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First published online 3 March 2004
doi: 10.1242/dev.01037

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Collagen IV is essential for basement membrane stability but dispensable for initiation of its assembly during early development

¹ Department of Experimental Medicine I, University Erlangen-Nürnberg, 91054 Erlangen, Germany
² Department of Ophthalmology, University Erlangen-Nürnberg, 91054 Erlangen, Germany
³ Department of Molecular Biology and Biochemistry, Okayama University Medical School, Okayama 700, Japan
⁴ Wellcome Trust Centre for Cell-Matrix Research, School of Biological Sciences, University of Manchester, Manchester M13 9PT, UK

View larger version (52K): [in a new window]   Fig. 1. Targeted inactivation of the Col4a1/2 locus. (A) The murine Col4a1/2 genes are arranged head-to-head with a common shared promoter element (P), exons (shaded squares) and relevant restriction sites (E, EcoRI; Xh, XhoI; Nh, NheI; H, HindIII) are indicated. The targeting construct containing the pgk/neomycin resistance cassette (Neo) and the mutant locus (bottom) are shown. Probes P1, P2 and the detected EcoRI fragments (arrows) are marked. (B) Genotyping of ES cells and newborns by Southern blotting. DNA was isolated, digested with EcoRI and hybridized with probe P1. The 15 kb and 9.5 kb bands are the wild-type and mutant alleles, respectively. (C) Genotyping of E10.5 embryos by PCR. The 0.7 and 0.4 kb fragments represent the mutant and wild-type alleles, respectively. Markers are indicated (kb). (D) Detection of 1(IV) and 2(IV) mRNAs in embryos at E9.5 and E10.5 by RT-PCR. Annexin A5 (Anxa5) was used as an internal control.

View larger version (36K): [in a new window]   Fig. 4. Deficiency of 1(IV)/2(IV) collagen is not compensated by the increased expression of other collagen IV chains. (A) Relative mRNA levels of collagen IV isoforms (3-6), laminin 1 chain (Ln-1) and nidogen-1 (Nd-1) were determined by quantitative RT-PCR in RNA isolated from individual wild-type (white bars) and 1(IV)/2(IV)-deficient embryos (black bars) from E9.5 and E10.5 littermates. The 3(IV), 4(IV) and 6(IV) mRNAs are not detectable or are present at only very low levels in any genotype. In mutant embryos no compensatory upregulation of 5(IV), laminin 1 chain and nidogen-1 is observed. Relative mRNA levels are expressed as a percentage of GAPDH expression. (B) No deposition of 5(IV) protein is observed by immunofluorescence in the Reichert's membrane of normal (+/+) or mutant embryos (–/–) at E10.5. A faint intracellular staining in trophoblast cells indicates the expression of low levels of non-secreted 5(IV) protein. Antibodies detecting laminins containing the 1, ß1 or 1 chains and 2(IV) collagen were used as positive controls. Scale bar: 10 µm.

View larger version (59K): [in a new window]   Fig. 2. Deficiency of collagen IV expression causes a variable degree of growth retardation in embryos at E9.5 (A), E10.5 (B) and E11.5 (C). The genotypes of representative littermates are indicated (+/+, +/–, –/–). Embryos at E11.5 show bleeding from the heart and dilation of blood vessels (arrows). Scale bars: 100 µm.

View larger version (121K): [in a new window]   Fig. 3. Basement membrane-like structures are deposited in the absence of collagen IV. Collagen IV (Col4), nidogen-1 (Nd-1) and laminins containing 1, ß1 or 1 subunits (Ln) were detected by immunohistochemistry at E10.5 in embryos (A) or in Reichert's membrane (B) with the indicated genotypes. Basement membrane discontinuities are indicated by arrows. N, notochord; P, pial basement membrane; E, epidermal basement membrane; RM, Reichert's membrane; T, trophoblast cells. Scale bars: 100 µm.

View larger version (103K): [in a new window]   Fig. 5. Defects of placental structures. Hematoxylin-Eosin staining of paraffin wax sections of deciduas (E11.5) from collagen IV-deficient (–/–) embryos and normal littermates (+/+). (A) The Reichert's membrane (RM) of collagen IV-deficient embryos appears thinner, and ruptures (arrows) are causing severe bleeding of maternal blood (arrowhead) into the yolk sac cavity. YS, yolk sac. (B) The development of the labyrinth layer of the placenta is retarded in mutant embryos (–/–) as compared to wild-type embryos (wt). The contact between maternal (open arrowheads) and embryonic blood (closed arrowheads) is reduced. Scale bar: 100 µm.

View larger version (44K): [in a new window]   Fig. 6. (A) Wild-type control. (B) Neuronal ectopias (arrows) are present in E11.5 mutant embryos because of the migration of neuronal cells through the pial basement membrane into the surrounding mesenchymal layers (arrows). N, neuroectoderm. Scale bars: 100 µm.

View larger version (63K): [in a new window]   Fig. 8. Ultrastructural abnormalities in basement membranes of collagen IV-deficient embryos. Epidermal (A) and pial basement (B) membranes in E10.5 mutant embryos (–/–) are amorphously deposited or absent when compared to normal controls. In some areas the deposited material detaches from the surface of cells and forms irregular folds (arrow) or is almost completely absent (arrowhead). (C) Reichert's membrane of mutant embryos (E10) is significantly thinner than in wild-type in comparable areas and shows an altered texture. Additionally, detachment from trophectoderm (T) and parietal endoderm cells (pE) is observed (*). Scale bars: 0.2 µm.

View larger version (113K): [in a new window]   Fig. 7. Deficiency of collagen IV does not affect the development of the vascular system, but causes aberrant organization of capillary structures. (A) The vascular system was detected by staining for ß-gal in E10.5 wild-type (+/+) and collagen IV-deficient (–/–) embryos on a Anxa5+/LacZ background. No gross differences are seen in whole-mount stainings. Scale bar: 1 mm. (B) The capillary plexus forming close to the pial basement membrane is less dense in mutant (–/–) embryos and capillaries entering the neuroectoderm are irregularly arranged (arrows) and detected in lower numbers in collagen IV-deficient embryos than in littermate controls (+/+). Scale bars: 100 µm.