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First published online 19 March 2009
doi: 10.1242/jcs.034736

Journal of Cell Science 122, 1145-1154 (2009)
Published by The Company of Biologists 2009
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NDST1-dependent heparan sulfate regulates BMP signaling and internalization in lung development

Zhonghua Hu1,*, Chaochen Wang1,*, Ying Xiao2, Nengyin Sheng3, Yibin Chen1, Ye Xu1, Liang Zhang1, Wei Mo1, Naihe Jing3 and Gengxi Hu1,{ddagger}

1 State Key Laboratory of Molecular Biology, Chinese Academy of Sciences, 200031 Shanghai, China
2 State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 200031 Shanghai, China
3 Laboratory of Molecular 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


Figure 1
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  		Fig. 1. Lung phenotype of Ndst1–/– mice. (A-H) Histological analysis of lung morphogenesis in mutant mice. (A,C,E,G) Hematoxylin and eosin-stained sections through wild-type lungs at 16.5 d.p.c. (A,E) and 18.5 d.p.c. (C,G). (B,D,F,H,J) sections through Ndst1–/– lungs at 16.5 d.p.c. (B,F) and 18.5 d.p.c. (D,H). (A-D) Low-magnification image of lung cells. (E-H) High-magnification image of lung cells. Arrowheads in G indicate the thin alveoli septa in wild-type lungs; the septa are thick in mutant lungs (indicated by arrowheads in H). (I-K) Increased lung cell proliferation in Ndst1–/– mice. Proliferating lung cells in 16.5 d.p.c. mice were labeled with BrdU and detected by antibody staining (I,J). BrdU-positive nuclei are stained black. Ndst–/– mice (J) have many more proliferating lung cells than the wild type (I). (K) BrdU incorporation calculated as the percentage of cells stained by BrdU in each field of vision from the lungs at 16.5 d.p.c. and 18.5 d.p.c. (P<0.01). Bars represent means + s.d. (L) RT-PCR analysis of NDST genes in WT and mutant (–/–) lungs at 17.5 d.p.c. Scale bars: 50 µm (E-H), 100 µm (A-D,I,J).

 

Figure 2
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  		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) were immunostained 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 with antibodies 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 18.5 d.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) AQP5 staining 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 mutant mice, 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 in normal littermates (O). (R-X) Caveolin-1 and SMA staining suggests that blood vessel structure is unchanged in Ndst1 mutant lungs. (Y) Real-time quantification of RNA transcripts of genes including Sftpa, Sftpb, Sftpc and Aqp5. **P<0.01. Bars represent means + s.d. Scale bars: 50 µm (C-F,H-K,O,P,R-U,W,X), 100 µm (A,B), 200 µm (M,N).

 

Figure 3
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  		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. Control sections (C,D,G,H) were stained with blocked serum instead of antibodies. Nuclei are stained with DAPI. (A,B) Labeling with antibodies against Smad1-P (red) reveals the expression level of Smad1-P is upregulated in mutant lungs. (E-F) Labeling with antibodies against Id1 (red) displays an upregulation of expression level of Id1. Scale bar: 100 µm. (I) Real-time quantification of RNA transcripts of genes Dlx5 and Tbx1 in lungs 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 of wild-type and mutant lungs at E17.5. β-actin is used as a reference for quantification.

 

Figure 4
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  		Fig. 4. Block of BMPR signaling rescues the defective differentiation of type I and type II epithelial cells in Ndst1 mutant lungs. Lung explants of 15.5 d.p.c. wild-type (A-H) and Ndst1–/– (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 indicates decreased lung cell proliferation after treatment with noggin. (C,D,K,L) Smad1-P in lungs is significantly 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 are stained with DAPI. Scale bar: 50 µm. (R) BrdU incorporation calculated as the percentage of cells stained by BrdU in each field of vision from the lungs in A, B, I and J. Error bars represent s.d.

 

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

 

Figure 6
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  		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) or pretreated 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: 25 µm. (U) Relative fluorescent signaling of each cell from A-P was quantified using confocal software from Leica microsystems. Results are means + s.d.

 

Figure 7
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  		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 embryos were 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 and heparin (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 protein SFTPC (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) is downregulated after treatment with heparin (red in N). Expression of SFTPC was upregulated in presence of both heparin and BMP4 (red in O) compared with BMP4 alone (red in P). However, expression of caveolin-1 remains unchanged (green in O,P). Nuclei are stained with DAPI. Scale bar: 50 µm. (Q) Lung explants were treated with 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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© The Company of Biologists Ltd 2009