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First published online 1 February 2006
doi: 10.1242/dev.02270

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Transgenic isolation of skeletal muscle and kidney defects in laminin ß2 mutant mice: implications for Pierson syndrome

¹ Renal Division, Washington University School of Medicine, St Louis, MO 63110, USA.
² Department of Cell Biology and Physiology, Washington University School of Medicine, St Louis, MO 63110, USA.
³ CROET, Oregon Health Sciences University, Portland, OR 97239, USA.

View larger version (13K): [in a new window]   Fig. 1. Structure of the transgenes. (A) The MCK-B2 transgene drives rat laminin ß2 expression from the mouse muscle creatine kinase (MCK) promoter, and transcription termination and polyadenylation signal sequences from SV40 (pA) ensure processing to mRNA. (B) The NEPH-B2 transgene drives ß2 expression from the mouse nephrin promoter. The ß2 cDNA and SV40 sequences were flanked by loxP sites for future manipulations.

View larger version (85K): [in a new window]   Fig. 2. Localization of endogenous and MCK-B2 transgene-derived laminin ß2. (A,B) In control skeletal muscle, endogenous mouse laminin ß2 (A) is concentrated at synapses (arrows) doubly labeled by -bungarotoxin (B); ß2 is also found in extrasynaptic regions of muscle fibers (A). (C,D) In MCK-B2 transgenics, antibody specific for transgene-derived rat ß2 (C) labels only synapses in skeletal muscle (arrows), identified by -bungarotoxin (D). (E-H) Transgene-derived rat laminin ß2 is also found in cardiac muscle BMs (E), in circular (cm) but not longitudinal (lm) smooth muscle or crypt (c) epithelial BMs of intestine (G), and weakly in large airway smooth muscle of lung (arrow in H) but not in alveolar (alv) BMs. No rat ß2 was detected in glomeruli (g) in kidney (F). Scale bars: 25 µm in A-D; 100 µm in E,H; 50 µm in F,G.

View larger version (127K): [in a new window]   Fig. 3. NEPH-B2 transgene-derived laminin ß2 accumulates specifically in the GBM. (A,B) Antibody specific for transgene-derived rat ß2 does not stain kidney glomeruli (g) from a control mouse (A) but stains GBM in kidney from NEPH-B2 transgenic mice (B). (C,D) NEPH-B2 transgene-derived ß2 is not deposited at skeletal muscle synapses (C) identified by double staining with -bungarotoxin (D). Scale bar: 50 µm in A,B; 20 µm in C,D.

View larger version (28K): [in a new window]   Fig. 4. Analysis of growth rates and urinary protein levels. (A) Growth curves (weight versus age) of control mice, Lamb2-/- mice and Lamb2-/- mice carrying transgenes. Lamb2-/- and Lamb2-/-; NEPH-B2 mice fail to surpass a weight of 4 g and die at 3-4 weeks of age. By contrast, Lamb2-/-; MCK-B2 mice grow at a rate similar to controls but die at 1 month of age. Lamb2-/-; MCK-B2; NEPH-B2 doubly transgenic mice exhibit normal weight gain and a long life. (B) Proteinuria in Lamb2-/-; MCK-B2 mice. Urine (1 µl) was analyzed by SDS-PAGE and stained with Coomassie Blue. M, markers; the pink band is albumin. Lanes 1, 5 and 6: urine from Lamb2+/-; MCK-B2 mice. Lanes 2, 4 and 7: urine from Lamb2-/-; MCK-B2 mice. Lane 3 was empty.

View larger version (85K): [in a new window]   Fig. 5. Restoration of proper synaptic architecture and BM composition in skeletal muscle from Lamb2-/-; MCK-B2 mice. (A,B) Colocalization of rat laminin ß2 (A) with -bungarotoxin (B) in a highly ramified skeletal muscle synapse. (C,D) Restoration of laminin 5 to synaptic BMs (C) identified by -bungarotoxin (D). (E,F) Laminin ß1 (E, green in F) is not associated with the -bungarotoxin-positive synaptic clefts (red in the merged image in F). Scale bar: 20 µm for A-F.

View larger version (139K): [in a new window]   Fig. 6. Ultrastructural analysis of neuromuscular junctions (NMJ), myotendinous junctions (MTJ) and glomerular filtration barriers. (A-D) A control synapse (A) shows a Schwann cell (s) capping the vesicle-rich nerve terminal (nt) adjacent to the muscle (m) endplate containing numerous junctional folds (jf). In the Lamb2-/- synapse (B), junctional folds are absent and the Schwann cell extends processes (arrow) between the nerve terminal and the muscle. Synaptic deposition of laminin ß2 in Lamb2-/-; MCK-B2 mice restores synaptic architecture to normal (C). In Lamb2-/-; NEPH-B2 mice, glomerular deposition of ß2 and prevention of proteinuria has no restorative effect on the synapse (D). (E-G) MTJ from a control (E) exhibits numerous infoldings of the muscle fiber (m) with continuous BMs. In the Lamb2-/- MTJ (F), infoldings are less complex, and the BMs (arrows) appear fuzzy. MCK-B2 transgene-derived ß2 restores much of the normal MTJ architecture (G). (H-K) Glomerular capillary segment from a control (H) shows the interdigitated podocyte foot processes (fp) adjacent to the GBM. Effaced foot processes (efp) are evident in the Lamb2-/- (I) and Lamb2-/-; MCK-B2 (J) mice, which are proteinuric. Deposition of rat ß2 into the GBM in Lamb2-/-; NEPH-B2 mice prevents proteinuria and foot process effacement (K). Scale bars in D and K are 1 µm for A-D and H-K, respectively; scale bar in G is 2 µm for E-G.

View larger version (110K): [in a new window]   Fig. 7. Functional and compositional analyses of MTJs. (A-D) Evans blue analysis reveals normal MTJ integrity in a control mouse diaphragm (A), but evidence of damage is observed in Lamb2-/- (B) and Lamb2-/-; MCK-B2 (C) mice at 3 weeks of age. A diaphragm from an older adult Lamb2-/-; MCK-B2; NEPH-B2 mouse shows no damage (D), suggesting eventual repair of MTJs. (E,F) Double staining for laminin ß2 (green) and laminin 2 (red) in intercostal muscles reveals concentration of ß2 at MTJs in both control (E) and Lamb2-/-; MCK-B2 (F) mice. (G,H) Double staining for mouse plus rat ß2 (G) and rat ß2 only (H) in a Lamb2+/-; MCK-B2 mouse shows that many MTJs contain little if any transgene-derived protein. (I-L) Double staining for total laminin ß2 (green) and 2 (red) in intercostal muscles from aged control (I,J) and Lamb2-/-; MCK-B2; NEPH-B2 (K,L) mice reveals efficient accumulation of ß2 at MTJs. Transgene-derived ß2 is missing from extra-junctional BMs in rescued mutant muscle fibers (F,K). Scale bars: in D, 1 mm for A-D; in H, 50 µm for A-I,K; in L, 20 µm for J,L.

View larger version (95K): [in a new window]   Fig. 8. Analysis of laminin ß chain composition in glomeruli. (A,B) In Lamb2+/-; NEPH-B2 mice, the GBM contains rat laminin ß2 (A), and laminin ß1 is restricted to the mesangial matrix (B). (C,D) In Lamb2-/- mice, no ß2 is present in the GBM (C); instead, ß1 is present (arrows in D). (E,F) In Lamb2-/-; NEPH-B2 mice, rat ß2 is deposited into the GBM (E), and ß1 is restricted to the mesangial matrix (F) as in controls. Scale bar: 25 µm.

View larger version (127K): [in a new window]   Fig. 9. Histological analysis of skeletal muscle and kidney. Aged control (A,C,E) and Lamb2-/-; MCK-B2; NEPH-B2 (B,D,F) mice. Paraffin sections were stained with PAS. No obvious pathology was observed in either skeletal muscle (A,B) or kidney (C,D; glomeruli at high power in E,F) from control or transgene-rescued mutant mice. Scale bar in B, 500 µm; in D, 200 µm; in F, 50 µm.

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