Abstract
Purpose :
The small Maf transcription factors (TFs) Mafg and Mafk belong to the basic leucine-zipper (bZIP) protein family. Previous data showed that Mafg-/-:Mafk+/- compound knockout (KO) mice develop cataract from age 4-month onward. Interestingly, Mafg-/-:Mafk-/- double KO mice exhibit lens defects significantly early, prior to birth. However, the role of Mafg and Mafk in embryonic lens development has not been examined. Therefore, we sought to characterize lens defects in embryonic stages in Mafg-/-:Mafk-/- double KO mice.
Methods :
Appropriate crosses were set-up to generate Mafg-/-:Mafk-/- mice and immunostaining and fluorescence microscopy was used for characterization of lens markers. High-throughput RNA-sequencing (RNA-seq) was performed on embryonic day (E) 16.5 Mafg-/-:Mafk-/- and control lenses in biological triplicates. Differentially expressed genes (DEGs) in E16.5 Mafg-/-:Mafk-/- lenses were validated using RT-qPCR or immunostaining.
Results :
At E16.5, the anterior epithelium of the lens (AEL) in Mafg-/-:Mafk-/- animals appears abnormally multilayered. Further, Mafg-/-:Mafk-/- lenses exhibit abnormal abundance of F-actin near the “fulcrum” region. To gain insights into the molecular basis of these defects, RNA-seq of Mafg-/-:Mafk-/- E16.5 lenses was performed. Over 200 DEGs were identified in Mafg-/-:Mafk-/- lenses. These DEGs were further prioritized based on iSyTE analysis, gene ontology (GO) analysis and relevance to lens biology. Among the prioritized candidates, Epha5 (Eph receptor A 5) is found to be significantly reduced in Mafg-/-:Mafk-/- lenses but not in control or Mafg-/-:Mafk+/- lenses. Because deletion of ephrin-A5 (Efna5), an established ligand of the Epha5 receptor, is shown to cause lens epithelial cell adhesion defects, it is possible that Epha5 reduction contributes to the multilayered AEL defect in Mafg-/-:Mafk-/- lenses. Further, other candidates with functional relevance to the cytoskeleton, cell cycle and extracellular matrix were also among the DEGs in Mafg-/-:Mafk-/- lenses and may contribute to the lens defects.
Conclusions :
These data demonstrate that Mafg and Mafk are necessary for proper development of the lens during embryogenesis. Further, these data uncover new downstream regulatory targets of Mafg and Mafk in the lens. Because these TFs are expressed beyond the lens, these new regulatory relationships may inform on small Maf-based control in non-lens tissues.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.