Christopher J. Potter

Akt regulates growth by directly phosphorylating Tsc2

The direct mechanism by which the serine/threonine kinase Akt (also known as protein kinase B (PKB)) regulates cell growth is unknown. Here, we report that Drosophila melanogaster Akt/PKB stimulates growth by phosphorylating the tuberous sclerosis complex 2 (Tsc2) tumour suppressor and inhibiting formation of a Tsc1–Tsc2 complex. We show that Akt/PKB directly phosphorylates Drosophila Tsc2 in vitro at the conserved residues, Ser 924 and Thr 1518. Mutation of these sites renders Tsc2 insensitive to Akt/PKB signalling, increasing the stability of the Tsc1–Tsc2 complex within the cell. Stimulating Akt/PKB signalling in vivo markedly increases cell growth/size, disrupts the Tsc1–Tsc2 complex and disturbs the distinct subcellular localization of Tsc1 and Tsc2. Furthermore, all Akt/PKB growth signals are blocked by expression of a Tsc2 mutant lacking Akt phosphorylation sites. Thus, Tsc2 seems to be the critical target of Akt in mediating growth signals for the insulin signalling pathway.

Drosophila Tsc1 Functions with Tsc2 to Antagonize Insulin Signaling in Regulating Cell Growth, Cell Proliferation, and Organ Size

Tuberous sclerosis complex is a dominant disorder that leads to the development of benign tumors in multiple organs. We have isolated a mutation in the Drosophila homolog of TSC1 (Tsc1). Cells mutant for Tsc1 are dramatically increased in size yet differentiate normally. Organ size is also increased in tissues that contain a majority of mutant cells. Clones of Tsc1 mutant cells in the imaginal discs undergo additional divisions but retain normal ploidy. We also show that the Tsc1 protein binds to Drosophila Tsc2 in vitro. Overexpression of Tsc1 or Tsc2 alone in the wing and eye has no effect, but co-overexpression leads to a decrease in cell size, cell number, and organ size. Genetic epistasis data are consistent with a model that Tsc1 and Tsc2 function together in the insulin signaling pathway.

Comprehensive maps of Drosophila higher olfactory centers: spatially segregated fruit and pheromone representation.

Summary In Drosophila, ∼50 classes of olfactory receptor neurons (ORNs) send axons to 50 corresponding glomeruli in the antennal lobe. Uniglomerular projection neurons (PNs) relay olfactory information to the mushroom body (MB) and lateral horn (LH). Here, we combine single-cell labeling and image registration to create high-resolution, quantitative maps of the MB and LH for 35 input PN channels and several groups of LH neurons. We find (1) PN inputs to the MB are stereotyped as previously shown for the LH; (2) PN partners of ORNs from different sensillar groups are clustered in the LH; (3) fruit odors are represented mostly in the posterior-dorsal LH, whereas candidate pheromone-responsive PNs project to the anterior-ventral LH; (4) dendrites of single LH neurons each overlap with specific subsets of PN axons. Our results suggest that the LH is organized according to biological values of olfactory input.

The Q System: A Repressible Binary System for Transgene Expression, Lineage Tracing, and Mosaic Analysis

We describe a new repressible binary expression system based on the regulatory genes from the Neurospora qa gene cluster. This Q system offers attractive features for transgene expression in Drosophila and mammalian cells: low basal expression in the absence of the transcriptional activator QF, high QF-induced expression, and QF repression by its repressor QS. Additionally, feeding flies quinic acid can relieve QS repression. The Q system offers many applications, including (1) intersectional logic gates with the GAL4 system for manipulating transgene expression patterns, (2) GAL4-independent MARCM analysis, and (3) coupled MARCM analysis to independently visualize and genetically manipulate siblings from any cell division. We demonstrate the utility of the Q system in determining cell division patterns of a neuronal lineage and gene function in cell growth and proliferation, and in dissecting neurons responsible for olfactory attraction. The Q system can be expanded to other uses in Drosophila and to any organism conducive to transgenesis.

Mechanisms of size control

The study of organ size control is a discipline of developmental biology that is largely unexplored. Although the size of an organ or organism depends largely on cell numbers and cell size, studies have found that the simple deregulation of cell proliferation or cell growth does not necessarily lead to changes in organ size. Recent genetic screens in Drosophila suggest that mutations that do affect organ size can be classified into three broad categories on the basis of their underlying effects: patterning, proliferation, and growth. Overall, experimental data suggest that organ size might be regulated by a total mass checkpoint mechanism which functions to link the regulation of cell size and cell proliferation. The mechanisms of organ size control could also be critical targets for evolutionary events or disease processes such as tumorigenesis.

A versatile in vivo system for directed dissection of gene expression patterns

Tissue-specific gene expression using the upstream activating sequence (UAS)–GAL4 binary system has facilitated genetic dissection of many biological processes in Drosophila melanogaster. Refining GAL4 expression patterns or independently manipulating multiple cell populations using additional binary systems are common experimental goals. To simplify these processes, we developed a convertible genetic platform, the integrase swappable in vivo targeting element (InSITE) system. This approach allows GAL4 to be replaced with any other sequence, placing different genetic effectors under the control of the same regulatory elements. Using InSITE, GAL4 can be replaced with LexA or QF, allowing an expression pattern to be repurposed. GAL4 can also be replaced with GAL80 or split-GAL4 hemi-drivers, allowing intersectional approaches to refine expression patterns. The exchanges occur through efficient in vivo manipulations, making it possible to generate many swaps in parallel. This system is modular, allowing future genetic tools to be easily incorporated into the existing framework.

Drosophila in cancer research: an expanding role

In recent years, Drosophila researchers have developed powerful genetic techniques that allow for the rapid identification and characterization of genes involved in tumor formation and development. The high level of gene and pathway conservation, the similarity of cellular processes and the emerging evidence of functional conservation of tumor suppressors between Drosophila and mammals, argue that studies of tumorigenesis in flies can directly contribute to the understanding of human cancer. In this review, we explore the historical and current roles of Drosophila in cancer research, as well as speculate on the future of Drosophila as a model to investigate cancer-related processes that are currently not well understood.

Plug-and-Play Genetic Access to Drosophila Cell Types Using Exchangeable Exon Cassettes

Genetically encoded effectors are important tools forxa0probing cellular function in living animals, but improved methods for directing their expression to specific cell types are required. Here, we introduce a simple, versatile method for achieving cell-type-specific expression of transgenes that leverages the untapped potential of coding introns (i.e., introns between coding exons). Our method couples the expression of a transgene to that of a native gene expressed in the cells of interest using intronically inserted plug-and-play cassettes (called Trojan exons) that carry a splice acceptor site followed by the coding sequences of T2A peptide and an effector transgene. We demonstrate the efficacy of this approach in Drosophila using lines containing suitable MiMIC (Minos-mediated integration cassette) transposons and a palette of Trojan exons capable of expressing a range of commonly used transcription factors. We also introduce an exchangeable, MiMIC-like Trojan exon construct that can be targeted to coding introns using the Crispr/Cas system.

Splinkerette PCR for Mapping Transposable Elements in Drosophila

Transposable elements (such as the P-element and piggyBac) have been used to introduce thousands of transgenic constructs into the Drosophila genome. These transgenic constructs serve many roles, from assaying gene/cell function, to controlling chromosome arm rearrangement. Knowing the precise genomic insertion site for the transposable element is often desired. This enables identification of genomic enhancer regions trapped by an enhancer trap, identification of the gene mutated by a transposon insertion, or simplifying recombination experiments. The most commonly used transgene mapping method is inverse PCR (iPCR). Although usually effective, limitations with iPCR hinder its ability to isolate flanking genomic DNA in complex genomic loci, such as those that contain natural transposons. Here we report the adaptation of the splinkerette PCR (spPCR) method for the isolation of flanking genomic DNA of any P-element or piggyBac. We report a simple and detailed protocol for spPCR. We use spPCR to 1) map a GAL4 enhancer trap located inside a natural transposon, pinpointing a master regulatory region for olfactory neuron expression in the brain; and 2) map all commonly used centromeric FRT insertion sites. The ease, efficiency, and efficacy of spPCR could make it a favored choice for the mapping of transposable element in Drosophila.

Leucine-rich repeat transmembrane proteins instruct discrete dendrite targeting in an olfactory map

Olfactory systems utilize discrete neural pathways to process and integrate odorant information. In Drosophila, axons of first-order olfactory receptor neurons (ORNs) and dendrites of second-order projection neurons (PNs) form class-specific synaptic connections at ∼50 glomeruli. The mechanisms underlying PN dendrite targeting to distinct glomeruli in a three-dimensional discrete neural map are unclear. We found that the leucine-rich repeat (LRR) transmembrane protein Capricious (Caps) was differentially expressed in different classes of PNs. Loss-of-function and gain-of-function studies indicated that Caps instructs the segregation of Caps-positive and Caps-negative PN dendrites to discrete glomerular targets. Moreover, Caps-mediated PN dendrite targeting was independent of presynaptic ORNs and did not involve homophilic interactions. The closely related protein Tartan was partially redundant with Caps. These LRR proteins are probably part of a combinatorial cell-surface code that instructs discrete olfactory map formation.