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. Author manuscript; available in PMC: 2009 Jun 24.
Published in final edited form as: J Neurosci. 2008 Jun 25;28(26):6714–6719. doi: 10.1523/JNEUROSCI.0326-08.2008

α6β4 integrin and dystroglycan cooperate to stabilize the myelin sheath

A Nodari 1, SC Previtali 2, G Dati 1,*, S Occhi 1, FA Court 1,**, C Colombelli 1, D Zambroni 1, G Dina 2, U Del Carro 2, K P Campbell 3, A Quattrini 2, L Wrabetz 1, ML Feltri 1
PMCID: PMC2597422  NIHMSID: NIHMS65497  PMID: 18579745

Abstract

Schwann cells integrate signals deriving from the axon and the basal lamina to myelinate peripheral nerves. Integrin α6β4 is a laminin receptor synthesized by Schwann cells and displayed apposed to the basal lamina. α6β4 integrin expression in Schwann cells is induced by axons at the onset of myelination, and rise in adulthood. The β4 chain has a uniquely long cytoplasmic domain that interacts with intermediate filaments such as dystonin, important in peripheral myelination. Furthermore, α6β4 integrin binds peripheral myelin protein 22, whose alteration causes the most common demyelinating hereditary neuropathy. All these data suggest a role for α6β4 integrin in peripheral nerve myelination. Here we show that ablating α6β4 integrin specifically in Schwann cells of transgenic mice does not affect peripheral nerve development, myelin formation, maturation or regeneration. However, consistent with maximal expression in adult nerves, α6β4 integrin-null myelin is more prone to abnormal folding with aging. When the laminin receptor dystroglycan is also ablated, major folding abnormalities occur, associated with acute demyelination in some peripheral nervous system districts. These data indicate that, similar to its role in skin, α6β4 integrin confers stability to myelin in peripheral nerves.

Keywords: α6β4 integrin, dystroglycan, myelin, Schwann cells, targeted mutagenesis, peripheral nervous system

INTRODUCTION

Schwann cells interact with peripheral axons to form myelinated or non-myelinated fibers. Spiraling and compaction of the glial membrane in myelin assures fast conduction of nerve impulses and involves a network of signals between axons, extracellular matrix and Schwann cells (reviewed in (Feltri and Wrabetz, 2005).

α6β4 integrin is a laminin receptor expressed by epithelia (Kajiji et al., 1989), endothelia (Kennel et al., 1992; Klein et al., 1993), thymocytes (Wadsworth et al., 1992) and Schwann cells (Einheber et al., 1993) and a component of hemidesmosomes (reviewed in (Quaranta and Jones, 1991). Multiple observations suggest that β4 integrin may be important in myelination. First, β4 integrin expression in Schwann cells is induced by axons during myelination (Einheber et al., 1993; Feltri et al., 1994). In contrast to α6β1 integrin that decreases during myelination (Einheber et al., 1993), α6β4 appears during myelin synthesis and its expression continues to increase in adulthood (Previtali et al., 2003b; Verheijen et al., 2003). Thus, Schwann cells may switch their laminin-binding integrins from α6β1 to α6β4 to promote or maintain myelination (Einheber et al., 1993). Second, β4 integrin has a long cytoplasmic domain, not homologous with other 3 integrins, that mediates signals through recruitment of Shc, and PI 3-kinase, affects MAPK and NF-kB nuclear translocation (reviewed in (Giancotti and Tarone, 2003) and interacts with intermediate filaments such as dystonin (Litjens et al., 2003), which has a Schwann cell autonomous role in myelination (Bernier et al., 1998). Fourth, α6β4 integrin in epithelia amplifies neuregulin signaling (Guo et al., 2006), which is crucial at multiple stages of Schwann cell development (reviewed in (Nave and Salzer, 2006). Finally α6β4 integrin interacts with PMP-22, a myelin protein mutated in Charcot-Marie-Tooth 1A neuropathy (Amici et al., 2006). Against a role for α6β4 integrin, myelination starts normally in newborn nerves and dorsal root ganglia explants from mice dying at post-natal day 1 (P1) due to β4 inactivation (Frei et al., 1999). However, β4 expression in Schwann cells is just beginning in P1 mice and follows, not precedes, the onset of myelination (Previtali et al., 2003b). Thus a role for β4 in myelin maturation or maintenance could not be addressed from these studies.

Therefore, we generated mice lacking β4 integrin in Schwann cells. Surprisingly, we find that absence of β4 integrin does not impact development, maturation or regeneration of myelinated fibers. β4 integrin contributes instead to stabilize myelin, as its absence causes an increase of age-related formation of myelin infoldings. Another laminin receptor, dystroglycan, is co-expressed with α6β4 integrin and its deletion causes abnormal folding of myelin sheaths (Saito et al., 2003). Deletion of both dystroglycan and β4 integrin aggravates the folding phenotype of dystroglycan mutants in ventral roots, causing a dramatic age-dependent de-myelination. Thus, the two receptors cooperate to stabilize myelin.

METHODS

For generation of β4 integrin “floxed” allele, antibodies used, PCR and rotarod see supplementary methods.

Generation of β4 integrin conditional null mice

P0Cre (mP0TOTACre) and DG floxed mice have been described (Feltri et al., 1999; Feltri et al., 2002; Moore et al., 2002). P0Cre, β4F/+ and DGF/+ lines were maintained by backcrosses with C57BL/6 mice. P0Cre mice used in the experiments were congenic in C57BL/6. Progeny used in this study was N3-N7 generations congenic in C57BL/6. In the experiments, littermates or mice deriving from the same parents were compared.

Immunohistochemistry on frozen nerves were conducted as described (Feltri et al., 2002). Immunoteasing was conducted as described (Occhi et al., 2005), except for S100/β1 integrin staining where acetone was omitted, fibers were permeabilized in 0.2% triton X-100 and stained immediately after dissection.

Western blotting was conducted under reducing conditions as described (Previtali et al., 2003b).

Morphology on semithin and ultrathin sections of nerves were conducted as described (Wrabetz et al., 2000).

Electrophysiological analysis was conducted as described (Occhi et al., 2005).

Crush injury was conducted as described (Quattrini et al., 1996).

BrdU incorporation and TUNEL assays were performed as described (Feltri et al., 2002).

Image acquisition

Images were acquired using confocal (Perkin Elmer UltraView ERS and Leica TCS-SP5) or fluorescence microscopes (Leica DM 5000 B). Image analysis was performed using Adobe Photoshop CS.

Statistical analysis

Statistical analysis was performed using Excel (Office X for Mac), Stat View (v5.0 for Mac) and SPSS v11 for Mac.

Results

Generation of a β4F -floxed allele and null mice

β4 integrin conditional or constitutive null mice were generated using the Cre/LoxP system. Constitutive null mice reproduced the skin blistering phenotype previously reported (Suppl. Fig. 1)(Dowling et al., 1996; van der Neut et al., 1996).

Schwann cell specific ablation in β4F/F//P0Cre mice does not impair myelinogenesis

Schwann cell specific ablation was achieved by mating β4F/F mice with mice that express Cre recombinase in Schwann cells (Feltri et al., 1999). In mutant sciatic nerves β4 protein was absent in Schwann, but present in perineurial cells (fig. 1A–E). Mutant mice did not show tremor, gait abnormalities, or atrophy (not shown). Rotarod performance and neurophysiology were normal (suppl. fig. 2), as well as morphology of sciatic nerve and spinal roots during development and up to 7 months of age (suppl. Fig. 3).

Fig. 1. Schwann cell specific inactivation of β4 integrin causes increased frequency of age-related myelin infoldings.

Fig. 1

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A) Western blots from sciatic nerve lysates. β4F/FP0Cre-P is a lysate deprived of the perineurium. B–E) Transversal section of sciatic nerves from β4F/+P0Cre (B, C) and β4F/FP0Cre (D, E) mice stained with anti-β4 integrin (B, D) and anti-neurofilament antibodies (in red C, E) show absence of β4 integrin protein in β4F/FP0Cre Schwann, but not perineurial cells. Bars = 50 μm.

F, G) Transverse sections of 18 month old sciatic (SN) and 12 month old digital nerves in the toes from wt (H) and β4 integrin mutants (I). J)Quantification of the number of infoldings (arrow in A) in 12 month old sciatic nerves as a percentage of the total number of myelinated fibers (wt = 1.02±0.128; ko = 3.40±0.454; p < 0.01 by paired t-test analysis; total 5114 fibers for wt and 3445 fibers for mutant, n= 3 animals per genotype. K) Serial transverse sections of a 12 month old β4 integrin null sciatic nerve demonstrate that folding originates near the nodes of Ranvier (NoR) and follows the fiber for 36–40 μm. Occasionally we observed myelin infoldings developing from the internode.

L) “chains” of infoldings near the node of Ranvier in mutant nerves seen by longitudinal sections. M) Electronmicroscopic analysis shows that the infoldings do not contain axonal structures, but rather amorphous substance. Bar in A = 25 μm; in D= 25 μm, in E 5 μm (left panel) and 1.6 μm (right panel).

Mutant Schwann cells display normal proliferation and survival and form normal axonal domains

Although in other systems β4 integrin controls cell cycle progression and amplifies neuregulin signaling (Guo et al., 2006), no significant differences between β4 integrin null and control nerves were detected by BrdU incorporation and TUNEL assay (suppl. Fig. 4).

Mutant fibers showed proper localization of voltage –gated sodium channels (Nav), phosphorylated ezrin-radixin-moesin (ERM), Caspr, neurofascin 155/186 and potassium channels (KV1.1) to appropriate nodal-paranodal and juxtaparanodal regions. Finally, the absence of β4 integrin did not alter the formation of Schmidt-Lantermann incisures (suppl. fig. 4).

Absence of β4 integrin does not influence nerve regeneration

To test if α6β4 integrin is involved in nerve repair after damage (Einheber et al., 1993; Niessen et al., 1994; Quattrini et al., 1996) we studied regeneration after sciatic nerve crush. Axonal regeneration and remyelination were not delayed at 15 or 21 days (suppl fig. 6) and 2 months (not shown) after crush. Finally neuromuscular junctions in soleus and sternocleidomastoid muscles of mutant mice at ten months of age were normal (data not shown).

β4 integrin conditional null mice have a late-onset increase in myelin folding

At 12 months of age mutant sciatic, digital nerves and anterior roots presented a progressive increase in the number of myelin in-foldings (fig 1). Myelin in-foldings may indicate myelin instability, as they occur normally with aging (Knox et al., 1989). Folding usually developed as myelin invaginations 2 –4 μm away from the node of Ranvier (fig. 1K) and appeared as rings of myelin inside the fiber that often formed chains (fig. 1L). Infoldings appeared to contain extracellular material (Fig 1M), suggesting that they are sites of myelin sheath/basal lamina invagination due to microtrauma. The absence of α6β4 likely impairs firm attachment of the Schwann cell to the basal lamina, which accelerates the age-related formation of abnormally folded myelin sheaths.

β4 integrin and dystroglycan have both redundant and non-redundant functions

Both α6β4 integrin and dystroglycan are laminin receptors whose expression is regulated similarly in development (Previtali et al., 2003b). Mice lacking Schwann cell dystroglycan present abnormalities of the nodes of Ranvier and late-onset myelin foldings (Saito et al., 2003; Occhi et al., 2005). To evaluate redundancy between the two receptors, we generated β4 integrin/DG double conditional null mice. Rotarod scores of six (not shown) and twelve month old β4 integrin/DG double null mice (before symptoms develop in single dystroglycan mice) were normal (suppl. fig. 5). Dystroglycan mice have a neuropathy with reduced nerve conduction velocity, increased F-wave latency and reduced motor action potential amplitude (Saito et al., 2003). Even if differences were not statistically significant, double mutant mice showed a further reduction of nerve conduction velocity and of the amplitude of the motor action potential, and an increase in F-wave latency (fig. 3).

Fig. 3. Excessive myelin folding in sciatic nerves, with myelin breakdown and degeneration in roots of double mutant mice.

Fig. 3

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A–M) Transversal semithin sections from 12 month old sciatic nerves and ventral roots. A–G) Both DG-null and double mutant nerves contain abnormal loops (e.g. in E), infoldings (e.g. in F) and outfoldings (e.g. in G). J–M) Ventral roots lacking dystroglycan show hypo-myelination (L). Hypo-myelination in double mutant mice is severe in ventral (M, vr and N), but not dorsal (dr) roots, with signs of acute de-myelination including macrophages (mφ) engulfing degenerated axons (O, arrowheads) and myelin breakdown products (mφ in O, P, Q), thin myelin sheaths (P) and de-myelinated axons (asterisks in P). Signs of re-myelination (onion bulbs, arrow in R) are present. Staining with anti-CD11b/CD18 (Mac-1) antibodies reveals the presence of macrophages infiltrating double mutant (I) but rarely wt (H) roots. T) The number of demyelinated axons was significantly higher in double than dystroglycan single mutant mice (p<0, 001 by Student’s t test, n= 4 DG- null and 6 double-null mice). Macrophage infiltration was higher in double mutants than in DG-null mice (p<0, 001 by Student’s t test, n = 6 DG-null mice, 6 double mutant mice). S) Neurophysiology in twelve month old β4 integrin/DG null mice and controls confirms that DG null mice have a neuropathy with reduced nerve conduction velocity, increased latencies of motor action potential (MAP) and of F-wave. These parameters worsen in double mutant mice, although the differences do not reach statistical significance. The reduction in MAP amplitude became statistically different in double mutants as compared to WT mice (p= 0,044 by paired t-test); n= 6 double null, 2 β4 integrin null, 4 DG null, 5 wt. Values and SEMs are indicated for each genotype. Bar= 30 μm in A–D, 50 μm in H–K, 25 μm in M–P.

Dystroglycan mutants showed defective clusterization of sodium channels at nodes of Ranvier (Saito et al., 2003; Occhi et al., 2005), which was not aggravated by the lack of β4 integrin (suppl. fig. 5). The distribution of Schmidt-Lantermann incisures, Caspr at paranodes and K+ channels at juxtaparanodes was normal in double null mice (suppl. fig. 5).

Absence of β4 integrin causes dislocation of potassium channels in the internode

Single and double β4 integrin/DG null myelinated internodes contained “deposits” of K+ channels, apparently associated with the inner mesaxon (fig. 2A). In β4 integrin/DG null myelinated fibers, the inner mesaxon itself, as evidenced by K+ channel staining, seemed disorganized (fig. 2A). This abnormality was specific to the absence of β4 integrin as it was not present in nerves lacking only dystroglycan.

Fig. 2. Abnormal K+ channel deposits in the internode and up-regulation of α7β1 integrin in mutant mice.

Fig. 2

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A) K+ channel staining reveals that the inner and outer mesaxon in β4 and β4/DG null internodes contain abnormal deposits (arrows) and loops (arrowhead). B) Quantification of deposits per internode demonstrates that they are specific to the absence of β4 integrin (WT 0.129±0.043, DG null 0.240±0.048, β4 integrin null 0.714±0.167 and β4 integrin/DG null 0.719±0.068, β4 integrin null vs wt p < 0.05 and β4 integrin/DG null vs DG null p < 0.001 by paired t-test). C) Drp2 staining on teased fibers from the indicated genotypes. D) Western blot from sciatic nerve lysates showing Drp2 expression. E. Staining with anti-β1 integrin antibodies show that β1 integrin is selectively localized in Cajal bands in wt fibers, but not in single and double mutant mice. F. β1 integrin is up-regulated in dystroglycan and double mutant mice by Western blot analysis (bars in graph indicate standard error of the mean), in parallel with the α7 subunit. In reducing condition, the α7 121 kD and the 35 kD proteolytic fragment are detected in nerves (Song et al., 1992). α6 integrin is not up-regulated in mutants, and almost absent in β4 mutants. Bars: 40μm in A and 35μm in C.

Drp2 clusters can form in the absence of both β4 integrin and dystroglycan

Drp2, a member of the dystrophin-glycoprotein complex (DGC), interacts with periaxin in Schwann cells and localizes in clusters along the myelin fiber (Sherman et al., 2001). In the absence of L-periaxin, Drp2 clusters are absent, whereas in dystroglycan-null nerves, Drp2 expression is reduced, but clusters can form and L-periaxin is normally expressed (Saito et al., 2003; Court et al., 2004). To test if α6β4 integrin contributes to Drp2 localization, we stained teased fibers from mutant mice. In β4 mutants, Drp2 was normally localized in the expected clustered fashion. In nerves lacking dystroglycan, or both dystroglycan and α6β4 integrin, Drp2 clusters were similarly small and disorganized (fig. 2C). By western blot analysis, the expression of Drp2 was reduced to similar extent in dystroglycan and double null mice (fig. 2D). L-periaxin expression was unchanged in all the mutants (data not shown). Thus absence of β4 integrin does not impair the formation of Drp2 clusters, even in the absence of dystroglycan.

Expression of α7β1 integrin in mutant mice

α6β1 and α7β1 integrins are other laminin receptors expressed by Schwann cells (Previtali et al., 2003a; Previtali et al., 2003b). β1 integrin protein was up-regulated by Western blot in sciatic nerves of mutants, especially double 34/dystroglycan null (fig. 2F). α7 integrin was up-regulated when dystroglycan was absent, whereas α6 integrin was not regulated in any mutant (fig. 2F). Instead, α6 integrin was almost absent from β4 mutant nerves, suggesting that normally the α6 subunit pairs with β4 and not β1 in adult nerves. In addition, the localization of β1 integrin, normally restricted to Cajal-bands found around DRP2 clusters, was diffuse in the outer Schwann cell surface in all mutants, becoming distributed in a manner now similar to β4 and dystroglycan (fig 2E and Court and Feltri, manuscript in preparation). Thus, α7β1 integrins can potentially compensate for the absence of α6β4 integrin and dystroglycan.

Severe folding abnormalities and demyelination in ventral roots from β4/DG double null mice

Morphologically, sciatic nerves from double β4 integrin/dystroglycan mutants presented abnormally folded myelin, with abnormal loops (E), in-folding (F) and out-folding (G) similar to those seen in single dystroglycan mice (figure 3), that increased in number with age (not shown).

Dorsal roots were not distinguishable between dystroglycan and double conditional null mice (not shown). Ventral roots from dystroglycan null mice presented hypomyelination, not previously reported (fig. 3L). This hypomyelination was more severe and widespread in double mutants (fig. 3M, vr), potentially explaining the increased F-wave latency. Hypomyelination became evident with age, and at 12 months included signs of acute de-myelination, with myelin degeneration, macrophage infiltration (fig. 3N–R) and re-myelination (onion bulbs fig. 3R). Macrophages were significantly more numerous in double than in dystroglycan single mutant (fig. 3I, T p< 0,001). Electron microscopy showed activated “foamy” macrophages containing myelin or axon debris, inside myelin sheaths and surrounding axons (fig 4 B–E), hypomyelination and un-compaction of myelin lamellae (fig 4H–K). Marcrophages often appeared in association with loose and redundant basal lamina (fig 4I), or were stripping the basal lamina away from myelinated fibers (fig 4 F, G). Longitudinal sections indicated that macrophages entered at paranodes (fig 4 M, N). These data suggest that lack of α6β4 integrin and dystroglycan causes abnormal myelin folding that coupled with poor attachment to the basal lamina eventually triggers acute de-myelination in roots. Thus dystroglycan and β4 integrin cooperate to maintain myelin integrity.

Fig. 4. Demyelination in ventral roots of double mutant mice.

Fig. 4

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Semitihin (A) and electromicroscopical (B–N) analyses of transverse (A–L) or longitudinal (M, N) sections through ventral roots from double mutants. A and B show the same macrophage (mφ) inside a myelinated axon. C shows a foamy macrophage between the axon (A) and myelin sheath (my). D, E, I and J show macrophages containing myelin debris. I) macrophage (mφ) found within the loose basal lamina (asterisks) of the Schwann cell. In F (and enlarged inset in G) a macrophage strips the basal lamina away (arrowheads) from the Schwann cell. H) uncompaction of myelin lamellae (arrows). K) de-myelinated fibers, L) re-myelinating figure. M (inset enlarged in N) shows a macrophage (mφ) penetrating through the paranode (pn). Bar= 30 μm in A; 1 μm in G and N; 1,5 μm in H; 2 μm in B–F, M, I and J; 3 μm in K, L.

DISCUSSION

A role for β4 integrin in myelination, maintenance or regeneration has long been suggested. To conclusively address the role of α6β4 integrin in myelin, we ablated it from Schwann cells of transgenic mice, alone or in combination with dystroglycan, another laminin receptor expressed by myelinating Schwann cells. We show that α6β4 integrin has a surprisingly minor role in nerve development, function or regeneration. However we provide evidence that α6β4 integrin and dystroglycan together are required to maintain the integrity of myelin sheaths.

Absence of α6β4 integrin in Schwann cells does not affect Schwann cell functions, but accelerates the folding of myelin sheaths seen in normal nerves with age

After extensive analysis, the only difference detected in β4 integrin mutant nerves was a slight increase in the frequency of myelin in-foldings, normally observed in myelinated fibers with age. These folds are scattered along the internode and concentrated in small chains at the paranodal-juxtaparanodal regions, and appear as regions where the myelin folds inward, possibly due to mechanical stress. The material contained inside the in-foldings appears of extracellular matrix origin. Although the phenotype is less dramatic, this is comparable to the structural role of attachment to the basal lamina that a6β4 integrin performs in keratinocytes (Dowling et al., 1996; van der Neut et al., 1996).

β4 integrin cooperates with dystroglycan to mantain stability of the myelin sheath

α6β4 integrin and dystroglycan are both expressed at the onset of myelination and link the cytoskeleton to laminins in the basal lamina. Dystroglycan null nerves present a late-onset instability of the myelin sheath with formation of myelin in/out-foldings resulting in motor impairment in older animals (Saito et al., 2003; Occhi et al., 2005). This neuropathy was more pronounced in the absence of both β4 integrin and dystroglycan, particularly in ventral roots of twelve month old mice where extensive myelin folding, hypomyelination and frequent signs of acute de-myelination, were observed. It is possible that in the absence of both receptors myelin instability reaches a threshold that precipitates myelin destruction, causing an inflammatory response and recruitment of inflammatory cells. Possibly, loose attachment to the basal lamina due to the lack of the two receptors also favors the entrance of macrophages, which were frequently observed associated with loose basal lamina, entering at paranodes and residing inside myelin sheaths.

β4 integrin interacts with Pmp22, but mutant mice have different severity of phenotypes

β4 integrin has recently been shown to form a complex with Pmp22 (Amici et al., 2006). Heterozygous loss-of-function mutations in PMP22 in mice and men cause a milder tomacular neuropathy, evident only after compression (Hereditary Neuropathy with liability to Pressure Palsy (HNPP). Heterozygous null mice for Pmp22 resemble HNPP pathology (Adlkofer et al., 1995), whereas homozygous null mice have a severe dys-and de-myelinating neuropathy with both hypomyelination and tomacula (Adlkofer et al., 1995; Amici et al., 2006). In these mice, an impaired organization of the basal lamina and a drastic reduction in the levels of β4 integrin was reported, suggesting a role for Pmp22 in stabilizing laminin-integrin interactions (Amici et al., 2006). A hypothesis arising from this observation is that laminin-integrin interactions at the basal lamina contribute to the stability of the myelin sheath. Even if β4 have a well organized and adherent basal lamina, and a milder phenotype than mice lacking one or two copies of Pmp-22, our report supports this view, and reinforces the idea that by maintaining adhesion to the basal lamina β4 integrin indirectly maintains the integrity of the myelin sheath.

Supplementary Material

Suppl. Fig. 1. Supplementary figure 1.

Construction and characterization of a ItgB4 floxed allele.

A) β4+is the β4 integrin wt locus, β4tk is the targeting vector. β4Fneo mice containing the neo cassette, surrounded by two Frt sites, were crossed with with Flpe mice to obtain β4 integrin “floxed” mice (β4F). Double diagonal lines on β4F indicate the end of the targeting vector. To generate constitutive or conditional β4− mice, β4F/F mice were crossed with either CMV-Cre or P0-Cre transgenic mice, respectively. As expected, expression of β4 integrin was not altered in β4F/F mice (not shown). B) Southern blots of ES cell clones positive for homologous recombination, after digestion of genomic DNA with XmnI (X in A). Blots were probed with probe A and probe B to confirm homologous recombination at the 3′ and 5′ arm, respectively. In the wt, both probe A and probe B gave a 14.1 Kb band. In the recombined clones, probe A gave a 3.9Kb band, whereas probe B gave a 11.4 Kb band. C-G) Constitutive β4 integrin-null mice. C) RT-PCR on E18.5 embryos from CMV-Cre β4 f/f mice using primers either on exon 2 or flanking exon 1 to 5. PCR product with primers in the exon 2 is 92 base pair (bp) long, and it is absent in the β4−/− mouse. PCR product with primers flanking exons 1 and 5 is 499 bp in the wt, and 380 bp (missing exon 2) in the β4−/− mouse. D-G) β4−/− mice manifest severe skin blistering. β4 integrin constitutive null (β4−/−) mice die a few hours after birth from respiratory and gastrointestinal failure, due to blistering caused by the separation of the dermal-epidermal junction (Dowling et al., 1996; van der Neut et al., 1996). Similarly, our β4 f/f//CMV-Cre transgenic mice recapitulated the null phenotype. In wt embryos, β4 integrin is normally expressed in keratinocytes at the dermal-epidermal layer, which is pseudocolored in blue in the bright field image (E). In β4−/− embryos β4 integrin is not expressed (F) and the dermal-epidermal layer detaches forming skin blisters (arrow in G). Thus, the β4floxed allele is efficiently recombined by CMV-Cre.

Suppl. Fig. 2. Supplementary. Fig. 2.

Normal behavior and neurophysiology in β4 integrin mutant mice.

Rotarod test and neurophysiology of six and twelve month old mice lacking β4 integrin in Schwann cells. A) Rotarod at six (left) and twelve (right) months of age. SEM are indicated for each trial. For each trial, P > 0.1 by paired t-test analysis n = 9 null and 6wt. B) Neurophysiology at six and ten months of age. Values and SEM are indicated; for each analysis, P > 0.1 by paired t-test analysis, n = 9 null and 6wt.

Suppl. Fig. 3. Supplentary. Fig 3.

Normal peripheral nerve development in β4 integrin mutant mice.

Morphological analysis of β4 integrin null nerves during development and in the adult. A–F) Transverse semithin sections of sciatic nerves of wt and mutant mice at P5 (A, B), P15 (C, D) and 7 months (E, F). No delays or abnormalities in myelination are present. Similarly, spinal roots did not present any abnormality in β4 integrin null mice up to 7 months of age (not shown). G–K) Electron microscopic analysis of sciatic nerves from wt and mutant mice. G) myelinated and non-myelinated fibers from β4 integrin null nerves have normal morphology. I, H) higher magnification image to demonstrate the presence of a continuous and adherent basal lamina and correct myelin compaction and periodicity in β4 integrin null nerves (J, K). Bars = 25 μm in A–F, 5 μm in G.

Suppl. Fig. 4. Supplementary Fig. 4.

Normal control of cell number and formation of axonal domains in mice lacking β4 integrin Schwann cells.

β4 integrin mRNA expression increases between E18.5 and early post-natal development, while β4 integrin protein is detectable after Schwann cells achieve a one-to-one relationship with axons (Previtali et al., 2003). During the same time (between E17.5 and P1 in the mouse), Schwann cells decrease proliferation and increase apoptosis to match the number of axons (reviewed in (Jessen and Mirsky, 2005). Since in other cellular systems β4 integrin is known to control cell cycle progression and to amplify neuregulin signaling (Guo et al., 2006), we evaluated proliferation and survival in mutant sciatic nerves by BrdU incorporation and TUNEL assay. A) Apoptotic index is indicated as the percentage of TUNEL positive nuclei over the total number of cigar-shaped Schwann cell nuclei in longitudinal sections of sciatic nerves (p > 0.1; n = 3 animals per genotype). B) Proliferating index is indicated as the percentage of BrdU incorporating cells over the total number of Schwann cell nuclei in longitudinal sections of sciatic nerves (p > 0.1 by paired t-test analysis, n = 3 animals per genotype). No significant differences were observed between β4 integrin null and wild type apoptotic or proliferative indexes in early post-natal nerves.

C) Immunofluorescence on teased fibers of β4 integrin null nerves does not reveal morphological abnormality in Schwann cell/axonal domains. Sodium clusters (Nav, C, in green) and phosphorylated ERM proteins (E, ERM-P) are enriched normally at nodes of Ranvier (arrows). Paranodal domains, labelled by Caspr staining (C, in red), are normal. Potassium channels are localized normally at the juxtaparanodes (KV1.1 D, in red). Neurofascins (155 and 186) are enriched normally at the nodal-paranodal region (F, in red). Finally, Schmidt-Lantermann incisures appear normal in morphology and number (G, in green F-actin staining). Bar = 10. μm in C, 30 μm in D, 40 μm in E, β4 μm in F, 47 μm in G.

Suppl. Fig 5. Supplementary figure 5. Rotarod performance and formation of axonal domains in β4 integrin/DG null mice.

A) Rotarod test on twelve month old β4 integrin/DG null animals and controls. Although double mutants perform more poorly that single DG null mice, they are not significantly different from wt mice. SEM are indicated for each trial. n = 16 double null, 2 β4 integrin null, 4 dg null, 12 wt. B–H. Immunofluorescence on teased fibers from DG null and β4 integrin/DG null mice. B,C) Schmidt-Lantermann incisures are normally spaced along the internodes, and Nav channels have similar degree of abnormality in the two genotypes (G, H). D, E) Caspr (red) and K+ (green) channels are normally localized at the paranodal/juxtaparanodal region in both single DG and double β4 DG null myelinated fibers. Bars = 47.6 μm in B–C, 11.4 μm in D–E, 14.7 in F–H.

Suppl. Fig.6. Supplementary figure 6. Normal regeneration of β4 mutant nerve.

Transverse semithin sections of crushed nerves of wt (A, B) and β4 integrin null (C, D) mice dissected at 15 (A,C) and 21 (B,D) days after crush. No differences in axonal regeneration and re-myelination between the two genotypes are evident. Further, Cholera toxin-B injection into the gastrocnemius muscle at T13 revealed normal presence of fluorescein-positive motor neurons in mutant spinal cords at T15 (data not shown).

Suppl. Methods

Acknowledgments

We thank C. Ferri for superb technical assistance, E. Fuchs (Rockefeller University, New York, USA) and J. Dowling (University of Michigan, Ann Arbor, USA) for the mβ4intλc clone, P. Orban (University of Montreal, Canada) for the pFlrt1 vector, V. Broccoli for TBV2 ES cells and Telethon Core Facility for Conditional Mutagenesis (San Raffaele Institute, Milan, Italy) for injecting targeted ES cells; P. Brophy and D. Sherman (University of Edinburgh, UK), E. Peles (Weizmann Institute, Rehovot, Israel), V. Lee, (University of Pennsylvania, Philadelphia, USA) and S. Kennel (Oak Ridge Laboratory, Oak Ridge, USA) for antibodies. Support was from the NIH (R01-NS045630 to MLF, R01-NS055256 to LW, and Senator Paul D. Wellstone Muscular Dystrophy Cooperative Research Center grant U54-NS053672 to KPC), Telethon Italia (GGP04019 to MLF and GGP071100 to LW), Istituto Superiore di Sanita’ e FIRB TissueNet (to AQ). K.P. Campbell is an investigator of the Howard Hughes Medical Institute.

BIBLIOGRAPHY

  1. Adlkofer K, Martini R, Aguzzi A, Zielasek J, Toyka KV, Suter U. Hypermyelination and demyelinating peripheral neuropathy in Pmp22-deficient mice. Nat Genet. 1995;11:274–280. doi: 10.1038/ng1195-274. [DOI] [PubMed] [Google Scholar]
  2. Amici SA, Dunn WA, Jr, Murphy AJ, Adams NC, Gale NW, Valenzuela DM, Yancopoulos GD, Notterpek L. Peripheral myelin protein 22 is in complex with alpha6beta4 integrin, and its absence alters the Schwann cell basal lamina. J Neurosci. 2006;26:1179–1189. doi: 10.1523/JNEUROSCI.2618-05.2006. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bernier G, De Repentigny Y, Mathieu M, David S, Kothary R. Dystonin is an essential component of the Schwann cell cytoskeleton at the time of myelination. Development. 1998;125:2135–2148. doi: 10.1242/dev.125.11.2135. [DOI] [PubMed] [Google Scholar]
  4. Court FA, Sherman DL, Pratt T, Garry EM, Ribchester RR, Cottrell DF, Fleetwood-Walker SM, Brophy PJ. Restricted growth of Schwann cells lacking Cajal bands slows conduction in myelinated nerves. Nature. 2004;431:191–95. doi: 10.1038/nature02841. [DOI] [PubMed] [Google Scholar]
  5. Dowling J, Yu QC, Fuchs E. Beta4 integrin is required for hemidesmosome formation, cell adhesion and cell survival. J Cell Biol. 1996;134:559–572. doi: 10.1083/jcb.134.2.559. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Einheber S, Milner TA, Giancotti F, Salzer JL. Axonal regulation of Schwann cell integrin expression suggests a role for alpha 6 beta 4 in myelination. J Cell Biol. 1993;123:1223–1236. doi: 10.1083/jcb.123.5.1223. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Feltri ML, Wrabetz L. Laminins and their receptors in Schwann cells and hereditary neuropathies. J Peripher Nerv Syst. 2005;10:128–143. doi: 10.1111/j.1085-9489.2005.0010204.x. [DOI] [PubMed] [Google Scholar]
  8. Feltri ML, D’Antonio M, Previtali S, Fasolini M, Messing A, Wrabetz L. P0-Cre transgenic mice for inactivation of adhesion molecules in Schwann cells. Ann N Y Acad Sci. 1999;883:116–123. [PubMed] [Google Scholar]
  9. Feltri ML, Scherer SS, Nemni R, Kamholz J, Vogelbacker H, Scott MO, Canal N, Quaranta V, Wrabetz L. Beta 4 integrin expression in myelinating Schwann cells is polarized, developmentally regulated and axonally dependent. Development. 1994;120:1287–1301. doi: 10.1242/dev.120.5.1287. [DOI] [PubMed] [Google Scholar]
  10. Feltri ML, Graus Porta D, Previtali SC, Nodari A, Migliavacca B, Cassetti A, Littlewood-Evans A, Reichardt LF, Messing A, Quattrini A, Mueller U, Wrabetz L. Conditional disruption of beta 1 integrin in Schwann cells impedes interactions with axons. J Cell Biol. 2002;156:199–209. doi: 10.1083/jcb.200109021. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Frei R, Dowling J, Carenini S, Fuchs E, Martini R. Myelin formation by Schwann cells in the absence of beta4 integrin. Glia. 1999;27:269–74. doi: 10.1002/(sici)1098-1136(199909)27:3<269::aid-glia8>3.0.co;2-2. [DOI] [PubMed] [Google Scholar]
  12. Giancotti FG, Tarone G. Positional control of cell fate through joint integrin/receptor protein kinase signaling. Annu Rev Cell Dev Biol. 2003;19:173–206. doi: 10.1146/annurev.cellbio.19.031103.133334. [DOI] [PubMed] [Google Scholar]
  13. Guo W, Pylayeva Y, Pepe A, Yoshioka T, Muller WJ, Inghirami G, Giancotti FG. Beta 4 integrin amplifies ErbB2 signaling to promote mammary tumorigenesis. Cell. 2006;126:489–502. doi: 10.1016/j.cell.2006.05.047. [DOI] [PubMed] [Google Scholar]
  14. Kajiji S, Tamura RN, Quaranta V. A novel integrin (alpha E beta 4) from human epithelial cells suggests a fourth family of integrin adhesion receptors. Embo J. 1989;8:673–680. doi: 10.1002/j.1460-2075.1989.tb03425.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Kennel SJ, Godfrey V, Ch’ang LY, Lankford TK, Foote LJ, Makkinje A. The beta 4 subunit of the integrin family is displayed on a restricted subset of endothelium in mice. J Cell Sci. 1992;101(Pt 1):145–150. doi: 10.1242/jcs.101.1.145. [DOI] [PubMed] [Google Scholar]
  16. Klein S, Giancotti FG, Presta M, Albelda SM, Buck CA, Rifkin DB. Basic fibroblast growth factor modulates integrin expression in microvascular endothelial cells. Mol Biol Cell. 1993;4:973–982. doi: 10.1091/mbc.4.10.973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Knox CA, Kokmen E, Dyck PJ. Morphometric alteration of rat myelinated fibers with aging. J Neuropathol Exp Neurol. 1989;48:119–139. doi: 10.1097/00005072-198903000-00001. [DOI] [PubMed] [Google Scholar]
  18. Litjens SH, Koster J, Kuikman I, van Wilpe S, de Pereda JM, Sonnenberg A. Specificity of binding of the plectin actin-binding domain to beta4 integrin. Mol Biol Cell. 2003;14:4039–4050. doi: 10.1091/mbc.E03-05-0268. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Moore SA, Saito F, Chen J, Michele DE, Henry MD, Messing A, Cohn RD, Ross-Barta SE, Westra S, Williamson RA, Hoshi T, Campbell KP. Deletion of brain dystroglycan recapitulates aspects of congenital muscular dystrophy. Nature. 2002;418:422–425. doi: 10.1038/nature00838. [DOI] [PubMed] [Google Scholar]
  20. Nave KA, Salzer JL. Axonal regulation of myelination by neuregulin 1. Curr Opin Neurobiol. 2006;16:492–500. doi: 10.1016/j.conb.2006.08.008. [DOI] [PubMed] [Google Scholar]
  21. Niessen CM, Cremona O, Daams H, Ferraresi S, Sonnenberg A, Marchisio PC. Expression of the integrin alpha 6 beta 4 in peripheral nerves: localization in Schwann and perineural cells and different variants of the beta 4 subunit. J Cell Sci. 1994;107(Pt 2):543–552. doi: 10.1242/jcs.107.2.543. [DOI] [PubMed] [Google Scholar]
  22. Occhi S, Zambroni D, Del Carro U, Amadio S, Sirkowski EE, Scherer SS, Campbell KP, Moore SA, Chen ZL, Strickland S, Di Muzio A, Uncini A, Wrabetz L, Feltri ML. Both laminin and Schwann cell dystroglycan are necessary for proper clustering of sodium channels at nodes of Ranvier. J Neurosci. 2005;25:9418–9427. doi: 10.1523/JNEUROSCI.2068-05.2005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Previtali SC, Dina G, Nodari A, Fasolini M, Wrabetz L, Mayer U, Feltri ML, Quattrini A. Schwann cells synthesize alpha7beta1 integrin which is dispensable for peripheral nerve development and myelination. Mol Cell Neurosci. 2003a;23:210–218. doi: 10.1016/s1044-7431(03)00014-9. [DOI] [PubMed] [Google Scholar]
  24. Previtali SC, Nodari A, Taveggia C, Pardini C, Dina G, Villa A, Wrabetz L, Quattrini A, Feltri ML. Expression of laminin receptors in schwann cell differentiation: evidence for distinct roles. J Neurosci. 2003b;23:5520–5530. doi: 10.1523/JNEUROSCI.23-13-05520.2003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Quaranta V, Jones JC. The internal affairs of an integrin. Trends Cell Biol. 1991;1:2–4. doi: 10.1016/0962-8924(91)90046-c. [DOI] [PubMed] [Google Scholar]
  26. Quattrini A, Previtali S, Feltri ML, Canal N, Nemni R, Wrabetz L. Beta 4 integrin and other Schwann cell markers in axonal neuropathy. Glia. 1996;17:294–306. doi: 10.1002/(SICI)1098-1136(199608)17:4<294::AID-GLIA4>3.0.CO;2-#. [DOI] [PubMed] [Google Scholar]
  27. Saito F, Moore SA, Barresi R, Henry MD, Messing A, Ross-Barta SE, Cohn RD, Williamson RA, Sluka KA, Sherman DL, Brophy PJ, Schmelzer JD, Low PA, Wrabetz L, Feltri ML, Campbell KP. Unique role of dystroglycan in peripheral nerve myelination, nodal structure, and sodium channel stabilization. Neuron. 2003;38:747–758. doi: 10.1016/s0896-6273(03)00301-5. [DOI] [PubMed] [Google Scholar]
  28. Sherman DL, Fabrizi C, Gillespie CS, Brophy PJ. Specific disruption of a schwann cell dystrophin-related protein complex in a demyelinating neuropathy. Neuron. 2001;30:677–687. doi: 10.1016/s0896-6273(01)00327-0. [DOI] [PubMed] [Google Scholar]
  29. Song WK, Wang W, Foster RF, Bielser DA, Kaufman SJ. H36-alpha 7 is a novel integrin alpha chain that is developmentally regulated during skeletal myogenesis. J Cell Biol. 1992;117:643–57. doi: 10.1083/jcb.117.3.643. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. van der Neut R, Krimpenfort P, Calafat J, Niessen CM, Sonnenberg A. Epithelial detachment due to absence of hemidesmosomes in integrin beta 4 null mice. Nat Genet. 1996;13:366–369. doi: 10.1038/ng0796-366. [DOI] [PubMed] [Google Scholar]
  31. Verheijen MH, Chrast R, Burrola P, Lemke G. Local regulation of fat metabolism in peripheral nerves. Genes Dev. 2003;17:2450–2464. doi: 10.1101/gad.1116203. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Wadsworth S, Halvorson MJ, Coligan JE. Developmentally regulated expression of the beta 4 integrin on immature mouse thymocytes. J Immunol. 1992;149:421–428. [PubMed] [Google Scholar]
  33. Wrabetz L, Feltri ML, Quattrini A, Imperiale D, Previtali S, D’Antonio M, Martini R, Yin X, Trapp BD, Zhou L, Chiu SY, Messing A. P(0) glycoprotein overexpression causes congenital hypomyelination of peripheral nerves. J Cell Biol. 2000;148:1021–1034. doi: 10.1083/jcb.148.5.1021. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Suppl. Fig. 1. Supplementary figure 1.

Construction and characterization of a ItgB4 floxed allele.

A) β4+is the β4 integrin wt locus, β4tk is the targeting vector. β4Fneo mice containing the neo cassette, surrounded by two Frt sites, were crossed with with Flpe mice to obtain β4 integrin “floxed” mice (β4F). Double diagonal lines on β4F indicate the end of the targeting vector. To generate constitutive or conditional β4− mice, β4F/F mice were crossed with either CMV-Cre or P0-Cre transgenic mice, respectively. As expected, expression of β4 integrin was not altered in β4F/F mice (not shown). B) Southern blots of ES cell clones positive for homologous recombination, after digestion of genomic DNA with XmnI (X in A). Blots were probed with probe A and probe B to confirm homologous recombination at the 3′ and 5′ arm, respectively. In the wt, both probe A and probe B gave a 14.1 Kb band. In the recombined clones, probe A gave a 3.9Kb band, whereas probe B gave a 11.4 Kb band. C-G) Constitutive β4 integrin-null mice. C) RT-PCR on E18.5 embryos from CMV-Cre β4 f/f mice using primers either on exon 2 or flanking exon 1 to 5. PCR product with primers in the exon 2 is 92 base pair (bp) long, and it is absent in the β4−/− mouse. PCR product with primers flanking exons 1 and 5 is 499 bp in the wt, and 380 bp (missing exon 2) in the β4−/− mouse. D-G) β4−/− mice manifest severe skin blistering. β4 integrin constitutive null (β4−/−) mice die a few hours after birth from respiratory and gastrointestinal failure, due to blistering caused by the separation of the dermal-epidermal junction (Dowling et al., 1996; van der Neut et al., 1996). Similarly, our β4 f/f//CMV-Cre transgenic mice recapitulated the null phenotype. In wt embryos, β4 integrin is normally expressed in keratinocytes at the dermal-epidermal layer, which is pseudocolored in blue in the bright field image (E). In β4−/− embryos β4 integrin is not expressed (F) and the dermal-epidermal layer detaches forming skin blisters (arrow in G). Thus, the β4floxed allele is efficiently recombined by CMV-Cre.

NIHMS65497-supplement-Suppl__Fig__1.tif (15.8MB, tif)
Suppl. Fig. 2. Supplementary. Fig. 2.

Normal behavior and neurophysiology in β4 integrin mutant mice.

Rotarod test and neurophysiology of six and twelve month old mice lacking β4 integrin in Schwann cells. A) Rotarod at six (left) and twelve (right) months of age. SEM are indicated for each trial. For each trial, P > 0.1 by paired t-test analysis n = 9 null and 6wt. B) Neurophysiology at six and ten months of age. Values and SEM are indicated; for each analysis, P > 0.1 by paired t-test analysis, n = 9 null and 6wt.

NIHMS65497-supplement-Suppl__Fig__2.tif (11.3MB, tif)
Suppl. Fig. 3. Supplentary. Fig 3.

Normal peripheral nerve development in β4 integrin mutant mice.

Morphological analysis of β4 integrin null nerves during development and in the adult. A–F) Transverse semithin sections of sciatic nerves of wt and mutant mice at P5 (A, B), P15 (C, D) and 7 months (E, F). No delays or abnormalities in myelination are present. Similarly, spinal roots did not present any abnormality in β4 integrin null mice up to 7 months of age (not shown). G–K) Electron microscopic analysis of sciatic nerves from wt and mutant mice. G) myelinated and non-myelinated fibers from β4 integrin null nerves have normal morphology. I, H) higher magnification image to demonstrate the presence of a continuous and adherent basal lamina and correct myelin compaction and periodicity in β4 integrin null nerves (J, K). Bars = 25 μm in A–F, 5 μm in G.

NIHMS65497-supplement-Suppl__Fig__3.tif (3.8MB, tif)
Suppl. Fig. 4. Supplementary Fig. 4.

Normal control of cell number and formation of axonal domains in mice lacking β4 integrin Schwann cells.

β4 integrin mRNA expression increases between E18.5 and early post-natal development, while β4 integrin protein is detectable after Schwann cells achieve a one-to-one relationship with axons (Previtali et al., 2003). During the same time (between E17.5 and P1 in the mouse), Schwann cells decrease proliferation and increase apoptosis to match the number of axons (reviewed in (Jessen and Mirsky, 2005). Since in other cellular systems β4 integrin is known to control cell cycle progression and to amplify neuregulin signaling (Guo et al., 2006), we evaluated proliferation and survival in mutant sciatic nerves by BrdU incorporation and TUNEL assay. A) Apoptotic index is indicated as the percentage of TUNEL positive nuclei over the total number of cigar-shaped Schwann cell nuclei in longitudinal sections of sciatic nerves (p > 0.1; n = 3 animals per genotype). B) Proliferating index is indicated as the percentage of BrdU incorporating cells over the total number of Schwann cell nuclei in longitudinal sections of sciatic nerves (p > 0.1 by paired t-test analysis, n = 3 animals per genotype). No significant differences were observed between β4 integrin null and wild type apoptotic or proliferative indexes in early post-natal nerves.

C) Immunofluorescence on teased fibers of β4 integrin null nerves does not reveal morphological abnormality in Schwann cell/axonal domains. Sodium clusters (Nav, C, in green) and phosphorylated ERM proteins (E, ERM-P) are enriched normally at nodes of Ranvier (arrows). Paranodal domains, labelled by Caspr staining (C, in red), are normal. Potassium channels are localized normally at the juxtaparanodes (KV1.1 D, in red). Neurofascins (155 and 186) are enriched normally at the nodal-paranodal region (F, in red). Finally, Schmidt-Lantermann incisures appear normal in morphology and number (G, in green F-actin staining). Bar = 10. μm in C, 30 μm in D, 40 μm in E, β4 μm in F, 47 μm in G.

NIHMS65497-supplement-Suppl__Fig__4.tif (61MB, tif)
Suppl. Fig 5. Supplementary figure 5. Rotarod performance and formation of axonal domains in β4 integrin/DG null mice.

A) Rotarod test on twelve month old β4 integrin/DG null animals and controls. Although double mutants perform more poorly that single DG null mice, they are not significantly different from wt mice. SEM are indicated for each trial. n = 16 double null, 2 β4 integrin null, 4 dg null, 12 wt. B–H. Immunofluorescence on teased fibers from DG null and β4 integrin/DG null mice. B,C) Schmidt-Lantermann incisures are normally spaced along the internodes, and Nav channels have similar degree of abnormality in the two genotypes (G, H). D, E) Caspr (red) and K+ (green) channels are normally localized at the paranodal/juxtaparanodal region in both single DG and double β4 DG null myelinated fibers. Bars = 47.6 μm in B–C, 11.4 μm in D–E, 14.7 in F–H.

NIHMS65497-supplement-Suppl__Fig_5.tif (79.8MB, tif)
Suppl. Fig.6. Supplementary figure 6. Normal regeneration of β4 mutant nerve.

Transverse semithin sections of crushed nerves of wt (A, B) and β4 integrin null (C, D) mice dissected at 15 (A,C) and 21 (B,D) days after crush. No differences in axonal regeneration and re-myelination between the two genotypes are evident. Further, Cholera toxin-B injection into the gastrocnemius muscle at T13 revealed normal presence of fluorescein-positive motor neurons in mutant spinal cords at T15 (data not shown).

NIHMS65497-supplement-Suppl__Fig_6.tif (10.2MB, tif)
Suppl. Methods
NIHMS65497-supplement-Suppl__Methods.doc (33KB, doc)

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