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Moffitt, J. R.

Publications and source records attributed to Moffitt, J. R..

At least 19 recordsLinked to original sources
bioRxiv2026

Early hematopoietic fate decisions are independent of bone marrow niche

Hematopoietic stem cells (HSCs) reside in specialized bone marrow microenvironments, termed niches, which regulate self-renewal and differentiation through local support programs. Although multiple anatomically distinct niches with unique cellular compositions have been described, it remains unclear to what degree these niches differentially shape HSC fate. In parallel, hematopoietic output is modulated with age, yet it remains unclear if this shift in HSC fate is driven by niche-specific modifications. To address these questions, we applied Multiplexed Error Robust Fluorescence in situ Hybridization to jointly define the cellular and spatial organization of the femoral marrow in young and aged mice. We find that while HSC niches are anatomically and compositionally distinct, stem cell lineage priming is not strongly associated with niche identity consistent with our demonstration that fate-shaping signals are provided redundantly across niches. Moreover, we find that while aging remodels the cellular composition of the marrow, reshapes cellular expression, and alters hematopoietic differentiation, HSC niches are preserved, and HSC fate remains niche agnostic. Together these findings separate anatomical niche identity from early HSC fate decisions and are consistent with a model in which hematopoietic support is distributed across different niche locations rather than partitioned among discrete sites.

immunology↗
bioRxiv2026

RNA-aware tissue preservation workflows for high-quality spatial transcriptomics

Image-based transcriptomic approaches can define, discover, and chart cell types and states within an array of tissues. However, measurement quality depends on RNA integrity, and the modern tissue preservation toolbox was not designed to protect this highly labile molecule. Here we leverage MERFISH to show that tissue-dependent differences in endogenous RNase activity can shape spatial transcriptomics data quality for different preservation methods and that RNase-activity-guided protocol optimization can improve data quality. In parallel, we introduce an RNA-aware pan-tissue preservation approach, Rapid Inhibition and Permanent Inactivation of Nucleases (RIPIN), that rapidly stabilizes samples with a broad-spectrum RNase inhibitor while permitting slow, chemical inactivation. RIPIN produces high-quality MERFISH measurements in all profiled human and mouse tissues, is compatible with clinical workflows, and is easily integrated with frozen or paraffin sectioning. By highlighting how RNA integrity can be lost during tissue processing, our work may inspire the next generation of RNA-aware histology methods.

genomics↗
bioRxiv2026

Barrier Immune Memory is Promoted by Intestinal Epithelial Cell Presentation of Injected Bacterial Antigens

The contributions of antigen compartmentalization to recognition differences between CD4 and CD8 T cells have long been appreciated, but little is known of how subcellular localization of different antigens expressed by a single pathogen impacts T cell immunity. By tracking a clonal CD4 T cell response to its cognate epitope shuttled between different virulence proteins of the enteropathogenic bacterium, Citrobacter rodentium (Cr), we find a remarkable bias in the magnitude and quality of the response contingent on whether antigen remains bacterially associated or is introduced into intestinal epithelial cells colonized by the bacterium. Only proteins injected into the cytosol of colonocytes via the type 3 secretion system (T3SS) of Cr were found to recruit robust antigen-specific T cell responses to the infected mucosa and give rise to CD4 resident memory T (Trm) cells that populate the mucosal epithelium--and this required direct presentation of these antigens by infected epithelial cells. Single-cell transcriptomic analyses revealed that sustained, bidirectional epithelial-T cell communication was required both to elicit epithelial barrier-protective T cell help and to promote transcriptional networks that program a tissue-residency rather than central memory fate. These results establish a central role for antigen presentation by non-professional APCs in controlling memory fate decisions by CD4 T cells, with important implications for development of successful mucosal vaccines.

immunology↗
bioRxiv2025

Brain-immune interactions generate pathogen-specific sickness states

In nature, animals encounter diverse pathogens that trigger specific peripheral defense programs and elicit sickness behavior, a set of stereotyped physiological and behavioral changes thought to promote host fitness. Most studies to date have relied on one or a few mouse models of infection, limiting insights into pathogen-specific neuroimmune interactions that generate sickness. We hypothesized that different pathogens might elicit distinct sickness states by engaging different cell types and brain circuits. Using inflammatory models representing bacterial, viral, allergic, parasitic or colitis conditions, we assessed sickness across scales: organismal - behavior and physiology; cellular - brain-wide neural activity; and molecular - single-cell in situ transcriptomics in hypothalamus areas associated with social and homeostatic functions affected during sickness. Remarkably, immune challenges elicited unique repertoires of changes across all scales. Our findings reveal pathogen-specific sickness states encoded by the brain across scales, thereby broadening our understanding of how infections make us sick.

neuroscience↗
bioRxiv2025

An image-based transcriptomics atlas reveals the regional and microbiota-dependent molecular, cellular, and spatial structure of the murine gut

The gastrointestinal environment is home to a massive diversity of diet-, host-, and microbiota-derived small molecules, collectively sensed by a remarkable variety of cells. To explore the cellular and spatial organization of sensation, we used MERFISH to profile receptor expression across 2.1 million cells in multiple regions of the murine gut under specific-pathogen-free (SPF) and germ-free (GF) conditions. This atlas revealed expected and novel cell types--including a candidate murine homolog of human BEST4 enterocytes--demonstrated cell-type regional specialization, discovered extensive location-dependent spatial fine-tuning in mucosal cell expression, and suggested cell-type specific mediators of the effects of microbiota-derived small molecules. In addition, this atlas revealed that, aside from immune cell abundance, many aspects of the murine gut are host-intrinsic and modified only modestly in the absence of a microbiota. Collectively, this atlas provides a valuable resource for understanding the cellular and spatial organization underlying small molecule sensation in the gut.

genomics↗
bioRxiv2025

Essentialome-Wide Multigenerational Imaging Reveals Mechanistic Origins of Cell Growth Laws

Escherichia coli is arguably the most thoroughly characterized organism, and yet [~]20% of its essential genes serve completely unknown functions,1,2 and many core quantitative physiological principles remain unexplained, including the famous nutrient growth law where cell volume seems to depend exponentially on growth rate. Here we develop a platform for massive, multi-generational Optical Pooled Screening (OPS)3-6, and apply it to link image-based phenotypes to genotypes for 133,000 CRISPRi knockdowns of essential genes, tracking tens of millions of lineages and analyzing 1.6 billion cells. Our multi-dimensional dynamic phenotypes correlate exceptionally well with known gene functions, allowing us to identify many unknown roles of essential genes. Quantifying the relation between growth and cell size in turn identifies three distinct variants of the bacterial growth laws, which we explain mechanistically by discovering a new role for (p)ppGpp and SpoT as a sensor of translation elongation. Finally, we propose and systematically test an exceedingly simple, passive mechanism for the nutrient growth law, based on triggering cell division through the accumulation of a protein that, unlike ribosomes, is not controlled by (p)ppGpp. The resulting hyperbolic growth law fits the data from our three variants even better than the previously proposed exponential relationship, and with fewer parameters.

systems biology↗
bioRxiv2025

In situ profiling of plasma cell clonality with image-based single-cell transcriptomics

Image-based single-cell transcriptomics can identify diverse cell types within intact tissues. However, in adaptive immunity, V(D)J recombination generates unique immune receptors within cells of the same type, leading to important functional variation that is not yet defined by these methods. Here we introduce B-cell-receptor multiplexed error robust fluorescence in situ hybridization (BCR-MERFISH), which distinguishes plasma cell clones based on V-gene usage in combination with transcriptome profiling. We demonstrate that BCR-MERFISH accurately identifies V-gene usage in cell culture and in mice with restricted or native plasma cell diversity. We then use BCR-MERFISH to reveal the microbiota-dependent changes in plasma cell abundance, clonal diversity, and public clonotype usage in the mouse gut and the non-uniform distribution of plasma cell clones along the mouse ileum. As tissue context is an essential modulator of plasma cell dynamics, we anticipate that BCR-MERFISH may offer new insights into a wide range of immunological questions.

immunology↗
bioRxiv2025

Protocol Optimization Improves the Performance of Multiplexed RNA Imaging

Spatial transcriptomics has emerged as a powerful tool to define the cellular structure of diverse tissues. One such method is multiplexed error robust fluorescence in situ hybridization (MERFISH). MERFISH identifies RNAs with error tolerant optical barcodes generated through sequential rounds of single-molecule fluorescence in situ hybridization (smFISH). MERFISH performance depends on a variety of protocol choices, yet their effect on performance has yet to be systematically examined. Here we explore a variety of properties to identify optimal choices for probe design, hybridization, buffer storage, and buffer composition. In each case, we introduce protocol modifications that can improve performance, and we show that, collectively, these modified protocols can improve MERFISH quality in both cell culture and tissue samples. As RNA FISH-based methods are used in many different contexts, we anticipate that the optimization experiments we present here may provide empirical design guidance for a broad range of methods.

genomics↗
bioRxiv2024

Identifying spatially variable genes by projecting to morphologically relevant curves

Spatial transcriptomics enables high-resolution gene expression measurements while preserving the two-dimensional spatial organization of the biological sample. A common objective in spatial transcriptomics data analysis is to identify spatially variable genes within predefined cell types or regions within the tissue. However, these regions are often implicitly one-dimensional, making standard two-dimensional coordinate-based methods less effective as they overlook the underlying tissue organization. Here we introduce a methodology grounded in spectral graph theory to elucidate a one-dimensional curve that effectively approximates the spatial coordinates of the examined sample. This curve is then used to establish a new coordinate system that reflects tissue morphology. We then develop a generalized additive model (GAM) to estimate spatial patterns which permits the detection of genes with variable expression in the new morphologically relevant coordinate system. Our approach directly models gene counts, thereby eliminating the need for normalization or transformations to satisfy normality assumptions. A second important advantage over existing hypothesis-testing approaches is that our method not only improves performance but also accurately estimates gene expression patterns and precisely pinpoints spatial loci where deviations from constant expression occur. We validate our approach through extensive simulation and by analyzing experimental data from multiple platforms such as Slide-seq and MERFISH. As an example of its ability to enable biological discovery, we demonstrate how our methodology enables the identification of novel interferon-related subpopulations in the mouse mucosa, as well as markers of inflammation-associated fibroblasts in a multi-sample spatial transcriptomic dataset.

bioinformatics↗
bioRxiv2024

Highly Multiplexed Spatial Transcriptomics in Bacteria

Single-cell decisions made in complex environments underlie many bacterial phenomena. Image-based transcriptomics approaches offer an avenue to study such behaviors, yet these approaches have been hindered by the massive density of bacterial mRNA. To overcome this challenge, we combine 1000-fold volumetric expansion with multiplexed error robust fluorescence in situ hybridization (MERFISH) to create bacterial-MERFISH. This method enables high-throughput, spatially resolved profiling of thousands of operons within individual bacteria. Using bacterial-MERFISH, we dissect the response of E. coli to carbon starvation, systematically map subcellular RNA organization, and chart the adaptation of a gut commensal B. thetaiotaomicron to micron-scale niches in the mammalian colon. We envision bacterial-MERFISH will be broadly applicable to the study of bacterial single-cell heterogeneity in diverse, spatially structured, and native environments.

microbiology↗
bioRxiv2024

Spatial transcriptomics of healthy and fibrotic human liver at single-cell resolution

Single-cell RNA sequencing (scRNA-seq) has advanced our understanding of cell types and their heterogeneity within the human liver, but the spatial organization at single-cell resolution has not yet been described. Here we apply multiplexed error robust fluorescent in situ hybridization (MERFISH) to map the zonal distribution of hepatocytes, resolve subsets of macrophage and mesenchymal populations, and investigate the relationship between hepatocyte ploidy and gene expression within the healthy human liver. We next integrated spatial information from MERFISH with the more complete transcriptome produced by single-nucleus RNA sequencing (snRNA-seq), revealing zonally enriched receptor-ligand interactions. Finally, analysis of fibrotic liver samples identified two hepatocyte populations that are not restricted to zonal distribution and expand with injury. Together these spatial maps of the healthy and fibrotic liver provide a deeper understanding of the cellular and spatial remodeling that drives disease which, in turn, could provide new avenues for intervention and further study.

genomics↗
bioRxiv2023

Spatial and temporal profiling of the complement system uncovered novel functions of the alternative complement pathway in brain development

Mounting evidence implicated the classical complement pathway (CP) in normal brain development, and the pathogenesis of neuropsychiatric and neurodegenerative diseases. However the source and regulation of complement in the brain remain unclear. Using MERFISH, a spatial transcriptomic method with single-cell resolution, we established a developmental brain atlas of the complement system. We showed that the brain synthesizes essential building blocks of the complement system locally with remarkable cellular and spatial heterogeneity. We provided transcriptional evidence supporting the presence of the alternative pathway (AP), but lack of lectin pathway (LP) activity in postnatal brain development. Cell type, temporal and spatial expression patterns of genes involved indicate non-redundant functions of the CP and AP. In addition, deficiency in Masp3-driven AP resulted in developmental and cognitive defects, indicating essential functions of the AP, an observation that highlights the necessity to disentangle differential involvement of the three complement activation pathways in development and diseases.

neuroscience↗
bioRxiv2023

Charting the cellular biogeography in colitis reveals fibroblast trajectories and coordinated spatial remodeling

Gut inflammation involves contributions from immune and non-immune cells, whose interactions are shaped by the spatial organization of the healthy gut and its remodeling during inflammation. The crosstalk between fibroblasts and immune cells is an important axis in this process, but our understanding has been challenged by incomplete cell-type definition and biogeography. To address this challenge, we used MERFISH to profile the expression of 940 genes in 1.35 million cells imaged across the onset and recovery from a mouse colitis model. We identified diverse cell populations; charted their spatial organization; and revealed their polarization or recruitment in inflammation. We found a staged progression of inflammation-associated tissue neighborhoods defined, in part, by multiple inflammation-associated fibroblasts, with unique expression profiles, spatial localization, cell-cell interactions, and healthy fibroblast origins. Similar signatures in ulcerative colitis suggest conserved human processes. Broadly, we provide a framework for understanding inflammation-induced remodeling in the gut and other tissues.

immunology↗
bioRxiv2023

Microbiome induced complement synthesized in the gut protects against enteric infections

Canonically, complement is a serum-based host defense system that protects against systemic microbial invasion. Little is known about the production and function of complement components on mucosal surfaces. Here we show gut complement component 3 (C3), central to complement function, is regulated by the composition of the microbiota in healthy humans and mice, leading to host-specific gut C3 levels. Stromal cells in intestinal lymphoid follicles (LFs) are the predominant source of intestinal C3. During enteric infection with Citrobacter rodentium or enterohemorrhagic Escherichia coli, luminal C3 levels increase significantly and are required for protection. C. rodentium is remarkably more invasive to the gut epithelium of C3-deficient mice than of wild-type mice. In the gut, C3-mediated phagocytosis of C. rodentium functions to clear pathogens. Our study reveals that variations in gut microbiota determine individuals intestinal mucosal C3 levels, dominantly produced by LF stromal cells, which directly correlate with protection against enteric infection. HighlightsO_LIGut complement component 3 (C3) is induced by the microbiome in healthy humans and mice at a microbiota-specific level. C_LIO_LIGut stromal cells located in intestinal lymphoid follicles are a major source of luminal C3 C_LIO_LIDuring enteric infections with Citrobacter rodentium or enterohemorrhagic Escherichia coli, gut luminal C3 levels increase and are required for protection. C_LIO_LIC. rodentium is significantly more invasive of the gut epithelium in C3-deficient mice when compared to WT mice. C_LIO_LIIn the gut, C3-mediated opsonophagocytosis of C. rodentium functions to clear pathogens. C_LI

immunology↗
bioRxiv2022

Understanding prefrontal cortex functions by decoding its molecular, cellular and circuit organization

The prefrontal cortex (PFC) is functionally one of the most complex regions of mammalian brain. Unlike other cortical areas that process single sensory modalities (like vision, touch, smell, etc.), the PFC integrates information across brain regions to regulate diverse functions ranging from cognition, emotion, executive action to even pain sensitivity. However, it is unclear how such diverse functions are organized at the cellular and circuit levels within the anatomical modules of the PFC. Here we employed spatially resolved single-cell transcriptome profiling to decode PFCs organizational heterogeneity. The results revealed that PFC has very distinct cell type composition relative to all neighboring cortical areas. Interestingly, PFC also adopts specialized transcriptional features, different from all neighbors, with differentially expressed genes regulating neuronal excitability. The projections to major subcortical targets of PFC emerge from combinations of neuron subclusters determined in a target-intrinsic fashion. These cellular and molecular features further segregated within subregions of PFC, alluding to the subregion-specific specialization of several PFC functions. Finally, using these unique cellular, molecular and projection properties, we identified distinct cell types and circuits in PFC that engage in pathogenesis of chronic pain. Collectively, we not only present a comprehensive organizational map of the PFC, critical for supporting its diverse functions, but also reveal the cluster and circuit identity of a pathway underlying chronic pain, a rapidly escalating healthcare challenge limited by molecular understanding of maladaptive PFC circuits. Major pointsO_LIPFC adopts unique cellular composition, distinct from other cortical areas C_LIO_LISelective transcriptomic features emerge in PFC to support its divergent functional portfolio C_LIO_LISubcortical projections of PFC assume target-intrinsic specification for innervating clusters C_LIO_LIA molecularly defined L5 projection neuron cluster (to PAG) potentially mediates chronic pain pathogenesis C_LI

neuroscience↗
bioRxiv2022

Spatial organization of the mouse retina at single cell resolution

The visual signal processing in the retina requires the precise organization of diverse neuronal types working in concert. We performed spatial transcriptomic profiling of over 100,000 cells from the mouse retina, uncovering the spatial distribution of all major retina cell types with over 100 cell subtypes. Our data revealed that the retina is organized in a laminar structure at the major cell type and subgroup level, both of which has strong correlation with the birth order of the cell. In contrast, overall random dispersion of cells within sub-laminar layers indicates that retinal mosaics are driven by dendritic field patterning rather than neuron soma placement. Through the integration of single cell transcriptomic and spatial data, we have generated the first comprehensive spatial single cell reference atlas of the mouse retina, a resource to the community and an essential step toward gaining a comprehensive understanding of the mechanism of retinal function. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/518972v1_ufig1.gif" ALT="Figure 1"> View larger version (76K): org.highwire.dtl.DTLVardef@1dc0eaforg.highwire.dtl.DTLVardef@480731org.highwire.dtl.DTLVardef@d0221borg.highwire.dtl.DTLVardef@6f5894_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗
bioRxiv2020

Spatial organization of the transcriptome in individual neurons

Neurons are highly polarized cells with complex neurite morphology. Spatial organization and local translation of RNAs in dendrites and axons play an important role in many neuronal functions. Here we performed super-resolution spatial profiling of RNAs inside individual neurons at the genome scale using multiplexed error-robust fluorescence in situ hybridization (MERFISH), and mapped the spatial organization of up to [~]4,200 RNA species (genes) across multiple length scales, ranging from sub-micrometer to millimeters. Our data generated a quantitative intra-neuronal atlas of RNAs with distinct transcriptome compositions in somata, dendrites, and axons, and revealed diverse sub-dendritic distribution patterns of RNAs. Moreover, our spatial analysis identified distinct groups of genes exhibiting specific spatial clustering of transcripts at the sub-micrometer scale that were dependent on protein synthesis and differentially dependent on synaptic activity. Overall, these data provide a rich resource for characterizing the subcellular organization of the transcriptome in neurons with high spatial resolution.

cell biology↗