Simultaneous single-cell lineage tracing and transcriptomics of the developing mammalian retina

Abstract

Purpose : The retina provides a valuable system for studying neuronal lineages and differentiation through development. Using a novel method we have designed for combining single-cell transcriptomics and lineage tracing, we aim to improve the resolution of developmental mapping of retinal cells as well as to provide comparative analysis of retinal lineages between mice and human iPSC-derived retinal organoids. With this technology, we hope to provide new insights into the transcriptional changes that accompany individual retinal cell-fate decisions and yield a deeper understanding of the mechanisms of retinal development and disease.

Methods : We have developed DREAM-seq (DNA/RNA extraction, amplification, and multiplexing), a high-throughput, multi-omic method for reading transcriptomic and targeted genomic output from single cells. Human iPS cells and mice have been barcoded using the homing guide RNA (hgRNA) system (Kalhor et al. Science 2018), thereby making possible continuous lineage tracing of individual cells throughout development. We are using DREAM-seq to read out single-cell transcriptomes and lineage barcodes at timepoints throughout development from both murine retinas and hPSC-derived retinal organoids. Novel bioinformatic strategies are being implemented in order to generate high-resolution single-cell lineage maps of retinal development.

Results : The methodology and model systems have been developed and validated for performing these lineage tracing studies. We are collecting data from organoids and murine retinas at a range of developmental timepoints, while concurrently developing and testing the necessary bioinformatic tools using in silico modeling and simulation of the data to reconstruct single-cell lineage relationships.

Conclusions : Using DREAM-seq for the parallel analysis of single-cell transcriptomes and lineages will provide us with a deeper understanding of developing retinal cell fates. This will allow us to identify the timing of cell fate decisions, detect rare or intermediate cell types that occur during retinal development, understand the mechanisms of developing cellular subtypes, and potentially identify targets for trans-differentiation. This level of detail will add to our current understanding of retinal development and have implications for treating retinal disease.

This is a 2021 ARVO Annual Meeting abstract.