Role of transcription factor activating protein-2 beta (AP-2β) in corneal epithelial cell fate determination and stratification

转录因子激活蛋白 2 beta (AP-2β) 在角膜上皮细胞命运决定和分层中的作用

• 批准号: 10683400
• 负责人: Judith A West-Mays
• 金额: $ 14.82万
• 依托单位: MCMASTER UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-01 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10683400
• 关键词: ATF2 gene; Address; BMP4; Birth; Blindness; Bone Morphogenetic Proteins; Burn injury; Cells; Chemicals; Communication; Conjunctival Epithelium; Cornea; Corneal Endothelium; Corneal Neovascularization; Corneal Opacity; Corneal Stroma; Data; Defect; Development; Disease; Epithelial Cells; Epithelium; Exhibits; Eye Development; Genetic Diseases; Human; Immunohistochemistry; Impairment; Infection; Inflammation; Injury; Invaded; Investigation; Irido-corneo-trabecular dysgenesis; Keratin; Knockout Mice; Label; Limbus Corneae; Maintenance; Mediating; Mesenchymal; Mesenchyme; Molecular Genetics; Mus; Mutant Strains Mice; Nature; Neural Crest; Neural Crest Cell; Nuclear; Pathogenesis; Pathologic; Pathology; Pattern; Peripheral; Phenotype; Physiologic pulse; Play; Population; Population Distributions; Population Dynamics; Proteins; Reagent; Reporting; Resources; Role; Series; Signal Pathway; Signal Transduction; Signaling Protein; Stratification; Stratified Epithelium; Stromal Cells; Surface; System; Techniques; Testing; Thinness; Topical application; Vascularization; WNT Signaling Pathway; beta catenin; bone morphogenetic protein 4; cell stroma; cell type; comparison control; corneal epithelial stem cells; corneal epithelium; daughter cell; epithelial stem cell; experimental study; gene regulatory network; innovation; keratin 12; limbal; mouse model; mutant; neovascularization; ocular surface; pluripotency; population migration; radiation burn; recombinase; single-cell RNA sequencing; stem cell biology; stem cell biomarkers; stem cell differentiation; stem cell niche; stem cell population; stem cells; transcription factor; treatment strategy

Corneal surface pathologies such as corneal conjunctivalization arise mainly from the loss of limbal epithelial stem cells (LESCs) and can lead to corneal neovascularization, opacity and ultimately, blindness. Although the role of neural crest cells (NCC)-derived periocular mesenchyme (POM) has been established in development of the corneal stroma, its role in development of the corneal limbus and corneal epithelium has yet to be determined. It has been shown that transcription factors including activating protein-2 beta (AP-2β) play key roles in the development and differentiation of the POM. However, the role of AP-2β in POM-mediated corneal epithelial development remains largely unknown, as AP-2β null mice die soon after birth. To address this, we have specifically deleted AP-2β in the NCC of mice, using the Wnt1Cre-recombinase system (AP-2β NCC KO). Two major defects observed in these mice were corneal thinning and vascularization and contributing to this phenotype were an absence of the corneal endothelium and impairment in corneal epithelial stratification. Our scRNA-seq analyses along with RNAscope and immunohistochemistry revealed an absence of keratin-12 (K12), a corneal epithelial marker, and expansion of keratin-15 (K15) and K13, conjunctival epithelial specific markers, into the corneal epithelium of the mutant when compared to controls. Further investigations revealed an absence of ABCB5, a LESC marker, from the limbal region of the mutants indicating a conjunctival-like phenotype due to the absence of AP-2β in the NCC. In addition, bone morphogenetic protein (BMP) 4, a key player in corneal mesenchymal-to-epithelial signaling known to be modulated by Wnt/β-catenin during corneal epithelial stratification, was absent in the epithelium of the mutants further suggesting a crucial role of AP-2β in regulating corneal epithelial cell fate and stratification. Thus, our overarching hypothesis is that expression of AP-2β in the POM is critical for corneal epithelial cell fate determination and stratification through modulation of the Wnt/β-catenin signaling pathway. In the current proposal we aim to determine: 1) the developmental timing and fate of LESC in the AP-2β NCC KO mutants and 2) whether Wnt/β-catenin/BMP4 –signaling axis-is disrupted in the mutant and contributes to the ocular surface defects. Overall, these studies will contribute to our understanding of the gene regulatory network (GRN) controlling corneal epithelial cell fate determination and stratification, and the pathogenesis of diseases marked by corneal thinning, neovascularization and opacification.

角膜表面病理（例如角膜结膜化）主要源于缘缘上皮的丧失 干细胞（LESC）可以导致角膜新血管形成，不透明性和最终失明。虽然 神经rest细胞（NCC）衍生的周期性间质（POM）的作用已在发展中建立 角膜基质，其在角膜边缘和角膜上皮的发育中的作用尚未确定。 已经表明，包括激活蛋白-2β（AP-2β）在内的转录因子在 POM的发展和差异。但是，AP-2β在POM介导的角膜上皮中的作用 由于AP-2β无效小鼠出生后不久死亡，发育仍然在很大程度上未知。为了解决这个问题，我们有 使用WNT1CRE聚合酶系统（AP-2βNCC KO），在小鼠NCC中的AP-2β。二 在这些小鼠中观察到的主要缺陷是角膜变薄和血管形成，并为此做出了贡献 表型是缺乏角膜内皮和角膜上皮分层的损害。我们的 SCRNA-SEQ与rNascope和免疫组织化学一起分析显示角蛋白12（K12）的吸收 角膜上皮标记，角蛋白15（K15）和K13的扩展，结膜上皮特异性标记， 与对照组相比，在突变体的角膜上皮中。进一步的调查显示 来自突变体的边缘区域的ABCB5，一种LESC标记，表明由于 NCC中没有AP-2β。此外，骨形态发生蛋白（BMP）4，角膜的关键参与者 在角膜上皮期间，wnt/β-catenin调节已知已知的间质至上皮信号传导 在突变体的上皮中不存在分层，进一步表明AP-2β在调节中起着至关重要的作用 角膜上皮细胞命运和分层。这就是我们的总体假设是AP-2β的表达 在POM中，通过调节测定角膜上皮细胞脂肪和分层至关重要 Wnt/β-catenin信号通路的。在当前的建议中，我们旨在确定：1） LESC在AP-2βNCC KO突变体中的时机和命运和2）Wnt/β-catenin/bmp4是否 - 信号轴IS 突变体中断并导致眼表面缺陷。总体而言，这些研究将有助于我们 了解控制角膜上皮细胞脂肪的基因调节网络（GRN）和 分层，以及由角膜稀疏，新血管形成和 无情。