Cell and tissue functions arise from complex interactions among numerous genes, and a systematic understanding of these functions requires isoform-resolved transcriptomic analysis of single cells with high spatial resolution. Here, we introduce an in situ RNA amplification method and its integration with multiplexed error-robust fluorescence in situ hybridization (MERFISH) to detect short RNA sequences and enable whole-transcriptome-scale, isoform-resolved spatial transcriptomics of individual cells in intact tissues. Using this approach, we imaged ∼33,000 distinct RNAs-including ∼23,000 genes and ∼10,000 isoforms-in the mouse brain. Our data enabled systematic analyses of region- and cell-type-specific gene programs and ligand-receptor-based cell-cell communications. These data further revealed rich spatial diversity and cell-type specificity in isoform usage across numerous genes, as well as brain structures particularly rich in isoform specificity. We anticipate broad application of this method for characterizing the molecular and cellular basis of tissue functions, unlocking previously inaccessible discoveries in cell and organismal biology.

Tissue biology involves an intricate balance between cell-intrinsic processes and interactions between cells organized in specific spatial patterns, which can be respectively captured by single-cell profiling methods, such as single-cell RNA-seq (scRNA-seq), and histology imaging data, such as Hematoxylin-and-Eosin (H&E) stains. While single-cell profiles provide rich molecular information, they can be challenging to collect routinely and do not have spatial resolution. Conversely, histological H&E assays have been a cornerstone of tissue pathology for decades, but do not directly report on molecular details, although the observed structure they capture arises from molecules and cells. Here, we leverage adversarial machine learning to develop SCHAF (Single-Cell omics from Histology Analysis Framework), to generate a tissue sample’s spatially-resolved single-cell omics dataset from its H&E histology image. We demonstrate SCHAF on two types of human tumors—from lung and metastatic breast cancer—training with matched samples analyzed by both sc/snRNA-seq and by H&E staining. SCHAF generated appropriate single-cell profiles from histology images in test data, related them spatially, and compared well to ground-truth scRNA-Seq, expert pathologist annotations, or direct MERFISH measurements. SCHAF opens the way to next-generation H&E2.0 analyses and an integrated understanding of cell and tissue biology in health and disease.

Cyclin-dependent kinase 4/6 inhibitors (CDK4/6i) have significantly improved the treatment of hormone receptor (HR+) positive breast cancer. Clinical trials have demonstrated that addition of CDK4/6i to endocrine therapy (ET) improves both progression-free and overall survival in patients with HR+ metastatic breast cancer (MBC). Nevertheless, their impact remains limited by the eventual development of resistance. While a number of resistance mechanisms have been identified using both preclinical model systems and translational interrogation of patient specimens, these are not apparent in all cases. As part of the Human Tumor Atlas Network, we integrated genomic (whole-exome sequencing; n=47), single-nucleus transcriptomic (snRNA-seq; n=56), and spatial expression profiling (MERFISH and multiplex immunofluorescence; n=22 and n=17, respectively) from 58 clinically annotated MBC biopsy specimens collected before or after treatment with CDK4/6i plus ET to dissect the roles of tumor-intrinsic programs and the tumor microenvironment (TME) in CDK4/6i resistance. Transcriptomic analysis of the tumor cell compartment with snRNA-seq not only extended prior findings including an association between expression of cell-cycle programs and CDK4/6i resistance, but importantly identified additional programs associated with response and resistance including ciliogenesis, immune evasion, and inflammation. Additionally, tumor-intrinsic gene programs related to epithelial-to-mesenchymal transition had a decreased activity in ESR1 mutant biopsies, a mutational feature of CDK4/6i-resistant MBC associated with upregulated estrogen receptor pathway activity. In the TME, CD8+ T cells were depleted in the CDK4/6i-resistant biopsies, both before and after treatment. In contrast, the CDK4/6i-sensitive biopsies displayed pro-inflammatory features including an increased number of CXCL9+/CXCL10+ macrophages and clustering of CD8+ T cells in the vicinity of the malignant cells spatially. Notably, MERFISH also demonstrated an association between spatial architecture and CDK4/6i response, with the malignant cells in the CDK4/6i-resistant biopsies dispersed spatially compared to the sensitive biopsies. In summary, to the best of our knowledge, this study represents the largest spatially-resolved single-cell MBC cohort with curated CDK4/6i response status established to date. Our findings offer insights into potential biomarkers for CDK4/6i response based on tumor-intrinsic and tumor-microenvironment mechanisms, as well as therapeutic opportunities. Junko Tsuji, Jorge Gómez Tejeda Zañudo, Timothy R. Blosser, Ha T. Vu, Danielle Firer, Nick Maus, Kathleen L. Pfaff, Billie A. Porter, Jason L. Weirather, Anne Carlisle, Allison M. Frangieh, Elliot Boblitt, Aaron R. Thorner, Karla E. Helvie, Isabella E. Kallassy, Laura K. DelloStritto, Ragnhild Laursen, François Aguet, Mendy Miller, Melissa E. Hughes, Nancy U. Lin, Sara M. Tolaney, Eliezer M. Van Allen, Samouil L. Farhi, Xiaowei Zhuang, Barbara Engelhardt, Scott J. Rodig, Alex K. Shalek, Aviv Regev, Bruce E. Johnson, Nikhil Wagle, Gad Getz, Daniel L. Abravanel. Spatially-resolved single-cell transcriptome landscape of response and resistance to CDK4/6 inhibitors in patients with hormone receptor-positive metastatic breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6605.

Immune checkpoint inhibitor (ICI) therapies have markedly improved the prognosis for patients with stage III & IV metastatic melanoma by prolonging progression-free and overall survival rates. However, the variability in immune evasion and resistance mechanisms presents significant challenges to the clinical efficacy of ICIs. This project aims to define drivers of immunotherapy response and resistance by employing advanced genomic, single-cell mRNA analyses, and spatial profiling techniques on tissue biopsies from metastatic melanoma patients. In this study, we developed a framework to analyze response and resistance, both intrinsic and acquired, via immune features in the tumor microenvironment in a standardized, uniformly processed, and deeply clinically annotated cohort of metastatic melanoma patients (n=61) treated with ICB as part of the human tumor atlas network (HTAN) initiative. From the tumor samples, we conducted single-nucleus RNA sequencing, and for a subset of the samples high-resolution spatial imaging (including protein mIHC, CODEX, and transcriptomics MERFISH). Standardized processing and data pipelines allowed for integrating genomic, transcriptomic, and spatial features to elucidate characteristics and mechanisms in tumor microenvironment and their relationships with resistance. Studies of the pretreatment samples demonstrated that CD4 and CD8 T cells, particularly TCF7+ CD8 T cells, are significantly more prevalent in responders to immunotherapy. Conversely, macrophages, especially Angio-TAMs, show higher levels of enrichment in non-responders. Moreover, the presence of B cells and follicular dendritic cells in non-lymph node biopsies supports the presence of tertiary lymphoid structures within the TME. Three levels of immune enrichment were identified through spatial analysis, and samples with more immune enrichment tend to have better responses. We also identified 10 recurrent cellular neighborhoods (RCNs) and found that RCN2,4, and 7 with high lymphocyte infiltration are significantly more enriched in responders than non-responders. In addition, RCN4 is associated with immune infiltration while RCN7 is TLS-like. Our findings indicate that patients with low immune infiltration exhibited enrichment of macrophages associated with the hypoxia and angiogenesis phenotypes, while patients with high immune infiltrates displayed enrichment in lymphocytes, particularly TCF7+ CD8+ T cells, confirming previous findings and indicating a robust T cell-mediated immune response. This project integrates genomic, transcriptomic, and spatial features to elucidate shared tumor and microenvironmental states and their relationships with resistance, and guide more personalized and effective treatment strategies for metastatic melanoma. Xinyu Cui, Giuseppe Tarantino, Yiwen He, Priyanka Solanky, Kathleen Pfaff, Aaron R. Thorner, Tyler J. Aprati, Boyang Zhang, Timothy Blosser, Emily Robitschek, Jiajia Chen, Junko Tsuji, Elliot Boblitt, Allison Frangieh, Hannah M. Faulkner, Marta Holovatska, Aleigha Lawless, Michael Manos, Karla Helvie, Tatyana Sharova, Dennie Frederick, James L. Fahey, Diego Villamarin, Sachi Krishna, Chanell Mangum, Ajit J. Nirmal, Domenic Abbondanza, Cai McCann, Bruce Johnson, Alex K. Shalek, Eliezer Van Allen, Xiaowei Zhuang, Ryan Sullivan, Barbara E. Engelhardt, Samouil L. Farhi, Scott Rodig, F. Stephen Hodi, Genevieve M. Boland, David Liu. Dissecting tumor-immune microenvironment in response and resistance to immune checkpoint blockade in metastatic melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4536.

Understanding the biological mechanisms underlying differential responses to immunotherapy is critical for advancing treatment strategies in metastatic melanoma. We analyzed single-nucleus RNA sequencing data from 66 melanoma samples, including 18 with matched spatial transcriptomics data via MERFISH, across metastatic sites such as lymph nodes, colon, skin, lung, brain, and liver. Our study aimed to identify tumor-intrinsic transcriptional programs and their association with durable clinical benefit. We employed non-negative matrix factorization to delineate distinct tumor programs within tumor cells. To assess the robustness and biological relevance of these programs, we conducted hallmark pathway analyses and utilized spatial transcriptomics to examine their associations with biopsy sites, response, and immune infiltration. Comparisons with published datasets were performed to characterize and ensure the stability and applicability of these programs. We identified five distinct tumor programs with differential enrichment between responders and non-responders. Tumor programs enriched in responders demonstrated strong associations with interferon alpha-beta signaling and antigen presentation pathways. This program was also enriched across diverse lymphocyte populations. Via MERFISH analysis, we classified patients into three categories based on the spatial distribution of immune cells relative to tumor borders: immune-enriched, immune-excluded, and immune-desert. Notably, the program enriched in responders was prevalent in the immune-excluded samples, suggesting that robust signaling pathways can drive therapeutic responses even in the absence of high immune infiltration. In contrast, a mesenchymal-like tumor program was predominant in non-responders, characterized by elevated epithelial-mesenchymal transition activity and diverse tumor-associated macrophage infiltration. This program was particularly enriched in colon metastases. Our findings underscore the importance of interferon signaling in driving therapy response and the role of mesenchymal-like programs in resistance. The unique association of immune-excluded but therapy-responsive samples with interferon-related pathways raises intriguing questions about tumor-immune dynamics. By integrating single-cell and spatial transcriptomics, this study provides insights into tumor heterogeneity and the tumor microenvironment, offering avenues for targeted therapeutic interventions. Future research will focus on spatial interactions, the characterization of immune subpopulations within tumor programs, and the dynamics of cell-cell communication between tumor and immune cells, aiming to refine and advance personalized treatment strategies. Priyanka Solanky, Giuseppe Tarantino, Yiwen He, Xinyu Cui, Kathleen Pfaff, Aaron R. Thorner, Tyler J. Aprati, Boyang Zhang, Timothy Blosser, Jiajia Chen, Junko Tsuji, Elliot Boblitt, Allison Frangieh, Hannah Mae Faulkner, Marta M. Holovatska, Aleigha R. Lawless, Michael Manos, Emily Robitschek, Karla Helvie, Tatyana Sharova, Dennie Frederick, James Liam Fahey, Diego Villamarin, Sachi Krishna, Chanell Mangum, Ajit J. Nirmal, Domenic Abbondanza, Cai McCann, Bruce Johnson, Alex Shalek, Eliezer Van Allen, Xiaowei Zhuang, Ryan J. Sullivan, Barbara Engelhardt, Sami Farhi, Scott J. Rodig, Stephen Hodi, Genevieve Boland, David Liu. Dissecting tumor-intrinsic programs and immune dynamics driving response to immunotherapy in metastatic melanoma through non-negative matrix factorization decomposition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6256.

Although metastatic disease is the leading cause of cancer-related deaths, its tumor microenvironment remains poorly characterized due to technical and biospecimen limitations. In this study, we assembled a multi-modal spatial and cellular map of 67 tumor biopsies from 60 patients with metastatic breast cancer across diverse clinicopathological features and nine anatomic sites with detailed clinical annotations. We combined single-cell or single-nucleus RNA sequencing for all biopsies with a panel of four spatial expression assays (Slide-seq, MERFISH, ExSeq and CODEX) and H&E staining of consecutive serial sections from up to 15 of these biopsies. We leveraged the coupled measurements to provide reference points for the utility and integration of different experimental techniques and used them to assess variability in cell type composition and expression as well as emerging spatial expression characteristics across clinicopathological and methodological diversity. Finally, we assessed spatial expression and co-localization features of macrophage populations, characterized three distinct spatial phenotypes of epithelial-to-mesenchymal transition and identified expression programs associated with local T cell infiltration versus exclusion, showcasing the potential of clinically relevant discovery in such maps. Single-nucleus and single-cell RNA sequencing plus spatial profiling with four methods of core biopsies from 60 patients with metastatic breast cancer reveal patient-specific gene expression programs of breast cancer metastases that are maintained across time, site of metastasis and spatial profiling method, with spatial phenotypes correlating with microenvironmental features.

Abstract Background: Immune checkpoint blockade (ICB) has revolutionized the treatment and prognosis of various cancers, including melanoma. Nevertheless, intrinsic or acquired resistance is common, and the interactions between tumor-intrinsic features and the microenvironment underlying these resistances remain unclear for most patients. Methods: In this study, we developed a framework to analyze response and resistance, both intrinsic and acquired, via tumor-intrinsic programs, immune features, and tumor-stromal-immune interactions in a standardized, uniformly processed, and deeply clinically annotated cohort of metastatic melanoma patients (n=61) treated with ICB as part of the NCI funded human tumor atlas network (HTAN) initiative. From the same tumor samples (pre-treatment (n=33) and post-progression (n=28)), we conducted bulk whole-exome sequencing (WES), single-nucleus RNA sequencing, and for a subset of the samples high-resolution spatial imaging (including protein mIHC, CODEX, and transcriptomics MERFISH). Standardized processing and data pipelines allowed for integration of genomic, transcriptomic, and spatial features to elucidate shared tumor and microenvironmental states and their relationships with resistance. Results: Our preliminary analysis on a subset of 28 samples identified an enrichment of B cells, plasma cells, and T follicular helper-like cells in the tumor microenvironment (TME) of patients sensitive to treatment. The TME of non-responders was predominantly characterized by macrophage/monocyte cell populations. Post-treatment responder samples showed an enrichment of CD4+ T cells. Genomic heterogeneity, assessed through WES, was confirmed as a feature of intrinsic resistance across different ICB treatment settings, while ploidy was associated with treatment response in pre-treatment, ipilimumab-naïve patients treated with PD-1 inhibitors. Comparing the immune compartments of samples with high and low heterogeneity revealed NK cell enrichment in samples with a high proportion of subclonal mutations. Conclusions and Future Directions: Overall, our work provides a high-resolution understanding of the tumor-stromal-immune interaction in metastatic melanoma, shedding light on the factors contributing to therapy response and resistance, and contributing a repository of data from deeply clinically annotated, multimodally characterized and uniformly processed patient samples. Ongoing spatial analysis will further enhance our understanding of these compartments at a spatial level. The results of our study have the potential to guide more personalized and effective treatment strategies for metastatic melanoma in the future. Citation Format: Giuseppe Tarantino, Yiwen He, Priyanka Solanky, Aaron Thorner, Tyler Aprati, Ryan Sullivan, Emily Robitschek, Timothy Blosser, Xiaowei Zhuang, Xiaowei Zhuang, Elliot Boblitt, Allison Frangieh, Marta Holovatska, Aleigha Lawless, Michael Manos, Kathleen Pfaff, Karla Helvie, Tatyana Sharova, Dennie Frederick, James Liam Fahey, Diego Villamarin, Sami Farhi, Scott Rodig, Bruce Johnson, Alex K. Shalek, Eliezer Van Allen, Stephen Hodi, Genevieve M. Boland, David Liu. Dissecting tumor-immune interaction in response and resistance to immune checkpoint blockade in metastatic melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2486.

Summary The cellular diversity and complex organization of the brain have hindered systematic characterization of age-related changes in its cellular and molecular architecture, limiting our ability to understand the mechanisms underlying its functional decline during aging. Here we generated a high-resolution cell atlas of brain aging within the frontal cortex and striatum using spatially resolved single-cell transcriptomics and quantified the changes in gene expression and spatial organization of major cell types in these brain regions over the lifespan of mice. We observed substantially more pronounced changes in the composition, gene expression and spatial organization of non-neuronal cells over neurons. Our data revealed molecular and spatial signatures of glial and immune cell activation during aging, particularly enriched in subcortical white matter, and identified both similarities and notable differences in cell activation patterns induced by aging and systemic inflammatory challenge. These results provide critical insights into age-related decline and inflammation in the brain.

Metastatic breast cancer (MBC) remains incurable due to inevitable development of therapeutic resistance. Although tumor cell intrinsic mechanisms of resistance in MBC are beginning to be elucidated by bulk sequencing studies, the roles of the tumor microenvironment and intratumor heterogeneity in therapeutic resistance remain underexplored due to both technological barriers and limited availability of samples. To comprehensively capture these characteristics we have adapted a research biopsy protocol to collect tissue for an array of single-cell and spatio-molecular assays whose performance we have optimized for MBC, including single-cell and single-nucleus RNA sequencing, Slide-Seq, Multiplexed Error-Robust FISH (MERFISH), Expansion Sequencing (ExSEQ), Co-detection by Indexing (CODEX) and Multiplexed Ion Beam Imaging (MIBI). To date, we have successfully performed single-cell or single-nucleus RNAseq in 67 MBC biopsies and generated detailed accompanying clinical annotations for each. These samples provide a representation of the clinicopathological diversity of MBC including different breast cancer subtypes (44 HR+/HER2-, 3 HR-/HER2+, 3 HR+/HER2+, 16 TNBC, 1 unknown), common anatomic sites of metastasis (37 liver, 9 axilla, 7 breast, 5 bone, 3 chest wall, 3 neck, 1 brain, 1 lung, 1 skin), metastatic presentations (53 recurrent, 14 de novo) and histologic subtypes in the breast (45 IDC, 7 ILC, 6 mixed, 3 DCIS, 1 mucinous, 5 unknown/NA). Following optimization, both single-cell and single-nucleus RNA seq perform well in these MBC biopsies recovering all expected cell types including the malignant, stromal (e.g. fibroblasts, endothelial cells), myeloid (e.g. monocytes, macrophages) and lymphoid compartments (e.g. T cells, B cells, NK cells) as well as relevant oncogenic programs (e.g. cell cycle programs in all compartments; EMT-like and ER signaling programs in the malignant compartment, immune checkpoint programs in the lymphoid compartment; and fibroblast activation and vascular homeostasis programs in the stromal compartment). In addition to differences between the two techniques, these data demonstrate substantial intratumor heterogeneity in cell type composition. For example in liver biopsies the average number of cells per sample compartment by single nucleus RNA-seq was 6745 malignant (56%, SD 4216), 4637 stromal (41%, SD 3727), 1196 lymphoid (8%, SD 1617) and 874 myeloid (6%, SD 852); in breast biopsies the average number of cells per compartment by single nucleus RNA-seq was 6421 malignant (70%, SD 3497), 1628 stromal (24%, SD 117), 333 lymphoid (4%, SD 170) and 213 myeloid (3%, SD 117). Additionally, we find both inter- and intra-tumor heterogeneity in expression patterns and programs including, for example, expression of ER, PR and HER2 within clinical receptor subtypes (log normalized counts for ER expression in tumor cells by single cell RNA-seq: HR+/HER2- 0.921 (SD 0.714); HR+/HER2+ 0.768 (SD 0.624); HR-/HER2+ 0.018 (SD 0.122); and HR-/HER2- 0.005 (SD 0.066). For a subset of 13 biopsies we are also completing the spatiomolecular characterization methods on serial sections of a single adjacent biopsy. This unique experimental setup was designed to enable efficient comparison and integration of these assays. In spite of differences between experimental techniques and readouts, cell typing can be approached by annotation transfer from matching single cell or single nucleus RNAseq data, enabling exploratory analyses including evaluation of spatial phenotypes and cell type colocalization. Overall, these single cell and spatial data afford a comprehensive atlas including cell types, cell states/programs, cell interactions and spatial organization in MBC lesions. Future analyses will include serial biopsies over time and integration of clinicopathologic data including therapeutic response and resistance. Citation Format: Daniel L Abravanel, Johanna Klughammer, Timothy Blosser, Yury Goltsev, Sizun Jiang, Yunjao Bai, Evan Murray, Shahar Alon, Yi Cui, Daniel R Goodwin, Anubhav Sinha, Ofir Cohen, Michal Slyper, Orr Ashenberg, Danielle Dionne, Judit Jané-Valbuena, Caroline BM Porter, Asa Segerstolpe, Julia Waldman, Sébastien Vigneau, Karla Helvie, Allison Frangieh, Laura DelloStritto, Miraj Patel, Jingyi We, Kathleen Pfaff, Nicole Cullen, Ana Lako, Madison Turner, Isaac Wakiro, Sara Napolitano, Abhay Kanodia, Rebecca Ortiz, Colin MacKichan, Stephanie Inga, Judy Chen, Aaron R Thorner, Asaf Rotem, Scott Rodig, Fei Chen, Edward S Boyden, Garry P Nolan, Xiaowei Zhuang, Orit Rozenblatt-Rosen, Bruce E Johnson, Aviv Regev, Nikhil Wagle. Spatio-molecular dissection of the breast cancer metastatic microenvironment [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr PD6-03.

The diversity and complex organization of cells in the brain have hindered systematic characterization of age-related changes in its cellular and molecular architecture, limiting our ability to understand the mechanisms underlying its functional decline during aging. Here, we generated a high-resolution cell atlas of brain aging within the frontal cortex and striatum using spatially resolved single-cell transcriptomics and quantified changes in gene expression and spatial organization of major cell types in these regions over the mouse lifespan. We observed substantially more pronounced changes in cell state, gene expression, and spatial organization of non-neuronal cells over neurons. Our data revealed molecular and spatial signatures of glial and immune cell activation during aging, particularly enriched in the subcortical white matter, and identified both similarities and notable differences in cell-activation patterns induced by aging and systemic inflammatory challenge. These results provide critical insights into age-related decline and inflammation in the brain.

To generate a comprehensive atlas characterizing neuroblastoma for the Human Tumor Atlas Pilot Project (HTAPP), we profiled tumor samples using molecular (single-cell/nucleus RNA-sequencing) and spatial transcriptomics and proteomics profiling technologies. Some of the spatial technologies used employ targeted approaches requiring pre-selection of a specific set of genes or proteins to be profiled. Designing targeted panels requires careful consideration in terms of the genes/probes or antibodies selected in order to identify the different cell types and programs in the tumor microenvironment and to gain biological/mechanistic insights. This document briefly describes our approach to designing a ~300-gene panel for HTAPP’s MERFISH profiling of neuroblastoma samples.

The nucleus accumbens (NAc) plays an important role in regulating multiple behaviors and its dysfunction has been linked to many neural disorders. However, the molecular, cellular and anatomic heterogeneity underlying its functional diversity remains incompletely understood. Here, we generate a cell census of the mouse NAc using high-throughput single cell RNA sequencing and multiplexed error-robust FISH, revealing a high level of cell heterogeneity in this brain region. We show that the transcriptional and spatial diversity of neuron subtypes underlie NAc’s anatomic and functional heterogeneity, and possibly contribute to the pathogenesis of different neurological disorders. These findings explain how the seemingly simple neuronal composition of the NAc achieves its highly heterogenous structure and diverse functions. Collectively, our study generates a spatially resolved cell taxonomy for understanding the NAc structure and function, which demonstrates the importance of combining molecular and spatial information in revealing the fundamental features of the nervous system.

The nucleus accumbens (NAc) plays an important role in regulating multiple behaviors, and its dysfunction has been linked to many neural disorders. However, the molecular, cellular and anatomic heterogeneity underlying its functional diversity remains incompletely understood. In this study, we generated a cell census of the mouse NAc using single-cell RNA sequencing and multiplexed error-robust fluorescence in situ hybridization, revealing a high level of cell heterogeneity in this brain region. Here we show that the transcriptional and spatial diversity of neuron subtypes underlie the NAc’s anatomic and functional heterogeneity. These findings explain how the seemingly simple neuronal composition of the NAc achieves its highly heterogenous structure and diverse functions. Collectively, our study generates a spatially resolved cell taxonomy for understanding the structure and function of the NAc, which demonstrates the importance of combining molecular and spatial information in revealing the fundamental features of the nervous system. The authors generated a cell census of mouse nucleus accumbens using single-cell RNA sequencing and multiplexed error-robust FISH. These data suggest that transcriptional and spatial diversity of neuron subtypes underlies nucleus accumbens anatomic and functional heterogeneity.

The thalamic reticular nucleus (TRN), the major source of thalamic inhibition, regulates thalamocortical interactions that are critical for sensory processing, attention and cognition 1 – 5 . TRN dysfunction has been linked to sensory abnormality, attention deficit and sleep disturbance across multiple neurodevelopmental disorders 6 – 9 . However, little is known about the organizational principles that underlie its divergent functions. Here we performed an integrative study linking single-cell molecular and electrophysiological features of the mouse TRN to connectivity and systems-level function. We found that cellular heterogeneity in the TRN is characterized by a transcriptomic gradient of two negatively correlated gene-expression profiles, each containing hundreds of genes. Neurons in the extremes of this transcriptomic gradient express mutually exclusive markers, exhibit core or shell-like anatomical structure and have distinct electrophysiological properties. The two TRN subpopulations make differential connections with the functionally distinct first-order and higher-order thalamic nuclei to form molecularly defined TRN–thalamus subnetworks. Selective perturbation of the two subnetworks in vivo revealed their differential role in regulating sleep. In sum, our study provides a comprehensive atlas of TRN neurons at single-cell resolution and links molecularly defined subnetworks to the functional organization of thalamocortical circuits.

This protocol describes the steps required to prepare glass coverslips for use in a MERFISH experiment. Out of the box, coverslips must be cleaned, silanized, coated with poly-D lysine, and covered with fiducial beads before it can be used to hold a sample for MERFISH imaging. This protocol is a part of a collection of protocols used in the profiling of OCT- embedded fresh-frozen metastatic breast cancer tissue samples with MERFISH for the Human Tumor Atlas Pilot Project (HTAPP).

Small RNA-guided protein complexes play an essential role in CRISPR-mediated immunity in prokaryotes. While these complexes initiate interference by flagging cognate invader DNA for destruction, recent evidence has implicated their involvement in new CRISPR memory formation, called priming, against mutated invader sequences. The mechanism by which the target recognition complex mediates these disparate responses-interference and priming-remains poorly understood. Using single-molecule FRET, we visualize how bona fide and mutated targets are differentially probed by E. coli Cascade. We observe that the recognition of bona fide targets is an ordered process that is tightly controlled for high fidelity. Mutated targets are recognized with low fidelity, which is featured by short-lived and PAM- and seed-independent binding by any segment of the crRNA. These dual roles of Cascade in immunity with distinct fidelities underpin CRISPR-Cas robustness, allowing for efficient degradation of bona fide targets and priming of mutated DNA targets.

Escherichia coli maintain different strategies to protect the cell against invading foreign DNA. In a recently discovered adaptive immune system, fragments of foreign DNA are integrated into specific loci on the bacterial genome, known as clustered regularly interspaced short palindromic repeats (CRISPR). Short CRISPR-derived RNAs (crRNAs) are incorporated into the CRISPR-associated complex for antiviral defence (Cascade) and guide the complex's search for the DNA of returning invaders, which is targeted for destruction upon binding. Recent studies have shown that Cascade must recognize both a target sequence and an immediately adjacent PAM (protospacer adjacent motif) sequence in order for successful targeting of the foreign DNA. The mechanism and structural dynamics of this target recognition and binding process, however, are not well understood. Here we report a single-molecule FRET (Förster resonance energy transfer) based assay to monitor in real time the target recognition process of Cascade. We observe that Cascade binds target DNAs that vary widely in sequence. Notably, a complementary target sequence with a correct PAM will be completely opened up by Cascade, whereas an identical sequence with a mutated PAM will be only partially opened. In addition, the latter exhibits a weaker affinity to Cascade. A sequence that contains a PAM but lacks a correct target sequence, still exhibits binding, but is not opened. Taken together, these results suggest that the discrimination of self from non-self occurs after promiscuous target binding. Our single-molecule study promises to reveal further mechanistic details of target DNA identification by the CRISPR immune system.

We describe a simple self-calibrating technique, incident-beam interference sweeping, for measuring the height of fluorescent labels. Using a tilted back-reflecting mirror and a scanning laser beam, a modulated fluorescence emission allows height determination of a label from a surface with a resolution of ∼3 nm. In addition, we show that the absolute distance of a label from the top-mounted mirror can be determined with a resolution of a few tens of nanometers over a micrometer range.

The packaging of eukaryotic genomes as chromatin restricts access to the DNA for critical processes such as replication, transcription, recombination, and repair. Thus, eukaryotic cells depend on a dynamic balance between genome compaction and access facilitated in part by chromatin remodeling enzymes (remodelers). Remodelers read epigenetic marks such as histone modifications and use the energy of ATP hydrolysis to assemble, disassemble, reposition, and modify the composition of nucleosomes. Single-molecule techniques enable characterization of transient intermediates formed during the remodeling reaction and can therefore uncover previously unobtainable insights into the mechanisms of chromatin remodeling. In this study, we developed a single-molecule fluorescence resonance energy transfer assay to study the dynamics of chromatin remodeling by human ACF, a prototypical member of the ISWI family of remodelers. ISWI remodelers are endowed with the ability to create regularly-spaced nucleosome arrays characteristic of transcriptionally silent heterochromatin. With the addition of ACF and ATP, nucleosomes exhibit gradual translocation along the DNA interrupted by kinetic pauses after approximately seven or three base pairs (bp) of translocation, thereby dividing the remodeling process into alternating translocation phases and pause phases. Moreover, we found that ACF is a highly processive and bidirectional nucleosome translocase capable of sliding a nucleosome back-and-forth for an average of 200 bp before dissociating. The nucleosome spacing activity of ISWI remodelers is regulated by two substrate features: (1) length of linker DNA, and (2) histone H4 N-terminal tail. Shortening the linker DNA or altering the H4 tail decrease the catalytic activity of ISWI remodelers with little effect on binding affinity. We discovered that the catalytic defects associated with these substrate modifications are attributed solely to changes in the pause phases and not the translocation phases, providing new insights into how nucleosomal features regulate chromatin remodelers.

ISWI-family enzymes remodel chromatin by sliding nucleosomes along DNA, but the nucleosome translocation mechanism remains unclear. Here we use single-molecule FRET to probe nucleosome translocation by ISWI-family remodelers. Distinct ISWI-family members translocate nucleosomes with a similar stepping pattern maintained by the catalytic subunit of the enzyme. Nucleosome remodeling begins with a 7 bp step of DNA translocation followed by 3 bp subsequent steps toward the exit side of nucleosomes. These multi-bp, compound steps are comprised of 1 bp substeps. DNA movement on the entry side of the nucleosome occurs only after 7 bp of exit-side translocation, and each entry-side step draws in a 3 bp equivalent of DNA that allows three additional base pairs to be moved to the exit side. Our results suggest a remodeling mechanism with well-defined coordination at different nucleosomal sites featuring DNA translocation toward the exit side in 1 bp steps preceding multi-bp steps of DNA movement on the entry side.