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First published online 2 November 2004
doi: 10.1242/jcs.01514

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Accumulation of type IV collagen in dilated ER leads to apoptosis in Hsp47-knockout mouse embryos via induction of CHOP

¹ Department of Molecular and Cellular Biology and CREST/JST, Institute for Frontier Medical Sciences, Kyoto University, Sakyo-ku, Kyoto 606-8397, Japan
² Department of Cell Biology, Nagahama Institute of Bio-Science and Technology, Nagahama, Shiga 526-0829, Japan
³ Institute for Molecular Science of Medicine, Aichi Medical University, Nagakute, Aichi 480-1195, Japan

View larger version (80K): [in a new window]   Fig. 1. Type IV collagen does not localize in the basement membrane (BM) regions of Hsp47–/– mutant mouse embryos at 9.5 days post coitus (dpc). (A-F) Wild-type (WT) and Hsp47–/– embryos were fixed with paraformaldehyde. Neighboring cryosections of the embryos were then analysed by staining with haematoxylin and eosin (A,B) and by immunostaining with anti-Hsp47 antibody and FITC-conjugated second antibody (C,D) or anti-type IV collagen antibody and rhodamine-conjugated second antibody (E,F). Arrows indicate BM regions. ME, mesenchyme; EP, epithelium. Bar, 50 µm.

View larger version (84K): [in a new window]   Fig. 2. Laminin and nidogen-1 localize normally in the BM regions of Hsp47–/– mutant mouse embryos at 9.5 dpc. (A-F) Neighboring sections of wild-type and Hsp47–/– embryos were analysed by immunostaining with antibodies against laminin (A,B), nidogen-1 (C,D) and fibronectin (E,F). Arrows indicate BM regions. Bar, 50 µm.

View larger version (77K): [in a new window]   Fig. 3. Type IV collagen does not localize in the BM regions of Hsp47–/– mutant mouse embryos at 10.5 dpc. (A-E) Neighboring sections of wild-type and Hsp47–/– embryos that included the neural tube region were stained with haematoxylin and eosin (A,B) or with antibodies against Hsp47 (C,D) or type IV collagen (E,F). Arrows indicate BM regions. Bar, 50 µm.

View larger version (72K): [in a new window]   Fig. 4. Staining with anti-laminin and anti-nidogen antibodies reveals that the BM of Hsp47–/– embryos at 10.5 dpc is fractured. (A-E) Neural tube regions of wild-type and Hsp47–/– embryos were analysed for the immunolocalization of laminin (A,B), nidogen-1 (C,D) and fibronectin (E,F). Arrows indicate BM regions. Bar, 50 µm.

View larger version (97K): [in a new window]   Fig. 5. Type IV collagen, but not laminin, is absent from the BM of Hsp47–/– embryos at 9.5 dpc, as determined by immunoelectron microscopy. (A-D) Cryosections of wild-type and Hsp47–/– embryos that include the BM and neighbouring epithelial cells were first incubated with antibodies against type IV collagen (A,B) or laminin (C,D) and then with secondary antibodies conjugated with gold particles. Ultrathin sections were analysed by electron microscopy. Arrows indicate BM regions. Note that the staining of the BM with gold particles is observed in panels A, C and D but not in panel B. EP, epithelial cells; ME, mesenchyme. Bar, 0.1 µm.

View larger version (138K): [in a new window]   Fig. 6. Type IV collagen accumulates in dilated ERs in epithelial cells of Hsp47–/– embryos at 9.5 dpc. (A,B) Sections of epithelial cells of wild-type (A) or Hsp47–/– (B) embryos were analysed by immunoelectron microscopy using an antibody against type IV collagen. Arrows indicate ERs that contain type IV collagen. Bar, 0.1 µm. (C,D) Large magnification view of the dilated ER in Hsp47–/– embryos. Arrowheads indicate ribosomes associated with the dilated ER membranes. Note that the ERs of the Hsp47–/– cells are highly dilated and exhibit large accumulations of type IV collagen. Bar, 10 nm.

View larger version (13K): [in a new window]   Fig. 7. Growth retardation of Hsp47–/– embryos. The head-to-tail lengths of wild-type (Hsp47+/+, n=3; Hsp47+/–, n=11) and Hsp47–/–mutant (n=5) mouse embryos at 10.5 dpc were measured, and the means and standard deviations are shown.

View larger version (86K): [in a new window]   Fig. 8. DNA isolated from Hsp47–/– embryos at 10.5 dpc, but not at 9.5 dpc, shows the ladder pattern that is typical of apoptosis. DNA extracted from wild-type and Hsp47–/– embryos at 9.5 and 10.5 dpc was analysed by agarose gel electrophoresis. The DNA ladder was observed only in Hsp47–/– embryos at 10.5 dpc (right side), although none of the embryos displayed DNA fragmentation at 9.5 dpc (left side). Size markers: A, lambda phage DNA digested with HindIII; B, x174 DNA digested with HaeIII.

View larger version (58K): [in a new window]   Fig. 9. DNA fragmentation was detected by TUNEL staining of sections of Hsp47–/– mutant mouse embryos at 10.5 dpc. (A-D) Sections of wild-type and Hsp47–/– embryos at 9.5 dpc (A,B) and 10.5 dpc (C,D) were analysed for DNA fragmentation by TUNEL staining. A strong signal was detected in Hsp47–/– embryos at 10.5 dpc (D) but not at 9.5 dpc (B), whereas no significant signals were detected in the wild-type embryos at either time point (A,C). Bar, 50 µm.

View larger version (46K): [in a new window]   Fig. 10. CHOP is upregulated in Hsp47–/– mutant mouse embryos at 10.5-11.0 dpc. The levels of CHOP (A), Hsp47 (B) and ß-actin (C) mRNAs in wild-type and Hsp47–/– embryos at 9.5, 10.5 and 11.0 dpc were analysed by RT-PCR using specific primers. CHOP mRNA levels were elevated in Hsp47–/– embryos at 10.5 and 11.0 dpc, but not at 9.5 dpc.

View larger version (12K): [in a new window]   Fig. 11. Quantitative RT-PCR analysis measuring CHOP mRNA levels in wild-type and Hsp47–/– embryos. Total RNA isolated from embryos at 9.5 dpc (A) and 10.5 dpc (B) were analysed by real time PCR using primers specific to CHOP and ß-actin in the presence of SYBER Green. Levels of amplified products were recorded throughout the PCR reaction, and the level of CHOP cDNA was normalized against that of ß-actin cDNA. The calculated CHOP mRNA levels of wild-type embryos were set at 1.0. Mean with standard deviations for 11-14 embryos are shown.

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