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: 10510823
• 负责人: Judith A West-Mays
• 金额: $ 14.88万
• 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/10510823
• 关键词: ATF2 gene; Address; BMP4; Birth; Blindness; Bone Morphogenetic Proteins; Burn injury; Cells; Chemicals; Conjunctival Epithelium; Cornea; Corneal Endothelium; Corneal Neovascularization; Corneal Opacity; Corneal Stroma; Data; Defect; Development; Disease; Epithelial; Epithelial Cells; Exhibits; Eye Development; Genetic Diseases; Human; Immunohistochemistry; Impairment; Infection; Inflammation; Injury; Investigation; Irido-corneo-trabecular dysgenesis; Keratin; Knockout Mice; Label; Lead; 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; Stromal Cells; Surface; System; Techniques; Testing; Thinness; Topical application; Vascularization; WNT Signaling Pathway; beta catenin; bone morphogenetic protein 4; cell stroma; cell type; 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.

角膜表面病变如角膜结膜化主要由角膜缘上皮细胞的丧失引起。 干细胞（LESC），并可导致角膜新生血管形成，混浊，并最终失明。虽然 神经嵴细胞（NCC）衍生的眼周间充质（POM）在眼内发育中的作用已被确立， 角膜基质、其在角膜利姆布斯和角膜上皮发育中的作用还有待确定。 研究表明，包括激活蛋白-2 β（AP-2β）在内的转录因子在细胞凋亡中起关键作用。 POM的发展和分化。然而，AP-2β在POM介导的角膜上皮细胞凋亡中的作用， 然而，发育仍然在很大程度上未知，因为AP-2β缺失小鼠在出生后不久死亡。为了解决这个问题，我们必须 使用Wnt 1Cre重组酶系统（AP-2β NCC KO），在小鼠NCC中特异性缺失AP-2β。两 在这些小鼠中观察到的主要缺陷是角膜变薄和血管化，并促成了这一点。 表型为角膜内皮缺失和角膜上皮分层受损。我们 scRNA-seq分析沿着RNAscope和免疫组织化学显示不存在角蛋白-12（K12）， 角膜上皮标志物，以及角蛋白-15（K15）和K13的扩增，结膜上皮特异性标志物， 与对照组相比，突变体的角膜上皮中。进一步的调查显示， 来自突变体的角膜缘区域的LESC标记物ABCB 5，指示结膜样表型，这是由于 NCC中AP-2β的缺失。此外，骨形态发生蛋白（BMP）4，在角膜上皮细胞中的关键作用， 间充质-上皮信号传导已知在角膜上皮细胞生长过程中由Wnt/β-连环蛋白调节 在突变体的上皮细胞中不存在，进一步表明AP-2β在调节细胞分化中的关键作用。 角膜上皮细胞的命运和分层。因此，我们的总体假设是AP-2β的表达 在POM中，通过调节角膜上皮细胞的命运决定和分层至关重要 Wnt/β-catenin信号通路。在目前的建议中，我们的目标是确定：1）发展 AP-2β NCC KO突变体中LESC的时间和命运，以及2）Wnt/β-catenin/BMP 4信号轴是否 在突变体中被破坏并导致眼表缺陷。总的来说，这些研究将有助于我们 了解控制角膜上皮细胞命运决定的基因调控网络（GRN）， 分层，以及以角膜变薄、新生血管形成和 不透明化