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.

Dynamics and Spatial Genomics of the Nascent Transcriptome by Intron seqFISH.

Visualization of the transcriptome and the nuclear organization in situ has been challenging for single-cell analysis. Here, we demonstrate a multiplexed single-molecule in situ method, intron seqFISH, that allows imaging of 10,421 genes at their nascent transcription active sites in single cells, followed by mRNA and lncRNA seqFISH and immunofluorescence. This nascent transcriptome-profiling method can identify different cell types and states with mouse embryonic stem cells and fibroblasts. The nascent sites of RNA synthesis tend to be localized on the surfaces of chromosome territories, and their organization in individual cells is highly variable. Surprisingly, the global nascent transcription oscillated asynchronously in individual cells with a period of 2 hr in mouse embryonic stem cells, as well as in fibroblasts. Together, spatial genomics of the nascent transcriptome by intron seqFISH reveals nuclear organizational principles and fast dynamics in single cells that are otherwise obscured.

Single-cell phenotyping within transparent intact tissue through whole-body clearing.

Understanding the structure-function relationships at cellular, circuit, and organ-wide scale requires 3D anatomical and phenotypical maps, currently unavailable for many organs across species. At the root of this knowledge gap is the absence of a method that enables whole-organ imaging. Herein, we present techniques for tissue clearing in which whole organs and bodies are rendered macromolecule-permeable and optically transparent, thereby exposing their cellular structure with intact connectivity. We describe PACT (passive clarity technique), a protocol for passive tissue clearing and immunostaining of intact organs; RIMS (refractive index matching solution), a mounting media for imaging thick tissue; and PARS (perfusion-assisted agent release in situ), a method for whole-body clearing and immunolabeling. We show that in rodents PACT, RIMS, and PARS are compatible with endogenous-fluorescence, immunohistochemistry, RNA single-molecule FISH, long-term storage, and microscopy with cellular and subcellular resolution. These methods are applicable for high-resolution, high-content mapping and phenotyping of normal and pathological elements within intact organs and bodies.

Integrated spatial genomics reveals global architecture of single nuclei.

Identifying the relationships between chromosome structures, nuclear bodies, chromatin states and gene expression is an overarching goal of nuclear-organization studies1-4. Because individual cells appear to be highly variable at all these levels5, it is essential to map different modalities in the same cells. Here we report the imaging of 3,660 chromosomal loci in single mouse embryonic stem (ES) cells using DNA seqFISH+, along with 17 chromatin marks and subnuclear structures by sequential immunofluorescence and the expression profile of 70 RNAs. Many loci were invariably associated with immunofluorescence marks in single mouse ES cells. These loci form 'fixed points' in the nuclear organizations of single cells and often appear on the surfaces of nuclear bodies and zones defined by combinatorial chromatin marks. Furthermore, highly expressed genes appear to be pre-positioned to active nuclear zones, independent of bursting dynamics in single cells. Our analysis also uncovered several distinct mouse ES cell subpopulations with characteristic combinatorial chromatin states. Using clonal analysis, we show that the global levels of some chromatin marks, such as H3 trimethylation at lysine 27 (H3K27me3) and macroH2A1 (mH2A1), are heritable over at least 3-4 generations, whereas other marks fluctuate on a faster time scale. This seqFISH+-based spatial multimodal approach can be used to explore nuclear organization and cell states in diverse biological systems.

Profiling the transcriptome with RNA SPOTs.

Single-molecule FISH (smFISH) has been the gold standard for quantifying individual transcript abundances. Here, we scale up multiplexed smFISH to the transcriptome level and profile 10,212 different mRNAs from mouse fibroblast and embryonic stem cells. This method, called RNA sequential probing of targets (SPOTs), provides an accurate, flexible, and low-cost alternative to sequencing for profiling transcriptomes.

Single-molecule RNA detection at depth by hybridization chain reaction and tissue hydrogel embedding and clearing.

Accurate and robust detection of mRNA molecules in thick tissue samples can reveal gene expression patterns in single cells within their native environment. Preserving spatial relationships while accessing the transcriptome of selected cells is a crucial feature for advancing many biological areas - from developmental biology to neuroscience. However, because of the high autofluorescence background of many tissue samples, it is difficult to detect single-molecule fluorescence in situ hybridization (smFISH) signals robustly in opaque thick samples. Here, we draw on principles from the emerging discipline of dynamic nucleic acid nanotechnology to develop a robust method for multi-color, multi-RNA imaging in deep tissues using single-molecule hybridization chain reaction (smHCR). Using this approach, single transcripts can be imaged using epifluorescence, confocal or selective plane illumination microscopy (SPIM) depending on the imaging depth required. We show that smHCR has high sensitivity in detecting mRNAs in cell culture and whole-mount zebrafish embryos, and that combined with SPIM and PACT (passive CLARITY technique) tissue hydrogel embedding and clearing, smHCR can detect single mRNAs deep within thick (0.5 mm) brain slices. By simultaneously achieving ∼20-fold signal amplification and diffraction-limited spatial resolution, smHCR offers a robust and versatile approach for detecting single mRNAs in situ, including in thick tissues where high background undermines the performance of unamplified smFISH.

Multiplexed Dynamic Imaging of Genomic Loci by Combined CRISPR Imaging and DNA Sequential FISH.

Visualization of chromosome dynamics allows the investigation of spatiotemporal chromatin organization and its role in gene regulation and other cellular processes. However, current approaches to label multiple genomic loci in live cells have a fundamental limitation in the number of loci that can be labeled and uniquely identified. Here we describe an approach we call "track first and identify later" for multiplexed visualization of chromosome dynamics by combining two techniques: CRISPR imaging and DNA sequential fluorescence in situ hybridization. Our approach first labels and tracks chromosomal loci in live cells with the CRISPR-Cas9 system, then barcodes those loci by DNA sequential fluorescence in situ hybridization in fixed cells and resolves their identities. We demonstrate our approach by tracking telomere dynamics, identifying 12 unique subtelomeric regions with variable detection efficiencies, and tracking back the telomere dynamics of respective chromosomes in mouse embryonic stem cells.

An automated live imaging platform for studying merozoite egress-invasion in malaria cultures.

Most cases of severe and fatal malaria are caused by the intraerythrocytic asexual reproduction cycle of Plasmodium falciparum. One of the most intriguing and least understood stages in this cycle is the brief preinvasion period during which dynamic merozoite-red-cell interactions align the merozoite apex in preparation for penetration. Studies of the molecular mechanisms involved in this process face formidable technical challenges, requiring multiple observations of merozoite egress-invasion sequences in live cultures under controlled experimental conditions, using high-resolution microscopy and a variety of fluorescent imaging tools. Here we describe a first successful step in the development of a fully automated, robotic imaging platform to enable such studies. Schizont-enriched live cultures of P. falciparum were set up on an inverted stage microscope with software-controlled motorized functions. By applying a variety of imaging filters and selection criteria, we identified infected red cells that were likely to rupture imminently, and recorded their coordinates. We developed a video-image analysis to detect and automatically record merozoite egress events in 100% of the 40 egress-invasion sequences recorded in this study. We observed a substantial polymorphism of the dynamic condition of pre-egress infected cells, probably reflecting asynchronies in the diversity of confluent processes leading to merozoite release.

Task-dependent Alteration in Delta Band Corticomuscular Coherence during Standing in Chronic Stroke Survivors.

Balance control is an important indicator of mobility and independence in activities of daily living. How the changes in functional integrity of corticospinal tract due to stroke affects the maintenance of upright stance remains to be known. We investigated the changes in functional coupling between the cortex and lower limb muscles during a challenging balance task over multiple frequency bands in chronic stroke survivors. Eleven stroke patients and nine healthy controls performed a challenging balance task. They stood on a computerized platform with/without somatosensory input distortion created by sway-referencing the support surface, thereby varying the difficulty levels of the task. We computed corticomuscular coherence between Cz (electroencephalography) and leg muscles and assessed balance performance using Berg Balance scale (BBS), Timed-up and go (TUG) and center of pressure (COP) measures. We found lower delta frequency band coherence in stroke patients when compared with healthy controls under medium difficulty condition for distal but not proximal leg muscles. For both groups, we found similar coherence at other frequency bands. On BBS and TUG, stroke patients showed poor balance. However, similar group differences were not consistently observed across COP measures. The presence of distal versus proximal effect suggests differences in the (re)organization of the corticospinal connections across the two muscles groups for balance control. We argue that the observed group difference in the delta coherence might be due to altered mechanisms for the detection of somatosensory modulation resulting from sway-referencing of the support platform for balance control.

Performance of Eosinophil Cationic Protein as a Biomarker in Asthmatic Children.

BACKGROUND: Although blood eosinophils are a frequently used marker of type 2 inflammation in children with asthma, their sensitivity is relatively poor. Additional markers of type 2 inflammation are needed. OBJECTIVE: We hypothesized that plasma concentrations of eosinophil cationic protein (ECP), a marker of eosinophil activation, would be useful for detection of type 2 inflammation and would predict poorer asthma outcomes over 1 year. METHODS: Children and adolescents 6 through 17 years (N = 256) with confirmed asthma completed a baseline visit and a follow-up visit at 12 months. A subset also underwent systemic corticosteroid responsiveness testing with intramuscular triamcinolone. Outcome measures at 12 months included uncontrolled asthma, lung function, and asthma exacerbations treated with systemic corticosteroids. RESULTS: Plasma ECP concentrations ranged from 0.03 to 413.61 ng/mL (median, 6.95 ng/mL) and were consistently associated with other markers of type 2 inflammation. At baseline, children in the highest ECP tertile had poorer asthma control, more airflow limitation, and more exacerbations, but also had greater symptom improvement with intramuscular triamcinolone. At 12 months, associations between the highest ECP tertile and exacerbations, but not lung function or asthma control, persisted after covariate adjustment. However, the sensitivity of ECP was modest and was not markedly different from that of blood eosinophil counts. CONCLUSION: Plasma ECP concentrations may be a useful marker of type 2 inflammation in children and may help identify those children at highest risk for recurrent exacerbations who could benefit from corticosteroid treatment. However, additional markers may be needed to improve sensitivity for outcome detection.

Single-cell nuclear architecture across cell types in the mouse brain.

Diverse cell types in tissues have distinct gene expression programs, chromatin states, and nuclear architectures. To correlate such multimodal information across thousands of single cells in mouse brain tissue sections, we use integrated spatial genomics, imaging thousands of genomic loci along with RNAs and epigenetic markers simultaneously in individual cells. We reveal that cell type­specific association and scaffolding of DNA loci around nuclear bodies organize the nuclear architecture and correlate with differential expression levels in different cell types. At the submegabase level, active and inactive X chromosomes access similar domain structures in single cells despite distinct epigenetic and expression states. This work represents a major step forward in linking single-cell three-dimensional nuclear architecture, gene expression, and epigenetic modifications in a native tissue context.

Identification of spatially associated subpopulations by combining scRNAseq and sequential fluorescence in situ hybridization data.

How intrinsic gene-regulatory networks interact with a cell's spatial environment to define its identity remains poorly understood. We developed an approach to distinguish between intrinsic and extrinsic effects on global gene expression by integrating analysis of sequencing-based and imaging-based single-cell transcriptomic profiles, using cross-platform cell type mapping combined with a hidden Markov random field model. We applied this approach to dissect the cell-type- and spatial-domain-associated heterogeneity in the mouse visual cortex region. Our analysis identified distinct spatially associated, cell-type-independent signatures in the glutamatergic and astrocyte cell compartments. Using these signatures to analyze single-cell RNA sequencing data, we identified previously unknown spatially associated subpopulations, which were validated by comparison with anatomical structures and Allen Brain Atlas images.

seqFISH Accurately Detects Transcripts in Single Cells and Reveals Robust Spatial Organization in the Hippocampus.

We recently applied multiplexed seqFISH to profile expressions of hundreds of genes at the single-cell level in situ (Shah et al., 2016) and provided a map of spatial heterogeneity within each subregion, reconciling previously contradictory descriptions of CA1 at lower spatial resolutions. The accompanying Matters Arising paper from Cembrowski and Spruston questions the spatial organization described for CA1 and raise concerns that the results were determined only by high expression, non-barcoded genes. In response, we show that the same robust spatial structure is observed when only lower average abundance genes measured by barcoded seqFISH are used. In fact, many genes with low average abundance are informative of cell states because they can be expressed strongly in specific subpopulation of cells. Our discussion highlights the importance of single-cell in situ analysis in resolving cellular and spatial heterogeneities otherwise lost in population-averaged measurements. This Matters Arising Response paper addresses the Cembrowski and Spruston (2017) Matters Arising paper, published concurrently in this issue of Neuron.

In Situ Transcription Profiling of Single Cells Reveals Spatial Organization of Cells in the Mouse Hippocampus.

Identifying the spatial organization of tissues at cellular resolution from single-cell gene expression profiles is essential to understanding biological systems. Using an in situ 3D multiplexed imaging method, seqFISH, we identify unique transcriptional states by quantifying and clustering up to 249 genes in 16,958 cells to examine whether the hippocampus is organized into transcriptionally distinct subregions. We identified distinct layers in the dentate gyrus corresponding to the granule cell layer and the subgranular zone and, contrary to previous reports, discovered that distinct subregions within the CA1 and CA3 are composed of unique combinations of cells in different transcriptional states. In addition, we found that the dorsal CA1 is relatively homogeneous at the single cell level, while ventral CA1 is highly heterogeneous. These structures and patterns are observed using different mice and different sets of genes. Together, these results demonstrate the power of seqFISH in transcriptional profiling of complex tissues.

Sequence polymorphism, segmental recombination and toggling amino acid residues within the DBL3X domain of the VAR2CSA placental malaria antigen.

Plasmodium falciparum malaria remains one of the world's foremost health problems, primarily in highly endemic regions such as Sub-Saharan Africa, where it is responsible for substantial morbidity, mortality and economic losses. Malaria is a significant cause of severe disease and death in pregnant women and newborns, with pathogenesis being associated with expression of a unique variant of the multidomain Plasmodium falciparum Erythrocyte Membrane Protein 1 (PfEMP1) called VAR2CSA. Here, we characterize the polymorphism of the DBL3X domain of VAR2CSA and identify regions under selective pressure among placental parasites from women living in endemic western Kenya. In addition to significant levels of polymorphism, our analysis reveals evidence for diversification through intra-segmental recombination and novel mutations that likely contributed to the high number of unique VAR2CSA sequence types identified in this study. Interestingly, we also identified a number of critical residues that may be implicated in immune evasion through switching (or toggling) to alternative amino acids, including an arginine residue within the predicted binding pocket in subdomain III, which was previously implicated in binding to placental CSA. Overall, these findings are important for understanding parasite diversity in pregnant women and will be useful for identifying epitopes and variants of DBL3X to be included in a vaccine against placental malaria.

The use of intensity-modulated radiation therapy photon beams for improving the dose uniformity of electron beams shaped with MLC.

Electrons are ideal for treating shallow tumors and sparing adjacent normal tissue. Conventionally, electron beams are collimated by cut-outs that are time-consuming to make and difficult to adapt to tumor shape throughout the course of treatment. We propose that electron cut-outs can be replaced using photon multileaf collimator (MLC). Two major problems of this approach are that the scattering of electrons causes penumbra widening because of a large air gap, and available commercial treatment planning systems (TPSs) do not support MLC-collimated electron beams. In this study, these difficulties were overcome by (1) modeling electron beams collimated by photon MLC for a commercial TPS, and (2) developing a technique to reduce electron beam penumbra by adding low-energy intensity-modulated radiation therapy (IMRT) photons (4 MV). We used blocks to simulate MLC shielding in the TPS. Inverse planning was used to optimize boost photon beams. This technique was applied to a parotid and a central nervous system (CNS) clinical case. Combined photon and electron plans were compared with conventional plans and verified using ion chamber, film, and a 2D diode array. Our studies showed that the beam penumbra for mixed beams with 90 cm source to surface distance (SSD) is comparable with electron applicators and cut-outs at 100 cm SSD. Our mixed-beam technique yielded more uniform dose to the planning target volume and lower doses to various organs at risk for both parotid and CNS clinical cases. The plans were verified with measurements, with more than 95% points passing the gamma criteria of 5% in dose difference and 5 mm for distance to agreement. In conclusion, the study has demonstrated the feasibility and potential advantage of using photon MLC to collimate electron beams with boost photon IMRT fields.

Cylinders vs. spheres: biofluid shear thinning in driven nanoparticle transport.

Increasingly, the research community applies magnetophoresis to micro and nanoscale particles for drug delivery applications and the nanoscale rheological characterization of complex biological materials. Of particular interest is the design and transport of these magnetic particles through entangled polymeric fluids commonly found in biological systems. We report the magnetophoretic transport of spherical and rod-shaped particles through viscoelastic, entangled solutions using lambda-phage DNA (λ-DNA) as a model system. In order to understand and predict the observed phenomena, we fully characterize three fundamental components: the magnetic field and field gradient, the shape and magnetic properties of the probe particles, and the macroscopic rheology of the solution. Particle velocities obtained in Newtonian solutions correspond to macroscale rheology, with forces calculated via Stokes Law. In λ-DNA solutions, nanorod velocities are 100 times larger than predicted by measured zero-shear viscosity. These results are consistent with particles experiencing transport through a shear thinning fluid, indicating magnetically driven transport in shear thinning may be especially effective and favor narrow diameter, high aspect ratio particles. A complete framework for designing single-particle magnetic-based delivery systems results when we combine a quantified magnetic system with qualified particles embedded in a characterized viscoelastic medium.