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Updated: May 6, 2026
Preparation and Culture of Rat Lens Epithelial Explants for Studying Terminal Differentiation
Published on: September 22, 2009
Identification and characterization of FGF2-dependent mRNA: microRNA networks during lens fiber cell differentiation
Louise Wolf1, Chun S Gao, Karen Gueta
1Department of Genetics, Albert Einstein College of Medicine, Bronx, New York 10461.
Abstract:
MicroRNAs (miRNAs) and fibroblast growth factor (FGF) signaling regulate a wide range of cellular functions, including cell specification, proliferation, migration, differentiation, and survival. In lens, both these systems control lens fiber cell differentiation; however, a possible link between these processes remains to be examined. Herein, the functional requirement for miRNAs in differentiating lens fiber cells was demonstrated via conditional inactivation of Dicer1 in mouse (Mus musculus) lens. To dissect the miRNA-dependent pathways during lens differentiation, we used a rat (Rattus norvegicus) lens epithelial explant system, induced by FGF2 to differentiate, followed by mRNA and miRNA expression profiling. Transcriptome and miRNome analysis identified extensive FGF2-regulated cellular responses that were both independent and dependent on miRNAs. We identified 131 FGF2-regulated miRNAs. Seventy-six of these miRNAs had at least two in silico predicted and inversely regulated target mRNAs. Genes modulated by the greatest number of FGF-regulated miRNAs include DNA-binding transcription factors Nfib, Nfat5/OREBP, c-Maf, Ets1, and N-Myc. Activated FGF signaling influenced bone morphogenetic factor/transforming growth factor-β, Notch, and Wnt signaling cascades implicated earlier in lens differentiation. Specific miRNA:mRNA interaction networks were predicted for c-Maf, N-Myc, and Nfib (DNA-binding transcription factors); Cnot6, Cpsf6, Dicer1, and Tnrc6b (RNA to miRNA processing); and Ash1l, Med1/PBP, and Kdm5b/Jarid1b/Plu1 (chromatin remodeling). Three miRNAs, including miR-143, miR-155, and miR-301a, down-regulated expression of c-Maf in the 3'-UTR luciferase reporter assays. These present studies demonstrate for the first time global impact of activated FGF signaling in lens cell culture system and predicted novel gene regulatory networks connected by multiple miRNAs that regulate lens differentiation.
Insights
MicroRNAs (miRNAs) and fibroblast growth factor (FGF) signaling are crucial for lens cell differentiation. This study reveals novel miRNA-mRNA networks regulated by FGF2, impacting key transcription factors and signaling pathways during lens development.
Area of Science:
- Molecular Biology
- Developmental Biology
- Genetics
Background:
- MicroRNAs (miRNAs) and fibroblast growth factor (FGF) signaling play vital roles in cellular functions, including lens fiber cell differentiation.
- The interplay between miRNAs and FGF signaling in lens differentiation requires further investigation.
Purpose of the Study:
- To investigate the functional requirement of miRNAs in differentiating lens fiber cells.
- To dissect miRNA-dependent pathways regulated by FGF2 during lens differentiation using a rat lens epithelial explant system.
- To identify novel miRNA:mRNA regulatory networks involved in lens development.
Main Methods:
- Conditional inactivation of Dicer1 in mouse lens to assess miRNA function.
- FGF2 induction of rat lens epithelial explants for differentiation.
- mRNA and miRNA expression profiling (transcriptome and miRNome analysis).
- In silico prediction and luciferase reporter assays to validate miRNA:mRNA interactions.
Main Results:
- FGF2 signaling extensively regulates cellular responses in lens cells, both dependently and independently of miRNAs.
- 131 FGF2-regulated miRNAs were identified, with 76 targeting at least two predicted mRNAs.
- Key target genes modulated by miRNAs include transcription factors like Nfib, Nfat5, c-Maf, Ets1, and N-Myc.
- FGF signaling impacts bone morphogenetic factor/transforming growth factor-β, Notch, and Wnt pathways.
- Novel miRNA:mRNA networks were predicted for transcription factors, RNA processing components, and chromatin remodelers.
- miR-143, miR-155, and miR-301a were shown to down-regulate c-Maf expression.
Conclusions:
- This study provides the first global analysis of activated FGF signaling in a lens cell culture system.
- Novel gene regulatory networks, mediated by multiple miRNAs, are predicted to control lens differentiation.
- miRNAs are essential regulators of FGF-driven lens fiber cell differentiation.
