- Methodology article
- Open Access
A new in vitro mouse oligodendrocyte precursor cell migration assay reveals a role for integrin-linked kinase in cell motility
© O’Meara et al. 2016
- Received: 23 July 2015
- Accepted: 24 January 2016
- Published: 1 February 2016
The decline of remyelination in chronic multiple sclerosis (MS) is in part attributed to inadequate oligodendrocyte precursor cell (OPC) migration, a process governed by the extracellular matrix (ECM). Elucidating the mechanisms underlying OPC migration is therefore an important step towards developing new therapeutic strategies to promote myelin repair. Many seminal OPC culture methods were established using rat-sourced cells, and these often need modification for use with mouse OPCs due to their sensitive nature. It is of interest to develop mouse OPC assays to leverage the abundant transgenic lines. To this end, we developed a new OPC migration method specifically suited for use with mouse-derived cells.
To validate its utility, we combined the new OPC migration assay with a conditional knockout approach to investigate the role of integrin-linked kinase (ILK) in OPC migration. ILK is a focal adhesion protein that stabilizes cellular adhesions to the extracellular matrix (ECM) by mediating a linkage between matrix-bound integrin receptors and the cytoskeleton. We identified ILK as a regulator of OPC migration on three permissive substrates. ILK loss produced an early, albeit transient, deficit in OPC migration on laminin matrix, while migration on fibronectin and polylysine was heavily reliant on ILK expression.
Inclusively, our work provides a new tool for studying mouse OPC migration and highlights the role of ILK in its regulation on ECM proteins relevant to MS.
- Mouse oligodendrocyte precursor cells
- Integrin-linked kinase
- Extracellular matrix
Oligodendrocytes (OLs) are responsible for generating myelin, a lipid-rich structure that envelops central nervous system (CNS) axons, allowing for rapid communication between neurons. OLs generate myelin as they differentiate by extending multiple processes that contact adjacent axons, forming concentric wrappings of lipid-rich OL membrane mostly devoid of cytoplasm and stabilized by myelin structural proteins . Prior to the onset of myelination, newborn OL precursor cells (OPCs) undergo a transient period of local proliferation followed by migration , their direction and extent governed by patterning molecules. Proper myelination is therefore highly dependent on the capacity of OPCs to migrate to target destinations and initiate the differentiation program. As such, researchers have endeavored to dissect the extracellular cues and the intracellular machinery governing OPC migration. This is especially relevant in neurological diseases like multiple sclerosis (MS) where the capacity for endogenous repair may be limited partly by the hampered migration of OPCs into lesions .
Over the years, our understanding of OL biology has vastly improved through the study of rat OLs in vitro, for which several isolation methods exist [4–6]. However, these strategies are often not ideal for isolating mouse OPCs for a number of reasons. First and foremost, mice possess fewer OPCs due to their smaller size. Primary mouse OPCs are also more difficult to isolate when compared to rat OPCs (reviewed by , and are less viable in vitro ). As such, researchers have endeavored to modify existing rat OPC protocols/assays to allow for their use with mouse-derived cells [7, 9–11] to take advantage of the broad spectrum of transgenic mouse lines.
We were interested in developing a mouse-optimized assay for studying OPC migration in vitro. Traditionally, OPC migration was typically assessed with the “transwell” or “agarose drop” assays . The transwell assay  measures cell migration through a porous membrane, whereas the agarose drop assay  measures radial migration out of a drop of low-melting temperature agarose. While effective with rat cells, certain aspects of these assays limit their applicability to mouse OPCs. In particular, the agarose drop assay calls for relatively long assay durations (days), which does not appease the limited viability of mouse OPCs. Both protocols also call for large numbers of OPCs, which are laborious to obtain from mice, thereby fuelling us to devise a method for assessing mouse OPC migration that is rapid and requires few cells.
We previously established a protocol for isolating mouse OPCs , and as a byproduct of the procedure, OPCs tend to form aggregates (henceforth termed OPCAs) suspended in the culture media. We show that a highly enriched population of OPCs efficiently emerges from OPCAs, and describe a method to quantifiably assess this phenomenon. By combining the OPCA assay with conditional knockout genetics, we reveal a role for the focal adhesion protein integrin-linked kinase (ILK) in OPC migration on three substrates. Inclusively, we provide a new tool for the study of mouse OPC motility in vitro, and validate its utility through use in identifying ILK as a mediator of OPC migration.
Animals used for this work were cared for according to the Canadian Council on Animal Care guidelines under University of Ottawa Animal Care Committee protocol number OGH-130. The floxed Ilk mouse line (Ilk fl/fl ; ) was graciously provided by Dr. René St-Arnaud (McGill University, Montreal, Canada). Ilk fl/fl mice were subsequently bred to homozygosity with the mT/mG reporter mouse strain  to yield the Ilk fl/fl ; mT/mG line.
Cell culture media
Mixed glial culture media was Dulbecco’s modified eagle medium (DMEM) supplemented with 1 % GlutaMAX (Life Technologies), 10 % fetal bovine serum (FBS), 0.33 % Penicillin–Streptomycin (P/S). From culture day 6 and onwards, the mixed glial culture media was supplemented with 5 µg/mL insulin.
Migration media was composed of 48–72 h conditioned mixed glial culture media (0.22 µm filtered), supplemented with 2 % B27 (Gibco), 100 µg/mL bovine serum albumin (BSA), 5 µg/mL insulin, 0.5 µg/mL Holo-transferrin, 60 ng/mL progesterone, 400 ng/mL 3,3′,5-triiodo-l-thyronine, 400 ng/mL l-thyroxine, 16 µg/mL putrescine, 5 ng/mL sodium selenite, 10 ng/µL platelet-derived growth factor (PDGF-AA; PeproTech), 50 ng/µL ciliary neurotrophic factor (CNTF; Peprotech) and 1 µg/mL aphidicolin (Sigma-Aldrich).
Conditional genetic ablation
In vitro recombination of the Ilk fl/fl and mT/mG alleles was achieved by treating mixed glial cultures with recombinant His-TAT (trans-activator of transcription)-NLS (nuclear localization sequence)-Cre recombinase (henceforth called TAT-Cre; Excellgen Incorporated). Briefly, the media was removed and the cultures were washed with PBS. TAT-Cre (or the corresponding vehicle control) was administered at 10 µL/mL in DMEM with 2 % B27 and incubated on the cultures for 1–2 h. This treatment was performed twice 48–72 h prior to OPCA isolation to promote a high level of recombination.
Immunostaining and microscopy
OPCAs were fixed with 3 % paraformaldehyde (PFA) and then either processed for indirect immunofluorescence microscopy, or mounted onto slides in DAKO medium after staining with Hoechst.
For indirect immunofluorescence, fixed OPCAs were permeabilized with 0.1 % Triton X-100 for 10 min at room temperature (RT), and washed with PBS. OPCAs were blocked with 10 % goat serum, and incubated overnight at 4 °C with primary antibodies against Olig2 (EMD Millipore) or PDGFR-α (provided by Dr. William Stallcup, Sanford Burnham Medical Research Institute, La Jolla, California). The following day, the samples were washed with PBS and incubated with Alexa fluor-conjugated secondary antibodies (invitrogen) for 45 min to 1 h at RT. Coverslips were subsequently washed with PBS, counterstained with Hoechst and mounted onto slides in DAKO medium.
Standard epifluorescent images were acquired with an Axio Imager M1 microscope equipped with an AxioCamHR HRm Rev.2 camera using EX Plan-Neofluar 10x/0.3 Ph1 or Fluar 5x/0.25 M27 objectives and Axiovision 4.8.2 software. Confocal microscopy was conducted with a Zeiss LSM 510 Meta DuoScan microscope using EC Plan-Neofluar 10×/0.3 M27, 20×/0.5 M27, 40×/1.3 Oil DIC M27 or Plan-Apochromat 63×/1.4 Oil DIC M27 objectives and Zen 8.0 software.
Phase contrast microscopy was performed with an Axiovert 200 M microscope fitted with an AxioCamHR HRm Rev.2 camera using LD Plan Neofluar 20×/0.4 Korr Ph2 or EC Plan-Neofluar 10×/0.25 Ph1 objectives and Axiovision 4.6 software.
Phase contrast images of OPCAs were acquired at time zero to record their original diameter. Migration assays were fixed at 4, 10 or 24 h and imaged by fluorescence microscopy. Using Photoshop, “exclusion zones” were digitally overlaid onto the residual OPCA core, serving to define the starting point of migration; only cells beyond the exclusion zone were considered to have truly migrated. For OPCAs seeded on laminin and fibronectin substrates, the size of the exclusion zone was 1.5 × the OPCA diameter at time zero. For the poly-d-lysine substrate, the exclusion zone was 1.75×, 2.0× and 2.75× the time zero OPCA diameter for 4, 10 and 24 h time points, respectively.
Concentric rings were then digitally centered over the exclusion zone, their diameter increment being 50, 100 and 200 µm for 4, 10 and 24 h time points for assays conducted on laminin. For the fibronectin substrate, the ring diameter increment was 20, 40 and 80 µm for the 4, 10 and 24 h time points, respectively. For the poly-d-lysine substrate, the ring increment for the 4, 10 and 24 h time points was 10, 20 and 40 µm, respectively. The number of OPCs that migrated to each concentric ring was quantified and represented as the percentage of OPCs in each ring. This can be considered a measure of migration distance or be represented as the total number of migrated OPCs, regardless of distance. We interpret this latter parameter as a function of the cell to initiate migration or polarize.
Mixed glial cultures were cooled on ice for 3 min, rinsed with ice cold PBS, and scraped into a commercial RIPA lysis buffer (cell signaling) supplemented with 2 mM PMSF. Lysates were left on ice for approximately 2 min, and centrifuged at 15,000 rpm for 5 min. Clarified lysates were transferred to new microfuge tubes and stored at −80 °C.
For SDS-PAGE, 30 µg of cell lysate per sample was resolved on a poly-acrylamide gel, and transferred to a PDVF membrane at 0.25 amps for 70 min. Membranes were blocked for approximately 1 h at RT with 5 % skim milk power in TBST (10 mM Tris–HCl pH 8.0, 150 mM NaCl, 0.1 % Tween-20). Primary antibodies against ILK (cell signaling) and GAPDH (Abcam) were incubated overnight in blocking buffer. Membranes were washed with TBST and probed with HRP-conjugated secondary antibody for 45 min. Membranes were then washed with PBS, and treated with ECL (Thermo Scientific). Resulting films were scanned with an EPSON Perfection 2450 PHOTO scanner, imported into ImageJ, and a box of standard dimensions was placed over each band to measure the mean gray value. Densitometric values for ILK were normalized to GAPDH as a loading control.
One “n” (i.e., experiment) was considered as data obtained from pooled biological material (i.e., cells) from mouse pups (usually 4–6) of a distinct litter. Differences in migration distance were assessed using two-way repeated measures ANOVA paired with Bonferroni multiple comparisons tests. All other data was tested using either unpaired two-tailed Student’s t tests or one-way ANOVA paired with Bonferroni multiple comparisons tests. For all statistical tests, “n” was equal to or greater than three, and differences in the mean were considered significant when p < 0.05.
An assay for investigating mouse OPC migration in vitro
In 1980, McCarthy and DeVellis published a seminal paper describing the isolation and propagation of primary rat OPCs in mixed glial cultures. In this method, neonatal cortical tissue is dissociated into a single cell suspension and seeded into tissue culture flasks. Over several days, astrocytes form a monolayer on the base of the flask, upon which OPCs proliferate. The OPCs are relatively loosely attached to this monolayer, and are susceptible to separation when flasks are shaken on an orbital rotator overnight at 37 °C. This procedure renders the suspension of OPCs in the culture medium, many of which form aggregates (OPCAs) due to their innate tendency to do so .
In our mouse adaptation of the McCarthy and deVellis method , we took advantage of this aggregation phenomenon to assess OPC migration. After the overnight shaking step, the medium containing the OPCAs was filtered through a 40 μm cell strainer, and then backwashed with migration media into a 50 mL conical tube (Fig. 1a–c). The backwashed media was then transferred to a Petri dish, and OPCAs were picked with a micropipette by aid of a stereomicroscope. As a result of the mechanism of their formation, OPCAs can be heterogeneous in shape, and care was taken in selecting the most uniform aggregates for migration experiments (Fig. 1d).
Once selected, OPCAs were seeded individually onto substrate-coated coverslips in 48–72 h conditioned mixed glial culture media supplemented with various OL survival factors (see “Methods” for details). The purpose of this media was to favor the maintenance of OPCs in a precursor state, as migration generally ceases in differentiated OLs. We also aimed to control OPC proliferation by including aphidicolin (mitotic inhibitor) in the migration media, as excessive proliferation could give the false impression of extensive migration.
Conditional ablation of ILK from OPCAs
To this end, we employed a mutant mouse possessing loxP sites flanking exons 5–12 of the endogenous Ilk gene (Ilk fl/fl ; Fig. 4b), where recombination presumably produces null alleles . We then bred Ilk fl/fl ;mT/mG double transgenics by crossing Ilk fl/fl mice with the mT/mG strain; a reporter line harboring a locus coding for near-ubiquitous expression of the fluorescent protein tdTomato . The action of Cre recombinase at this locus ablates tdTomato, while at the same time inducing EGFP (Fig. 4c), thereby permitting identification of Cre-recombined cells. Therefore, recombined cells should express EGFP and be devoid of ILK, while non-recombined cells remain tdTomato+ and retain ILK expression.
Mixed glial cultures were then established from Ilk fl/fl ;mT/mG mice and treated with cell-permeable TAT-Cre recombinase or vehicle control (see “Methods” for details). As expected, TAT-Cre administration led to a reduction in total ILK protein in Ilk fl/fl ;mT/mG cultures as compared with vehicle-treated controls (Fig. 4d). We then isolated OPCAs from TAT-Cre treated cultures and allowed OPCs to migrate outwards for 10 h. Cre-recombined OPCs could be identified by their expression of EGFP, although non-recombined (tdTomato+) OPCs were still a significant component of the TAT-Cre treated OPCA (Fig. 4e). In sum, our work shows that the OPCA assay can be used in conjunction with conditional knockout genetics, and we next aimed to utilize this strategy to investigate the role of ILK in OPC migration.
Loss of ILK mildly impairs OPC migration on laminin
Merosin, a blanket term for α2-chain containing laminin (Ln) proteins (Ln-2, Ln-4 and Ln-13;  influences various aspects of OL biology including growth factor-mediated survival [27, 28], morphological development , and CNS myelination . Notably, myelinating CNS white matter tracts express the Ln α2-chain [30, 31], which is also upregulated after experimentally induced demyelination ; reviewed by . In addition, Ln promotes OPC migration [34–36], rendering Ln signaling pathways appealing pharmacological targets for overcoming the inhibitory barriers impeding OPC infiltration into MS lesions.
ILK loss severely disrupts OPC migration on fibronectin
Fibronectin (Fn) is a migration-permissive substrate for OPCs [34, 37], but evidence suggests that Fn aggregation impedes remyelination in MS . This underscores the importance of elucidating the signaling pathways that govern OPC migration on Fn, as their pharmacological manipulation may influence the degree to which OPCs infiltrate MS lesions. OL-lineage cells express the αvβ1, αvβ3, αvβ5 and αvβ8 integrin receptors which recognize the Arg-Gly-Asp (RGD) integrin binding sequence intrinsic to Fn and other ECM molecules [37, 39]. As ILK is recruited to β1 and β3 integrin tails to stabilize actin at cell–matrix contacts, we hypothesized that ILK-depleted OPCs would suffer from defective migration on Fn substrates.
ILK is required for OPC migration on polylysine matrix
Poly-d-lysine (PDL) is a molecule that carries a net positive charge and interacts with anionic cell membrane domains to mediate adhesion . While polylysine itself does not mediate cell adhesion via the integrins , it can promote the deposition of cell culture media-borne proteins , some of which may activate integrins to regulate migration. PDL can be thought of as a substrate that provides both a non-specific charge-based affinity for cells, and one that facilitates veritable receptor-ligand interactions.
In this report, we demonstrate a role for ILK in OPC migration by utilizing a newly developed method tailored for mouse-derived cells. We show ILK regulates OPC migration on Ln and Fn, two ECM proteins present in demyelinated lesions [32, 38] as well as on a polylysine matrix. Impaired OPC infiltration into MS lesions is thought to partly underlie the lack of regeneration seen in chronic MS, emphasizing the need to understand the mechanics governing OPC migration, especially when pursuing remyelination therapies. Our work contributes to this increasing understanding, while also providing a new tool for investigating OPC migration that is amenable to both constitutive and conditional knockout/transgenic mouse lines.
An assay to study mouse OPC migration in vitro
The use of mouse OPCs in basic research is becoming increasingly popular, a trend surely reflecting the accessibility of mutant lines and technological advancements in their generation. However, when compared to rat-sourced cells, the utility of mouse OPCs is limited as they are more difficult to isolate  and do not survive as well in culture . This imposes constraints on the commonly used “agarose drop” and “transwell” migration assays, emphasizing the need for methods that account for the shortcomings of mouse OPCs. We estimate that an OPCA is composed of approximately 300-1000 cells, and a typical preparation yields roughly 50–100 OPCAs. Additionally, migration can be measured in as little as 4 h rather than several days, as is the case for the agarose drop assay. Our assay therefore accommodates the limitations of mouse OPCs by calling for few cells and requiring relatively short assay durations.
Our method bears resemblance to the “oligosphere” assay, originally described by . In this method, rat OPCs are purified using a Percoll gradient, and seeded onto uncoated tissue culture vessels. Over several days, suspended OPCs form spherical aggregates reminiscent of neurospheres, while their OL identity is maintained through use of N1 supplemented B104-conditioned media. When seeded onto polyornithine substrates, OPCs emerge from oligospheres in a radial fashion as in our OPCA assay. This method has been applied to elegantly demonstrate that polysialylation of neural cell adhesion molecule (PSA-NCAM) favours OPC migration , and been leveraged to identify the importance of metalloproteases in OPC migration on CNS matrices . To modify the assay for use with mouse cells,  increased the concentration of insulin and progesterone in the oligosphere culture media to enhance OPC proliferation and/or ameliorate viability .
One major benefit of the OPCA assay over the oligosphere method is that aggregate generation requires less researcher manipulation. To generate OPC aggregates in the OPCA assay, mixed glial cultures are simply shaken on an orbital rotator overnight in a tissue culture incubator. To generate oligospheres however, multiple 18 h differential adhesion steps need be conducted . Our method also describes an efficient means to conditionally ablate/modify genes using LoxP technology, which is of high value when investigating genes whose constitutive loss leads to embryonic lethality.
Another feature of the OPCA method is its potential use in live cell imaging, facilitating the observation of OPC migration in real-time. In our hands however, we have experienced phototoxicity as a result of visualizing fluorescent proteins (e.g., EGFP, tdTomato) in live OPCs (data not shown). In addition, live imaging may not be suitable when assessing the effect of photosensitive compounds on OPC migration.
ILK in OPC migration on various substrates
Interactions between the ECM and OL-bound integrin receptors govern numerous aspects of OL biology (reviewed by . OPCs express the α6β1 integrin Ln receptor along with the αvβ1 and αvβ3 integrin Fn receptors , to which ILK binds at β1 and β3 cytoplasmic tails [19, 20]. The impaired migration of Ilk −/− OPCs on Ln and Fn matrices likely results from perturbed stability of connections between the ECM and the actin cytoskeleton (Fig. 4a). Disrupting these adhesion complexes would likely translate to difficulty stabilizing a leading process, and compromise the cell’s ability to utilize focal contacts to transduce cytoskeletal contractile forces required for migration.
Interestingly, ILK loss produced only a mild deficit in OPC motility on Ln, in that migration was lagging at 4 h, but had recovered at the 10 and 24 h time points. As ILK stabilizes α6β1 integrins on Ln, our findings are in agreement with previous studies showing a role for α6β1 integrin only during the early phases of OPC migration , with no significant contribution in the long term . These data insinuate a more significant role for α6β1 integrin in other aspects of OL biology, as it is the major β1-type receptor expressed by OLs, and is consistently expressed throughout OL development . Accordingly, extension of OL myelin membrane , and CNS myelination [45, 46] are regulated by Ln-α6β1 integrin interactions, while similar functions have been identified for ILK [24, 25]. The Ln-α6β1-ILK complex may therefore be more relevant in post-migratory OL development, while only mildly contributing to Ln-directed motility. The observation that efficient migration still occurred on Ln in the absence of ILK suggests alternate signaling mechanisms predominantly drive migration on this substrate.
We offer the possibility that OPCs express additional, ILK-independent receptors that contribute to OPC migration on Ln. Existence of such receptors could explain how Ilk −/− OPCs are only mildly incapacitated in their migration on Ln, and ultimately travel to the same degree as control cells. One candidate is dystroglycan, a Ln binding protein expressed by OL-lineage cells , with a function in OL process outgrowth . Dystroglycan localizes to OL adhesion contact sites, and co-precipitates with focal adhesion kinase . Recent work has also shown that nestin-Cre driven deletion of dystroglycan negatively impacts cerebellar granule neuron migration , offering a parallel function for dystroglycan in OPCs, as neurons and OLs share much of the same migratory mechanisms . The role of dystroglycan (and/or other Ln adhesion molecules) in OPC migration remains an open question for investigation, and may provide an explanation for the migration-permissive character of Ln.
In contrast to Ln, for which α6β1 is the exclusive integrin receptor, OPCs express the Fn-sensitive αvβ1, αvβ3, αvβ5 and αvβ8 integrins . With regard to our work, αvβ3 and αvβ1 are most relevant, as ILK exclusively binds β1 and β3 cytosolic tails [19, 20].  previously demonstrated a role for αvβ3 in OPC migration on Fn, while, in contrast, αvβ1 was determined dispensable. A role for β1 integrin in OPC migration on Fn was suggested by Tiwari-Woodruff and colleagues in 2001, although in their study, migration was assessed after 4 days in vitro, a point at which OPC proliferation may confound observations. Rather, in line with , we favor the concept that the migratory phenotype manifested by Ilk −/− OPCs on Fn is primarily a product of perturbed signaling/stability at Fn-αvβ3-actin connections. Importantly, this does not discount a role for αvβ1 integrin in OPC migration in a broader context, as this receptor significantly mediates OPC migration on astrocyte-produced ECM , a substrate surely composed of various integrin ligands. Ilk −/− OPCs were not completely ablated in their ability to migrate on Fn, suggesting that receptors other than αvβ3 and αvβ1 may be at play. Of note,  previously found no role for αvβ5 in OPC migration on Fn, leaving the less-characterized αvβ8 integrin receptor with a possible function in this process .
In contrast to Ln and Fn that bind specific receptors on the cell surface, the adhesion of cells to PDL is mediated by charge interactions between anionic membrane domains and the positively charged nature of polylysine . Polylysine is not thought to engage integrins, although it is capable of binding ECM proteins present in cell culture media . We propose the impaired migration of Ilk −/− OPCs on PDL is a result of this latter phenomenon, where ILK-devoid cells are compromised in their ability to engage PDL-immobilized ligands contributed by the media. Fetal bovine serum, which makes up 10 % of our migration media, is known to contain the ECM protein vitronectin. Similar to Fn, vitronectin also possesses the arginine-glycine-aspartic acid (RGD) motif recognized by many integrin receptors . In fact, vitronectin is believed to be the major cell-attachment protein present in fetal bovine serum . OLs express αvβ1, αvβ3, αvβ5 and αvβ8 integrins, which all possess affinity for vitronectin [53–56]. As ILK only binds β1 and β3 integrins, we can speculate that defective migration of Ilk −/− OPCs on PDL is caused partly by the reduced stability of αvβ1, αvβ3 integrin adhesions responsible for stabilizing connections between vitronectin and the cytoskeleton.
Cell signaling in OPC migration and relevance to MS therapy development
Oligodendrocyte precursor cells (OPCs) migrate extensively prior to the onset of central nervous system (CNS) myelination  a process governed in part by the extracellular matrix (ECM) during both normal development and remyelination of demyelinated lesions . Endogenous lesion repair in MS decreases with disease progression, a consequence of reduced OPC proliferation, differentiation and/or migration . Of note, OPCs accumulate at the periphery of early MS lesions , which is thought to be a result of lesion-derived inhibitory factors impeding OPC recruitment inwards [60–64]. Elucidating the molecular mechanisms that govern OPC migration will therefore facilitate the design of therapeutics aimed at promoting OPC infiltration into MS lesions.
We have previously shown that ILK-devoid OLs possess elevated RhoA activity, a phenomenon associated with a disorganized actin cytoskeleton . This observation agrees with previous work from other cell systems [65, 66]; reviewed by [21, 22], where ILK loss leads to an upregulation in RhoA activity, while concomitantly reducing cell–matrix adhesions . Conversely, reducing RhoA activity promotes reorganization of focal contacts , and diminishes actomyosin contractility to facilitate migration (reviewed by . It is quite likely then, that an ideal level of RhoA activity exists that promotes persistent directional migration when properly localized subcellularly. In such a case, localized RhoA activity would promote cell migration by preventing the extension of numerous leading lamellipodia, ensuring focal contacts remain dynamic and by controlling the cleavage of trailing-edge adhesions. If Ilk −/− OPCs do suffer from elevated RhoA activity, the dysregulation of these cellular processes could explain their compromised migratory capacity. While we did not test this hypothesis, it is an intriguing direction for future research.
The RhoA signaling axis is becoming of increasing interest with regard to MS therapeutics. Other proteins such as Netrin-1, Slit2 and NG2 mediate OPC migration through modulation of RhoA signaling [70–72]. RhoA pathway downregulation by pharmacological means enhances morphological development of OL-lineage cells [25, 73, 74], even in the presence of OL-inhibitory factors [75, 76]. Blockade of LINGO-1, an upstream regulator of RhoA (reviewed by , not only facilitates OL differentiation in vitro  but also enhances remyelination in vivo . As multiple signaling pathways converge on RhoA to regulate OL biology, as well as the fact that RhoA-ROCK pathway inhibitors are currently in use for other CNS ailments , their efficacy in promoting remyelination in MS disease models remains an appealing direction for future investigation.
Here, we present a new OPC migration assay tailored specifically for use with mouse-derived cells, which we name the OPCA assay. OPCAs are highly enriched for OPCs, and when seeded onto Ln, Fn or polylysine, rapid outward migration occurs. We further show that the OPCA method is amenable to conditional gene ablation through use of TAT-Cre recombinase to deplete ILK from OPCAs generated from Ilk fl/fl mice. ILK-devoid OPCs were heavily impaired at migrating on Fn or polylysine matrices, while only mildly delayed in their migration on Ln. Inclusively, our work identifies ILK as a player in OPC migration, and provides a new tool for the study of mouse OPC motility in vitro.
RWO developed the OPC migration assay, performed all of the studies, participated in the analysis, performed the statistical analysis and drafted the manuscript. SEC and JPM helped perform some of the experiments. RK participated in the design of the study and coordination, and helped to draft the manuscript. All authors read and approved the final manuscript.
The authors would like to thank Dr. René St-Arnaud and Dr. Steffany Bennett for generous donation of transgenic mice. We also thank Dr. William Stallcup for providing the PDGFR-α antibody. This work was supported by a grant to RK from the Multiple Sclerosis Society of Canada. RWO and JPM are both recipients of the Frederick Banting and Charles Best Canadian Institutes of Health Research Doctoral Award, SEC is a recipient of an Ontario Graduate Scholarship and RK is a recipient of a University Health Research Chair from the University of Ottawa.
The authors declare that they have no competing interests.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
- Snaidero N, Möbius W, Czopka T, Hekking LHP, Mathisen C, Verkleij D, Goebbels S, Edgar J, Merkler D, Lyons DA, Nave K-A, Simons M. Myelin membrane wrapping of CNS axons by PI(3,4,5)P3-dependent polarized growth at the inner tongue. Cell. 2014;156:277–90.View ArticlePubMedGoogle Scholar
- Noll E, Miller RH. Oligodendrocyte precursors originate at the ventral ventricular zone dorsal to the ventral midline region in the embryonic rat spinal cord. Development. 1993;118:563–73.PubMedGoogle Scholar
- Keough MB, Yong VW. Remyelination therapy for multiple sclerosis. Neurotherapeutics. 2013;10:44–54.PubMed CentralView ArticlePubMedGoogle Scholar
- McCarthy KD, de Vellis J. Preparation of separate astroglial and oligodendroglial cell cultures from rat cerebral tissue. J Cell Biol. 1980;85:890–902.View ArticlePubMedGoogle Scholar
- Avellana-Adalid V, Nait-Oumesmar B, Lachapelle F, Baron-Van Evercooren A. Expansion of rat oligodendrocyte progenitors into proliferative “oligospheres” that retain differentiation potential. J Neurosci Res. 1996;45:558–70.View ArticlePubMedGoogle Scholar
- Stallcup WB, Beasley L. Bipotential glial precursor cells of the optic nerve express the NG2 prtoglycan. J Neurosci. 1987;7(9):2737–44.PubMedGoogle Scholar
- Chen Y, Balasubramaniyan V, Peng J, Hurlock EC, Tallquist M, Li J, Lu QR. Isolation and culture of rat and mouse oligodendrocyte precursor cells. Nat Protoc. 2007;2:1044–51.View ArticlePubMedGoogle Scholar
- Horiuchi M, Lindsten T, Pleasure D, Itoh T. Differing in vitro survival dependency of mouse and rat NG2 + oligodendroglial progenitor cells. J Neurosci Res. 2010;88:957–70.PubMed CentralPubMedGoogle Scholar
- O’Meara RW, Ryan SD, Colognato H, Kothary R. Derivation of enriched oligodendrocyte cultures and oligodendrocyte/neuron myelinating co-cultures from post-natal murine tissues. J Vis Exp. 2011.Google Scholar
- Pedraza CE, Monk R, Lei J, Hao Q, Macklin WB. Production, characterization, and efficient transfection of highly pure oligodendrocyte precursor cultures from mouse embryonic neural progenitors. Glia. 2008;56:1339–52.PubMed CentralView ArticlePubMedGoogle Scholar
- Vitry S, Avellana-Adalid V, Hardy R, Lachapelle F, Barone-Van Evercooren A. Mouse oligospheres: from pre-progenitors to functional oligodendrocytes. J Neurosci Res. 1999;58(6):735–51.View ArticlePubMedGoogle Scholar
- Frost EE, Milner R, Ffrench-Constant C. Migration assays for oligodendrocyte precursor cells. Methods in molecular biology. 2000;139:265–78.PubMedGoogle Scholar
- Boyden S. The chemotactic effect of mixtures of antibody and antigen on polymorphonuclear leucocytes. J Exp Med. 1962;115:453–66.PubMed CentralView ArticlePubMedGoogle Scholar
- Varani J, Orr W, Ward PA. A comparison of the migration patterns of normal and malignant cells in two assay systems. Am J Pathol. 1978;90:159–72.PubMed CentralPubMedGoogle Scholar
- Terpstra L, Prud’homme J, Arabian A, Takeda S, Karsenty G, Dedhar S, St-Arnaud R. Reduced chondrocyte proliferation and chondrodysplasia in mice lacking the integrin-linked kinase in chondrocytes. J Cell Biol. 2003;162:139–48.PubMed CentralView ArticlePubMedGoogle Scholar
- Muzumdar MD, Tasic B, Miyamichi K, Li L, Luo L. A global double-fluorescent Cre reporter mouse. Genesis. 2007;2000(45):593–605.View ArticleGoogle Scholar
- Nishiyama A, Komitova M, Suzuki R, Zhu X. Polydendrocytes (NG2 cells): multifunctional cells with lineage plasticity. Nat Rev Neurosci. 2009;10:9–22.View ArticlePubMedGoogle Scholar
- Milner R, Ffrench-Constant C. A developmental analysis of oligodendroglial integrins in primary cells: changes in alpha v-associated beta subunits during differentiation. Development. 1994;120:3497–506.PubMedGoogle Scholar
- Hannigan GE, Leung-Hagesteijn C, Fitz-Gibbon L, Coppolino MG, Radeva G, Filmus J, Bell JC, Dedhar S. Regulation of cell adhesion and anchorage-dependent growth by a new beta 1-integrin-linked protein kinase. Nature. 1996;379:91–6.View ArticlePubMedGoogle Scholar
- Pasquet J-M, Noury M, Nurden AT. Evidence that the platelet integrin alphaIIb beta3 is regulated by the integrin-linked kinase, ILK, in a PI3-kinase dependent pathway. Thromb Haemost. 2002;88:115–22.PubMedGoogle Scholar
- Ghatak S, Morgner J, Wickström SA. ILK: a pseudokinase with a unique function in the integrin-actin linkage. Biochem Soc Trans. 2013;41:995–1001.View ArticlePubMedGoogle Scholar
- Wickström SA, Lange A, Montanez E, Fässler R. The ILK/PINCH/parvin complex: the kinase is dead, long live the pseudokinase. EMBO J. 2010;29:281–91.PubMed CentralView ArticlePubMedGoogle Scholar
- Legate KR, Montañez E, Kudlacek O, Fässler R. ILK, PINCH and parvin: the tIPP of integrin signalling. Nat Rev Mol Cell Biol. 2006;7:20–31.View ArticlePubMedGoogle Scholar
- Chun SJ, Rasband MN, Sidman RL, Habib AA, Vartanian T. Integrin-linked kinase is required for laminin-2-induced oligodendrocyte cell spreading and CNS myelination. J Cell Biol. 2003;163:397–408.PubMed CentralView ArticlePubMedGoogle Scholar
- O’Meara RW, Michalski J-P, Anderson C, Bhanot K, Rippstein P, Kothary R. Integrin-linked kinase regulates process extension in oligodendrocytes via control of actin cytoskeletal dynamics. J Neurosci. 2013;33:9781–93.View ArticlePubMedGoogle Scholar
- Wewer UM, Engvall E. Merosin/laminin-2 and muscular dystrophy. Neuromuscul Disord. 1996;6:409–18.View ArticlePubMedGoogle Scholar
- Decker L, ffrench-Constant C. Lipid rafts and integrin activation regulate oligodendrocyte survival. J Neurosci. 2004;24:3816–25.View ArticlePubMedGoogle Scholar
- Laursen LS, Chan CW, ffrench-Constant C. An integrin-contactin complex regulates CNS myelination by differential Fyn phosphorylation. J Neurosci. 2009;29:9174–85.PubMed CentralView ArticlePubMedGoogle Scholar
- Buttery P, Ffrench-Constant C. Laminin-2/integrin interactions enhance myelin membrane formation by oligodendocytes. Mol Cell Neurosci. 1999;14:199–212.View ArticlePubMedGoogle Scholar
- Colognato H, Baron W, Avellana-Adalid V, Relvas JB, Baron-Van Evercooren A, Georges-Labouesse E, ffrench-Constant C. CNS integrins switch growth factor signalling to promote target-dependent survival. Nat Cell Biol. 2002;4:833–41.View ArticlePubMedGoogle Scholar
- Morissette N, Carbonetto S. Laminin alpha 2 chain (M chain) is found within the pathway of avian and murine retinal projections. J Neurosci. 1995;15:8067–82.PubMedGoogle Scholar
- Zhao C, Fancy SPJ, Franklin RJM, ffrench-Constant C. Up-regulation of oligodendrocyte precursor cell alphaV integrin and its extracellular ligands during central nervous system remyelination. J Neurosci Res. 2009;87:3447–55.View ArticlePubMedGoogle Scholar
- Colognato H, Tzvetanova ID. Glia unglued: how signals from the extracellular matrix regulate the development of myelinating glia. Developmental neurobiology. 2011;71:924–55.View ArticlePubMedGoogle Scholar
- Frost E, Kiernan BW, Faissner A, ffrench-Constant C. Regulation of oligodendrocyte precursor migration by extracellular matrix: evidence for substrate-specific inhibition of migration by tenascin-C. Dev Neurosci. 1996;18:266–73.View ArticlePubMedGoogle Scholar
- Niehaus A, Stegmüller J, Diers-Fenger M, Trotter J. Cell-surface glycoprotein of oligodendrocyte progenitors involved in migration. J Neurosci. 1999;19:4948–61.PubMedGoogle Scholar
- Schmidt C, Ohlemeyer C, Labrakakis C, Walter T, Kettenmann H, Schnitzer J. Analysis of motile oligodendrocyte precursor cells in vitro and in brain slices. Glia. 1997;20:284–98.View ArticlePubMedGoogle Scholar
- Milner R, Edwards G, Streuli C, Ffrench-Constant C. A role in migration for the alpha V beta 1 integrin expressed on oligodendrocyte precursors. J Neurosci. 1996;16:7240–52.PubMedGoogle Scholar
- Stoffels JMJ, de Jonge JC, Stancic M, Nomden A, van Strien ME, Ma D, Sisková Z, Maier O, Ffrench-Constant C, Franklin RJM, Hoekstra D, Zhao C, Baron W. Fibronectin aggregation in multiple sclerosis lesions impairs remyelination. Brain. 2013;136:116–31.View ArticlePubMedGoogle Scholar
- Gudz TI, Komuro H, Macklin WB. Glutamate stimulates oligodendrocyte progenitor migration mediated via an alphav integrin/myelin proteolipid protein complex. J Neurosci. 2006;26:2458–66.View ArticlePubMedGoogle Scholar
- Mazia D, Schatten G, Sale W. Adhesion of cells to surfaces coated with polylysine. Applications to electron microscopy. J Cell Biol. 1975;66:198–200.View ArticlePubMedGoogle Scholar
- Machesky LM, Hall A. Role of actin polymerization and adhesion to extracellular matrix in Rac- and Rho-induced cytoskeletal reorganization. J Cell Biol. 1997;138:913–26.PubMed CentralView ArticlePubMedGoogle Scholar
- Mao X, Peng H, Ling J, Friis T, Whittaker AK, Crawford R, Xiao Y. Enhanced human bone marrow stromal cell affinity for modified poly(L-lactide) surfaces by the upregulation of adhesion molecular genes. Biomaterials. 2009;30:6903–11.View ArticlePubMedGoogle Scholar
- Decker L, Avellana-Adalid V, Nait-Oumesmar B, Durbec P, Baron-Van Evercooren A. Oligodendrocyte precursor migration and differentiation: combined effects of PSA residues, growth factors, and substrates. Mol Cell Neurosci. 2000;16:422–39.View ArticlePubMedGoogle Scholar
- Amberger VR, Avellana-Adalid V, Hensel T, Baron-Van Evercooren A, Schwab ME. Oligodendrocyte-type 2 astrocyte progenitors use a metalloendoprotease to spread and migrate on CNS myelin. Eur J Neurosci. 1997;9(1):151–62.View ArticlePubMedGoogle Scholar
- Câmara J, Wang Z, Nunes-Fonseca C, Friedman HC, Grove M, Sherman DL, Komiyama NH, Grant SG, Brophy PJ, Peterson A, Ffrench-Constant C. Integrin-mediated axoglial interactions initiate myelination in the central nervous system. J Cell Biol. 2009;185:699–712.PubMed CentralView ArticlePubMedGoogle Scholar
- Lee KK, De Repentigny Y, Saulnier R, Rippstein P, Macklin WB, Kothary R. Dominant-negative beta1 integrin mice have region-specific myelin defects accompanied by alterations in MAPK activity. Glia. 2006;53:836–44.View ArticlePubMedGoogle Scholar
- Colognato H, Galvin J, Wang Z, Relucio J, Nguyen T, Harrison D, Yurchenco PD, Ffrench-Constant C. Identification of dystroglycan as a second laminin receptor in oligodendrocytes, with a role in myelination. Development. 2007;134:1723–36.View ArticlePubMedGoogle Scholar
- Eyermann C, Czaplinski K, Colognato H. Dystroglycan promotes filopodial formation and process branching in differentiating oligodendroglia. J Neurochem. 2012;120:928–47.PubMedGoogle Scholar
- Nguyen H, Ostendorf AP, Satz JS, Westra S, Ross-Barta SE, Campbell KP, Moore SA. Glial scaffold required for cerebellar granule cell migration is dependent on dystroglycan function as a receptor for basement membrane proteins. Acta Neuropathol Commun. 2013;1:58.PubMed CentralView ArticlePubMedGoogle Scholar
- De Castro F, Bribián A. The molecular orchestra of the migration of oligodendrocyte precursors during development. Brain Res Brain Res Rev. 2005;49:227–41.View ArticlePubMedGoogle Scholar
- Ruoslahti E. RGD and other recognition sequences for integrins. Annu Rev Cell Dev Biol. 1996;12:697–715.View ArticlePubMedGoogle Scholar
- Hayman EG, Pierschbacher MD, Suzuki S, Ruoslahti E. Vitronectin—a major cell attachment-promoting protein in fetal bovine serum. Exp Cell Res. 1985;160(2):245–58.View ArticlePubMedGoogle Scholar
- Bodary SC, McLean JW. The integrin beta 1 subunit associates with the vitronectin receptor alpha v subunit to form a novel vitronectin receptor in a human embryonic kidney cell line. J Biol Chem. 1990;265:5938–41.PubMedGoogle Scholar
- Nishimura SL, Sheppard D, Pytela R. Integrin alpha v beta 8. Interaction with vitronectin and functional divergence of the beta 8 cytoplasmic domain. J Biol Chem. 1994;269(28):708–15.Google Scholar
- Pytela R, Pierschbacher MD, Ruoslahti E. A 125.115-kDa cell surface receptor specific for vitronectin interacts with the arginine-glycine-aspartic acid adhesion sequence derived from fibronectin. Proc Natl Acad Sci USA. 1985;82:5766–70.PubMed CentralView ArticlePubMedGoogle Scholar
- Wayner EA, Orlando RA, Cheresh DA. Integrins alpha v beta 3 and alpha v beta 5 contribute to cell attachment to vitronectin but differentially distribute on the cell surface. J Cell Biol. 1991;113(4):919–29.View ArticlePubMedGoogle Scholar
- De Castro F, Bribián A, Ortega MC. Regulation of oligodendrocyte precursor migration during development, in adulthood and in pathology. Cell Mol Life Sci. 2013;70:4355–68.View ArticlePubMedGoogle Scholar
- Lau LW, Cua R, Keough MB, Haylock-Jacobs S, Yong VW. Pathophysiology of the brain extracellular matrix: a new target for remyelination. Nat Rev Neurosci. 2013;14:722–9.View ArticlePubMedGoogle Scholar
- Kuhlmann T, Miron V, Cui Q, Cuo Q, Wegner C, Antel J, Brück W. Differentiation block of oligodendroglial progenitor cells as a cause for remyelination failure in chronic multiple sclerosis. Brain. 2008;131:1749–58.View ArticlePubMedGoogle Scholar
- Bin JM, Rajasekharan S, Kuhlmann T, Hanes I, Marcal N, Han D, Rodrigues SP, Leong SY, Newcombe J, Antel JP, Kennedy TE. Full-length and fragmented netrin-1 in multiple sclerosis plaques are inhibitors of oligodendrocyte precursor cell migration. Am J Pathol. 2013;183:673–80.View ArticlePubMedGoogle Scholar
- Boyd A, Zhang H, Williams A. Insufficient OPC migration into demyelinated lesions is a cause of poor remyelination in MS and mouse models. Acta Neuropathol. 2013;125:841–59.PubMed CentralView ArticlePubMedGoogle Scholar
- Clemente D, Ortega MC, Arenzana FJ, de Castro F. FGF-2 and Anosmin-1 are selectively expressed in different types of multiple sclerosis lesions. J Neurosci. 2011;31:14899–909.View ArticlePubMedGoogle Scholar
- Sobel RA, Ahmed AS. White matter extracellular matrix chondroitin sulfate/dermatan sulfate proteoglycans in multiple sclerosis. J Neuropathol Exp Neurol. 2001;60:1198–207.View ArticlePubMedGoogle Scholar
- Williams A, Piaton G, Aigrot M-S, Belhadi A, Théaudin M, Petermann F, Thomas J-L, Zalc B, Lubetzki C. Semaphorin 3A and 3F: key players in myelin repair in multiple sclerosis? Brain. 2007;130:2554–65.View ArticlePubMedGoogle Scholar
- Blumbach K, Zweers MC, Brunner G, Peters AS, Schmitz M, Schulz J-N, Schild A, Denton CP, Sakai T, Fässler R, Krieg T, Eckes B. Defective granulation tissue formation in mice with specific ablation of integrin-linked kinase in fibroblasts—role of TGFβ1 levels and RhoA activity. J Cell Sci. 2010;123:3872–83.View ArticlePubMedGoogle Scholar
- Kogata N, Tribe RM, Fässler R, Way M, Adams RH. Integrin-linked kinase controls vascular wall formation by negatively regulating Rho/ROCK-mediated vascular smooth muscle cell contraction. Genes Dev. 2009;23:2278–83.PubMed CentralView ArticlePubMedGoogle Scholar
- Elad N, Volberg T, Patla I, Hirschfeld-Warneken V, Grashoff C, Spatz JP, Fässler R, Geiger B, Medalia O. The role of integrin-linked kinase in the molecular architecture of focal adhesions. J Cell Sci. 2013;126:4099–107.View ArticlePubMedGoogle Scholar
- Lavelin I, Wolfenson H, Patla I, Henis YI, Medalia O, Volberg T, Livne A, Kam Z, Geiger B. Differential effect of actomyosin relaxation on the dynamic properties of focal adhesion proteins. PLoS One. 2014;9(2):e90269.View ArticleGoogle Scholar
- Lauffenburger DA, Horwitz AF. Cell migration: a physically integrated molecular process. Cell. 1996;84:359–69.View ArticlePubMedGoogle Scholar
- Binamé F, Sakry D, Dimou L, Jolivel V, Trotter J. NG2 regulates directional migration of oligodendrocyte precursor cells via Rho GTPases and polarity complex proteins. J Neurosci. 2013;33:10858–74.View ArticlePubMedGoogle Scholar
- Liu X, Lu Y, Zhang Y, Li Y, Zhou J, Yuan Y, Gao X, Su Z, He C. Slit2 regulates the dispersal of oligodendrocyte precursor cells via Fyn/RhoA signaling. J Biol Chem. 2012;287:17503–16.PubMed CentralView ArticlePubMedGoogle Scholar
- Rajasekharan S, Bin JM, Antel JP, Kennedy TE. A central role for RhoA during oligodendroglial maturation in the switch from netrin-1-mediated chemorepulsion to process elaboration. J Neurochem. 2010;113:1589–97.PubMedGoogle Scholar
- Liang X, Draghi NA, Resh MD. Signaling from integrins to Fyn to Rho family GTPases regulates morphologic differentiation of oligodendrocytes. J Neurosci. 2004;24:7140–9.View ArticlePubMedGoogle Scholar
- Wolf RM, Wilkes JJ, Chao MV, Resh MD. Tyrosine phosphorylation of p190 RhoGAP by Fyn regulates oligodendrocyte differentiation. J Neurobiol. 2001;49:62–78.View ArticlePubMedGoogle Scholar
- Baer AS, Syed YA, Kang SU, Mitteregger D, Vig R, Ffrench-Constant C, Franklin RJM, Altmann F, Lubec G, Kotter MR. Myelin-mediated inhibition of oligodendrocyte precursor differentiation can be overcome by pharmacological modulation of Fyn-RhoA and protein kinase C signalling. Brain. 2009;132:465–81.PubMed CentralView ArticlePubMedGoogle Scholar
- Siebert JR, Osterhout DJ. The inhibitory effects of chondroitin sulfate proteoglycans on oligodendrocytes. J Neurochem. 2011;119:176–88.View ArticlePubMedGoogle Scholar
- Mi S, Pepinsky RB, Cadavid D. Blocking LINGO-1 as a therapy to promote CNS repair: from concept to the clinic. CNS Drugs. 2013;27:493–503.View ArticlePubMedGoogle Scholar
- Mi S, Miller RH, Lee X, Scott ML, Shulag-Morskaya S, Shao Z, Chang J, Thill G, Levesque M, Zhang M, Hession C, Sah D, Trapp B, He Z, Jung V, McCoy JM, Pepinsky RB. LINGO-1 negatively regulates myelination by oligodendrocytes. Nat Neurosci. 2005;8:745–51.View ArticlePubMedGoogle Scholar
- Mi S, Miller RH, Tang W, Lee X, Hu B, Wu W, Zhang Y, Shields CB, Zhang Y, Miklasz S, Shea D, Mason J, Franklin RJM, Ji B, Shao Z, Chédotal A, Bernard F, Roulois A, Xu J, Jung V, Pepinsky B. Promotion of central nervous system remyelination by induced differentiation of oligodendrocyte precursor cells. Ann Neurol. 2009;65:304–15.View ArticlePubMedGoogle Scholar
- Chen M, Liu A, Ouyang Y, Huang Y, Chao X, Pi R. Fasudil and its analogs: a new powerful weapon in the long war against central nervous system disorders? Expert Opin Investig Drugs. 2013;22:537–50.View ArticlePubMedGoogle Scholar