We describe an engineered family of highly antigenic molecules based on GFP-like fluorescent proteins. These molecules contain numerous copies of peptide epitopes and simultaneously bind IgG antibodies at each location. These 'spaghetti monster' fluorescent proteins (smFPs) distributed well in neurons, notably into small dendrites, spines and axons. smFP immunolabeling localized weakly expressed proteins not well resolved with traditional epitope tags. By varying epitope and scaffold, we generated a diverse family of mutually orthogonal antigens. In cultured neurons and mouse and fly brains, smFP probes allowed robust, orthogonal multicolor visualization of proteins, cell populations and neuropil. smFP variants complement existing tracers and greatly increase the number of simultaneous imaging channels, and they performed well in advanced preparations such as array tomography, super-resolution fluorescence imaging and electron microscopy. In living cells, the probes improved single-molecule image tracking and increased yield for RNA-seq. These probes facilitate new experiments in connectomics, transcriptomics and protein localization.

Many tools are available to analyse genomes but are often challenging to use in a cell type-specific context. We have developed a method similar to the isolation of nuclei tagged in a specific cell type (INTACT) technique [Deal,R.B. and Henikoff,S. (2010) A simple method for gene expression and chromatin profiling of individual cell types within a tissue. Dev. Cell, 18, 1030-1040; Steiner,F.A., Talbert,P.B., Kasinathan,S., Deal,R.B. and Henikoff,S. (2012) Cell-type-specific nuclei purification from whole animals for genome-wide expression and chromatin profiling. Genome Res., doi:10.1101/gr.131748.111], first developed in plants, for use in Drosophila neurons. We profile gene expression and histone modifications in Kenyon cells and octopaminergic neurons in the adult brain. In addition to recovering known gene expression differences, we also observe significant cell type-specific chromatin modifications. In particular, a small subset of differentially expressed genes exhibits a striking anti-correlation between repressive and activating histone modifications. These genes are enriched for transcription factors, recovering those known to regulate mushroom body identity and predicting analogous regulators of octopaminergic neurons. Our results suggest that applying INTACT to specific neuronal populations can illuminate the transcriptional regulatory networks that underlie neuronal cell identity.

The anatomy of many neural circuits is being characterized with increasing resolution, but their molecular properties remain mostly unknown. Here, we characterize gene expression patterns in distinct neural cell types of the Drosophila visual system using genetic lines to access individual cell types, the TAPIN-seq method to measure their transcriptomes, and a probabilistic method to interpret these measurements. We used these tools to build a resource of high-resolution transcriptomes for 100 driver lines covering 67 cell types, available at http://www.opticlobe.com. Combining these transcriptomes with recently reported connectomes helps characterize how information is transmitted and processed across a range of scales, from individual synapses to circuit pathways. We describe examples that include identifying neurotransmitters, including cases of apparent co-release, generating functional hypotheses based on receptor expression, as well as identifying strong commonalities between different cell types.

Rod and cone photoreceptors are highly similar in many respects but they have important functional and molecular differences. Here, we investigate genome-wide patterns of DNA methylation and chromatin accessibility in mouse rods and cones and correlate differences in these features with gene expression, histone marks, transcription factor binding, and DNA sequence motifs. Loss of NR2E3 in rods shifts their epigenomes to a more cone-like state. The data further reveal wide differences in DNA methylation between retinal photoreceptors and brain neurons. Surprisingly, we also find a substantial fraction of DNA hypo-methylated regions in adult rods that are not in active chromatin. Many of these regions exhibit hallmarks of regulatory regions that were active earlier in neuronal development, suggesting that these regions could remain undermethylated due to the highly compact chromatin in mature rods. This work defines the epigenomic landscapes of rods and cones, revealing features relevant to photoreceptor development and function.