selenite airways (tr, trachea). (ITS) (21). As determined by MTT assay, Fstl1 increased the proliferation of mesenchymal cells (Fig. 2D). Fstl1 also strongly promoted ITS-induced Col2a1 mRNA expression (6.7 fold) by sections of E18.5 homozygous trachea revealed a striking de- −/− using quantitative RT-PCR (qRT-PCR). Collectively, Fstl1 is formed lumen. Fstl1 tracheas were enlarged at the upper and essential for all chondrogenic steps in the development of the lower levels but narrowed at the middle level, and their inner upper respiratory tract. Deletion of Fstl1 limits the proliferation margins were irregular, with many small folds sometimes ac- and differentiation of cartilaginous precursors, resulting in mal- − − companied by protuberances in the tracheal aperture (Fig. 2A formed rings during tracheal development. However, Fstl1 / and Fig. S2A). At birth [postnatal day (P) 0], apart from a few trachea with impaired cartilage rings does not develop significant − − instances of tracheal stenosis, most lumens of Fstl1 / tracheas stenosis, indicating that tracheal defects may not be the main − − (>80%) were larger than their WT controls (Fig. 2A and Fig. cause of respiratory failure in Fstl1 / neonates. S2A). These larger tracheas could be observed as early as E15.5 Lung Atelectasis in Fstl1-Deficient Mice. Gross phenotype of lung (Fig. S2B). Another consistent observation was a profound dis- − − − − / organization of the Fstl1 / tracheal epithelium (Fig. S2C). was largely normal in neonatal Fstl1 pups. Their lungs had − − E15.5 and E18.5 Fstl1 / trachea also displayed interrupted similar size and the correct number of lobes (Fig. 1C), and showed similar lung/body dry weight ratio compared with their WT lit- or truncated cartilages, whereas their WT littermates showed C- − − termates (Fstl1 / , 0.47%, n =7;Fstl1+/+, 0.48%, n = 10; P > shaped cartilage rings (Fig. 2A and Fig. S2 A and B). The larynx and − − 0.05). The striking abnormality of Fstl1 / lungs was their con- the trachea were greatly disorganized in all homozygous fetuses densed appearance with a few big air bubbles in the distal airways (Fig. 2B). The number of tracheal rings formed was reduced +/+ −/− (Fig. 1C). Histological examination of WT lungs displayed many (Fstl1 , 14 rings, n =15;Fstl1 , four to eight rings, n = 10) and small distal saccules with thin septa (E18.5) and showed normal − − the rings did not grow and extend as dorsally as those in WT saccular expansion at birth (P0). By contrast, E18.5 Fstl1 / lungs samples. These impaired cartilages failed to provide the airway with had a 60% reduction in air sac spaces and thickened hypercellular − − a rigid skeletal support, resulting in the soft and flabby tracheal tubes. intersaccular septa (Fstl1 / , 30.5 ± 2.0 μm; Fstl1+/+, 15.5 ± 2.2 Immunohistochemistry (IHC) analysis also revealed extremely at- μm, n =4;P < 0.05; Fig. 3A). At birth (P0), they were atelectatic tenuated type II collagen signals at E13.5 and E15.5 (Fig. 2C), and characterized by areas of poorly expanded sacs as well as − − showing the defective cartilaginous differentiation in Fstl1 / mice. some compensating overexpanding bronchioli (Fig. 3A and Fig. S3A). This condensed appearance started from the saccular stage (E17.5 to approximately P0; Fig. S3A).

Fig. 3. Abnormal lung morphogenesis and lung epithelial cell hyperplasia BIOLOGY − − in Fstl1 / mice. (A) Histological analysis of embryos (E18.5) and newborn

− − DEVELOPMENTAL Fig. 2. Tracheal malformation in Fstl1 / mice. (A) Transverse sections of the pups (P0) revealed normal inﬂated alveoli with thinner septa in WT, but − − middle portion of trachea from a WT and two homozygous mice before thickened hypercellular septa with reduced airspaces in Fstl1 / . (Scale bars, (E18.5) and after breath (P0) showed an increase in the lumen diameter 50 μm.) (B) Distribution of TTF1 by IHC. (Scale bars, 100 μm.) The percentage (asterisk), dispersion and discontinuity of cartilage ring (arrow), and disor- of the total examined sac area of the lung section or the percentage of the ganization of the epithelial layer in the mutants. (Scale bars, 100 μm.) (B) TTF1-positive cells in the total cells of the lung are shown in the top right Alcian blue staining revealed impaired banding pattern of tracheal C-ring corner of each diagram (P < 0.05). (C) Quantiﬁcation of cell proliferation by − − cartilage in all mutant skeletal preparations (ventral views). (C). Sections p-HH3 immunostaining in E15.5 WT and Fstl1 / lungs. The graph represents stained with an antibody against type II collagen (arrows). (Scale bars, 100 the mean of four independent experiments showing the comparison of the μm.) (D). Effects of stable overexpression of Fstl1 on cell proliferation of percentages of p-HH3–positive cells in the epithelium and mesenchyme ATDC5 cells with MTT assays. between WT and Fstl1−/− lungs.

Geng et al. PNAS | April 26, 2011 | vol. 108 | no. 17 | 7059 Downloaded by guest on September 26, 2021 − − − − To determine whether the hypercellularity of Fstl1 / lungs is mRNA level of Cc10 were comparable between WT and Fstl1 / caused by increased relative numbers of epithelial cells, we lungs (Fig. 4 A and B). − − performed IHC with antibody to TTF1, which marks all lung To analyze distal epithelial differentiation in Fstl1 / lungs, we − − epithelial cells. At E18.5, Fstl1 / lungs had a 29% increase in the next performed IHC with antibodies specific for AEC-I cells number of TTF1-positive epithelial cells (Fig. 3B) compared with (T1α) or AEC-II cells (SP-C). In contrast to the increased ex- WT controls. Sections were also stained with a phosphohistone pression of SP-C, the percentage of T1α staining coverage was −/− H3 (p-HH3) monoclonal antibody to detect cells undergoing decreased in the lung saccules of Fstl1 mice (4.8 ± 1.7%, n =4) − − mitosis. At E15.5, Fstl1 / lungs showed a 19% increase in the compared with WT controls (12.2 ± 3.2%, n =4;P < 0.05; Fig. number of p-HH3–positive cells in the epithelial compartment 4A). qRT-PCR analyses showed a marked decrease in mRNA α but a slight reduction in the mesenchymal compartment (Fig. 3C expression levels of both T1 and Aqp5 (AEC-I cell marker) in −/− fi and Fig. S3B). By E18.5, few stained cells were present in either E18.5 Fstl1 lungs (Fig. 4B), con rming less differentiation of − − Fstl1 / or WT lungs (Fig. S3B). BrdU incorporation showed AEC-I cells. fi We further examined the lung epithelium from E18.5 WT and a similar result (Fig. S3 C and D). In addition, no signi cant cell −/− −/− Fstl1 mice at the ultrastructural level. Squamous AEC-I cells death was observed by TUNEL staining in either Fstl1 or WT −/− lungs. Thus, Fstl1 deficiency increases epithelial cell proliferation, were clearly visible in WT lungs whereas it was lacking in Fstl1 − − resulting in a hypercellular phenotype of Fstl1 / lungs. saccules (Fig. 4C). Although the cuboidal AEC-II cells were observed in both WT and mutant lungs, the WT cells were mature Impaired Distal Epithelial Differentiation in Fstl1−/− Lung. To de- with many apical microvilli and lamellar bodies. Surfactant materials were observed within the saccular spaces. By contrast, termine whether the epithelial differentiation occurred properly − − − − / in hypercellular septa of Fstl1 / embryos, markers for proximal most cuboidal AEC-II cells lining Fstl1 saccules were immature (CC10) and distal (SP-C) epithelium were used to immunostain with smaller apical microvilli, fewer developing lamellar bodies, − − sections of E18.5 lungs. Both markers were present in the Fstl1 / and dispersed cytoplasmic glycogen (Fig. 4C). The glycogen- enriched immature AEC-II cells were further determined by lungs, suggesting that the absence of Fstl1 activity does not in- – terfere with the differentiation of specialized lung epithelial cells increased periodic acid Schiff (PAS) staining in the saccular epi- − − thelium (Fig. S3E), compared with WT controls. Taken together, along a proximal–distal axis. However, Fstl1 / lungs had a higher − − deletion of Fstl1 is associated with impaired distal epithelial dif- percentage of SP-C–positive staining cells (Fstl1 / , 24.98 ± 3.6%; ferentiation, as manifested by the promoted differentiation but Fstl1+/+, 12.65 ± 0.23%, n =4;P < 0.01; Fig. 4A), which was − − delayed maturation of AEC-II cells and the less differentiated consistent with the higher Sftpc mRNA level in Fstl1 / lungs AEC-I cells. This structural immaturation of saccular epithelium − − compared with WT lungs (Fig. 4B). The expression pattern and causes respiratory failure of Fstl1 / neonates.

Surfactant Dysfunction of Immature AEC-II Cells in Fstl1−/− Mice. Mature AEC-II cells are responsible for the production and secretion of the lung surfactants that are crucial in lowering the surface tension in the lung and thereby preventing end-expiratory atelectasis (22). An insufﬁcient production of surfactants, espe- cially the two surfactant-associated proteins (SP-B and SP-C), in both infants and adults, has already been reported to be associated with respiratory distress syndrome (7, 23). We next analyzed − − the expression and secretion of SP-B and SP-C in Fstl1 / lungs. Western blot analysis (Fig. 4D) showed that deletion of Fstl1 slightly affected the expression of pro-SP-B/SP-C from E18.5 lung tissues. However, production of mature SP-B/SP-C was strikingly − − decreased in Fstl1 / lungs. These data suggest that absence of Fstl1 activity interferes with SP-B/SP-C mature processing in AEC-II. The reduced surfactant production of immature AEC-II − − cells led to Fstl1 / lung atelectasis.

Fstl1 Binds to BMP4 and Negatively Regulates Smad Signaling in Vitro. To determine the molecular mechanisms whereby the absence of Fstl1 activity results in the phenotypes described earlier, we examined the Smad-mediated TGF-β/activin/BMP signaling. − − Fig. 4. Impaired alveolar epithelial cell differentiation/maturation in Fstl1 / Western blotting using lung extracts showed higher phosphoryla- − − lungs. (A) Expression of differentiation marker genes for lung epithelial cells tion levels of Smad1/5/8 (Fig. 5A) from E18.5 Fstl1 / lungs than in E18.5 embryos. (Scale bars, 50 μm.) (B) The relative expression levels of −/− those from WT littermates, whereas similar levels of phosphor- differentiation marker genes in E18.5 Fstl1 lungs as determined by qRT- Smad2 were observed between the genotypes (Fig. S4A). These PCR. Data represent the mean ± SEM in triplicates. (C) Transmission EM of the lung septa of E18.5 embryos shown at low magnification (LM) and high data indicate that the modulating effects of Fstl1 on BMP/Smad1/ magnification (HM). Squamous AEC-I cell in WT was closely opposite to 5/8 signaling are during the saccular lung development. We then densely stained capillary endothelial cell creating thin blood–air barrier examined the role of Fstl1 by using in vitro cultured Hep3B cells. (Upper Left). Cuboidal AEC-II cells in WT lungs contained many lamellar As shown in Fig. 5B, phosphorylation of Smad1 induced by BMP4 bodies (arrows) and apical microvilli (Lower Left). Surfactants (asterisk) were was inhibited by recombinant Fstl1 protein or Fstl1 over- visible in the saccular spaces (Upper Left). The blood–air barrier was signifi- expression. In agreement with this, Fstl1 inhibited the BMP4- cantly thicker with increased numbers of undifferentiated cuboidal epithelial − − induced expression of the reporters BRE-luciferase (Fig. 5C)or cells in Fstl1 / (Upper Right). These cells were immature with dispersed cytoplasmic glycogen and some small lamellar bodies (arrows; Lower Right). GCCG-luciferase (Fig. S4B) in a dose-dependent manner. Mean- (Scale bars: Upper,10μm; Lower,2μm.) (D) Western blotting of pro–SP-C and while, Fstl1 featured a comparable activity to other BMP anta- pro–SP-B, mature SP-C, and mature SP-B proteins in extracts of whole lungs gonists, such as Noggin and Follistatin (Fig. 5B and C), further taken from WT and Fstl1−/− embryos at E18.5. supporting its negative role in regulating BMP4/Smad signaling.

7060 | www.pnas.org/cgi/doi/10.1073/pnas.1007293108 Geng et al. Downloaded by guest on September 26, 2021 Fig. 5. Fstl1 modulated BMP4/Smad1/5/8 signaling via binding to BMP4. (A) Phosphorylated Smad1/5 in lung tissues from WT and Fstl1−/− embryos at E18.5. (B) Fstl1 inhibited BMP4-induced phosphorylation of Smad1/5 in Hep3B cells. Recombinant Fstl1 protein (25, 100 ng/mL) or transient overexpression of Fstl1 (pc-Fstl1, 1 μg) inhibited Smad1/5 phosphorylation induced by BMP4 (20 ng/mL). Noggin (25 ng/mL) and pc-Follistatin (Fst) (1 μg) were used as positive controls because they are known BMP antagonists. After 1 h of BMP4 treatment, cells were harvested for immunoblotting. (C) Fstl1 inhibited BMP4-induced expression of the reporter BRE-luciferase ac- tivities in Hep3B cells. The construct of BRE-luciferase reporter (0.3 μg) was Fig. 6. Reducing BMP4 signaling activity rescued atelectasis phenotype of cotransfected with pc-Fstl1 (0.1 μg, 0.2 μg) or treated with recombinant Fstl1 − − Fstl1 / embryonic lung explants. (A) Noggin increased saccular dilation of protein (25 ng/mL or 100 ng/mL). After 16 h of BMP4 treatment, cells were − − − − Fstl1 / fetal lung explants. E15.5 lung explants from WT and Fstl1 / litter- harvested for luciferase assay. Pc-Fst (0.1 μg) or Noggin (25 ng/mL) were used mates were cultured for 48 to 54 h in absence (CTL) or presence of Noggin as control. The data represent the mean ± SEM of three independent (500 ng/mL). H&E staining of explant sections shown at low (Upper) and high experiments after normalized to Renilla activity. (D) Sensorgrams of SPR − − (Lower) magnification. Saccular dilation was shown in Noggin treated-Fstl1 / analyses show the binding of Fstl1 to BMP4 or to TGF-β1, not to activin A; explants. The percentage of the total examined sac area of the lung section is EGF and NGF were used as negative controls. (E) Fstl1 binds to type II re- shown in the top right corner of each diagram (Upper). (Scale bar, 100 μm.) (B) ceptor of BMP4 using a pull-down assay. The Ni-Fstl1-BMPRII protein com- Fstl1 (100 ng/mL) inhibited BMP4-increased SFTPC expression in A549 cells. plex was immunoblotted with anti-Flag antibody to confirm the presence of The data represent the mean ± SEM of three independent experiments. (C) BMPRII (Upper). Each blot was further developed with anti-Myc antibody to Summary of the role of Fstl1 in embryonic lung development. Fstl1 modulates confirm the presence of Fstl1 (Lower). BMP4-induced Smad-1/5/8 activity and inhibits pro–SP-C expression.

As Fstl1 is a secreted protein, we reasoned that it should function at the ligand/receptor level. To test this possibility, we phenotype with 54% less dilation of saccular airways compared with WT controls (Fig. 6A), similar to the 60% reduction in the sac used constitutively active BMP type I receptors (caALK1 and − − spaces of in vivo E18.5 Fstl1 / lungs (Fig. 3A). Addition of Noggin caALK6), which can activate Smad1/5/8 independently of ligands −/− and type II receptors. Overexpression of Fstl1 did not inhibit increased the saccular spaces of Fstl1 explant, with 17% less caALK1/6-induced activation of GCCG-luciferase (Fig. S4C), dilation than that of WT control, indicating the atelectatic defect −/− fi suggesting that Fstl1 acts upstream of BMP type I receptors. We seen in Fstl1 lungs was partially rescued (Fig. 6A). Bright- eld then determined whether Fstl1 could bind to either BMP4 or microscopy images of saccular airways at the explant periphery BMP4 receptors. The binding affinity of Myc-His-tagged mouse showed a similar result (Fig S5 A and B). Furthermore, we ob- Fstl1 to BMP4 was determined by surface plasmon resonance served that Fstl1 had similar inhibitory effect of Noggin on BMP4- induced SFTPC mRNA expression in A549 cells (Fig. 6B). (SPR) analysis. Kinetic measurements using different concen- − − Additionally, Noggin reduced Sftpc mRNA expression in Fstl1 / trations of Fstl1 yielded a Kd of 7.2 nM for BMP4 (Fig. 5D). There explants (Fig S5C). These data demonstrate a functional link be- was weak binding of Fstl1 to TGF-β1(Kd of 36 nM), but no binding to activin A, nerve growth factor, or epidermal growth tween the Fstl1 and BMP4-dependent signaling pathways (Fig. factor. These data indicate that Fstl1 directly and specifically 6C) during embryonic saccular lung development. binds BMP4. We further examined whether Fstl1 could bind to Discussion BMP receptors. Fstl1 could pull down the BMP type II receptor (BMPRII; Fig. 5E), but not the type I receptor, ALK6 (Fig. S4D). The lung is derived from an outpocketing of the foregut endoderm As Fstl1 has the ability to interact with both BMP4 and its type II into the mesenchyme of the fetal thorax. Its formation depends receptors, we deduced that there is competition between BMP upon complex interactions between epithelial–mesenchymal cells and Fstl1 in receptor binding. Indeed, BMP4 competed with Fstl1 via paracrine factors. The present study provides evidence for the in binding with BMPRII (Fig. S4E). We therefore concluded that critical role of the secreted glycoprotein Fstl1 in mouse lung Fstl1 negatively regulates BMP4 signaling through interfering and trachea development. Targeted inactivation of the Fstl1 gene – resulted in tracheal flaccidity, saccular septal hyperplasia, end- with the ligand receptor interaction. BIOLOGY expiratory atelectasis, impairments of distal saccular epithelial cell Reducing BMP Signaling Activity Rescues Pulmonary Atelectasis of differentiation and maturation, and, ultimately, failure of lung DEVELOPMENTAL − − Fstl1−/− Mice. If increased BMP/Smad signaling in Fstl1 / lung function. Mechanistically, Fstl1 executes its functions partially plays a causative role in the development of atelectasis, it is through interaction with BMP4 to negatively modulate BMP4/ expected that reducing BMP signaling would ameliorate or even Smad signaling. − − rescue the atelectasis in Fstl1 / lungs. To test this possibility, we Mice lacking Fstl1 showed malformed tracheal cartilage rings. used an E15.5 embryonic mouse lung explant model of saccular Malformations of laryngotracheal cartilage in human infants with stage development (24) and added Noggin, an antagonist of BMP congenital airway anomalies can cause death (25). However, to ligand, to reduce BMP signaling activity. After 48 to 54 h of ex vivo date, the genetic basis of this malformation has not been estab- − − culture, sections of Fstl1 / explants showed condensed/atelectatic lished. Our model suggests Fstl1 as a candidate gene for correct

Geng et al. PNAS | April 26, 2011 | vol. 108 | no. 17 | 7061 Downloaded by guest on September 26, 2021 laryngotracheal cartilage formation. During cartilaginous de- ing affinity for activin (34). Although, Fstl1 does not bind activin, velopment, Fstl1 might promote the proliferation of committed it binds BMP4 with a Kd similar to that of Follistatin. It is unclear mesenchymal cells in cartilage primordia, or help these precursor whether Fstl1 binds BMP4 via FS domain in a manner similar to cells to differentiate into chondrocytes. The role of Fstl1 in Follistatin (14, 34). chondrogenesis is supported by the evidence of overall skeletal BMP4 signaling has been implicated in the regulation of mor- − − defects in Fstl1 / mice. Interestingly, without the rigid skeleton phologically correct development of lung, specifically distal epi- − − support, Fstl1 / mice did not develop tracheal stenosis, which has thelial differentiation (11, 12). In the current mouse model, we been seen in the neonatal lethality of mice deficient in Wnt5a (26) observed up-regulation of phospho-Smad1/5 together with the − − and Hoxa5 (27). Instead, Fstl1 / mice had enlarged tracheal increased SP-C–positive staining AEC-II cells in atelectatic − − lumens. Similar observations were reported in Hoxa-3 mutants Fstl1 / lungs. Reducing BMP signaling activity by Noggin par- (28). It is unclear whether other tracheal tissue components tially rescued the collapsed saccules and reversed the increased − − are involved. Sftpc mRNA levels in Fstl1 / lung explants. The defective Target deletion of Fstl1 causes inhibition of saccular structural phenotype is, at least in some respects, similar to those reports maturation. This abnormal phenotype is characterized by failure for impaired BMP activity (12), supporting our hypothesis that in progression of saccular septa thinning, which is necessary for Fstl1 affects distal lung epithelial differentiation partially − − gas exchange. In Fstl1 / mice, saccules were lined by a cuboidal through modulating BMP signaling. Fstl1 may orchestrate with epithelium that lacked squamous AEC-I cells. Fstl1 inactivation other BMP antagonists via their individual spatiotemporal ex- appeared to increase lung cellular proliferation (29, 30), but these pression patterns and control BMP4 signaling that is required cuboidal cells were immature SP-C–positive AEC-II–like cells for distal epithelial differentiation. containing large glycogen droplets and some LB bodies. Our We have shown here that targeted deletion of the Fstl1 gene in model is different from many genetically immature lung pheno- mice caused neonatal death from tracheal impairment and sac- types, such as Foxa2-null mice (31) and Pten-null mice (32), in cular immaturity. Fstl1 is essential for tracheal cartilage formation which numerous proliferative epithelial cells in the thickened and peripheral lung epithelial differentiation and maturation. We saccular septa are often accompanied with the inhibition of AEC- further demonstrated that Fstl1 interacted with BMP4 and regu- II cell differentiation as reflected by the reduced Sftpc expression lated lung AEC-II cell differentiation by negatively regulating and few LB bodies. The exact molecular mechanisms responsible BMP4/Smad1/5/8 signaling. Taken together, Fstl1 acts as a BMP4 for this specific activity of Fstl1 remain to be elucidated. On the signaling antagonist to modulate saccular maturation. The precise contrary, our model is in agreement with the hypothesis that mechanisms that Fstl1 regulates tracheal cartilage formation and AEC-I cells are differentiated from AEC-II cells (6) and further alveolar epithelial cell differentiation, if and how Fstl1 regulates suggests that the requirement of mature AEC-II cells. β − − other signaling pathways, such as Wnt and TGF- 1, during lung Structural immaturation of Fstl1 / lungs was accompanied by morphogenesis, and the interplay among Fstl1-BMP4, Fstl1-Wnt, biochemical immaturation. The productions of mature SP-B/C in and Fstl1-TGF-β1, are actively pursued in our laboratories. This −/− Fstl1 lungs were significantly decreased. Clinically, respiratory study and our continuing efforts will provide insight into the failure of neonates mostly resulted from insufficient production of mechanism coordinating the TGF-β superfamily in regulating or- surfactant (7, 33). Infants born with congenital SP-B or SP-C ganogenesis and into the understanding of the molecular mecha- deficiency (33) and mice with a targeted deletion of the SP-B gene nisms of congenital tracheal and lung anomalies in human, and (23) succumb to respiratory distress syndrome. The production of would provide new strategies for new therapeutic developments. mature SP-B/SP-C requires specific multistep proteolytic cleav- ages as pro–SP-B/C are trafficked through the regulated secretory Experimental Procedures −/− fl route (22). Unfortunately, the mechanisms underlying this are Generation of Fstl1 Mice. Fstl1 ox/+ mice (129S4 × C57BL/6J) were gen- largely unknown. We postulate that Fstl1 may play a role in sur- erated by standard homologous recombination. In these mice, Fstl1 exon 2 fl factant processing. The structural and biochemical immaturation encoding the signal peptide was flanked by loxP sequences. Fstl1 ox/+ mice − − of Fstl1 / lungs causes saccular collapse (pulmonary atelectasis), were then mated to EIIa-Cre transgenic mice (FVB/N; Jackson Laboratory). which is the main cause for neonatal death. Deletion of exon 2 results in loss of its signal peptide and disrupts its ORF, leading to loss of Fstl1 expression. The chimeric offspring were backcrossed In the present study, we provided several lines of evidence that − with C57BL/6J to generate Fstl1+/ mice, which then were intercrossed for Fstl1 may modulate BMP signaling. Although its orthologues in fi −/− fi production of Fstl1 de cient (Fstl1 ) mice. zebra sh, zfstl 1/2, have been observed to function redundantly to SI Experimental Procedures provides additional details on the generation of fi − − nog1 and chd to antagonize BMP activity during zebra sh de- Fstl1 / mice, as well as details on genotyping and Southern analysis, Western velopment (16, 17), effects of Fstl1 on BMP signaling are still blotting, pull-down assay, qRT-PCR, morphological analysis, cell culture and under discussion (18). No evidence of the interaction between saccular explant culture, luciferase assay, SPR analysis, and statistical analysis. Fstl1 and TGF-β superfamily proteins has been proposed. Our in vitro data showed that Fstl1 can directly bind BMP4 and exert its ACKNOWLEDGMENTS. We thank Xu Zhang for providing critical reagent; and function by interfering with the BMP4/BMPRII complex and Zhengping Xu and Henry Y. Keutmann for providing human Fst eukaryotic expression plasmid (pc-Fst). This work was supported by Ministry of Science and negatively regulate downstream Smad signaling. This is similar Technology (China) Grants 2007CB947100, 2009CB522101, and 2010CB833706; to the function of its paralogue Follistatin. Follistatin is a well and National Natural Science Foundation of China Grants 30671094, 30771130, known TGF-β superfamily antagonist protein, with a high bind- 31071241, and 30930050.

1. Morrisey EE, Hogan BL (2010) Preparing for the ﬁrst breath: genetic and cellular 7. Mendelson CR (2000) Role of transcription factors in fetal lung development and mechanisms in lung development. Dev Cell 18:8–23. surfactant protein gene expression. Annu Rev Physiol 62:875–915. 2. Maeda Y, Davé V, Whitsett JA (2007) Transcriptional control of lung morphogenesis. 8. Zhou L, Dey CR, Wert SE, Whitsett JA (1996) Arrested lung morphogenesis in Physiol Rev 87:219–244. transgenic mice bearing an SP-C-TGF-beta 1 chimeric gene. Dev Biol 175:227–238. 3. Rossant J, Tam PPL (2002) Mouse Development: Patterning, Morphogenesis, and 9. Zhao J, et al. (1996) Abrogation of transforming growth factor-beta type II receptor Organogenesis (Elsevier, Amsterdam). stimulates embryonic mouse lung branching morphogenesis in culture. Dev Biol 180: 4. Que J, Choi M, Ziel JW, Klingensmith J, Hogan BL (2006) Morphogenesis of the trachea 242–257. and esophagus: Current players and new roles for noggin and Bmps. Differentiation 10. Zhao J, Lee M, Smith S, Warburton D (1998) Abrogation of Smad3 and Smad2 or of 74:422–437. Smad4 gene expression positively regulates murine embryonic lung branching 5. Hogan BL, Yingling JM (1998) Epithelial/mesenchymal interactions and branching morphogenesis in culture. Dev Biol 194:182–195. morphogenesis of the lung. Curr Opin Genet Dev 8:481–486. 11. Bellusci S, Henderson R, Winnier G, Oikawa T, Hogan BL (1996) Evidence from normal 6. Warburton D, et al. (2000) The molecular basis of lung morphogenesis. Mech Dev 92: expression and targeted misexpression that bone morphogenetic protein (Bmp-4) 55–81. plays a role in mouse embryonic lung morphogenesis. Development 122:1693–1702.

7062 | www.pnas.org/cgi/doi/10.1073/pnas.1007293108 Geng et al. Downloaded by guest on September 26, 2021 12. Weaver M, Yingling JM, Dunn NR, Bellusci S, Hogan BL (1999) Bmp signaling regulates 23. Clark JC, et al. (1995) Targeted disruption of the surfactant protein B gene disrupts proximal-distal differentiation of endoderm in mouse lung development. Development surfactant homeostasis, causing respiratory failure in newborn mice. Proc Natl Acad 126:4005–4015. Sci USA 92:7794–7798. 13. Shibanuma M, Mashimo J, Mita A, Kuroki T, Nose K (1993) Cloning from a mouse 24. Benjamin JT, et al. (2009) The role of integrin alpha8beta1 in fetal lung osteoblastic cell line of a set of transforming-growth-factor-beta 1-regulated genes, one morphogenesis and injury. Dev Biol 335:407–417. of which seems to encode a follistatin-related polypeptide. Eur J Biochem 217:13–19. 25. Carden KA, Boiselle PM, Waltz DA, Ernst A (2005) Tracheomalacia and 14. Hambrock HO, et al. (2004) Structural characterization of TSC-36/Flik: Analysis of two tracheobronchomalacia in children and adults: An in-depth review. Chest 127: – charge isoforms. J Biol Chem 279:11727–11735. 984 1005. 15. Zhou J, et al. (2006) Identiﬁcation of a follistatin-related protein from the tick 26. Li C, Xiao J, Hormi K, Borok Z, Minoo P (2002) Wnt5a participates in distal lung morphogenesis. Dev Biol 248:68–81. Haemaphysalis longicornis and its effect on tick oviposition. Gene 372:191–198. 27. Aubin J, Lemieux M, Tremblay M, Bérard J, Jeannotte L (1997) Early postnatal lethality 16. Dal-Pra S, Fürthauer M, Van-Celst J, Thisse B, Thisse C (2006) Noggin1 and Follistatin-like2 in Hoxa-5 mutant mice is attributable to respiratory tract defects. Dev Biol 192: function redundantly to Chordin to antagonize BMP activity. Dev Biol 298:514–526. 432–445. 17. Esterberg R, Delalande JM, Fritz A (2008) Tailbud-derived Bmp4 drives proliferation 28. Manley NR, Capecchi MR (1995) The role of Hoxa-3 in mouse thymus and thyroid and inhibits maturation of zebraﬁsh chordamesoderm. Development 135:3891–3901. development. Development 121:1989–2003. 18. Umezu T, Yamanouchi H, Iida Y, Miura M, Tomooka Y (2010) Follistatin-like-1, 29. Sumitomo K, et al. (2000) Expression of a TGF-beta1 inducible gene, TSC-36, causes a diffusible mesenchymal factor determines the fate of epithelium. Proc Natl Acad Sci growth inhibition in human lung cancer cell lines. Cancer Lett 155:37–46. USA 107:4601–4606. 30. Chan QK, et al. (2009) Tumor suppressor effect of follistatin-like 1 in ovarian and 19. Adams D, Larman B, Oxburgh L (2007) Developmental expression of mouse Follistatin- endometrial carcinogenesis: A differential expression and functional analysis. like 1 (Fstl1): Dynamic regulation during organogenesis of the kidney and lung. Gene Carcinogenesis 30:114–121. – Expr Patterns 7:491 500. 31. Wan H, et al. (2004) Foxa2 is required for transition to air breathing at birth. Proc Natl 20. Wagner EF, Karsenty G (2001) Genetic control of skeletal development. Curr Opin Acad Sci USA 101:14449–14454. Genet Dev 11:527–532. 32. Yanagi S, et al. (2007) Pten controls lung morphogenesis, bronchioalveolar stem cells, 21. Shukunami C, et al. (1996) Chondrogenic differentiation of clonal mouse embryonic and onset of lung adenocarcinomas in mice. J Clin Invest 117:2929–2940. cell line ATDC5 in vitro: differentiation-dependent gene expression of parathyroid 33. Whitsett JA, Weaver TE (2002) Hydrophobic surfactant proteins in lung function and hormone (PTH)/PTH-related peptide receptor. J Cell Biol 133:457–468. disease. N Engl J Med 347:2141–2148. 22. Mulugeta S, Beers MF (2006) Surfactant protein C: Its unique properties and emerging 34. Rider CC, Mulloy B (2010) Bone morphogenetic protein and growth differentiation immunomodulatory role in the lung. Microbes Infect 8:2317–2323. factor cytokine families and their protein antagonists. Biochem J 429:1–12. BIOLOGY DEVELOPMENTAL

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