A Transcriptomics Analysis of the Regulation of Fiber Cell Differentiation in the Absence of FGFRs and PTEN

The addition of 50% vitreous humor to the media bathing lens epithelial explants results in the induction of both lens fiber cell differentiation and a significant immune/inflammatory response. Although Fgfr loss blocks differentiation in lens epithelial explants, this loss can be partially rescued by deleting Pten, demonstrating an epistatic relationship between FGFR-signaling and PTEN. To investigate the functions of Fgfrs and Pten during lens fiber cell differentiation, we utilized a lens epithelial explant system and conducted RNA sequencing on vitreous humor-exposed explants lacking Fgfrs, or Pten or both Fgfrs and Pten. We found that Fgfr loss impairs both vitreous-induced differentiation and inflammation while the additional loss of Pten restores these responses. Additionally, vitreous exposure triggers a less pronounced immune response in explants lacking both Fgfrs and Pten than in wildtype explants, but a stronger immune response than in explants lacking only Fgfrs. Furthermore, transcriptomic analysis suggested that PDGFR-signaling in FGFR-deficient explants might be required to mediate the rescue of vitreous-induced fiber cell differentiation in explants lacking both Fgfrs and Pten. The blockage of ß- crystallin induction in explants lacking both Fgfrs and Pten in the presence of a PDGFR-inhibitor supports this hypothesis. Our findings demonstrate that a wide array of genes associated with fiber cell differentiation are downstream of FGFR-signaling and that the vitreous-induced immune responses also depend on FGFR-signaling. Our data also demonstrates that many of the vitreous-induced gene expression changes Fgfr-deficient explants can be rescued in explants lacking both Fgfrs and Pten. Overall design: Lens epithelial explants from 8-day oldFVB/N-strainlens epithelia were prepared from mice of four different genotypes. These were 1) wildtype (FVB), 2) homozygous fornull alleles ofFgfr3and loxP- flanked alleles forFgfr1and Fgfr2(TKO), 3) homozygous for loxP-flanked alleles of Pten (PTEN), and TKO explants that also were homozygous for the loxP-flanked alleles of Pten (QKO). Explants were cultured in M-199 media containing 0.1% BSA and a 1% antibiotic-antimycotic solution and incubated at 37°C in a humidified chamber with 5% CO2. Recombinant adenoviral vectors delivered Cre recombinase (used to delete loxP-flanked alleles) to explants at a concentration of 1 X 10 6 PFU per dish containing six explants each. 24 hours after explanting, the media was replaced with fresh media mixed at a 1:1 ratio with bovine vitreous. RNA was extracted from lens epithelial explants and subjected to RNA-seq at four stages as follows:(1) 1 day after Cre addition (2) 5 days after Cre addition (3) 10 days after Cre addition. The differentially expressed genes (DEGs) were identified between different conditions.