Comparative, transcriptome analysis of self-organizing optic tissues

Abstract

Embryonic stem (ES) cells have a remarkable capacity to self-organize complex, multi-layered optic cups in vitro via a culture technique called SFEBq. During both SFEBq and in vivo optic cup development, Rax (Rx) expressing neural retina epithelial (NRE) tissues utilize Fgf and Wnt/β-catenin signalling pathways to differentiate into neural retina (NR) and retinal-pigmented epithelial (RPE) tissues, respectively. How these signaling pathways affect gene expression during optic tissue formation has remained largely unknown, especially at the transcriptome scale. Here, we address this question using RNA-Seq. We generated Rx+ optic tissue using SFEBq, exposed these tissues to either Fgf or Wnt/β-catenin stimulation, and assayed their gene expression across multiple time points using RNA-Seq. This comparative dataset will help elucidate how Fgf and Wnt/β-catenin signaling affect gene expression during optic tissue differentiation and will help inform future efforts to optimize in vitro optic tissue culture technology.

Figure 1. Optic cup development, gene expression, and schematic diagram of SFEBq culture.

(a) Schematic diagram of murine eye development from embryonic days E8—E11, a time in which the neural retina (NR) and retinal pigmented epithelial (RPE) tissues emerge. (b) Cryosections of an E10.5 mouse embryo underwent immunohistochemistry, selected gene expression displayed in red, DAPI shown in yellow, Actin shown in blue. Scale bar 100 μm. (c) Pseudocoloured E10.5 cryosection showing developing NR, RPE and lens. (d) Schematic diagram of SFEBq, a technique that generates ES-cell aggregates with a peripheral layer of Rx+ optic progenitor tissue. (e) Cryosections of Day 10 SFEBq Rx:GFP aggregate with a continuous peripheral layer of optic progenitor tissue underwent immunohistochemistry. Marker gene expression displayed in red, DAPI shown in yellow, Actin shown in blue. Scale bar 100 μm. (f) Pseudocoloured diagram of a Day 10 SFEBq aggregate with differentiating NR-like and RPE-like tissues.

Figure 2

Generation of RPE-like and NR-like tissues in vitro using Wnt/β-catenin or Fgf stimulation. (a) Schematic of marker gene expression and signaling pathways that promote neural retina epithelial (NRE) tissue to form neural retina (NR) or retinal pigmented epithelial (RPE) tissues. (b) Transillumination (Trans) and fluorescent images of a Day 10 SFEBq aggregate generated from Rx::GFP//TOP::DsRed ES cells. The TOP promoter (‘TCF/LEF optimized promoter’) drives DsRed expression downstream of Wnt/β-catenin signaling. Scale bar 100 μm. (c) Immunohistochemistry was performed on cryosections of Day 10 SFEBq Rx::GFP//TOP::DsRed aggregates, closed white arrow showing the overlap of TOP::DsRed and Mitf staining. Scale bar shows 100 μm. (d) Montage of images taken from Data Citation 1, showing Day 10 Rx::GFP+//TOP::DsRed tissue in the presence of Wnt/β-catenin (+Wnt) or Fgf stimulation media over 5 days. (e,f) Immunohistochemistry was performed on Day 15 explants cultured with Wnt-stimulating media (e) or Fgf-stimulating media (f). Scale bars 100 μm. Wnt stimulation produces aggregates that are majority Mitf+ and Chx10- where as Fgf stimulation produces aggregates that are majority Chx10+ with some aggregates showing small patches of Mitf+ cells (open white arrow).

Figure 3. RNA-Seq-based transcriptome analysis of Wnt/β-catenin or Fgf stimulated Day 10 Rx::GFP+ SFEBq tissue.

(a) Schematic diagram of RNA-Seq experimental design and sample collection. (b) Electrophoretic quality control of sample total RNA and prepared libraries using, respectively, the RNA Pico Kit and High Sensitivity DNA Assay Kit using Bioanalyzer (Agilent). Leftmost lanes show marker ladders in base pairs (bp) (c) Circos plot showing the genomic coverage of the mapped reads of a sample from each group. (d) Global expression profiling using PCA analysis. (e) Heatmaps showing the expression of known NRE, RPE, NR, Fgf-target, and Wnt-target genes (see Technical Validation).