Correspondence to Bruce L. Patton: pattonb{at}ohsu.edu
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Introduction |
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Peripheral myelination is a concerted process in which Schwann cell proliferation, axon defasciculation, and myelin assembly overlap (Webster, 1971; Martin and Webster, 1973; Webster et al., 1973; Stewart et al., 1993). Premyelinating Schwann cells cover fascicles of cotargeted axons. Their proliferation rate initially matches axonal growth, but increases during myelination to supply Schwann cells for individual axons, at perinatal ages in rodents. Progeny invade fascicles after longitudinal division, which increases Schwann cell density along subsets of axons. Invading cells often transiently ensheath several axons, but retract all but one process and myelinate a single axon. Recurrence of these events ultimately reduces fascicles to axons lacking promyelinating signals, which are defasciculated but remain unmyelinated by the final Schwann cell progeny. Webster described the progressive defasciculation and myelination of peripheral axons as radial sorting, and proposed that Schwann cell proliferation is intimately involved in the commitment of longitudinal cohorts to defasciculate and ensheath subjacent axons (Webster, 1971; Martin and Webster, 1973; Webster et al., 1973). Although neuregulins have been identified as key signals for Schwann cell proliferation (Garratt et al., 2000), molecular mechanisms that accelerate perinatal proliferation and propel radial sorting are not known.
The one factor known to have specific roles in radial sorting is Ln-2 (merosin), a major component of the Schwann cell surface basal lamina (BL). Lns comprise a family of ß
heterotrimers. Loss of Ln-2 through mutations in the
2 chain causes a complex neuromuscular disease including peripheral dysmyelination. In the most studied dy and dy2J strains of Ln
2 mutant mice, peripheral nerves contain bundles of unsheathed axons that resemble embryonic fascicles (Bradley and Jenkison, 1973; Biscoe et al., 1974). This unique pattern of dysmyelination presumably represents incomplete radial sorting and has therefore been termed "amyelination."
Mechanistic hypotheses for amyelination presume endoneurial BLs are necessary for Schwann cell motility and/or differentiation during rapid remodeling (Madrid et al., 1975; Bunge, 1993; Feltri et al., 2002; Chen and Strickland, 2003). Lns that self-polymerize, including Ln-2, are the key structural component of BLs (Yurchenco et al., 2004), and Ln-2deficient Schwann cells form patchy, discontinuous BLs (Madrid et al., 1975). However, only spinal roots and cranial nerves are severely amyelinated in dy and dy2J mice; sciatic nerves are partially affected and brachial nerves are nearly normal (Bradley and Jenkison, 1975; Stirling, 1975; Weinberg et al., 1975). One possibility is that BL structure and Ln have limited roles in radial sorting, only critical in large nerves. Alternatively, loss of Ln-2 may be partially compensated by isoforms containing the 1,
4, and
5 chains. Ln
1 is absent in normal nerves, but is expressed in dy2J sciatic nerves; lack of
1 expression in dy2J spinal roots may account for severe amyelination there (Previtali et al., 2003b). Ln
5 is selectively expressed in roots (Nakagawa et al., 2001), which could interfere with
1-Ln heterotrimer assembly in dy2J. Ln
4 is normally low in mature nerves, but is up-regulated in developing nerves and
2-deficient nerves (Patton et al., 1997, 1999; Nakagawa et al., 2001). Targeted deletion of the Ln
1 chain causes more widespread peripheral dysmyelination than occurs in dy mice, consistent with roles for multiple isoforms (Chen and Strickland, 2003). Here, we address independent and combined roles of Lns containing the
2,
4, and
5 chains.
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Results |
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Second, polyaxonal myelination (Fig. 1, l, m, and o; Table I) was common in Lama4/ but rare in dy2J, consistent with previous observations in dy (Okada et al., 1977). Most small bundles (25 axons) in Lama4/ tibial nerves were "myelinated." Conversely, dy2J nerves contained many islands of ensheathed-but-not-myelinated axons, which were rare in Lama4/ (Fig. 1 o, right). Superficially similar to Remak bundles, islands were less condensed, more numerous, and included mixed caliber axons. They appear to be abnormal transitional structures, intermediate between amyelinated bundles and properly myelinated axons, which preferentially appear in dy2J nerves when fascicles contain few axons. Thus, although Ln-2 and -8 both promote axon sorting, they have decidedly unequal roles in roots, and dissimilar roles in the transition from premyelinating to myelinating Schwann cell phenotype.
Redundancy and compensation in the BL
To ask if Ln-2 and -8 independently incorporate into endoneurial BLs, we stained cryostat sections of normal and mutant sciatic nerves with Ln chainspecific antibodies. In normal nerves, Ln 4 was coconcentrated with the
2, ß1, and
1 chains at ab-axonal Schwann cell surfaces; none were detected at axonal surfaces (Fig. 3, d, e, m, and n; unpublished data). Detection of
4 varied considerably with antibody, but endoneurial BLs stained weakly compared with perineurium, as shown previously (Patton et al., 1997; Nakagawa et al., 2001). Loss of
4 did not affect staining for
2 at any postnatal age (Fig. 3 u; unpublished data). Similarly,
4 staining was not decreased in dy2J nerve; indeed, levels increased relative to controls, confirming previous results (Patton et al., 1997, 1999; Nakagawa et al., 2001) with additional reagents. In addition, staining for entactin, perlecan, agrin, and collagen IV was unaffected in either mutant (Fig. 3, gi, pr, and ya'; unpublished data). Thus, amyelination-inducing mutations in
2 and
4 do not act by inhibiting expression of their counterpart specifically, or disrupting the molecular composition of endoneurial BLs generally. We found no morphological or histological defects in double-heterozygous Lama2dy2J/+;Lama4+/ offspring (not depicted). Therefore, Ln-2 and Ln-8 each contribute a distinct activity necessary to complete radial sorting.
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Several aspects of Ln 1 expression were inconsistent with roles in radial sorting. First,
1 expression and myelination were poorly correlated in adult dy2J (69 wk).
1 was undetectable on many myelinated fibers (Fig. 3 k), and was absent from any brachial or sciatic fibers in 4 of 8 dy2J mice. Several
1 antibodies, which strongly stained CNS pial surfaces included as controls, gave similar results. Second, we were unable to detect
1 in dy2J nerves before P14, when radial sorting has largely ended even in mutants (Fig. 5, d and e; unpublished data). Third,
1 was absent from partially myelinated nerves in Lama4/ (Fig. 3 t) and
2-null dy3K mice (Fig. 5 f). Fourth, levels of
1 were highest in severely amyelinated dy2J/
4null nerves (Fig. 5, gi), indicating endogenous
1 expression is insufficient for radial sorting. As Ln-1 (
1ß1
1) is elsewhere implicated in BL assembly (Yurchenco et al., 2004), the heterogeneous expression of Ln
1 we observed in dy2J may account for the variable integrity of endoneurial BLs present (but not often acknowledged) in this strain. In dy2J/
4null nerves,
1 was predominantly associated with myelinated fibers (Fig. 5, h and i), many of which contained well-formed BLs (Fig. 4 k). Pre- and promyelinating dy2J/a4null Schwann cells lacked BLs (Fig. 4 k). Thus, Ln
1 may not promote radial sorting because
2-deficient Schwann cells express it after myelination.
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Cell and molecular mechanisms
Amyelination has been thought to derive in large measure from disruption of Schwann cell BLs (Madrid et al., 1975; Bunge et al., 1986; Eldridge et al., 1989; Feltri et al., 2002). However, amyelination in Lama4/ was not accompanied by disruption of endoneurial BLs, on either myelinating or premyelinating Schwann cells (Fig. 3 d'; unpublished data). Moreover, Schwann cells in dy2J/a4null roots ensheathed and myelinated all axons despite lacking even a trace of endoneurial BLs (Fig. 4 l). Promyelinating Schwann cells in dy2J/a4null/TgA5 nerves also lacked BLs (Fig. 5 n). Thus, BLs are neither sufficient (in Lama4/) or necessary (in dy2J/a4null) for Schwann cells to complete radial sorting.
Therefore, we considered potential signaling roles for Lns. Several lines of research suggest signaling through Ln receptors might promote Schwann cell proliferation during myelination. Radial sorting is closely coupled to Schwann cell mitosis (Bradley and Asbury, 1970; Webster et al., 1973), amyelinated regions of Ln 2 mutant nerves have fewer Schwann cells than normal (Bray and Aguayo, 1975; Okada et al., 1976), and Ln-1 promotes Schwann cell proliferation in vitro (Porter et al., 1987). First, we asked if Schwann cell deficits correlate specifically with amyelination, or with loss of Ln-2 or BL structure. We found Schwann cell deficits accompanied amyelination in Lama4/, and large deficits accompanied severe amyelination in dy2J/
4nulls (Fig. 6 a). In transverse sections of Lama4/ tibial nerve, deficits in myelinated axons (24 ± 2%; calculated from Table I) and Schwann cells (30 ± 13%; Fig. 6 a) were proportional. In teased Lama4/ nerve preparations, myelinated fibers had normal numbers of Schwann cells (Fig. 6, b and c; controls, 12 ± 4 nuclei/field at 1000x; Lama4/, 14 ± 5). Thus, Schwann cell deficits specifically accrue from amyelinated axons.
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To ask if Ln-2 and -8 promote proliferation directly, we cultured primary Schwann cells on substrates containing purified isoforms (Fig. 7, ad). Populations plated at moderate densities on Ln-1, -2, and -8 expanded at similar rates, doubling the rate on uncoated surfaces. When Ln concentration or cell density were limiting, proliferation was significantly faster on Ln-8 than on Ln-1 or -2. These data extend previous studies with Ln-1 (Porter et al., 1987) to suggest that Ln-2 and -8 promote Schwann cell proliferation in concert with autocrine growth factors. The results are consistent with the early hypothesis that increasing cell density activates Schwann cells to invade fascicles and ensheath axons (Martin and Webster, 1973).
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Discussion |
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Decreased proliferation and Schwann cell deficits occur in Ln 2-deficient nerves (Bradley and Jenkison, 1973; Bray and Aguayo, 1975; Stirling, 1975; Perkins et al., 1980). However, perinatal proliferation is reportedly normal in mice with Schwann cellspecific loss of Ln
1 or integrin ß1, both of which are amyelinated (Feltri et al., 2002; Chen and Strickland, 2003). Interestingly, disruption of floxed Ln
1 alleles occurred unexpectedly in the Schwann cell lineage at E17, through Cre expression off a CaM kinase II (CaMKII) promoter (Chen and Strickland, 2003). As this coincides with the onset of Ln-dependent proliferation we identify, CaMKII-Cre expression could begin in Ln-dependent progeny. Defects in CaMKIICre/Ln
1-deficient nerves would be consistent with additional roles for Ln-2 and -8 in the invasion of fascicles and/or ensheathment of axons, as suggested previously (Chen and Strickland, 2003). Similarly, ß1 integrins could preferentially mediate these later roles (Feltri et al., 2002).
Our results suggest Ln-2 and -8 coordinate axonal ensheathment by promyelinating Schwann cells. Lama4/ Schwann cells prematurely myelinated bundles of axons, before completing their separation. dy2J Schwann cells lingered between ensheathing multiple axons and myelinating single axons. In principle, both activating and inhibitory mechanisms could underlie these results. For example, Ln-8 may promote the formation of multiple ensheathing Schwann cell processes, or simply retard myelin formation; loss of either function could produce polyaxonal myelination. Similarly, Ln-2 may promote myelin formation, and/or limit the formation of axon-ensheathing processes. Regardless, the results provide an initial insight into the mystery of how each Schwann cell manages to myelinate a single axon.
Molecular mechanisms
Ln-2 and -8 do not fully compensate each other's loss in vivo, and have distinct binding and proliferative activities for Schwann cells in vitro, suggesting distinct receptors mediate their actions. Schwann cells express several Ln-binding integrins and dystroglycan (Previtali et al., 2001). Early steps of myelination depend greatly on ß1-integrins (Fernandez-Valle et al., 1994; Feltri et al., 2002), and not dystroglycan (Saito et al., 2003). Amyelination caused by loss of integrin ß1 (partial in distal nerves; nearly absent in roots) now appears to largely phenocopy loss of Ln-8 rather than Ln-2. The major ß1-integrin in developing Schwann cells is integrin 6ß1. As blocking antibody to integrin
6 inhibited Schwann cell binding to Ln-8 and not Ln-2 (Fig. 7), the simplest interpretation at present is that integrin
6ß1 primarily mediates the effects of Ln-8 in radial sorting. Ln-2 engages additional receptors, as adding its loss to
4-deficiency (i.e., in dy2J/
4null and Ln
2/
4-DKO) produces amyelination far exceeding that in ß1-integrindeficient nerves. Testing in vivo roles for integrin
6ß4, expressed by perinatal Schwann cells, may require tissue-specific mutations, as mice lacking these subunits die at birth (Dowling et al., 1996; Georges-Labouesse et al., 1996).
Endoneurial BLs
The idea that Schwann cell BLs are necessary for the proper defasciculation and myelination of axons in developing nerves (Madrid et al., 1975) has endured for nearly 30 years (Bunge, 1993; Feltri et al., 2002; Chen and Strickland, 2003). In muscle, disruption of myofiber BLs likely initiates 2-deficient myodegeneration (Moll et al., 2001; Durbeej and Campbell, 2002). However, our results show no correlation in nerves between radial sorting and endoneurial BL integrity. First,
4-deficiency has no ulterior effect on endoneurial BL structure or composition, but causes the same degree of sciatic amyelination as
2-deficiency. Second, Ln-8 promotes considerable sorting and myelination without BLs. For example, radial sorting and myelination in dy2J brachial nerves is nearly normal, develops without BLs, and is almost entirely dependent on Ln-8 (dy2J/
4null brachial nerves are severely amyelinated). The inability of Ln-8 to promote BL assembly is consistent with Ln
4 lacking amino-terminal domains required for heterotrimer polymerization (Yurchenco et al., 2004). Third, all axons in dy2J/
4null spinal roots are sorted and myelinated without endoneurial BL formation. Fourth, transgenic Ln
5 promotes myelination in dy2J/
4null sciatic nerves without forming BLs on the pre- and promyelinating Schwann cells involved in radial sorting. This last result is curious as
5-Lns are expected to promote BL formation, but is consistent with observations in
2-deficient spinal roots, which contain
5 but lack BLs (Madrid et al., 1975; Weinberg et al., 1975; Nakagawa et al., 2001). In sum, endoneurial BLs are neither necessary to achieve complete radial sorting nor sufficient to prevent amyelination. That BL integrity is irrelevant to the initial myelination of axons brings mammalian myelination into line with amphibians, in which Schwann cells acquire BLs after myelination (Webster and Billings, 1972).
Therefore, it seems likely that signaling through Ln receptors regulates Schwann cell activation during myelination. In this context, it is worth reconsidering the dy2J isoform of Ln-2 (Xu et al., 1994). Amyelination in dy2J/a4null and Ln2/
4-DKO sciatic nerves were similarly severe, revealing that Ln-2(dy2J) is nearly inactive. Yet, the amino-terminal Lama2dy2J mutation specifically impairs the ability of Ln-2(dy2J) to polymerize and scaffold BLs, and does not prevent binding to cell surface receptors (Colognato and Yurchenco, 1999), which seems to argue strongly for the importance of BL structure. To reconcile these results with the above view that BLs are not required for sorting, we suggest that short-arm interactions between Ln-2 heterotrimers are critical for Ln-2 to activate its Schwann cell receptors, possibly through receptor aggregation. Further, we speculate that transgenic expression of Ln
5 promotes radial sorting without BL assembly by stabilizing short-arm interactions with Ln-2(dy2J) and restoring the activation of Ln-2 receptors. Ln-8, which lacks an
-chain short arm, may paradoxically promote the severe amyelination in dy2J roots by diluting Ln-10/Ln-2(dy2J) interactions.
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Materials and methods |
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Genotypes were identified by PCR off tail-tip DNA, using the following sense (-S) and antisense (-AS) primers. Lama4wt-S: 5'-GGCAGGCGTCCCAGTGTC-3'; -AS: 5'-CAACAAAGTTGCAACTTGGGCTC-3'. Lama4null-S: 5'-AGCGTACCCTCCCACCCAC-3'; -AS: 5'-GCTAAAGCGCATGCTCCAGACTG-3', in PGK promoter. Lama2wt-S: 5'-CCAGATTGCCTACGTAATTG-3'; -AS: 5'-CCTCTCCATTTTCTAAAG-3'. Lama2dy3K-S: 5'-CTTTCAGATTGCATTGCAAGC-3'; -AS: 5'-TCGTTTGTTCGGATCCGTCG-3'. Lama2dy2J-S: 5'-TCCTGCTGTCCTGAATCTTG-3'; -AS: 5'-AGGCTCATGAGTCCTTTG-3'. dy2J offspring were typed similar to (Vilquin et al., 2000). Touch-down PCR across the point mutation site (annealing from 60°C to 52°C in 10 cycles plus 25 cycles at 50°C) was followed by NdeI digestion to produce 164- and 109-bp fragments from dy2J but not wild-type product. Digestion ambiguities were resolved by HypCH4 III, which cuts wild-type but not dy2J product.
Antibodies
Rat mAbs 198 and 200 to Ln 1 were from Lydia Sorokin (Sorokin et al., 1992; Lund University, Lund, Sweden). Rabbit antibodies to Ln
1 and
2 (Rambukkana et al., 1997) were from Peter Yurchenco (Robert Wood Johnson Medical School, Piscataway, NJ); anti-
1 was generated against EHS Ln-1, affinity purified against E3 fragment, and cross-adsorbed against E8 fragment. mAbs to
2 (4H8-2; Qbiogene), ß1 (MAB1928; CHEMICON International), and
1 (MAB1914; CHEMICON International) were purchased. Rabbit and guinea pig antibodies to Ln
4,
5, and ß2 are described elsewhere (Miner et al., 1997). A pAb to purified human Ln-8 (Fig. 8), and mAb 1G5 raised to an
4 LG1-domain fusion protein, were generated in Lama4/ mice; each labels Ln-8 on blots (Fig. 5 a) and all
4-rich BLs in normal mice, but no BLs in Lama4/. Rabbit antibodies to integrin ß1D and agrin were gifts from Eva Engvall (Burnham Institute, La Jolla, CA) and David Glass (corporate license from Regeneron Pharmaceuticals), respectively. Other purchased antibodies: MAB1946 (entactin), MAB1948 (perlecan), AB1920 (integrin
3), and MAB1982 (integrin
6) from CHEMICON International; GoH3 (integrin
6; Beckman Coulter); CD29 (integrin ß1; BD Biosciences); VIA4-1 (
-dystroglycan; Upstate Biotechnology); ß-dystroglycan and ß-sarcoglycan (NovoCastra); neurofilament (2H3; Developmental Studies Hybridoma Bank repository, Ames, Iowa); S100 (Neomarkers); Ki67 (Vector Laboratories); and Alexa 488 (Molecular Probes, Inc.), Cy3-, and Cy5-conjugated (Jackson ImmunoResearch Laboratories) second antibodies.
Lns
Mouse Ln-1 and human Ln-2 were from CHEMICON International. Human Ln-8 was purified from T98G cellconditioned media using nondenaturing methods. Protein precipitated by 40% ammonium sulfate was bound to a DEAE-Sepharose Fast-Flow FPLC column (Amersham Biosciences) in imidazole (10 mM; pH 7.0) and eluted with a 0.051.0-M NaCl gradient. Pooled fractions containing Ln-8 (0.40.5 M NaCl) by dot-blot assay were dialyzed (5 mM phosphate and 50 mM NaCl, pH 7.3) and repurified by DEAE-Sepharose. The second DEAE pool was concentrated (Aquacide; Calbiochem) and size-fractionated by FPLC through Superose 6 HR. The final pool contained a single major protein complex at 680 kDl on silver-stained SDS-PAGE nonreducing gels (Fig. 5 a). In this material, immunoreactivity for Ln 4, ß1, and
1 chains comigrated, and
1,
2,
5, and ß2 were not detectable.
Histology
For resin sections, killed animals were perfused with 3% (wt/vol) PFA, 1% (vol/vol) glutaraldehyde, in PBS; nerves were incubated overnight at 4°C in 4% PFA, 4% glutaraldehyde in 0.1 M cacodylate; 1-mm pieces were post-fixed 1 h in 1% OsO4, dehydrated through ethanol, and embedded in Epon. Semithin sections (0.5 µm) were stained with toluidine blue (1% in alcohol) and imaged by digital color photomicroscopy. Ultrathin sections (90 nm) stained with uranyl acetate were imaged by transmission EM. Quantitation of myelination patterns was performed on photographic montages of transverse sections of the entire tibial nerve. Myelinated fibers were counted from semithin sections; nonmyelinated axons were counted from ultrathin sections on Formvar-coated hole grids photographed at 2,00010,000x.
Immunohistochemistry was done as described previously (Miner et al., 1997), using 810-µm cryostat sections cut from OCT-embedded unfixed tissue snap frozen in 150°C 2-methylbutane, or teased sciatic nerves prepared by gently spreading 12-mm segments on subbed slides. Ln 4 and ß2 epitopes required denaturation (Miner et al., 1997). In brief, sections were incubated overnight with antibodies diluted in PBS containing 5% (wt/vol) BSA, washed in PBS, and bound antibodies detected with species-specific, fluorescent second antibodies (1 h). Teased fibers were prefixed for 15 min with 2% PFA, cleared with 0.1 M glycine, and stained in PBS with 5% BSA and 0.5% Triton X-100. Hoechst 33258 (Molecular Probes, Inc.) was added to mounting medium to visualize nuclei. TUNEL staining was performed according to kit directions (Roche; product 1684795). Myelin was visible by intrinsic fluorescence (Ex365 nm/Em450 nm). Images were made at ambient temperature with PlanApo 60x (1.4 NA) oil-immersion lenses on BX microscopes (Olympus), using either a DC 350F camera and IM50 acquisition software (Leica) or an FV300 confocal scan head (Olympus). Multiply stained images were colorized and superimposed in Photoshop 6.0. Quantitation of Schwann cell nuclei was performed strictly on transverse sections of PFA-fixed medial sciatic nerves frozen in situ (the thigh). All nerve nuclei (Hoechst, total; Ki67, proliferating; TUNEL, apoptotic) not residing in perineurial sheaths (distinguished by integrin
6 counterstaining and morphology) were counted in digital images taken at 400x without background subtraction.
Cell culture
Proliferation assays used Schwann cells (9298% S100-positive) freshly prepared from desheathed E13 chick sciatic nerves (Patton et al., 1998). Plastic 96-wells were coated with poly-L-lysine (0.1 mg/ml, 1 h; Sigma-Aldrich), then Ln-1, -2, or -8 in PBS (for 8 h at 4°C; concentrations in Fig. 7). Cells were plated at indicated densities in 0.2 ml DME and 10% FCS, and were incubated at 37°C. Population levels were measured 3 d later, by release with 0.1 ml of 0.05% trypsin, 10 mM EDTA, and duplicate hemocytometer readings. Experiments included triplicate wells for each condition. In control experiments, fed by 50% medium replacement at d 3, cells remained adherent to all substrates at d 5, when TUNEL assay (Roche) labeled <2% of cells. Adhesion assays used Schwann cells cultured 10 d or less after preparation from P4 mice. Schwann cells were enriched (>97% S100-reactive) by complement-mediated lysis of fibroblasts with anti-Thy1.1 antibody (TN-26; Sigma-Aldrich), and expanded in DME, 10% FCS, and recombinant heregulin (10 ng/ml; Sigma-Aldrich). Constellations of substrate spots of Ln-1, -2, -8 (10 µg/ml PBS), and poly-D-lysine (100 µg/ml) were formed on tissue culture plastic by incubating 5-µl aliquots overnight at 4°C in a humid box. Sulforhodamine (20 µg/ml) was included to identify spot boundaries by fluorescence. Substrates were washed (PBS), blocked 412 h with 10 mg/ml Ig-free BSA, and preincubated for 30 min in DME with or without anti-Ln antibodies. Cells were collected from growth plates by minimal trypsin treatment, thrice washed in DME, resuspended to 0.5 x 106/ml in DME containing 3 mg/ml Ig-free BSA with or without blocking antibodies, preincubated 30 min at 4°C, and incubated with substrates (23,000 cells/cm2) for 2 h at 37°C. After washing with PBS, bound cells were fixed (3% PFA in PBS) and coverslipped in medium containing Hoechst. Cells were counted at 400x with phase and fluorescence optics and an eyepiece reticule. Averages were calculated from four contiguous image fields, nearly spanning each spot. Reported values show mean ± SEM for three assays.
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Acknowledgments |
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Work was funded by grants from the National Institutes of Health (NS40759 to B.L. Patton; GM60432 to J.H. Miner; and NS39550 and RR00163 to L.S. Sherman), the Muscular Dystrophy Association (to B.L. Patton), and the March of Dimes (to J.H. Miner).
Submitted: 29 November 2004
Accepted: 23 December 2004
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References |
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