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，如角膜结膜形成，主要是由于角膜缘上皮的丧失引起的。 干细胞(LESCs)可导致角膜新生血管、混浊，最终导致失明。尽管 神经脊细胞(NCC)来源的眼周间充质(POM)在眼球发育中的作用已被证实 角膜基质及其在角膜缘和角膜上皮发育中的作用尚不清楚。 已有研究表明，包括激活蛋白-2β(AP-2β)在内的转录因子在细胞周期调控中起着关键作用。 POM的发展和分化。然而，AP-2β在多孔膜介导的角膜上皮细胞中的作用 由于AP-2β缺失小鼠在出生后不久就会死亡，因此发育情况在很大程度上仍不清楚。为了解决这个问题，我们有 利用WNT1Cre重组酶系统(AP-2ββNCC KO)，在小鼠鼻咽癌组织中特异性缺失AP-2 DNA。二 在这些小鼠身上观察到的主要缺陷是角膜变薄和血管形成，这与此有关 表型为角膜上皮细胞缺失和角膜上皮层叠受损。我们的 ScRNA-seq分析以及RNAScope和免疫组织化学显示角蛋白-12(K12)缺失。 角膜上皮标志物，以及角蛋白-15(K15)和K13的扩张，结膜上皮特异性标志物， 当与对照组相比时，突变体的角膜上皮细胞。进一步的调查显示， ABCB5，一个LESC标记，来自突变体的角膜缘区，表明由于 鼻咽癌组织中AP-2β缺失。此外，在角膜中起关键作用的骨形成蛋白(BMP)4 Wnt/β-catenin调控角膜上皮细胞间充质-上皮信号转导的研究 在突变体的上皮中没有复层，进一步表明AP-2β在调节 角膜上皮细胞的命运和层积。因此，我们的首要假设是AP-2β的表达 在POM中是决定角膜上皮细胞命运和调控分层的关键 Wnt/β-连环蛋白信号通路的表达。在目前的提案中，我们的目标是确定：1)发展 AP-2βNCC KO突变体中LESC的时序和命运以及2)Wnt/β-catenin/BMP4-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的 对控制角膜上皮细胞命运的基因调控网络(GRN)的理解 分层，以及以角膜变薄、新生血管和角膜为标志的疾病的发病机制 混浊。