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50 Janelia Publications

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    09/23/19 | Mamo decodes hierarchical temporal gradients into terminal neuronal fate.
    Liu L, Long X, Yang C, Miyares RL, Sugino K, Singer RH, Lee T
    Elife. 2019 Sep 23;8:. doi: 10.7554/eLife.48056

    Temporal patterning is a seminal method of expanding neuronal diversity. Here we unravel a mechanism decoding neural stem cell temporal gene expression and transforming it into discrete neuronal fates. This mechanism is characterized by hierarchical gene expression. First, neuroblasts express opposing temporal gradients of RNA-binding proteins, Imp and Syp. These proteins promote or inhibit translation, yielding a descending neuronal gradient. Together, first and second-layer temporal factors define a temporal expression window of BTB-zinc finger nuclear protein, Mamo. The precise temporal induction of Mamo is achieved via both transcriptional and post-transcriptional regulation. Finally, Mamo is essential for the temporally defined, terminal identity of α'/β' mushroom body neurons and identity maintenance. We describe a straightforward paradigm of temporal fate specification where diverse neuronal fates are defined via integrating multiple layers of gene regulation. The neurodevelopmental roles of orthologous/related mammalian genes suggest a fundamental conservation of this mechanism in brain development.

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    05/30/19 | CLADES: a programmable sequence of reporters for lineage analysis
    Garcia-Marques J, Yang C, Espinosa-Medina I, Koyama M, Lee T
    bioRxiv. 2019 May 30:. doi:

    We present CLADES (Cell Lineage Access Driven by an Edition Sequence), a technology for cell lineage studies based on CRISPR/Cas9. CLADES relies on a system of genetic switches to activate and inactivate reporter genes in a pre-determined order. Targeting CLADES to progenitor cells allows the progeny to inherit a sequential cascade of reporters, coupling birth order with reporter expression. This gives us temporal resolution of lineage development that can 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, marking each generation with the corresponding combination of reporters. CLADES thus 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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    03/26/19 | Neurotransmitter identity is acquired in a lineage-restricted manner in the Drosophila CNS.
    Lacin H, Chen H, Long X, Singer RH, Lee T, Truman JW
    Elife. 2019 Mar 26;8:. doi: 10.7554/eLife.43701

    The vast majority of the adult fly ventral nerve cord is composed of 34 hemilineages, which are clusters of lineally related neurons. Neurons in these hemilineages use one of the three fast-acting neurotransmitters (acetylcholine, GABA, or glutamate) for communication. We generated a comprehensive neurotransmitter usage map for the entire ventral nerve cord. We did not find any cases of neurons using more than one neurotransmitter, but found that the acetylcholine specific gene ChAT is transcribed in many glutamatergic and GABAergic neurons, but these transcripts typically do not leave the nucleus and are not translated. Importantly, our work uncovered a simple rule: All neurons within a hemilineage use the same neurotransmitter. Thus, neurotransmitter identity is acquired at the stem cell level. Our detailed transmitter- usage/lineage identity map will be a great resource for studying the developmental basis of behavior and deciphering how neuronal circuits function to regulate behavior.

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    11/27/18 | Temporal control of Drosophila central nervous system development.
    Miyares RL, Lee T
    Current Opinion in Neurobiology. 2018 Nov 27;56:24-32. doi: 10.1016/j.conb.2018.10.016

    A complex nervous system requires precise numbers of various neuronal types produced with exquisite spatiotemporal control. This striking diversity is generated by a limited number of neural stem cells (NSC), where spatial and temporal patterning intersect. Drosophila is a genetically tractable model system that has significant advantages for studying stem cell biology and neuronal fate specification. Here we review the latest findings in the rich literature of temporal patterning of neuronal identity in the Drosophila central nervous system. Rapidly changing consecutive transcription factors expressed in NSCs specify short series of neurons with considerable differences. More slowly progressing changes are orchestrated by NSC intrinsic temporal factor gradients which integrate extrinsic signals to coordinate nervous system and organismal development.

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    07/31/18 | High-resolution transcriptional dissection of in vivo Atoh1-mediated hair cell conversion in mature cochleae identifies Isl1 as a co-reprogramming factor.
    Yamashita T, Zheng F, Finkelstein D, Kellard Z, Carter R, Rosencrance CD, Sugino K, Easton J, Gawad C, Zuo J
    PLoS Genetics. 2018 07;14(7):e1007552. doi: 10.1371/journal.pgen.1007552

    In vivo direct conversion of differentiated cells holds promise for regenerative medicine; however, improving the conversion efficiency and producing functional target cells remain challenging. Ectopic Atoh1 expression in non-sensory supporting cells (SCs) in mouse cochleae induces their partial conversion to hair cells (HCs) at low efficiency. Here, we performed single-cell RNA sequencing of whole mouse sensory epithelia harvested at multiple time points after conditional overexpression of Atoh1. Pseudotemporal ordering revealed that converted HCs (cHCs) are present along a conversion continuum that correlates with both endogenous and exogenous Atoh1 expression. Bulk sequencing of isolated cell populations and single-cell qPCR confirmed 51 transcription factors, including Isl1, are differentially expressed among cHCs, SCs and HCs. In transgenic mice, co-overexpression of Atoh1 and Isl1 enhanced the HC conversion efficiency. Together, our study shows how high-resolution transcriptional profiling of direct cell conversion can identify co-reprogramming factors required for efficient conversion.

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    05/15/18 | Lineage-guided Notch-dependent gliogenesis by multi-potent progenitors.
    Ren Q, Awasaki T, Wang Y, Huang Y, Lee T
    Development (Cambridge, England). 2018 May 15:. doi: 10.1242/dev.160127

    Macroglial cells in the central nervous system exhibit regional specialization and carry out region-specific functions. Diverse glial cells arise from specific progenitors in specific spatiotemporal patterns. This raises an interesting possibility that there exist glial precursors with distinct developmental fates, which govern region-specific gliogenesis. Here we mapped the glial progeny produced by the type II neuroblasts, which, like vertebrate radial glia cells, yield both neurons and glia via intermediate neural progenitors (INPs). Distinct type II neuroblasts produce different characteristic sets of glia. A single INP can make both astrocyte-like and ensheathing glia, which co-occupy a relatively restrictive subdomain. Blocking apoptosis uncovers further lineage distinctions in the specification, proliferation, and survival of glial precursors. Both the switch from neurogenesis to gliogenesis and the subsequent glial expansion depend on Notch signaling. Taken together, lineage origins preconfigure the development of individual glial precursors with involvement of serial Notch actions in promoting gliogenesis.

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    09/05/17 | Dissection of the Drosophila neuropeptide F circuit using a high-throughput two-choice assay.
    Shao L, Saver M, Chung P, Ren Q, Lee T, Kent CF, Heberlein U
    Proceedings of the National Academy of Sciences of the United States of America. 2017 Sep 05;114(38):e8091-9. doi: 10.1073/pnas.1710552114

    In their classic experiments, Olds and Milner showed that rats learn to lever press to receive an electric stimulus in specific brain regions. This led to the identification of mammalian reward centers. Our interest in defining the neuronal substrates of reward perception in the fruit fly Drosophila melanogaster prompted us to develop a simpler experimental approach wherein flies could implement behavior that induces self-stimulation of specific neurons in their brains. The high-throughput assay employs optogenetic activation of neurons when the fly occupies a specific area of a behavioral chamber, and the flies' preferential occupation of this area reflects their choosing to experience optogenetic stimulation. Flies in which neuropeptide F (NPF) neurons are activated display preference for the illuminated side of the chamber. We show that optogenetic activation of NPF neuron is rewarding in olfactory conditioning experiments and that the preference for NPF neuron activation is dependent on NPF signaling. Finally, we identify a small subset of NPF-expressing neurons located in the dorsomedial posterior brain that are sufficient to elicit preference in our assay. This assay provides the means for carrying out unbiased screens to map reward neurons in flies.

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    08/29/17 | Imp and Syp RNA-binding proteins govern decommissioning of Drosophila neural stem cells.
    Yang C, Samuels TJ, Huang Y, Yang L, Ish-Horowicz D, Davis I, Lee T
    Development (Cambridge, England). 2017 Aug 29;144(19):3454-64. doi: 10.1242/dev.149500

    The termination of the proliferation of Drosophila neural stem cells, also known as neuroblasts (NBs), requires a "decommissioning" phase that is controlled in a lineage-specific manner. Most NBs, with the exception of those of the Mushroom body (MB), are decommissioned by the ecdysone receptor and mediator complex causing them to shrink during metamorphosis, followed by nuclear accumulation of Prospero and cell cycle exit. Here, we demonstrate that the levels of Imp and Syp RNA-binding proteins regulate NB decommissioning. Descending Imp and ascending Syp expression have been shown to regulate neuronal temporal fate. We show that Imp levels decline slower in the MB than other central brain NBs. MB NBs continue to express Imp into pupation, and the presence of Imp prevents decommissioning partly by inhibiting the mediator complex. Late-larval induction of transgenic Imp prevents many non-MB NBs from decommissioning in early pupae. Moreover, the presence of abundant Syp in aged NBs permits Prospero accumulation that, in turn, promotes cell cycle exit. Together our results reveal that progeny temporal fate and progenitor decommissioning are co-regulated in protracted neuronal lineages.

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    04/10/17 | Stem cell-intrinsic, seven-up-triggered temporal factor gradients diversify intermediate neural progenitors.
    Ren Q, Yang C, Liu Z, Sugino K, Mok K, He Y, Ito M, Nern A, Otsuna H, Lee T
    Current Biology : CB. 2017 Apr 10;27(9):1303-13. doi: 10.1016/j.cub.2017.03.047

    Building a sizable, complex brain requires both cellular expansion and diversification. One mechanism to achieve these goals is production of multiple transiently amplifying intermediate neural progenitors (INPs) from a single neural stem cell. Like mammalian neural stem cells, Drosophila type II neuroblasts utilize INPs to produce neurons and glia. Within a given lineage, the consecutively born INPs produce morphologically distinct progeny, presumably due to differential inheritance of temporal factors. To uncover the underlying temporal fating mechanisms, we profiled type II neuroblasts' transcriptome across time. Our results reveal opposing temporal gradients of Imp and Syp RNA-binding proteins (descending and ascending, respectively). Maintaining high Imp throughout serial INP production expands the number of neurons and glia with early temporal fate at the expense of cells with late fate. Conversely, precocious upregulation of Syp reduces the number of cells with early fate. Furthermore, we reveal that the transcription factor Seven-up initiates progression of the Imp/Syp gradients. Interestingly, neuroblasts that maintain initial Imp/Syp levels can still yield progeny with a small range of early fates. We therefore propose that the Seven-up-initiated Imp/Syp gradients create coarse temporal windows within type II neuroblasts to pattern INPs, which subsequently undergo fine-tuned subtemporal patterning.

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    01/23/17 | Wiring the Drosophila brain with individually tailored neural lineages.
    Lee T
    Current Biology : CB. 2017 Jan 23;27(2):R77-R82. doi: 10.1016/j.cub.2016.12.026

    A complex brain consists of multiple intricate neural networks assembled from distinct sets of input and output neurons as well as region-specific local interneurons. Within a given anatomical set, there exist diverse neuronal types that can vary in morphology, neural physiology, and modes of neurotransmission. The genetic programs that guide specification of neuronal types during neurogenesis preconfigure the brain. This is best demonstrated in the Drosophila central brain, which is composed of ∼100 pairs of individually tailored neuronal lineages. Each neuronal lineage (the neurons/glia produced from a single stem cell) can contain multiple morphological classes of neurons that can consist of many analogous neuronal types. The detailed patterns of neuronal diversification are lineage-specific and can differ drastically even among neighboring neuronal lineages. Furthermore, the interrelationships between neuronal lineages and neural networks are complex. These phenomena underscore the importance of tracking all neuronal lineages in understanding brain development and evolution.

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