α6β1 and α7β1 integrins are required in Schwann cells to sort axons

Abstract

During development, Schwann cells extend lamellipodia-like processes to segregate large- and small-caliber axons during the process of radial sorting. Radial sorting is a prerequisite for myelination and is arrested in human neuropathies because of laminin deficiency. Experiments in mice using targeted mutagenesis have confirmed that laminins 211, 411, and receptors containing the β1 integrin subunit are required for radial sorting; however, which of the 11 α integrins that can pair with β1 forms the functional receptor is unknown. Here we conditionally deleted all the α subunits that form predominant laminin-binding β1 integrins in Schwann cells and show that only α6β1 and α7β1 integrins are required and that α7β1 compensates for the absence of α6β1 during development. The absence of either α7β1 or α6β1 integrin impairs the ability of Schwann cells to spread and to bind laminin 211 or 411, potentially explaining the failure to extend cytoplasmic processes around axons to sort them. However, double α6/α7 integrin mutants show only a subset of the abnormalities found in mutants lacking all β1 integrins, and a milder phenotype. Double-mutant Schwann cells can properly activate all the major signaling pathways associated with radial sorting and show normal Schwann cell proliferation and survival. Thus, α6β1 and α7β1 are the laminin-binding integrins required for axonal sorting, but other Schwann cell β1 integrins, possibly those that do not bind laminins, may also contribute to radial sorting during peripheral nerve development.

Figure 1.

Efficient genomic recombination and Schwann cell-specific deletion of α6 integrin in α6 integrin^F/F P0Cre mutant nerves. A, Constitutive deletion of the α6 allele in α6^−/− mice causes detachment between epidermis and dermis (epidermolysis bullosa) resulting from the absence of α6 integrin (red arrows). The white arrow in wild-type indicates the normal α6 staining at the dermal–epidermal junction. Arrowheads point to a non-specific staining in the stratum corneus. B, Genomic DNA from tail samples of wild-type (α6^+/+), α6^F/+, α6^F/F, and α6^F/− mice were amplified using primers AH065 (placed upstream of the 5′ loxP site), AHM4 and AHM3 (flanking the 3′ loxP site) and generated the expected WT (120 base pairs), unrecombined floxed (154 base pair), and recombined floxed (1000 base pair) bands. PCR on genomic DNA from α6 integrin^F/F and α6 integrin^F/F P0Cre sciatic nerves (SN) at P1 and P28 amplified the floxed band and the recombined floxed band only in the presence of P0Cre at P1 and P28. C, D, Western blotting and immunohistochemistry in α6 integrin^F/F P0Cre^+/− and control sciatic nerves at P1, P5, and P28. α6 integrin is absent in mutant nerves at P28 and markedly reduced by P1. One representative experiment of three is shown; different animals or pools of animals were used for each experiment. C, β-tubulin was used as a loading control. D, Green represents α6 integrin. Red represents neurofilament. Blue represents nuclei (DAPI). Of note, α6 integrin in mutant adult nerves is still present in blood vessels (*) and in the perineurium (**), where P0Cre is not expressed. Scale bars: A, 300 μm; D, 35 μm.

Figure 2.

α6 integrin ablation in Schwann cells does not lead to radial sorting defects in α6 integrin^F/F P0Cre sciatic nerves and spinal roots. Semithin sections of sciatic nerves (SN), dorsal (DR), and ventral roots (VR) from wild-type and α6 integrin^F/F P0Cre mutant mice (cKO) at P5 and P28 are shown. No bundles of naked axons are present in mutant mice, indicating normal radial sorting. Scale bar, 10 μm.

Figure 3.

Absence of α6 integrin does not lead to radial sorting arrest and subsequent myelination defects in Schwann cell-DRG neuron explants. Schwann cell–DRG neuron explants were prepared from wild-type and α6 integrin constitutive null embryos, and induced to myelinate for 10 d. A, Wild-type and α6-deficient (α6KO) explants were immunostained for α6 integrin (green), neurofilament (red), and nuclei (DAPI, blue); α6 protein is expressed in Schwann cells from wild-type explants, but it is absent in Schwann cells from deficient mice. B, Wild-type and α6 integrin-null explants were immunostained for MBP (green), neurofilament (red), and nuclei (DAPI, blue). Both wild-type and α6 integrin-null Schwann cells can myelinate axons to a similar extent. One representative of three experiments is shown. C, Western blot analysis of total protein extracts from wild-type and α6 integrin mutant explants shows a similar amount of P0 protein in null samples and controls. β-tubulin was used as a loading control. One representative of three experiments is shown. D, Quantification of myelin internodes in wild-type and α6 integrin-null explants in three independent experiments; in each experiment, different pools of DRGs were used. The number of myelin segments is comparable between the two genotypes. Error bars indicate SEM. Statistical significance was measured by Student's t test. Scale bars: A, 35 μm; B, 130 μm.

Figure 4.

The absence of α6 integrin in Schwann cells causes ensheathment abnormalities in sciatic nerves. Electron microscopy analysis performed on α6 integrin^F/F P0Cre sciatic nerves at P28 revealed the presence of hypermyelination (A, B, arrowheads), hypomyelination (C, F, **), axons <1 μm inappropriately myelinated (E, arrows), sometimes by a thick myelin sheath (F, G, arrowheads), Remak bundles containing axons >1 μm (C, D, F, *), axons >1 μm that were sorted but not myelinated (H, *), and occasional signs of axonal degeneration (I, L, arrows). K, Quantification of these abnormalities showed that ∼25% of fibers were hypermyelinated (g-ratio < 0.6) and ∼5% of fibers were hypomyelinated (g-ratio > 0.8) in mutant nerves. Densities are indicated per 100 μm². Error bars indicate 2 SEs. *p < 0.05 (Mann–Whitney test). Scale bars: A–C, 2 μm; D–K, 1 μm.

Figure 5.

α3β1 integrin is dispensable in peripheral nerve development and is neither redundant nor compensatory for α6 integrin. A, Western blot analysis of total protein extracts from wild-type and α3 integrin^F/F P0Cre sciatic nerves shows that the α3 protein (arrow) is absent from mutant sciatic nerves at P28, but a residual level of the protein is still present in P5 mutant nerves. *An aspecific band was detected in all samples. Calnexin was used as a loading control. Sciatic nerve pools from different animals were used for each sample. B, Western blot analysis of total protein extracts from wild-type and α6 integrin^F/F P0Cre sciatic nerves shows that α3 integrin expression is comparable between wild-type and mutant sciatic nerves; thus, the absence of α6 integrin does not cause a compensatory upregulation of α3 integrin. Calnexin was used as a loading control. Sciatic nerve pools from different animals were used for each sample. C, D, Semithin sections of sciatic nerves (SN), dorsal (DR), and ventral roots (VR) from wild-type, α3, and double α3/α6 integrin conditional null mice (α3 integrin^F/F, α6 integrin^F/F P0Cre) at P28. Single α3 and double α3/α6 integrin mutant sciatic nerve and root morphology is comparable with control nerves. Scale bar, 10 μm.

Figure 6.

α7 integrin protein is expressed earlier and upregulated in sciatic nerves from α6 conditional null mice during development. A, Top, Immunoprecipitation of β1 integrin from wild-type and α6 conditional-null sciatic nerves at P5; blotting with β1 integrin antibody shows similar amounts of β1 integrin in wild-type and mutant sciatic nerves (SN) and significant enrichment compared with inputs (first two lanes). Omission of the primary antibody on wild-type samples was used as a negative control. Bottom, Mass spectrometry analysis was performed on four bands identified on the silver-stained immunoprecipitates (arrows). α7 was identified by mass spectrometry only in α6 mutant samples. B, C, Western blot analysis of total protein lysates from wild-type and mutant sciatic nerves (B) or DRG explants (C) validates a strong upregulation of α7 integrin when α6 integrin is absent in vivo and in vitro. α6 cKO, Schwann cell conditional knockout in B; α6 KO, general knockout in C. B, α7 integrin-deficient sciatic nerve lysate (α7 KO) was used as negative control for α7 integrin. One representative experiment of three is shown; nerves (or DRGs) were pooled from different animals in each experiment. D, RNA extraction was performed on P5 wild-type and mutant sciatic nerves; and α7 integrin mRNA levels were assessed by real-time PCR. Error bars indicate SEM. The experiment was done in triplicate; nerves were pooled from different animals in each experiment. E, Frozen sections of wild-type and α6 mutant sciatic nerves at P1, P5, and P28 immunostained for α7 integrin (red) and neurofilament (green). α7 integrin is expressed in Schwann cells only after myelination in WT nerves, but beginning at P1 in α6 integrin-null nerves. Magnified insets, Endoneurial α7 integrin staining comes from Schwann cells. Scale bar, 35 μm.

Figure 7.

Double α6/α7 mutant sciatic nerves have defective radial sorting throughout development. A–L, Semithin sections from wild-type, α6 integrin Schwann cell conditional null nerves (α6 cKO), α7 integrin-null nerves (α7 KO), and double α6/α7 integrin mutant nerves (α6/α7 KO) at P5, P15, and P28. In contrast to the normal morphology of single-mutant nerves, double-mutant sciatic nerves present many bundles of naked axons (D, H, L, arrows), evidence of defective radial sorting. Scale bar, 10 μm. B, Electron microscopy analysis of double α6/α7 mutant sciatic nerves at P28 reveals the presence of bundles of naked and unsorted axons of mixed caliber (M, N), basal lamina detachment (O, arrows), hypermyelinated axons (P, *), and occasional axonal degeneration (N, *). Scale bars: M, N, P, 2 μm; O, 1 μm.

Figure 8.

Double α6/α7 mutant sciatic nerves do not show significant alterations in signaling, proliferation, and survival at the time of axonal sorting. A, B, Schwann cells from α6 and α7 integrin-null mice were isolated from DRG explant cultures, plated on laminin 111, and stained for S100. Schwann cells area was measured by ImageJ software: both α6 and α7 null Schwann cells spread significantly less than wild-type cells on laminin. **p < 0.01 (Student's t test). C, Pull-down assay of Pak-binding-domain GST for Rac1 was performed on pools of 6–10 P5 sciatic nerves from WT and mutant mice. Active proteins were normalized to total Rac1, and equal loading was verified by β-tubulin. One of two experiments is shown. D–G, Western blot analysis on total protein extracts from three pools of 10–16 P5 sciatic nerves from mice of the indicated genotype reveals no significant alterations in the phosphorylation levels of ERK (D), AKT (Ser473) (E), Src (Tyr416) (F), and Fak (Tyr397) (G) in double-mutants compared with controls. Calnexin was used as a loading control. One representative experiment of three is shown. Error bars indicate SEM. The statistical significance of the various replicates was measured by Student's t test. H, I, Frozen sections of mutant sciatic nerves at P5, P15, and P28 were analyzed by TUNEL or immunostained for phosphorylated histone3 (P-H3). The percentage of P-H3 (H) or TUNEL (G) positive nuclei was calculated for each genotype. No differences in the level of Schwann cell apoptosis or proliferation were observed, except for an increase in Schwann cell proliferation at P15 in α7 null and double α6/α7 null nerves. Experiments were performed in triplicate; different pools of animals for each experiment were used. Error bars indicate SEM. *p < 0.05 (Student's t test).

Figure 9.

Deletion of α6 or α7 integrin in Schwann cells reduces binding to laminins 411 and 211, respectively. DRG explant cultures from wild-type, α6 integrin (A, E), and α7 integrin (C, G) null embryos were cultured for 5 d, then treated with laminin 211 or 411 at 37°C for 3 h to allow binding of laminins to the basal Schwann cell surface. After washing, cells were immunostained for α2 or α4 laminin chains (green), neurofilament (red), and DAPI (blue). Staining of untreated cells detected low levels of endogenous laminins. Laminin 211 binds less to α7 null explants, and laminin 411 binds less to α6 null samples. One representative experiment of three is shown. B, H, Semiquantitative estimation of the relative amount of fluorescence (green represents laminin; blue represents DAPI) in WT, α6KO, and α7KO explants to which laminins were added was performed with ImageJ software. Scale bar, 15 μm. Error bars indicate SEM. *p < 0.05 (Student's t test). **p < 0.001 (Student's t test).