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First published online 11 November 2008
doi: 10.1242/jcs.031047

Journal of Cell Science 121, 3901-3911 (2008)
Published by The Company of Biologists 2008
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Identity, developmental restriction and reactivity of extralaminar cells capping mammalian neuromuscular junctions

Felipe A. Court1,2,*, Thomas H. Gillingwater1,2, Shona Melrose2, Diane L. Sherman2, Kay N. Greenshields3, A. Jennifer Morton4, John B. Harris5, Hugh J. Willison3 and Richard R. Ribchester1,2,{ddagger}

1 Euan MacDonald Centre for Motor Neurone Disease Research, The University of Edinburgh, 1 George Square, Edinburgh EH8 9JZ, UK
2 Centre for Neuroscience Research, The University of Edinburgh, Summerhall, Edinburgh EH9 1QH, UK
3 Division of Clinical Neuroscience, Glasgow Biomedical Research Centre, University of Glasgow, 120 University Place, Glasgow G12 8TA, UK
4 Department of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge CB2 1PD, UK
5 Institute of Neuroscience Faculty of Medical Sciences, University of Newcastle upon Tyne, Framlington Place, Newcastle upon Tyne NE2 4HH, UK


Figure 1
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  		Fig. 1. Immunostaining of mouse triangularis sterni muscles with 2166 antibody reveals capping cells at NMJs. (A) Low-power confocal projection showing numerous 2166-positive interstitial cells and a prominent subpopulation in the vicinity of NMJs. (B) High-power confocal projection images of NMJ-capping cells (green), juxtaposed with motor endplates counterstained with TRITC-{alpha}-BTX (red). (C) Triple-stained NMJ with axon stained with neurofilament antibodies (red), NMJ-capping cell (green) and motor endplate AChR stained with Alexa Fluor 647-{alpha}-BTX (blue). (D,E) Other interstitial and endothelial cells with positive 2166 immunostaining were scattered at low density throughout the muscles. (F) Quantification of density of NMJ-capping cells at motor endplates. All endplates showed at least one and most were associated with only one NMJ-capping cell; no NMJ was associated with more than three of these cells. Scale bars: 100 µm (A); 20 µm (E, applies also to B-D).

 

Figure 2
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  		Fig. 2. NMJ-capping cells are not Schwann cells but express collagen-synthesising enzyme and neuregulin. (A) Confocal projection images of a TS muscle endplate in an S100-eGFP transgenic mouse, showing endogenous fluorescence in terminal Schwann cells (green) overlying endplate ACh receptors (blue). The right panel shows superimposition of 2166 immunostaining (red). (B) Immunostaining for the collagen-synthesising enzyme rPH of an NMJ-capping cell in rat TS muscle. (C) Immunostaining of a NMJ-capping cell in mouse triangularis sterni muscle using HM-24 antibody, which recognises GGFII/neuregulin. Scale bar: 50 µm.

 

Figure 3
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  		Fig. 3. NMJ-capping cells are positive for CD34 antibody and CTB and reside outside the synaptic basal lamina. (A,B) Confocal projections of an NMJ-capping cell in a mouse triangularis sterni muscle immunostained with 2166 (green) and CD34 (red) antibodies and with endplate AChR counterstained (blue). Lower panels in A show green and red channels separately; Panels 1 and 2 in B show orthogonal projections at the optical cuts indicated in the uppermost panel. (C) Transverse section of a mouse TS muscle immunostained with 2166 antibody (green spots), laminin antibody (red) and endplate ACh receptor (blue). The immunostained processes of the NMJ-capping cells clearly lie outside the basal lamina. (D) Fluorescent conjugates of CTB also stain cells similar in form and location to NMJ-capping cells. Right panel shows the red channel indicating endplate ACh receptors only. Scale bars: 50 µm.

 

Figure 4
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  		Fig. 4. NMJ-capping cells almost completely cover motor endplates. Electron micrographs and 3D surface-rendered reconstructions of serially sectioned electron micrographs showing the relationship between two putative NMJ-capping cells, motor nerve terminal, terminal Schwann cell and skeletal muscle fibre at a neuromuscular junction from mouse TS muscle. (A,B) Sections 84 and 75, respectively, from a 300-section series through a single NMJ. The soma and thin cytoplasmic processes of both NMJ-capping cells at this NMJ can be seen above the terminal Schwann cell and nerve terminal, lying outside the basal lamina. Each of the main cell types has been outlined on these electron micrographs (motor nerve terminal, green; muscle fibre, brown, terminal Schwann cell, yellow; 2166-positive cells, magenta and lilac). (C) Surface rendering of reconstructed serial electron micrographs from the NMJ shown in A and B revealed two NMJ-capping cells (magenta and lilac) overlying a single terminal Schwann cell (yellow) and a motor nerve terminal (green) synapsing with a skeletal muscle fibre (brown). a, Superior view of the nerve terminal and skeletal muscle fibre; b, nerve terminal only; c, nerve terminal and terminal Schwann cell; d, nerve terminal, Schwann cell and two NMJ-capping cells; e, nerve terminal and two NMJ-capping cells; f, two NMJ-capping cells; g/h, the two NMJ-capping cells shown in isolation; i, orthogonal view of the reconstructed NMJ-capping cells. Animations of the volume rendering of these two cells are presented in supplementary material Movies 6 and 7. The NMJ-capping cells cover approximately 60% of the nerve terminal at this NMJ. In contrast to the terminal Schwann cell, the veil-like processes of both NMJ-capping cells extend beyond the immediate boundaries of the endplate. (D) Transmission electron micrograph through a neuromuscular junction in the rat diaphragm. The motor nerve terminal and terminal Schwann cell are covered by a slender process from a putative NMJ-capping cell (arrows). A high resolution image is reproduced in supplementary material Fig. S3. Scale bar: 2.5 µm.

 

Figure 5
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  		Fig. 5. NMJ-capping cells become restricted to the endplate zone postnatally but spread following denervation in adults. (A-C) 2166 immunostaining (green) is diffuse at birth but becomes progressively concentrated near NMJs over the following 1-2 weeks. (D-F) Denervation in adults triggers nestin reactivity and sprouting in terminal Schwann cells within 3 days but no discernible sprouting or nestin immunoreactivity is seen 1 day after axotomy. By contrast, extensive spreading of 2166 immunostaining occurs within 1 day of axotomy (see also supplementary material Fig. S4). At 3 days, some of the Schwann cell sprouts were associated with 2166-positive links between motor endplates (arrowhead) (see also supplementary material Fig. S5). (G) Immunostaining following injection of a BrdU pulse shows BrdU positive staining (red) of nuclei (blue) in both junctional and non-junctional 2166-positive cells (green), suggesting that NMJ-capping cells both divide and spread from endplates following axotomy. Arrowheads indicate BrdU-positive nuclei. Dotted lines indicate the location of three motor endplates, visualized by Alexa-Fluor-647-conjugated {alpha}-bungarotoxin (not shown). Scale bar: 100 µm (A-C); 50 µm (D-G).

 

Figure 6
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  		Fig. 6. NMJ-capping cells also spread in paralysed and atrophic muscles. (A,B) Profusion of 2166 immunoreactivity (green) is seen within 1 day of injection of a locally paralysing dose of botulinum toxin type-A (Botox) but terminal Schwann-cell reactivity and sprouting, assessed by nestin immunostaining (red), is not observed until after least 6 days of paralysis in TS muscles. (C,D) Similar profusion and spread of NMJ-capping cells is observed in constitutively atrophic muscles of 15-week-old R6/2 transgenic mice. Littermate controls show normal, endplate-localised staining and distribution of NMJ-capping cells. Scale bar: 50 µm.

 

Figure 7
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  		Fig. 7. Reactive NMJ-capping cells are associated with tenascin-C expression but do not require tenascin-C in order to localise, react or spread from NMJs. (A-C) Tenascin-C immunoreactivity (red) spread from the endplate region from 3-6 days after axotomy (left panels) and these tenascin-C-rich regions coincide with the distribution of NMJ-capping cells (green, right panels; AChR counterstained blue). (D,E) Selective X-Gal staining of intramuscular nerve and motor endplates in heterozygous tenascin-C-null mutant mice with lacZ substitution, 5 days after axotomy. The area shown in E corresponds to the area bounded by the boxed outline in D. Unoperated muscles were X-Gal negative (not shown). (F,G) NMJ-capping cells are also localised to NMJ in homozygous tenascin-C-null mutant mice and these cells spread from endplates 3 days after axotomy, as in wild-type mice. Scale bar in C, right: 50 µm (A-C), 100 µm (D), 30 µm (E). Scale bars in F and G: 50 µm.

 

Figure 8
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  		Fig. 8. NMJ-capping cells are integral, plastic cellular components of the NMJ. (A) Relationship between four main cell types proposed to constitute the mammalian NMJ: muscle fibre, motor nerve terminal, terminal (perisynaptic) Schwann cell and NMJ-capping cells, named here `kranocytes' (see Discussion). (B) Stages in kranocyte reactivity to denervation, paralysis or muscle atrophy that could indicate a permissive function for these cells in terminal Schwann cell and axonal sprouting. Kranocyte sprouting is the first reaction, followed by terminal Schwann cell sprouting, then axonal sprouting.

 

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© The Company of Biologists Ltd 2008