Article Figures & Data
Figures
- Figure 1.
Analysis of the nestin-Cre mice. Nestin-Cre mice were crossed with Rosa26lacZflox/flox mice. The Cre recombination pattern was analyzed by LacZ staining (blue). a, Whole-mount staining at E10.5. LacZ was expressed throughout the entire neural tube (arrow). b, c, Sections of P3 animals. In transversal sections through spinal cord at forelimb (b) and hindlimb (c) levels LacZ expression was detected in the spinal cord. It was pronounced in motor neuron pools of the ventral horn (b, arrowhead). LacZ expression was also evident in dorsal root ganglia (asterisk). d, Higher magnification view of a transversal section through spinal cord at P3. Sections were stained with Nissl (violet) to label motor neuron nuclei and with LacZ (blue). Essentially all motor neurons (arrowheads) were lacZ-positive. Scale bars: a, 200 μm; b, c, 100 μm; d, 30 μm.
- Figure 2.
Expression and inactivation ofβ1 integrin expression in motor neurons. a, Transverse sections from E11.5 HB9-GFP embryos were analyzed for GFP fluorescence (green) and β1 expression (red). The GFP transgene is expressed specifically in the cell bodies and projections of spinal motor neurons. Prominent β1 expression was detected in motor neurons (thick arrows), in DRG neurons (thin arrows), and in skin (arrowheads). b, Transverse sections from E11.5 wild-type and nestin-Itgb1Ko animals were stained with antibodies to β-tubulin to visualize neurons and with antibodies to β1 integrin. The β1 expression was diminished drastically in motor neurons (thick arrows), DRG neurons (thin arrows), and peripheral nerve bundles (asterisks), but not in skin (arrowhead). c, Western blot analysis for the β1 protein. Extracts were prepared from the neural tube dissected from E12.5 wild-type and nestin-Itgb1Ko embryos. The β1 protein was readily detectable in wild-type mice, but not in mutant mice. Scale bars: a, left, 200 μm; a, right, 25 μm; b, 200 μm.
- Figure 3.
Neurite outgrowth assay in vitro. a, Phase-contrast and fluorescent images of spinal motor neuron explants prepared from E10.5 wild-type and nestin-Itgb1Ko embryos plated on poly-d-lysine/LN-coated coverslips. Wild-type explants formed numerous processes. In explants derived from nestin-Itgb1Ko embryos the neurite outgrowth was strongly impaired, and neurites formed fascicles (bottompanel). b, The growth of β1-deficient motor neurons was not impaired on cocultured fibroblasts. c, The mean number of neurites per explant on LN substrates was reduced significantly in nestin-Itgb1Ko as compared with wild-type embryos. d, The results of the means of neurite lengths per explant on LN substrates were calculated, demonstrating a strong reduction in neurite length between wild-type and nestin-Itgb1Ko explants; ***p = 0.0012. Error bars indicate SEM. The SEM was determined and a Student t test was performed. e, Cumulative frequency distribution plot of neurite length on LN substrates shows a shift for nestin-Itgb1Ko explants toward shorter neurite lengths. f, Cumulative frequency distribution plot of neurite length on fibroblast substrates shows no difference in neurite length between wild-type and nestin-Itgb1Ko explants. Scale bars: a, b, 120 μm.
- Figure 4.
Analysis of axonal projections in vivo. a, Side views of E12.5 wild-type and nestin-Itgb1Ko embryos stained in whole mount with an antibody to neurofilament to visualize axonal projections in limbs (arrowheads) and the intercostal areas (asterisks). Staining occasionally was observed within the body cavity of the eviscerated embryos, likely caused by nonspecific trapping of antibodies (arrows). b, Top view of one-half of a diaphragm from a E16.5 wild-type and nestin-Itgb1Ko embryo stained for neurofilament (green) to reveal motor neurons. The phrenic nerve and its side branches that terminated in the central endplate zone (arrowheads) were present in wild-type and nestin-Itgb1Ko embryos. c, Higher magnification view of the diaphragm shown in b. Neurofilament staining is shown in green and α-bungarotoxin staining in red. AChRs were clustered at synaptic sites in the medial part of the muscle. Scale bars: a, 400 μm; b, 100 μm; c, 50 μm.
- Figure 5.
Analysis of presynaptic and postsynaptic differentiation. Sections of gastrocnemius muscle from 4-week-old wild-type and nestin-Itgb1Ko mice were stained with TRITC-α-bungarotoxin (red) and antibodies against presynaptic and postsynaptic proteins (green). a, The postsynaptic markers rapsyn (RAP), utrophin (UTP),β-dystroglycan (β-DG), and integrin α7B (Itg-α7B) were colocalized with AChR clusters at synaptic sites in wild-type and nestin-Itgb1Ko mice. b, GFAP-positive terminal Schwann cells apposing AChR clusters were clearly visible in longitudinal sections. c, Synaptophysin (SYN) was enriched in areas that were in register with AChR clusters in the postsynaptic membrane (arrowheads). Scale bars, 5 μm.
- Figure 6.
Defective innervation of β1-deficient muscle. a-f, Anti-neurofilament staining of the phrenic nerve innervating diaphragm muscle in wild-type and HSA-Itgb1Ko embryos. a, b, At E15.5 the motor axons extended beyond the medial endplate zone in the mutants(arrowheads). c,d, Severe nerve defasciculation and overgrowth were evident in mutant muscle at E17.5. e,f, The defects were pronounced even more at P0. In the mutants the axons occasionally failed to respect muscle boundaries and grew onto the tendon organ. Note that some axons had abnormal nerve terminals with a round morphology (d, f, arrowheads). Scale bars: a, b, 200 μm; c-f, 300 μm.
- Figure 7.
Defects in AChR clustering and presynaptic differentiation. Diaphragm muscles from wild-type and HSA-Itgb1Ko embryos were stained with antibodies to neurofilament (green) and with TRITC-α-bungarotoxin (red) to reveal motor neurons and AChRs, respectively. a, At E14.5 the clustering of AChRs was observed in a broad endplate band in muscle of wild-type and HSA-Itgb1Ko embryos. Small aneural AChR clusters were present (arrowheads) in muscle of wild-type and mutant animals, whereas bigger clusters that were opposed by nerve were visible only in wild-type animals (asterisks). b, The size distribution of AChR clusters was compared in diaphragm muscle from wild-type and HSA-Itgb1Ko embryos. (Figure legend continues.) (Figure legend continued.) Wild-type mice had a higher percentage of clusters >12 μm and fewer clusters <2 μm. Error bars indicate SEM. c, By E15.5 the AChR clusters in muscle in wild-type embryos had grown in size and were concentrated in the endplate zone, opposing flat terminal nerve boutons. In contrast, muscle of HSA-Itgb1Ko embryos displayed only a few and small AChR clusters, which were opposed by nerve terminals with a ball-like shape (arrowheads). d, At E17.5 some dispersed AChR clusters were present in muscle of mutant animals (arrowheads). e, At P0 all AChR clusters in muscle of wild-type embryos were opposed by nerve. Only a few small and weakly stained AChR clusters remained in the muscle of mutant animals. Motor axons grew extensively in P0 mutants. Scale bars: a, 100 μm; c, left, 100 μm; c, right, 5 μm; d, e, 100 μm.
- Figure 8.
The β1-deficient primary myotubes are responsive to recombinant agrin. Myotubes were generated in vitro with cells from wild-type (a, c, e, g) or HSA-Itgb1Ko embryos (b, d, f, h). Some myotubes (c-h) were treated for 24 hr with recombinant neural agrin. Cultures were stained with TRITC-α-bungarotoxin (a-d, red) or antibodies to utrophin (e-h, green) and DAPI (blue). Wild-type and β1-deficient myotubes formed few AChR clusters in the absence of recombinant agrin (a, b, arrow). Wild-type and β1-deficient myotubes were similarly responsive to recombinant agrin (c, d, arrows), and utrophin was concentrated at AChR clusters (e-h, arrows). Scale bar, 20 μm.
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