Limits...
LIM Homeobox Domain 2 Is Required for Corneal Epithelial Homeostasis.

Sartaj R, Chee RI, Yang J, Wan P, Liu A, Guaiquil V, Fuchs E, Rosenblatt MI - Stem Cells (2016)

Bottom Line: Immunodetection on corneal sections were used to visualize conjunctivalization, a sign of limbal barrier failure.Cell based assays showed that Lhx2cKO derived corneal epithelial cells have a significantly lower capacity to form colonies over time and delayed wound-healing recovery when compared to wildtype cells.We conclude that Lhx2 is required for maintenance of the corneal epithelial cell compartment and the limbal barrier.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology and Visual Sciences, Illinois Eye and Ear Infirmary, University of Illinois at Chicago, Chicago, Illinois, USA.

No MeSH data available.


Related in: MedlinePlus

Lhx2 is expressed in the central cornea and limbus.Lhx2 wholemount cornea and conjunctival nuclear staining (red) were detected on Lhx2eGFP corneal tissue in central, limbal, and conjunctival tissue (A). GFP expression of Lhx2eGFP in corneal wholemounts was detected in the central cornea and spanned the entire limbal region, while conjunctival tissue highly expressed Lhx2eGFP(B). In tissue cross sections, GFP‐positive cells were detected in the basal layer of the central corneal epithelium. In the peripheral cornea where the limbus is located, GFP expression was more abundant including suprabasal and basal layers. Basal cells of the conjunctiva stained positive for GFP as well (C). In human tissue, LHX2 nuclear staining in the central cornea localized to few cells in the basal layer, and included basal and suprabasal cells in the limbus. The conjunctiva distinctly expressed LHX2 in basal cells (D). Using quantitative reverse transcriptase polymerase chain reaction analysis, expression of GFP and Lhx2 was found significantly higher in murine epithelial cells originating from the limbus when compared with those from the central cornea (GFP =  4.5‐fold higher, Lhx2 =  1.6‐fold higher vs. central corneal epithelium) (E). Similarly, human LHX2 was expressed at significantly higher levels in limbal versus central corneal epithelium (32‐fold higher) (F). White arrows indicate Lhx2eGFP + and hLHX2 + cells, respectively; yellow dashed line separates the central cornea, from the limbus, conjunctiva, and sclera. Scale bar = 50 µm for images in (A–D). Data are means ± SE (human n = 3, mouse n = 4). *, p ≤ 0.05. Abbreviations: cc, central cornea; cj, conjunctiva; DAPI, * * *; GFP, green fluorescent protein; l, limbus; sc, sclera.
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stem2257-fig-0001: Lhx2 is expressed in the central cornea and limbus.Lhx2 wholemount cornea and conjunctival nuclear staining (red) were detected on Lhx2eGFP corneal tissue in central, limbal, and conjunctival tissue (A). GFP expression of Lhx2eGFP in corneal wholemounts was detected in the central cornea and spanned the entire limbal region, while conjunctival tissue highly expressed Lhx2eGFP(B). In tissue cross sections, GFP‐positive cells were detected in the basal layer of the central corneal epithelium. In the peripheral cornea where the limbus is located, GFP expression was more abundant including suprabasal and basal layers. Basal cells of the conjunctiva stained positive for GFP as well (C). In human tissue, LHX2 nuclear staining in the central cornea localized to few cells in the basal layer, and included basal and suprabasal cells in the limbus. The conjunctiva distinctly expressed LHX2 in basal cells (D). Using quantitative reverse transcriptase polymerase chain reaction analysis, expression of GFP and Lhx2 was found significantly higher in murine epithelial cells originating from the limbus when compared with those from the central cornea (GFP =  4.5‐fold higher, Lhx2 =  1.6‐fold higher vs. central corneal epithelium) (E). Similarly, human LHX2 was expressed at significantly higher levels in limbal versus central corneal epithelium (32‐fold higher) (F). White arrows indicate Lhx2eGFP + and hLHX2 + cells, respectively; yellow dashed line separates the central cornea, from the limbus, conjunctiva, and sclera. Scale bar = 50 µm for images in (A–D). Data are means ± SE (human n = 3, mouse n = 4). *, p ≤ 0.05. Abbreviations: cc, central cornea; cj, conjunctiva; DAPI, * * *; GFP, green fluorescent protein; l, limbus; sc, sclera.

Mentions: To detect Lhx2 expression, we used a mouse genetics approach where the Lhx2 gene promoter was fused to eGFP and could be detected by direct fluorescence or immunostaining for GFP. Our studies on wholemount cornea, limbus, and conjunctival tissue demonstrated consistent localization that was diffusely distributed in central cornea, with more focal expression in the limbus, and high levels of expression in the conjunctival tissue at 20 weeks old. There was a distinct area of localization at the limbus which was not seen in adjacent sclera (Fig. 1A, 1B indicated by dashed yellow lines). GFP expression in the cornea is found in few central basal cells and in all epithelial layers of the limbus, which showed high expression (Fig. 1C, yellow line separate central cornea from limbus). The localization of human followed the same expression pattern as observed in the mouse. The central cornea expressed Lhx2 in few basal epithelial cells, whereas limbal corneal expression included basal and a few suprabasal layers. The conjunctival expression, as seen in mouse was highly pronounced in the basal layer and included few cells above (Fig. 1D). To corroborate the immunostaining, we isolated cells from both areas of the cornea and performed a RT‐PCR analysis. We found that both the expression of GFP and Lhx2 were significantly higher in the limbus as compared to central cornea (4.5 and 0.6 fold respectively) (Fig. 1E). We also investigated the relevance of the findings in mice to humans and found that this preferential expression of Lhx2 in limbal versus central cornea was even more pronounced in human cornea. Real‐time PCR demonstrated a 32‐fold higher levels of Lhx2 in human limbus versus central corneal epithelium (Fig. 1F).


LIM Homeobox Domain 2 Is Required for Corneal Epithelial Homeostasis.

Sartaj R, Chee RI, Yang J, Wan P, Liu A, Guaiquil V, Fuchs E, Rosenblatt MI - Stem Cells (2016)

Lhx2 is expressed in the central cornea and limbus.Lhx2 wholemount cornea and conjunctival nuclear staining (red) were detected on Lhx2eGFP corneal tissue in central, limbal, and conjunctival tissue (A). GFP expression of Lhx2eGFP in corneal wholemounts was detected in the central cornea and spanned the entire limbal region, while conjunctival tissue highly expressed Lhx2eGFP(B). In tissue cross sections, GFP‐positive cells were detected in the basal layer of the central corneal epithelium. In the peripheral cornea where the limbus is located, GFP expression was more abundant including suprabasal and basal layers. Basal cells of the conjunctiva stained positive for GFP as well (C). In human tissue, LHX2 nuclear staining in the central cornea localized to few cells in the basal layer, and included basal and suprabasal cells in the limbus. The conjunctiva distinctly expressed LHX2 in basal cells (D). Using quantitative reverse transcriptase polymerase chain reaction analysis, expression of GFP and Lhx2 was found significantly higher in murine epithelial cells originating from the limbus when compared with those from the central cornea (GFP =  4.5‐fold higher, Lhx2 =  1.6‐fold higher vs. central corneal epithelium) (E). Similarly, human LHX2 was expressed at significantly higher levels in limbal versus central corneal epithelium (32‐fold higher) (F). White arrows indicate Lhx2eGFP + and hLHX2 + cells, respectively; yellow dashed line separates the central cornea, from the limbus, conjunctiva, and sclera. Scale bar = 50 µm for images in (A–D). Data are means ± SE (human n = 3, mouse n = 4). *, p ≤ 0.05. Abbreviations: cc, central cornea; cj, conjunctiva; DAPI, * * *; GFP, green fluorescent protein; l, limbus; sc, sclera.
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stem2257-fig-0001: Lhx2 is expressed in the central cornea and limbus.Lhx2 wholemount cornea and conjunctival nuclear staining (red) were detected on Lhx2eGFP corneal tissue in central, limbal, and conjunctival tissue (A). GFP expression of Lhx2eGFP in corneal wholemounts was detected in the central cornea and spanned the entire limbal region, while conjunctival tissue highly expressed Lhx2eGFP(B). In tissue cross sections, GFP‐positive cells were detected in the basal layer of the central corneal epithelium. In the peripheral cornea where the limbus is located, GFP expression was more abundant including suprabasal and basal layers. Basal cells of the conjunctiva stained positive for GFP as well (C). In human tissue, LHX2 nuclear staining in the central cornea localized to few cells in the basal layer, and included basal and suprabasal cells in the limbus. The conjunctiva distinctly expressed LHX2 in basal cells (D). Using quantitative reverse transcriptase polymerase chain reaction analysis, expression of GFP and Lhx2 was found significantly higher in murine epithelial cells originating from the limbus when compared with those from the central cornea (GFP =  4.5‐fold higher, Lhx2 =  1.6‐fold higher vs. central corneal epithelium) (E). Similarly, human LHX2 was expressed at significantly higher levels in limbal versus central corneal epithelium (32‐fold higher) (F). White arrows indicate Lhx2eGFP + and hLHX2 + cells, respectively; yellow dashed line separates the central cornea, from the limbus, conjunctiva, and sclera. Scale bar = 50 µm for images in (A–D). Data are means ± SE (human n = 3, mouse n = 4). *, p ≤ 0.05. Abbreviations: cc, central cornea; cj, conjunctiva; DAPI, * * *; GFP, green fluorescent protein; l, limbus; sc, sclera.
Mentions: To detect Lhx2 expression, we used a mouse genetics approach where the Lhx2 gene promoter was fused to eGFP and could be detected by direct fluorescence or immunostaining for GFP. Our studies on wholemount cornea, limbus, and conjunctival tissue demonstrated consistent localization that was diffusely distributed in central cornea, with more focal expression in the limbus, and high levels of expression in the conjunctival tissue at 20 weeks old. There was a distinct area of localization at the limbus which was not seen in adjacent sclera (Fig. 1A, 1B indicated by dashed yellow lines). GFP expression in the cornea is found in few central basal cells and in all epithelial layers of the limbus, which showed high expression (Fig. 1C, yellow line separate central cornea from limbus). The localization of human followed the same expression pattern as observed in the mouse. The central cornea expressed Lhx2 in few basal epithelial cells, whereas limbal corneal expression included basal and a few suprabasal layers. The conjunctival expression, as seen in mouse was highly pronounced in the basal layer and included few cells above (Fig. 1D). To corroborate the immunostaining, we isolated cells from both areas of the cornea and performed a RT‐PCR analysis. We found that both the expression of GFP and Lhx2 were significantly higher in the limbus as compared to central cornea (4.5 and 0.6 fold respectively) (Fig. 1E). We also investigated the relevance of the findings in mice to humans and found that this preferential expression of Lhx2 in limbal versus central cornea was even more pronounced in human cornea. Real‐time PCR demonstrated a 32‐fold higher levels of Lhx2 in human limbus versus central corneal epithelium (Fig. 1F).

Bottom Line: Immunodetection on corneal sections were used to visualize conjunctivalization, a sign of limbal barrier failure.Cell based assays showed that Lhx2cKO derived corneal epithelial cells have a significantly lower capacity to form colonies over time and delayed wound-healing recovery when compared to wildtype cells.We conclude that Lhx2 is required for maintenance of the corneal epithelial cell compartment and the limbal barrier.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology and Visual Sciences, Illinois Eye and Ear Infirmary, University of Illinois at Chicago, Chicago, Illinois, USA.

No MeSH data available.


Related in: MedlinePlus