Single-cell Profiling of Developing and Mature Retinal Neurons

Summary

A method for the isolation of single retinal cells and subsequent amplification of their cDNAs is described. Single-cell transcriptomics reveals the degree of cellular heterogeneity present in a tissue and uncovers new marker genes for rare cell populations. The accompanying protocol can be adjusted to suit many different cell types.

Abstract

Highly specialized, but exceedingly small populations of cells play important roles in many tissues. The identification of cell-type specific markers and gene expression programs for extremely rare cell subsets has been a challenge using standard whole-tissue approaches. Gene expression profiling of individual cells allows for unprecedented access to cell types that comprise only a small percentage of the total tissue^1-7. In addition, this technique can be used to examine the gene expression programs that are transiently expressed in small numbers of cells during dynamic developmental transitions⁸.

This issue of cellular diversity arises repeatedly in the central nervous system (CNS) where neuronal connections can occur between quite diverse cells⁹. The exact number of distinct cell types is not precisely known, but it has been estimated that there may be as many as 1000 different types in the cortex itself¹⁰. The function(s) of complex neural circuits may rely on some of the rare neuronal types and the genes they express. By identifying new markers and helping to molecularly classify different neurons, the single-cell approach is particularly useful in the analysis of cell types in the nervous system. It may also help to elucidate mechanisms of neural development by identifying differentially expressed genes and gene pathways during early stages of neuronal progenitor development.

As a simple, easily accessed tissue with considerable neuronal diversity, the vertebrate retina is an excellent model system for studying the processes of cellular development, neuronal differentiation and neuronal diversification. However, as in other parts of the CNS, this cellular diversity can present a problem for determining the genetic pathways that drive retinal progenitors to adopt a specific cell fate, especially given that rod photoreceptors make up the majority of the total retinal cell population¹¹. Here we report a method for the identification of the transcripts expressed in single retinal cells (Figure 1). The single-cell profiling technique allows for the assessment of the amount of heterogeneity present within different cellular populations of the retina^2,4,5,12. In addition, this method has revealed a host of new candidate genes that may play role(s) in the cell fate decision-making processes that occur in subsets of retinal progenitor cells⁸. With some simple adjustments to the protocol, this technique can be utilized for many different tissues and cell types.

Protocol

1. Cell Dissociation

1. A flowchart outlining the protocol is shown is Figure 1. For the catalog numbers of the particular reagents used throughout this protocol, please refer to Table 1. Dissect the retina in a PBS bath. During the dissection, it is best to remove the vitreous and the lens since keeping them with the retina can impede the dissociation. It is not always critically important to remove all of the retinal pigment epithelium (RPE) and in some instances it may be impossible to completely remove it. However, for single cell profiling experiments of photoreceptors, the RPE should be removed. Failure to remove the RP....

Discussion

An ever-expanding number of studies are revealing robust cell-to-cell variability in populations that were believed to be more homogeneous with regard to their gene expression^6,8. In at least one instance, this gene expression "noise" has been shown to play an important biological function¹³. Gene expression differences between individual cells are obscured using traditional whole-tissue methods. These experiments generate the expression profile of an "average" cell, which may not be representative<.......