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First published online February 8, 2006
doi: 10.1242/10.1242/jcs.02780

Journal of Cell Science 119, 625-635 (2006)
Published by The Company of Biologists 2006
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Glycosylation catalyzed by lysyl hydroxylase 3 is essential for basement membranes

Heli Ruotsalainen1,*, Laura Sipilä1,*, Miia Vapola1, Raija Sormunen2, Antti M. Salo1, Lahja Uitto1, Derry K. Mercer3, Simon P. Robins3, Maija Risteli1, Attila Aszodi4, Reinhard Fässler4 and Raili Myllylä1,{ddagger}

1 Department of Biochemistry, Biocenter Oulu, University of Oulu, FI-90014 Oulu, Finland
2 Department of Pathology, Biocenter Oulu, University of Oulu, FI-90014 Oulu, Finland
3 Rowell Research Institute, Greenburn Road, Bucksburn, Aberdeen, AB21 9SB, UK
4 Department of Molecular Medicine, Max Planck Institute of Biochemistry, Am Klopferspitz 18, 82152 Martinsried, Germany


Figure 1
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  		Fig. 1. Targeted manipulations of the Plod3 gene by homologous recombination. (A) Targeting strategy showing the wild-type allele and the targeted Plod3 alleles. Knockout (k/o) denotes the knockout of LH3, and hypomorph (hy/hy) and LH mutant (m/m) the LH-mutated LH3 with or without the Neo selection cassette, respectively. The homologous arms of the targeting construct are indicated as segments of a line. The positions of the 5' external probe for Southern blot analyses are indicated as gray bars. Note that exons 8-17 are not shown. Northern analysis of LH3 (B) and LH1 and LH2 (D) levels in the embryos of each mouse line. The mRNA levels of LH3, LH2 and LH1 were determined from poly(A)+ RNA isolated from E9.5-E13.5 embryos. LH3, LH2, LH1 and ß-actin (loading control) mRNA were hybridized with radioactively labeled cDNA probes. (C) Western analysis of hypomorphic LH3 and LH mutant E13.5 embryos with LH3 antibody. The positions of molecular size markers are indicated.

 

Figure 2
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  		Fig. 2. E9.5 knockout (A, k/o) and hypomorphic (B, hy/hy) embryos, and E12.5 hypomorphic embryos (C, hy/hy) demonstrate retarded developmental size when compared with wild-type embryos (+/+). Arrows indicate blood vessel dilations in the region of the sinus venosus (A,B) or in the head (C). A blister in the hypomorphic embryo is marked with an arrowhead (C).

 

Figure 3
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  		Fig. 3. Type IV collagen and laminin immunofluorescent staining of E9.5 wild-type (+/+) and homozygous embryos of knockout (k/o), hypomorphic (hy/hy) and LH mutant (m/m) mouse lines. In the knockout, the type IV collagen staining was mostly inside the cells (insert in A, panel 2). (C) EM figures of the BM (arrows) of the neural tube from wild-type, knockout, hypomorphic and LH mutant embryos at E9.5. In the knockout, the BM was absent (panel 2), whereas in the hypomorph it was discontinuous (panel 3, arrowhead) and amorphous material was also detected in the extracellular space (panel 3,*) when compared to the wild type (panel 1). The basement membrane of LH mutant was comparable to the wild-type (C4). (D) Fragments of BM were detected under the thin endothelial cells in wild-type (arrows in panel 1) and LH mutant (not shown) embryos at E9.5, but it was not detected in knockout (panel 2, arrowhead) and hypomorphic (not shown) embryos, leading to the ruptured cell layer (panel 3, arrow). The ER was dilated in homozygous knockout (panel 4, {blacksquare}) and hypomorphic (not shown) embryos. Bars, 10 µm (A,B); 0.1 µm (C); 0.2 µm (D, panels 1-3); 1 µm (D, panel 4).

 

Figure 4
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  		Fig. 4. EM analysis of the LH mutant newborn skin. The BM of the homozygous (m/m) newborn skin showed thinner lamina densa (B, arrows) than wild-type (+/+) littermates (A). The structure of collagen bundles was looser and less organized in the homozygotes (D,F) than in the wild type (C,E). Collagen fibrils in the homozygotes were covered with diffuse material (* in D and F). Bars, 0.2 µm.

 

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© The Company of Biologists Ltd 2006