Christianto Antonius, Mongan Maureen, Xiao Bo, Wang Qin, Puga Alvaro, Robinson Michael L, Xia Ying
Department of Environmental and Public Health Sciences, University of Cincinnati College of Medicine, Cincinnati, OH.
Department of Biology, Miami University, Miami, OH.
Mol Vis. 2025 Mar 28;31:85-97. eCollection 2025.
DNA methyltransferase 1 (DNMT1) is a crucial enzyme for the development of the retina and lens in the eye, but its roles in the cornea and eyelids are yet to be investigated.
Ocular surface epithelium (OSE)-specific knockout mice, denoted as , were generated. Prenatal eye tissues were characterized by hematoxylin and eosin staining; DNMT1 expression, DNA methylation, epithelial differentiation and cell-cell junctions were determined by immunohistochemistry; proliferation was assessed by 5-ethynyl 2´-deoxyuridin labeling and apoptosis evaluated by terminal deoxynucleotidyl transferase dUTP nick-end labeling assay. Keratinocytes derived from mice were infected with adenoviruses carrying either green fluorescent protein or Cre recombinase to obtain wild-type and deficient cells. In these cells, expression and epithelial terminal differentiation were evaluated by real-time PCR and/or western blotting; adherence junction and apoptosis were assessed by immunohistochemistry; proliferation was determined by 5-ethynyl 2´-deoxyuridin labeling; transcription factor activities were determined by luciferase reporter assays.
The abundant DNMT1 expression and cytosine methylation (5meC) detected in the ocular surface epithelia of wild-type embryos were largely diminished in that of embryos. Besides lens degeneration, the fetuses had severe abnormalities of the cornea and eyelids. The surface epithelial cells and stromal keratocytes in the knockout corneas were distorted and the eyelids failed to fuse in the knockout embryos, resulting in an eye-open-at-birth phenotype. At the cellular level, DNMT1-deficient OSE had normal proliferation but increased apoptosis and aberrant cell junctions. In addition, the knockout corneal epithelia failed to express corneal-specific keratin 12, and the knockout eyelid epithelia had increased expression of keratin 10, indicating accelerated terminal differentiation. In vitro studies validated that DNMT1 was required for epithelial cell survival, terminal differentiation and cell junctions, and further identified signaling pathways aberrantly activated by its ablation.
DNMT1 maintains survival and differentiation of corneal and eyelid epithelium for the development of the eye.
DNA甲基转移酶1（DNMT1）是眼睛视网膜和晶状体发育的关键酶，但其在角膜和眼睑中的作用尚待研究。
构建眼表上皮（OSE）特异性敲除小鼠，记为 。用苏木精和伊红染色对产前眼组织进行特征分析；通过免疫组织化学测定DNMT1表达、DNA甲基化、上皮分化和细胞间连接；通过5-乙炔基-2'-脱氧尿苷标记评估增殖，通过末端脱氧核苷酸转移酶dUTP缺口末端标记法评估凋亡。用携带绿色荧光蛋白或Cre重组酶的腺病毒感染源自 小鼠的角质形成细胞，以获得野生型和缺陷型细胞。在这些细胞中，通过实时PCR和/或蛋白质印迹评估 表达和上皮终末分化；通过免疫组织化学评估黏附连接和凋亡；通过5-乙炔基-2'-脱氧尿苷标记测定增殖；通过荧光素酶报告基因测定法测定转录因子活性。
在野生型胚胎的眼表上皮中检测到的丰富DNMT1表达和胞嘧啶甲基化（5meC）在 胚胎的眼表上皮中大大减少。除晶状体变性外， 胎儿的角膜和眼睑有严重异常。敲除角膜中的表面上皮细胞和基质角膜细胞变形，敲除胚胎中的眼睑未能融合，导致出生时睁眼的表型。在细胞水平上，DNMT1缺陷的OSE增殖正常，但凋亡增加且细胞连接异常。此外，敲除角膜上皮未能表达角膜特异性角蛋白12，敲除眼睑上皮角蛋白10的表达增加，表明终末分化加速。体外研究证实，DNMT1是上皮细胞存活、终末分化和细胞连接所必需的，并进一步确定了其缺失异常激活的信号通路。
DNMT1维持角膜和眼睑上皮的存活和分化以促进眼睛发育。
