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doi: 10.1242/10.1242/jcs.00128

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Defects in keratinocyte activation during wound healing in the syndecan-1-deficient mouse

¹ Department of Anatomy and Cell Biology, The George Washington University Medical School, Washington DC 20037, USA
² Department of Ophthalmology, The George Washington University Medical School, Washington DC 20037, USA
³ Division of Developmental and Newborn Biology, Department of Pediatrics, Harvard Medical School, Boston, MA 02115, USA
⁴ Department of Pathology, The George Washington University Medical School, Washington DC 20037, USA

View larger version (40K): [in a new window]   Fig. 1. Synd1ko mice express no synd1 mRNA or protein, and their epithelial tissues appear normal. (A) The targeting vector was a MboI/SalI fragment with homology to the endogenous gene and includes 1.4 kb of the 5' flanking sequence, the entire 0.3 kb first exon and 5.3 kb of the first large intron. Positive (PGK neo) and negative (MC1-tk) selection markers were used. The disrupted allele was 8.6 kb versus the wild-type allele, which was 7.1 kb. (B) Using a 3' probe, Southern blot analyses of tail DNA isolated from two litters of mice included wildtypes (+/+), heterozygotes (+/-) and homozygotes (-/-) for the disrupted gene. (C) Northern blots show that, when equal amounts of RNA isolated from wild-type and -/- mouse skin are run out on gels and transferred to nitrocellulose, the synd1ko mice have no detectable synd1 mRNA, whereas the wild-type mice do. Replicate blots probed with a synd4 cDNA, serving as a positive control for RNA quality, confirm the presence of synd4 mRNA in both wild-type and -/- tissues, but there is no upregulation in the synd1ko tissues. RNA sizes are indicated on the right; synd1 mRNA is present as two discrete bands of 2.6 and 3.4 kb, whereas synd4 mRNA is 2.6 kb. (D) Using confocal microscopy, skin and corneal tissues from wild-type and synd1ko mice were evaluated to determine the degree of colocalization of synd1 and ß4 integrin, 3 integrin and E-cadherin or 9 integrin and ZO-1. Merged images are presented. Although E-cadherin is expressed at the lateral membranes of basal cells, it is less abundant at this site, and the large amount of 3 integrin in basal cells makes it difficult to document E-cadherin—3-integrin co-expression in the basal cells in these merged images. For the 9/ZO-1 merged images of the corneal limbal region, the central cornea is located to the left and the conjunctiva is to the right. No differences in the localization of integrins, E-cadherin or ZO-1 between wild-type and synd1ko tissues are observed. Bar in D, 90 µm.

View larger version (92K): [in a new window]   Fig. 2. The absence of synd1 leads to delayed closure of corneal epithelial scrape wounds, which is associated with a failure to increase cell proliferation after wounding. The numbers of open wounds and corneas used to generate these data are presented in the methods section. (A) Representative photomicrographs obtained after wounding at the indicated times (d, day) are depicted. The delay in closure of epithelial wounds is statistically significant (P<0.05) at 24 and 36 hours (asterisk) as determined using the Mantel-Haenszel Chi-Squared test. (B) Data from the experiment described in A were used to create a graph showing the time (hr, hour) of 50% wound closure. There is an 8-hour delay in the 50% wound closure time in synd1ko mice compared with wild-type mice. (C) Cell proliferation was assessed using BrdU. Data show that, although wild-type corneal epithelial cells increase proliferation at 24 hours after wounding, the synd1ko animals fail to respond to the corneal wound by increasing their proliferation rate at any time point analyzed. Data for the synd1ko are significantly (P<0.05) different from wildtype at 24 hours. Surprisingly, there is also a significant (P<0.05) increase in proliferation rate in the unwounded synd1ko mice compared with that in unwounded wild-type mice. Data are compared using the unpaired t-test, and asterisks indicate significant differences between groups. (D) Histology shows that migration in the synd1ko mouse cornea is associated with a transient increase in inflammatory cells in the corneal stroma and a failure to restratify properly by 2 weeks. 18 hours after wounding, more inflammatory cells are observed beneath the migrating synd1ko epithelial sheet than in the wildtype. Arrows indicate the leading edge, and the arrowheads indicate inflammatory cells; direction of cell migration is from left to right. Bar, 60 µm.

View larger version (114K): [in a new window]   Fig. 3. Although ß4 integrin localization appears normal during migration, subtle differences in the localization of 9 integrin emerge between the wild-type and synd1ko corneas immediately prior to wound closure. (A) Unwounded tissues and tissues taken at 18 hours after wounding are double-labeled to detect synd1 and ß4 integrin simultaneously. Asterisks indicate the regions shown at higher magnification at the right — both as doubly and individually labeled images to allow for better determination of the regions of overlap. Arrows indicate the tip of the leading edge; migration occurs from left to right. Bar, 80 µm in lower magnification images at the left and 25 µm in higher magnification images at the right. (B) En face views of whole mounts of corneas from control or wounded mice have been treated with propidium iodide to highlight nuclei and simultaneously stained with an antibody to detect 9 integrin. 9 integrin is absent from the central corneas of unwounded wild-type animals but present at the limbal region as seen in the inset. Similar results are seen in the synd1ko mouse. When matched for the size of the remaining wound, wild-type corneas show more 9 integrin around epithelial cells close to the leading edge (asterisks) as well as at sites where seams form as advancing epithelial sheet edges merge (arrows) compared to the synd1ko corneas. Trapping of sera at the leading edge within wound opening occurs in some samples and is not specific. Bar, 150 µm.

View larger version (60K): [in a new window]   Fig. 4. Tight junctions are functional after wounding in the synd1 ko mouse cornea despite the persistent failure of ZO-1 to relocalize exclusively to the apical-most cell layers in the wounded synd1 ko corneas. (A) Immunofluorescence microscopy was performed to colocalize ZO-1 and 9 integrin, and ZO-1 and ß4 integrin at 12 weeks after wounding in wild-type and synd1 ko tissues. Age-matched unwounded synd1 ko corneas were also analyzed. For all corneas shown, the limbal images are oriented with their conjunctiva to the left and their central cornea to the right. The central cornea of a wild-type mouse 12 weeks after wounding and the unwounded, age-matched, synd1 ko mouse cornea at 5 months of age show polarized localization of ZO-1. However, in both of the synd1 ko corneas shown 12 weeks after wounding, ZO-1 can still be seen at non-apical cell layers. 9 integrin is upregulated in response to aging in wild-type and synd1 ko corneal epithelial basal cells, and this occurs with or without wounding. ß4 integrin is present at the basal cell basement membrane zone in the central cornea in both unwounded and wounded wild-type and synd1 ko mice. Bar, 75 µm. (B) Biotin penetration was assayed in wild-type and synd1 ko corneas 4 weeks after wounding as an indication of tight junction integrity. Biotin was detected with rhodamin-avidin, and sections were simultaneously stained for ZO-1 using a goat anti-rat secondary antibody conjugated with Alexa 488. To facilitate comparison with the results presented for ZO-1 in A, Adobe Photoshop was used to invert the colors of the rhodamin-avidin to green and the Alexa-488 to red. In both wild-type and synd1 ko corneas, biotin did not penetrate past ZO-1 -positive cell layers, indicating that tight junctions were functional in the synd1 ko corneas. Bar, 75 µm.

View larger version (176K): [in a new window]   Fig. 5. Synd1 ko mice show delayed re-epithelialization of full-thickness cutaneous wounds. Histology performed on fixed tissues shows that as soon as 5 days (d) after wounding, signs of defective re-epithelialization are detected in the synd1 ko mice compared to wildtype. Low magnification shows wound margins 5 days after wounding, and high magnification shows the poor overall morphology of the synd1 ko tissue compared with wild-type at 5, 7 and 9 days after wounding. Bar, 300 µm in the lower magnification image at day 5, bar, 50 µm in images at days 5 and 7, and bar, 25 µm in the images at day 9.

View larger version (57K): [in a new window]   Fig. 6. Cell proliferation and integrin expression are altered in syndko mice during wound healing in response to dermabrasion. (A) Dermabrasion was performed on wild-type and synd1ko mouse back skin after removal of hair; and mice were sacrificed 2 and 5 days after hair removal. Synd1ko mouse have deeper and more inflamed wounds at both 2 and 5 days compared with the wildtype. (B) Cell proliferation was analyzed in unwounded mouse back skin from which hair had been removed 2 or 5 days previously and in dermabraded mouse skin at 2 and 5 days. Although the variation among both individual mice and within the same mouse after wounding is substantial, analyses of over 85 visual fields per sample for 2 days and over 30 visual fields for the 5-day samples yield data that are significant (P>0.05) at both 2 and 5 days by the unpaired t-test. Further, unwounded synd1ko and wild-type skin have significantly different cell proliferation rates at both 2 and 5 days after hair removal. (C) Colocalization of 9 integrin and ZO-1 indicates that unwounded skin from which hair had been removed 2 days previously has a normal distribution of ZO-1. Both wild-type and synd1ko mouse back skin show basal cell localization for 9 integrin. This distribution is similar to that observed in control skin taken from the lip, as shown in Fig. 1D. 2 days after dermabrasion, there is an increase in the number of cell layers expressing 9 integrin in the wild-type skin. The increase is less dramatic in the wounded skin of synd1ko mice. In both wild-type and synd1ko skin, ZO-1 localization over the involved skin areas is discontinuous. (D) Colocalization of ß4 integrin with synd1, and 3 integrin with E-cadherin, is shown in wounded skin 2 days after dermabrasion in wild-type and synd1ko mice. There are no differences in the localization of ß4 integrin, and synd1 is not present in the synd1ko tissues. 3 integrin and E-cadherin are also not differentially affected by wounding in synd1ko mice. Bar in C and D, 75 µm. (E) Immunoblots of skin tissue extracts from unwounded and dermabraded mouse skin show differences in integrin expression after wounding. Samples were normalized on the basis of their protein concentration; tissues were extracted from two mice per variable, and dermabrasion repeated twice. cont., extracts from tissues immediately after sacrifice and removal of hair; u, extracts from tissues from which hair had been removed 2 days prior to sacrifice; w, extracts from tissues from animals that had been wounded 2 days prior to sacrifice.