1145Research Article

IntroductionLung epithelium and mesenchyme generate essential secreted

proteins for each other and thus coordinate lung embryonic

morphogenesis. Multiple factors, including bone morphogenetic

proteins (BMPs), fibroblast growth factors (FGFs) and hedgehog

are reportedly involved in lung formation. Inhibition of BMP

signaling with the BMP antagonist, noggin (NOGG) or dominant-

negative BMP receptor (dnAlk6) alters expression of FOXJ1,

uteroglobin (UTER/CC10) and pulmonary surfactant-associatedprotein C (PSPC/SFTPC) and causes a severe reduction in distal

lung epithelial cell types and an increase in proximal cell types

(Weaver et al., 1999). Ectopic expression of gremlin (GREM1),

another antagonist of BMP, results in disruption of the proximal-

distal pattern in embryonic lung (Lu et al., 2001). In addition,

deletion of BMPR1a or BMP4 in mouse lung epithelium leads to

reduction in number of type II pneumocyte and a decrease in

epithelial proliferation (Eblaghie et al., 2006). Misexpression of

BMP4 also causes a decrease of type II cells and inhibition of

epithelial proliferation, along with cell death in the mesenchyme

during lung development (Bellusci et al., 1996). Abnormal septa

in alveolar were observed in mice with deletions of FGFR3 and

FGFR4 (Weinstein et al., 1998). Overexpression of Sonic hedgehog

leads to an abundance of mesenchyme and loss of typical alveoli

(Bellusci et al., 1997). Additional signals such as EGF, Wnt and

TGFβ are also important in lung development.

A number of signaling pathways including BMP, FGF, hedgehog

and Wnt depend on heparan sulfate proteoglycans (HSPGs) (Hacker

et al., 2005; Lin, 2004). HSPGs are macromolecules composed of

heparan sulfate glycosaminoglycan (GAG) side chains covalently

bound to core proteins. Biosynthesis of heparan sulfate (HS) is

initiated from a chain composed of repeated D-glucuronic acid

(GlcUA) N-acetyl-D-glucosamine (GlcNAc) residues. The

glucosaminoglycan chains first undergo N-deacetylation and N-

sulfation of selected GlcNAc residues by GlcNAc N-deacetylase/N-

sulfotransferase (NDST) (Lindahl et al., 1998; Salmivirta et al.,

1996). The subsequent modifications, such as C-5 epimerization of

GlcA to iduronic acid (IdoA) and O-sulfation at various positions,

are dependent on the prior N-sulfation of GlcN units created by

NDST (Esko and Selleck, 2002; Lindahl et al., 1998). NDST has

a key role in the modification of the HS polysaccharide chain.

Genes encoding four known NDST isozymes, Ndst1-Ndst4, have

been identified in mammals (Aikawa and Esko, 1999; Aikawa et

al., 2001; Eriksson et al., 1994; Hashimoto et al., 1992; Kusche-

Gullberg et al., 1998). Ndst1 and Ndst2 are expressed ubiquitously

in both embryonic and adult mice, whereas Ndst3 and Ndst4 are

mostly expressed during embryonic development (Ford-Perriss et

al., 2002; Grobe et al., 2002; Miettinen et al., 1997; Yabe et al.,

2005). Mice lacking Ndst2 have abnormal mast cells without

properly sulphated heparin and mast-cell proteases (Forsberg et al.,

1999; Humphries et al., 1999), and those lacking Ndst3 have no

obvious phenotype (Grobe et al., 2002). However, disruption of Ndst1results in severe malformations in lung, brain, cranial facial, lens,

vascular, skeletal, and lacrimal gland development (Abramsson et

al., 2007; Fan et al., 2000; Grobe et al., 2005; Hu et al., 2007; Pan

et al., 2008; Pan et al., 2006; Ringvall et al., 2000). Thus, NDST1

is an essential NDST isozyme in mouse embryonic development.

Previously we reported that Ndst1 mutant mice developed

atelectasis and respiratory distress and died shortly after birth (Fan

Heparan sulfate proteoglycans (HSPGs) are required for various

signaling pathways, one of which is the bone morphogenetic

protein (BMP) signaling pathway. N-deacetylase/N-

sulfotransferase-1 (NDST1) participates in synthesizing heparan

sulfate (HS) chains of HSPGs, and is involved in bone and lung

development. Here, we report that in spite of the redundant

expression of Ndst2, Ndst3 and Ndst4 genes, Ndst1–/– mice

display defective differentiation of lung cells and increased cell

proliferation. Loss of Ndst1 in the lung enhances downstream

BMP signaling in vivo. Noggin, which is an antagonist of BMP,

can rescue the Ndst1–/– lung morphogenetic defects in explant

cultures. Further studies in vitro indicated that loss of Ndst1significantly impairs BMP internalization by decreasing BMP

binding to endogenous HS. Exogenous heparin can rescue both

the BMP signaling and BMP internalization abnormalities in

Ndst1–/– lung. Thus, we propose that HS regulates BMP signaling

by controlling the balance between BMP binding to HS, and

that BMP receptors and NDST1-dependent modification are

essential for this process. The results suggest that NDST1-

dependent HS is essential for proper functioning of BMP in

embryonic lung development.

Supplementary material available online at

http://jcs.biologists.org/cgi/content/full/122/8/1145/DC1

Key words: NDST1, BMP signaling, Lung development

Summary

NDST1-dependent heparan sulfate regulates BMPsignaling and internalization in lung developmentZhonghua Hu1,*, Chaochen Wang1,*, Ying Xiao2, Nengyin Sheng3, Yibin Chen1, Ye Xu1, Liang Zhang1,Wei Mo1, Naihe Jing3 and Gengxi Hu1,‡

1State Key Laboratory of Molecular Biology, 2State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica and 3Laboratory ofMolecular Cell Biology, Key Laboratory of Stem Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institute for Biological Sciences,Chinese Academy of Sciences, 200031 Shanghai, China*These authors contributed equally to this work‡Author for correspondence (e-mail: hgxgene@sunm.shcnc.ac.cn)

Accepted 2 December 2008Journal of Cell Science 122, 1145-1154 Published by The Company of Biologists 2009doi:10.1242/jcs.034736

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et al., 2000). However, the detailed developmental defects and the

underlying mechanisms of NDST1-depedent HS modulation of

signaling pathways remain unclear. Here, we report that BMP

signaling is affected in Ndst1 mutant lung, which could be one of

the causes of defective lung development.

ResultsAbnormal lung morphogenesis in Ndst1 mutantsMouse lung arises from the laryngotracheal groove at 9.5 days post

coitum (d.p.c.). Terminal sacs and vascularization develop in the

period of 16.5-17.5 d.p.c. After 17.5 d.p.c., the number of terminal

sacs and vascularization increase and type I and type II cells

differentiate (Warburton et al., 2000). Previous studies have

demonstrated that Ndst1-null mice develop pulmonary hypoplasia

and neonatal respiratory distress (Fan et al., 2000; Ringvall et al.,

2000). To further characterize the phenotype of the mutants,

histological examination of embryonic lung development in mutant

mice was performed by hemotoxylin and eosin staining lung

sections of mice at 16.5 and 18.5 d.p.c. At 16.5 d.p.c., the terminal

sacs were less dilated in lungs of Ndst1 mutants than those in the

wild type (Fig. 1A,B,E,F). The mesenchyma in 16.5 d.p.c. mutant

lungs was also thicker than that in their wild-type littermates (Fig.

1A,B). At 18.5 d.p.c., mutant lungs exhibited less dilated sacs and

thicker septa compared with wild-type lungs (Fig. 1C,D,G,H).

Furthermore, BrdU labeling of 16.5 and 18.5 d.p.c. embryos

indicated that mutant lungs had many more proliferative cells than

normal littermates (Fig. 1I-K), consistent with the observation that

mesenchyma and septa were thicker in mutant lungs than in wild-

type lungs.

Expression of NDST genes during lung developmentAlthough NDST1 is essential for lung morphogenesis, it is not the

only NDST protein expressed during lung development. Lung

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samples from Ndst1 mutants and normal littermates were examined

for expression levels of NDST genes by RT-PCR. At 17.5 d.p.c.,

all four NDST isozymes were expressed in normal lungs. In the

Ndst1–/– lung, expressions of Ndst2, Ndst3 and Ndst4 mRNA

transcripts were upregulated (Fig. 1L), suggesting a potential

redundant effect among the NDST enzymes. Because it is

considered to be the less prevalent phenotype in Ndst2 or Ndst3mutant mice (Forsberg et al., 1999; Grobe et al., 2002; Humphries

et al., 1999), NDST1 might be the most important isoform in the

NDST family. The phenotype observed in Ndst1–/– mice might form

as a result of lack of NDST1 function that cannot be compensated

by other members of this enzyme family.

Defective differentiation of lung cells in Ndst1 mutantsNeonatal respiratory distress observed in Ndst1 mutants might be

caused by reduced production of surfactant proteins (Fan et al.,

2000). Immunostaining analyses revealed a striking reduction in

expression of two surfactant proteins, SFTPC and SFTPA in lungs

of Ndst1-knockout mice. At 16.5 d.p.c., staining of SFTPC was

specifically detected in the distal epithelium (Fig. 2C), whereas

staining of SFTPA was detected throughout the epithelium, including

proximal and distal parts, in lungs of wild-type mice (Fig. 2A).

Significantly, both proteins were barely detectable in mutant lungs

(Fig. 2B,D). Furthermore, analysis of the mutant lungs at 18.5 d.p.c.

revealed a notable decrease of SFTPC-positive cells (Fig. 2E-G).

In parallel, real-time RT-PCR assay indicated that mRNA levels of

Sftpc, Sftpa and Sftpb were significantly reduced in mutant lung at

17.5 d.p.c. (Fig. 2Y). These observations imply that inactivation of

Ndst1 leads to defective development of distal epithelium and

immaturity of type II alveolar cells, which fail to produce surfactant

proteins.

Type I alveolar cells were also found to be immature in Ndst1-

null mice. From 17.5 d.p.c., type I alveolar cells arise from their

Fig. 1. Lung phenotype of Ndst1–/–

mice. (A-H) Histological analysis oflung morphogenesis in mutant mice.(A,C,E,G) Hematoxylin and eosin-stained sections through wild-typelungs at 16.5 d.p.c. (A,E) and 18.5d.p.c. (C,G). (B,D,F,H,J) sectionsthrough Ndst1–/– lungs at 16.5 d.p.c.(B,F) and 18.5 d.p.c. (D,H).(A-D) Low-magnification image oflung cells. (E-H) High-magnificationimage of lung cells. Arrowheads in Gindicate the thin alveoli septa in wild-type lungs; the septa are thick inmutant lungs (indicated by arrowheadsin H). (I-K) Increased lung cellproliferation in Ndst1–/– mice.Proliferating lung cells in 16.5 d.p.c.mice were labeled with BrdU anddetected by antibody staining (I,J).BrdU-positive nuclei are stainedblack. Ndst–/– mice (J) have manymore proliferating lung cells than thewild type (I). (K) BrdU incorporationcalculated as the percentage of cellsstained by BrdU in each field of visionfrom the lungs at 16.5 d.p.c. and 18.5d.p.c. (P<0.01). Bars represent means+ s.d. (L) RT-PCR analysis of NDSTgenes in WT and mutant (–/–) lungs at17.5 d.p.c. Scale bars: 50μm (E-H),100μm (A-D,I,J).

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1147NDST1 regulates BMP signaling

precursor cells. Both cell types characteristically express aquaporin-

5 (AQP5), a water channel protein (Krane et al., 1999; Kreda et

al., 2001). Immunofluorescent examination of 16.5 and 18.5 d.p.c.

mutant mouse lungs indicated that the number of AQP5-positive

cells was significantly reduced in Ndst1-null mice (Fig. 2H-L).

Consistently, the mRNA level of Aqp5 was also reduced in lungs

of 17.5 d.p.c. mutant animals (Fig. 2Y).

The expression pattern of the proximal bronchiole epithelium

marker CC10 (UTER/Clara Cell 10 protein), did not alter between

Ndst1 mutant and control lungs (Fig. 2M,N). Immunofluorescent

examination revealed that the number of CC10-immunoreactive

cells did not change in mutant lungs (Fig. 2O-Q). However, the

bronchioles lining with these cells were less dilated in mutant lungs

than in normal littermates (Fig. 2O,P), suggesting that development

of proximal bronchiole epithelium was affected by Ndst1inactivation.

There is a close relationship between blood vessel and lung

structural development. Immunofluorescent examination showed

that the mutant lungs had no significant alteration in distributions

of caveolin-1 (CAV1) and α-smooth muscle actin (SMA), which

are molecular markers of blood vessels (Fig. 2R-V for caveolin-1;

Fig. 2W,X for SMA). It appears that inactivation of Ndst1 does not

affect the lung blood vessel development.

BMP signaling pathway is upregulated in Ndst1–/– lungsThe embryonic lung in Grem1–/– mice exhibits an abnormal

‘proximalized’ phenotype, which is caused by BMP-signaling

dysregulation (Michos et al., 2004). Thus, BMP signaling was

examined in Ndst1–/– lungs. Binding of BMPs to preformed

heteromeric receptor complexes results in the phosphorylation of

Smad proteins, and subsequent stimulation of expression of Id1(Hollnagel et al., 1999; Nohe et al., 2002), Dlx5 (Holleville et al.,

2003; Miyama et al., 1999) and Tbx1 (Bachiller et al., 2003).

Phosphorylated Smad1 (Smad1-P) was highly upregulated in lungs

of 16.5 d.p.c. mutant mice (Fig. 3A,B), and upregulation of ID1

protein was evident at 18.5 d.p.c. (Fig. 3E,F). Interestingly,

overexpressed ID1 in mutant lungs seemed to localize not only in

the nucleus, but also in the cytoplasm. A similar phenomenon was

Fig. 2. Defective differentiation of lung cells in Ndst1–/– mice. Sections of wild-type (A,C,E,H,J,M,O,R,T,W) and Ndst1 mutant lungs (B,D,F,I,K,N,P,S,U,X) wereimmunostained with antibodies as indicated. Nuclei are stained with hematoxylin (A,B) or DAPI (C-F,H-K,O,P,R-U,W,X). The percentage of cells that stained withantibodies against SFTPC (G), AQP5 (L), CC10 (Q) and caveolin-1(V) were calculated from six sections from three lungs of each genotype at 16.5 d.p.c. and 18.5d.p.c. (C-F) Labeling with antibodies against SFTPC indicates that there are fewer type II alveolar cells (red) in mutant lungs than in the wild type. (H-K) AQP5staining reveals there are fewer type I alveolar cells or their precursor cells in mutant lungs than in the wild type. (M-P) Bronchioles in wild-type, as well as in mutantmice, are lined with CC10-immunoreactive Clara cells (brown in M,N; green in O,P). However, the bronchioles are smaller and less dilated in mutant lungs (P) than innormal littermates (O). (R-X) Caveolin-1 and SMA staining suggests that blood vessel structure is unchanged in Ndst1 mutant lungs. (Y) Real-time quantification ofRNA transcripts of genes including Sftpa, Sftpb, Sftpc and Aqp5. **P<0.01. Bars represent means + s.d. Scale bars: 50μm (C-F,H-K,O,P,R-U,W,X), 100μm (A,B),200μm (M,N).

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observed in small cell lung cancer with upregulated ID1, although

the reason for this was not clear (Kamalian et al., 2008). mRNA

levels of both Dlx5 and Tbx1 were also increased in lungs of Ndst1-

deficient animals (Fig. 3I). All these data demonstrate that the BMP-

signaling pathway is upregulated in Ndst1–/– lungs. However,

mRNA levels of Bmp2, Bmp4, Bmp5 and Bmp7 were not changed

in Ndst1 mutant lungs (Fig. 3J), indicating that the upregulation of

BMP signaling in the mutants was not caused by an increased BMPs.

Blockade of BMP signaling rescues the defectivedifferentiation of type I and type II cells in Ndst1-null miceTo determine whether dysregulated BMP signaling caused the

defective differentiation of type I and type II cells in Ndst1-null

lungs, Noggin, a BMP antagonist, was applied to block BMP

signaling. Similarly to wild-type lung explants (Fig. 4A-H),

treatment of 15.5 d.p.c. Ndst1–/– lungs with noggin resulted in

significantly downregulated expression of Smad1-P protein (Fig.

4K,L), and upregulation of SFTPC (Fig. 4M,N) and AQP5 (Fig.

4O,P). Furthermore, BrdU labeling indicated that cell proliferation

was decreased in the presence of noggin (Fig. 4I,J). These results

demonstrate that block of BMP signaling could rescue the

developmental failure in type I and type II cells, or their precursor

cells, in mutant lungs. And it reinforced the idea that upregulation

of BMP signaling contributes to the defective lung development in

Ndst1 mutants. However, exogenous noggin inhibited the

proliferation of both wild-type and mutant lungs (Fig. 4R), indicating

that a physiological concentration of BMP is essential for cellular

processes, including DNA synthesis and mitosis.

Decreased binding of BMP2 and BMP4 to endogenous HS inNdst1 mutant lungs Since NDST1 catalyzes the first modification step in biosynthesis

of HS and the HS structure in most basement membrane is affected

in Ndst1–/– mice, the HS chain in mutant lungs is therefore probably

affected. Thus, endogenous HS in wild-type and mutant lungs was

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detected using an antibody that reacts with O-sulfated N-acetylated

glucosamine residues of HSPGs (Fig. 5A-D). Wild-type lungs

showed a strong signal, whereas no signal was detected in mutant

lungs. This demonstrates that loss of NDST1 causes failure in the

synthesis of normal HS chains, which is consistent with a previous

report in the Ndst-1–/– lens (Pan et al., 2006). BMPs are reportedly

involved in lung morphogenesis, and bind to heparin. To investigate

how the BMP-signaling pathway is regulated in Ndst1 mutant lungs,

histochemical assays were performed to test the interaction between

secreted proteins and endogenous HS in mutant lungs. At 18.5 d.p.c.,

the binding of BMP2 and BMP4 was less in mutant lungs than that

in normal littermates (Fig. 5E-H), suggesting that the capacity of

the secreted BMP proteins to bind to HS was decreased in Ndst1–/–

lungs. Pre-treatment of lung sections with heparitinase greatly

reduced the binding in Ndst1-null mice and wild-type littermates

(Fig. 5I,J), indicating that the binding of these secreted proteins is

indeed HS dependent.

HS-dependent binding of BMP to cell surface is essential forBMP internalization in lung cellsBased on our data and previous reports (Ruppert et al., 1996; Fisher

et al., 2006; Jiao et al., 2007), HS seems to have an inhibitory role

in the BMP-signaling pathway. Thus HS-dependent BMP binding

appears to be distinct from receptor-dependent BMP binding. To

study the function of HS-dependent BMP binding to the cell surface,

BMP internalization was monitored in lung epithelial cells. In

cultured wild-type lung epithelial cells, heparitinase treatment

significantly reduced BMP binding to the cell surface and its

consequent internalization (Fig. 6A,C,M,O,U). This demonstrates

that HS-dependent binding of BMP is essential for BMP

internalization.

To test whether BMP receptors are involved in BMP

internalization, noggin, which inhibits BMP signaling by binding

to BMPs and preventing their interaction with receptors (Smith and

Harland, 1992; Zimmerman et al., 1996), was applied to

Fig. 3. BMP-signaling pathway is upregulated in Ndst1–/– lungs.(A-H) Sections of wild-type (A,C,E,G) and Ndst1 mutant lungs(B,D,F,H) immunostained with antibodies as indicated. Controlsections (C,D,G,H) were stained with blocked serum instead ofantibodies. Nuclei are stained with DAPI. (A,B) Labeling withantibodies against Smad1-P (red) reveals the expression level ofSmad1-P is upregulated in mutant lungs. (E-F) Labeling withantibodies against Id1 (red) displays an upregulation ofexpression level of Id1. Scale bar: 100μm. (I) Real-timequantification of RNA transcripts of genes Dlx5 and Tbx1 inlungs from 17.5 d.p.c. mice. **P<0.01. Bars represent means +s.d. (J) Gene expression assayed by RT-PCR of total RNA ofwild-type and mutant lungs at E17.5. β-actin is used as areference for quantification.

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internalization assays. BMP2 binding to the cell

surface and internalization in normal and Ndst1mutant lung epithelium were not changed in the

presence of noggin (Fig. 6E,G). It is conceivable that

BMP internalization occurs mainly via binding to HS

chains rather than binding to BMP receptors. In

Ndst1–/– lung cells, the binding of BMPs and

consequent internalization was also markedly

attenuated (Fig. 6B,D,F,H,U), indicating the necessity

of NDST1-dependent HS modification during this

process. Similar results were obtained with

mesenchymal cells (Fig. 6Q-T).

It was unexpected that exogenous heparin could

largely rescue BMP2 binding to the cell surface and

consequent internalization in Ndst1–/– mice (Fig.

6J,L,U), whereas its effect on wild-type cells was

much less significant (Fig. 6I,K,U). It seems that not

only cell surface HS, but also extracellular HS,

participates in the internalization of BMP.

Exogenous heparin could also rescue thephenotype of Ndst1–/– lung Since exogenous heparin could rescue the BMP

binding and internalization, it might be also able to

rescue the dysregulated BMP signalling and

consequent morphogenetic defects in Ndst1–/– lungs.

Treatment of 15.5 d.p.c. wild-type and Ndst1–/– lung

explants with 10 μg/ml exogenous heparin, which has

a higher content of N-sulfation than HS, reduced the

expression of Smad1-P (Fig. 7A,B,G,H) and enhanced

the expression of SFTPC and AQP5 (Fig. 7C-F,I-L).

The findings confirm that impaired BMP signaling

with N-sulfation or heparin can facilitate the

differentiation of type I and type II alveolar cells.

Consistently, in wild-type lungs, BMP4 and heparin

together led to the decreased expression of Smad1-P(Fig. 7M,N) and increased expression of SFTPC,

compared with that in lungs treated with BMP4 alone

(red in Fig. 7O,P), but did not affect the expression

of caveolin-1 (Fig. 7O,P, green).

Western blot assays were also performed. Similarly

to results of the histochemical assays, Ndst1–/– lung

explants displayed a much higher level of

phosphorylation of Smad1 than wild-type lung

explants, whereas treatment with noggin or heparin

dramatically reduced this abnormal high level (Fig.

7Q). This confirmed the inhibitory function of heparin

in BMP signaling.

DiscussionNDSTs might compensate for each other in knockout miceHere, we found that loss of Ndst1 in lung results in defective BMP

signaling even with the redundantly enhanced expression of Ndst2,

Ndst3 and Ndst4, whereas FGF signalling, but not hedgehog

signalling, was also affected in mutant lungs (supplementary

material Figs S1 and S2). Ndst1-null mice display severe brain and

facial defects, which might be consistent with impaired sonic

hedgehog (Shh) and FGF interaction with mutant HS (Grobe et al.,

2005) in some mutants. It is also possible that only part of NDST1’s

function could be compensated by other NDST isozymes in lung

development. For instance, it is possible that the hedgehog signaling

in lung was compensated. Although multiple abnormalities

previously described in Ndst1–/– mice were not observed in Ndst2or Ndst3 mutant mice, it is likely that other isoforms of the NDST

enzyme family might have compensated for the loss of Ndst2 or

Ndst3. Considering that NDST1 modulates FGF signaling, but not

BMP and Wnt during lens development (Abramsson et al., 2007;

Pan et al., 2006), compensation between NDSTs seemed to be tissue

dependent. Moreover, in lungs, we found that blood vessel formation

was not affected by loss of NDST1 whereas differentiation of lung

epithelium cells was. This implies that the compensation might even

be cell-type dependent.

Fig. 4. Block of BMPR signaling rescues the defective differentiation of type I and type IIepithelial cells in Ndst1 mutant lungs. Lung explants of 15.5 d.p.c. wild-type (A-H) andNdst1–/– (I-P) mice embryos were cultured for 3 days with control medium (A,C,E,G,I,K,M,O)or medium supplemented with noggin (B,D,F,H,J,L,N,P). (A,B,I,J) BrdU labeling indicatesdecreased lung cell proliferation after treatment with noggin. (C,D,K,L) Smad1-P in lungs issignificantly downregulated after treatment with noggin. (E-H,M-P) SFTPC (red in E,F,M,N)and AQP5 (red in G,H,O,P) in lungs are upregulated after treatment with noggin. Nuclei arestained with DAPI. Scale bar: 50μm. (R) BrdU incorporation calculated as the percentage ofcells stained by BrdU in each field of vision from the lungs in A, B, I and J. Error barsrepresent s.d.

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NDST1-dependent modification is essential for HS modulationof the BMP-signaling pathway and BMP internalizationHere, we propose that dysregulation of BMP signaling pathway

contributes to the defective morphogenesis in Ndst1-null lung.

Consistently, similar abnormal septation of the lung airway

epithelium was observed in embryonic lung when GREM1, an

antagonist of BMP, was deficient (Michos et al., 2004). By contrast,

overexpression of BMP4 in embryonic distal lung epithelium

results in thicker mesenchyma than observed in control and in the

‘emphysematous’ phenotype (Bellusci et al., 1996). Nog–/– mice

exhibit abnormal morphology with a malformed and truncated lobe

(Weaver et al., 2003). There are several possible explanations for

these discrepancies. First, endogenous GREM1 is expressed in

proximal airway epithelium (Weaver et al., 1999), whereas noggin

is normally expressed in the distal mesenchyme (Lu et al., 2001).

By inhibition of BMP signaling, GREM1 and noggin might have

different roles in spatial and temporal regulation of lung

development. Thus, the phenotypes observed in these mice models

result from deficiencies in partial epithelium or mesenchyme

Journal of Cell Science 122 (8)

differentiation alone. Similarly, ectopic expression of BMP4 in distal

epithelium had no obvious affect on the epithelium in the proximal

airway (Bellusci et al., 1996). HS plays an upstream regulatory role

in the BMP-signaling pathway, and thereby modulates the

differentiation of all the epithelium and mesenchyme. To determine

how HS does this, further conditional gene knockout studies are

needed. In addition, depending on concentration, BMP promotes

mesenchyme proliferation or death (Bellusci et al., 1996). In Bmp4transgenic mice, a high level of BMP4 resulted in cell death and

inhibition of cell proliferation. However, in our model and in

GREM1-deficient mice, cell proliferation increased, probably

because of the occurrence of endogenous BMP at proper

concentrations. Finally, HS modulates not only BMP signaling, but

also other important pathways during lung development, including

the FGF-signaling pathway (supplementary material Fig. S1). Thus,

the phenotype we report here might result from dysregulation of

several signaling pathways.

Accordingly, we asked how NDST1-dependent HS was involved

in the BMP-signaling pathway. It is reported that HS binds to the

N-terminal of BMP-2 and modulates the function of BMP2 in chick

limb bud assays (Ruppert et al., 1996). In C2C12 myoblast cells,

blockade of cell surface HSPG sulfation or removal of the GAG

chain enhanced BMP2 signaling and bioactivity, which could be

attenuated by exogenous heparin (Jiao et al., 2007). In this study,

we further proved that the binding of HS to BMP inhibits BMP

signalling, rather than facilitates it, in lung development. In

particular, NDST1-dependent modification is essential for the

activity of HS in the regulation of BMP-signaling pathways. In

addition to regulating BMP binding, HS also has a role in binding

noggin to the cell surface. Therefore, reduced noggin binding in

Ndst1 mutant lungs might also contribute to the hyperactivity of

the BMP-signaling pathway (Viviano et al., 2004).

Recent studies have implied that cell surface HSPGs are also

involved in cellular internalization of proteins (Belting, 2003; Jiao

et al., 2007; Payne et al., 2007). Live-cell imaging indicates that

HSPGs mediate cationic ligand internalization via a clathrin- and

caveolin-independent, but flotillin- and dynamin-dependent pathway

(Payne et al., 2007). Our data further demonstrate that the NDST1-

dependent HS modifications, including N-deacetylation and N-

sulfation, are required for BMP internalization. We also observed

that HS-mediated BMP internalization might be independent of

BMP receptors (Fig. 6C,G). More interestingly, we found that

exogenous heparin could partly rescue the BMP internalization

defects caused by loss of NDST1 (Fig. 6J,L). This implies that, not

only cell-surface HS, but also extracellular heparin or HS, also

contribute to BMP internalization, which has not been reported

before.

Taken together, it is postulated that BMPs that bind to HS prefer

to internalise, whereas BMPs that bind to receptors prefer to activate

downstream signaling. The balance between BMP binding to HS

and to receptors might control BMP signaling.

Compared with our previous work on Ndst1 mutant bone (Hu et

al., 2007), the activity of HS in modulating signaling pathways

seems variable in different tissues. First, unlike in embryonic lung,

exogenous heparin might not be able to rescue the morphogenetic

defects in Ndst1–/– embryonic limb bone. Second, FGF1 binding to

the cell surface is attenuated in mutant lung but not in mutant bone.

It is conceivable that other factors participate in the process of HS

binding to cytokines.

In summary, previous studies on the Ndst1-knockout mouse

indicate that HS could regulate various secreted ligands in different

Fig. 5. Reduced binding capacity of BMP2 and BMP4 to endogenous HS inNdst1 mutant lungs. (A-D) Sections of wild-type (A) and Ndst1 mutant lungs (B)were immunostained with antibody HepSS-1. Control sections (C,D) werestained with blocked serum instead of antibody. (E-J) In situ HS binding assaysfor lung sections from 18.5 d.p.c. mice reveal that binding ability of BMP2 andBMP4 to HS is decreased in Ndst1–/– mice (F,H) compared with that of normallittermates (E,G). (I,J) BMP2 binding following pretreatment with heparitinase.Scale bars: 100μm.

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tissue contexts. Therefore, the tissue-specific regulation of HS and

secreted ligands might contribute to the observation that HS plays

a vital role in different developmental tissues via various signaling

pathways. In this study, we provide a mouse model for exploring

the function of HS in BMP signaling. We point out the different

roles of HS in regulating BMP signaling and BMP internalization,

and thus present an explanation for the mechanistic involvement

of NDST1-dependent HS in modulation of the BMP-signaling

pathway during lung development,

In addition to insights into developmental regulation, our study

also has important clinical implications. We found an inhibitory

effect of heparin on the modulation of BMP signalling, which might

provide an explanation for the clinical observations that heparin

can improve outcome in small-cell lung cancer (SCLC) (Altinbas

et al., 2004; Lebeau et al., 1994), in which BMP8 is overexpressed

(Henderson et al., 2005).

Materials and MethodsMiceThe generation of the Ndst1-deficient mice and molecular examination by PCR todistinguish wild-type and mutant Ndst1 alleles have been reported previously (Fanet al., 2000). All mice used in this study were bred and maintained at Shanghai Instituteof Biological Sciences under specific pathogen-free conditions in accordance withinstitutional guidelines.

Histological and immunohistochemical analysisEmbryonic lungs were fixed overnight in 4% buffered formaldehyde at 4°C andembedded in paraffin for sectioning. For histological analysis, 5 μm sections were

Fig. 6. NDST1-dependent HS binds BMP2 and mediates BMP2 internalization. (A-T) Epithelial cells (A-P) or mesenchymal cells (Q-T) were untreated (control) orpretreated with: noggin for 1 hour, heparin for 1 hour, or heparitinase for 4 hours. The cells were incubated with BMP2 pre-labeled with goat anti-BMP2 (30 minutes,4°C) and then fixed to detect surface-bound BMP2 (A,B,E,F,I,J,M,N,Q,R) or transferred to 37°C for 1 hour (C,D,G,H,K,L,O,P,S,T) to allow BMP2 internalization.BMP2 was detected with FITC-conjugated anti-goat IgG (green), and cell nuclei were stained with DAPI. Scale bar: 25μm. (U) Relative fluorescent signaling of eachcell from A-P was quantified using confocal software from Leica microsystems. Results are means + s.d.

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stained with hematoxylin and eosin, mounted in xylene-based medium and

photographed. For immunohistochemistry, 5 μm tissue sections were pretreated with

10 mM sodium citrate buffer (pH 6.0) at 95°C, and then incubated overnight at 4°C

with anti-CC10 (T-18), anti-SFTPA (N-19), anti-SFTPC (M-20), anti-AQP5 (G-19),

anti-caveolin-1 (N-20), anti-Smad1-P (Ser463-Ser465), anti-Id1 (C-20), anti-Patched

(G-19), anti-Gli1 (N-16) (all from Santa Cruz Biotechnology, Santa Cruz, CA), anti-

SMA (Sigma, St Louis, MO) or anti-HepSS-1 (Seikagaku Corp., Tokyo, Japan). For

enzymatic staining, biotinylated secondary antibodies and the ABC staining system

(Santa Cruz) were applied. The images were captured on a cooled CCD camera (SPOT

II, Diagnostic Instruments, Sterling Heights, MI) on an Olympus BX51 microscope.

For immunofluorescence, secondary antibodies conjugated with appropriate

fluorochrome, FITC, Cy3, Cy5 (Jackson ImmunoResearch, West Grove, PA), were

used. Digital images were analyses by confocal laser-scanning microscope (Leica

SP2 system) and processed using Adobe Photoshop. At least three embryos for each

genotype were analyzed for each antibody.

RT-PCRTotal RNA was isolated from lungs of 17.5 d.p.c. Ndst1–/– and wild-type embryos

using Trizol Reagent (Invitrogen, Paisley, UK), and then reverse transcribed with

Super-scriptase (Invitrogen). The primers used in PCR assays were Ndst1 (forward,

5�-CTG CCC TGG CGT GCC TCC-3�; reverse, 5�-TGG GCC GTG TCA CAT AGA

GCA GT-3�); Ndst3 (forward, 5�-TCA CAT GCA GCC CCA CCT CTT-3�; reverse,

5�-GCT CCC CTC CAT GAA TAC TCT TGT-3�); Ndst2, Ndst4 (Pan et al., 2006);

Bmp2 (forward, 5�-TCT TCC GGG AAC AGA TAC AGG-3�; reverse, 5�-TCT CCT

CTA AAT GGG CCA CTT-3�); Bmp4 (Zhang et al., 2007), β-actin (Actb) (forward,

5�-CTG GCT GGC CGG GAC CTG ACA-3�; reverse, 5�-ACC GCT CGT TGC

CAA TAG TGA TGA-3�). Actb was used as an internal control for quantification.

Real-time PCR assays were performed on a DNA Engine Opticon 2 (MJ Research,

Watertown, MA) using the DyNAmo SYBR Green qPCR kit (FinnzymesOy, Espoo,

Finland). The data were expressed as relative mRNA (gene) copies, which were

normalized to the expression level of Actb. The following primers were used: Aqp5(forward, 5�-AGC CTT ATC CAT TGG CTT GTC-3�; reverse, 5�-TGA GAG GGG

CTG AAC CGA T-3�); SftpA (Okubo and Hogan, 2004); SftpB (forward, 5�-ACG

TCC TCT GGA AGC CTT CA-3�; reverse, 5�-TGT CTT CTT GGA GCC ACA

ACA G-3�); SftpC (forward, 5�-ACC CTG TGT GGA GAG CTA CCA-3�; reverse,

5�-TTT GCG GAG GGT CTT TCC T-3�); Dlx5 (forward, 5�-GTC CCA AGC ATC

CGA TCC G-3�; reverse, 5�-GCT TTG CCA TAA GAA GCA GAG G-3�); Tbx1(forward, 5�-AGG CAG ACG AAT GTT CCC C-3�; reverse, 5�-GCT TGT CAT

CTA CGG GCA CA-3�).

BrdU labeling and detectionMouse embryos were labeled with BrdU (BrdU labeling and detection kit II, Roche,

Germany) by intraperitoneal injection of 10 mM BrdU (1-2 ml per 100 g body weight)

into pregnant females 1 hour before sacrifice. Cultured lung explants were treated

with 100 μM BrdU for 1.5 hours before harvesting, and then were fixed in 95%

ethanol at 4°C and embedded in paraffin. Antibody staining of embryo sections was

carried out according to the manufacturer’s instructions. The number of BrdU-positive

nuclei and total cells in each field of vision was estimated from eight sections from

three animals for each genotype.

In situ HS-binding assaysThe assays were performed on paraffin-embedded sections essentially as previously

described for cryosections (Chang et al., 2000; Friedl et al., 1997). Briefly, after

blocking, sections were incubated with 15 nM BMP2, 15 nM BMP4 and 30 nM

FGF1 (all from R&D Systems, Wiesbaden, Germany), respectively. Then, sections

were incubated with anti-BMP2, anti-BMP4, anti-FGF1 antibodies (all from R&D

Systems) and stained using the ABC staining system (Santa Cruz).

Organ culture of embryonic lung explantsMouse embryonic lungs were cultured essentially as previously described (Dean et

al., 2005; del Moral et al., 2006). Briefly, lungs were isolated from 15.5 d.p.c. crosses

of Ndst1+/– mice. They were placed in a six-well plate on an 8 μm Nucleopore

membrane floating in 1 ml BGJ-B medium (GibcoBRL, Grand Island, NY) with

antibiotic/antimycotic (Life Technologies, Paisley, UK) and 0.1% BSA, and were

maintained at 37°C in a humidified 5% CO2 incubator. Lung explants were cultured

in medium supplemented with 10 μg/ml heparin (Sigma) or with 500 ng/ml noggin

(R&D Systems) for 3 days and were compared with explants cultured in BGJ-B

medium. The medium was changed every day.

Primary lung epithelial and mesenchymal cell culturesEpithelial and mesenchymal cell cultures were obtained by differential adhesion as

previously described (Lebeche et al., 1999). Briefly, whole lungs were dissected at

15.5 d.p.c. and digested with 0.4% dispase 0.8% Collagenase (37°C, 60 minutes;

GibcoBRL) to give rise to single cells. The resulting filtered suspension was plated

in 30 mm dishes and incubated at 37°C in a humidified 5% CO2 incubator for 1 hour

for differential adhesion. The supernatant containing epithelium cells was removed

and centrifuged at 1000 r.p.m. for 10 minutes at room temperature. The cell pellet

was resuspended in Dulbecco’s modified Eagle’s medium (DMEM) with 10% heat-

inactivated fetal bovine serum, and plated in dishes. The mesenchymal cells attached

to the dish were washed with PBS and cultured with fresh medium.

Fig. 7. Exogenous heparin negatively regulates BMP signaling in lung explants. Lung explants of 15.5 d.p.c. wild-type (A-F, Q-T) and Ndst1–/– (G-L) mice embryoswere cultured for 3 days with control medium (A,C,E,G,I,K) or medium supplemented with heparin (B,D,F,H,J,L), BMP4 (M,O) or simultaneously with BMP4 andheparin (N,P). The expression levels of protein Smad1-P (red in A,B,G,H) in lungs are downregulated in the presence of heparin. The expression levels of proteinSFTPC (red in C,D,I,J) and AQP5 (red in E,F,K,L) in lungs are upregulated in the presence of heparin. Smad phosphorylation in response to BMP4 (red in M) isdownregulated after treatment with heparin (red in N). Expression of SFTPC was upregulated in presence of both heparin and BMP4 (red in O) compared with BMP4alone (red in P). However, expression of caveolin-1 remains unchanged (green in O,P). Nuclei are stained with DAPI. Scale bar: 50μm. (Q) Lung explants were treatedwith noggin and heparin for 3 days and then collected for western blot. Samples were immunoblotted with indicated antibodies; β-actin was used as an internal control.

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BMP internalization assaysBMP2 internalization was performed mostly as previously described (Jiao et al., 2007).Briefly, BMP2 (12 μl of 400 ng/ml; R&D systems) was incubated with goat anti-BMP2 antibody (40 μl of 500 ng/ml; Santa Cruz) for 30 minutes at 37°C to formthe BMP2-anti-BMP2 complex. Lung epithelial or mesenchymal cells, seeded onglass coverslips in 24-well plates, were incubated with BMP2-anti-BMP2 complexat 4°C for 30 minutes. After a wash with ice-cold serum-free DMEM, the cells wereincubated at 37°C for 30 minutes. Cells were then incubated with FITC-conjugatedsecondary antibody (Jackson ImmunoResearch) and 4,6-diamidino-2-phenylindole(DAPI) and examined by confocal laser-scanning microscope (Leica SP2 system)after being fixed.

Western blotLung explants were homogenized and lysed after treated with noggin and heparin.Then lysates were collected after brief concentration. Immunoblotting was performedas described previously (Huang et al., 2002) with primary antibodies against Smad1-P (Cell Signaling Technology), Smad1 (a kind gift from Yeguang Chen, TsinghuaUniversity, Beijing, China) and β-actin (Sigma).

Statistical analysisThe Student’s t-test was used to determine levels of difference between groups, andP values for significance were set to 0.05. Values for all measurements were expressedas the means ± s.d.

We are grateful to Xinhua Lin and Xiaoyan Ding for helpfuldiscussions and Yeguang Chen for supplying antibody against Smad1.This work was supported by Minster for Science and Technology GrantG1998051007 and Chinese High-Tech R&D Program (863)-2001AA231011 and 0022Z2002.

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