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Poster Full Abstracts - Cell Death
Poster board number is above title. The first author is the presenter
212
298A
A screening for autophagic genes in
Drosophila melanogaster
.
Ahrum Jin
1,2
, Joonho Choe
1
, Thomas Neufeld
2
. 1) Department of Biological Sciences,
Korea Advanced Institute of Science and Technology, Daejeon, South Korea; 2) Department of Genetics, Cell Biology and Development, University of
Minnesota, Minneapolis, MN.
Autophagy is known as a ‘self-eating mechanism’ which is conserved from yeasts to mammals. When autophagy is induced, cytoplasmic components are
sequestered in double-membrane vesicles called autophagosomes and degraded after fusing with lysosomes. Autophagy is involved in various cellular
processes such as the starvation response, immune response, development, maintenance of cellular homeostasis (protein turnover and removal of damaged
organelles), cell survival and death and the oxidative stress response. During the process of autophagy, the ubiquitin-related protein Atg8/LC3 is cleaved and
conjugated with phosphatidylethanolamine. This conversion is reflected in Western blot by the appearance of two bands (LC3-I and LC3-II) that change in
intensity as the rate of autophagy increases. We used this LC3 conversion assay in an RNAi screen in S2 cells to identify novel genes required for autophagy
regulation. From ~1500 genes screened, we obtained ~100 hits with an altered LC3-I/II ratio. We have performed secondary screening in vivo using
Drosophila RNAi fly lines, and identified 17 genes that affect autophagy activity in the larval fat body. We will describe the characterization of these genes
in autophagy regulation.
299B
Regulation of neural stem cell fate in Drosophila cell death mutants.
RICHA ARYA, Ying Tan, Hsiao-Yu Huang, Megumu Yamada-Mabuchi, Kristin
White. CBRC, MGH/HARVARD, CHARLESTOWN, MA.
In mammals and non-mammalian systems, many neural stem cells (neuroblasts or NBs) are eliminated by apoptosis once they generate a stereotyped set of
progeny cells. However the mechanism by which specific subset of NBs is selected for death is largely unknown. We are studying the spatial and temporal
regulation of developmental apoptosis, using Drosophila NBs as a model. Our genetic studies showed that rpr and grim are required for normal apoptosis of
NBs in the abdominal region of the ventral nerve cord. In rpr-grim mutants NBs survive and divide to produce large numbers of neuronal progeny resulting
in neural hypertrophy. Furthermore we identified a cis-regulatory region (NBRR) between rpr and grim that controls the expression of these genes in the
doomed NBs. Whole embryo chip-chip and chip-seq data and in silico analysis strongly suggest the presence of many transcription factor binding sites
within the NBRR. Based on these binding analyses we selected a 5kb region (enhancer 1or enh1) to generate GFP reporter transgenic flies, to ask how this
regulatory region activates apoptosis in doomed cells. We analyzed enh1-GFP during development and found that it is expressed in a subset of abdominal
NBs in the late embryo, likely to be those that will die. In the larvae, the three abdominal NBs that normally survive in each hemisegment start expressing
low levels of enh1-GFP in early L2. This expression peaks at mid-third instar, when these NBs die. Surprisingly, some thoracic NBs that normally do not
undergo apoptosis also express enh1-GFP in the larvae, indicating either a missing repressor or the presence of survival signal in these cells. To identify the
upstream regulators of the NBRR we performed an RNAi screen for factors that affect the expression of enh1-GFP. In a preliminary screen we identified
three transcription factors that regulate the expression of enh1-GFP in abdominal neuroblasts. Further studies will describe how upstream pathways are
integrated to regulate the expression of apoptotic effectors to eliminate individual cells during development.
300C
Investigating a role of dHb9-positive motor neurons in eclosion behavior.
David S Conway, Soumya Banerjee, Marcus Toral, Alexander Busch, Joyce
Fernandes. Zoology, Miami Univ, Oxford, OH.
The nervous system of is remodeling extensively during metamorphosis from a larval framework to develop adult specific neural circuits which drive adult
specific behaviors. One of the major changes that takes place is the shift of locomotor control from the entire body (crawling larvae) to the thorax (walking
and flying adult). The adult abdominal segment is still essential for carrying out other adult specific behaviors like mating and eclosion. The eclosion
behavior is mediated by persistent muscle fibers (PMFs), which are retained from the larval stage into the adult stage, and are degraded shortly after the
eclosion behavior is complete. We specifically study PMFs 12 and 13, in segments A1 and A2, which have been implicated in eclosion behavior (Kimura
and Truman, 1990). These PMFs are innervated by motor neurons that express the transcription factor dHb9, as shown by previous work in our lab. We
intend to test the hypothesis that dHb9+ motor neurons are essential for bringing about the eclosion behavior. Apoptosis will be induced or prevented in
dHb9+ motor neurons during the pupal stage using the targeted expression of the genes Reaper and p35 respectively. The onset of the expression of these
genes will be manipulated through the use of the Gal4/Gal80 temporal control system. The resulting adults will be examined and classified according to their
eclosion behavior, normal eclosion, late eclosion or absence of eclosion. For the targeted reaper expression, we will examine the presence or absence of
dHb9 driven GFP expression in the ventral ganglion and the body wall to determine the extent to which dHb9 cells are eliminated. A preliminary trial of this
experiment showed that flies in which Reaper was activated failed to eclose 25% of the time, as compared to control flies failing to eclose 14% of the time
(n=60 animals). Current work is aimed at completing this study and to examine any co-relations of the three categories of eclosion to the extent of
eliminating dHb9-positive neurons.
301A
A Genetic Screen to Identify Cell Death Regulators In the
Drosophila
Ovary.
Tatevik Keshishyan, Jeremy Nguyen, Olivia Rudnicki, Michelle Gammill,
Jemma Taipan, Sarah Durrin, Luz Ceballos, Aileen Leung, Elizabeth Tanner, Jeanne Peterson, Kim McCall. Boston University, Boston, MA.
Cell death is an essential mechanism for the survival and development of many organisms. Excessive cell death can lead to degenerative disorders such as
Parkinson’s disease, while too little cell death can result in diseases such as cancer.
Drosophila
oogenesis is an excellent model system for studying
programmed cell death (PCD). One type of programmed cell death occurs during mid-stage oogenesis in response to nutrient deprivation. Cell death also
occurs during late-stage oogenesis when nurse cells, which contain contents essential for development of an embryo, dump their cytoplasmic contents into a
developing oocyte and are then eliminated through PCD. We performed an unbiased mis-expression screen based on EY P element lines to identify
regulators of cell death. Over-expression in the germline was accomplished by crossing to nanos-GAL4. Of the 1200 lines we have screened, 27 fly lines
consistently showed abnormal cell death phenotypes. These abnormal phenotypes included persisting nurse cell (NC) nuclei in late oogenesis, death-resistant
NCs in mid-oogenesis, and excessive degeneration of egg chambers. The affected genes function in processes such as DNA/RNA binding, cytoskeleton
arrangement, cell signaling, and mitochondrial events. To further analyze the causes of the abnormal phenotypes, staining has been performed to determine