A versatile viral toolkit for functional discovery in the nervous system.

The ability to precisely control transgene expression is essential for basic research and clinical applications. Adeno-associated viruses (AAVs) are non-pathogenic and can be used to drive stable expression in virtually any tissue, cell type, or species, but their limited genomic payload results in a trade-off between the transgenes that can be incorporated and the complexity of the regulatory elements controlling their expression. Resolving these competing imperatives in complex experiments inevitably results in compromises. Here, we assemble an optimized viral toolkit (VTK) that addresses these limitations and allows for efficient combinatorial targeting of cell types. Moreover, their modular design explicitly enables further refinements. We achieve this in compact vectors by integrating structural improvements of AAV vectors with innovative molecular tools. We illustrate the potential of this approach through a systematic demonstration of their utility for targeting cell types and querying their biology using a wide array of genetically encoded tools.

Genetic and epigenetic coordination of cortical interneuron development.

One of the hallmarks of the cerebral cortex is the extreme diversity of interneurons1-3. The two largest subtypes of cortical interneurons, parvalbumin- and somatostatin-positive cells, are morphologically and functionally distinct in adulthood but arise from common lineages within the medial ganglionic eminence4-11. This makes them an attractive model for studying the generation of cell diversity. Here we examine how developmental changes in transcription and chromatin structure enable these cells to acquire distinct identities in the mouse cortex. Generic interneuron features are first detected upon cell cycle exit through the opening of chromatin at distal elements. By constructing cell-type-specific gene regulatory networks, we observed that parvalbumin- and somatostatin-positive cells initiate distinct programs upon settling within the cortex. We used these networks to model the differential transcriptional requirement of a shared regulator, Mef2c, and confirmed the accuracy of our predictions through experimental loss-of-function experiments. We therefore reveal how a common molecular program diverges to enable these neuronal subtypes to acquire highly specialized properties by adulthood. Our methods provide a framework for examining the emergence of cellular diversity, as well as for quantifying and predicting the effect of candidate genes on cell-type-specific development.

Cellular birthdate predicts laminar and regional cholinergic projection topography in the forebrain.

The basal forebrain cholinergic system projects broadly throughout the cortex and constitutes a critical source of neuromodulation for arousal and attention. Traditionally, this system was thought to function diffusely. However, recent studies have revealed a high degree of spatiotemporal specificity in cholinergic signaling. How the organization of cholinergic afferents confers this level of precision remains unknown. Here, using intersectional genetic fate mapping, we demonstrate that cholinergic fibers within the mouse cortex exhibit remarkable laminar and regional specificity and that this is organized in accordance with cellular birthdate. Strikingly, birthdated cholinergic projections within the cortex follow an inside-out pattern of innervation. While early born cholinergic populations target deep layers, late born ones innervate superficial laminae. We also find that birthdate predicts cholinergic innervation patterns within the amygdala, hippocampus, and prefrontal cortex. Our work reveals previously unappreciated specificity within the cholinergic system and the developmental logic by which these circuits are assembled.

Viral manipulation of functionally distinct interneurons in mice, non-human primates and humans.

Recent success in identifying gene-regulatory elements in the context of recombinant adeno-associated virus vectors has enabled cell-type-restricted gene expression. However, within the cerebral cortex these tools are largely limited to broad classes of neurons. To overcome this limitation, we developed a strategy that led to the identification of multiple new enhancers to target functionally distinct neuronal subtypes. By investigating the regulatory landscape of the disease gene Scn1a, we discovered enhancers selective for parvalbumin (PV) and vasoactive intestinal peptide-expressing interneurons. Demonstrating the functional utility of these elements, we show that the PV-specific enhancer allowed for the selective targeting and manipulation of these neurons across vertebrate species, including humans. Finally, we demonstrate that our selection method is generalizable and characterizes additional PV-specific enhancers with exquisite specificity within distinct brain regions. Altogether, these viral tools can be used for cell-type-specific circuit manipulation and hold considerable promise for use in therapeutic interventions.

Characterizing chromatin landscape from aggregate and single-cell genomic assays using flexible duration modeling.

ATAC-seq has become a leading technology for probing the chromatin landscape of single and aggregated cells. Distilling functional regions from ATAC-seq presents diverse analysis challenges. Methods commonly used to analyze chromatin accessibility datasets are adapted from algorithms designed to process different experimental technologies, disregarding the statistical and biological differences intrinsic to the ATAC-seq technology. Here, we present a Bayesian statistical approach that uses latent space models to better model accessible regions, termed ChromA. ChromA annotates chromatin landscape by integrating information from replicates, producing a consensus de-noised annotation of chromatin accessibility. ChromA can analyze single cell ATAC-seq data, correcting many biases generated by the sparse sampling inherent in single cell technologies. We validate ChromA on multiple technologies and biological systems, including mouse and human immune cells, establishing ChromA as a top performing general platform for mapping the chromatin landscape in different cellular populations from diverse experimental designs.

Developmental diversification of cortical inhibitory interneurons.

Diverse subsets of cortical interneurons have vital roles in higher-order brain functions. To investigate how this diversity is generated, here we used single-cell RNA sequencing to profile the transcriptomes of mouse cells collected along a developmental time course. Heterogeneity within mitotic progenitors in the ganglionic eminences is driven by a highly conserved maturation trajectory, alongside eminence-specific transcription factor expression that seeds the emergence of later diversity. Upon becoming postmitotic, progenitors diverge and differentiate into transcriptionally distinct states, including an interneuron precursor state. By integrating datasets across developmental time points, we identified shared sources of transcriptomic heterogeneity between adult interneurons and their precursors, and uncovered the embryonic emergence of cardinal interneuron subtypes. Our analysis revealed that the transcription factor Mef2c, which is linked to various neuropsychiatric and neurodevelopmental disorders, delineates early precursors of parvalbumin-expressing neurons, and is essential for their development. These findings shed new light on the molecular diversification of early inhibitory precursors, and identify gene modules that may influence the specification of human interneuron subtypes.

Developmental specification of forebrain cholinergic neurons.

Striatal cholinergic interneurons and basal forebrain cholinergic projection neurons, which together comprise the forebrain cholinergic system, regulate attention, memory, reward pathways, and motor activity through the neuromodulation of multiple brain circuits. The importance of these neurons in the etiology of neurocognitive disorders has been well documented, but our understanding of their specification during embryogenesis is still incomplete. All forebrain cholinergic projection neurons and interneurons appear to share a common developmental origin in the embryonic ventral telencephalon, a region that also gives rise to GABAergic projection neurons and interneurons. Significant progress has been made in identifying the key intrinsic and extrinsic factors that promote a cholinergic fate in this precursor population. However, how cholinergic interneurons and projection neurons differentiate from one another during development, as well as how distinct developmental programs contribute to heterogeneity within those two classes, is not yet well understood. In this review we summarize the transcription factors and signaling molecules known to play a role in the specification and early development of striatal and basal forebrain cholinergic neurons. We also discuss the heterogeneity of these populations and its possible developmental origins.