Multimodal Analysis of Cell Types in a Hypothalamic Node Controlling Social Behavior

The ventrolateral subdivision of the ventromedial hypothalamus (VMHvl) contains ∼4,000 neurons that project to multiple targets and control innate social behaviors including aggression and mounting. However, the number of cell types in VMHvl and their relationship to connectivity and behavioral function are unknown. We performed single-cell RNA sequencing using two independent platforms—SMART-seq (∼4,500 neurons) and 10x (∼78,000 neurons)—and investigated correspondence between transcriptomic identity and axonal projections or behavioral activation, respectively. Canonical correlation analysis (CCA) identified 17 transcriptomic types (T-types), including several sexually dimorphic clusters, the majority of which were validated by seqFISH. Immediate early gene analysis identified T-types exhibiting preferential responses to intruder males versus females but only rare examples of behavior-specific activation. Unexpectedly, many VMHvl T-types comprise a mixed population of neurons with different projection target preferences. Overall our analysis revealed that, surprisingly, few VMHvl T-types exhibit a clear correspondence with behavior-specific activation and connectivity

Enhancer viruses and a transgenic platform for combinatorial cell subclass-specific labeling

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

Multimodal Analysis of Cell Types in a Hypothalamic Node Controlling Social Behavior

The ventrolateral subdivision of the ventromedial hypothalamus (VMHvl) contains ∼4,000 neurons that project to multiple targets and control innate social behaviors including aggression and mounting. However, the number of cell types in VMHvl and their relationship to connectivity and behavioral function are unknown. We performed single-cell RNA sequencing using two independent platforms—SMART-seq (∼4,500 neurons) and 10x (∼78,000 neurons)—and investigated correspondence between transcriptomic identity and axonal projections or behavioral activation, respectively. Canonical correlation analysis (CCA) identified 17 transcriptomic types (T-types), including several sexually dimorphic clusters, the majority of which were validated by seqFISH. Immediate early gene analysis identified T-types exhibiting preferential responses to intruder males versus females but only rare examples of behavior-specific activation. Unexpectedly, many VMHvl T-types comprise a mixed population of neurons with different projection target preferences. Overall our analysis revealed that, surprisingly, few VMHvl T-types exhibit a clear correspondence with behavior-specific activation and connectivity

Functional enhancer elements drive subclass-selective expression from mouse to primate neocortex

Viral genetic tools to target specific brain cell types in humans and non-genetic model organisms will transform basic neuroscience and targeted gene therapy. Here we used comparative epigenetics to identify thousands of human neuronal subclass-specific putative enhancers to regulate viral tools, and 34% of these were conserved in mouse. We established an AAV platform to evaluate cellular specificity of functional enhancers by multiplexed fluorescent in situ hybridization (FISH) and single cell RNA sequencing. Initial testing in mouse neocortex yields a functional enhancer discovery success rate of over 30%. We identify enhancers with specificity for excitatory and inhibitory classes and subclasses including PVALB, LAMP5, and VIP/LAMP5 cells, some of which maintain specificity in vivo or ex vivo in monkey and human neocortex. Finally, functional enhancers can be proximal or distal to cellular marker genes, conserved or divergent across species, and could yield brain-wide specificity greater than the most selective marker genes

Comparative cellular analysis of motor cortex in human, marmoset and mouse

The primary motor cortex (M1) is essential for voluntary fine-motor control and is functionally conserved across mammals1. Here, using high-throughput transcriptomic and epigenomic profiling of more than 450,000 single nuclei in humans, marmoset monkeys and mice, we demonstrate a broadly conserved cellular makeup of this region, with similarities that mirror evolutionary distance and are consistent between the transcriptome and epigenome. The core conserved molecular identities of neuronal and non-neuronal cell types allow us to generate a cross-species consensus classification of cell types, and to infer conserved properties of cell types across species. Despite the overall conservation, however, many species-dependent specializations are apparent, including differences in cell-type proportions, gene expression, DNA methylation and chromatin state. Few cell-type marker genes are conserved across species, revealing a short list of candidate genes and regulatory mechanisms that are responsible for conserved features of homologous cell types, such as the GABAergic chandelier cells. This consensus transcriptomic classification allows us to use patch-seq (a combination of whole-cell patch-clamp recordings, RNA sequencing and morphological characterization) to identify corticospinal Betz cells from layer 5 in non-human primates and humans, and to characterize their highly specialized physiology and anatomy. These findings highlight the robust molecular underpinnings of cell-type diversity in M1 across mammals, and point to the genes and regulatory pathways responsible for the functional identity of cell types and their species-specific adaptations

Simultaneous trimodal single-cell measurement of transcripts, epitopes, and chromatin accessibility using TEA-seq

Single-cell measurements of cellular characteristics have been instrumental in understanding the heterogeneous pathways that drive differentiation, cellular responses to signals, and human disease. Recent advances have allowed paired capture of protein abundance and transcriptomic state, but a lack of epigenetic information in these assays has left a missing link to gene regulation. Using the heterogeneous mixture of cells in human peripheral blood as a test case, we developed a novel scATAC-seq workflow that increases signal-to-noise and allows paired measurement of cell surface markers and chromatin accessibility: integrated cellular indexing of chromatin landscape and epitopes, called ICICLE-seq. We extended this approach using a droplet-based multiomics platform to develop a trimodal assay that simultaneously measures transcriptomics (scRNA-seq), epitopes, and chromatin accessibility (scATAC-seq) from thousands of single cells, which we term TEA-seq. Together, these multimodal single-cell assays provide a novel toolkit to identify type-specific gene regulation and expression grounded in phenotypically defined cell types

A comprehensive platform for analyzing longitudinal multi-omics data

The analysis of longitudinal bulk and single-cell multi-omics data is a highly complex task. Here, the authors introduce PALMO, a software platform with five modules to analyse longitudinal bulk and single-cell multi-omics data, which is extensively tested in external datasets that include multiple omics modalities

All-conjugated block copolymers: from controlled synthesis to chiral expression

In the last two decades, the field of conjugated polymers (CPs) expanded rapidly. Besides the discovery of several controlled chain growth polymerizations and the expansion of the monomer scope, also research toward more complex CPs was performed. Copolymers, e.g. block, gradient and random copolymers, and polymers with different topologies, e.g. hyperbranched, star, circular, graft and toothbrush polymers, were synthesized, but the potential of this field remains large. Because the material properties do not only depend on the molecular structure, but also on the supramolecular structure, also the morphology of CPs is one of the frequently studied topics. For conjugated homopolymers, the aggregation behavior is already well described, but for more complex CPs, like block copolymers, a lot of blind spots remain. Therefore, this dissertation focusses on the controlled synthesis and (chiral) aggregation behavior of conjugated block copolymers. In the first part, the influence of branching of the side chain on the controlled polymerization of 3-alkylthiophenes was investigated. It was found that it reduces the propagation rate constant, leading to remarkably low dispersities. Also the maximal DP that could be obtained in a controlled manner was determined to be 150 and nona-stage polymers were synthesized. Further, it was established that a branched side chain diminishes the long term controlled character of the polymerization and that decomplexation of the Ni-catalyst from the polymer backbone was the main termination reaction. The second part deals with the controlled synthesis and (chiral) aggregation behavior of conjugated triblock copolymers. Triblock copolymers consisting of solely thiophene units, as well as triblock copolymers containing selenophene and (a)chiral thiophene units were synthesized in a controlled manner and by investigating their aggregation behavior, it was found that this is influenced by the order of the blocks. In the third part, block copolymers of electronically different monomers were synthesized. Their chiral aggregation behavior was studied in order to investigate whether block copolymers can be designed to exhibit a set of properties, which cannot be achieved in homopolymers, by transferring properties of one block to the other. In a stepwise approach, a poly(fluorene)-b-poly(thiophene) block copolymer, that exhibits β-phase aggregation and chiral expression (two aggregation features which cannot be obtained together in homopolymers), was designed. In the final part, the influence of the nature and the position of defects on the chiral expression in P3ATs was investigated. Polymers with one head-to-head (HH) or tail-to-tail (TT) defect in predetermined positions and one regioregular polymer were synthesized and their chiral aggregation behavior was compared. It was found that the largest chiral expression was obtained when the defect is situated at the beginning of the polymer chain. A TT defect always decreases the chiral expression, but an HH defect can cause an increased chiral expression compared with a complete regioregular polymer. Also, the effect of mixing polymers with a defect in different positions was investigated. For the polymers with an HH defect, no significant influence was observed, but for the polymers with a TT defect, the chiral expression was relatively lowered or enlarged, depending on the type of mixture that was made. This shows that not only the nature or the position of a defect influences the chiral expression, but also the mixing of polymers with a defect in different positions.status: publishe

Single-nucleus and single-cell transcriptomes compared in matched cortical cell types.

Transcriptomic profiling of complex tissues by single-nucleus RNA-sequencing (snRNA-seq) affords some advantages over single-cell RNA-sequencing (scRNA-seq). snRNA-seq provides less biased cellular coverage, does not appear to suffer cell isolation-based transcriptional artifacts, and can be applied to archived frozen specimens. We used well-matched snRNA-seq and scRNA-seq datasets from mouse visual cortex to compare cell type detection. Although more transcripts are detected in individual whole cells (~11,000 genes) than nuclei (~7,000 genes), we demonstrate that closely related neuronal cell types can be similarly discriminated with both methods if intronic sequences are included in snRNA-seq analysis. We estimate that the nuclear proportion of total cellular mRNA varies from 20% to over 50% for large and small pyramidal neurons, respectively. Together, these results illustrate the high information content of nuclear RNA for characterization of cellular diversity in brain tissues