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6 Janelia Publications

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    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.

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    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.

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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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    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.

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