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Lee Tzumin Lab / Publications
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44 Publications

Showing 1-10 of 44 results
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:. 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:. 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:. 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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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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