Abstract
Purpose :
Although photoreceptor gene expression has been studied in the contexts of normal postnatal development and rd1 degeneration using RNA-seq, the genetic drivers regulating these processes remain unknown. Kinetics studies of photoreceptor degeneration suggest that the trigger initiating cell death is stochastic, and signal from a stochastic driver is likely to be masked when pooling populations of cells, in which each individual cell is at a different stage on the path to degeneration. We hypothesize that these stochastic drivers of cell death may be better identified and characterized using single cell RNA-seq.
Methods :
Fluorescence activated cell sorting was used to isolate single CD73+ rod photoreceptors from wildtype (wt) and rd1 mice. Smart-seq2 was used to prepare full length cDNA from single cells for Illumina sequencing library preparation. 94 single photoreceptors were sequenced from both wt and rd1 at postnatal days (P) 5, 8, 11, 14, and 28 (wt only), for a total of 846 single cells. A branched pseudotemporal trajectory representing photoreceptors undergoing normal development and rd1 degeneration was reconstructed using Monocle2. Candidate driver genes were identified via branched expression analysis modeling and experimentally tested using qPCR.
Results :
Principal component analysis formed distinct clusters based on both age and strain. Pseudotemporal ordering reconstructed a trajectory in which all photoreceptors progressed together up to a distinct point, after which wt and rd1 photoreceptors diverged along two different trajectories. The wt trajectory recapitulated known features of normal postnatal photoreceptor development. The rd1 trajectory upregulated markers of cell stress and downregulated markers of photoreceptor maturity. The rd1 photoreceptors isolated from P5 and P8 also showed precocious development relative to their wt counterparts.
Conclusions :
Single cell RNA-seq reconstructed highly resolved gene expression trajectories for photoreceptors undergoing normal postnatal development and rd1 degeneration, revealing novel insights into the genetic mechanisms driving these processes.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.