All Notch1-/- eyes including those with advanced corneal pathology (Figure 4 B1 4). The percentage of goblet cells in the conjunctiva was measured on impression cytology samples and likewise found to be similar (P = 0.118) (Figure 4 B5). Therefore, in our model, the loss of Notch1 on the ocular surface did not appear to adversely affect the conjunctival goblet cells.Tight junction formation is delayed in Notch1-/- corneal epithelial cells in vitroGiven that multiple factors on the ocular surface (e.g. trauma, inflammation) can contribute to the loss of the barrier, we proceeded to examine the expression of tight junctions in vitro. Primary mouse corneal epithelial cells from conditional Notch1-/- and WT mice were grown in low calcium serum free media and both treated with 4-OHT for 48 hours. Successful deletion of Notch1 was confirmed by Western blot (Figure 5C). To induce differentiation and tight junction formation, we raised the calcium to 1mM after which the expression pattern of the tight junction protein ZO-1 was examined. At 12 hours after calcium switch, WT cells demonstrated a more continuous staining pattern at the cell-cell junctions (consistent with organized tight junction structures) compared to Notch1 deleted cells which showed a predominance of non-continuous staining pattern (Figure 5A,B). This difference was quantified by measuring the mean fluorescence intensity of ZO-1 staining at the cell membranes using a previously published method [37]. The results indicated a significantly higher intensity of membrane staining in WT (arbitrarily set at 100 ) compared to Notch1-/- cells (56 ) (P <0.001) (Figure 5D). With longer exposure to the high calcium condition (24-48 hours), 1315463 the Notch1 deleted cells were able to develop a more continuousAqueous tear production is increased in conditional Notch1-/- KS-176 biological activity miceGiven the importance of the tear film in the corneal epithelial barrier function, we proceeded to examine the aqueous tearNotch1 and Corneal Epithelial BarrierFigure 3. Notch1-/- corneal epithelium is delayed in recovering its barrier function after wounding. Fluorescein staining of WT (A1-A4) and Notch1-/- (B1-B4) eyes immediately (0 h), 24, 72 and 96 hours after 2.0 mm central corneal epithelial debridement wounds; indicating a delay in barrier recovery in Notch1-/- eyes compared to WT. LC-biotin barrier function test at 96 hours postwounding showing that the WT cornea is impermeable to LC-biotin (C1) while in the Notch1-/- cornea LC-biotin has penetrated into the stroma (C2). LC-biotin staining showed penetration of the molecule into the stroma in 100 (7/7) of Notch1-/- mice compared to only 28.6 (2/7) of WT littermates at 96 hours (p=0.021). (C3). Oil Red O staining confirmed the presence of oil 76932-56-4 producing meibomian glands (green arrow head) in both WT (D1) and Notch1-/- (D2) at one week after 4-OHT treatment. Red: rhodamine, Blue: DAPI; scale bar: 100 .doi: 10.1371/journal.pone.0069113.gpattern of ZO-1 staining (mean fluorescent intensity for Notch1-/- was 117.6?2.9 vs 122.4?8.3 for WT, P = 0.429) suggesting that tight junction formation can ultimately be compensated by other means in vitro.DiscussionIn this study we have demonstrated an essential role for Notch1 in the corneal epithelial barrier particularly its recovery after wounding. This is consistent with our previous studieswhich demonstrated that Notch1 is down-regulated during initial stages of wound healing to promote epithelial migration, after which it must.All Notch1-/- eyes including those with advanced corneal pathology (Figure 4 B1 4). The percentage of goblet cells in the conjunctiva was measured on impression cytology samples and likewise found to be similar (P = 0.118) (Figure 4 B5). Therefore, in our model, the loss of Notch1 on the ocular surface did not appear to adversely affect the conjunctival goblet cells.Tight junction formation is delayed in Notch1-/- corneal epithelial cells in vitroGiven that multiple factors on the ocular surface (e.g. trauma, inflammation) can contribute to the loss of the barrier, we proceeded to examine the expression of tight junctions in vitro. Primary mouse corneal epithelial cells from conditional Notch1-/- and WT mice were grown in low calcium serum free media and both treated with 4-OHT for 48 hours. Successful deletion of Notch1 was confirmed by Western blot (Figure 5C). To induce differentiation and tight junction formation, we raised the calcium to 1mM after which the expression pattern of the tight junction protein ZO-1 was examined. At 12 hours after calcium switch, WT cells demonstrated a more continuous staining pattern at the cell-cell junctions (consistent with organized tight junction structures) compared to Notch1 deleted cells which showed a predominance of non-continuous staining pattern (Figure 5A,B). This difference was quantified by measuring the mean fluorescence intensity of ZO-1 staining at the cell membranes using a previously published method [37]. The results indicated a significantly higher intensity of membrane staining in WT (arbitrarily set at 100 ) compared to Notch1-/- cells (56 ) (P <0.001) (Figure 5D). With longer exposure to the high calcium condition (24-48 hours), 1315463 the Notch1 deleted cells were able to develop a more continuousAqueous tear production is increased in conditional Notch1-/- miceGiven the importance of the tear film in the corneal epithelial barrier function, we proceeded to examine the aqueous tearNotch1 and Corneal Epithelial BarrierFigure 3. Notch1-/- corneal epithelium is delayed in recovering its barrier function after wounding. Fluorescein staining of WT (A1-A4) and Notch1-/- (B1-B4) eyes immediately (0 h), 24, 72 and 96 hours after 2.0 mm central corneal epithelial debridement wounds; indicating a delay in barrier recovery in Notch1-/- eyes compared to WT. LC-biotin barrier function test at 96 hours postwounding showing that the WT cornea is impermeable to LC-biotin (C1) while in the Notch1-/- cornea LC-biotin has penetrated into the stroma (C2). LC-biotin staining showed penetration of the molecule into the stroma in 100 (7/7) of Notch1-/- mice compared to only 28.6 (2/7) of WT littermates at 96 hours (p=0.021). (C3). Oil Red O staining confirmed the presence of oil producing meibomian glands (green arrow head) in both WT (D1) and Notch1-/- (D2) at one week after 4-OHT treatment. Red: rhodamine, Blue: DAPI; scale bar: 100 .doi: 10.1371/journal.pone.0069113.gpattern of ZO-1 staining (mean fluorescent intensity for Notch1-/- was 117.6?2.9 vs 122.4?8.3 for WT, P = 0.429) suggesting that tight junction formation can ultimately be compensated by other means in vitro.DiscussionIn this study we have demonstrated an essential role for Notch1 in the corneal epithelial barrier particularly its recovery after wounding. This is consistent with our previous studieswhich demonstrated that Notch1 is down-regulated during initial stages of wound healing to promote epithelial migration, after which it must.