Summary
The rapid pace of cell type identification by new single-cell analysis methods has not been met with efficient experimental access to the newly discovered types. To enable flexible and efficient access to specific neural populations in the mouse cortex, we collected chromatin accessibility data from individual cells and clustered the single-cell data to identify enhancers specific for cell classes and subclasses. When cloned into adeno-associated viruses (AAVs) and delivered to the brain by retro-orbital injections, these enhancers drive transgene expression in specific cell subclasses in the cortex. We characterize several enhancer viruses in detail to show that they result in labeling of different projection neuron subclasses in mouse cortex, and that one of them can be used to label the homologous projection neuron subclass in human cortical slices. To enable the combinatorial labeling of more than one cell type by enhancer viruses, we developed a three-color Cre-, Flp- and Nigri-recombinase dependent reporter mouse line, Ai213. The delivery of three enhancer viruses driving these recombinases via a single retroorbital injection into a single Ai213 transgenic mouse results in labeling of three different neuronal classes/subclasses in the same brain tissue. This approach combines unprecedented flexibility with specificity for investigation of cell types in the mouse brain and beyond.
Competing Interest Statement
The authors have declared no competing interest.
Footnotes
This preprint has been updated from original biorXiv submission. The following are major changes: Version 3: 1.Characterization of triple-reporter mouse line Ai213. 2.Description and characterization of additional viral constructs: enhanced versions of mscRE4-based constructs, additional L6-targeted enhancers, additional high-specificity L5 PT-targeted enhancers. 3.Viral infection experiments in human slice culture demonstrating cross-species functionality of L5 PT enhancers. 4.RNAscope experiments to validate specificity and completeness of several enhancer viruses 5.Removal of coembedding analyses with Cusanovich, Hill, et al. (2018) data. It's a great dataset, but fit less with the theme of the manuscript as we shift more towards additional genetic tools. Version 2: 1.Correction of GM12878 cell culturing and collection methods. GM12878 is a suspension cell line, not adherent. Thanks to Darren Cusanovich for identifying this error. 2.Addition of GM12878 data from Pliner, et al. (2018) to Supp Fig 4 and updates to some calculations. This better shows the high quality of current sci-ATAC-seq methods. Thanks to Jay Shendure and Darren Cusanovich for recommending this addition. 3.Updated analysis of Cusanovich, Hill, et al. (2018) data in Supp Fig 7 by changing the label metadata column selected. Thanks to Andrew Hill for recommending these higher-resolution labels. 4.Proper citation of Cusanovich, Hill et al. (2018), and discussion of the prior work in Drosophila in Cusanovich, Reddington, Garfield et al. (2018). Thanks to Jay Shendure for identifying these omissions. 5.Listing of R packages used for analysis.
