A comprehensive spatial-temporal transcriptomic analysis of differentiating nascent mouse lens epithelial and fiber cells

Abstract

Elucidation of both the molecular composition and organization of the ocular lens is a prerequisite to understand its development, function, pathology, regenerative capacity, as well as to model lens development and disease using in vitro differentiation of pluripotent stem cells. Lens is comprised of the anterior lens epithelium and posterior lens fibers, which form the bulk of the lens. Lens fibers differentiate from lens epithelial cells through cell cycle exit-coupled differentiation that includes cellular elongation, accumulation of crystallins, cytoskeleton and membrane remodeling, and degradation of organelles within the central region of the lens. Here, we profiled spatiotemporal expression dynamics of both mRNAs and non-coding RNAs from microdissected mouse nascent lens epithelium and lens fibers at four developmental time points (embryonic [E] day 14.5, E16.5, E18.5, and P0.5) by RNA-seq. During this critical time window, multiple complex biosynthetic and catabolic processes generate the molecular and structural foundation for lens transparency. Throughout this developmental window, 3544 and 3518 genes show consistently and significantly greater expression in the nascent lens epithelium and fibers, respectively. Comprehensive data analysis confirmed major roles of FGF-MAPK, Wnt/β-catenin, PI3K/AKT, TGF-β, and BMP signaling pathways and revealed significant novel contributions of mTOR, EIF2, EIF4, and p70S6K signaling in lens formation. Unbiased motif analysis within promoter regions of these genes with consistent expression changes between epithelium and fiber cells revealed an enrichment for both established (e.g. E2Fs, Etv5, Hsf4, c-Maf, MafG, MafK, N-Myc, and Pax6) transcription factors and a number of novel regulators of lens formation, such as Arntl2, Dmrta2, Stat5a, Stat5b, and Tulp3. In conclusion, the present RNA-seq data serves as a comprehensive reference resource for deciphering molecular principles of normal mammalian lens differentiation, mapping a full spectrum of signaling pathways and DNA-binding transcription factors operating in both lens compartments, and predicting novel pathways required to establish lens transparency.

Identification of Novel Gata3 Distal Enhancers Active in Mouse Embryonic Lens

“…1E-F). Gata3 expression was no longer observed at E18.5 or in the neonatal lenses (data not shown), consistent with our recent RNA-seq studies of mouse embryonic (E14.5, E16.5, and E18.5) and newborn lenses (Zhao et al, 2018). Taken together, our data confirm that in the embryonic lens, Gata3 expression is restricted to the cells that undergo cell cycle exit and lens differentiation.…”

“…Gata3 was also observed in the prospective lens ectoderm and optic vesicle in chick (Sheng and Stern, 1999) and Zebrafish (Yao et al, 2014). Interestingly, Gata3 expression is downregulated after E16.5 and is not visible at E18.5 or in neonatal lens (data not shown), nor is it detectable by RNA-seq data analysis (Zhao et al, 2018). In mice, the requirement of Gata3 in global placodal development is still unclear and needs further investigation; nevertheless, inactivation of Gata3 by Pax6-cre (Wolf et al, 2013) disrupts lens development after the formation of the lens vesicle (Martynova et al, unpublished data).…”

“…In mice, the requirement of Gata3 in global placodal development is still unclear and needs further investigation; nevertheless, inactivation of Gata3 by Pax6-cre (Wolf et al, 2013) disrupts lens development after the formation of the lens vesicle (Martynova et al, unpublished data). Interestingly, Gata3 expression is downregulated after E16.5 and is not visible at E18.5 or in neonatal lens (data not shown), nor is it detectable by RNA-seq data analysis (Zhao et al, 2018).…”

RNA sequencing-based transcriptomic profiles of embryonic lens development for cataract gene discovery

“…In addition to validating these known autophagy-related genes, our lens RNA-seq data identifies high expression in the lens of new candidate genes such as Atg4c , Atg9a , Ctsd , Pacs2 and Wipi1 (Table S9). Finally, in agreement with previous reports (Zhao et al 2018), we find that the autophagy/mitophagy-related genes Bnip3 , Gabarapl1 , Mtor , Pink1 , Plekhm1 , and Rblcc1 show an overall increase in expression with progression of lens development (Table S1, Table S5). …”

“…Further, at stages E12.5 and E14.5, “Biosynthesis of unsaturated fatty acids” was enriched (Tables S7, S8), while only stage E16.5 showed enrichment of the gene groups for “Basement membrane” and “Phospholipid biosynthetic process”, which correlates with lens differentiation (Table S9). Moreover, gene groups for “autophagosome assembly” and “mitophagy” were identified only at E16.5; genes identified in these autophagy/mitophagy-related GO clusters, for example Atg4a , Atg7 , Pink1 , have been previously reported in the lens (Brennan et al 2012) and were also identified by a recent RNA-seq based study of isolated lens fiber cells (Zhao et al 2018). In addition to validating these known autophagy-related genes, our lens RNA-seq data identifies high expression in the lens of new candidate genes such as Atg4c , Atg9a , Ctsd , Pacs2 and Wipi1 (Table S9).…”