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50 Janelia Publications

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    09/08/16 | Cell class-lineage analysis reveals sexually dimorphic lineage compositions in the Drosophila brain.
    Ren Q, Awasaki T, Huang Y, Liu Z, Lee T
    Current Biology : CB. 2016 Sep 08;26(19):2583-93. doi: 10.1016/j.cub.2016.07.086

    The morphology and physiology of neurons are directed by developmental decisions made within their lines of descent from single stem cells. Distinct stem cells may produce neurons having shared properties that define their cell class, such as the type of secreted neurotransmitter. The relationship between cell class and lineage is complex. Here we developed the transgenic cell class-lineage intersection (CLIn) system to assign cells of a particular class to specific lineages within the Drosophila brain. CLIn also enables birth-order analysis and genetic manipulation of particular cell classes arising from particular lineages. We demonstrated the power of CLIn in the context of the eight central brain type II lineages, which produce highly diverse progeny through intermediate neural progenitors. We mapped 18 dopaminergic neurons from three distinct clusters to six type II lineages that show lineage-characteristic neurite trajectories. In addition, morphologically distinct dopaminergic neurons are produced within a given lineage, and they arise in an invariant sequence. We also identified type II lineages that produce doublesex- and fruitless-expressing neurons and examined whether female-specific apoptosis in these lineages accounts for the lower number of these neurons in the female brain. Blocking apoptosis in these lineages resulted in more cells in both sexes with males still carrying more cells than females. This argues that sex-specific stem cell fate together with differential progeny apoptosis contribute to the final sexual dimorphism.

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    02/01/16 | Transcriptomes of lineage-specific Drosophila neuroblasts profiled by genetic targeting and robotic sorting.
    Yang C, Fu C, Sugino K, Liu Z, Ren Q, Liu L, Yao X, Lee LP, Lee T
    Development (Cambridge, England). 2016 Feb 1;143(3):411-21. doi: 10.1242/dev.129163

    A brain consists of numerous distinct neurons arising from a limited number of progenitors, called neuroblasts in Drosophila. Each neuroblast produces a specific neuronal lineage. To unravel the transcriptional networks that underlie the development of distinct neuroblast lineages, we marked and isolated lineage-specific neuroblasts for RNA sequencing. We labeled particular neuroblasts throughout neurogenesis by activating a conditional neuroblast driver in specific lineages using various intersection strategies. The targeted neuroblasts were efficiently recovered using a custom-built device for robotic single-cell picking. Transcriptome analysis of mushroom body, antennal lobe and type II neuroblasts compared with non-selective neuroblasts, neurons and glia revealed a rich repertoire of transcription factors expressed among neuroblasts in diverse patterns. Besides transcription factors that are likely to be pan-neuroblast, many transcription factors exist that are selectively enriched or repressed in certain neuroblasts. The unique combinations of transcription factors present in different neuroblasts may govern the diverse lineage-specific neuron fates.

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    10/16/15 | Opposing intrinsic temporal gradients guide neural stem cell production of varied neuronal fates.
    Liu Z, Yang C, Sugino K, Fu C, Liu L, Yao X, Lee LP, Lee T
    Science (New York, N.Y.). 2015 Oct 16;350(6258):317-20. doi: 10.1126/science.aad1886

    Neural stem cells show age-dependent developmental potentials, as evidenced by their production of distinct neuron types at different developmental times. Drosophila neuroblasts produce long, stereotyped lineages of neurons. We searched for factors that could regulate neural temporal fate by RNA-sequencing lineage-specific neuroblasts at various developmental times. We found that two RNA-binding proteins, IGF-II mRNA-binding protein (Imp) and Syncrip (Syp), display opposing high-to-low and low-to-high temporal gradients with lineage-specific temporal dynamics. Imp and Syp promote early and late fates, respectively, in both a slowly progressing and a rapidly changing lineage. Imp and Syp control neuronal fates in the mushroom body lineages by regulating the temporal transcription factor Chinmo translation. Together, the opposing Imp/Syp gradients encode stem cell age, specifying multiple cell fates within a lineage.

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    03/01/15 | An enhanced gene targeting toolkit for Drosophila: golic+.
    Chen H, Huang Y, Pfeiffer BD, Yao X, Lee T
    Genetics. 2015 Mar;199(3):683-94. doi: 10.1534/genetics.114.173716

    Ends-out gene targeting allows seamless replacement of endogenous genes with engineered DNA fragments by homologous recombination, thus creating designer "genes" in the endogenous locus. Conventional gene targeting in Drosophila involves targeting with the preintegrated donor DNA in the larval primordial germ cells. Here we report G: ene targeting during O: ogenesis with L: ethality I: nhibitor and C: RISPR/Cas (Golic+), which improves on all major steps in such transgene-based gene targeting systems. First, donor DNA is integrated into precharacterized attP sites for efficient flip-out. Second, FLP, I-SceI, and Cas9 are specifically expressed in cystoblasts, which arise continuously from female germline stem cells, thereby providing a continual source of independent targeting events in each offspring. Third, a repressor-based lethality selection is implemented to facilitate screening for correct targeting events. Altogether, Golic+ realizes high-efficiency ends-out gene targeting in ovarian cystoblasts, which can be readily scaled up to achieve high-throughput genome editing.

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    07/22/14 | Optimized CRISPR/Cas tools for efficient germline and somatic genome engineering in Drosophila.
    Port F, Chen H, Lee T, Bullock SL
    Proceedings of the National Academy of Sciences of the United States of America. 2014 Jul 22;111(29):E2967-76. doi: 10.1073/pnas.1405500111

    The type II clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system has emerged recently as a powerful method to manipulate the genomes of various organisms. Here, we report a toolbox for high-efficiency genome engineering of Drosophila melanogaster consisting of transgenic Cas9 lines and versatile guide RNA (gRNA) expression plasmids. Systematic evaluation reveals Cas9 lines with ubiquitous or germ-line-restricted patterns of activity. We also demonstrate differential activity of the same gRNA expressed from different U6 snRNA promoters, with the previously untested U6:3 promoter giving the most potent effect. An appropriate combination of Cas9 and gRNA allows targeting of essential and nonessential genes with transmission rates ranging from 25-100%. We also demonstrate that our optimized CRISPR/Cas tools can be used for offset nicking-based mutagenesis. Furthermore, in combination with oligonucleotide or long double-stranded donor templates, our reagents allow precise genome editing by homology-directed repair with rates that make selection markers unnecessary. Last, we demonstrate a novel application of CRISPR/Cas-mediated technology in revealing loss-of-function phenotypes in somatic cells following efficient biallelic targeting by Cas9 expressed in a ubiquitous or tissue-restricted manner. Our CRISPR/Cas tools will facilitate the rapid evaluation of mutant phenotypes of specific genes and the precise modification of the genome with single-nucleotide precision. Our results also pave the way for high-throughput genetic screening with CRISPR/Cas.

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    04/01/14 | Making Drosophila lineage-restricted drivers via patterned recombination in neuroblasts.
    Awasaki T, Kao C, Lee Y, Yang C, Huang Y, Pfeiffer BD, Luan H, Jing X, Huang Y, He Y, Schroeder MD, Kuzin A, Brody T, Zugates CT, Odenwald WF, Lee T
    Nature Neuroscience. 2014 Apr;17(4):631-7. doi: 10.1038/nn.3654

    The Drosophila cerebrum originates from about 100 neuroblasts per hemisphere, with each neuroblast producing a characteristic set of neurons. Neurons from a neuroblast are often so diverse that many neuron types remain unexplored. We developed new genetic tools that target neuroblasts and their diverse descendants, increasing our ability to study fly brain structure and development. Common enhancer-based drivers label neurons on the basis of terminal identities rather than origins, which provides limited labeling in the heterogeneous neuronal lineages. We successfully converted conventional drivers that are temporarily expressed in neuroblasts, into drivers expressed in all subsequent neuroblast progeny. One technique involves immortalizing GAL4 expression in neuroblasts and their descendants. Another depends on loss of the GAL4 repressor, GAL80, from neuroblasts during early neurogenesis. Furthermore, we expanded the diversity of MARCM-based reagents and established another site-specific mitotic recombination system. Our transgenic tools can be combined to map individual neurons in specific lineages of various genotypes.

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    02/23/14 | Sparse, decorrelated odor coding in the mushroom body enhances learned odor discrimination.
    Lin AC, Bygrave AM, de Calignon A, Lee T, Miesenböck G
    Nature Neuroscience. 2014 Feb 23;17(4):559-68. doi: 10.1038/nn.3660

    Sparse coding may be a general strategy of neural systems for augmenting memory capacity. In Drosophila melanogaster, sparse odor coding by the Kenyon cells of the mushroom body is thought to generate a large number of precisely addressable locations for the storage of odor-specific memories. However, it remains untested how sparse coding relates to behavioral performance. Here we demonstrate that sparseness is controlled by a negative feedback circuit between Kenyon cells and the GABAergic anterior paired lateral (APL) neuron. Systematic activation and blockade of each leg of this feedback circuit showed that Kenyon cells activated APL and APL inhibited Kenyon cells. Disrupting the Kenyon cell-APL feedback loop decreased the sparseness of Kenyon cell odor responses, increased inter-odor correlations and prevented flies from learning to discriminate similar, but not dissimilar, odors. These results suggest that feedback inhibition suppresses Kenyon cell activity to maintain sparse, decorrelated odor coding and thus the odor specificity of memories.

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    01/01/14 | Drosophila intermediate neural progenitors produce lineage-dependent related series of diverse neurons.
    Wang Y, Yang JS, Johnston R, Ren Q, Lee Y, Luan H, Brody T, Odenwald WF, Lee T
    Development. 2014 Jan;141:253-8. doi: 10.1242/dev.103069

    Drosophila type II neuroblasts (NBs), like mammalian neural stem cells, deposit neurons through intermediate neural progenitors (INPs) that can each produce a series of neurons. Both type II NBs and INPs exhibit age-dependent expression of various transcription factors, potentially specifying an array of diverse neurons by combinatorial temporal patterning. Not knowing which mature neurons are made by specific INPs, however, conceals the actual variety of neuron types and limits further molecular studies. Here we mapped neurons derived from specific type II NB lineages and found that sibling INPs produced a morphologically similar but temporally regulated series of distinct neuron types. This suggests a common fate diversification program operating within each INP that is modulated by NB age to generate slightly different sets of diverse neurons based on the INP birth order. Analogous mechanisms might underlie the expansion of neuron diversity via INPs in mammalian brain.

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    01/01/14 | Generating mosaics for lineage analysis in flies.
    Lee T
    Wiley Interdisciplinary Reviews - Developmental Biology. 2014 Jan;3(1):69-81. doi: 10.1002/wdev.122

    By generating and studying mosaic organisms, we are learning how intricate tissues form as cells proliferate and diversify through organism development. FLP/FRT-mediated site-specific mitotic recombination permits the generation of mosaic flies with efficiency and control. With heat-inducible or tissue-specific FLP transgenes at our disposal, we can engineer mosaics carrying clones of homozygous cells that come from specific pools of heterozygous precursors. This permits detailed cell lineage analysis followed by mosaic analysis of gene functions in the underlying developmental processes. Expression of transgenes (e.g., reporters) only in the homozygous cells enables mosaic analysis in the complex nervous system. Tracing neuronal lineages by using mosaics revolutionized mechanistic studies of neuronal diversification and differentiation, exemplifying the power of genetic mosaics in developmental biology. WIREs Dev Biol 2014, 3:69–81. doi: 10.1002/wdev.122

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    09/19/13 | Extremes of lineage plasticity in the Drosophila brain.
    Lin S, Marin EC, Yang C, Kao C, Apenteng BA, Huang Y, O’Connor MB, Truman JW, Lee T
    Current Biology. 2013 Sep 19;23(19):1908-13. doi: 10.1016/j.cub.2013.07.074

    An often-overlooked aspect of neural plasticity is the plasticity of neuronal composition, in which the numbers of neurons of particular classes are altered in response to environment and experience. The Drosophila brain features several well-characterized lineages in which a single neuroblast gives rise to multiple neuronal classes in a stereotyped sequence during development [1]. We find that in the intrinsic mushroom body neuron lineage, the numbers for each class are highly plastic, depending on the timing of temporal fate transitions and the rate of neuroblast proliferation. For example, mushroom body neuroblast cycling can continue under starvation conditions, uncoupled from temporal fate transitions that depend on extrinsic cues reflecting organismal growth and development. In contrast, the proliferation rates of antennal lobe lineages are closely associated with organismal development, and their temporal fate changes appear to be cell cycle-dependent, such that the same numbers and types of uniglomerular projection neurons innervate the antennal lobe following various perturbations. We propose that this surprising difference in plasticity for these brain lineages is adaptive, given their respective roles as parallel processors versus discrete carriers of olfactory information.

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