Michael Buszczak

The carnegie protein trap library: a versatile tool for Drosophila developmental studies.

Metazoan physiology depends on intricate patterns of gene expression that remain poorly known. Using transposon mutagenesis in Drosophila, we constructed a library of 7404 protein trap and enhancer trap lines, the Carnegie collection, to facilitate gene expression mapping at single-cell resolution. By sequencing the genomic insertion sites, determining splicing patterns downstream of the enhanced green fluorescent protein (EGFP) exon, and analyzing expression patterns in the ovary and salivary gland, we found that 600–900 different genes are trapped in our collection. A core set of 244 lines trapped different identifiable protein isoforms, while insertions likely to act as GFP-enhancer traps were found in 256 additional genes. At least 8 novel genes were also identified. Our results demonstrate that the Carnegie collection will be useful as a discovery tool in diverse areas of cell and developmental biology and suggest new strategies for greatly increasing the coverage of the Drosophila proteome with protein trap insertions.

The Drosophila melanogaster Cajal body

Cajal bodies (CBs) are nuclear organelles that are usually identified by the marker protein p80-coilin. Because no orthologue of coilin is known in Drosophila melanogaster, we identified D. melanogaster CBs using probes for other components that are relatively diagnostic for CBs in vertebrate cells. U85 small CB–specific RNA, U2 small nuclear RNA, the survival of motor neurons protein, and fibrillarin occur together in a nuclear body that is closely associated with the nucleolus. Based on its similarity to CBs in other organisms, we refer to this structure as the D. melanogaster CB. Surprisingly, the D. melanogaster U7 small nuclear RNP resides in a separate nuclear body, which we call the histone locus body (HLB). The HLB is invariably colocalized with the histone gene locus. Thus, canonical CB components are distributed into at least two nuclear bodies in D. melanogaster. The identification of these nuclear bodies now permits a broad range of questions to be asked about CB structure and function in a genetically tractable organism.

Exploring strategies for protein trapping in Drosophila

The use of fluorescent protein tags has had a huge impact on cell biological studies in virtually every experimental system. Incorporation of coding sequence for fluorescent proteins such as green fluorescent protein (GFP) into genes at their endogenous chromosomal position is especially useful for generating GFP-fusion proteins that provide accurate cellular and subcellular expression data. We tested modifications of a transposon-based protein trap screening procedure in Drosophila to optimize the rate of recovering useful protein traps and their analysis. Transposons carrying the GFP-coding sequence flanked by splice acceptor and donor sequences were mobilized, and new insertions that resulted in production of GFP were captured using an automated embryo sorter. Individual stocks were established, GFP expression was analyzed during oogenesis, and insertion sites were determined by sequencing genomic DNA flanking the insertions. The resulting collection includes lines with protein traps in which GFP was spliced into mRNAs and embedded within endogenous proteins or enhancer traps in which GFP expression depended on splicing into transposon-derived RNA. We report a total of 335 genes associated with protein or enhancer traps and a web-accessible database for viewing molecular information and expression data for these genes.

Flytrap, a database documenting a GFP protein-trap insertion screen in Drosophila melanogaster

Flytrap is a web-enabled relational database of transposable element insertions in Drosophila melanogaster. A green fluorescent protein (GFP) artificial exon carried by a transposable P-element is mobilized and inserted into a host gene intron creating a GFP fusion protein. The sequence of the tagged gene is determined by sequencing inverse-PCR products derived from genomic DNA. Flytrap contains two principle data types: micrographs of protein localization and a cellular component ontology, based on rules derived from the Gene Ontology consortium (http://www.geneontology.org), describing protein localization. Flytrap also has links to gene information contained in Flybase (http:// flybase.bio.indiana.edu). The system is designed to accept submissions of micrographs and descriptions from any type of tissue (e.g. wing imaginal disk, ovary) and at any stage of development. Insertion lines can be searched using a number of queries, including Berkeley Drosophila Genome Project (BDGP) numbers and protein localization. In addition, Flytrap provides online order forms linked to each insertion line so that users may request any line generated from this project. Flytrap may be accessed from the homepage at http://flytrap.med. yale.edu.

Drosophila stem cells share a common requirement for the histone H2B ubiquitin protease scrawny.

Stem cells within diverse tissues share the need for a chromatin configuration that promotes self-renewal, yet few chromatin proteins are known to regulate multiple types of stem cells. We describe a Drosophila gene, scrawny (scny), encoding a ubiquitin-specific protease, which is required in germline, epithelial, and intestinal stem cells. Like its yeast relative UBP10, Scrawny deubiquitylates histone H2B and functions in gene silencing. Consistent with previous studies of this conserved pathway of chromatin regulation, scny mutant cells have elevated levels of ubiquitinylated H2B and trimethylated H3K4. Our findings suggest that inhibiting H2B ubiquitylation through scny represents a common mechanism within stem cells that is used to repress the premature expression of key differentiation genes, including Notch target genes.

Searching Chromatin for Stem Cell Identity

Stem cells encapsulate the fundamental problem of metazoan biology in miniature: How do cells establish and maintain their fates? Increasing evidence indicates that stem cell chromatin activates proliferation genes and represses differentiation genes. Understanding how these configurations are stabilized by Polycomb group proteins will advance our understanding of embryonic development, tissue homeostasis, regeneration, aging, and oncogenesis.

Eggs to die for: Cell death during Drosophila oogenesis

Extensive programmed cell death occurs in the female germline of many species ranging from C. elegans to humans. One purpose for germline apoptosis is to remove defective cells unable to develop into fertile eggs. In addition, recent work suggests that the death of specific germline cells may also play a vital role by providing essential nutrients to the surviving oocytes. In Drosophila, the genetic control of germline apoptosis and the proteins that carry out the death sentences are beginning to emerge from studies of female sterile mutations. These studies suggest that the morphological changes that occur during the late stages of Drosophila oogenesis may be initiated and driven by a modified form of programmed cell death. Cell Death and Differentiation (2000) 7, 1071–1074

Systematic Discovery of Rab GTPases with Synaptic Functions in Drosophila

BACKGROUND Neurons require highly specialized intracellular membrane trafficking, especially at synapses. Rab GTPases are considered master regulators of membrane trafficking in all cells, and only very few Rabs have known neuron-specific functions. Here, we present the first systematic characterization of neuronal expression, subcellular localization, and function of Rab GTPases in an organism with a brain. RESULTS We report the surprising discovery that half of all Drosophila Rabs function specifically or predominantly in distinct subsets of neurons in the brain. Furthermore, functional profiling of the GTP/GDP-bound states reveals that these neuronal Rabs are almost exclusively active at synapses and the majority of these synaptic Rabs specifically mark synaptic recycling endosomal compartments. Our profiling strategy is based on Gal4 knockins in large genomic fragments that are additionally designed to generate mutants by ends-out homologous recombination. We generated 36 large genomic targeting vectors and transgenic rab-Gal4 fly strains for 25 rab genes. Proof-of-principle knockout of the synaptic rab27 reveals a sleep phenotype that matches its cell-specific expression. CONCLUSIONS Our findings suggest that up to half of all Drosophila Rabs exert specialized synaptic functions. The tools presented here allow systematic functional studies of these Rabs and provide a method that is applicable to any large gene family in Drosophila.

Changes in rRNA Transcription Influence Proliferation and Cell Fate Within a Stem Cell Lineage

Germline Pol I RNA polymerase I (Pol I)–directed ribosomal RNA (rRNA) transcription has been extensively studied in mammalian cell lines and yeast. However, the functional significance of cell-specific regulation of Pol I transcription within developmental contexts in vivo remains unclear. Zhang et al. (p. 298) characterized a Drosophila Pol I regulatory complex and found that germline stem cells (GSCs) of the ovary exhibited increased levels of rRNA transcription relative to their immediate daughter cells. High levels of rRNA expression promoted GSC proliferation, with attenuation of Pol I activity showing effects during early germ cell differentiation. The RNA polymerase I regulatory complex promotes dynamic regulation of ribosomal RNA synthesis within the Drosophila germline. Ribosome biogenesis drives cell growth and proliferation, but mechanisms that modulate this process within specific lineages remain poorly understood. Here, we identify a Drosophila RNA polymerase I (Pol I) regulatory complex composed of Under-developed (Udd), TAF1B, and a TAF1C-like factor. Disruption of udd or TAF1B results in reduced ovarian germline stem cell (GSC) proliferation. Female GSCs display high levels of ribosomal RNA (rRNA) transcription, and Udd becomes enriched in GSCs relative to their differentiating daughters. Increasing Pol I transcription delays differentiation, whereas reducing rRNA production induces both morphological changes that accompany multicellular cyst formation and specific decreased expression of the bone morphogenetic protein (BMP) pathway component Mad. These findings demonstrate that modulating rRNA synthesis fosters changes in the cell fate, growth, and proliferation of female Drosophila GSCs and their daughters.

New components of the Drosophila fusome suggest it plays novel roles in signaling and transport

The fusome plays an essential role in prefollicular germ cell development within insects such as Drosophila melanogaster. Alpha-spectrin and the adducin-like protein Hu-li tai shao (Hts) are required to maintain fusome integrity, synchronize asymmetric cystocyte mitoses, form interconnected 16-cell germline cysts, and specify the initial cell as the oocyte. By screening a library of protein trap lines, we identified 14 new fusome-enriched proteins, including many associated with its characteristic vesicles. Our studies reveal that fusomes change during development and contain recycling endosomal and lysosomal compartments in females but not males. A significant number of fusome components are dispensable, because genetic disruption of tropomodulin, ferritin-1 heavy chain, or scribble, does not alter fusome structure or female fertility. In contrast, rab11 is required to maintain the germline stem cells, and to maintain the vesicle content of the spectrosome, suggesting that the fusome mediates intercellular signals that depend on the recycling endosome.