Abstract
Purpose :
Lens epithelial explants respond to vitreous humor by differentiating into fiber cells but this differentiation response is lost when Fgfrs are deleted. Simultaneous loss of both Fgfrs and Pten restores the ability of vitreous to induce lens fiber cell differentiation. To better understand the mechanism by which FGFRs contribute to lens fiber cell differentiation, we undertook a transcriptomic analysis of lens epithelial explants lacking Fgfrs, Pten or both Fgfrs and Pten following exposure to vitreous.
Methods :
P8 FVB/N lens epithelia, homozygous for null alleles of Fgfr3 and loxP-flanked alleles for Fgfr1, Fgfr2 and Pten (QKO), or Fgfrs only (TKO) or Pten only were cultured in serum-free M-199 media and incubated at 37°C in a humidified chamber with 5% CO2. Adenoviral vectors delivered Cre recombinase to explants at a concentration of 1 X 106 PFU per dish containing six explants each. 24 hours after explanting, media was replaced with fresh media mixed at a 1:1 ratio with bovine vitreous. RNA was extracted from explants and subjected to RNA-seq 1 day after Cre removal and 5 days after Cre removal. The explants prepared from wild-type (WT) FVB/N lenses were used as a control. The differentially expressed genes (DEGs) were identified between different conditions.
Results :
As expected, the loss of Fgfrs led to the downregulation of many genes associated with fiber cell differentiation relative to WT including Gja8, Lim2 and Mip. Lens fiber cell-associated genes (Mip, Crybb2, Gja8, Tmod1, Hsf4, Bnip3, Jag1) were significantly upregulated in QKO explants compared to WT explants. Interstingly, even in the absence of Fgfrs and Pten, MAPK associated genes (Spry1, Ets1, Myc, Etv5, Etv1, Ets2, Etv4, Spry4) were significantly uprgelated in QKO samples. Some chromatin remodelers (H2bc3, H2ac6, H2bc15, H2bc11) were also upregulated in QKO samples compared to WT samples. The removal of Pten alone increased the expression of many genes associated with lens epithelium including Foxe3, Mme, Sulf1 and Pdgfra.
Conclusions :
Following exposure to vitreous, the explants lacking Fgfrs and Pten exhibited an overall gene expression response closer to WT explants than those explants lacking Fgfrs alone.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.