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Kirchner, Rory

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Kirchner

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Rory

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Kirchner, Rory
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    Single-cell transcriptomics of the developing lateral geniculate nucleus reveals insights into circuit assembly and refinement
    (National Academy of Sciences, 2018) Kalish, Brian; Cheadle, Lucas; Hrvatin, Sinisa; Nagy, M. Aurel; Rivera, Samuel; Crow, Megan; Gillis, Jesse; Kirchner, Rory; Greenberg, Michael
    Coordinated changes in gene expression underlie the early patterning and cell-type specification of the central nervous system. However, much less is known about how such changes contribute to later stages of circuit assembly and refinement. In this study, we employ single-cell RNA sequencing to develop a detailed, whole-transcriptome resource of gene expression across four time points in the developing dorsal lateral geniculate nucleus (LGN), a visual structure in the brain that undergoes a well-characterized program of postnatal circuit development. This approach identifies markers defining the major LGN cell types, including excitatory relay neurons, oligodendrocytes, astrocytes, microglia, and endothelial cells. Most cell types exhibit significant transcriptional changes across development, dynamically expressing genes involved in distinct processes including retinotopic mapping, synaptogenesis, myelination, and synaptic refinement. Our data suggest that genes associated with synapse and circuit development are expressed in a larger proportion of nonneuronal cell types than previously appreciated. Furthermore, we used this single-cell expression atlas to identify the Prkcd-Cre mouse line as a tool for selective manipulation of relay neurons during a late stage of sensory-driven synaptic refinement. This transcriptomic resource provides a cellular map of gene expression across several cell types of the LGN, and offers insight into the molecular mechanisms of circuit development in the postnatal brain.
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    GEMINI: Integrative Exploration of Genetic Variation and Genome Annotations
    (Public Library of Science, 2013) Paila, Umadevi; Chapman, Brad; Kirchner, Rory; Quinlan, Aaron R.
    Modern DNA sequencing technologies enable geneticists to rapidly identify genetic variation among many human genomes. However, isolating the minority of variants underlying disease remains an important, yet formidable challenge for medical genetics. We have developed GEMINI (GEnome MINIng), a flexible software package for exploring all forms of human genetic variation. Unlike existing tools, GEMINI integrates genetic variation with a diverse and adaptable set of genome annotations (e.g., dbSNP, ENCODE, UCSC, ClinVar, KEGG) into a unified database to facilitate interpretation and data exploration. Whereas other methods provide an inflexible set of variant filters or prioritization methods, GEMINI allows researchers to compose complex queries based on sample genotypes, inheritance patterns, and both pre-installed and custom genome annotations. GEMINI also provides methods for ad hoc queries and data exploration, a simple programming interface for custom analyses that leverage the underlying database, and both command line and graphical tools for common analyses. We demonstrate GEMINI's utility for exploring variation in personal genomes and family based genetic studies, and illustrate its ability to scale to studies involving thousands of human samples. GEMINI is designed for reproducibility and flexibility and our goal is to provide researchers with a standard framework for medical genomics.
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    edgeRun: an R package for sensitive, functionally relevant differential expression discovery using an unconditional exact test
    (Oxford University Press, 2015) Dimont, Emmanuel; Shi, Jiantao; Kirchner, Rory; Hide, Winston
    Summary: Next-generation sequencing platforms for measuring digital expression such as RNA-Seq are displacing traditional microarray-based methods in biological experiments. The detection of differentially expressed genes between groups of biological conditions has led to the development of numerous bioinformatics tools, but so far, few exploit the expanded dynamic range afforded by the new technologies. We present edgeRun, an R package that implements an unconditional exact test that is a more powerful version of the exact test in edgeR. This increase in power is especially pronounced for experiments with as few as two replicates per condition, for genes with low total expression and with large biological coefficient of variation. In comparison with a panel of other tools, edgeRun consistently captures functionally similar differentially expressed genes. Availability and implementation: The package is freely available under the MIT license from CRAN (http://cran.r-project.org/web/packages/edgeRun). Contact: edimont@mail.harvard.edu Supplementary information: Supplementary data are available at Bioinformatics online.
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    In Vitro Effects of Lead on Gene Expression in Neural Stem Cells and Associations between Up-regulated Genes and Cognitive Scores in Children
    (National Institute of Environmental Health Sciences, 2016) Wagner, Peter J.; Park, Hae-Ryung; Wang, Zhaoxi; Kirchner, Rory; Wei, Yongyue; Su, Li; Stanfield, Kirstie; Guilarte, Tomas R.; Wright, Robert O.; Christiani, David C.; Lu, Quan
    Background: Lead (Pb) adversely affects neurodevelopment in children. Neural stem cells (NSCs) play an essential role in shaping the developing brain, yet little is known about how Pb perturbs NSC functions and whether such perturbation contributes to impaired neurodevelopment. Objectives: We aimed to identify Pb-induced transcriptomic changes in NSCs and to link these changes to neurodevelopmental outcomes in children who were exposed to Pb. Methods: We performed RNA-seq-based transcriptomic profiling in human NSCs treated with 1 μM Pb. We used qRT-PCR, Western blotting, ELISA, and ChIP (chromatin immunoprecipitation) to characterize Pb-induced gene up-regulation. Through interrogation of a genome-wide association study, we examined the association of gene variants with neurodevelopment outcomes in the ELEMENT birth cohort. Results: We identified 19 genes with significantly altered expression, including many known targets of NRF2—the master transcriptional factor for the oxidative stress response. Pb induced the expression of SPP1 (secreted phosphoprotein 1), which has known neuroprotective effects. We demonstrated that SPP1 is a novel direct NRF2 target gene. Single nucleotide polymorphisms (SNPs) (rs12641001) in the regulatory region of SPP1 exhibited a statistically significant association (p = 0.005) with the Cognitive Development Index (CDI). Conclusion: Our findings revealed that Pb induces an NRF2-dependent transcriptional response in neural stem cells and identified SPP1 up-regulation as a potential novel mechanism linking Pb exposure with neural stem cell function and neurodevelopment in children. Citation: Wagner PJ, Park HR, Wang Z, Kirchner R, Wei Y, Su L, Stanfield K, Guilarte TR, Wright RO, Christiani DC, Lu Q. 2017. In vitro effects of lead on gene expression in neural stem cells and associations between up-regulated genes and cognitive scores in children. Environ Health Perspect 125:721–729; http://dx.doi.org/10.1289/EHP265
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    Sequencing of Captive Target Transcripts Identifies the Network of Regulated Genes and Functions of Primate-Specific miR-522
    (Elsevier BV, 2014) Tan, Shen Mynn; Kirchner, Rory; Jin, Jingmin; Hofmann, Oliver; McReynolds, Larry; Hide, Winston; Lieberman, Judy
    Identifying microRNA (miRNA)-regulated genes is key to understanding miRNA function. However, many miRNA recognition elements (MREs) do not follow canonical “seed” base-pairing rules, making identification of bona fide targets challenging. Here, we apply an unbiased sequencing-based systems approach to characterize miR-522, a member of the oncogenic primate-specific chromosome 19 miRNA cluster, highly expressed in poorly differentiated cancers. To identify miRNA targets, we sequenced full-length transcripts captured by a biotinylated miRNA mimic. Within these targets, mostly noncanonical MREs were identified by sequencing RNase-resistant fragments. miR-522 overexpression reduced mRNA, protein levels, and luciferase activity of >70% of a random list of candidate target genes and MREs. Bioinformatic analysis suggested that miR-522 regulates cell proliferation, detachment, migration, and epithelial-mesenchymal transition. miR-522 induces G1 cell-cycle arrest and causes cells to detach without anoikis, become invasive, and express mesenchymal genes. Thus, our method provides a simple but effective technique for identifying miRNA-regulated genes and biological function.
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    Subcellular transcriptomes and proteomes of developing axon projections in the cerebral cortex
    (Springer Science and Business Media LLC, 2019-01) Poulopoulos, Alexandros; Murphy, Alexander; Ozkan, Abdulkadir; Davis, Christopher; Hatch, John; Kirchner, Rory; Macklis, Jeffrey
    The development of neural circuits relies on axon projections establishing diverse, yet well-defined, connections between areas of the nervous system. Each projection is formed by growth cones—subcellular specializations at the tips of growing axons, encompassing sets of molecules that control projection-specific growth, guidance, and target selection. To investigate the set of molecules within native growth cones that form specific connections, here we developed growth cone sorting and subcellular RNA–proteome mapping, an approach that identifies and quantifies local transcriptomes and proteomes from labelled growth cones of single projections in vivo. Using this approach on the developing callosal projection of the mouse cerebral cortex, we mapped molecular enrichments in trans-hemispheric growth cones relative to their parent cell bodies, producing paired subcellular proteomes and transcriptomes from single neuron subtypes directly from the brain. These data provide generalizable proof-of-principle for this approach, and reveal molecular specializations of the growth cone, including accumulations of the growth-regulating kinase mTOR, together with mRNAs that contain mTOR-dependent motifs. These findings illuminate the relationships between subcellular distributions of RNA and protein in developing projection neurons, and provide a systems-level approach for the discovery of subtype- and stage-specific molecular substrates of circuit wiring, miswiring, and the potential for regeneration.
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    ALS-Implicated Protein TDP-43 Sustains Levels of STMN2, a Mediator of Motor Neuron Growth and Repair
    (Springer Nature, 2019-01-14) Limone, Francesco; Guerra San Juan, Irune; Burberry, Aaron; Kirchner, Rory; Chen, Kuchuan; Eggan, Kevin; Klim, Joseph; Williams, Luis; Davis-Dusenbery, Brandi N; Mordes, Daniel; Steinbaugh, Michael; Gamage, Kanchana; Moccia, Rob; Cassel, Seth; Wainger, Brian; Woolf, Clifford
    The discovery that TDP-43 mutations cause familial ALS and that many patients display pathological TDP-43 mislocalization has nominated altered RNA metabolism as a potential disease mechanism. Despite its importance, the identity of RNAs regulated by TDP-43 in motor neurons remains poorly understood. Here, we report transcripts whose abundances in human motor neurons are sensitive to TDP-43 depletion. Notably, we found STMN2, which encodes a microtubule regulator, declined after TDP-43 knockdown, in patient-specific motor neurons, following TDP-43 mislocalization, and in the postmortem patient spinal cords. Loss of STMN2 upon reduced TDP-43 function was due to the emergence of a cryptic exon, which is of substantial functional importance, as we further demonstrate that STMN2 is necessary for both axonal outgrowth and repair. Importantly, post-translational stabilization of STMN2 could rescue neurite outgrowth and axon regeneration deficits induced by TDP-43 depletion. We propose restoring STMN2 expression warrants future examination as an ALS therapeutic strategy.