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.

角膜表面病变如角膜结膜化主要是由于角膜缘上皮的丧失引起的 干细胞（LESC）会导致角膜新生血管形成、混浊并最终导致失明。 神经嵴细胞 (NCC) 衍生的眼周间充质 (POM) 在 角膜基质，其在角膜缘和角膜上皮发育中的作用尚未确定。 研究表明，包括激活蛋白 2 β (AP-2β) 在内的转录因子在 然而，AP-2β在POM介导的角膜上皮中的作用。 发育仍然很大程度上未知，因为 AP-2β 缺失小鼠在出生后不久就会死亡。 使用 Wnt1Cre 重组酶系统（AP-2β NCC KO）特异性删除小鼠 NCC 中的 AP-2β。 在这些小鼠中观察到的主要缺陷是角膜变薄和血管化，并导致了这种情况 表型是角膜内皮缺失和角膜上皮分层受损。 scRNA-seq 分析以及 RNAscope 和免疫组织化学显示不存在 keratin-12 (K12)， 角膜上皮标记物，以及角蛋白 15 (K15) 和 K13 的扩展，结膜上皮特异性标记物， 与对照相比，进一步的研究表明突变体的角膜上皮不存在。 ABCB5（一种 LESC 标记）来自突变体的角膜缘区域，表明由于以下因素导致结膜样表型 NCC 中缺乏 AP-2β 此外，骨形态发生蛋白 (BMP) 4（角膜中的关键角色）。 已知角膜上皮细胞过程中 Wnt/β-catenin 调节间充质到上皮信号传导 突变体上皮中不存在分层现象，进一步表明 AP-2β 在调节中发挥着关键作用 因此，我们的首要假设是 AP-2β 的表达。 POM 中的 POM 对于角膜上皮细胞的命运决定和通过调节进行分层至关重要 在当前的提案中，我们的目标是确定：1​​）发育。 AP-2β NCC KO 突变体中 LESC 的时间和命运以及 2) Wnt/β-连环蛋白/BMP4 –信号轴是否是 总的来说，这些研究将有助于我们的研究。 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。