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64 Publications

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    09/02/22 | Tracing and Manipulating Drosophila Cell Lineages Based on CRISPR: CaSSA and CLADES.
    Garcia-Marques J, Lee T
    Methods in Molecular Biology. 2022 Sep 02;2540:201-217. doi: 10.1007/978-1-0716-2541-5_9

    Cell lineage defines the mitotic connection between cells that make up an organism. Mapping these connections in relation to cell identity offers an extraordinary insight into the mechanisms underlying normal and pathological development. The analysis of molecular determinants involved in the acquisition of cell identity requires gaining experimental access to precise parts of cell lineages. Recently, we have developed CaSSA and CLADES, a new technology based on CRISPR that allows targeting and labeling specific lineage branches. Here we discuss how to better exploit this technology for lineage studies in Drosophila, with an emphasis on neuronal specification.

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    06/12/22 | Metamorphosis of memory circuits in Drosophila reveal a strategy for evolving a larval brain.
    James W. Truman , Jacquelyn Price , Rosa L. Miyares , Tzumin Lee
    bioRxiv. 2022 Jun 12:. doi: 10.1101/2022.06.09.495452

    Insects like Drosophila produce a second brain adapted to the form and behavior of a larva. Neurons for both larval and adult brains are produced by the same stem cells (neuroblasts) but the larva possesses only the earliest born neurons produced from each. To understand how a functional larval brain is made from this reduced set of neurons, we examined the origins and metamorphic fates of the neurons of the larval and adult mushroom body circuits. The adult mushroom body core is built sequentially of γ Kenyon cells, that form a medial lobe, followed by α’β’, and αβ Kenyon cells that form additional medial lobes and two vertical lobes. Extrinsic input (MBINs) and output (MBONs) neurons divide this core into computational compartments. The larval mushroom body contains only γ neurons. Its medial lobe compartments are roughly homologous to those of the adult and same MBONs are used for both. The larval vertical lobe, however, is an analogous “facsimile” that uses a larval-specific branch on the γ neurons to make up for the missing α’β’, and αβ neurons. The extrinsic cells for the facsimile are early-born neurons that trans-differentiate to serve a mushroom body function in the larva and then shift to other brain circuits in the adult. These findings are discussed in the context of the evolution of a larval brain in insects with complete metamorphosis.

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    05/23/22 | Hormone-controlled changes in the differentiation state of post-mitotic neurons.
    Lai Y, Miyares RL, Liu L, Chu S, Lee T, Yu H
    Current Biology. 2022 May 23;32(10):2341-2348. doi: 10.1016/j.cub.2022.04.027

    While we think of neurons as having a fixed identity, many show spectacular plasticity. Metamorphosis drives massive changes in the fly brain; neurons that persist into adulthood often change in response to the steroid hormone ecdysone. Besides driving remodeling, ecdysone signaling can also alter the differentiation status of neurons. The three sequentially born subtypes of mushroom body (MB) Kenyon cells (γ, followed by α'/β', and finally α/β) serve as a model of temporal fating. γ neurons are also used as a model of remodeling during metamorphosis. As γ neurons are the only functional Kenyon cells in the larval brain, they serve the function of all three adult subtypes. Correspondingly, larval γ neurons have a similar morphology to α'/β' and α/β neurons-their axons project dorsally and medially. During metamorphosis, γ neurons remodel to form a single medial projection. Both temporal fate changes and defects in remodeling therefore alter γ-neuron morphology in similar ways. Mamo, a broad-complex, tramtrack, and bric-à-brac/poxvirus and zinc finger (BTB/POZ) transcription factor critical for temporal specification of α'/β' neurons, was recently described as essential for γ remodeling. In a previous study, we noticed a change in the number of adult Kenyon cells expressing γ-specific markers when mamo was manipulated. These data implied a role for Mamo in γ-neuron fate specification, yet mamo is not expressed in γ neurons until pupariation, well past γ specification. This indicates that mamo has a later role in ensuring that γ neurons express the correct Kenyon cell subtype-specific genes in the adult brain.

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    07/23/21 | YAP1 nuclear efflux and transcriptional reprograming follow membrane diminution upon VSV-G-induced cell fusion.
    Feliciano D, Ott CM, Isabel Espinosa Medina , 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.

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    04/01/21 | The art of lineage tracing: From worm to human.
    Garcia-Marques J, Isabel Espinosa Medina , 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.

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    12/01/20 | A programmable sequence of reporters for lineage analysis.
    Garcia-Marques J, Isabel Espinosa Medina , 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.

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    11/26/20 | The art of lineage tracing: from worm to human.
    Garcia-Marques J, Isabel Espinosa Medina , 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.

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    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 .

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    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.

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    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.

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