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Specific ablation of the nidogen-binding site in the laminin 1 chain interferes with kidney and lung development

¹ Max-Planck-Institute for Biochemistry, Department of Protein Chemistry, 82152 Martinsried, Germany
² Institute for Histology, University of Göttingen, 37075 Göttingen, Germany
³ University of Pittsburgh, Department of Neurobiology, Pittsburgh, PA 15261, USA
⁴ Institute for Biochemistry, University of Cologne, 50931 Cologne, Germany
⁵ University of Manchester, Wellcome Trust Centre for Cell-Matrix Research, Manchester M13 9PT, UK

View larger version (43K): [in a new window]   Fig. 1. Generation and analysis of ES cells and mice lacking the nidogen-binding module 1III4. (A) Restriction map for the genomic region of domain III of the Lamc1 gene. The sequence coding for the nidogen-binding module 1III4 is depicted in gray. The targeting vector and the resulting targeted allele after homologous recombination in ES cells are shown below. The final targeted allele was achieved by Cre-mediated deletion of the selection cassette. Predicted sizes of genomic fragments after EcoRV restriction and Southern blotting, using the externally located 5' probe, are indicated. A, ApaI; B, BamHI; EV, EcoRV; H, HindIII; Nh, NheI. (B) Southern blot analysis of EcoRV digests of genomic DNA from respective ES cell clones (left panel) and F2 embryos at 18.5 dpc (right panel). A fragment of 7.5 kb is detected in the wild type. In addition, a 4.5 kb fragment is seen in heterozygous cells, which is further reduced in size to 2.7 kb after transient transfection with Cre (+/–cre). Homozygous mutant embryos were identified by the appearance of a single 2.7 kb band (right panel), whereas homozygous ES cells showed both a 4.5 and a 2.7 kb band (left panel). (C) RT-PCR analysis of RNA isolated from wild-type (+/+), heterozygous (+/–) and homozygous mutant (–/–) ES cells. Primer pairs spanning the deletion site resulted in the expected size of fragments for the wild-type (369 bp) and mutated (201 bp) laminin 1 chain (top panel). In all genotypes, the downstream located modules 1III6-7 (middle panel) are normally transcribed.

View larger version (76K): [in a new window]   Fig. 2. Rotary shadowing electron microscopy demonstrates the absence of nidogen in homozygous mutant (–/–) laminin compared with the wild-type (+/+) form (arrowheads). Scale bar: 50 nm.

View larger version (33K): [in a new window]   Fig. 3. Bilateral renal agenesis in laminin 1III4-deficient mice. Urogenital tracts were dissected from male (A-C) and female (D,E) embryos at 18.5 dpc. Eighty percent of the homozygotes display bilateral renal agenesis (C,E). Testis (C) and ovary (E) are normally present, but ureter, vas deferens and uterus are lacking. Ten percent of the homozygous mutant animals develop two kidneys (B). The remaining of the urogenital system appears normal, except the epididymis, which has a vesicular appearance (arrowhead in B). Ad, adrenal gland; B, bladder; Ep, epididymis; K, kidney; O, ovary; Ov, oviduct; Ut; uterus; T, testis.

View larger version (184K): [in a new window]   Fig. 4. Metanephric kidney development in 13.5 and 11.5 dpc embryos. Morphology of representative sagittal H&E stained sections of 13.5 (A-C) and 11.5 dpc (D-E) embryos. At 13.5 dpc, the metanephric kidney is well developed in wild-type embryos (A) and a few homozygous mutant embryos (C). In the majority of mutant embryos, the metanephros is lacking and only remnants of the metanephric mesenchyme (arrow) are visible (B). At 11.5 dpc, the ureteric bud has invaded the mesenchyme and bifurcated once, surrounded by condensed metanephric mesenchyme in wild-type (D) and a few mutant embryos (F). In the majority of mutant animals, only uninduced metanephric mesenchyme is present (E). Ad, adrenal gland; CM, condensed metanephric mesenchyme; M, metanephric mesenchyme; Met, metanephric kidney; UB, ureteric bud. Scale bars: 150 µm in A-C; 50 µm in D-F.

View larger version (142K): [in a new window]   Fig. 5. Wolffian duct in 11 dpc embryos. The mesonephric kidney is comparably developed in 1III4-deficient (B) and in wild-type (A) embryos (arrows). (C,D) Growth of the Wolffian duct (arrowheads) can be followed in consecutive sagittal sections throughout the urogenital ridge in wild-type (C) but not in mutant (D) embryos. A representative sections of the blind-ending Wolffian duct in mutant embryos is shown in D. Confocal microscopy after immunostaining for laminin (E-G) and BrdU (red label in G) reveals continuous basement membranes structures in the controls (E) and mutants (F), which are, however, partially interrupted (arrows) in deeper layers of the section (G). Pax2 (H,I) is normally present in the Wolffian duct in the mutants (I) compared with littermate control embryos (H). Scale bars: 100 µm in A,B; 250 µm in C,D; 40 µm in E-G; 50 µm in H,I.

View larger version (150K): [in a new window]   Fig. 6. Kidney abnormalities in 18.5 dpc embryos that are associated with the mutation. The outer cortical layer in the mutant embryos (B) is indistinguishable from the controls (A). Note, however, the reduced number of tubuli (asterisk) in the mutants (B). (C-E) Glomerular defects in the mutants are visible in the inner cortex with the glomerular tufts showing mild (D) to severe (E) capillary aneurysms compared with normal appearance (C). Mutant male embryos display a hydronephric kidney with a thin kidney parenchyma (F). (G) Consecutive transverse section of the same animal as shown in F, demonstrating fusion (arrowhead) of the ureter with the vas deferens. Ur, ureter; Vas Def, vas deferens. Scale bars: 100 µm in A,B; 25 µm in C-E; 250 µm in F,G.

View larger version (108K): [in a new window]   Fig. 7. Analysis of basement membrane components in 18.5 dpc tissues. Double-label immunofluorescence analysis (A-D) of nidogen 1 (A,B) and nidogen 2 (C,D) in kidney sections demonstrates strongly reduced levels of nidogen 1 immunoreactivity in the mutants (B), whereas nidogen 2 (D) is normally present. (E,F) Localization of the laminin 1 chain to basement membrane structures is unaffected through the mutation (F) compared with controls (E). (G) Immunoblot analysis of extracts prepared from lung tissues shows comparable amounts of intact nidogen 1 in mutant lysates. LN, purified laminin-nidogen complex as control. Scale bar: 50 µm in A-F.

View larger version (110K): [in a new window]   Fig. 8. Histology of lung tissue from 1III4-deficient embryos. Representative sections of the lungs of mutant (B,D,F,H) and control (A,C,E,G) embryos through comparable regions are shown. Note the smaller and more compact size of the mutant lung (B) compared with controls (A) at 18.5 dpc. At higher magnification, it is apparent that the alveoli (asterisk) in 1III4-deficient embryos (D) are only poorly developed with marked mesenchymal thickening around the terminal air spaces compared with wild type (C). At 13.5 dpc, no apparent differences in lung development are visible in mutant (F) when compared with wild-type (E) embryos. Staining for surfactant protein C demonstrates that differentiation of airway epithelial precursor cells is not defective (G,H). Br, Bronchiole; H, heart; L, lung. Scale bars: 1 mm in A,B; 250 µm in C,D; 100 µm in E,F; 50 µm in G,H.

View larger version (182K): [in a new window]   Fig. 9. Ultrastructural analysis of the elongating Wolffian duct and lung. Ultrastructural analysis demonstrates a well-developed basement membrane separating the air-blood space of the alveoli (arrows) in control tissue (A) at 18.5 dpc. In the mutant lungs, normal (B), amorphously deposited (C) and missing (B) basement membranes are present (arrows). Note the cell protrusion when basement membranes are missing (B). At 10.5 dpc, basement membranes of the elongating Wolffian duct show discontinuities (asterisk) next to normal electron dense structures (arrows) in the mutant (E) when compared with controls (D). ery, erythrocyte. Scale bars: 100 nm in A-C; 50 nm in D,E.