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In JoVE (1)
Other Publications (6)
Articles by Bruce H. Reed in JoVE
JoVE General
The Preparation of Drosophila Embryos for Live-Imaging Using the Hanging Drop Protocol
Department of Biology, University of Waterloo
A simple, inexpensive, and effective method of preparing Drosophila embryos for live-imaging analysis is presented. Our protocol provides humidity and gas exchange and does not compress the Drosophila embryo. This method is suitable for GFP-based live imaging of Drosophila embryos using a stereomicroscope or upright compound microscope.
Other articles by Bruce H. Reed on PubMed
The Anaphase Promoting Complex/cyclosome is Required During Development for Modified Cell Cycles
Animals and plants use modified cell cycles to achieve particular developmental strategies. In one common example, most animals and plants have tissues in which the cells become polyploid or polytene by means of an S-G cycle, but the mechanism by which mitosis is inhibited in the endo cycle is not understood. The Drosophila morula (mr) gene regulates variant cell cycles, because in addition to disrupting the archetypal cycle (G1-S-G2-M), mr mutations affect the rapid embryonic (S-M) divisions as well as the endo cycle (S-G) that produces polyploid cells. In dividing cells mr mutations cause a metaphase arrest, and endo cycling nurse cells inappropriately reenter mitosis in mr mutants. We show mr encodes the APC2 subunit of the anaphase promoting complex/cyclosome. This finding demonstrates that anaphase promoting complex/cyclosome is required not only in proliferating cells but also to block mitosis in some endo cycles. The mr mutants further indicate that transient mitotic functions in endo cycles change chromosome morphology from polytene to polyploid.
Regulation of Maternal Transcript Destabilization During Egg Activation in Drosophila
In animals, the transfer of developmental control from maternal RNAs and proteins to zygotically derived products occurs at the midblastula transition. This is accompanied by the destabilization of a subset of maternal transcripts. In Drosophila, maternal transcript destabilization occurs in the absence of fertilization and requires specific cis-acting instability elements. We show here that egg activation is necessary and sufficient to trigger transcript destabilization. We have identified 13 maternal-effect lethal loci that, when mutated, result in failure of maternal transcript degradation. All mutants identified are defective in one or more additional processes associated with egg activation. These include vitelline membrane reorganization, cortical microtubule depolymerization, translation of maternal mRNA, completion of meiosis, and chromosome condensation (the S-to-M transition) after meiosis. The least pleiotropic class of transcript destabilization mutants consists of three genes: pan gu, plutonium, and giant nuclei. These three genes regulate the S-to-M transition at the end of meiosis and are thought to be required for the maintenance of cyclin-dependent kinase (CDK) activity during this cell cycle transition. Consistent with a possible functional connection between this S-to-M transition and transcript destabilization, we show that in vitro-activated eggs, which exhibit aberrant postmeiotic chromosome condensation, fail to initiate transcript degradation. Several genetic tests exclude the possibility that reduction of CDK/cyclin complex activity per se is responsible for the failure to trigger transcript destabilization in these mutants. We propose that the trigger for transcript destabilization occurs coincidently with the S-to-M transition at the end of meiosis and that pan gu, plutonium, and giant nuclei regulate maternal transcript destabilization independent of their role in cell cycle regulation.
Integrin-dependent Apposition of Drosophila Extraembryonic Membranes Promotes Morphogenesis and Prevents Anoikis
Two extraembryonic tissues form early in Drosophila development. One, the amnioserosa, has been implicated in the morphogenetic processes of germ band retraction and dorsal closure. The developmental role of the other, the yolk sac, is obscure.
Dose-sensitive Autosomal Modifiers Identify Candidate Genes for Tissue Autonomous and Tissue Nonautonomous Regulation by the Drosophila Nuclear Zinc-finger Protein, Hindsight
The nuclear zinc-finger protein encoded by the hindsight (hnt) locus regulates several cellular processes in Drosophila epithelia, including the Jun N-terminal kinase (JNK) signaling pathway and actin polymerization. Defects in these molecular pathways may underlie the abnormal cellular interactions, loss of epithelial integrity, and apoptosis that occurs in hnt mutants, in turn causing failure of morphogenetic processes such as germ band retraction and dorsal closure in the embryo. To define the genetic pathways regulated by hnt, 124 deficiencies on the second and third chromosomes and 14 duplications on the second chromosome were assayed for dose-sensitive modification of a temperature-sensitive rough eye phenotype caused by the viable allele, hntpeb; 29 interacting regions were identified. Subsequently, 438 P-element-induced lethal mutations mapping to these regions and 12 candidate genes were tested for genetic interaction, leading to identification of 63 dominant modifier loci. A subset of the identified mutants also dominantly modify hnt308-induced embryonic lethality and thus represent general rather than tissue-specific interactors. General interactors include loci encoding transcription factors, actin-binding proteins, signal transduction proteins, and components of the extracellular matrix. Expression of several interactors was assessed in hnt mutant tissue. Five genes--apontic (apt), Delta (Dl), decapentaplegic (dpp), karst (kst), and puckered (puc)--are regulated tissue autonomously and, thus, may be direct transcriptional targets of HNT. Three of these genes--apt, Dl, and dpp--are also regulated nonautonomously in adjacent non-HNT-expressing tissues. The expression of several additional interactors--viking (vkg), Cg25, and laminin-alpha (LanA)-is affected only in a nonautonomous manner.
Autophagy Promotes Caspase-dependent Cell Death During Drosophila Development
The relationship between autophagic cell death and apoptosis is a poorly understood aspect of programmed cell death (PCD). We have examined this relationship by studying the elimination of an extra-embryonic tissue, known as the amnioserosa (AS), during Drosophila development. The AS becomes autophagic during the final stages of embryogenesis; ultimately, however, the elimination of the AS involves caspase-dependent nuclear fragmentation, tissue dissociation and engulfment by phagocytic macrophages. Mutants that are defective in the activation or execution of caspase-dependent PCD fail to degrade and eliminate the AS but show no abatement in AS autophagy. Sustained autophagy does not, therefore, necessarily result in cell death. Surprisingly, the downregulation of autophagy also results in a persistent AS phenotype and reduced cell death. Conversely, upregulation of autophagy results in caspase-dependent premature AS dissociation. These observations are consistent with the interpretation that autophagy is a prerequisite for caspase-dependent cell death in the AS.
Autophagy Can Promote but is Not Required for Epithelial Cell Extrusion in the Amnioserosa of the Drosophila Embryo
During Drosophila embryogenesis the majority of the extra-embryonic epithelium known as the amnioserosa (AS) undergoes programmed cell death (PCD) following the completion of the morphogenetic process of dorsal closure. Approximately ten percent of AS cells, however, are eliminated during dorsal closure by extrusion from the epithelium. Using biosensors that report autophagy and caspase activity in vivo, we demonstrate that AS cell extrusion occurs in the context of elevated autophagy and caspase activation. Furthermore, we evaluate AS extrusion rates, autophagy, and caspase activation in embryos in which caspase activity or autophagy are altered by genetic manipulation. This includes using the GAL4/UAS system to drive expression of p35, reaper, dINR (ACT) and Atg1 in the AS; we also analyze embryos lacking both maternal and zygotic expression of Atg1. Based on our results we suggest that autophagy can promote, but is not required for, epithelial extrusion and caspase activation in the amnioserosa.
