Wiechmann AF , Ceresa BP , Howard EW .

EY021497 NEI NIH HHS , R01 EY021497 NEI NIH HHS

	Figure 1. MMP-2 and TIMP are co-localized in Xenopus leavis surface corneal epithelium sections. Cryostat sections of corneas obtained during the late dark period (NIGHT: two hrs before lights on) or early light period (DAY; two hrs after lights on) were immunolabeled with MMP-2 and or/TIMP-2 antibodies and analyzed with confocal microscopy. (A) At nighttime, MMP-2 immunoreactivity (red) was localized in cell clusters mainly to the surface layer of CE cells (arrow), with some labeling also present in the underlying sub-superficial layer of cells. Immunolabeling in the deeper layers of the CE was very sparse. (B) As negative controls, corneal sections obtained at night were processed for immunohistochemistry in the absence of primary antibody, and no specific immunoreactivity was observed (arrow). (C) At early daytime, intensity of MMP-2 immunoreactivity (red) was generally diminished relative to the night time levels (arrow). (D) At late nighttime, TIMP-2 immunoreactivity (green) was localized in cell clusters mainly to the surface layer of CE cells (arrow), with some labeling also present in the underlying sub-superficial layer of cells. Immunolabeling in the deeper layers of the CE was very sparse. (E) As negative controls, corneal sections obtained during the daytime were processed for immunohistochemistry in the absence of primary antibody, and no specific immunoreactivity was observed (arrow). (F) At early daytime, intensity of TIMP-2 immunoreactivity (green) was generally higher relative to the nighttime levels (arrow), and some labeling also present in the underlying sub-superficial layer of cells, inverse of the temporal pattern observed with MMP-2 in A and C. (G) Double-label immunocytochemistry of MMP-2 (red) and TIMP-2 (green) of nighttime corneas revealed co-localization of the two proteins. Yellow indicates regions of co-localization of the red and green signal (arrows). (H) During the early daytime, MMP-2 (red) and TIMP-2 (green) displayed a similar yellow co-localization (arrow) as seen at nighttime. Note that the two proteins were either expressed together (arrow) or not at all (arrowhead). Sections were stained with DAPI, which stained the nuclei blue. Scale bar = 20 µm. doi:10.1371/journal.pone.0113810.g001
	Figure 2. In situ zymography demonstrates MMP gelatinase activity in Xenopus surface corneal epithelium. The presence of green fluorescence with confocal microscopy indicates the presence of gelatinase enzyme activity in unfixed cornea sections obtained during the late dark period (NIGHT: two hrs before lights on) or early light period (DAY; two hrs after lights on). (A) Late at night, MMP activity (green) was present in the surface layers of the CE (arrow) with some labeling also present in the deeper layers of the CE. In the early daytime, MMP activity (green) in the surface CE (arrow) appeared to be lower early in the light period with some labeling persisting in the deeper layers of the cornea. (C) Inclusion of a specific inhibitor of MMP-2 and MMP-9 (1.0 µm phenanthroline) in the incubation mixture blocked most of the gelatinase activity. Sections were stained with DAPI, which stained the nuclei blue. Scale bar = 20 µm. doi:10.1371/journal.pone.0113810.g002
	Figure 3. Tight junction proteins occludin and claudin-4 are co-expressed in Xenopus corneal epithelium lateral membranes and are disrupted at night. Double-label confocal immunocytochemistry was performed on whole flat-mounted preparations of Xenopus corneas that were obtained from animals in the late afternoon (DAY; 9 hours after lights on in a 12L:12D cycle) and in the late night (NIGHT; 3 hours before lights on). (A) In the late afternoon, occludin (green) was uniformly primarily localized to the lateral membranes of the surface CE. (B) The same specimen as in A was labeled for the presence of claudin-4 (red), and was also uniformly primarily localized to the lateral membranes of the surface CE. (C) Merged green/red images from A and B demonstrate a high degree of co-localization in the surface cell CE lateral membranes, as indicated by the yellow signal. (D) In the late night, occludin (green) was localized to the CE surface cell lateral membranes as during the day, but the pattern of labeling was often interrupted in some clusters of cells (arrows). (E) The same specimen as in D was labeled for the presence of claudin-4 (red), and was also localized to the CE surface cell lateral membranes, but the pattern of labeling was also interrupted in the same clusters of cells (arrows). (F) Merged green/red images from D and F demonstrate a high degree of co-localization in the surface cell CE lateral membranes, as indicated by the yellow signal, with a similar level of disruption a discrete loci. Specimens were stained with the blue nuclear DAPI stain. Scale bar = 20 µm. doi:10.1371/journal.pone.0113810.g003
	Figure 4. Occludin expression on Xenopus surface CE is disrupted at night, and is inversely associated with MMP-2 expression. Flat-mounted whole corneas were double-labeled for localization of occludin and MMP-2 immunoreactivity in the late afternoon (DAY; 9 hours after lights on in a 12L:12D cycle) and in the late night (NIGHT; 3 hours before lights on). (A) In the late afternoon, occludin (green) immunolabeling of the lateral membranes of the surface CE was generally intact, and some sporadic labeling of MMP-2 (red) was also present on the lateral membranes and/or cytoplasm (arrows) which was sometimes co-localized (yellow) with occludin. (B) During the late night, most of the surface CE cells exhibited intact occludin (green) labeling on their lateral membranes (double asterisks), but there were also many clusters of surface CE cells that lacked occludin immunoreactivity in their lateral membranes (single asterisk). In neighboring cells of some occludin-negative clusters, there was a gap between the occludin-labeled lateral membranes, indicating the disruption of tight junctions between the cells (arrows). MMP-2 (red) was very often expressed in the cells that exhibited gaps between the lateral membranes. Also, in these areas of high MMP-2 expression, upward-folding flaps of surface cells were lifting from the CE surface, representing cells in the act of desquamation (arrowheads). (C) In some cell clusters that were almost devoid of occludin labeling on their lateral membranes at nighttime (presumptive former sub-superficial cells; asterisks), intense occludin (green) immunoreactivity was observed in perinuclear intracytoplasmic compartments. The neighboring surface CE cells express high levels of MMP-2 (red) that are in the process of desquamation (arrows). There was also considerable co-localization (yellow) of occludin and MMP-2 on the lateral membranes between neighboring cells. Specimens were stained with the blue nuclear DAPI stain. Scale bar = 20 µm. doi:10.1371/journal.pone.0113810.g004
	Figure 5. Claudin-4 expression on Xenopus surface CE is disrupted at night, and is inversely associated with MMP-2 expression. Flat-mounted whole corneas were double-labeled for localization of claudin-4 and MMP-2 immunoreactivity in the late afternoon (DAY; 9 hours after lights on in a 12L:12D cycle) and in the late night (NIGHT; 3 hours before lights on). (A) In the late afternoon, claudin-4 (green) immunolabeling of the lateral membranes of the surface CE was generally intact, and some sporadic labeling of MMP-2 (red) was also present on the lateral membranes and/or cytoplasm (arrows) which was sometimes co-localized (yellow) with occludin. (B) During the late night, most of the surface CE cells exhibited intact claudin-4 (green) labeling on their lateral membranes, but there were also many clusters of surface CE cells in which the claudin-4 immunoreactivity was disrupted (arrows) and was accompanied by high levels of MMP-2 immunoreactivity. Specimens were stained with the blue nuclear DAPI stain. Scale bar = 20 µm. doi:10.1371/journal.pone.0113810.g005
	Figure 6. Preservation of occludin expression is positively associated with TIMP-2 on surface CE and exhibits day/night changes. Flat-mounted whole corneas were double-labeled for localization of claudin-4 and TIMP-2 immunoreactivity in the late afternoon (DAY; 9 hours after lights on in a 12L:12D cycle) and in the late night (NIGHT; 3 hours before lights on). (A) During the daytime, occludin (green) immunolabeling of the CE surface cell lateral membranes was generally intact. Small clusters of surface and sub-superficial CE cells exhibited TIMP-2 immunoreactivity in intracytoplasmic compartments (arrows). (B) At nighttime, TIMP-2 (red) immunoreactivity in the surface CE was very low, and occludin (green) immunoreactivity was disrupted (arrows) in some cell clusters (asterisks). (C) A cornea retrieved in the late afternoon (DAY) had an unidentified pathology in which almost all CE was eroded, leaving the stromal layer at the corneal surface. A small patch of about 20 CE cells persisted as a single monolayer above the stromal layer. The surviving CE cells displayed occludin (green) immunolabeling on their lateral membranes, and very intense immunolabeling of TIMP-2 (red) in their cytoplasm (arrows). There was some high non-specific green labeling of the connective tissue surface. Specimens were stained with the blue nuclear DAPI stain. Scale bar = 20 µm. doi:10.1371/journal.pone.0113810.g006
	Figure 7. Membrane type 1-MMP is expressed in surface CE cells and associates with lateral membranes at nighttime. Flat-mounted whole corneas were double-labeled for localization of claudin-4 and membrane type 1-MMP (MT1-MMP) immunoreactivity in the late afternoon (DAY; 9 hours after lights on in a 12L:12D cycle) and in the late night (NIGHT; 3 hours before lights on). (A) In the late afternoon, claudin-4 (red) immunolabeling of the surface CE cell lateral membranes was generally intact and uniform. MT1-MMP (green) labeling, when present, was observed almost exclusively in cytoplasmic compartments of the surface CE. (B, C) Two examples are presented to illustrate that during the late night, a majority of the surface CE cells exhibited intact claudin-4 (green) labeling on their lateral membranes, but there were also many clusters of surface CE cells in which the claudin-4 immunoreactivity was disrupted. Also at nighttime, MT1-MMP labeling was present both in the cytoplasm and intermittently associated with the surface CE cell lateral membranes (arrows). The membrane co-localization of the red claudin-4 and green MT1-MMP resulted in a merged yellow labeling of the lateral membranes in some areas (arrows). Specimens were stained with the blue nuclear DAPI stain. Scale bar = 20 µm. doi:10.1371/journal.pone.0113810.g007
	Figure 8. MMP-2 and TIMP-2 expression levels are inversely correlated during the light/dark cycle in surface corneal epithelium. Representative confocal images (N = 4) of each sample group were analyzed for day/night changes in immunolabeling intensity. MMP-2 immunolabeling intensity was significantly higher at nighttime than during the day (P<0.01), whereas TIMP-2 intensity was significantly lower at nighttime compared to the day (P<0.01; Two-tailed Student's t-test). MT1-MMP levels appeared unchanged between the light and dark cycle. The lower nighttime levels of occludin was not statistically significant, although the day/night change in claudin-4 expression was significant (P<0.05). doi:10.1371/journal.pone.0113810.g008

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