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First published online March 18, 2009
doi: 10.1242/10.1242/jcs.040378

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The extracellular-matrix protein matrilin 2 participates in peripheral nerve regeneration

¹ Centre for Molecular Neurobiology, University of Hamburg, Falkenried 94, D-20251 Hamburg, Germany
² Human Genetics Division, University of Southampton, Tremona Road, Southampton SO16 6YD, UK
³ Center for Biochemistry, Medical Faculty, University of Cologne, Joseph-Stelzmann-Str. 52, D-50931 Cologne, Germany
⁴ Max Planck Institute of Biochemistry, Department of Molecular Medicine, Am Klopferspitz 18, D-82152 Martinsried, Germany
⁵ Department of Otorhinolaryngology, Friedrich-Schiller-University Jena, Lessingstrasse 2, D-07740 Jena, Germany

View larger version (84K): [in this window] [in a new window]   Fig. 1. Different expression pattern of matrilin 2 in wild-type and ErbB3–/– embryos, and analysis of matrilin-2 expression levels in different cell types. (A) Semi-quantitative RT-PCR using cDNA prepared from DRG of wild-type and ErbB3-mutant embryos. GAPDH was used as an internal control. At comparable GAPDH levels, much higher levels of matrilin-2-specific amplicons were present in wild type compared with mutant extracts. (B) Analysis of matrilin-2 expression by semi-quantitative RT-PCR. Matrilin 2 is expressed in the rat SC line (S16) and primary embryonic SCs, whereas there is no expression of matrilin 2 in neural crest stem cells (Monc) (precursors of embryonic SCs). (C-F) In situ hybridization with 35S-labelled matrilin-2-specific riboprobe on transverse cryosections of mouse embryos (E12-18). Arrowheads indicate the expression of matrilin 2 within DRG of wild-type embryos, whereas the arrows indicate the expression in SCs aligning axonal projections. Note the absence of the expression along axonal projections in the ErbB3 mutant embryo at E12 (arrow in D). As almost all ErBb3-deficient embryos die at E12.5 and the PNS in surviving mutants is progressively degenerating, no older stages were analysed. Scale bars: 100 µm (in C for C,D); 125 µm (E); 175 µm (F).

View larger version (87K): [in this window] [in a new window]   Fig. 2. Comparison of migration and adhesion of S16 cells on different extracellular matrix substrates. (A) Effect of matrilin 2 on SC adhesion. SC suspensions were incubated for 30 minutes in wells of 24-well tissue culture dishes coated with different ECM substrates. After washing, the adherent cells were fixed and counted using phase-contrast microscopy. Results are expressed as a percentage of the number of cells adhering to laminin, which was normalized to 100%. Statistical analysis showed a significant increase (***P<0.001) of the numbers of cells adhering to different substrates versus non-coated (nc) dishes. Note that also the differences in adhesion between laminin and fibronectin or matrilin 2 are significant. (B,C) S16 SCs migration on different coating substrates determined by the agarose drop migration assay. (B) Mean distances of SCs migration on different ECM substrates, fibronectin, laminin and matrilin 2. All ECM molecules promoted SCs migration compared with the uncoated control substrate (***P<0.001). In addition, the distances of migration on matrilin-2 surfaces were significantly increased (***P<0.001) in comparison with laminin- or fibronectin-coated surfaces. **P<0.01. (C) Cell migration was analyzed using phase-contrast microscopy. Scale bar: 100 µm.

View larger version (61K): [in this window] [in a new window]   Fig. 3. Matrilin 2 promotes SC migration from embryonic DRG. (A) Cell migration from wild-type DRG on different ECM substrates. Cells allowed to migrate for 24 hours on different substrates were visualized by fluorescence microscopy after incubation with Sox10-specific antibodies. More cells migrate out of DRG on matrilin 2 and a mixture of matrilin 2 + laminin compared with just laminin. Scale bar: 250 µm. (B) Mean distances of SC migration on different substrates. Migration was quantified by measuring the distance from the edge of a ganglion to the leading edge of migrating SCs on photomicrographs. Significant differences are indicated (***P<0.001; *P<0.05). (C) Mean numbers of migrating cells expressed as fold difference compared with PDO. Significant differences are indicated. In addition, the numbers of migrating cells on a mixture of laminin and matrilin 2 are significantly elevated (***P<0.001) compared with migration on laminin or matrilin 2 alone. (D) Migration of ESCs from DRG prepared from matrilin-2-deficient and wild-type embryos. Cells were allowed to migrate on laminin and matrilin-2-coated surfaces with or without addition of matrilin 2 (2 µg/ml) to the medium for 72 hours. Mean numbers of migrating cells expressed as fold difference compared with wild-type on laminin. Significant differences are indicated (***P<0.001). Addition of matrilin 2 to the culture medium had no significant effect on wild-type cultures (data not shown). (E) DRG from wild-type (+/+) and matrilin-2-deficient (–/–) embryos grown on laminin for 72 hours without and with the addition of matrilin 2 to the culture medium (+mat). Note the increase in migrating SCs (Sox10-positive) in matrilin-2-deficient cultures on laminin after addition of matrilin 2 (+mat) compared with the mutant culture without matrilin 2. Scale bar: 250 µm.

View larger version (38K): [in this window] [in a new window]   Fig. 4. Matrilin 2 promotes axonal outgrowth of DRG neurons but does not affect branching. (A) Axonal outgrowth from wild-type DRG cultures on different ECM substrates. Axons were allowed to grow for 24 hours on different substrates and were then visualized with NF160-specific antibodies. Significant differences are indicated (***P<0.001; *P<0.05). (B) Axon outgrowth from DRG neurons prepared from wt (+/+) and matrilin-2-deficient (–/–) embryos. Axons were grown for 48 hours on laminin (lam.) or matrilin-2-coated surfaces (mat.) with (+matrilin 2) or without the addition of matrilin 2 to the culture medium. After visualization using NF200-specific antibodies, the lengths of axons were measured. Significant differences are indicated (***P<0.001; *P<0.05). Note that axonal outgrowth on matrilin-2-coated surfaces showed no statistical differences and that addition of matrilin 2 to the medium on laminin could not fully rescue the mutants. (C-E) Axonal branching of wild-type DRG neurons on different ECM substrates. Dissociated DRG neurons were prepared from wild-type pups (P0-P3). Morphology of neurites of dissociated sensory neurons grown for 12 hours on laminin (C), matrilin 2 (D) and mixture of laminin and matrilin 2 (E) visualized by β-tubulin (Tuj1) immunostaining. Scale bar: 25 µm. (F) Quantitative analysis of the mean number of branching points per main neurites demonstrated similar ratios of branching points. Note, however, that there is a slight difference in branching points between laminin and a mixture of laminin and matrilin 2 (*P<0.05).

View larger version (155K): [in this window] [in a new window]   Fig. 5. Growth patterns of DRG neuron axons on different substrates analysed by a stripe assay. DRG explants were grown for 24 hours on 22 mm glasses that had been coated in stripes with laminin (L), poly-l-lysine (P), matrilin 2 (M), or laminin and matrilin 2 (L/M), fixed and immunostained using NF antibodies. Within one experiment, DRG axons were grown on slides coated with alternating stripes of laminin and poly-l-lysine (PLL) (A), matrilin 2 and PLL (B), matrilin 2 and laminin (C) or laminin and a mixture of laminin and matrilin 2 (D).

View larger version (69K): [in this window] [in a new window]   Fig. 6. Upregulation of matrilin 2 and 4 in nerves after lesion. Matrilin-2 expression analysed by immunofluorescence staining of transverse sections from damaged (A,C) and intact (B,D) femoral (A,B) and sciatic (C,D) nerves dissected from adult wild-type mice 3 or 5 days after transection of the femoral or sciatic nerve, respectively. Matrilin-4 expression analysed by immunofluorescence staining of transverse sections from damaged (E) and intact (F) sciatic nerve 7 days after transection. (A-F) Note the strong upregulation upon injury (n=4 mice per experiment). Scale bars: 100 µm. (G) Upregulation of matrilin 2 in damaged sciatic nerves 7 days after nerve injury revealed by western blotting. Samples from intact nerves, as well as proximal and distal stumps of injured nerves, were analysed using antibodies against matrilin 2 and β-actin. (H) RT-PCR analysis was performed on lesioned femoral nerves from wild-type (+/+) and matrilin-2-deficient adult (–/–) animals 5 days after nerve lesion. Note the higher expression of matrilin 4 in samples from lesioned nerve from mutant animals compared with wild-type animals. Expression of Ddost served as an internal control.

View larger version (78K): [in this window] [in a new window]   Fig. 7. Immunohistochemical analysis of matrilin-2 expression following nerve injury. Transverse sections from the proximal (A,C,E,G) and distal (B,D,F,H) nerve stumps 7 days after nerve transection in adult mice. (A,B) Co-immunostaining using anti-NF antibody to visualize axons (green) and anti-matrilin-2 (red) antibodies. Note the mutually exclusive pattern revealing the absence of matrilin-2 expression in axons. (C,D) Matrilin 2 (green) is also not detectable in myelin sheaths visualized with an antibody against myelin basic protein (MBP, red). (E-H) Matrilin 2 (red) colocalizes with the SC marker p75 (green) (E,F) and matrilin 2 (green) is also detectable in basement membranes, as indicated by its colocalization with nidogen 1 (red) (G,H). In A-F, cell nuclei were visualized with DAPI (blue). Shown are representative staining examples performed on sections from four independent wild-type mice. Scale bars: 20 µm in A,B,E-H; 10 µm in C,D.

View larger version (16K): [in this window] [in a new window]   Fig. 8. Time course and degree of motor recovery after femoral nerve lesion in matrilin-2-deficient (matrilin 2–/–) and wild-type (matrilin 2+/+) mice. Shown are heel-tail angles (A), foot-base angles (B) and stance recovery indices (C) at different time-points after nerve transection and repair. Asterisks indicate significant differences between matrilin-2-deficient and wild-type mice (n=7 per group) at the respective time point (*P<0.05).

View larger version (19K): [in this window] [in a new window]   Fig. 9. Morphological evaluation of muscle reinnervation and speed of regeneration and Schwann cell proliferation. (A) Numbers of motoneurons innervating the motor or sensory branches only, or both branches, as well as the total number of regenerated motoneurons 3 months after nerve repair. (B) Relative degrees of myelination estimated by g-ratios (axon/fibre diameter ratios) and diameters of regenerated axons (C) evaluated in semi-thin sections from regenerated nerves cut 2 mm distal from the site of transaction 3 months after nerve repair. (D) Analysis of axonal regrowth 5 days after nerve lesion. Numbers of regenerated axons, identified by β-tubulin immunofluorescence, present in transverse sections of femoral nerves collected at distances of 3, 4 and 5 mm distal to lesion site. Asterisks indicate significant differences between matrilin-2-deficient and wild-type mice (**P<0.01) in numbers of Tuj1-positive axons 5 mm distal from the site of transection. (E) Numbers of proliferating SCs in the distal stumps 3 days (3 dpl), 5 days (5 dpl) and 7 days (7 dpl) after lesion in wild-type (+/+) and matrilin-2-deficient (–/–) adult mice. Animals were sacrificed following a 4-hour pulse of BrdU applied by injection. Transverse sections of femoral nerves distal to the site of lesion were incubated with antibodies against p75 (SC marker) and BrdU. Only cells positive for both signals were counted. No statistically significant differences were observed between mutant and control at any time point. Similar results were obtained when antibodies against Ki-67 (proliferation marker) were used instead of BrdU.

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