Main Menu (Mobile)- Block

Main Menu - Block

custom | custom

Search Results

general_search_page-panel_pane_1 | views_panes

50 Janelia Publications

Showing 1-10 of 50 results
Your Criteria:
    07/23/21 | YAP1 nuclear efflux and transcriptional reprograming follow membrane diminution upon VSV-G-induced cell fusion.
    Feliciano D, Ott CM, Espinosa-Medina I, Weigel AV, Benedetti L, Milano KM, Tang Z, Lee T, Kliman HJ, Guller SM, Lippincott-Schwartz J
    Nature Communications. 2021 Jul 23;12(1):4502. doi: 10.1038/s41467-021-24708-2

    Cells in many tissues, such as bone, muscle, and placenta, fuse into syncytia to acquire new functions and transcriptional programs. While it is known that fused cells are specialized, it is unclear whether cell-fusion itself contributes to programmatic-changes that generate the new cellular state. Here, we address this by employing a fusogen-mediated, cell-fusion system to create syncytia from undifferentiated cells. RNA-Seq analysis reveals VSV-G-induced cell fusion precedes transcriptional changes. To gain mechanistic insights, we measure the plasma membrane surface area after cell-fusion and observe it diminishes through increases in endocytosis. Consequently, glucose transporters internalize, and cytoplasmic glucose and ATP transiently decrease. This reduced energetic state activates AMPK, which inhibits YAP1, causing transcriptional-reprogramming and cell-cycle arrest. Impairing either endocytosis or AMPK activity prevents YAP1 inhibition and cell-cycle arrest after fusion. Together, these data demonstrate plasma membrane diminishment upon cell-fusion causes transient nutrient stress that may promote transcriptional-reprogramming independent from extrinsic cues.

    View Publication Page
    04/01/21 | The art of lineage tracing: From worm to human.
    Garcia-Marques J, Espinosa-Medina I, Lee T
    Progress in Neurobiology. 2021 Apr;199:101966. doi: 10.1016/j.pneurobio.2020.101966

    Reconstructing the genealogy of every cell that makes up an organism remains a long-standing challenge in developmental biology. Besides its relevance for understanding the mechanisms underlying normal and pathological development, resolving the lineage origin of cell types will be crucial to create these types on-demand. Multiple strategies have been deployed towards the problem of lineage tracing, ranging from direct observation to sophisticated genetic approaches. Here we discuss the achievements and limitations of past and current technology. Finally, we speculate about the future of lineage tracing and how to reach the next milestones in the field.

    View Publication Page
    12/01/20 | A programmable sequence of reporters for lineage analysis.
    Garcia-Marques J, Espinosa-Medina I, Ku K, Yang C, Koyama M, Yu H, Lee T
    Nature Neuroscience. 2020 Dec 01;23(12):1618-28. doi: 10.1038/s41593-020-0676-9

    We present CLADES (cell lineage access driven by an edition sequence), a technology for cell lineage studies based on CRISPR-Cas9 techniques. CLADES relies on a system of genetic switches to activate and inactivate reporter genes in a predetermined order. Targeting CLADES to progenitor cells allows the progeny to inherit a sequential cascade of reporters, thereby coupling birth order to reporter expression. This system, which can also be temporally induced by heat shock, enables the temporal resolution of lineage development and can therefore be used to deconstruct an extended cell lineage by tracking the reporters expressed in the progeny. When targeted to the germ line, the same cascade progresses across animal generations, predominantly marking each generation with the corresponding combination of reporters. CLADES therefore offers an innovative strategy for making programmable cascades of genes that can be used for genetic manipulation or to record serial biological events.

    View Publication Page
    11/26/20 | The art of lineage tracing: from worm to human.
    Garcia-Marques J, Espinosa-Medina I, Lee T
    Progress in Neurobiology. 2020 Nov 26:101966. doi: 10.1016/j.pneurobio.2020.101966

    Reconstructing the genealogy of every cell that makes up an organism remains a long-standing challenge in developmental biology. Besides its relevance for understanding the mechanisms underlying normal and pathological development, resolving the lineage origin of cell types will be crucial to create these types on-demand. Multiple strategies have been deployed towards the problem of lineage tracing, ranging from direct observation to sophisticated genetic approaches. Here we discuss the achievements and limitations of past and current technology. Finally, we speculate about the future of lineage tracing and how to reach the next milestones in the field.

    View Publication Page
    05/28/20 | Enhanced Golic+: Highly effective CRISPR gene targeting and transgene HACKing in .
    Chen H, Yao X, Ren Q, Chang C, Liu L, Miyares RL, Lee T
    Development. 2020 May 28:. doi: 10.1242/dev.181974

    Gene targeting is an incredibly valuable technique. Sometimes however, it can also be extremely challenging for various intrinsic reasons (e.g. low target accessibility or nature/extent of gene modification). To bypass these barriers, we designed a transgene-based system in Drosophila that increases the number of independent gene targeting events while at the same time enriching for correctly targeted progeny. Unfortunately, with particularly challenging gene targeting experiments, our original design yielded numerous false positives. Here we deliver a much-improved technique named Enhanced Golic+ (E-Golic+). E-Golic+ incorporates genetic modifications to tighten lethality-based selection while simultaneously boosting efficiency. With E-Golic+, we easily achieve previously unattainable gene targeting. Additionally, we built an E-Golic+ based, high-efficiency genetic pipeline for transgene swapping. We demonstrate its utility by transforming GAL4 enhancer-trap lines into tissue-specific Cas9-expressing lines. Given the superior efficiency, specificity and scalability, E-Golic+ promises to expedite development of additional sophisticated genetic/genomic tools in .

    View Publication Page
    05/01/20 | Neuronal upregulation of Prospero protein is driven by alternative mRNA polyadenylation and Syncrip-mediated mRNA stabilisation.
    Samuels TJ, Arava Y, Järvelin AI, Robertson F, Lee JY, Yang L, Yang C, Lee T, Ish-Horowicz D, Davis I
    Biology Open. 2020 May;9(5):. doi: 10.1242/bio.049684

    During and vertebrate brain development, the conserved transcription factor Prospero/Prox1 is an important regulator of the transition between proliferation and differentiation. Prospero level is low in neural stem cells and their immediate progeny, but is upregulated in larval neurons and it is unknown how this process is controlled. Here, we use single molecule fluorescent hybridisation to show that larval neurons selectively transcribe a long mRNA isoform containing a 15 kb 3' untranslated region, which is bound in the brain by the conserved RNA-binding protein Syncrip/hnRNPQ. Syncrip binding increases the mRNA stability of the long isoform, which allows an upregulation of Prospero protein production. Adult flies selectively lacking the long isoform show abnormal behaviour that could result from impaired locomotor or neurological activity. Our findings highlight a regulatory strategy involving alternative polyadenylation followed by differential post-transcriptional regulation.

    View Publication Page
    04/07/20 | Conservation and divergence of related neuronal lineages in the central brain.
    Lee Y, Yang C, Miyares RL, Huang Y, He Y, Ren Q, Chen H, Kawase T, Ito M, Otsuna H, Sugino K, Aso Y, Ito K, Lee T
    eLife. 2020 Apr 07;9:. doi: 10.7554/eLife.53518

    Wiring a complex brain requires many neurons with intricate cell specificity, generated by a limited number of neural stem cells. central brain lineages are a predetermined series of neurons, born in a specific order. To understand how lineage identity translates to neuron morphology, we mapped 18 central brain lineages. While we found large aggregate differences between lineages, we also discovered shared patterns of morphological diversification. Lineage identity plus Notch-mediated sister fate govern primary neuron trajectories, whereas temporal fate diversifies terminal elaborations. Further, morphological neuron types may arise repeatedly, interspersed with other types. Despite the complexity, related lineages produce similar neuron types in comparable temporal patterns. Different stem cells even yield two identical series of dopaminergic neuron types, but with unrelated sister neurons. Together, these phenomena suggest that straightforward rules drive incredible neuronal complexity, and that large changes in morphology can result from relatively simple fating mechanisms.

    View Publication Page
    03/18/20 | CAMIO: a transgenic CRISPR pipeline to create diverse targeted genome deletions in Drosophila.
    Chen H, Marques JG, Sugino K, Wei D, Miyares RL, Lee T
    Nucleic Acids Research. 2020 Mar 18:. doi: 10.1093/nar/gkaa177

    The genome is the blueprint for an organism. Interrogating the genome, especially locating critical cis-regulatory elements, requires deletion analysis. This is conventionally performed using synthetic constructs, making it cumbersome and non-physiological. Thus, we created Cas9-mediated Arrayed Mutagenesis of Individual Offspring (CAMIO) to achieve comprehensive analysis of a targeted region of native DNA. CAMIO utilizes CRISPR that is spatially restricted to generate independent deletions in the intact Drosophila genome. Controlled by recombination, a single guide RNA is stochastically chosen from a set targeting a specific DNA region. Combining two sets increases variability, leading to either indels at 1-2 target sites or inter-target deletions. Cas9 restriction to male germ cells elicits autonomous double-strand-break repair, consequently creating offspring with diverse mutations. Thus, from a single population cross, we can obtain a deletion matrix covering a large expanse of DNA at both coarse and fine resolution. We demonstrate the ease and power of CAMIO by mapping 5'UTR sequences crucial for chinmo's post-transcriptional regulation.

    View Publication Page
    12/01/19 | High-throughput dense reconstruction of cell lineages.
    Espinosa-Medina I, Garcia-Marques J, Cepko C, Lee T
    Open Biology. 2019 Dec 01;9(12):190229. doi: 10.1098/rsob.190229

    The first meeting exclusively dedicated to the 'High-throughput dense reconstruction of cell lineages' took place at Janelia Research Campus (Howard Hughes Medical Institute) from 14 to 18 April 2019. Organized by Tzumin Lee, Connie Cepko, Jorge Garcia-Marques and Isabel Espinosa-Medina, this meeting echoed the recent eruption of new tools that allow the reconstruction of lineages based on the phylogenetic analysis of DNA mutations induced during development. Combined with single-cell RNA sequencing, these tools promise to solve the lineage of complex model organisms at single-cell resolution. Here, we compile the conference consensus on the technological and computational challenges emerging from the use of the new strategies, as well as potential solutions.

    View Publication Page
    10/23/19 | Unlimited genetic switches for cell-type-specific manipulation.
    Garcia-Marques J, Yang C, Espinosa-Medina I, Mok K, Koyama M, Lee T
    Neuron. 2019 Oct 23;104(2):227-38. doi: https://doi.org/10.1016/j.neuron.2019.07.005

    Gaining independent genetic access to discrete cell types is critical to interrogate their biological functions as well as to deliver precise gene therapy. Transcriptomics has allowed us to profile cell populations with extraordinary precision, revealing that cell types are typically defined by a unique combination of genetic markers. Given the lack of adequate tools to target cell types based on multiple markers, most cell types remain inaccessible to genetic manipulation. Here we present CaSSA, a platform to create unlimited genetic switches based on CRISPR/Cas9 (Ca) and the DNA repair mechanism known as single-strand annealing (SSA). CaSSA allows engineering of independent genetic switches, each responding to a specific gRNA. Expressing multiple gRNAs in specific patterns enables multiplex cell-type-specific manipulations and combinatorial genetic targeting. CaSSA is a new genetic tool that conceptually works as an unlimited number of recombinases and will facilitate genetic access to cell types in diverse organisms.

    View Publication Page