SUMMARY Patterns of gene expression can be used to characterize and classify neuronal types. It is challenging, however, to generate taxonomies that fulfill the essential criteria of being comprehensive, harmonizing with conventional classification schemes, and lacking superfluous subdivisions of genuine types. To address these challenges, we used massively parallel single-cell RNA profiling and optimized computational methods on a heterogeneous class of neurons, mouse retinal bipolar cells (BCs). From a population of ~25,000 BCs we derived a molecular classification that identified 15 types including all types observed previously, and two novel types, one of which has a non-canonical morphology and position. We validated the classification scheme and identified dozens of novel markers using methods that match molecular expression to cell morphology. This work provides a systematic methodology for achieving comprehensive molecular classification of neurons, identifies novel neuronal types, and uncovers transcriptional differences that distinguish types within a class.

The large number of candidate genes made available by comprehensive genome analysis requires that relatively rapid techniques for the study of function be developed. Here, we report a rapid and convenient electroporation method for both gain-and loss-offunction studies in vivo and in vitro in the rodent retina. Plasmid DNA directly injected into the subretinal space of neonatal rodent pups was taken up by a significant fraction of exposed cells after several pulses of high voltage. With this technique, GFP expression vectors were efficiently transfected into retinal cells with little damage to the operated pups. Transfected GFP allowed clear visualization of cell morphologies, and the expression persisted for at least 50 days. DNA-based RNA interference vectors directed against two transcription factors important in photoreceptor development led to photoreceptor phenotypes similar to those of the corresponding knockout mice. Reporter constructs carrying retinal cell type-specific promoters were readily introduced into the retina in vivo, where they exhibited the appropriate expression patterns. Plasmid DNA was also efficiently transfected into retinal explants in vitro by high-voltage pulses.

In the vertebrate central nervous system, the retina has been a useful model for studies of cell fate determination. Recent results from studies conducted in vitro and in vivo suggest a model of retinal development in which both the progenitor cells and the environment change over time. The model is based upon the notion that the mitotic cells within the retina change in their response properties, or "competence", during development. These changes presage the ordered appearance of distinct cell types during development and appear to be necessary for the production of the distinct cell types. As the response properties of the cells change, so too do the environmental signals that the cells encounter. Together, intrinsic properties and extrinsic cues direct the choice of cell fate.