Andrabi, M., Kuraku, S., Takata, N., Sasai, Y., and Love, N.R. (2015). Comparative, transcriptome analysis of self-organizing optic tissues. Sci. Data 2, 150030.
Summary: This paper discusses an RNASeq study of optic organoids, focusing on describing in-depth methods. The goal was to examine transcriptional regulation downstream of Fgf and Wnt signaling during differentiation of neural retina (NR) and retinal-pigmented epithelium (RPE). These pathways were already known to play important roles in NR and RPE development, but transcriptional effects had not been elucidated. Several known targets of Wnt and Fgf and markers of NR and RPE were found to be modulated according to expectations. Furthermore, publication of the RNASeq dataset may lead to discovery of additional targets not previously identified. This could guide developmental studies and lead to further optimization of optic organoid method.
Basic methods: The optic organoid method was the basis of the inner ear organoid method and begins similarly with the dissociation of mouse ES cells and reaggregation in serum-free medium using U-bottom 96-well plates (3000 cells per well) on day 1. Matrigel was added at 4% (v/v) on day 2. (Interestingly, the original method published in 2011 called for 2% Matrigel, but this paper reports that an increased concentration results in a continuous epithelium of retinal precursors (Rx+) rather than a confined, budding optic cup-like structure.)
On day 10, this epithelium was dissected via forceps and either collected for RNASeq or cultured further in a maturation medium. Wnt or Fgf signaling was modulated between day 10 and day 15 using Wnt pathway agonist CHIR99201 (3 uM on day 10, then 1 uM from day 11 on) or Fgf2 (5 ng/mL) + 10% FBS. Media was exchanged on days 11, 12, and 14, and additional samples were collected for RNASeq on days 12 and 15.
Important RNASeq parameters: 3 replicates per sample, 100 bp paired-end reads, RINs > 7, Reference genome mm10
Main experimental questions:
- Day 10 vs day 12 comparison: What genes change expression levels after stimulation of competent Rx+ tissue with Wnt or Fgf?
- Day 12 vs day 15 comparison: What genes change expression during maturation?
- Day 12 Wnt vs day 12 Fgf2 or day 15 Wnt vs day 15 Fgf2: How are transcriptome profiles of RPE- and NR-like tissues different?
Why it’s important: We are planning several RNASeq experiments, and this study is a useful model for our analyses. Since the inner ear organoid method was based on the optic organoid method, it makes sense to refer to this study as a model for RNASeq experiment design in an organoid system. The journal (Scientific Data) seeks to publish datasets and in-depth descriptions of the methods used to generate them in order to improve reproducibility. Therefore, this paper is a good resource as I’m learning to do the bioinformatics to analyze and present the results from our RNASeq experiments. I also think it’s important to note the rationale: They already knew Fgf and Wnt would be important but wanted to know more about downstream transcriptional effects.