Mitogen-Activated Protein Kinase Kinase Kinase 1 Overexpression Disrupts Development of the Ocular Surface Epithelium

Abstract

Mitogen-Activated Protein Kinase Kinase Kinase 1 (MAP3K1) is a key signaling molecule essential for eyelid closure during embryogenesis. In mice, Map3k1 knockout leads to a fully penetrant eye-open at birth (EOB) phenotype due to disrupted MAPK signaling, abnormal epithelial differentiation, and morphogenesis. To further explore the roles of MAP3K1 in ocular development, we generated a Cre-inducible gain-of-function transgenic mouse, designated as Map3k1^TG, and crossed it with Lens epithelial (Le)-Cre mice to drive MAP3K1 overexpression in developing ocular surface epithelium (OSE). Map3k1^TG;Le-Cre embryos exhibited ocular defects including premature eyelid closure, lens degeneration, and corneal edema. While corneal epithelial differentiation remained intact, the lens epithelium degenerated with lens formation compromised. Eyelid epithelium was markedly thickened, containing cells with aberrant keratin (K)14/K10 co-expression. Genetic rescue experiments revealed that Map3k1^TG;Le-Cre restored eyelid closure in Map3k1 knockout mice, whereas MAP3K1 deficiency attenuated the epithelial thickening caused by transgene expression. Mechanistically, MAP3K1 overexpression enhanced c-Jun phosphorylation in vivo and activated JNK-c-Jun, WNT, TGFβ, and Notch signaling and promoted keratinocyte proliferation and migration in vitro. These findings highlight a dose-sensitive role for MAP3K1 in regulating epithelial proliferation, differentiation, and morphogenesis during eyelid development.

Keywords: MAP3K1 gain-of-function; epithelial morphogenesis; eyelid closure; ocular surface epithelium.

Figure 1

Characterization of the Map3k1^TG mouse model for OSE-specific overexpression. (a) Schematic diagram of the transgenic construct showing the CAG promoter, loxP-flanked STOP cassette, and V5- and TurboID-tagged Map3k1 cDNA, with primer positions indicated. (b) Genotyping PCR of genomic DNA from control (Ctl) and transgenic (TG) mice. (c) Immunofluorescence of E15.5 eye sections showing V5-MAP3K1 expression (green) in Map3k1^TG/+;Le-Cre but not Map3k1^TG/+ embryos. Nuclei were counterstained with Hoechst (blue). Dash lines mark basement membrane of the eyelid epithelium; red arrowheads point at V5-MAP3K1-positive staining. (d) High-magnification image of V5-MAP3K1 (red) in suprabasal eyelid epithelial cells, co-stained with E-cadherin (green). Punctate V5 signal localizes to cytoplasm (arrowheads) and plasma membrane (arrows). Abbreviations: CO: cornea; LE: lens; RE: retina; EL: eyelid.

Figure 2

MAP3K1 overexpression alters corneal and lens development. (a) H&E staining of E15.5 and E17.5 eye sections from Map3k1^TG/+ and Map3k1^TG/+;Le-Cre embryos/fetuses and images were captured at low magnification. (b,e) High magnification views of cornea and lens. (c,d) Immunostaining for K12 (green, suprabasal) and K14 (green, basal) in epithelium, ZO-1 in endothelium, and α-SMA in stroma of E15.5 corneas. Arrowheads point at staining positive cells. (f) Quantification of lens epithelial cell number. (g) EdU incorporation for cell proliferation. (h) TUNEL-positive apoptotic cells. Data represent mean ± s.e.m. from ≥3 sections per embryo, with ≥3 embryos per genotype. *** p < 0.001. Abbreviations: CO, cornea, EL, eyelid, RE, retina, LE, lens, cep, corneal epithelium; cst, stroma; cen, endothelium; lep, lens epithelium; lfb, lens fibers. Dash lines mark the basement membrane of epithelium.

Figure 3

MAP3K1 overexpression affects eyelid morphogenesis. (a) Gross images of E15.5 embryonic eyes showing smaller eye openings in Map3k1^TG/+;Le-Cre. White arrowheads mark canthi of the eyelids. (b) Quantification of open-eye area. (c) H&E staining of eyelid epithelium. (d) Measurement of epithelial thickness. (e) Immunostaining for K14 (top) and K10 (bottom). (f) Quantification of EdU-positive and TUNEL-positive cells. Dash lines mark basement membrane of eyelid epithelium. Data represent mean ± s.e.m. from ≥3 sections and ≥3 embryos per genotype. ** p < 0.01; *** p < 0.001.

Figure 4

Severity of eye defects correlates with MAP3K1 expression level. (a,b) Immunostaining for V5-MAP3K1 in Map3k1^TG/TG;Le-Cre E15.5 eyelids. Arrowheads point at V5-positive signals. (c) H&E staining of eye sections at E15.5 and E17.5. Red arrows mark eyelid leading edge. (d) K12 and K14 immunostaining of corneal epithelium. (e) Quantification of open-eye area. (f) Eyelid epithelium thickness. (g) TUNEL staining and (h) quantification of apoptotic cells. Dash lines mark basement membrane of the epithelium. Data represent mean ± s.e.m. from ≥3 sections and ≥3 embryos per genotype. ** p < 0.01.

Figure 5

MAP3K1 overexpression rescues the EOB phenotype in Map3k1^−/− mice. (a) Gross eye images of Map3k1^−/−;Map3k1^TG/+ and Map3k1^−/−;Map3k1^TG/+;Le-Cre fetuses at E16.5. White arrowheads mark canthi of the eyelids. (b) H&E staining and (c) high-magnification images of eyelid epithelium. Yellow dash lines mark basement membrane of eyelid epithelium. Red arrows point at eyelid leading edge/fusion junction. (d) Quantification of epithelial thickness. Data represent mean ± s.e.m. from ≥3 sections and ≥3 embryos per genotype. *** p < 0.001. Abbreviations: CO: cornea; LE: lens; RE: retina; EL: eyelid.

Figure 6

MAP3K1 overexpression activates multiple signaling pathways in keratinocytes. (a) Western blots detecting V5-MAP3K1, endogenous MAP3K1, p-JNK, p-ERK, p-p38, p-c-Jun, and β-actin in Ad-GFP- or Ad-Cre-infected cells. (b) Densitometric quantification of phospho-proteins. (c) Proliferation measured by EdU incorporation. (d) Representative images of wound healing assays in Ad-GFP- and Ad-Cre-infected cells and (e) quantification of wound areas at 0 and 24 h of wounding. (f) AP-1, TCF, BRE, CBF1/RBP, and SRE reporter activities. (g) Immunostaining for p-c-Jun (green, arrowheads) in E15.5 eyelid epithelium; Hoechst (blue) labels nuclei and dash lines mark basement membrane. Data represent mean ± s.e.m. from ≥3 biological replicates. ** p < 0.01; *** p < 0.001.