Person: Moffitt, Jeffrey
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Moffitt
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Jeffrey
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Moffitt, Jeffrey
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Publication Molecular, Spatial, and Functional Single-Cell Profiling of the Hypothalamic Preoptic Region(American Association for the Advancement of Science (AAAS), 2018-11-16) Moffitt, Jeffrey; Bambah-Mukku, Dhananjay; Eichhorn, Stephen W.; Vaughn, Eric; Shekhar, Karthik; Perez, Julio D.; Rubinstein, Nimrod D.; Hao, Junjie; Regev, Aviv; Dulac, Catherine; Zhuang, XiaoweiThe hypothalamus controls essential social behaviors and homeostatic functions. However, the cellular architecture of hypothalamic nuclei, including the molecular identity, spatial organization, and function of distinct cell types, is poorly understood. Here, we developed an imaging-based in situ cell type identification and mapping method and combined it with single-cell RNA-sequencing to create a molecularly annotated and spatially resolved cell atlas of the mouse hypothalamic preoptic region. We profiled ~1 million cells, identified ~70 neuronal populations characterized by distinct neuromodulatory signatures and spatial organizations, and defined specific neuronal populations activated during social behaviors in male and female mice, providing a high-resolution framework for mechanistic investigation of behavior circuits. The approach described opens a new avenue for the construction of cell atlases in diverse tissues and organisms.Publication Author Correction: Multiplexed imaging of high-density libraries of RNAs with MERFISH and expansion microscopy(Nature Publishing Group UK, 2018) Wang, Guiping; Moffitt, Jeffrey; Zhuang, XiaoweiPublication High-throughput, image-based screening of pooled genetic variant libraries(2018) Emanuel, George; Moffitt, Jeffrey; Zhuang, XiaoweiImage-based, high-throughput screening of genetic perturbations will advance both biology and biotechnology. We report a high-throughput screening method that allows diverse genotypes and corresponding phenotypes to be imaged in numerous individual cells. We achieve genotyping by introducing barcoded genetic variants into cells and using massively multiplexed FISH to measure the barcodes. We demonstrated this method by screening mutants of the fluorescent protein YFAST, yielding brighter and more photostable YFAST variants.Publication Multiplexed imaging of high-density libraries of RNAs with MERFISH and expansion microscopy(Nature Publishing Group UK, 2018) Wang, Guiping; Moffitt, Jeffrey; Zhuang, XiaoweiAs an image-based single-cell transcriptomics approach, multiplexed error-robust fluorescence in situ hybridization (MERFISH) allows hundreds to thousands of RNA species to be identified, counted and localized in individual cells while preserving the native spatial context of RNAs. In MERFISH, RNAs are identified via a combinatorial labeling approach that encodes RNA species with error-robust barcodes followed by sequential rounds of single-molecule FISH (smFISH) to read out these barcodes. The accuracy of RNA identification relies on spatially separated signals from individual RNA molecules, which limits the density of RNAs that can be measured and makes the multiplexed imaging of a large number of high-abundance RNAs challenging. Here we report an approach that combines MERFISH and expansion microscopy to substantially increase the total density of RNAs that can be measured. Using this approach, we demonstrate accurate identification and counting of RNAs, with a near 100% detection efficiency, in a ~130-RNA library composed of many high-abundance RNAs, the total density of which is more than 10 fold higher than previously reported. In parallel, we demonstrate the combination of MERFISH with immunofluorescence in expanded samples. These advances increase the versatility of MERFISH and will facilitate its application to a wide range of biological problems.Publication The single-cell chemostat: an agarose-based, microfluidic device for high-throughput, single-cell studies of bacteria and bacterial communities(Royal Society of Chemistry (RSC), 2012) Moffitt, Jeffrey; Lee, Jeffrey B.; Cluzel, PhilippeOptical microscopy of single bacteria growing on solid agarose support is a powerful method for studying the natural heterogeneity in growth and gene expression. While the material properties of agarose make it an excellent substrate for such studies, the sheer number of exponentially growing cells eventually overwhelms the agarose pad, which fundamentally limits the duration and the throughput of measurements. Here we overcome the limitations of exponential growth by patterning agarose pads on the sub-micron-scale. Linear tracks constrain the growth of bacteria into a high density array of linear micro-colonies. Buffer flow through microfluidic lines washes away excess cells and delivers fresh nutrient buffer. Densely patterned tracks allow us to cultivate and image hundreds of thousands of cells on a single agarose pad over 30-40 generations, which drastically increases single-cell measurement throughput. In addition, we show that patterned agarose can facilitate single-cell measurements within bacterial communities. As a proof-of-principle, we study a community of E. coli auxotrophs that can complement the amino acid deficiencies of one another. We find that the growth rate of colonies of one strain decreases sharply with the distance to colonies of the complementary strain over distances of only a few cell lengths. Because patterned agarose pads maintain cells in a chemostatic environment in which every cell can be imaged, we term our device the single-cell chemostat. High-throughput measurements of single cells growing chemostatically should greatly facilitate the study of a variety of microbial behaviours.Publication Statistical Analysis of Molecular Signal Recording(Public Library of Science, 2013) Glaser, Joshua I.; Zamft, Bradley M.; Marblestone, Adam Henry; Moffitt, Jeffrey; Tyo, Keith; Boyden, Edward S.; Church, George; Kording, Konrad P.A molecular device that records time-varying signals would enable new approaches in neuroscience. We have recently proposed such a device, termed a “molecular ticker tape”, in which an engineered DNA polymerase (DNAP) writes time-varying signals into DNA in the form of nucleotide misincorporation patterns. Here, we define a theoretical framework quantifying the expected capabilities of molecular ticker tapes as a function of experimental parameters. We present a decoding algorithm for estimating time-dependent input signals, and DNAP kinetic parameters, directly from misincorporation rates as determined by sequencing. We explore the requirements for accurate signal decoding, particularly the constraints on (1) the polymerase biochemical parameters, and (2) the amplitude, temporal resolution, and duration of the time-varying input signals. Our results suggest that molecular recording devices with kinetic properties similar to natural polymerases could be used to perform experiments in which neural activity is compared across several experimental conditions, and that devices engineered by combining favorable biochemical properties from multiple known polymerases could potentially measure faster phenomena such as slow synchronization of neuronal oscillations. Sophisticated engineering of DNAPs is likely required to achieve molecular recording of neuronal activity with single-spike temporal resolution over experimentally relevant timescales.Publication Super-resolution imaging reveals distinct chromatin folding for different epigenetic states(2015) Boettiger, Alistair; Bintu, Bogdan; Moffitt, Jeffrey; Wang, Siyuan; Beliveau, Brian; Fudenberg, Geoffrey; Imakaev, Maxim; Mirny, Leonid A.; Wu, Chao-ting; Zhuang, XiaoweiMetazoan genomes are spatially organized at multiple scales, from packaging of DNA around individual nucleosomes to segregation of whole chromosomes into distinct territories1–5. At the intermediate scale of kilobases to megabases, which encompasses the sizes of genes, gene clusters and regulatory domains, the three-dimensional (3D) organization of DNA is implicated in multiple gene regulatory mechanisms2–4,6–8, but understanding this organization remains a challenge. At this scale, the genome is partitioned into domains of different epigenetic states that are essential for regulating gene expression9–11. Here, we investigate the 3D organization of chromatin in different epigenetic states using super-resolution imaging. We classified genomic domains in Drosophila cells into transcriptionally active, inactive, or Polycomb-repressed states and observed distinct chromatin organizations for each state. Remarkably, all three types of chromatin domains exhibit power-law scaling between their physical sizes in 3D and their domain lengths, but each type has a distinct scaling exponent. Polycomb-repressed chromatin shows the densest packing and most intriguing folding behaviour in which packing density increases with domain length. Distinct from the self-similar organization displayed by transcriptionally active and inactive chromatin, the Polycomb-repressed domains are characterized by a high degree of chromatin intermixing within the domain. Moreover, compared to inactive domains, Polycomb-repressed domains spatially exclude neighbouring active chromatin to a much stronger degree. Computational modelling and knockdown experiments suggest that reversible chromatin interactions mediated by Polycomb-group proteins plays an important role in these unique packaging properties of the repressed chromatin. Taken together, our super-resolution images reveal distinct chromatin packaging for different epigenetic states at the kilobase-to-megabase scale, a length scale that is directly relevant to genome regulation.Publication Spatial organization shapes the turnover of a bacterial transcriptome(eLife Sciences Publications, Ltd, 2016) Moffitt, Jeffrey; Pandey, Shristi; Boettiger, Alistair; Wang, Siyuan; Zhuang, XiaoweiSpatial organization of the transcriptome has emerged as a powerful means for regulating the post-transcriptional fate of RNA in eukaryotes; however, whether prokaryotes use RNA spatial organization as a mechanism for post-transcriptional regulation remains unclear. Here we used super-resolution microscopy to image the E. coli transcriptome and observed a genome-wide spatial organization of RNA: mRNAs encoding inner-membrane proteins are enriched at the membrane, whereas mRNAs encoding outer-membrane, cytoplasmic and periplasmic proteins are distributed throughout the cytoplasm. Membrane enrichment is caused by co-translational insertion of signal peptides recognized by the signal-recognition particle. Time-resolved RNA-sequencing revealed that degradation rates of inner-membrane-protein mRNAs are on average greater that those of the other mRNAs and that this selective destabilization of inner-membrane-protein mRNAs is abolished by dissociating the RNA degradosome from the membrane. Together, these results demonstrate that the bacterial transcriptome is spatially organized and suggest that this organization shapes the post-transcriptional dynamics of mRNAs. DOI: http://dx.doi.org/10.7554/eLife.13065.001Publication Robust Circadian Oscillations in Growing Cyanobacteria Require Transcriptional Feedback(American Association for the Advancement of Science, 2013) Teng, Shu-Wen; Mukherji, Shankar; Moffitt, Jeffrey; de Buyl, Sophie; O'Shea, ErinThe remarkably stable circadian oscillations of single cyanobacteria enable a population of growing cells to maintain synchrony for weeks. The cyanobacterial pacemaker is a posttranslational regulation (PTR) circuit that generates circadian oscillations in the phosphorylation state of the clock protein KaiC. Layered on top of the PTR is transcriptional-translational feedback regulation (TTR), common to all circadian systems, consisting of a negative feedback loop in which KaiC regulates its own production. We found that the PTR circuit is sufficient to generate oscillations in growing cyanobacteria. However, in the absence of TTR, individual oscillators were less stable and synchrony was not maintained in a population of cells. Experimentally constrained mathematical modeling reproduced sustained oscillations in the PTR circuit alone and demonstrated the importance of TTR for oscillator synchrony.Publication MAFG-Driven Astrocytes Promote CNS Inflammation(Springer Science and Business Media LLC, 2020-02-12) Wheeler, Michael; Clark, Iain; Tjon, Emily; Li, Zhaorong; Zandee, Stephanie E. J.; Couturier, Charles P.; Watson, Brianna; Scalisi, Giulia; Alkwai, Sarah; Rothhammer, Veit; Rotem, Assaf; Heyman, John; Thaploo, Shravan; Sanmarco, Liliana; Ragoussis, Jiannis; Weitz, David; Petrecca, Kevin; Moffitt, Jeffrey; Becher, Burkhard; Antel, Jack P.; Prat, Alexandre; Quintana, Francisco