Transcriptomic and Epigenetic Regulation of Fiber Cell Differentiation in Murine Ocular Lens

Lens epithelial explants serve as a valuable in vitro model for studying cellular processes related to lens development and differentiation. Despite significant research, key mechanisms underlying lens fiber cell differentiation and related signaling pathways remain unclear. This dissertation aims to address this gap by investigating the roles of key genes, transcription factors, and microRNAs in lens development and fiber cell differentiation through multiple studies involving RNA-sequencing, ATAC-sequencing, and other molecular biology techniques.Chapter 2 focuses on the influence of vitreous humor on lens epithelial explants, revealing that it increases chromatin accessibility and upregulates genes related to lens fiber cell differentiation while downregulating those associated with lens epithelial cells. The study's unbiased analysis indicated that RUNX, SOX, and TEAD transcription factors might drive these gene expression changes, providing a basis for further exploration.Chapter 3 investigates the role of Fgfrs and Pten in lens fiber cell differentiation and immune responses using RNA-sequencing on explants lacking Fgfrs, Pten, or both. The results show that the loss of Fgfr signaling impairs vitreous-induced fiber differentiation and immune responses, while the loss of Pten can partially rescue these effects. Gene set enrichment analysis suggested that PDGFR-signaling might mediate this rescue, which was confirmed with immunohistochemistry showing beta crystallin expression, indicating fiber cell differentiation.Chapter 4 explores the functional roles of specific microRNAs in lens development. A comprehensive analysis of miRNA transcripts revealed that the loss of miR-26 leads to postnatal cataracts and significant changes in gene expression, with abnormal increases in genes related to neural development, inflammation, and epithelial-to-mesenchymal transition. This demonstrates that miR-26 is crucial for normal lens development and cataract prevention.Chapter 5 examines mutations in FOXE3, a key transcription factor in the lens epithelium. Using CRISPR/Cas9 genome editing, the study generated three novel mutant alleles, uncovering varying degrees of lens-related abnormalities, including cataracts and anterior segment dysgenesis. RNA-seq analysis of homozygous mutants revealed a significant downregulation of genes associated with lens fiber cell differentiation and an upregulation of genes linked to neural and retinal differentiation.Together, these chapters highlight critical aspects of lens development and fiber cell differentiation, demonstrating the complex interplay between key signaling pathways, transcription factors, and microRNAs. The findings suggest future research directions, such as exploring the role of PDGFR in fiber cell differentiation and investigating chromatin architecture changes using ATAC-sequencing, to further understand the mechanisms driving lens development and differentiation.