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Allan C Spradling

Publications and source records attributed to Allan C Spradling.

15 recordsLinked to original sources
PubMed2006

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.

Animals↗
PubMed2006

Mouse oocytes within germ cell cysts and primordial follicles contain a Balbiani body.

The Balbiani body or mitochondrial cloud is a large distinctive organelle aggregate found in developing oocytes of many species, but its presence in the mouse has been controversial. Using confocal and electron microscopy, we report that a Balbiani body does arise in mouse neonatal germline cysts and oocytes of primordial follicles but disperses as follicles begin to grow. The mouse Balbiani body contains a core of Golgi elements surrounded by mitochondria and associated endoplasmic reticulum. Because of their stage specificity and perinuclear rather than spherical distribution, these clustered Balbiani body mitochondria may have been missed previously. The Balbiani body also contains Trailer hitch, a widely conserved member of a protein complex that associates with endoplasmic reticulum/Golgi-like vesicles and transports specific RNAs during Drosophila oogenesis. Our results provide evidence that mouse oocytes develop using molecular and developmental mechanisms widely conserved throughout the animal kingdom.

Amino Acid Sequence↗
PubMed2006

Breaking out of the mold: diversity within adult stem cells and their niches.

During the past several years, it has become increasingly possible to study adult stem cells in their native territories within tissues. These studies have provided new evidence for the existence of stem cells in the breast, muscle, lung and kidney and have led to a deeper understanding of the best-known stem cells in Drosophila and mice. Tissue stem cells are turning out to be diverse, with varying division rates, lineage lengths, and mechanisms of regulation. In addition, stem cells are now known to engage in a wide variety of interactions with neighboring cells and extracellular matrices, and to respond to various neural and hormonal signals. Stem cell niches are also diverse, sometimes harboring multiple stem cell types. Internally, a stem cell's chromatin and cytoskeletal organization play key roles. Understanding how stem cells and their progeny are controlled will illuminate fundamental biological mechanisms that govern the construction and maintenance of tissues within metazoan animals.

Aging↗
PubMed2006

Milton controls the early acquisition of mitochondria by Drosophila oocytes.

Mitochondria in many species enter the young oocyte en mass from interconnected germ cells to generate the large aggregate known as the Balbiani body. Organelles and germ plasm components frequently associate with this structure. Balbiani body mitochondria are thought to populate the germ line, ensuring that their genomes will be inherited preferentially. We find that milton, a gene whose product was previously shown to associate with Kinesin and to mediate axonal transport of mitochondria, is needed to form a normal Balbiani body. In addition, germ cells mutant for some milton or Kinesin heavy chain (Khc) alleles transport mitochondria to the oocyte prematurely and excessively, without disturbing Balbiani body-associated components. Our observations show that the oocyte acquires the majority of its mitochondria by competitive bidirectional transport along microtubules mediated by the Milton adaptor. These experiments provide a molecular explanation for Balbiani body formation and, surprisingly, show that viable fertile offspring can be obtained from eggs in which the normal program of mitochondrial acquisition has been severely perturbed.

Animals↗
PubMed2006

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.

Animals↗
PubMed2006

The Drosophila P68 RNA helicase regulates transcriptional deactivation by promoting RNA release from chromatin.

Terminating a gene's activity requires that pre-existing transcripts be matured or destroyed and that the local chromatin structure be returned to an inactive configuration. Here we show that the Drosophila homolog of the mammalian P68 RNA helicase plays a novel role in RNA export and gene deactivation. p68 mutations phenotypically resemble mutations in small bristles (sbr), the Drosophila homolog of the human mRNA export factor NXF1. Full-length hsp70 mRNA accumulates in the nucleus near its sites of transcription following heat shock of p68 homozygotes, and hsp70 gene shutdown is delayed. Unstressed mutant larvae show similar defects in transcript accumulation and gene repression at diverse loci, and we find that p68 mutations are allelic to Lighten-up, a known suppressor of position effect variegation. Our observations reveal a strong connection between transcript clearance and gene repression. P68 may be needed to rapidly remove transcripts from a gene before its activity can be shut down and its chromatin reset to an inactive state.

Blotting, Northern↗
PubMed2005

Drosophila poly(ADP-ribose) glycohydrolase mediates chromatin structure and SIR2-dependent silencing.

Protein ADP ribosylation catalyzed by cellular poly(ADP-ribose) polymerases (PARPs) and tankyrases modulates chromatin structure, telomere elongation, DNA repair, and the transcription of genes involved in stress resistance, hormone responses, and immunity. Using Drosophila genetic tools, we characterize the expression and function of poly(ADP-ribose) glycohydrolase (PARG), the primary enzyme responsible for degrading protein-bound ADP-ribose moieties. Strongly increasing or decreasing PARG levels mimics the effects of Parp mutation, supporting PARG's postulated roles in vivo both in removing ADP-ribose adducts and in facilitating multiple activity cycles by individual PARP molecules. PARP is largely absent from euchromatin in PARG mutants, but accumulates in large nuclear bodies that may be involved in protein recycling. Reducing the level of either PARG or the silencing protein SIR2 weakens copia transcriptional repression. In the absence of PARG, SIR2 is mislocalized and hypermodified. We propose that PARP and PARG promote chromatin silencing at least in part by regulating the localization and function of SIR2 and possibly other nuclear proteins.

Adenosine Diphosphate Ribose↗
PubMed2005

The expression profile of purified Drosophila germline stem cells.

We developed a method to highly purify germline stem cells (GSCs) from the Drosophila ovary, one of the best understood types of adult stem cell. GSCs express variant isoforms of general transcriptional components, translation initiation factors, and several variant ribosomal proteins, including RpL22, a protein enriched in several mammalian stem cells. These novel isoforms may help regulate stem cell gene expression because a reversion assay indicated that at least four were specific for GSCs. By comparative analysis, we identify additional genes enriched in GSCs, including Psc, the Drosophila homolog of the Bmi-1 Polycomb group gene, as well as genes that may delay cytokinesis in pre-meiotic germ cells. By comparing GSCs arrested by BMP over-expression and bam mutation, we hypothesize that mRNA utilization is modulated in differentiating GSC daughters. Our findings suggest that Drosophila and mammalian stem cells utilize at least two regulatory mechanisms in common.

Animals↗
PubMed2005

The Drosophila ovarian and testis stem cell niches: similar somatic stem cells and signals.

The stem cell niches at the apex of Drosophila ovaries and testes have been viewed as distinct in two major respects. While both contain germline stem cells, the testis niche also contains "cyst progenitor" stem cells, which divide to produce somatic cells that encase developing germ cells. Moreover, while both niches utilize BMP signaling, the testis niche requires a key JAK/STAT signal. We now show, by lineage marking, that the ovarian niche also contains a second type of stem cell. These "escort stem cells" morphologically resemble testis cyst progenitor cells and their daughters encase developing cysts before undergoing apoptosis at the time of follicle formation. In addition, we show that JAK/STAT signaling also plays a critical role in ovarian niche function, and acts within escort cells. These observations reveal striking similarities in the stem cell niches of male and female gonads, and suggest that they are largely governed by common mechanisms.

Animals↗
PubMed2004

Alpha-endosulfine, a potential regulator of insulin secretion, is required for adult tissue growth control in Drosophila.

Alpha-endosulfine is a small protein that has been proposed to regulate ion channel activity and insulin secretion, but in vivo studies have been lacking. We have previously established the Drosophila ovary as a model system in which to study adult tissue growth regulation, and demonstrated a role of the insulin pathway in the proliferative response of ovarian cells to nutritional changes. Here, we find that the Drosophila alpha-endosulfine (dendos) gene, whose protein is expressed in germline and somatic cells of the ovary, as well as in the brain and certain regions of the intestine, is also required for this response. This requirement is non-cell autonomous, which is consistent with a role of dendos in secretion of Drosophila insulin-like peptides (DILPs), required for the proliferative response to nutritional changes. Our results show that dendos is also required for a distinct process in oogenesis, namely, the osmotic regulation of stage 14 oocytes, and that this requirement is cell autonomous, consistent with the role in ion channel regulation suggested by studies of the mammalian homologues.

Amino Acid Sequence↗
PubMed2004

The BDGP gene disruption project: single transposon insertions associated with 40% of Drosophila genes.

The Berkeley Drosophila Genome Project (BDGP) strives to disrupt each Drosophila gene by the insertion of a single transposable element. As part of this effort, transposons in >30,000 fly strains were localized and analyzed relative to predicted Drosophila gene structures. Approximately 6300 lines that maximize genomic coverage were selected to be sent to the Bloomington Stock Center for public distribution, bringing the size of the BDGP gene disruption collection to 7140 lines. It now includes individual lines predicted to disrupt 5362 of the 13,666 currently annotated Drosophila genes (39%). Other lines contain an insertion at least 2 kb from others in the collection and likely mutate additional incompletely annotated or uncharacterized genes and chromosomal regulatory elements. The remaining strains contain insertions likely to disrupt alternative gene promoters or to allow gene misexpression. The expanded BDGP gene disruption collection provides a public resource that will facilitate the application of Drosophila genetics to diverse biological problems. Finally, the project reveals new insight into how transposons interact with a eukaryotic genome and helps define optimal strategies for using insertional mutagenesis as a genomic tool.

Animals↗
PubMed2003

A Balbiani body and the fusome mediate mitochondrial inheritance during Drosophila oogenesis.

Maternally inherited mitochondria and other cytoplasmic organelles play essential roles supporting the development of early embryos and their germ cells. Using methods that resolve individual organelles, we studied the origin of oocyte and germ plasm-associated mitochondria during Drosophila oogenesis. Mitochondria partition equally on the spindle during germline stem cell and cystocyte divisions. Subsequently, a fraction of cyst mitochondria and Golgi vesicles associates with the fusome, moves through the ring canals, and enters the oocyte in a large mass that resembles the Balbiani bodies of Xenopus, humans and diverse other species. Some mRNAs, including oskar RNA, specifically associate with the oocyte fusome and a region of the Balbiani body prior to becoming localized. Balbiani body development requires an intact fusome and microtubule cytoskeleton as it is blocked by mutations in hu-li tai shao, while egalitarian mutant follicles accumulate a large mitochondrial aggregate in all 16 cyst cells. Initially, the Balbiani body supplies virtually all the mitochondria of the oocyte, including those used to form germ plasm, because the oocyte ring canals specifically block inward mitochondrial transport until the time of nurse cell dumping. Our findings reveal new similarities between oogenesis in Drosophila and vertebrates, and support our hypothesis that developing oocytes contain specific mechanisms to ensure that germ plasm is endowed with highly functional organelles.

Animals↗
PubMed2002

The Drosophila heterochromatic gene encoding poly(ADP-ribose) polymerase (PARP) is required to modulate chromatin structure during development.

Poly(ADP-ribose) polymerase (PARP) is a major NAD-dependent modifying enzyme that mediates important steps in DNA repair, transcription, and apoptosis, but its role during development is poorly understood. We found that a single Drosophila Parp gene spans more than 150 kb of transposon-rich centromeric heterochromatin and produces several differentially spliced transcripts, including a novel isoform, PARP-e, predicted to encode a protein lacking enzymatic activity. An insertion mutation near the upstream promoter for Parp-e disrupts all Parp expression. Heterochromatic but not euchromatic sequences become hypersensitive to micrococcal nuclease, nucleoli fail to form, and transcript levels of the copia retrotransposon are elevated more than 50-fold; the variegated expression of certain transgenes is dominantly enhanced. Larval lethality can be rescued and PARP activity restored by expressing a cDNA encoding PARP-e. We propose that PARP-e autoregulates Parp transcription by influencing the chromatin structure of its heterochromatic environment. Our results indicate that Parp plays a fundamental role organizing the structure of Drosophila chromatin.

Animals↗