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Jiawen Xiang, Adam Faranda, Yan Wang, Samuel G Novo, David W Li, Melinda K Duncan; ATF4 is required for both the maintenance of the lens epithelium and late lens fiber cell differentiation. Invest. Ophthalmol. Vis. Sci. 2021;62(8):2085.
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© ARVO (1962-2015); The Authors (2016-present)
ATF4 is a key transcription factor implicated in diverse biological processes -including the unfolded protein response and regulation of amino acid homeostasis. It was reported over two decades ago that ATF4 null mice have defects in lens development, although there is controversy about whether the primary defect manifested in the lens fiber or epithelial cell. While several reports have found that ATF4 levels upregulate in the lens when the secretory pathway is overloaded with unfolded protein, the function of ATF4 in the healthy lens is largely unknown. Here, we take an unbiased approach to elucidate ATF4 function in the developing lens.
RNA-seq was performed on E16.5 embryonic lenses obtained from ATF4 knock out homozygous (ATF4-/-) and WT embryos, followed by iPathwayGuide bioinformatics analysis. E16.5 ATF4-/- and WT embryonic lens phenotype was detected by hematoxylin and eosin staining.
The central lens epithelium of E16.5 ATF4-/- mice is disorganized while vacuoles are seen throughout the lens fibers (Figure 1.A). RNAseq revealed that 562 genes were differentially expressed (DEGs) in E16.5 ATF4-/- lenses, with 173 upregulated and 389 downregulated. The downregulated genes included lens epithelium markers like Foxe3 and E-Cadherin (Cdh1) as well as late lens fiber differentiation markers like Crybb2, Birc7 and Dnase2b. Pathway analysis of these DEGs revealed that genes regulating several different metabolic pathways were downregulated, including at least 17 genes known to regulate amino acid synthesis and transport (including Aldoc, Pycr1 and Phgdh) and at least 7 genes known to be involved with glucagon signaling and insulin secretion including Gcg, Pygl, Camk2a and Abcc8(Figure 1.B). Immunostaining has confirmed that E-cadherin downregulates in while α-SMA (Acta2) upregulates in the ATF4-/- lens at the protein level.
Our data demonstrate that ATF4 is required for both lens epithelial cell maintenance and the terminal differentiation of lens fiber cells during late embryonic development. As the downregulated genes are highly enriched in those encoding proteins important for cellular metabolism, we are hypothesizing that ATF4 is required to establish the unique metabolic requirements of the lens.
This is a 2021 ARVO Annual Meeting abstract.
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