Biology subjects

Wong, W.

Publications and source records attributed to Wong, W..

At least 55 records · Page 3Linked to original sources

bioRxiv2023

Evolution of promoter-proximal pausing enabled a new layer of transcription control

Promoter-proximal pausing of RNA polymerase II (Pol II) is a key regulatory step during transcription. Despite the central role of pausing in gene regulation, we do not understand the evolutionary processes that led to the emergence of Pol II pausing or its transition to a rate-limiting step actively controlled by transcription factors. Here we analyzed transcription in species across the tree of life. Unicellular eukaryotes display a slow acceleration of Pol II near transcription start sites that transitioned to a longer-lived, focused pause in metazoans. This event coincided with the evolution of new subunits in the NELF and 7SK complexes. Depletion of NELF in mammals shifted the promoter-proximal buildup of Pol II from the pause site into the early gene body and compromised transcriptional activation for a set of heat shock genes. Our work details the evolutionary history of Pol II pausing and sheds light on how new transcriptional regulatory mechanisms evolve.

evolutionary biology↗

bioRxiv2023

Cell-type-specific inhibitory circuitry from a connectomic census of mouse visual cortex

Mammalian cortex features a vast diversity of neuronal cell types, each with characteristic anatomical, molecular and functional properties. Synaptic connectivity powerfully shapes how each cell type participates in the cortical circuit, but mapping connectivity rules at the resolution of distinct cell types remains difficult. Here, we used millimeter-scale volumetric electron microscopy1 to investigate the connectivity of all inhibitory neurons across a densely-segmented neuronal population of 1352 cells spanning all layers of mouse visual cortex, producing a wiring diagram of inhibitory connections with more than 70,000 synapses. Taking a data-driven approach inspired by classical neuroanatomy, we classified inhibitory neurons based on the relative targeting of dendritic compartments and other inhibitory cells and developed a novel classification of excitatory neurons based on the morphological and synaptic input properties. The synaptic connectivity between inhibitory cells revealed a novel class of disinhibitory specialist targeting basket cells, in addition to familiar subclasses. Analysis of the inhibitory connectivity onto excitatory neurons found widespread specificity, with many interneurons exhibiting differential targeting of certain subpopulations spatially intermingled with other potential targets. Inhibitory targeting was organized into "motif groups," diverse sets of cells that collectively target both perisomatic and dendritic compartments of the same excitatory targets. Collectively, our analysis identified new organizing principles for cortical inhibition and will serve as a foundation for linking modern multimodal neuronal atlases with the cortical wiring diagram.

neuroscience↗

bioRxiv2022

Large-scale unsupervised discovery of excitatory morphological cell types in mouse visual cortex

Neurons in the neocortex exhibit astonishing morphological diversity which is critical for properly wiring neural circuits and giving neurons their functional properties. However, the organizational principles underlying this morphological diversity remain an open question. Here, we took a data-driven approach using graph-based machine learning methods to obtain a low-dimensional morphological "bar code" describing more than 30,000 excitatory neurons in mouse visual areas V1, AL and RL that were reconstructed from the millimeter scale MICrONS serial-section electron microscopy volume. Contrary to previous classifications into discrete morphological types (m-types), our data-driven approach suggests that the morphological landscape of cortical excitatory neurons is better described as a continuum, with a few notable exceptions in layers 5 and 6. Dendritic morphologies in layers 2-3 exhibited a trend towards a decreasing width of the dendritic arbor and a smaller tuft with increasing cortical depth. Inter-area differences were most evident in layer 4, where V1 contained more atufted neurons than higher visual areas. Moreover, we discovered neurons in V1 on the border to layer 5 which avoided deeper layers with their dendrites. In summary, we suggest that excitatory neurons morphological diversity is better understood by considering axes of variation than using distinct m-types.

neuroscience↗

bioRxiv2022

A microwell platform for high-throughput longitudinal phenotyping and selective retrieval of organoids

Organoids are powerful experimental models for studying the ontogeny and progression of diseases including cancer. Organoids are conventionally cultured in bulk using an extracellular matrix mimic. However, organoids in bulk culture physically overlap, making it impossible to track the growth of individual organoids over time in high throughput. Moreover, local spatial variations in bulk matrix properties make it difficult to assess whether observed phenotypic heterogeneity between organoids results from intrinsic cell differences or microenvironment variability. Here, we developed a microwell-based method that enables high-throughput quantification of image-based parameters for organoids grown from single cells, which can be retrieved from their microwells for sequencing and molecular profiling. Coupled with a deep-learning image processing pipeline, we characterized phenotypic traits including growth rates, cellular movement, and apical-basal polarity in two CRISPR-engineered human gastric organoid models, identifying genomic changes associated with increased growth rate and changes in accessibility and expression correlated with apical-basal polarity.

bioengineering↗

bioRxiv2022

An adhesive signaling axis regulates the establishment of the cortical glial scaffold.

The mature mammalian cortex is composed of six architecturally and functionally distinct layers. Two key steps in the assembly of this layered structure are the initial establishment of the glial scaffold and the subsequent migration of post-mitotic neurons to their final position. These processes involve the precise and timely regulation of adhesion and detachment of neural cells from their substrates. Although much is known about the roles of adhesive substrates during neuronal migration and the formation of the glial scaffold, less is understood about how these signals are interpreted and integrated within these neural cells. Here, we provide in vivo evidence that Cas proteins, a family of cytoplasmic adaptors, serve a functional and redundant role during cortical lamination. Cas triple conditional knock-out (Cas TcKO) mice display severe cortical phenotypes that feature cobblestone malformations. Molecular epistasis and genetic experiments suggest that Cas proteins act downstream of transmembrane Dystroglycan and {beta}1-Integrin in a radial glial cell-autonomous manner. Overall, these data establish a new and essential role for Cas adaptor proteins during the formation of cortical circuits and reveal a signaling axis controlling cortical scaffold formation.

neuroscience↗

bioRxiv2022

Quantitative Census of Local Somatic Features in Mouse Visual Cortex

Mammalian neocortex contains a highly diverse set of cell types. These types have been mapped systematically using a variety of molecular, electrophysiological and morphological approaches. Each modality offers new perspectives on the variation of biological processes underlying cell type specialization. Cellular scale electron microscopy (EM) provides dense ultrastructural examination and an unbiased perspective into the subcellular organization of brain cells, including their synaptic connectivity and nanometer scale morphology. It also presents a clear challenge for analysis to identify cell-types in data that contains tens of thousands of neurons, most of which have incomplete reconstructions. To address this challenge, we present the first systematic survey of the somatic region of all cells within a cubic millimeter of cortex using quantitative features obtained from EM. This analysis demonstrates a surprising sufficiency of the perisomatic region to identify cell-types, including types defined primarily based on their connectivity patterns. We then describe how this classification facilitates cell type specific connectivity characterization and locating cells with rare connectivity patterns in the dataset.

neuroscience↗

bioRxiv2022

Visualization of a Limonene Synthesis Metabolon inside Living Bacteria by Hyperspectral SRS Microscopy

Metabolons consisting of cellular structure elements and sequential metabolic enzymes are expected to be involved in diverse biological processes. However, direct visualization of metabolons in prokaryotic cells is still challenging. In this study, we report direct visualization of concentrated subcellular regions of limonene synthesis inside single engineered Escherichia coli by using hyperspectral stimulated Raman scattering (hSRS) microscopy. Equipped with spectral unmixing, hSRS imaging provides a reliable method to quantify intracellular limonene content. In E. coli strains with a complete limonene synthesis pathway, intracellular limonene is found locally concentrated and colocalized with proteins. Furthermore, dual-modality SRS and two-photon fluorescence imaging showed colocalization of limonene and GFP-fused limonene synthase. Significance StatementMonitoring biosynthesis activity at the single-cell level is key to metabolic engineering but is still difficult to achieve in a label-free manner. Using hyperspectral stimulated Raman scattering imaging in the 670-900 cm-1 region, we visualized localized limonene synthesis inside engineered E. coli. The colocalization of limonene and GFP-fused limonene synthase was confirmed by co-registered stimulated Raman scattering and two-photon fluorescence images. Our finding suggests a limonene synthesis metabolon with a polar distribution inside the cells. This finding expands our knowledge of de novo limonene biosynthesis in engineered bacteria and highlights the potential of SRS chemical imaging in metabolic engineering research.

biophysics↗

bioRxiv2022

Programmable Mixed-Signal Biocomputers in Mammalian Cells

Living cells perform sophisticated computations that guide them toward discrete states. Synthetic genetic circuits are powerful tools for programing these computations, where transcription-regulatory networks and DNA recombination are the two dominant paradigms for implementing these systems. While each strategy exhibits unique strengths and weaknesses, integrating both into one seamless design framework would enable advanced gene circuit designs intractable with either approach alone. Here, we present Computation via Recombinase Assisted Transcriptional Effectors (CREATE), which leverages site-specific recombination to perform robust logic on discreet computational layers and programmable transcription factors that connect these layers, allowing individual calculations to contribute toward larger operations. We demonstrate the functionality of CREATE by producing sophisticated circuits using a simple plug- and-play framework, including 189 2-input-3-output circuits, modular digital-to-analog signal converters, a 2-bit multiplier circuit, and a digital and analog mixed-signal generator. This work establishes CREATE as a versatile platform for programming complex signal processing systems capable of high-fidelity logic computation and tunable control over circuit output levels. One-Sentence SummaryWe present a minimal and robust genetic circuit platform for programming cells with sophisticated signal processing capabilities.

synthetic biology↗

bioRxiv2021

Petascale neural circuit reconstruction: automated methods

3D electron microscopy (EM) has been successful at mapping invertebrate nervous systems, but the approach has been limited to small chunks of mammalian brains. To scale up to larger volumes, we have built a computational pipeline for processing petascale image datasets acquired by serial section EM, a popular form of 3D EM. The pipeline employs convolutional nets to compute the nonsmooth transformations required to align images of serial sections containing numerous cracks and folds, detect neuronal boundaries, label voxels as axon, dendrite, soma, and other semantic categories, and detect synapses and assign them to presynaptic and postsynaptic segments. The output of neuronal boundary detection is segmented by mean affinity agglomeration with semantic and size constraints. Pipeline operations are implemented by leveraging distributed and cloud computing. Intermediate results of the pipeline are held in cloud storage, and can be effortlessly viewed as images, which aids debugging. We applied the pipeline to create an automated reconstruction of an EM image volume spanning four visual cortical areas of a mouse brain. Code for the pipeline is publicly available, as is the reconstructed volume.

neuroscience↗

bioRxiv2021

Single-cell chemical imaging of engineered strains reveals heterogeneity in fatty acid production

Understanding metabolic heterogeneity is critical for optimizing microbial production of valuable chemicals, but requires tools that can quantify metabolites at the single-cell level over time. Here, we develop longitudinal hyperspectral stimulated Raman scattering (SRS) chemical imaging to directly visualize free fatty acids in engineered Escherichia coli over many cell cycles. We also develop compositional analysis to determine the chain length and unsaturation of the fatty acids in living cells. Our method reveals substantial heterogeneity in fatty acid production among and within colonies that emerges over the course of many generations. Interestingly, the strains display distinct types of production heterogeneity in an enzyme-dependent manner. By pairing time-lapse and SRS imaging, we examine the relationship between growth and production at the single-cell level. Single-cell quantification does not show a significant growth-production tradeoff in a strain that exhibits high production heterogeneity. Our results demonstrate that cell-to-cell production heterogeneity is pervasive and provide a means to link single-cell and population-level production.

synthetic biology↗

bioRxiv2021

Raman Needle Arthroscopy for In Vivo Molecular Assessment of Cartilage

The development of treatments for osteoarthritis (OA) is burdened by the lack of standardized biomarkers of cartilage health that can be applied in clinical trials. We present a novel arthroscopic Raman probe that can "optically biopsy" cartilage and quantify key ECM biomarkers for determining cartilage composition, structure, and material properties in health and disease. Technological and analytical innovations to optimize Raman analysis include: 1) multivariate decomposition of cartilage Raman spectra into ECM-constituent-specific biomarkers (glycosaminoglycan [GAG], collagen [COL], water [H2O] scores), and 2) multiplexed polarized Raman spectroscopy to quantify superficial zone collagen anisotropy via a PLS-DA-derived Raman collagen alignment factor (RCAF). Raman measurements were performed on a series of ex vivo cartilage models: 1) chemically GAG-depleted bovine cartilage explants (n=40), 2) mechanically abraded bovine cartilage explants (n=30), 3) aging human cartilage explants (n=14), and 4) anatomical-site-varied ovine osteochondral explants (n=6). Derived Raman GAG score biomarkers predicted 95%, 66%, and 96% of the variation in GAG content of GAG-depleted bovine explants, human explants, and ovine explants, respectively (p<0.001). RCAF values were significantly different for explants with abrasion-induced superficial zone collagen loss (p<0.001). The multivariate linear regression of Raman-derived ECM biomarkers (GAG and H2O scores) predicted 94% of the variation in elastic modulus of ovine explants (p<0.001). Finally, we demonstrated the first in vivo Raman arthroscopy assessment of an ovine femoral condyle through intraarticular entry into the synovial capsule. This work advances Raman arthroscopy towards a transformative low cost, minimally invasive diagnostic platform for objective monitoring of treatment outcomes from emerging OA therapies.

bioengineering↗

bioRxiv2021

Oligodendrocyte precursor cells prune axons in the mouse neocortex

Neurons in the developing brain undergo extensive structural refinement as nascent circuits adopt their mature form1. This transformation is facilitated by the engulfment and degradation of excess axonal branches and inappropriate synapses by surrounding glial cells, including microglia and astrocytes2,3. However, the small size of phagocytic organelles and the complex, highly ramified morphology of glia has made it difficult to determine the contribution of these and other glial cell types to this process. Here, we used large scale, serial electron microscopy (ssEM) with computational volume segmentation to reconstruct the complete 3D morphologies of distinct glial types in the mouse visual cortex. Unexpectedly, we discovered that the fine processes of oligodendrocyte precursor cells (OPCs), a population of abundant, highly dynamic glial progenitors4, frequently surrounded terminal axon branches and included numerous phagolysosomes (PLs) containing fragments of axons and presynaptic terminals. Single- nucleus RNA sequencing indicated that cortical OPCs express key phagocytic genes, as well as neuronal transcripts, consistent with active axonal engulfment. PLs were ten times more abundant in OPCs than in microglia in P36 mice, and declined with age and lineage progression, suggesting that OPCs contribute very substantially to refinement of neuronal circuits during later phases of cortical development.

neuroscience↗

bioRxiv2021

Clinically-Driven Design of Synthetic Gene Regulatory Programs in Human Cells

Synthetic biology seeks to enable the rational design of regulatory molecules and circuits to reprogram cellular behavior. The application of this approach to human cells could lead to powerful gene and cell-based therapies that provide transformative ways to combat complex diseases. To date, however, synthetic genetic circuits are challenging to implement in clinically-relevant cell types and their components often present translational incompatibilities, greatly limiting the feasibility, efficacy and safety of this approach. Here, using a clinically-driven design process, we developed a toolkit of programmable synthetic transcription regulators that feature a compact human protein-based design, enable precise genome-orthogonal regulation, and can be modulated by FDA-approved small molecules. We demonstrate the toolkit by engineering therapeutic human immune cells with genetic programs that enable titratable production of immunotherapeutics, drug-regulated control of tumor killing in vivo and in 3D spheroid models, and the first multi-channel synthetic switch for independent control of immunotherapeutic genes. Our work establishes a powerful platform for engineering custom gene expression programs in mammalian cells with the potential to accelerate clinical translation of synthetic systems.

synthetic biology↗

bioRxiv2020

Pancreatic Ductal Adenocarcinoma Comprises Coexisting Regulatory States with both Common and Distinct Dependencies

Despite extensive efforts, reproducible assessment of pancreatic ductal adenocarcinoma (PDA) heterogeneity and plasticity at the single cell level remains elusive. Systematic, network-based analysis of regulatory protein activity in single cells identified three PDA Developmental Lineages (PDLs), coexisting in virtually all tumors, whose transcriptional states are mechanistically driven by aberrant activation of Master Regulator (MR) proteins associated with gastrointestinal lineages (GLS state), morphogen and EMT pathways (MOS state), and acinar-to-ductal metaplasia (ALS state), respectively. Each PDL is further subdivided into sub-states characterized by low vs. high MAPK pathway activity. This taxonomy was remarkably conserved across multiple cohorts, cell lines, and PDX models, and harmonized with bulk profile analyses. Cross-state plasticity and MR essentiality was confirmed by barcode-based lineage tracing and CRISPR/Cas9 assays, respectively, while MR ectopic expression induced PDL transdifferentiation. Together these data provide a mechanistic foundation for PDA heterogeneity and a roadmap for targeting PDA cellular subtypes.

cancer biology↗

bioRxiv2020

Multiscale and multimodal reconstruction of cortical structure and function

We present a semi-automated reconstruction of L2/3 mouse primary visual cortex from 3 million cubic microns of electron microscopic images, including pyramidal and inhibitory neurons, astrocytes, microglia, oligodendrocytes and precursors, pericytes, vasculature, mitochondria, and synapses. Visual responses of a subset of pyramidal cells are included. The data are being made publicly available, along with tools for programmatic and 3D interactive access. The density of synaptic inputs onto inhibitory neurons varies across cell classes and compartments. We uncover a compartment-specific correlation between mitochondrial coverage and synapse density. Frequencies of connectivity motifs in the graph of pyramidal cells are predicted quite accurately from node degrees using the configuration model of random graphs. Cells receiving more connections from nearby cells exhibit stronger and more reliable visual responses. These example findings illustrate the resources utility for relating structure and function of cortical circuits as well as for neuronal cell biology.

neuroscience↗

bioRxiv2020

HiC-DC+: systematic 3D interaction calls and differential analysis for Hi-C and HiChIP

We present HiC-DC+, a software tool for Hi-C/Hi-ChIP interaction calling and differential analysis using an efficient implementation of the HiC-DC statistical framework. HiC-DC+ integrates with popular preprocessing and visualization tools, includes TAD and A/B compartment callers, and outperformed existing tools in H3K27ac HiChIP benchmarking as validated by CRISPRi-FlowFISH. Differential HiC-DC+ analysis recovered global principles of 3D organization during cohesin perturbation and differentiation, including TAD aggregation, enhancer hubs, and promoter-enhancer loop dynamics.

bioinformatics↗

bioRxiv2020

The early evolution of oral poliovirus vaccine is shaped by strong positive selection and tight transmission bottlenecks

The evolution of circulating vaccine-derived polioviruses (cVDPV) from components of the live-attenuated oral poliovirus vaccine (OPV) presents a major challenge to global polio eradication. This process has largely been characterized by consensus sequencing of isolates collected from routine surveillance, and little is known about the early evolution of OPV within vaccinated hosts. These early events are critical steps in the progression of OPV to cVDPV. Here, we use whole genome, high depth of coverage sequencing to define the evolutionary trajectories of monovalent type 2 OPV in a cluster-randomized trial of polio vaccines in Matlab, Bangladesh. By sequencing 416 longitudinal samples from 219 mOPV2 recipients and 81 samples from 52 household contacts, we were able to examine the extent of convergent evolution in vaccine recipients and track the amount of viral diversity transmitted to new hosts. Using time-series data from a synchronized point of vaccine administration, we identify strong positive selection of reversion mutations at three known attenuating sites within two months post-vaccination. Beyond these three recognized "gate-keeper" mutations, we identify 19 mutations that exhibit significant parallelism across vaccine recipients, providing evidence for early positive selection not previously detected by phylogenetic inference. An analysis of shared genetic variants in samples from vaccinated individuals and their household contacts suggests a tight effective bottleneck during transmission. The absence of positively selected variants among household contacts across the cohort suggests that this tight bottleneck limits the transmission of these early adaptive mutations. Together, our results highlight the distinct evolutionary dynamics of live attenuated virus vaccines and have important implications for the success of novel OPV2 and other next generation approaches. SignificanceThe emergence of circulating vaccine-derived polioviruses (cVDPV) through evolution of the oral polio vaccine (OPV) poses a significant obstacle to global eradication. Understanding the genetic changes in OPV that occur as it evolves and transmits in populations is important for preventing future cVDPV outbreaks. Little is known about the early events in VDPV evolution and the selective forces that drive them. We used high depth-of-coverage genome sequencing to assess the within-host evolutionary dynamics of monovalent type 2 OPV in a vaccine trial in Matlab, Bangladesh. We leverage longitudinal sampling from vaccine recipients and household contacts to identify mutations that arise in parallel across individuals and estimate the size of the transmission bottleneck. We find evidence for strong positive selection on key sites in the capsid and the 5 noncoding region, many of which have not been previously identified. Our results also suggest that narrow transmission bottlenecks can constrain the spread of mutations selected within individuals. These results provide important insights into how OPV variants spread in populations and are highly relevant for ongoing poliovirus surveillance and the design of improved polio vaccines.

microbiology↗

bioRxiv2020

ExBoX: a simple Boolean exclusion strategy to drive expression in neurons

The advent of modern single-cell biology has revealed the striking molecular diversity of cell populations once thought to be more homogeneous. This newly appreciated complexity has made intersectional genetic approaches essential to understanding and probing cellular heterogeneity at the functional level. Here we build on previous knowledge to develop a simple AAV-based approach to define specific subpopulations of cells by Boolean exclusion logic (AND NOT). This Expression by Boolean Exclusion (ExBoX) system encodes for a gene of interest which is turned On by a particular recombinase (Cre or FlpO) and turned Off by another. ExBoX allows for the specific transcription of a gene of interest in cells expressing only the activating recombinase, but not in cells expressing both. We show the ability of the ExBoX system to tightly regulate expression of fluorescent reporters in vitro and in vivo, and further demonstrate the adaptability of the system by achieving expression of a variety of virally-delivered coding sequences in the mouse brain. This simple strategy will expand the molecular toolkit available for cell- and time-specific gene expression in a variety of systems. Summary statementUbina et al. describe the generation of a novel AAV-based intersectional approach to define and target specific subpopulations of cells in time and space via Expression by Boolean Exclusion (ExBoX).

neuroscience↗