- Research article
- Open Access
The roles of calcium signaling and ERK1/2 phosphorylation in a Pax6 +/-mouse model of epithelial wound-healing delay
- Lucy J Leiper†1,
- Petr Walczysko†1,
- Romana Kucerova1,
- Jingxing Ou1,
- Lynne J Shanley1,
- Diane Lawson1,
- John V Forrester1,
- Colin D McCaig1,
- Min Zhao1 and
- J Martin Collinson1Email author
© Leiper et al; licensee BioMed Central Ltd. 2006
- Received: 01 February 2006
- Accepted: 16 August 2006
- Published: 16 August 2006
Congenital aniridia caused by heterozygousity at the PAX6 locus is associated with ocular surface disease including keratopathy. It is not clear whether the keratopathy is a direct result of reduced PAX6 gene dosage in the cornea itself, or due to recurrent corneal trauma secondary to defects such as dry eye caused by loss of PAX6 in other tissues. We investigated the hypothesis that reducing Pax6 gene dosage leads to corneal wound-healing defects. and assayed the immediate molecular responses to wounding in wild-type and mutant corneal epithelial cells.
Pax6 +/- mouse corneal epithelia exhibited a 2-hour delay in their response to wounding, but subsequently the cells migrated normally to repair the wound. Both Pax6 +/+ and Pax6 +/- epithelia activated immediate wound-induced waves of intracellular calcium signaling. However, the intensity and speed of propagation of the calcium wave, mediated by release from intracellular stores, was reduced in Pax6 +/- cells. Initiation and propagation of the calcium wave could be largely decoupled, and both phases of the calcium wave responses were required for wound healing. Wounded cells phosphorylated the extracellular signal-related kinases 1/2 (phospho-ERK1/2). ERK1/2 activation was shown to be required for rapid initiation of wound healing, but had only a minor effect on the rate of cell migration in a healing epithelial sheet. Addition of exogenous epidermal growth factor (EGF) to wounded Pax6 +/- cells restored the calcium wave, increased ERK1/2 activation and restored the immediate healing response to wild-type levels.
The study links Pax6 deficiency to a previously overlooked wound-healing delay. It demonstrates that defective calcium signaling in Pax6 +/- cells underlies this delay, and shows that it can be pharmacologically corrected. ERK1/2 phosphorylation is required for the rapid initiation of wound healing. A model is presented whereby minor abrasions, which are quickly healed in normal corneas, transiently persist in aniridic patients, compromising the corneal stroma.
- Epidermal Growth Factor
- Wound Healing
- Corneal Epithelium
- Wound Edge
- Corneal Epithelial Cell
The transcription factor PAX6 is required for development and maintenance of several human ocular tissues, including the corneal epithelium. The gene PAX6 was identified by positional cloning as the defective gene in human aniridia and its associated ocular complications [1–3].
Heterozygous deficiency in PAX6 leads to ocular surface disease characterized by corneal opacification (aniridia-related keratopathy; ARK), and the Pax6 +/- mouse model displays all the morphologic defects of human ARK [4–7]. ARK in Pax6 +/- mice and humans is characterized by the presence of thin, fragile vacuolated corneal epithelia [4, 6]. There is ingrowth of blood vessels into the cornea, sporadic appearance of goblet cells [8, 9], and infiltration by inflammatory cells . The cell biology that links Pax6 dosage to changes in cellular behavior remains poorly understood. Maintenance of the structural integrity of the corneal epithelium is essential for corneal transparency. Impaired re-epithelialization after wounding increases the risk of infection, exacerbates inflammation, and undermines normal stromal remodeling . The cumulative effect of very small corneal scars following chronic failure of wound repair may result in loss of transparency. It has recently been suggested that ARK may be due to corneal epithelial fragility combined with an abnormal wound-healing response , resulting in degeneration of the cornea.
Epithelial sheets react to mechanical injuries via a complex process that relies on coordinated proliferation and migration into the denuded area, matrix deposition and tissue remodeling [13–16]. Wound-induced migration and proliferation are regulated by cytokines, extracellular matrix molecules, and growth factors including insulin-like growth factor, keratinocyte growth factor, hepatocyte growth factor and epidermal growth factor (EGF) [17–22].
One of the first reactions to injury of the cell monolayer is an intracellular calcium rise spreading as a wave from the injury site to the neighboring cells. This has been reported in a range of cell systems, mainly in cell lines [23–29]. The wound-elicited calcium wave stimulates cell proliferation after injury, and is required for further healing processes . The intensity of the calcium response quantitatively controls the subsequent rate of healing in some epithelial cells, and remodeled calcium signaling may underlie disease states such as diabetes, hypertension, heart disease, manic depression and Alzheimer's disease [30–32].
Alongside the calcium wave, the other early response of epithelial cells to wounding is at least one slower wave of mitogen-activated protein kinase (MAPK) ERK1/2 phosphorylation . The migration of at least some epithelial cell types appears to be ERK1/2-driven . Addition of EGF activates ERK1/2 in a 'wild-type' rabbit corneal epithelial cell line , and exerts positive effects on epithelial wound healing by promoting migration and mitosis [17, 29, 36–38]. In corneal epithelial cells, EGF exhibits a positive effect on proliferation via orchestrated calcium influx from intracellular calcium stores and extracellular space [29, 39].
Work by our laboratory and several others have investigated the roles of Pax6 in control of cell adhesion in development and disease, but the downstream cell-signaling pathways have received less attention. We hypothesised that cell signaling may be faulty. As a model for the reactive cellular pathways that may be controlled by Pax6, we have developed an in vitro wounding assay for murine primary corneal epithelial cells and have investigated the roles of Pax6 in corneal healing. We find that unlike wild-type, Pax6 +/- cells fail to commence healing within the first 2 hours after wounding, and that wound-induced waves of calcium and phospho-ERK1/2 signaling are required for rapid response to wounding. Propagation of the wound-induced calcium wave is defective in Pax6 +/- cells, but addition of EGF restores calcium signaling and promotes ERK1/2 phosphorylation, rescuing their rapid response to wounding.
Wound healing is delayed in Pax6 +/-corneal epithelial cultures
Calcium response to wounding
Calcium wave initiation and propagation
In separate experiments, intracellular calcium stores were depleted by 1 μM thapsigargin, which inhibits the Ca2+ATPase pump of the endoplasmic reticulum. Following injury, cells directly adjacent to the wound edge rapidly increased [Ca2+]i, but subsequent wave propagation was prevented (Figure 4A,C; see Additional file 3). Wave initiation and propagation in murine corneal epithelial cells can therefore be decoupled. Intracellular Ca2+ is required only for wave propagation.
When either extracellular calcium or intracellular Ca2+ stores were depleted, both Pax6 +/+ and Pax6 +/- cells failed to repair wounds (Figure 4D). The rate of healing in untreated Pax6 +/+ controls was 26.6 ± 1.5 μm/h compared with 6.3 ± 3.6 μm/h in thapsigargin-treated cultures (t-test: p < 0.0001) and 8.8 ± 2.9 μm/h in calcium-free media (t-test, p < 0.0001). Hence, both phases of the Ca2+ response are required for coordinated wound closure.
Propagation is via P2Y receptors, but ATP cannot rescue mutant cells
EGF can restore the calcium wave and rescue wound-healing defects
ERK1/2 response to injury
Pax6 +/- cells also elicited a phospho-ERK1/2 response to wounding (Figure 7C). Using immunocytochemistry, it appeared that ERK1/2 phosphorylation in Pax6 +/- cells was stimulated by addition of EGF 90 seconds prior to wounding. This was confirmed by Western blotting (Figure 7D,E). EGF had no effect on the levels of Pax6 protein in cultures, assayed by Western blotting (Figure 7E).
We investigated the requirement for ERK1/2 phosphorylation for wound healing. Wild-type and Pax6 +/- cells were injured in the presence of 10 μM U0126 to inhibit the activated ERK1/2 pathway. It was found that addition of U0126 specifically prevented migration of Pax6 +/+ cells within the first 2 hours after wounding (t-test: Pax6 +/+ controls versus Pax6 +/+ U0126-treated, p = 0.007; n = 17 control, 23 drug-treated) (Figure 7C). In contrast, migration of wild-type cells 2–6 hours after wounding in the presence of U0126 was relatively normal (mean 20.7 μm/h), although still marginally significantly slower than the untreated controls (32.4 μm/h; t-test: Pax6 +/+ controls versus Pax6 +/+ U0126-treated, p = 0.039; n = 17 control, n = 15 drug-treated) (Figure 7C). To determine whether this confined effect was caused by degradation of the drug after the first 2 hours of incubation with the cells, the experiment was repeated with replacement of the culture medium with fresh medium containing 10 μM U0126 every 2 hours. This had no additional effect; the rate of Pax6 +/+ cell migration 2–6 hours after wounding with addition of fresh drug at 2 and 4 hours (21.9 μm/h, n = 8) was not significantly different that of experimental groups with a single addition of drug prior to wounding (20.7 μm/h, n = 15, as above; t-test, p = 0.81). Similarly, U0126 had no effect on wound healing by Pax6 +/- cells. In the first 2 hours after wounding, untreated cells showed negligible rates of migration, as described previously, and addition of 10 μM U0126 did not change that. During hours 2–6 after wounding, U0126-treated Pax6 +/- cells healed at 18.1 ± 3.30 μm/h, with or without addition of fresh drug – slower than untreated Pax6 +/- controls (34.5 ± 3.57 μm/h) but not significantly different from drug-treated Pax6 +/+ cells (t-test, p = 0.51) (Figure 7D).
Consistent with the transient ERK1/2 activation response to wounding, we conclude that ERK1/2 phosphorylation within the first 30 minutes is required for the initiation of cell migration. In the absence of ERK1/2 phosphorylation, Pax6 +/+ cells experience a wound-healing delay, but all cells, whatever their Pax6 genotype, can overcome this block and start migration after about 2 hours. It is clear that there are other signaling systems that can elicit a wounding response, which were not investigated here.
Pax6 +/- mice represent an excellent model of human PAX6 +/- aniridia-related keratopathy. Clinically, the symptoms of ARK are consistent with an altered wound-healing response . Pax6 directly binds matrix metalloproteinase-9 (MMP-9, also known as gelB) promoter to upregulate MMP-9 in wounded corneas . MMP-9 is one of a family of proteins required for tissue remodeling during wound repair. Experiments carried out previously on intact Pax6 +/- corneas suggested in a non-quantitative manner that Pax6 +/- corneas do heal [47, 48], but investigated cell migration on a diseased stroma, which may modulate wound healing [49, 50]. The etiology of ARK is complicated, because anterior eye development is coordinated to some extent by the lens, which is very sensitive to Pax6 dosage , and although the corneal epithelium expresses high levels of Pax6 throughout life, the stroma also expresses Pax6 around birth [5, 52]. Corneal epithelial dysgenesis in Pax6 mutants might be the result of intrinsic defects within the epithelial cells themselves or the fault of defective tissue-tissue interactions in the eye. The extent to which tissue-tissue interactions contribute to corneal epithelial dysgenesis can be addressed by isolating the epithelial cells. If cultured mutant corneal epithelial cells were phenotypically and molecularly normal, we would infer that the corneal epithelial defects were secondary to a problem with Pax6 dosage elsewhere in the eye. To identify the primary defects that underlie the etiology of the disease, we therefore investigated wound healing in Pax6 +/- corneal epithelial primary culture. We developed and validated an in vitro system for wound healing in primary murine corneal epithelia. Cells formed Pax6-positive confluent epithelial sheets, and we demonstrated that they show physiological calcium and MAPK responses to injury. The results of the investigation were surprising, with fundamental implications for the links between transcription-factor action, cell signaling, cell behavior, and disease.
Pax6 dosage and wound healing
Pax6 +/- corneal epithelial cultures expressed substantial quantities of Pax6, estimated to be about 60–70% that of wild-type. Pax6 autoregulates its own expression , and there is no direct 1:1 relationship between Pax6 gene dosage and the levels of protein present in the cells . Overexpression of Pax6 in corneal epithelial cells inhibits cell-cycle progression , and the anterior eye is particularly sensitive to dosage of important transcription factors, with <70–80% or >~150% activity leading to dysgenesis . Furthermore, corneal epithelial cells are derived from Pax6-positive stem-like cells in the corneal periphery (limbal stem cells) , and heterozygosity at the PAX6 locus compromises these cells  and leads to the production of corneal epithelia that may not be fully differentiated . We consider, therefore, that abnormal behavior of Pax6 +/- corneal epithelial cells in culture may relate to a failure of full differentiation that occurred in vivo days or weeks before.
We showed that wound healing was delayed for 2 hours in mutant cells, but that once healing began, the cells moved as fast as wild-type. Rates of cell migration, at 25–50 μm/h for wild-type cells, are similar to those obtained by Song et al for wild-type rat corneas in vivo .
While this paper was under review, a new paper  showed that in ex vivo whole-eye culture, Pax6 +/- corneas healed faster than wild-type over a 24-hour period. The first 2 hours of wound healing were not studied by Ramaesh et al , levels of Pax6 were not assayed, and the calcium or MAPK response to wounding in the epithelium was not studied. As described above, in the whole-eye culture system, the corneal epithelial migration is modulated by the presence of an inflamed stroma, which would be expected to accelerate cell migration over extended periods of healing. Another important difference between the experiments is the size of the wounds, which were very large (1000–1200 μm) in the study by Ramaesh et al , encompassing up to half the cornea and encroaching on the limbal region. Larger wounds in Pax6 +/- corneal epithelia healed faster than smaller wounds (presumably because larger wounds were closer to the source of proliferating limbal stem cells), and extrapolation from figure 2 in that study  suggested that wounds smaller than 500 μm in diameter would heal more slowly than wild-type. Wounds in our system are smaller and more physiological, rarely larger than 200 μm, so there is therefore no immediate contradiction between our work and this new study; however, the situation appears to be more complicated than that presented by either paper, and there is clearly more work needed.
Our data show that there is no defect per se in the ability of mutant cells to migrate normally, providing they get the correct signals. The delay in wound healing was, however, intrinsic to the epithelia, so we investigated the immediate responses to wound healing in mutant cells.
Calcium wave and healing rate
Calcium response, genotype and wound healing.
No Ca2+ o
No Ca2+ i
Pax6 +/- + ATP
Pax6 +/- + EGF
EGF signaling and the calcium and ERK responses
In gastric mucosal epithelia, extracellular signaling (in this case, by Sonic hedgehog) elevates intracellular calcium, which in turn activates ERK, leading to cell proliferation . The activation of ERK pathways by calcium signaling in response to extracellular signals has also been observed in a number of neural cell types [61–63]. Wounding of both Pax6 +/+ and Pax6 +/- corneal epithelia elicited ERK1/2 phosphorylation in a dynamic manner around the wound within 10 minutes. By 30 minutes after wounding, however, pERK1/2 immunolabeling was restricted primarily to nuclei of cells immediately adjacent to the wound edge. The pattern of immunolabeling was identical to that found previously by Matsubayashi et al  upon wounding of MDCK epithelial cells. They showed that inhibition of the ERK1/2 signalling pathway reduced wound healing by about 50% in the 6 hours after wounding. We also found this; over the whole 6 hours of the experiment, mean ± SEM rate of wound healing of untreated wild-type cells was 27.6 ± 2.8 μm/h, compared with 13.8 ± 2.2 μm/h for wild-type cells treated with U0126. However, we have extended the conclusions of Matsubayashi et al with a finer temporal assay of rates of wound healing, and show that although ERK1/2 inhibition does reduce the rate of cell migration during healing, ERK1/2 phosphorylation is primarily required for the rapid response to wounding within the first 2 hours.
The distribution of EGFR is abnormal in Pax6 +/- corneal epithelia , and our data demonstrate that the calcium reaction in response to EGF of unwounded Pax6 +/- cells is significantly lower than in Pax6 +/+. Addition of exogenous EGF to wounded Pax6 +/- cells enhanced both the wound-elicited calcium wave and ERK1/2 phosphorylation, and restored the immediate migration response to wounding to wild-type levels without affecting the rate of migration. This suggests that Pax6 +/- EGF signaling is defective, perhaps due to reduced autocrine EGF production or failure of normal EGFR activity. EGF is known to enhance capacitative calcium entry (CCE) in corneal epithelial cells . Because CCE is known to be quantitatively coupled with the migration speed in mucosal epithelial wound repair [31, 32], this suggests a possible mechanistic explanation of positive EGF action on the wound-healing rates via calcium in our system.
EGF also acts via ERK1/2 to downregulate Pax6 and promote proliferation in rabbit corneal epithelial cells [39, 64]. We detected no change in Pax6 levels following EGF addition to Pax6 +/- cells, but this is not inconsistent with the results of Li and Lu , because our study investigates the cellular responses within the first few minutes of wounding, whereas changes in Pax6 protein levels would be expected to take much longer than this. Furthermore, in the present study, it is unlikely that cell proliferation affects wound-healing rates, because the changes in cell proliferation take several hours (2–3 days in the study of Li and Lu ). We have detected no difference in 5-bromodeoxyuridine uptake between primary cultures with different Pax6 dosages, and very little cell proliferation is noted in the first 2 h after wounding (Kitazawa et al ; JMC, unpublished observations), whereas Pax6 +/+ cells, and Pax6 +/- cells with exogenous EGF, begin to migrate within the first few minutes. Our data suggest that EGF signaling functions via enhanced calcium and ERK1/2 signaling to initiate cell migration.
Wound healing in Pax6 +/-corneas
Based on our data, we divide wound healing into an immediate period of 2 hours when calcium and MAPK signaling occur to initiate a rapid migratory response, and the subsequent 'post-initial' coordinated movement of epithelial sheets when cell migration is maintained. In the hours subsequent to this, cell proliferation is initiated; this last process is also modulated by Pax6 and the role of Pax6 in this regard has been studied previously .
We propose a model, based on our data combined with previous studies cited earlier, which postulates that Pax6 dosage does not affect the rate of corneal epithelial cell migration per se on a neutral substrate. In contrast, the primary defect of the Pax6 +/- corneal epithelium is the initial failure to respond to wounding. We have shown that upon wounding a corneal epithelium, external calcium enters cells around the wound, and this is quantitative similar in both wild-type and Pax6 +/- cells. Further propagation of calcium rise to cells away from the wound is required for the wound response, which is realized by cell-cell signaling via P2Y receptors and involves predominantly calcium from intracellular stores. We have shown that this process is inefficient in Pax6 +/- cells, and that secondary messenger ERK1/2 signaling pathways must also be activated following wounding to initiate immediate cell migration. Calcium signaling occurs in the first 2 minutes, whereas ERK1/2 signaling is expected to peak about 3–10 minutes after wounding . Both calcium and ERK responses are sensitive to EGF, which is present in tears at 1–10 ng/ml , and we show that calcium response to EGF is diminished in heterozygotes (possibly due to mislocalisation of EGFR in the mutant epithelium ). Together, these cell-signaling problems decelerate the response of mutant cells to small physiological wounds. In vivo, the chronic delay in sealing small holes caused by minor abrasion or other cell loss may be sufficient to allow the corneal stroma to become compromised, leading to opacity. We therefore link a transcription factor mutation, via defects in calcium signaling, to a cellular abnormality that underlies human disease.
Microarray study in the lens suggested that as many as 5000 genes may be regulated by Pax6 . It was perhaps expected therefore that the links between Pax6 and cell behavior would be pleiotropic, with a complex etiology intractable to mechanistic investigation. Our data suggest, however, that this is not the case, and that the link between genetics of disease and pharmacological correction (in this case via EGF) may not be so esoteric.
Wound healing is in part a recapitulation of important developmental pathways,  and it is very likely that the genetic pathways investigated here recapitulate genetic pathways that are controlled by Pax6 during development. We hypothesize that in other organ systems where Pax6 and calcium signaling are both known to be required, such as axon guidance [69, 70], Pax6 dosage may directly control the kinetics of the calcium response of cells to their external environment.
Propagation of wound-induced signaling via calcium release from intracellular stores is inefficient in Pax6 +/- corneal epithelial cells, and this underlies an intrinsic wound-healing delay. Pax6 dosage does not, however, affect the rate of cell migration in healing epithelia, 2–6 hours after wounding. MAP kinase signaling via phosphorylation of ERK1/2 is required for the immediate wound-healing response, but has only a marginal effect on the rate of cell migration in a healing epithelium. Addition of EGF prior to wounding rescues the calcium response in mutant cells and exaggerates the levels of ERK1/2 phosphorylation, restoring rapid wound-induced cell migration. Failure to respond quickly to minor wounding in fragile Pax6 +/- corneal epithelia could lead to the observed chronic corneal degeneration in patients, but our data suggest that pharmacological manipulation of the EGF pathway is a potential therapeutic strategy.
Mice and cell culture
Pax6 +/Sey-Neu (Pax6 +/-) mice  were maintained on the CBA/Ca genetic background by heterozygous mating. Corneas were dissected from Pax6 +/+ and Pax6 +/- littermates 8–10 weeks old. A small proportion of heterozygous corneas were rejected for use because they exhibited severe ocular surface damage. Previous work had demonstrated that adult murine Pax6 +/- corneal epithelia are cytokeratin-12-positive with only minor incursion of goblet cells, and no other conjunctival contamination was detected .
Studies were performed using primary monolayer corneal epithelial cells. Corneas were isolated and cultured according to the method of Hazlett et al . Central corneal buttons, free of conjunctival cells, were washed in phosphate-buffered saline (PBS) containing 500 U/ml penicillin G and 500 μg/ml streptomycin for 10 minutes and transferred to 35-mm plastic dishes or glass coverslips as appropriate, epithelium-side up, for 5 minutes before addition of complete culture medium (15 ml keratinocyte basal medium (Cambrex, UK), 19 ml DMEM:F12, 5 ml fetal bovine serum (12.5% v/v), 125 μg/ml gentamycin, 125 μM 2-β-mercaptoethanol and 25 mM HEPES). The corneal explant was removed after 14 days and cultures were used 2–3 days later. Corneal epithelial cells cultured in this way have previously been shown proteomically to be very similar to fresh corneal epithelia, and express major corneal epithelial markers .
Chemicals and reagents
Thapsigargin and suramin were from Calbiochem (Nottingham, Nottinghamshire, UK); MEK inhibitor U0126 from Promega (Southampton, Hampshire, UK); EGF and ATP from Sigma-Aldrich (Poole, Dorset, UK); anti-phospho-ERK1/2 from NE Biolabs (Hitchin, Hertfordshire, UK). Anti-Pax6 monoclonal antibody developed by A. Kawakami was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD (University of Iowa, IA, USA). Fluorescent probes were from Invitrogen (Paisley, UK).
Calcium imaging was performed in modified Hanks' balanced salt solution (mHBSS; 2.5 mM CaCl2, 150 mM NaCl, 4 mM KCl, 1 mM MgCl2, 10 mg/ml D-glucose, 20 mM HEPES). For experiments in Ca2+-free medium, the CaCl2 was replaced with 1 mM EGTA.
Murine corneal epithelial cells grown on glass coverslips were incubated for 45 minutes in mHBSS with 8.8 μM Fluo-3 AM in dimethyl sulfoxide (final concentration 0.2%) and Pluronic F-127 (final concentration 0.02%). Drugs were preincubated with the cells in mHBSS during the Fluo-3 incubation. Cells were washed twice with mHBSS (Ca-free or with drugs when appropriate) and positioned on a Zeiss Axiovert S100T microscope with a 100-W mercury arc lamp, a cooled CCD camera (Quantix, Photometrics) and 10 × objective. For excitation, an FITC filter (480/30 nm) was used. No emission filter was used. Images were captured at least 15 seconds before injury (an approximately circular wound using a 3G needle) to establish the baseline of fluorescence reading at 2-second intervals for 180 seconds. For experiments in which EGF was added prior to wounding, imaging started 70 seconds after EGF addition, and the cells were injured after further 20 seconds.
Ca2+ dynamics were evaluated using the changes in fluorescence intensity of calcium probe Fluo-3. MetaMorph (Universal Imaging Corp.) and Image J http://rsb.info.nih.gov/ij/ software were used. The data are presented as percentage change in the fluorescence intensity at each time point (F) to the first time point (F0) reading; the term ((F - F0)/F0) × 100 was used to represent this percentage change. In captured images, the second and sixth cell rows from the wound edge were drawn. The second-row intensity was assessed in order to study the calcium dynamics in cells adjacent to the wound. The first cell row on the wound edge was not evaluated because it included damaged cells. The fluorescence intensity of the sixth cell row was used to investigate the spread of calcium waves from the wound edge into the neighboring tissue.
Rate of wound healing in epithelial cultures
Small wounds were introduced into confluent Pax6 +/+ and Pax6 +/- monolayers in 35-mm dishes using a fire-polished glass pipette. The initial wound size and shape were similar and therefore shared the same mechanistic features in healing. The distance across each wound in four places was measured using calibrated eyepiece graticules every hour for 6 hours or until wounds were completely closed. Cells were incubated at 37°C, 5% CO2 at all times. Rate of wound healing was expressed in μm/h.
For inhibition of phospho-ERK1/2 signaling, 10 μM U0126 (made up as a 10 mM solution in DMSO) was added to the cells in culture 15 minutes before wounding unless stated otherwise.
Cultures were fixed in PBS containing 4% w/v paraformaldehyde, permeabilised in methanol for 10 minutes at -20°C, and rinsed three times with PBS before blocking for 30 minutes (blocking buffer: 0.3% BSA, 4% normal goat serum, PBS). Cells were incubated in primary antibody (Pax6 diluted 1:50, Phospho-ERK1/2 diluted 1:300, both in blocking buffer) overnight at 4°C. After three 5-minute washes in PBS, secondary antibody (Alexa fluor 488 goat anti-mouse, 1:200 in blocking buffer) was added for 1 hour. Following three 5-minute washes in PBS, cells were mounted in Vectashield (Vector Laboratories).
Unpaired Student's t-test was used for assessment of significance of differences between groups. The non-parametric Mann-Whitney test was used when the standard deviations of compared groups were significantly different.
We thank John D. West and Thaya Ramaesh for helpful discussions and advice. LJL was funded by Wellcome Trust grant 074127 to JMC and JO by Newlife BDF grant 04/07 to JMC. PW and LJS are funded by Wellcome Trust grants 068012 and 068932 to MZ. DL was funded by Scottish Hospitals Endowment Research Trust grant RG46/03 to JMC.
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