The 2nd International Insect Hormone Workshop Sunday, July 12, 2015 16:00 – 20:00 20:00 Registration Dinner Monday, July 13, 2015 7:45 – 8:45 8:45 – 9:00 Breakfast Welcome: Kirst King-­‐Jones and Kim Rewitz Session I: Neuroendocrine Regulation of Hormonal Processes (Chair: Kim Rewitz) 9:00 – 9:25 9:25 – 9:50 9:50 – 10:15 10:15 – 10:40 10:40 – 11:10 Julien Colombani, Ditte S. Andersen, Laura Boulan, Emilie Boone, Nuria Romero, Michael Texada and Pierre Léopold. Growth coordination mechanisms. Yuko Shimada-­‐Niwa, Eisuke Imura and Ryusuke Niwa. The mapping and functional analysis of the prothoracic gland innervating neurons controlling ecdysteroid biosynthesis in Drosophila melanogaster: SE0PG neurons and beyond. The Drosophila peptidergic connectome. Michael J. Texada and James W. Truman. Derya Deveci, Nuria M. Romero, Francisco A. Martin, Pierre Léopold. A genetic screen to uncover new signals controlling Drosophila juvenile-­‐maturation transition. Coffee break Session II: Regulation of Ecdysone Biosynthesis (Chair: Ryusuke Niwa) 11:10 – 11:35 11:35 – 12:00 12:30 Yuya Ohhara and Naoki Yamanaka. Endoreplication in the prothoracic gland underlies developmental checkpoint mechanism. Qiuxiang Ou, Brian Phelps, Arash Bashirullah and Kirst King-­‐Jones. Discovery of genes essential for heme biosynthesis in the Drosophila ring gland. Lunch I Session II (continued): Regulation of Ecdysone Biosynthesis (Chair: Ryusuke Niwa) 14:30 – 14:55 14:55 – 15:20 15:20 – 15:45 15:45 – 16:10 16:10 – 16:35 16:35 – 17:00 20:00 E. Thomas Danielsen, Morten E. Moller, Naoki Yamanaka, Kirst King-­‐Jones, Michael B. O’Connor and Kim F. Rewitz. A genome-­‐wide in vivo RNAi screen in Drosophila identifies regulators of cholesterol-­‐dependent steroid production. Jie Zeng, Qiuxiang Ou and Kirst King-­‐Jones. Snail is a prothoracic gland-­‐specific transcription factor required for ecdysone production in Drosophila larvae. Skarlatos G. Dedos, Alexandros Alexandratos, Panagiotis Moulos, Ioannis Nellas and Konstantinos Mavridis. Plasma membrane receptors in prothoracic gland cells of Bombyx mori. Coffee break Jennifer Gerlach, Dominik Steiger and Peter Gallant. The growth regulator Myc affects ecdysone and developmental transitions. Tatsuya Komura-­‐Kawa , Outa Uryu , Keiko Hirota, Yuko Shimada-­‐Niwa, Rieko Yamauchi, MaryJane Shimell, Tetsuro Shinoda, Akiyoshi Fukamizu, Michael B. O’Connor and Ryusuke Niwa. Ouija board is the zinc finger transcription factor controlling ecdysteroid biosynthesis through specific regulation of spookier in Drosophila. Dinner Tuesday, July 14, 2015 8:00 – 9:00 Breakfast Session III: Nutrients and Metabolism (Chair: Kirst King-­‐Jones) 9:00 – 9:25 9:25 – 9:50 9:50 – 10:15 10:15 – 10:40 Marko Brankatschk, Theresia Gutmann, Uenal Coskun and Suzanne Eaton. The viable temperature range of Drosophila is controlled by diet. Takashi Koyama and Christen K. Mirth. Nutritional control of body size through FoxO-­‐
Ultraspiracle mediated ecdysone biosynthesis. Martina Gáliková, Max Diesner, Peter Klepsatel, Philip Hehlert, Yanjun Xu, Iris Bickmeyer, Reinhard Predel and Ronald P. Kühnlein. Functions of adipokinetic hormone and adipokinetic hormone precursor related peptide in Drosophila melanogaster. Elizabeth Rideout, Abhishek Ghosh and Savraj Grewal. Nutrition-­‐ and sex-­‐dependent regulation of insulin signaling and growth in Drosophila. II 10:40 – 11:10 11:10 – 11:35 11:35 – 12:00 12:00 – 12:25 12:30 14:30 – 16:30 20:00 Coffee break A. Scopelliti, C. Bauer, J.B. Cordero, O.J. Sansom and M. Vidal. Drosophila enteroendocrine cells are involved in nutrient sensing and regulate systemic metabolism through bursicon secretion. Zhangwu Zhao. Regulation of Drosophila circadian rhythms by miRNA let-­‐7 is mediated by a regulatory cycle. Marion Hartl, Dafni Hadjieconomou, Sophie Austin and Irene Miguel-­‐Aliaga. Control of feeding behaviour and metabolic physiology by peptidergic enteric neurons. Lunch Poster Session Dinner Wednesday, July 15, 2015 8:00 – 9:00 Breakfast Session IV: Juvenile Hormone Signaling (Chair: Marek Jindra) 9:00 – 9:25 9:25 – 9:50 9:50 – 10:15 10:15 – 10:45 10:45 – 11:10 11:10 – 11:35 Sheng Li. Recent advances in juvenile hormone signaling in Drosophila. Pengcheng Liu, Reyhaneh Ojani and Jinsong Zhu. Protein phosphorylation is involved in juvenile hormone signal transduction. Beata Greb-­‐Markiewicz, Natalia Banaś, Daria Sadowska, Jakub Godlewski, Mirosław Zarębski and Andrzej Ożyhar. Juvenile Hormone dependent transport of the Drosophila Germ cell expressed (GCE) protein. Coffee break Takaaki Daimon, Miwa Uchibori and Tetsuro Shinoda. Dispensable roles for juvenile hormones in holometabolous lifecycle: insights from knockout Bombyx. Rebecca F. Spokony, Robert Arthur, Christopher D. Brown, Alec Victorsen, Jennifer R. Moran, Matthew Kirkey, Jeffrey Gersh and Kevin P. White. Genome-­‐wide mapping of hormone-­‐sensitive Methoprene-­‐tolerant binding sites Drosophila melanogaster. III 11:35 – 12:00 12:00 – 12:25 12:30 Qiangqiang Jia and Sheng Li. Regulation of fat body cell dissociation by JH and 20E via regulating expression of Mmps. Marek Jindra, Pavel Jedlicka and Robert Hanus. Zooming in on the JH receptor: Stereoisomer selectivity for natural and synthetic ligands. Lunch Afternoon & Evening off (Dinner on your own) Thursday, July 16, 2015 8:00 – 9:00 Breakfast Session V. Hormonal Regulation of Organ Growth/Remodeling (Chair: James Truman) 9:00 – 9:25 9:25 – 9:50 9:50 – 10:15 10:15 – 10:45 10:45 – 11:10 Andrew J. Andres, Kathryn M. Lantz, Marwa Al-­‐Karawi, Pawel Parafianowicz and Benjamin F. B. Costintino. Using the Drosophila salivary gland to model exocrine secretion. James W. Truman, Susana Tae and the FlyLight Imaging Project. Hormones and the cellular morphogenesis of the Drosophila nervous system. Yuya Kaieda, Ryota Masuda and Hajime Ono. Expression of a glue protein, Salivary gland secretion 3, is triggered by steroid hormone independently from developmental timeline in Drosophila melanogaster. Coffee break Leire Herboso, Marisa M. Oliveira, Enric Ureña, Ana Talamillo, Coralia Pérez, Monika González, David Martín, James D. Sutherland, Alexander W. Shingleton, Christen K. Mirth and Rosa Barrio. Contribution of ecdysone to imaginal discs growth. PLUS: Introduction to COST BM1307 PROTEOSTASIS (Rosa Barrio) 11:10 – 11:35 11:35 – 12:00 12:30 Natalie A. Dye and Suzanne Eaton. Ecdysone directly stimulates growth of the Drosophila wing. Lynn M. Riddiford, Aljoscha Nern and James W. Truman. Juvenile hormone and development of the optic lobe in Drosophila. Lunch IV Session VI: Developmental Timing and Metamorphosis (Chair: Michael O’Connor) 14:30 – 14:55 14:55 – 15:20 15:20 – 15:45 15:45 – 16:10 16:10 – 16:35 16:35 – 17:00 17:00 – 17:25 20:00 Jacob S. Jaszczak, Jacob B. Wolpe, Anh Q. Dao and Adrian Halme. Coordination of growth and development during regeneration. Enric Ureña, Xavier Franch-­‐Marro and David Martin. Genetic interactions between E93, Krüppel homolog-­‐1 and Broad-­‐Complex transcription factors underlie the formation of the holometabolous pupa. Ken-­‐ichi Hironaka and Yoshihiro Morishita. Optimal growth schedule of holometabolous insects. Coffee break Hitoshi Ueda, Haruka Nishida, Abdel-­‐Rahman Sultan, Kazutaka Akagi, Takumi Nakayama and Moustafa Sarhan. Unstable character of transcriptional repressor Blimp-­‐1 contributes to accurate time measuring system for pupation timing in Drosophila melanogaster. Silvia Chafino, Enric Ureña, Elena Casacuberta, David Martin and Xavier Franch-­‐Marro. Attainment of critical weight is necessary for E93 up-­‐regulation in Tribolium castaneum. Xianyu Lin, Na Yu and Guy Smagghe. RNAi of insulin pathway genes in larval stages of Tribolium castaneum demonstrates regulation in feeding and metamorphosis. Dinner Friday, July 17, 2015 8:00 – 9:00 Breakfast Session VII: Insulin Signaling (Chair: Pierre Leopold) 9:00 – 9:25 Naoki Okamoto and Takashi Nishimura. Signaling relay and feedback mechanisms control the nutrient dependent production of insulin-­‐like peptides in Drosophila. 9:25 – 9:50 Hiroko Sano, Akira Nakamura, Michael J. Texada, James W. Truman, Hiroshi Ishimoto, Azusa Kamikouchi, Yutaka Nibu, Kazuhiko Kume, Takanori Ida and Masayasu Kojima. The nutrient-­‐responsive hormone CCHamide-­‐2 controls growth by regulating insulin-­‐like peptides in the brain of Drosophila melanogaster. 9:50 – 10:15 Hua Yan, Comzit Opachaloemphan, Giacomo Mancini, Steven Shen, Roberto Bonasio, Jürgen Liebig, Shelley Berger, Claude Desplan and Danny Reinberg. Regulation of longevity in castes of ants. V 10:15-­‐10:30 10:30 – 11:00 11:00 – 11:25 11:25 – 11:50 11:50 – 12:15 12:15 – 12:40 12:45 Neha Agrawal, Renald Delanoue, Alessandra Mauri and Pierre Léopold. The Drosophila TNF Eiger is an adipokine that mediates nutrient response. Coffee break Jan Provaznik, Milena Damulewicz, Martin Pivarci, Ivan Fiala, Joanna Kotwica-­‐Rolinska, Hanka Vaneckova and David Dolezel. Insulin signaling cascade in linden bug, Pyrrhocoris apterus (Heteroptera). Jason Clements, Kurt Buhler, Sofie De Groef, Harry Heimberg and Patrick Callaerts. The retinal determination gene network is required for proper development and function of insulin-­‐producing cells. Kurt Buhler, Jason Clements, Korneel Hens, Magali de Bruyn, Yiyun Chen, Feng Wang, Rui Chen and Patrick Callaerts. Ecdysone regulates insulin signaling in the insulin-­‐
producing cells of Drosophila to determine normal growth and developmental timing. Kiyoshi Hiruma and Yu Kaneko. Pupal commitment of a single-­‐celled Verson’s gland is induced in a two-­‐step process by the insulin and TOR/Akt signals, which occurs gradually, not on an all-­‐or-­‐none basis. Lunch Session VIII. Hormones and Reproduction (Chair: Alexander Raikhel) 15:00 – 15:25 15:25 – 15:50 15:50 – 16:15 Sourav Roy, Tusar T. Saha, Lisa K. Johnson, Bo Zhao, Jisu Ha and Alexander S. Raikhel. Regulation of gene expression patterns in mosquito reproduction. Tomotsune Ameku and Ryusuke Niwa. Mating-­‐induced increase in female germline stem cells requires ovarian ecdysteroid biosynthesis and neuronal sex peptide signaling in Drosophila melanogaster. Alexander S. Raikhel, Yuan Hou, Xue-­‐Li Wang, Tusar T. Saha, Sourav Roy, Bo Zhao and Zhen Zou. Critical roles of juvenile hormone receptor Methoprene-­‐tolerant and ecdysone receptor in temporal coordination of mosquito metabolism. Closing remarks 16:30 – 17:30 Business meeting 19:00 Karlson lecture (Michael B. O’Connor) 20:00 Dinner/banquet VI Saturday, July 18, 2015 Safe travels… VII ORAL PRESENTATIONS: ABSTRACTS Growth coordination mechanisms
Julien Colombani (1-3), Ditte S. Andersen (1-3), Laura Boulan (1-3), Emilie Boone (13), Nuria Romero (1-3), Michael Texada (4) and Pierre Leopold (1-3)
(1) University of Nice-Sophia Antipolis, Institute of Biology Valrose, Parc Valrose, 06108 Nice, France. (2) CNRS,
Institute of Biology Valrose, Parc Valrose, 06108 Nice, France. (3) INSERM, Institute of Biology Valrose, Parc
Valrose, 06108 Nice, France. (4) HHMI Janelia Research Campus, 19700 Helix Drive, Ashburn, VA 20147, USA.
Early transplantation and grafting experiments suggest that body organs follow
autonomous growth trajectories, therefore pointing to the need for coordination
mechanisms to produce fit individuals with proper proportions. We recently identified
Drosophila insulin-like peptide 8 (Dilp8) as a relaxin/insulin-like peptide secreted from
growing tissues that plays central role in coordinating growth between organs and
coupling organ growth with animal maturation. Deciphering Dilp8 function in growth
coordination relies on the identification of the receptor and tissues relaying Dilp8
signalling. Using a genetic approach, we show here that the orphan receptor leucinerich repeat-containing G-coupled receptor 3 (Lgr3), a member of the highly conserved
family of relaxin family peptide receptors (RXFPs), mediates the checkpoint function
of Dilp8 for entry into maturation. We functionally identify two pairs of Lgr3-positive
brain neurons that are required to induce the developmental delay observed upon
ectopic Dilp8 expression. These neurons are located in the pars intercerebralis, an
important neuroendocrine area in the brain and act as an intermediate relay in the
circuitry that ultimately controls the release of the moulting steroid ecdysone.
Reducing Lgr3 levels in these neurons results in adult flies exhibiting increased
fluctuating bilateral asymmetry, therefore recapitulating the phenotype of dilp8
mutants. Our work reveals a novel Dilp8/Lgr3 neuronal circuitry involved in a
feedback mechanism that ensures coordination between organ growth and
developmental transitions and prevents developmental variability.
1 The mapping and functional analysis of the prothoracic glandinnervating neurons controlling ecdysteroid biosynthesis in
Drosophila melanogaster: SE0PG neurons and beyond
Yuko Shimada-Niwa (1), Eisuke Imura (1), and Ryusuke Niwa (1, 2)
(1) Graduate School of Life and Environmental Sciences, University of Tsukuba, Japan. (2) PRESTO, JST, Japan.
The temporal transition of insect development is flexibly coordinated in the context of
the nutrient environment, and this coordination is essential for insects to increase
their survival fitness and reproductive success. An insect steroid, ecdysteroid, has a
central role in controlling the developmental transition from one stage to the next,
epitomized by molting and metamorphosis. Importantly, the timing of ecdysteroid
biosynthesis is tightly coupled to nutrient availability, and the underlying molecular
mechanism remains to be elucidated.
During the larval stages, ecdysteroid is synthesized from dietary cholesterol in the
endocrine organ called the prothoracic gland (PG). We found that a subset of
serotonergic neurons “SE0PG” directly innervates the PG and shares a tract with the
stomatogastric nervous system. Their cell bodies are located nearby the
subesophagal ganglion, known as the insect feeding center, implying that these
neurons receive food-related signals. Interestingly, the projecting neurites
morphologically responded to nutrient conditions: on the yeast-poor food conditions,
the neurites hardly projected to the PG cells and the timing of pupariation was
delayed. Moreover, reduced activity of the serotonergic neurons or of serotonin
signaling in the PG strongly correlates with a delayed developmental transition. The
yeast-poor food condition suppressed the delay, supporting our hypothesis that
SE0PG neurons mediate some nutrient signals to the PG. Our results suggest that
serotonergic neurons form a link between the nutrient environment and the internal
endocrine system by adaptively tuning the timing of ecdysteroid biosynthesis.
Given that ecdysteroid biosynthesis is affected by various environmental conditions
other than nutrition, we assume if there are other types of the PG-innervating
neurons. To obtain a complete set of neurons innervating the PG, we utilized the
Janelia-GAL4 database to screen about 80 lines. Based on the study of Siegmund
and Korge 2001, we classified these neurons into several groups, which include
uncharacterized neurons so far. The latest results of the mapping of the PGinnervating neurons will be represented.
2 The Drosophila Peptidergic Connectome
Michael J. Texada and James W. Truman
HHMI Janelia Research Campus, 19700 Helix Drive, Ashburn, VA 20147.
Generating behavioral patterns that are appropriate to an animal’s acute
circumstances – e.g., developmental, nutritional, and stress states – is important for
survival; likewise, the components or phases of any given behavioral pattern must
be organized properly. One robust multi-phase behavioral program – insect ecdysis
– has been shown to be governed by a cascade of peptide neuromodulators that
firstly influence each other’s release to correctly organize the stages of the ecdysis
sequence and secondly regulate the activity of downstream circuitry to drive
behavioral performance.
Hormone receptors, unlike their peptide ligands, are expressed at low levels and are
difficult to detect directly. To sidestep this problem, we have created BAC-based
receptor-GAL4 lines to allow genetic access to cells expressing the ~60 known or
potential peptide-binding GPCRs and transmembrane guanylyl cyclases. These
constructs retain large flanking sequences, UTRs, and introns; thus their expression
is likely an accurate reflection of endogenous target-gene activity. Indeed, their
patterns match well with tissue-level FlyAtlas expression data (Chintapalli et al.,
Nature Genetics, 2007) as well as with known aspects of receptor expression;
therefore, our tools produce biologically relevant information.
By co-staining receptors and peptides in the wandering third-instar larva, we have
assembled a map of the “modulatory connectome.” This map recapitulates the
known elements of the ecdysis- modulatory machinery and adds novel elements.
The ecdysis machinery is not a well-isolated subnetwork within the connectome,
however, but rather is contained within a dense mesh of other hormones and
subnetworks. Several peptidergic cell types appear to be modulatory “hubs”
integrating many hormonal signals; conversely, others communicate broadly to
many other peptidergic and non-peptidergic cells, indicating a role in setting the
modulatory tone of broad swaths of the nervous system. Our lines have also allowed
us to identify sensory, inter-, and motor neurons that receive and process
information in a modulator-sensitive way. With our collection of tools, the activity of
these cells can be visualized using genetically encoded biosensors or perturbed in
different ways to throw light on many aspects of neuronal processing and the
production of adaptive behaviors.
3 A genetic screen to uncover new signals controlling Drosophila
juvenile-maturation transition
Derya Deveci (1-3), Nuria M. Romero (1-3), Francisco A. Martin (4), Pierre Léopold
(1-3)
(1) University of Nice-Sophia Antipolis, Institute of Biology Valrose, Parc Valrose, 06108 Nice, France. (2) CNRS,
Institute of Biology Valrose, Parc Valrose, 06108 Nice, France. (3) INSERM, Institute of Biology Valrose, Parc
Valrose, 06108 Nice, France. (4) Instituto Cajal (CSIC), Avda. Doctor Arce 37, 28002 Madrid, Spain.
Deciphering the regulatory mechanisms controlling final body size is a fundamental
question in biology. In species with so called “determinate growth”, final body size is
fixed at the transition from growing juvenile to sexually mature adult, a process
known as juvenile maturation transition (JMT). Various external cues such as light,
nutrition, oxygen and temperature play a role in the timing of this transition.
However, it remains largely unknown which internal sensory mechanisms are involved
in the coupling and integration of these cues for the subsequent activation of the
cascade of events leading to JMT. In vertebrates, the onset of JMT is triggered by a
peak of steroid hormones. In Drosophila melanogaster, and in various other
holometabolous insects, a similar mechanism takes place. A peak of
prothoracicotropic hormone (PTTH) produced by two pairs of neurons leads to the
production of the insect steroid hormone ecdysone. PTTH is one of the first known
signals to activate the cascade of events leading to juvenile maturation transition in
D. melanogaster. If PTTH production is blocked, a delay is observed in this transition
whereas this transition is accelerated upon PTTH over expression, indicating the
importance of PTTH neurons in the integration of internal and/or external cues
(Yamanaka, Romero, et al. 2013, Rewitz, et al. 2009, McBrayer, et al. 2007). In order
to understand the role of PTTH neurons in controlling JMT, we have conducted a
biased RNAi screen in the PTTH neurons for genes whose knocked down delays JMT.
By using Gene Ontology (GO) we have selected ~1300 predicted membraneassociated protein encoding genes as well as transcription factors specifically
expressed in the brain. In a primary screen, we used a strong Gal4 driver expressed
in PTTH neurons as well as in some other neurons (NP0423-Gal4), from which we
identified 270 hits.
Next, we subjected these hits to a second round of screening using a driver specific
for PTTH neuron (PTTH-Gal4), which markedly narrowed the number of positive hits
to 36. We obtained 6 hits with unknown function or domain, 9 hits involved in
synapsis or neuronal function, 8 hits involved in protein modification, 4 hits with
heterogeneous functions, 3 nuclear receptors and 9 hits with receptor function. The
detailed study of putative receptors controlling the function of PTTH neurons should
help us uncover new signals integrated by the PTTH neurons that control the JMT.
4 Endoreplication in the prothoracic gland underlies developmental
checkpoint mechanism
Yuya Ohhara and Naoki Yamanaka
Department of Entomology, University of California, Riverside, 900 University Ave, Riverside, CA 92521, USA.
Upregulated production and release of ecdysone, the immediate precursor of the
molting hormone 20E, is a key to the timing of adult development in holometabolous
insects. Production of ecdysone in the larval prothoracic gland (PG) is regulated by
multiple extracellular signals, but a unified view of how the upregulation of
ecdysteroidogenesis in the PG is initiated is still missing. Here we provide evidence
that endoreplication in the PG is necessary for initiating ecdysteroidogenesis that
triggers metamorphosis in the fruit fly Drosophila melanogaster.
Endocycling of the PG cells is regulated by a checkpoint mechanism that monitors the
resource availability in the extracellular environment. We propose that a cell cycle
checkpoint mechanism is thus integrated into a developmental checkpoint mechanism
in insects and an irreversible DNA amplification in the PG leads to the irreversible
commitment of the PG to ecdysteroidogenesis.
5 Discovery of genes essential for heme biosynthesis in the Drosophila
ring gland
Qiuxiang Ou (1), Brian Phelps (1), Arash Bashirullah (2), Kirst King-Jones (1)
(1) Department of Biological Sciences, University of Alberta, Edmonton, Canada. (2) Department of Pharmacy,
University of Wisconsin-Madison, USA.
Heme is an essential molecule found in all life forms. Steroid hormones are ancient
signaling molecules that are produced from sterol precursors through the catalytic
activity of mostly cytochrome P450 enzymes. All P450 enzymes require heme as a
cofactor. Microarray and RNA-Seq data from our lab indicates that P450 genes are
among the most highly expressed genes in the Drosophila ring gland, even when no
steroid hormone (ecdysone) peak is produced. Despite these incredibly high basal
levels, some P450 genes are induced transcriptionally >100fold prior to a major
ecdysone peak, suggesting that the demand for heme production must be coordinated
accordingly to match P450 protein synthesis. However, little is known about the
mechanisms by which heme synthesis is regulated and coordinated with ecdysone
production in the prothoracic gland (PG).
To identify genes essential for heme biosynthesis and its regulation in PG cells, we
carried out a PG-specific, genome-wide RNAi screen and examined larvae with
developmental defects for inappropriate heme precursor accumulation. An inability to
complete heme production usually results in a substantial accumulation of fluorescent
heme precursors known as protoporphyrins, a feature that we exploited for our
screen. Using this strategy, we identified 34 hits, which included transcription factors,
signaling molecules, and mitochondrial components. On the molecular level, impaired
heme biosynthesis causes strong transcriptional upregulation of ALAS in vertebrates,
a gene that encodes the rate-limiting enzyme for heme biosynthesis. Consistent with
this, Drosophila ALAS is strongly upregulated in the RNAi lines we identified. In
addition, we have shown that the nuclear receptor DHR51 is required for the
upregulation of ALAS, in line with a previous finding that DHR51 binds heme in vitro
and thus might function as a heme sensor in vivo. Together, these data shed light on
a new layer of steroid hormone regulation through the control of heme synthesis in
the Drosophila prothoracic gland.
6 A genome-wide in vivo RNAi screen in Drosophila identifies regulators
of cholesterol-dependent steroid production
E. Thomas Danielsen (1) ($), Morten E. Moller (1) ($), Naoki Yamanaka (2), Kirst
King-Jones (3), Michael B. O’Connor (4) and Kim F. Rewitz (1). ($: equal
contribution).
(1) University of Copenhagen, Denmark. (2) University of California, Riverside, USA. (3) University of Alberta,
Canada. (4) University of Minnesota, USA.
Steroid hormones are signal molecules synthesized from sterols that regulate a
variety of processes during embryogenesis, postembryonic development and
reproduction. Although steroid synthesis in endocrine cells requires uptake of
cholesterol, the mechanisms regulating cholesterol uptake, storage and availability for
steroid production are poorly understood. We have performed a genome-wide in vivo
RNAi screen to uncover the genes required for steroid production in the endocrine
steroid-producing cells of Drosophila. Using this approach, we reduced the expression
of 12,504 genes in the steroid producing prothoracic gland (PG) cells to investigate
their potential role in steroid biosynthesis.
Several genes previously shown to be required for delivery of cholesterol for
steroidogenesis were identified including genes involved in the Niemann pick type C
disease, scavenger receptors and lipid droplet proteins. Cholesterol is a low density
lipoprotein (LDL)-derived lipid, taken up by cells through endocytosis and stored as
cholesteryl ester in lipid droplets. To identify novel genes involved in cholesterol
uptake and transportation, we performed a secondary screen to analyze which of
genes identified in our primary screen for steroidogenesis that are specifically involved
in cholesterol trafficking. In this screen we identified 24 novel genes that seem to be
involved in regulating cholesterol uptake and trafficking for steroid production.
Loss of genes identified in our screen caused defects in formation of cholesterol-rich
lipid droplets indicating that the genes are involved in uptake, trafficking or storage of
cholesterol. Among the pathways identified, we show that genes involved in fatty acid
elongation and sphingolipid biosynthesis are required for endosomal trafficking of
cholesterol, which is coordinated by TOR and depends on autophagosomal trafficking.
Many of these novel genes have conserved human homologs that have been
associated with diseases that involve dysregulation of cholesterol homeostasis and
steroid signaling.
7 Snail is a prothoracic gland-specific transcription factor required for
ecdysone production in Drosophila larvae
Jie Zeng, Qiuxiang Ou, Kirst King-Jones
Department of Biological Sciences, University of Alberta, Edmonton, Alberta, Canada, T6G 2E9.
Our research focuses on exploring novel signaling pathways controlling the production
of steroid hormones in Drosophila melanogaster. The major steroid hormones in
Drosophila (ecdysteroids, hereafter ecdysone) are released as pulses throughout the
fly’s life cycle, thereby triggering developmental transitions such as the molts or
puparium formation. During larval stages, ecdysone is produced in the prothoracic
gland (PG), which is part of the ring gland, the principal neuroendocrine organ during
Drosophila development. In an effort to identify novel regulators of ecdysone
production, our lab carried out ring gland-specific microarrays, which identified ~120
genes that are specifically expressed in this tissue, one of which was Snail. Snail has
well known functions during Drosophila embryonic development, however, we show
here that snail is also dynamically expressed in the ring gland during postembryonic
stages, with a characteristic single peak around 6-8 hours prior to the L2-L3 molt,
contrasted with a gradual decline of snail expression during the L3 stage. PG-specific
RNAi against snail leads to larval arrest (~20% L2 and ~80% L3 arrest), which are
phenotypes commonly associated with reduced ecdysone signaling. Providing
extraneous 20E in the diet was able to partially rescue the RNAi phenotype.
Moreover, qPCR analysis showed that loss-of-snail function in the PG lowered
transcript levels of the genes related to ecdysone production (torso, phantom and
spookier). Loss-of-snail function also affected the growth of PG cells, where cells
appeared to stop growing during the L3 stage when compared to controls. These data
suggested that snail might be required to maintain PG growth and morphology, and
was also necessary for normal ecdysone production. However, it remains unclear
whether Snail regulates ecdysone production via direct or indirect mechanisms.
As a first step towards identifying processes controlled by Snail in the PG, we are
currently carrying out ring gland-specific Next-generation RNA sequencing of samples
that express snail-RNAi or snail cDNA in the ring gland. This will allow us to identify
candidate target genes that we can compare to known targets of snail in the embryo
and thus recognize processes controlled by this transcription factor specifically in the
PG.
8 Plasma membrane receptors in prothoracic gland cells of Bombyx mori
Skarlatos G. Dedos, Alexandros Alexandratos, Panagiotis Moulos, Ioannis Nellas,
Konstantinos Mavridis
Department of Biology, National and Kapodistrian University of Athens, Panepistimioupoli Zografou, 157 84, Athens,
Greece.
An in-depth analysis of plasma membrane (PM) receptors in the prothoracic gland
(PG) cells of the silkworm, Bombyx mori, was carried out throughout the 5th instar
and the first day of the pupal stage. The aim of the study was to correlate signalling
pathway dynamics with ecdysteroidogenic activity of these cells and thus interpret
the inherent variations in ecdysteroid secretion that govern the development of this
insect. We used proteomic analysis of liquid chromatography-tandem mass
spectrometry generated peptide fragments, whole-transcriptome analysis of Illumina
RNA sequencing data and quantitative PCR to determine the expression of a wide
spectrum of PM receptors in PG cells. Our combined approach identified a large
number of G protein-coupled receptors as well as receptor tyrosine kinases,
transforming growth factor β receptor serine/threonine kinases, receptor tyrosine
phosphatases, receptor guanylate cyclases and integrins, whose expression varied
throughout the examined developmental period. Among other PM receptors, we have
identified and determined the expression levels of genes coding for receptors
involved in the Wnt/Wingless signalling pathway, the Hedgehog signalling pathway
and the Notch signalling pathway. Contrary to the commonly employed relative
quantification of transcript levels using housekeeping genes, transcript levels of three
(3) housekeeping genes (GAPDH, Actin A3 and RPL32) varied widely throughout the
examined developmental stage, with peaks and troughs of expression incongruous
with the ecdysteroidogenic activity of PG cells.
Besides questioning the methodological approach used in published data, this finding
necessitated the use of absolute quantification of transcript levels by generating
standard curves using Bombyx mori prothoracicotropic hormone receptor (Torso)
recombinant DNA as a reference. Using this approach, we generated heat maps of
expression of genes that code for several dozens of known or yet unidentified PM
receptors. Distinct patterns of expression were identified for several genes
throughout the examined developmental stage, raising questions pertinent to the
exact role each gene plays in these cells. The generated heat maps of expression can
pinpoint precisely the timing and combination of extracellular signals that shape the
ecdysteroidogenic activity of PG cells on each day of the examined developmental
stage.
9 The growth regulator Myc affects ecdysone and developmental
transitions
Jennifer Gerlach, Dominik Steiger and Peter Gallant
Theodor Boveri Institute, Biocenter, University of Würzburg, Würzburg, Germany.
The principal molting hormone of Drosophila is ecdysone. It is synthesized from
dietary cholesterol in the prothoracic gland and afterwards converted to the active
form 20- hydroxyecdysone in peripheral tissues which controls several
developmental transitions. The timing of development is also influenced by larval
growth, but the molecular connections between growth and timing are not entirely
understood yet. Here, we describe a new link between ecdysone-controlled
developmental timing and the regulator of growth Myc. Myc is normally thought to
bind together with its dimerization partner Max to E-box sequences and activate the
transcription of target genes. We now find a Max-independent effect of Myc on larval
development. Ubiquitous overexpression of Myc in max null mutants kills larvae
during the L2 to L3 molt; overexpression of Myc in these mutants after L3 ecdysis
results in a complete block of pupariation and produces L3 larvae that survive for up
to 33 days. These developmental blocks presumably reflect a failure in ecdysone
synthesis, since they can be overcome by feeding of 20-hydroxyecdysone and since
the levels of the ecdysone biosynthetic genes are strongly decreased in the affected
animals. We are currently investigating the molecular links between Myc and the
halloween genes and exploring the physiological context of this regulation.
10 Ouija board is the zinc finger transcription factor controlling
ecdysteroid biosynthesis through specific regulation of spookier in
Drosophila
Tatsuya Komura-Kawa (1), Outa Uryu (1), Keiko Hirota (1, 2), Yuko Shimada-Niwa (1,
2), Rieko Yamauchi (1, 2), MaryJane Shimell (3), Tetsuro Shinoda (4), Akiyoshi
Fukamizu (1, 2), Michael B. O’Connor (3), and Ryusuke Niwa (1, 5)
(1) Graduate School of Life and Environmental Sciences, University of Tsukuba, Japan. (2) Life Science Center,
Tsukuba Advanced Research Alliance, University of Tsukuba, Japan. (3) Department of Genetics, Cell Biology and
Development, University of Minnesota, USA. (4) National Institute of Agrobiological Sciences, Japan. (5) PRESTO,
JST, Japan.
Ecdysteroids are coordinately synthesized from dietary cholesterol via a series of
hydroxylation and oxidation steps in the prothoracic gland (PG) during development.
The steroidogenic function of the PG cells is defined by the restricted expression of
several enzyme genes that are required for converting ecdysteroid intermediates. The
cell-type specific expression pattern implies a tight transcriptional regulation of the
biosynthetic enzymes in the PG. However, the PG specific transcriptional regulatory
networks have not yet been fully elucidated.
Here, we report the C2H2-type zinc finger transcription factor gene ouija board
(ouib), which is predominantly expressed in the PG. Genetic null mutants of ouib
result in larval arrest and ecdysteroid deficiency. ouib mutants exhibit a strong
reduction in the expression of only one ecdysteroidogenic enzyme gene, spookier.
Ouib protein binds to a specific response element in the spookier enhancer.
Remarkably, the ouib mutant phenotype is fully rescued by over- expression of the
spookier paralog. These results suggest that Ouib is involved in the PG specific
transcriptional regulation of ecdysteroid biosynthesis. Our work also illustrates that a
certain transcription factor is required for expression of a very small subset of
steroidogenic gene(s) involved in specific catalytic step(s), providing a novel insight
into the transcriptional regulatory mechanism of developmental timing in organisms.
In this presentation, a role of other C2H2-type zinc finger transcription factors in
controlling ecdysteroid biosynthesis will also be discussed.
11 The viable temperature range of Drosophila is controlled by diet
Marko Brankatschk (1), Theresia Gutmann (2), Uenal Coskun (2) and Suzanne Eaton
(1)
(1) Max Planck Institute of Molecular Cell Biology and Genetics. (2) Mediziniches Theoretischces Zentrum,
Technische Universität Dresden, Dresden, Germany.
Like other cold-blooded animals, Drosophila melanogaster can develop and survive
over a broad but limited range of temperatures. Increasing temperature proportionally
increases the rate of development throughout most of the viable temperature range.
But this proportionality does not hold near the upper and lower limits of viable
temperature range. What sets these limits is unknown. Here, we show that Drosophila
prefer plants to yeast as a food source at low temperature. Feeding on plants lowers
the temperature range over which development is possible, and enables adults to
survive subzero temperatures. Plant lipids contain more unsaturated fatty acids than
those of yeast, and this is directly reflected in the tissue lipid composition of Drosophila
that feed on plants. Liposomes prepared from lipids of plant-fed animals remain fluid at
lower temperatures than those from animals fed with yeast, suggesting that the lower
limit of the viable temperature range is determined by membrane fluidity. In contrast,
yeast--fed animals can develop at higher temperatures than plant-fed animals. This is
a consequence of elevated systemic insulin signalling in animals fed with yeast.
Although plant and yeast-based diets contain similar numbers of calories from
carbohydrate protein and fat, yeast lipids promote release of Drosophila Insulin-like
peptides whereas plant lipids do not. dILP release is induced because yeast, but not
plant, lipids promote transport of the lipoprotein LTP across the blood brain barrier to
neurons that connect to and active insulin producing cells (IPC’s). This increases dILP
release and speeds development on yeast food. Artificially activating dILP release in
plant-fed animals increases their developmental rate and enables them to develop at
higher temperatures. This suggests that the ability to increase developmental rate with
temperature is key to survival and is limited by the rate of nutrient uptake. Increasing
the rate of development in the presence of yeast may also allow Drosophila to rapidly
exploit at transient dietary resource. 12 Nutritional Control of Body Size through FoxO-Ultraspiracle Mediated
Ecdysone Biosynthesis
Takashi Koyama and Christen K. Mirth
Instituto Gulbenkian de Ciência, Rua da Quinta Grande, 6, Oeiras 2780-156, Portugal.
Despite their fundamental importance for body size regulation, the mechanisms that
stop growth are poorly understood. In Drosophila melanogaster, growth ceases in
response to a peak of the molting hormone ecdysone that coincides with a nutritiondependent checkpoint, critical weight. Previous studies indicate that insulin/insulinlike growth factor signaling (IIS)/Target of Rapamycin (TOR) signaling in the
prothoracic glands (PGs) regulates ecdysone biosynthesis and critical weight. Here
we elucidate a mechanism through which this occurs. We show that Forkhead Box
class O (FoxO), a negative regulator of IIS/TOR, directly interacts with Ultraspiracle
(Usp), part of the ecdysone receptor. While overexpressing FoxO in the PGs delays
ecdysone biosynthesis and critical weight, disrupting FoxO-Usp binding reduces these
delays.
Further, feeding ecdysone to larvae eliminates the effects of critical weight. Thus,
nutrition controls ecdysone biosynthesis partially via FoxO-Usp prior to critical
weight, ensuring that growth only stops once larvae have achieved a target
nutritional status.
13 Functions of adipokinetic hormone and adipokinetic hormone
precursor related peptide in Drosophila melanogaster
Martina Gáliková (1), Max Diesner (2), Peter Klepsatel (1), Philip Hehlert (1), Yanjun
Xu (1), Iris Bickmeyer (1), Reinhard Predel (2) and Ronald P. Kühnlein (1)
(1) Max-Planck-Institut für biophysikalische Chemie, Research Group Molecular Physiology, Am Faßberg 11, D37077 Göttingen, Germany. (2) Universität zu Köln, Institut für Zoologie, Zülpicher Str. 47b, D-50674 Cologne,
Germany.
Survival in a changing environment requires timely mobilization of energy reserves
for a variety of processes ranging from basal metabolism to locomotion or
reproduction. In insects, different contexts of energy mobilization employ the same
regulatory system, signaling via adipokinetic hormones (AKHs). These hormones arise
from processing of pre-prohormones coding for AKH and an additional peptide termed
APRP with so far unknown function.
To study the AKH and APRP functions in Drosophila, we created an AKH-specific and
AKH - APRP double mutants. These mutants enabled us to address for the first time
specific physiological functions of AKH and APRP in fundamental biological processes
like development, reproduction, metabolism and stress response. Based on the
comparisons with previous studies in other insect species, where mostly injections of
the AKH peptide or RNAi approaches have been used, our study revealed a surprising
divergence in the engagement of AKH in several crucial processes requiring
mobilization of energy reserves. We show that in Drosophila, AKH signaling is
dispensable for ontogenesis, locomotion, oogenesis and metabolism of stored energy
reserves before and during metamorphosis. However, during Drosophila adulthood,
AKH signaling acquires important metabolic roles like regulation of stored lipids and
circulating, but not stored carbohydrates. Next to these metabolic functions, AKH
signaling in fly modulates also stress response, including adaptation to nutritional and
oxidative stress.
14 Nutrition- and sex-dependent regulation of insulin signaling and
growth in Drosophila
Elizabeth Rideout, Abhishek Ghosh, Savraj Grewal
Clark Smith Brain Tumour Centre, Southern Alberta Cancer Research Institute, Department of Biochemistry and
Molecular Biology. University of Calgary, Calgary, Alberta T2N 4N1.
The conserved insulin/insulin-like growth factor signaling (IIS) pathway is a major
endocrine regulator of body growth and development in Drosophila. Recent work
has emphasized how inter-organ communication can control of IIS in response to
different environmental and genetic cues. Here I present our work on regulation of
IIS by two cues important for growth: nutrients and sex.
We find that dietary nutrients control IIS in part through tissue-specific control of
tRNA and rRNA synthesis by TOR kinase signaling. TOR stimulates tRNA synthesis
by inhibiting Maf1, a pol III repressor, and stimulates tRNA synthesis via activation
of TIF-IA, a pol I transcription factor. Control of rRNA and tRNA synthesis can exert
non cell-autonomous effects on overall body growth and development - stimulation
of tRNA synthesis in larval fat larval fat body leads to enhanced body growth; in
contrast, rRNA synthesis is required in larval muscle to control overall body growth.
In both cases, the growth effects involve changes in expression and release of
insulin-like peptides from the CNS resulting in altered systemic IIS.
Almost all animals show sex differences in body size. For example, in Drosophila,
females are significantly larger than males. We show that the sex determination
gene transformer (tra) contributes to promoting increased female body growth. Tra
is expressed only in females where it has classically been shown to control sexual
differentiation and to determine secondary sexual characteristics and sex-specific
behaviour in adults. We find that loss of Tra in female larvae decreases final body
size, while ectopic Tra expression in males increases body size. We also observe
that Tra expression in the female fat body and the central nervous system (CNS)
promotes larger female body size in a non cell-autonomous manner. In particular,
Tra function depends on insulin signaling and Tra expression in the female fat body
promotes the secretion of insulin-like peptides from the CNS.
15 Drosophila enteroendocrine cells are involved in nutrient sensing and
regulate systemic metabolism through bursicon secretion.
A. Scopelliti, C. Bauer, JB. Cordero, OJ. Sansom, M. Vidal
Cancer Research UK Beatson Institute, Garscube Estate, Switchback Road, Bearsden, Glasgow, G61 1BD.
The heterodimeric hormone Bursicon, made by the alpha and the beta subunits, is a
well-known mediator of post molting events during arthropod development. We
recently described a novel paracrine signaling system in the adult midgut, in which
the enteroendocrine (ee) cells secrete bursicon alpha, which acts as a paracrine factor
on the visceral muscle through its receptor DLGR2 (a GPCR encoded by rk locus), to
constrain proliferation of intestinal stem cells.
While Bursicon expression seems to be restricted exclusively to a subpopulation of ee
cells, dLGR2 is widely expressed in several organs, implying that bursicon alpha can
exert additional endocrine effects on peripheral tissues.
We observed that guts from fasting animals displayed elevated bursicon alpha
immunoreactivity in ee cells. This effect is reversed upon re-feeding with sucrose but
not with a protein only diet, suggesting that bursicon alpha is secreted in response to
carbohydrate availability in the diet.
Flies mutants for bursicon alpha or dLgr2 show a compound phenotype, which include
hypersensitivity to starvation. This sensitivity is partially dependent on their inability
to mobilize carbohydrates stored as glycogen.
Moreover, despite the hyperphagic behavior and the reduced locomotor activity,
mutant flies show a strong hypoglycemia and a gradual depletion of lipid storage over
time, suggesting an abnormal increase of energy expenditure.
Overall, our data suggest that Bursicon mediates an endocrine axis connecting
nutrient sensing to systemic metabolism.
16 Regulation of Drosophila circadian rhythms by miRNA let-7 is
mediated by a regulatory cycle
Zhangwu Zhao
Department of Entomology, College of Agriculture and Biotechnology, China Agricultural University. 2 Yuanmingyuan
West Road, Haidian district, Beijing, 100193, China.
MicroRNA-mediated post-transcriptional regulations are increasingly recognized as
important components of the circadian rhythm. Here we identify microRNA let-7, part
of the Drosophila let- 7-Complex, as a regulator of circadian rhythms mediated by a
circadian regulatory cycle.
Overexpression of let-7 in clock neurons lengthens circadian period and its deletion
attenuates the morning activity peak as well as molecular oscillation. Let-7 regulates
the circadian rhythm via repression of CLOCKWORK ORANGE (CWO). Conversely, upregulated cwo in cwo-expressing cells can rescue the phenotype of let-7-Complex
overexpression. Moreover, circadian prothoracicotropic hormone (PTTH) and CLOCK
regulated 20-OH ecdysteroid signaling contribute to the circadian expression of let-7
through the 20-OH ecdysteroid receptor. Thus, we find a regulatory cycle involving
PTTH, a direct target of CLOCK, and PTTH-driven miRNA let-7.
17 Control of Feeding Behaviour and Metabolic Physiology by
Peptidergic Enteric Neurons
Marion Hartl, Dafni Hadjieconomou, Sophie Austin and Irene Miguel-Aliaga
Imperial College, London, W12 0NN, U. K.
Feeding is largely determined by two factors: the presence of potential food sources
and the motivational state of an animal. The latter is modulated by a complex range
of internal signals including the nutritional state. The mechanisms that relay internal
state play a key role in modulating feeding behaviour and maintaining metabolic
homeostasis, but remain incompletely understood. Early experiments in the blowfly
provided proof of principle that neuronal communication between the central
nervous system and the gut has drastic effects on food intake (Evans and Dethier,
1957; Dethier and Bodenstein, 1958; Dethier and Gelperin, 1967; Belzer, 1978).
However, the relevant neuronal populations and their mode of action remain to be
characterised. Previous work in the lab has shown that changes in the activity of
gut-innervating neurons can affect food intake in Drosophila (Cognigni et al (2011)
Cell Metab and unpublished). However, comprehensive description of the anatomy,
neuropeptidergic profile and homeostatic functions of enteric neurons is currently
lacking.
Taking advantage of the genetic tractability of Drosophila melanogaster, we have
focused on its anterior midgut to acquire comprehensive knowledge of its
innervation. To this end, we have been screening the latest generation of Gal4 lines
in combination with photoactivatable GFP and Flybow-based technologies. We have
determined the contribution of central and peripheral neurons to gut innervation,
and have found lines with restricted expression in peptidergic neurons. Thus,
besides the epithelial enteroendocrine cells, gut-innervating neurons provide a
heterogeneous source of neuropeptides with potential intestinal and/or systemic
roles.
This approach has also generated genetic tools to target specific central and
peripheral gut- innervating neurons, allowing further functional characterisation. We
have developed new physiological assays and adapted others previously used in
larger insects. These now allow us to simultaneously monitor changes in intestinal
physiology and feeding decisions. We have combined these assays with genetic
manipulations of enteric neuron activity. We will present our initial results on how
different aspects of intestinal physiology can be modulated by subsets of peptidergic
neurons.
18 Recent advances in juvenile hormone signaling in Drosophila
Sheng Li
Key Laboratory of Insect Developmental and Evolutionary Biology, Institute of Plant Physiology and Ecology,
Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China.
Juvenile hormones (JH) and 20-hydroxyecdysone (20E) coordinately control insect
molting and metamorphosis. One major function of JH is to inhibit some actions of
20E during the larval stages and therefore, and JH is usually referred to as the “status
quo” hormone. In the fruit fly, Drosophila melanogaster, a dipteran insect, the “status
quo” action of JH is subtle but functionally important.
Based on a genetic screen to isolate mutants that are resistant to a JH agonist
(methoprene) in Drosophila, Methoprene-tolerant (Met), a transcription factor
belonging to the bHLH-PAS family, was suggested to be involved in JH action. Met
forms homodimers or heterodimers with its paralog, Germ-cell expressed (Gce), and
JH reduces this dimerization. Although both the Met and the gce null mutant are
viable, the Met gce double mutant dies during the larval-pupal transition, resembling
what is seen in the JH-deficient animal. Functionally, Met and Gce mediate JH action
to prevent the 20E-triggered metamorphic events and thus maintaining the “status
quo” action. Moreover, Met binds to JH at physiological concentrations in vitro,
suggesting strongly that Met is a bona fide JH receptor.
Krüppel homolog 1 (Kr-h1), encoding a zinc-finger transcription factor, is a JH
primary-response gene. Met and Gce transduce JH signals to induce Kr-h1 expression
via JH response element (JHRE) containing E-box-like motifs. Moreover, Kr-h1
mediates JH signals to repress expression of br, a 20E primary-response gene,
suggesting that Kr-h1 plays a role to mediate JH-20E crosstalk.
Taiman, a bHLH-PAS family transcriptional regulator, directly interacts with Met, acts
as a potential partner molecule of Met to mediate JH-induced Kr-h1 expression. The
chaperone protein, heat shock protein 83 (Hsp83), was identified as one of the
proteins bound to the JHRE in the presence of JH. Hsp83 modulates JH signaling
through mediating the JH binding and nuclear localization of Met.
Methyl farnesoate (MF) is the immediate precursor of JH III in the JH biosynthetic
pathway and lacks the epoxide moiety characteristic of JHs. MF plays a dual role in
regulating Drosophila metamorphosis: exerting its anti-metamorphic effects indirectly
after conversion to JHB3 and acting as a hormone itself through a direct interaction
with Met and Gce, the two JH receptors. Taking the advantage of Drosophila genetics
and other techniques, it should be the right time to understand the mystery of the
“status quo” action of JH.
Protein phosphorylation is involved in juvenile hormone signal
transduction
19 Pengcheng Liu, Reyhaneh Ojani, Jinsong Zhu
Department of Biochemistry, Virginia Tech, 313 Engel Hall, Blacksburg, VA 24061, USA.
Juvenile hormone (JH) is a key regulator of a wide diversity of developmental and
physiological events in insects. The mechanisms by which JH exerts pleiotropic
functions are probably manifold and integrated in nature to ensure proper hormonal
responses. We found that JH activated the phospholipase C (PLC) pathway in Aedes
aegypti mosquitoes, and quickly increased the levels of inositol 1,4,5-trisphosphate,
diacylglycerol and intracellular calcium, leading to activation of calcium/calmodulindependent protein kinase II (CaMKII) and protein kinase C (PKC). Moreover, the PLC
pathway was essential for the JH-induced phosphorylation of the intracellular JH
receptor MET and its partner TAI. Inactivation of the PLC pathway in mosquitoes
significantly decreased the binding of MET and TAI to JH response elements,
considerably reducing the expression of JH target genes. These results imply that the
genomic response to JH is governed by both MET and an unidentified membrane JH
receptor that acts upstream of PLC. In addition to transcriptional regulation, our
recent study suggests that JH may also regulate RNA splicing in target tissues.
Serine/arginine-rich (SR) splicing factors can either activate or repress splicing,
depending on their phosphorylation state and their interaction partners.
Phosphorylation of individual SR factors was drastically different between the newly
emerged adult mosquitoes and maturing adult mosquitoes. In vitro tissue culture
experiments indicated that the change in SR protein phosphorylation was induced by
JH and was accompanied with alternative splicing of a number of genes. The signaling
pathways that regulate the phosphorylation of SR proteins remain to be elucidated.
Nevertheless, these findings add new dimensions to the complexity of signal
transduction initiated by JH.
20 Juvenile Hormone dependent transport of the Drosophila Germ cellexpressed (GCE) protein.
Beata Greb-Markiewicz (1), Natalia Banaś (1), Daria Sadowska (1), Jakub Godlewski
(2),
Mirosław Zarębski (3), Andrzej Ożyhar (1)
(1) Department of Biochemistry, Faculty of Chemistry, Wrocław University of Technology, Wybrzeże Wyspiańskiego
27, 50-370 Wrocław, Poland. (2) Department of Neurosurgery, Brigham and Woman's Hospital, Harvard Medical
School, Harvard Institutes of Medicine, 4 Blackfan Circle, MA 02115, Boston, USA. (3) Department of Cell
Biophysics, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Gronostajowa 7, 30387 Kraków, Poland.
Insect development is regulated by two hormones: juvenile hormone (JH) and 20hydroxyecdysone. It has been proved that methoprene tolerant protein (MET) is a
long time unknown juvenile hormone receptor. MET belongs to the family of bHLHPAS transcription factors, critical regulators of gene expression networks. In contrast
to other insects that depend on MET to survival, Drosophila posses a MET paralog
germ cell-expressed (GCE). Recently, it has been shown that functions of MET and
GCE are only partially redundant and tissue specific. The bHLH-PAS transcription
factors functional activity is often correlated with shuttling between nucleus and
cytoplasm according to the masking and unmasking by interacting partners, signals
directing localization of these proteins. Previously, we identified nuclear localization
signals (NLSs) and nuclear export signals (NESs) for MET [Greb-Markiewicz et al.,
2011]. Until now however, no data has been presented on the GCE intracellular
shuttling and localization signals.
In order to determine the sequences of NLSs and NESs in GCE, we prepared series of
deletion and point mutants tagged by yellow fluorescence potein (YFP), expressed in
mammalian cells and observed with confocal microscopy system. Contrary to nuclear
localization of MET, we observe GCE in both compartments of the cell. We
demonstrate that GCE possess specific pattern of localization signals, only partially
consistent with presented previously for MET. Additionally, we demonstrate influence
of JH presence on subcellular distribution of GCE. We conclude that GCE similarly to
MET is a cytoplasm-nucleus shuttling protein with a complicated control system of
localization which vary between proteins. Workshop presentation will discuss results
in details.
This work was supported by a statutory activity subsidy from the Polish Ministry of
Science and Higher Education for the Faculty of Chemistry of Wrocław University of
Technology.
Greb-Markiewicz, B., Orłowski, M., Dobrucki, J., Ożyhar, A., 2011. Sequences that
direct subcellular traffic of the Drosophila methoprene-tolerant protein (MET) are
located predominantly in the PAS domains. Mol. Cell. Endocrinol. 345, 16–26.
21 Dispensable roles for juvenile hormones in holometabolous
lifecycle: insights from knockout Bombyx
Takaaki Daimon, Miwa Uchibori, and Tetsuro Shinoda
National Institute of Agrobiological Sciences. Owashi 1-2, Tsukuba city, Ibaraki, 305-8634, Japan.
Insect juvenile hormones (JHs) prevent precocious metamorphosis and allow the
larvae to undergo multiple rounds of “status quo” molts. However, little is known
about the roles for JHs during embryonic and very early larval stages. We here report
generation and characterization of knockout silkworms (Bombyx mori) having null
mutations in JH biosynthesis or JH receptor genes. We generated a knockout mutant
of JH acid methyltransferase (JHAMT), which catalyzes the last step of JH biosynthesis
in lepidopteran insects, as well as the two JH receptor genes in Bombyx, Met1 and
Met2, using TALENs. We found that embryonic growth and morphogenesis are largely
independent of JHs in Bombyx and, even in the absence of JHs or JH signaling, pupal
characters are not formed in the first or second instar larvae, and precocious
metamorphosis is induced after the second instar at the earliest. We also showed that
a pupal specifier gene broad, which is dramatically upregulated in late stage of the
last larval instar, is essential for pupal commitment in the epidermis by mosaic
analysis. Importantly, the mRNA expression level of broad, which is considered to be
repressed by JHs, remained at very low basal levels during the early larval instars of
JH- or JH signaling-deficient knockouts. Therefore, our study suggest that the longbelieved paradigm that JHs maintain the juvenile status throughout the larval life
should be revised because the larval status can be maintained by a JH-independent
mechanism in very early larval instars. We propose that the lack of competence for
metamorphosis during the early larval stages may be attributable to the absence of
an unidentified broad-inducing factor, or competence factor. We think the presence of
this factor has long been overlooked because its action may be “concealed” by JHs.
22 Genome-wide mapping of hormone-sensitive Methoprene-tolerant
binding sites Drosophila melanogaster
Rebecca F. Spokony (1), Robert Arthur (2), Christopher D. Brown (3), Alec Victorsen
(2), Jennifer R. Moran (2), Matthew Kirkey (2), Jeffrey Gersh (2), Kevin P. White (2)
(1) Baruch College, CUNY, New York NY 10010. (2) Institute for Genomics and Systems Biology, University of
Chicago, Chicago IL 60637. (3) Perelman School of Medicine, University of Pennsylvania, Philadelphia PA 19104.
Methoprene-tolerant (Met) is one of two paralogous juvenile hormone (JH) receptors
in Drosophila melanogaster. Its mode of molecular action is poorly understood. By
recombineering a 110 kb BAC genomic fragment, we made a line expressing eGFP
tagged MET regulated by its endogenous cis-genomic region (including 30 kb
upstream of the Met promoter) to examine where MET binds during mid-prepual
development, when JH titer is normally low. Then we exposed these pupae to the JH
mimic (JHM) methoprene and measured the change in MET binding. 1322 MET
binding sites were sensitive to a four hour methoprene treatment, beginning at the
white prepupal (WPP) stage. We mapped the DNA regions showing changes in MET
binding to the nearest upstream and downstream promoters in 1495 different genes.
16% of these genes show transcriptional changes following JHM application.
Methoprene-sensitive MET binding sites mapped to Eip75 and Kr-h1, which are known
JH-inducible genes in several insect species. The top Gene Ontology (GO) category
(using ProfCom at the BioProfiling.de web portal) for genes near binding sites with
increased MET occupancy following JH treatment is “Open Tracheal System
Development” (p<.01). For genes near sites showing lower MET binding, the top GO
category is “Imaginal Disc Derived Wing Morphogenesis”, “Compound Eye
Development”, followed by “Inter-Male Aggressive Behavior” (p<.01). These GO
categories are intriguing since methoprene-sensitive phenotypes include several
morphogenesis defects, and certain Met alleles show dramatic, nonconditional
compound eye defects. We hypothesize that some of these target genes underlie
methoprene sensitive phenotypes in Drosophila. We are currently conducting a
Genome-Wide Association Study (GWAS) on methropene sensitivity using the DGRP
lines to linkmethoprene-sensitive MET binding sites with various sensitivity
phenotypes. A transgenic line containing an eGFP tagged version of the paralogous JH
receptor, gce, is in progress.
23 Regulation of fat body cell dissociation by JH and 20E via regulating
expression of Mmps
Qiangqiang Jia and Sheng Li
Key Laboratory of Insect Developmental and Evolutionary Biology, Institute of Plant Physiology and Ecology,
Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China.
During Drosophila metamorphosis, the single-cell layer of fat body tissues gradually
dissociates into individual cells. We found that two matrix metalloproteinases (MMPs),
Mmp1 and Mmp2, are both required for fat body cell dissociation. Mmp1 was found to
preferentially cleave DE-cadherin-mediated cell-cell junctions, while Mmp2 was shown
to preferentially degrade basement membrane (BM) components and thus destroy
cell-BM junctions. Direct 20-hydroxyecdysone (20E) treatment does not induce fat
body cell dissociation, but expression of a dominant negative form of EcR to block 20E
signaling in the fat body was sufficient to inhibit this process. βftz-f1, the competent
factor of 20E signaling, promotes fat body cell dissociation by inducing expression of
Mmp1 and Mmp2 in both Drosophila fat body and Kc cells. βftz-f1 binding sites exist
in the promoter regions of both Mmp genes. Meanwhile, premature fat body cell
dissociation occurs in the juvenile hormone (JH) deficient animals, the Met Gce double
mutant, and the Kr-h1 mutant. Moreover, Kr-h1 overexpression delayed fat body cell
dissociation by reducing expression of Mmp1 and Mmp2 in both Drosophila fat body
and Kc cells. Our results show that fat body cell dissociation is coordinately regulated
of by JH and 20E via regulating expression of Mmps.
24 Zooming in on the JH receptor: Stereoisomer selectivity for natural
and synthetic ligands
Marek Jindra (1), Pavel Jedlicka (2), Robert Hanus (2)
(1) Biology Center CAS, Ceske Budejovice, Czech Republic. (2) Institute of Organic Chemistry and Biochemistry CAS,
Prague, Czech Republic.
Ecdysteroids and juvenile hormones (JHs) are non-peptide signals of equal
importance to arthropod development and reproduction. However, defining an
intracellular receptor for JH was lagging 20 years behind finding of the ecdysone
receptor (EcR), and over four decades after the discovery of the sesquiterpenoid
structure of JH. Converging evidence attributes the JH receptor role to a unique bHLHPAS protein called Met in most insects, or Met and its paralog Gce in Drosophila. While
the interaction of EcR with its steroid ligand has been resolved at the atomic level,
details of binding and activation of Met/Gce by JH are virtually unknown. Here, we will
first demonstrate in vivo genetic evidence that binding of an activating ligand is
indeed necessary for Met/Gce to sustain normal Drosophila development, thus firmly
establishing Met/Gce in the JH receptor role. Interestingly, Met/Gce can be activated
by binding to natural ligands, such as the JH precursor methyl farnesoate, and to
synthetic JH mimics of diverse chemistries. Such a remarkable flexibility in the ligand
preference of Met/Gce may parallel a similarly wide spectrum of ligands that activate
a related mammalian bHLH-PAS protein, the Aryl hydrocarbon receptor (AhR, dioxin
receptor). Despite the apparent flexibility of the ligand-binding pocket of Met/Gce, we
have been able to show that the JH receptor strictly discriminates between multiple
geometric and optical isomers derived from the same native JHs or synthetic JHmimicking compounds.
Importantly, those stereoisomers that best activated the receptor in vitro also were
more effective when tested in live insects. These results show that the JH receptor
Met/Gce possesses a highly stereoselective ligand-binding pocket for specific,
biologically active ligands.
25 Using the Drosophila salivary gland to model exocrine secretion
Andrew J. Andres, Kathryn M. Lantz, Marwa Al-Karawi, Pawel Parafianowicz, and
Benjamin F. B. Costintino
School of Life Sciences, University of Nevada Las Vegas, 4505 Maryland Parkway, Las Vegas, NV 89154, USA.
The larval salivary gland of Drosophila melanogaster is an excellent model in which to
study how one target tissue responds differently to temporally specific pulses of
steroid hormone. In the middle of the third instar the gland responds to a small pulse
of 20-hyroxyecdysone (20E) by inducing the expression of the Sgs (glue) genes; at
the end of the third instar the tissue responds to the pre-metamorphic pulse of
hormone by secreting those glue proteins into the lumen; and at the end of the
prepupal period the gland is histolyzed by yet another pulse of steroid. In an effort to
understand how the pre-metamorphic pulse of 20E triggers the regulated secretion of
the glue granules, we have analyzed the transcriptome of salivary glands before and
after 20E exposure. Our work indicates that the hormone stimulates multiple triggers
that contribute to the physiological response of the tissue. Using a combination of ex
vivo organ culture, live imaging of glands loaded with fluorescently tagged proteins,
and tissue-specific silencing of candidate genes, we are beginning to assemble those
pathways. We show 20E stimulates Calcium signaling, membrane targeting, and
cytoskeletal reorganizations, all of which are needed for glue granules to be secreted
against a concentration gradient into the limited luminal space of the salivary gland.
Because many of these same molecules have been implicated in the regulated
exocytosis of cargoes from many other exocrine systems controlled by different
stimuli, we expect that the molecular characterization of 20E-stimulated glue
secretion will contribute to our fundamental understanding of how these types of
tissues respond to important signals during a physiological or developmental
response.
26 Hormones and the Cellular Morphogenesis of the Drosophila Nervous
System.
James W. Truman, Susana Tae, and the FlyLight Imaging Project
Janelia Research Campus, Howard Hughes Medical Institute, 19700 Helix Drive, Ashburn, VA 20147 USA.
Ecdysone evokes profound tissue remodeling at metamorphosis as larval organ
systems are transformed into their adult counterparts. This transformation is extreme
in higher flies, such as Drosophila, in which the tissue remodeling occurs almost
exclusively through cellular replacement. The striking exception to this pattern is the
central nervous system. Although the CNS shows abundant cell death and extensive
addition of new neurons, a central core of the adult CNS is provided by neurons that
are carried forward from the larva. In response to 20E, these larval neurons undergo
an extreme cellular remodeling, as they transition from their larval to their adult
forms. Most studies on this neuronal remodeling have been carried out on easily
accessible neurons such as motoneurons and sensory neurons but relatively little
attention paid to the interneurons. Consequently, it is not known how variable is the
remodeling response [and is this variability encoded in the hormone response
pathways], whether cellular persistence means that there is a similar persistence of
circuit motifs from larva to adult, and if functional requirements in one stage place
constraints on the functional capacity of neurons in the other?
Using GAL4 enhancer lines and multi-color flip out techniques, we have been
constructing an atlas of neuronal types for the Drosophila larval CNS. The cells of this
light-level atlas are being linked to their counterparts in an EM reconstruction of the
wiring of the larval CNS directed by the Albert Cardona group. These EM and light
microscope based datasets are then being linked to a collection of driver lines to cover
most of the ~2000 neuron types in the larval CNS. These specific lines are being
constructed using a split-GAL4 intersection strategy. One use of these neuron-specific
lines has been as an efficient way of tracking the fates of interneurons through
metamorphosis using a flip-out strategy that results in the constitutive activation of
LexA in specific larval cells, with the ability to subsequently visualize them at all
subsequent stages using a LexAOp-GFP reporter. The talk will examine the extent of
cellular preservation amongst interneuron populations through metamorphosis and
how strategies of cellular and circuit remodeling may be adapted for the very different
functions of the nervous systems of the two stages.
27 Expression of a glue protein, Salivary gland secretion 3, is triggered by
steroid hormone independently from developmental timeline in
Drosophila melanogaster
Yuya Kaieda, Ryota Masuda, Hajime Ono
Graduate School of Agriculture, Kyoto University, Kyoto, 606-8502, Japan.
The proper development of animals usually follows as programmed timeline. One of
cues to regulate developmental timing is steroid hormone. During the steroid
hormone biosynthesis in insects, ecdysone (E) is synthesized in the prothoracic gland
(PG) and hydroxylated to the molting hormone, 20-hydroxyecdysone (20E), in
peripheral tissues. In Drosophila melanogaster, definite peaks of 20E are observed at
the time of developmental transitions including molting and metamorphosis. Besides,
three small peaks of 20E are observed at the early third larval instar (L3) stage prior
to the large peak around the time of pupariation. One of the early peaks is required
for expression of genes coding for glue proteins in the salivary glands. The regulation
of expression of a glue protein, Salivary gland secretion 3 (Sgs3), by 20E has been
well characterized as a model for 20E-regulated gene cascades.
To characterize each 20E titer during larval stage, we temporally over-expressed an
ecdysteroid- inactivating enzyme, CYP18A1, in the PG using the GeneSwitch-GAL4
system. In this system, E/20E was inactivated when animals were fed with a
progesterone analog, RU486. When L1 larvae were fed with RU486, all animals died
at L1 stage. This developmental arrest was restored by feeding E or 20E, indicating
that application of RU486 could inactivate E/20E. We examined the onset of Sgs3
expression in the salivary glands using animals containing transgene expressing
Sgs3:GFP fusion protein. By feeding RU486 to L3 larvae, a half of them underwent
prolonged L3 stage and then died. When these prolonged-L3 larvae were fed with 20E
at 48h after L3 ecdysis corresponding to timing of the onset of pupariation, most of
them died without pupariation but Sgs3-GFP expression was observed. In the case of
L2 larvae, more than half of them died after prolong L2 stage by feeding RU486.
These larvae mostly molted to L3 stage by feeding 20E at 0, 6 or 12h after L2
ecdysis. In contrast, when these larvae were fed with 20E at 18 or 24h after L2
ecdysis, most of them did not molt, but Sgs-GFP expression was observed even at
prolonged L2 stage. Sgs3-GFP expression was also triggered by 20E application in
prolonged L1 larvae fed with RU486. These results suggest that 20E-responsiveness
for Sgs3 expression in the salivary glands cells can be acquired even in aberration of
developmental timeline.
28 Contribution of ecdysone to imaginal discs growth
Leire Herboso (1), Marisa M. Oliveira (2), Enric Ureña (3), Ana Talamillo (1),
Coralia Pérez (1), Monika González (1), David Martín (3), James D. Sutherland
(1), Alexander W. Shingleton (4), Christen K. Mirth (2) and Rosa Barrio (1).
(1) CIC bioGUNE, Derio, Bizkaia, Spain. (2) Instituto Gulbenkian de Ciência, Rua da Quinta Grande, 6, 2780-156
Oeiras, Portugal. (3) Institute of Evolutionary Biology, CSIC–Universitat Pompeu Fabra, 08003 Barcelona, Spain.
(4) Lake Forest College, 555 North Sheridan Road, Lake Forest, IL 60045, USA.
Animals have a determined species-specific body size, which results from the
combined action of hormones and signaling pathways regulating growth rate and
duration. In Drosophila, the steroid hormone ecdysone regulates developmental
transitions, thereby regulating the duration of the growth period. Here we show that
ecdysone promotes the growth of imaginal discs in mid-third instar larvae, as imaginal
discs from larvae with reduced or no ecdysone synthesis are smaller than wild type
due to smaller and fewer cells. Our data provide new insights into the relationship
between insulin/insulin-like/Tor and ecdysone pathways in the control of organ
growth. The role of ecdysone in growth control is conserved in hemimetabolous
insects.
29 Ecdysone directly stimulates growth of the Drosophila wing
Natalie A. Dye and Suzanne Eaton
MPI-CBG, Pfotenhauerstrasse 108, 01307 Dresden, Germany.
Ecdysteroids have well characterized functions in regulating the major developmental
transitions in arthropods. In Drosophila, peaks of ecdysteroid biosynthesis regulate
larval molting and pupariation. The ecdysone peak just prior to pupariation is known
to initiate the unfolding and expansion of imaginal tissues like the wing disc. Here, we
show that lower levels of ecdysone present during the third larval instar have a
different effect on imaginal discs: they directly promote normal growth. We
demonstrate that genetically blocking ecdysone production by the ring gland during
the third instar inhibits proliferation in the wing disc and reduces wing disc size. The
effect is mimicked by expressing a dominant negative version of the ecdysone
receptor in the wing disc, suggesting that ecydsone acts directly on the wing to
promote its growth. We also found that low levels of ecdysone are sufficient to
support long-term proliferation of dissected wing discs in culture. To characterize the
pattern and dynamics of growth in culture, we performed spinning disc microscopy on
wing discs expressing E-Cadherin-GFP and imaged at cellular resolution. Previous
clonal analysis has shown that in vivo the wing grows in a reproducibly oriented
pattern, and analysis of spindle orientation in fixed tissue has suggested that oriented
cell divisions underlies this oriented tissue growth. We found that wing discs cultured
without ecdysone but with insulin, which has been previously shown to stimulate cell
divisions in culture, do not exhibit oriented tissue growth. In contrast, wing discs
cultured with ecdysone exhibit a tissue growth pattern that is consistent with in vivo
clonal analysis. We quantitatively analyzed the contributions of cell divisions, cell
rearrangements and cell deformations to tissue growth during ecdysone culture.
Interestingly, this analysis reveals that cell divisions are not the only process
underlying oriented growth in the wing disc; oriented cell rearrangements and cell
shape changes make quantitatively similar contributions. Ecdysone may act to
stimulate growth, at least in part, by maintaining morphogen signaling in the disc, as
accumulation of Wingless and Hedgehog depends on ecdysone both in culture and in
vivo. Our findings identify a mechanism for imaginal growth regulation that helps to
integrate local cues with hormonal signals, and also help overcome a long-standing
technical barrier to studying tissue growth in this important model system.
30 Juvenile hormone and development of the optic lobe in Drosophila
Lynn M. Riddiford, Aljoscha Nern, and James W. Truman
Janelia Research Campus, Howard Hughes Medical Institute, 19700 Helix Drive, Ashburn, VA 20175, USA.
In Drosophila melanogaster, the reappearance of juvenile hormone (JH) at the time of
pupariation is necessary to prevent ecdysteroid-induced premature differentiation of
the optic lobe during the prepupal period (Riddiford et al., Development, 2010). Now
we show that JH must then disappear before the onset of adult development to allow
normal adult differentiation. One manifestation of its continued presence is the
suppression of the ecdysone receptor EcR-B1 that normally occurs in the optic lobe
during early adult development. Using different GAL4 lines to mark specific
interneuron types in the optic lobe, we find that many types are sensitive to JH but a
few are not. An interesting contrast is provided by two of the main lamina
interneurons, L1 and L5. The maturation of L5 is insensitive to JH treatment while
that of L1 is suppressed by JH application at pupariation (P0), except for the very
oldest L1 neurons at the posterior edge of the lamina. Treatment with JH at
successively later times results in progressively more cells being able to mature until
all of them are JH-insensitive by 24 hr after pupariation. Although the L5 cells are
able to mature regardless of the time of JH treatment, at the early treatment times
most cells lack a proximal arbor which is the main site of L1 contact. With
progressively later treatments more L5s acquire this synaptic arbor in step with the
L1 cells that can mature. We think that the JH effect on L5 is indirect and mediated
through its inhibition of L1 maturation. The loss of JH sensitivity in the optic lobe
correlates with the appearance of EcR-B1. By contrast, the up-regulation of Broad Z3
is unaffected by JH. Experiments are underway to determine whether the suppression
of EcR-B1 and/or the up-regulation of Kr-h1 are involved in the JH disruption of adult
differentiation in the optic lobe and how these may relate to L1 and L5.
31 Coordination of growth and development during regeneration
Jacob S. Jaszczak, Jacob B. Wolpe, Anh Q. Dao, and Adrian Halme
Department of Cell Biology, University of Virginia School of Medicine, 1340 Jefferson Park Avenue, Charlottesville, VA
22908.
Our laboratory has been working to address a basic issue of metazoan growth
regulation: How distinct tissues coordinate growth in order to produce adults with
consistent proportion. During Drosophila melanogaster larval development, damage
to imaginal discs activates a regeneration checkpoint through Dilp8 release from
damaged tissues. This produces both a delay in developmental timing and slows the
growth of undamaged tissues, coordinating regeneration of the damaged tissue with
developmental progression and overall growth. We have demonstrated that Dilp8dependent growth coordination between regenerating and undamaged tissues, but
not developmental delay, requires the activity of nitric oxide synthase (NOS) in the
prothoracic gland. NOS limits the growth of undamaged tissues by reducing ecdysone
biosynthesis – a requirement for imaginal disc growth during both the regenerative
checkpoint and normal development.
Therefore, NOS activity in the prothoracic gland translates information about the
growth of individual tissues into coordinated tissue growth through regulation of
endocrine signals.
Consistent with this model, we have identified a putative Dilp8 receptor that is
required in the PG to regulate growth during regenerative checkpoint activation, and
also functions in the larval brain to regulate growth and developmental timing. We are
currently characterizing this receptor’s activity and association with Dilp8.
32 Genetic interactions between E93, Krüppel homolog-1 and BroadComplex transcription factors underlie the formation of the
holometabolous pupa
Enric Ureña, Xavier Franch-Marro and David Martin
Institute of Evolutionary Biology (CSIC-Universitat Pompeu Fabra), Passeig Marítim de la Barceloneta 37-49, 08003
Barcelona, Spain.
Complete metamorphosis (Holometaboly) is a key innovation that underlies the
spectacular success of holometabolous insects. Holometabola form a monophyletic
group that evolved from ancestors that exhibit hemimetabolous development
(Hemimetaboly). Unfortunately, the molecular mechanisms underlying this crucial
transition, including the occurrence of the holometabolan-specific pupal stage, are
poorly understood. Despite the differences, the genetic switch between juvenile and
adult programs in both types of insects relies on the disappearance of juvenile
hormone (JH), which prevents metamorphosis until the final juvenile stage through
the induction of the anti-metamorphic krüppel-homolog 1 (Kr-h1) transcription factor.
A second important metamorphic gene is Broad-complex (Br-C), although its role, in
contrast to Kr-h1, is specific of holometabolus insects in the formation of the pupal
form. In addition to these two genes, our group has recently identified E93 as the
critical transcription factor that promotes adult metamorphosis in both
hemimetabolous and holometabolous insects, thus acting as the conserved adult
specifier in all winged insects (1).
Using gene silencing by RNAi in the German cockroach Blattella germanica, the red
flour beetle Tribolium castaneum and the fruit fly Drosophila melanogaster, we show
that depletion of E93 prevents adult metamorphosis and induces endless repetitions
of nymphal molts in hemimetabolous insects and the repetition of the pupal program
in holometabolous insects. In addition to promoting adult differentiation, we show
that E93 is also required to repress the expression of Kr-h1 and Br-C during the last
immature stages of hemimetabolous and holometabolous insects ensuring the
accurate transition to the adult forms. Finally, we demonstrate that critical changes in
the expression and in the regulatory interactions between E93, Kr-h1 and Br-C factors
during the final larval stage of holometabolous insects have played a critical role in
facilitating the formation of the pupal stage. In particular, we show that the transient
pulse of Kr-h1 at the end of the larval development prevents the direct transformation
of larval tissues to adult ones by maintaining low levels of E93 during this period and,
in doing so, allowing the strong up-regulation of TcBr-C and the occurrence of a new
developmental stage, the pupa.
(1) Ureña, E., Manjón, C., Franch-Marro, X. and Martin, D. (2014) Proc. Natl. Acad.
Sci. USA 111(19): 7024-7029.
33 Optimal growth schedule of holometabolous insects
Ken-ichi Hironaka and Yoshihiro Morishita
RIKEN Center for Developmental biology (CDB), 2-2-3 Minatojima-minamimachi, Chuo-ku, Kobe, Hyogo 650-0047,
Japan.
A holometabolous insect larva becomes committed to metamorphosis when it
reaches a critical weight. Although physiological mechanism involved in this process
is well-studied, adaptive significance of the critical weight is still unclear. Here, we
develop a life history model for holometabolous insects and analyzed it with optimal
control theory. Without explicit assumption of the critical weight, optimal growth
schedule is biphasic, showing that the existence of the critical weight is a natural
consequence of optimal energy allocation. Our model can estimate the optimal timing
of the critical weight from a few physiological parameters, that are in good
agreement with observation in Drosophila melanogaster and Manduca sexta.
Furthermore, our model also accurately predicts developmental response to
environmental variation observed in those species. These results suggest that the
critical weight has an important role not only in commitment to metamorphosis but
also in predictive adaptive response to environmental change.
34 Unstable character of transcriptional repressor Blimp-1 contributes to
accurate time measuring system for pupation timing in Drosophila
melanogaster
Hitoshi Ueda, Haruka Nishida, Abdel-Rahman Sultan, Kazutaka Akagi, Takumi
Nakayama and Moustafa Sarhan
The Graduated School of Natural Science and Technology, Okayama University, Japan.
In Drosophila melanogaster, pupation occurs about 12 hours after puparium
formation at the standard culture condition at 25 °C. We have been studying timer
system to determine pupation timing and have shown the importance of the
regulation of the ftz-f1 gene encoding transcriptional activator by the transcriptional
repressor Blimp-1. Here we address the mechanism to determine pupation timing
accurately.
First we showed that the expression timing of the ftz-f1 gene is critical to determine
pupation timing, indicating that control of the expression timing of the ftz-f1 gene is
important to understand the accuracy of the system. Next, we tried to change factors
which affect the expression of the ftz-f1 gene and effect on the accuracy of the
pupation timing was observed. The results revealed that the expression levels of
these factors did not have strong effect. In contrast, the stability of Blimp-1 protein
gave obvious effect on the accuracy of pupation timing. We expect that unstable
character of Blimp-1 protein contribute to the accuracy of the time measuring system
for pupation.
35 Attainment of critical weight is necessary for E93 up-regulation in
Tribolium castaneum
Silvia Chafino, Enric Ureña, Elena Casacuberta, David Martin and Xavier Franch-Marro
Institute of Evolutionary Biology (CSIC-Universitat Pompeu Fabra), Passeig Marítim de la Barceloneta 37-49, 08003
Barcelona, Spain.
Insects have undergone extensive evolution in their development since their
origination from arthropod ancestors, such that three types of metamorphosis have
emerged: ametaboly, hemimetaboly and holometaboly. These three forms of
metamorphosis represent an evolving sequence from the primitive ametabolous
(direct-developing) to hemimetabolous (incomplete metamorphosis) to the most
derived holometabolous type of metamorphosis (complete metamorphosis). Two
events are critical to promote complete metamorphosis during the onset of the last
larval instar in holometabolous species: (i) the up-regulation of E93, which induces
adult differentiation, and (ii) the disappearance of JH and the anti-metamorphic factor
Kr-h1. Despite the importance of both events, however, the identity of the stagespecific signal(s) that control such events is poorly understood. One of the principal
candidates to exert this function is the Critical Weight (CW). In holometabolous
insects, the CW has been defined as the minimal size threshold that is necessary to
end larval development and initiate metamorphosis. It has been reported that once
critical weight is reached, JH titer declines, thus, down-regulating its target gene Krh1 to allow metamorphosis. Interestingly, we have also observed that CW attainment
also correlates with the up-regulation of E93, suggesting a functional link between
E93 and CW. To address this crucial question, we have used the beetle Tribolium
castaneum as a model as this insect presents a variable number of larval stages.
Under our rearing conditions, pupation takes place after seven larval instars (L1-L7).
However, when we rear L7 larvae under poor-nutritional conditions, these larvae
undergo new larval molting (until reaching L10) instead of pupating.
Interestingly, under these conditions expression of E93 is not activated and Kr-h1
expression remains high. Remarkably, depletion of E93 in the last larval stage L7
Tribolium induces molting into new larvae. Therefore, our results show a strong
relationship between the CW and the up- regulation of E93 to allow metamorphosis.
36 RNAi of insulin pathway genes in larval stages of Tribolium castaneum
demonstrates regulation in feeding and metamorphosis
Xianyu Lin, Na Yu, Guy Smagghe
Department of Crop Protection, Faculty of Bioscience Engineering, Ghent University, Coupure links 653, 9000 Ghent,
Belgium.
Insects require energy and nutrients from their food to support development.
Insulin/TOR signaling pathway is considered to be a nutrient sensitive pathway,
however, our recent studies reveal an important role of this pathway in the food
intake regulation in the red flour beetle, Tribolium castaneum. Reducing insulin
receptor (InR) expression using RNAi markedly decreased food intake during larval
stage. This effect concurred with an increase of SK expression. In addition, TORsilenced larvae formed smaller pupae and showed defects on the body after pupation
with typically reduced adult appendages. In FOXO level-reduced insects, it was of
great interest to observe a delay of metamorphosis with 4 days. Here we investigated
the expression of genes coding for enzymes in the biosynthesis of ecdysteroids and
juvenile hormone (JH).
The data are discussed in relation to the crosstalk of different insect hormone
signaling pathways in the regulation of food intake and metamorphosis.
37 Signaling relay and feedback mechanisms control the nutrientdependent production of insulin-like peptides in Drosophila
Naoki Okamoto & Takashi Nishimura
Laboratory for Growth Control Signaling, RIKEN Center for Developmental Biology, Japan.
Members of the insulin-like peptides (ILPs) play key biological roles in the regulation
of growth, metabolism, developmental timing, fecundity and lifespan under the
control of nutrients in a wide variety of metazoans. Although functions of ILPs are well
understood, the mechanisms underlying how organisms sense the nutrient status and
thereby control the production of ILPs still remain largely unknown. In the fruit fly
Drosophila, ILPs (Dilps) are mainly produced by brain neuroendocrine cells known as
insulin-producing cells (IPCs). To better understand the interface between nutrient
status and Dilp production in the IPCs, we focused on the expression of dilp5, one of
the IPC-derived Dilps which gene expression is tightly regulated by nutrient
availability.
Our previous work revealed that transcription factors Dachshund (Dac) and Eyeless
(Ey) synergistically and directly promote dilp5 expression in the IPCs (Okamoto et al.,
PNAS 2012). However, it remained unclear how these two transcription factors are
involved in the nutrient- dependent expression of dilp5.
Here, we show that transcription factor Forkhead box O (FoxO) is a critical
transcription factor that negatively regulates the nutrient-dependent dilp5 expression.
FoxO is highly expressed in the IPCs, and its localization is tightly regulated by
nutrient availability. In the starved condition, FoxO localizes at nucleus and directly
binds to Ey, thereby competing the interaction between Ey and Dac. We further found
that the FoxO activity in IPCs is mainly regulated by receptor tyrosine kinase
Anaplastic lymphoma kinase (Alk), through PI3K signaling pathway. The Alk ligand
Jelly belly (Jeb) is produced by cholinergic neurons and directs a signal to the IPCs in
a non-cell autonomous manner. Furthermore, Dilp6 produced in the surface glia acts
as a nutrient signal to remotely regulate dilp5 expression through Jeb. The secreted
Dilps into the hemolymph from the IPCs in turn amplifies the production of Dilp6
together with the amino acid signals in the surface glia. Together, these results
provide a molecular framework to explain how the production of an endocrine
hormone in specific tissue coordinates with environmental conditions.
38 The nutrient-responsive hormone CCHamide-2 controls growth by
regulating insulin-like peptides in the brain of Drosophila
melanogaster
Hiroko Sano (1)*, Akira Nakamura (2), Michael J. Texada (3), James W. Truman
(3), Hiroshi Ishimoto (4), Azusa Kamikouchi (4), (5), Yutaka Nibu (6), Kazuhiko
Kume (7), Takanori Ida (8), and Masayasu Kojima (1)
(1) Department of Molecular Genetics, Institute of Life Science, Kurume University, Kurume, Fukuoka 839-0864,
Japan(2) Department of Germline Development, Institute of Molecular Embryology and Genetics, Kumamoto
University, Kumamoto 860-0811, Japan(3) Janelia Research Campus, Howard Hughes Medical Institute, Ashburn,
VA 20147, USA(4) Graduate School of Science, Nagoya University, Nagoya, Aichi 464-8602, Japan(5) Precursory
Research for Embryonic Science and Technology, Japan Science and Technology Agency, Tokyo 102-0076, Japan(6)
Department of Cell and Developmental Biology, Weill Medical College of Cornell University, New York, NY 10065,
USA(7) Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya, Aichi 467-8603, Japan(8)
Division for Searching and Identification of Bioactive Peptides, Department of Bioactive Peptides, Frontier Science
Research Center, University of Miyazaki, Miyazaki, Miyazaki 889-1692, Japan.
The coordination of growth with nutritional status is essential for proper development
and physiology. Nutritional information is mostly perceived by peripheral organs
before being relayed to the brain, which modulates physiological responses. Hormonal
signaling ensures this organ-to- organ communication, and the failure of endocrine
regulation in humans can cause diseases including obesity and diabetes. In Drosophila
melanogaster, the fat body (adipose tissue) has been suggested to play an important
role in coupling growth with nutritional status. Here, we show that the peripheral
tissue-derived peptide hormone CCHamide-2 (CCHa2) acts as a nutrient- dependent
regulator of Drosophila insulin-like peptides (Dilps). A BAC-based transgenic reporter
revealed strong expression of CCHa2 receptor (CCHa2-R) in insulin-producing cells
(IPCs) in the brain. Calcium imaging of brain explants and IPC-specific CCHa2-R
knockdown demonstrated that peripheral-tissue derived CCHa2 directly activates
IPCs. Interestingly, genetic disruption of either CCHa2 or CCHa2-R caused almost
identical defects in larval growth and developmental timing.
Consistent with these phenotypes, the expression of dilp5, and the release of both
Dilp2 and Dilp5, were severely reduced. Furthermore, transcription of CCHa2 is
altered in response to nutritional levels, particularly of glucose. These findings
demonstrate that CCHa2 and CCHa2-R form a direct link between peripheral tissues
and the brain, and that this pathway is essential for the coordination of systemic
growth with nutritional availability.
39 Regulation of longevity in castes of ants
Hua Yan (1), Comzit Opachaloemphan (1), Giacomo Mancini (2), Steven Shen (1, 3),
Roberto Bonasio (4, 5), Jürgen Liebig (6), Shelley Berger (4, 5, 7, 8), Claude Desplan
(2, 9) Danny Reinberg (1, 10)
(1) Department of Biochemistry and Molecular Pharmacology, New York University School of Medicine, New York, NY
10016, USA. (2) Department of Biology, New York University, 100 Washington Square East, New York, NY 10003,
USA. (3) Center for Health Informatics and Bioinformatics, New York University School of Medicine, New York, 10016,
USA. (4) Department of Cell and Developmental Biology, University of Pennsylvania Perelman School of Medicine,
Philadelphia, Pennsylvania 19104, USA. (5) University of Pennsylvania Epigenetics Program, Philadelphia,
Pennsylvania 19104, USA. (6) School of Life Sciences, Arizona State University, Tempe, Arizona 85283, USA. (7)
Department of Biology, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA. (8) Department of
Genetics, University of Pennsylvania Perelman School of Medicine, Philadelphia, Pennsylvania 19104, USA. (9) New
York University Abu Dhabi, Center for Genomics and Systems Biology, Abu Dhabi, United Arab Emirates. (10)
Howard Hughes Medical Institute, New York University School of Medicine, New York, NY 10016, USA.
Ants live in a highly organized and social environment. In an ant colony, the queen
and workers are almost genetically identical, but exhibit striking differences in
behavior and lifespan. In contrast to most social insect species, adult individuals in
the ant Harpegnathos saltator display strong phenotypic plasticity: The loss of
reproductive individuals in a colony triggers dominance fight among workers, whereby
some workers become reproductive gamergates and exhibit queen-like behavior.
Notably, the worker to gamergate transition extends the lifespan by approximately 5
fold. In most animals, longevity is associated with decreased insulin signaling, which
suppresses reproduction. Therefore, the positive correlation between longevity and
reproduction in social insects raises an intriguing question: How does the insulin
pathway regulate reproductive longevity? In H. saltator, there are two insulin-like
peptides (ilps). The expression of both ilps is upregulated during the worker to
gamergate transition. Interestingly, we found that one ilp may functionally inhibit
insulin signaling when overexpressed in Drosophila. This finding suggests that ilps in
H. saltator may differentially regulate reproduction and longevity, thereby enabling
gamergates to keep reproducing in their long lives.
40 The Drosophila TNF Eiger is an adipokine that mediates nutrient
response
Neha Agrawal (1-3), Renald Delanoue (1-3), Alessandra Mauri (1-3) and Pierre Léopold
(1-3)
(1) University of Nice-Sophia Antipolis, Institute of Biology Valrose, Parc Valrose, 06108 Nice, France. (2) CNRS,
Institute of Biology Valrose, Parc Valrose, 06108 Nice, France. (3) INSERM, Institute of Biology Valrose, Parc
Valrose, 06108 Nice, France. Adaptation of organisms to ever-changing nutritional environments relies on sensor
tissues and systemic signals. Identification of these signals would help understand the
physiological cross-talk between organs contributing to growth and metabolic
homeostasis. Here we show that Eiger, the Drosophila TNF-a, acts as a metabolic
hormone mediating nutrient response by remotely acting on brain insulin-producing
cells (IPCs). In condition of nutrient shortage, a metalloprotease of the TNF-alpha
converting enzyme (TACE) family is active in fat body (adipose-like) cells, allowing the
cleavage and release of adipose Eiger in the hemolymph. In the brain IPCs, Eiger
activates its receptor Grindelwald leading to JNK-dependent inhibition of insulin
production. Therefore, we have identified a humoral connexion between the fat body
and the brain insulin-producing cells relying on TNF-alpha signaling and mediating
adaptive response to nutrient deprivation. 41 Insulin signaling cascade in linden bug, Pyrrhocoris apterus
(Heteroptera)
Jan Provaznik (1, 2) ($), Milena Damulewicz (1) ($), Martin Pivarci (1, 2) ($), Ivan
Fiala (1, 2), Joanna Kotwica-Rolinska (1), Hanka Vaneckova (1), and David Dolezel
(1, 2)
[$: equal contribution]
(1) Biology Center, Academy of Sciences of the Czech Republic, 37005 Ceske Budejovice, Czech Republic. (2) Faculty
of Science, University of South Bohemia, 37005 Ceske Budejovice, Czech Republic.
The linden bug, Pyrrhocoris apterus, served as a powerful model of insect
endocrinology for decades. However, our knowledge of actual genes coding for
neurohormones and their receptors was limited. Therefore we have used massively
parallel sequencing and peptidomic approaches to identify genes, transcripts and
corresponding processed neuropeptides.
In silico quest for neurohormone receptors identified dozens of GPCRs, Eclosion
hormone receptor and a few Tyrosin Kinase receptors. Remarkably, we have identified
3 homologs of insulin receptors (InRs) in P. apterus. All these 3 InRs contain
characteristic domains: Receptor L- domains, Furin-like cysteine rich region,
Fibronectin type III and Tyrosin kinase. Phylogenetic analysis revealed these InRs are
results of two independent gene duplications. First duplication event happened early
in insect evolution resulting in two distinct InR clades observed in most (but not all)
insect orders. Second (recent) gene duplication is specific to P. apterus, where the
original intron-containing gene is branching together with its derived intronless
homolog.
Despite evolutionary relationship we were able to design two specific non-overlapping
dsRNA fragments for every P. apterus’s InR which then selectively knocked down
expression of targeted transcript without affecting expression of two remaining
paralogs. RNA interference of 3 insulin receptors, 2 insulin-like peptides (ligands) and
insulin receptor substrate (Chico) significantly reduced reproduction even after
individual gene knockdown, suggesting these multiple InRs paralogs are not
redundant. Organ culture experiments on isolated fat bodies then identified specific
role for particular InR in regulating vitellogenin expression. These data suggest that
multiple InRs can function in tissue specific manner.
42 The Retinal Determination Gene Network is Required for Proper
Development and Function of Insulin-Producing Cells
Jason Clements (1) ($), Kurt Buhler$ (1) ($), Sofie De Groef (2), Harry Heimberg
(2), Patrick Callaerts (1) [$ equal contribution]
(1) Laboratory of Behavioral and Developmental Genetics, Center for Human Genetics, K.U.Leuven & Center for
the Biology of Disease, VIB, Leuven, Belgium. (2) Beta Cell Neogenesis Laboratory, Diabetes Research Center, Vrije
Universiteit Brussel, Brussels, Belgium.
The Drosophila insulin-producing cells (IPCs) are a cluster of 14 neurons located in
the pars intercerebralis of the brain. These neurons produce three insulin-like
peptides (Dilp2, -3, and -5), which are secreted and known to have roles in growth
and development, metabolism, longevity, reproduction, and behavior. Previous work
by us and others has demonstrated a requirement for Eyeless and Dachshund, two
transcription factors in the retinal determination (RD) gene network, in IPC
development and regulation of dilp transcription. Furthermore, in a screen by
Buhler, Clements et al. examining the role of putative Eyeless transcriptional targets
in IPC development and function, the RD genes homothorax and tiptop were also
found be required in the IPCs. In light of these data, we have explored the
requirement of the complete RD network in the development and function of the IPC
neurons. We find that the majority of the RD network is expressed in the IPC
neurons, and that these genes are required for the regulation of neuronal
differentiation and control of dilp transcription. In addition, we have investigated the
expression of the homologous RD genes in the mouse pancreas, and demonstrate
that a number of these transcripts are up-regulated in a mouse β cell differentiation
model.
43 Ecdysone Regulates Insulin Signaling in the Insulin-Producing Cells
of Drosophila to Determine Normal Growth and Developmental
Timing
Kurt Buhler (1) ($), Jason Clements (1) ($), Korneel Hens (2), Magali de Bruyn (1),
Yiyun Chen (3), Feng Wang (3), Rui Chen (3), Patrick Callaerts (1) [$: equal
contribution]
(1) Laboratory of Behavioral and Developmental Genetics, Center for Human Genetics, K.U.Leuven & Center for
the Biology of Disease, VIB, Leuven, Belgium. (2) Centre for Neural Circuits and Behaviour, The University of
Oxford, Tinsley Building, Mansfield Road, Oxford OX1 3SR, UK. (3) Program in Developmental Biology &
Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030, USA.
Growth and developmental timing are processes coupled by many intersecting
hormonal and other signalling cascades during development of an organism. In
Drosophila, the Drosophila insulin-like peptides (Dilps) produced by the insulinproducing cells (IPCs) are key factors in the regulation of metabolic and growth
homeostasis, whereas maturation is determined by cascading peaks of the steroid
hormone Ecdysone. While these hormone signals intersect in both peripheral tissues
and the Ecdysone-producing Prothoracic gland, nothing is known about the role of
Ecdysone signalling in the IPCs themselves. We show or the first time that Ecdysone
signaling components are necessary for the development and function of the IPCs,
where they regulate transcription of dilp3 and dilp5 and orchestrate normal growth
and development. This suggests the existence of an autoregulatory loop between
the insulin and Ecdysone-producing tissues to mediate developmental growth and
progression.
44 Pupal commitment of a single-celled Verson’s gland is induced in a
two-step process by the insulin and TOR/Akt signals, which occurs
gradually, not on an all-or-none basis
Kiyoshi Hiruma and Yu Kaneko
Faculty of Agriculture and Life Sciences, Hirosaki University, Hirosaki 036-8561, Japan.
During the pupal metamorphosis in insects, cellular commitment for pupal
differentiation must precede before its differentiation. The pupal commitment of
Bombyx mori epidermis occurs from day 3 to day 6 5th (last) instar larvae in
response to the gradual increase in ecdysteroid titer in the absence of juvenile
hormone (JH). Although pupal commitment of epidermis occurs gradually over three
days, it is uncertain whether or not the commitment of an individual epidermal cell
occurs in the same fashion. A single-celled Verson’s gland in Bombyx mori, grows
bigger than an adult Drosophila, expressed a number of larval- and pupal-specific
genes, and we were able to use them as molecular markers for pupal commitment of
a single cell. Pupal commitment of Verson’s glands occurred during the feeding stage,
1-3 days after the last larval ecdysis, the time at which little ecdysteroid and JH were
detected. When we used a day 2 5th Verson’s gland, which expressed some larvalbut no pupal-specific genes, for the commitment assay, the transcripts of both type of
genes were co-expressed in a single secretory cell. Therefore, pupal commitment of a
cell does not occur on an all-or-none basis, but rather gradually.
Application of JH and starvation prevented its commitment, so that nutrition is an
important factor rather than ecdysone for the process. Injection of insulin to starved
larvae prevented the expression of larval-specific genes but had no effect on pupalspecific genes. Therefore, insulin prevents larval-specific genes to express but is
unable to induce pupal commitment. Suppression of Akt by RNAi prevented the pupal
commitment but InR RNAi did not, and the suppression of both InR and Akt had little
effect on larval commitment. Therefore, part of the nutritional signal might be due to
the TOR signaling pathway. RNAi of TOR had the same effect as Akt RNAi on the
commitment, but TOR had little effect on the Akt expression so that TOR is likely
responsible for the phosphorylation of Akt. Based on these results, pupal commitment
of a Verson’s gland is composed of two different steps. In the absence of JH, part of
the insulin signal that is stimulated by nutrition tears down the larval status, and then
the TOR signal activates Akt and the activated Akt induces final pupal commitment.
Supported by JSPS.
45 Regulation of Gene expression Patterns in Mosquito Reproduction
Sourav Roy, Tusar T Saha, Lisa K Johnson, Bo Zhao, Jisu Ha and Alexander S Raikhel
Department of Entomology, University of California, Riverside 900 University Ave., Riverside, CA 9252.
In multicellular organisms, development, growth and reproduction require coordinated
expression of numerous functional and regulatory genes. Insects represent
outstanding model organisms for studying regulatory mechanisms of synchronized
gene expression due to their rapid development and reproduction. Diseasetransmitting female mosquitoes have adapted uniquely for ingestion and utilization of
the huge blood meal required for rapid reproductive events to complete egg
development within a 72-h period. We investigated the network of regulatory factors
mediating sequential gene expression in the fat body, a multifunctional organ
analogous to the vertebrate liver and adipose tissue. Transcriptomic and
bioinformatics analyses revealed that ~7500 genes are differentially expressed, most
of this occurs in four sequential waves over the 72-h reproductive period, within the
fat body. Using a combination of RNA interference gene silencing and an in-vitro
organ culture, we identified the major regulators responsible for up and
downregulation of the co-expressed gene sets. We detected the first wave of gene
activation, regulated by amino acids (AAs), between 3 h and 12 h post-blood meal
(PBM); genes within this set are later repressed by 20-hydroxyecdysone (20E),
through its receptor EcR. During the second wave, between 12 h and 36 h PBM, most
genes are highly upregulated by a synergistic action of AAs, 20E and EcR. The
expressions of these genes decrease with a decline in the 20E titer; the nuclear
receptor HR3 augments the downregulation. Between 36 h and 48 h PBM, the third
wave of gene activation—regulated mainly by HR3—occurs. These genes were found
to be downregulated by 20E and EcR during the early period PBM, and by juvenile
hormone (JH) through its receptor Methoprene-tolerant (Met) during the later stage
PBM. JH and Met were found to be the major regulators for the final wave of gene
activation between 48 h and 72 h PBM, and representatives of this gene set were
found to be repressed by AAs during the early period PBM. We found that insulin has
a limited role during this period—activating just the yolk protein precursor genes,
which are a subset of the second co-expressed gene set—and it is active only in
combination with AAs and 20E. Taken together, our study provides a better
understanding of the complexity of the regulatory mechanisms responsible for the
temporal coordination of gene expression during reproduction in the female Aedes
aegypti mosquito.
46 Mating-induced increase in female germline stem cells requires
ovarian ecdysteroid biosynthesis and neuronal sex peptide
signaling in Drosophila melanogaster
Tomotsune Ameku and Ryusuke Niwa
1-1-1 Tennodai, Tsukuba-shi, Ibaraki-ken, Japan.
Gametogenesis and mating are two essential components of animal reproduction.
Gametogenesis is modulated by the need for gametes, yet little is known of how
mating, a process that consumes gametes, may modulate the process of
gametogenesis. Here, we show a relationship between mating stimulus and female
germline stem cell (GSC) proliferation via the major insect steroid hormone
ecdysteroid in the fruit fly Drosophila melanogaster. We found that mating stimulated
an increase in the number of GSCs and ecdysteroid levels in the adult ovary. To
examine whether mating-induced ovarian ecdysteroid biosynthesis is crucial for
inducing GSC proliferation, we established adult ovary-specific RNAi animals for
neverland (nvd), which encodes the ecdysteroidogenic enzyme responsible for
converting dietary cholesterol into 7- dehydrocholesterol (7dC). nvd RNAi animals did
not exhibit a mating-induced increase in female GSC number. In addition, the GSC
phenotype was rescued by an oral administration of 7-dC or 20-hydroxyecdysone
(20E). Moreover, we showed that a mating-induced GSC proliferation was regulated
by the male seminal fluid peptide called a Sex peptide (SP) and its receptor SPR, the
important signaling pathway that transduces mating stimulus to a specific subset of
female neurons. SPR RNAi female flies did not show a mating-induced increase in
either ovarian ecdysteroid level or female GSC number. Importantly, the GSC
phenotype in SPR RNAi animals was rescued by an oral administration of 20E. All of
our data suggest that a mating-induced increase in female GSC number is regulated
by the ovarian ecdysteroid whose biosynthesis is positively controlled by neuronal SPSPR signaling in mated female flies. This is the first study to show that GSC
proliferation is under the control of the characterized neuroendocrine system in
response to external stimulus, mating.
47 Critical Roles of Juvenile Hormone Receptor Methoprene-tolerant and
Ecdysone Receptor in Temporal Coordination of Mosquito Metabolism
Alexander S. Raikhel (1), Yuan Hou (2), (3), Xue-Li Wang (2, 3), Tusar T. Saha (1),
Sourav Roy (1), Bo Zhao (1), and Zhen Zou (2)
(1) Department of Entomology and Institute for Integrative Genome Biology, University of California Riverside,
Riverside, CA 92521, USA. (2) State Key Laboratory of Integrated Management of Pest Insects and Rodents, Institute
of Zoology, Chinese Academy of Sciences, Beijing 100101, China. (3) University of Chinese Academy of Sciences,
Beijing 100049, China.
Hematophagous mosquitoes serve as vectors of multiple devastating human diseases
and many unique physiological features contribute to their incredible evolutionary
success. These functions place high-energy demands, and metabolism must be
synchronized with the needs of a reproducing female mosquito. Transcriptomic
profiling have shown that genes encoding enzymes of carbohydrate and lipid
pathways are dramatically repressed at late post eclosion stage and sharply increased
at post-blood meal stage in the mosquito fat body. Consistent with changes in
metabolic genes and enzymes, glycogen, glucose, trehalose and other secondary
metabolites are also periodically accumulated and degraded during the reproductive
cycle. Triacylglycerols, which represent another important energy storage form in the
mosquito fat body, follow a similar tendency. On the other hand, ATP that is
generated by catabolism of secondary metabolites showed an opposite trend. We
used RNA interference approach for the juvenile hormone and ecdysone receptors,
Met and EcR, coupled with transcriptomics and metabolomics analyses to show that
these hormone receptors function as major regulatory switches coordinating
metabolism with the differing energy requirements of the female mosquito throughout
its reproductive cycle. Our study demonstrates how, by hormonally regulated
metabolic reprogramming, an insect adapts to drastic and rapid physiological
changes.
48 KARLSON LECTURE 2015:
Michael B. O’Connor
Defining the molecular mechanisms that modulate metamorphic timing
in Drosophila
Naoki Yamanaka (1,2), Xeuyang Pan (1), MaryJane Shimell (1), Michael B. O’Connor
(1)
(1) Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, USA 55455. (2)
Department of Entomology, University of California, Riverside, USA 92521.
Neuroendocrine circuits play key roles in timing the initiation of juvenile to adult
transitions in many organisms. In insects, release of the neuropeptide prothoraciotropic
hormone (PTTH) from a pair of neurons in each brain hemisphere is thought to be key to
generate pulses of the steroid hormone ecdysone (E) and its active derivative 20hydroxyecdysone (20E) that initiates metamorphosis. PTTH binds to Torso, a RTK that
transduces signals through RAS/RAF/ER, to mediate transcriptional up regulation of E
biosynthetic enzymes in the prothoracic gland (PG). We have eliminated PTTH production
in Drosophila by two methods: a) genetic ablation or mispecfication of the PTTH producing
neurons (PG neurons) and b) through TALEN generated null mutations. PTTH null
mutations produce a 1-day delay during third instar development while genetic ablation or
misspecification of the PG neurons produces a 5-6 day delay. In PTTH null mutants,
transcription of most biosynthetic enzymes is delayed but levels eventually reach
approximately 60% of maximum induction. In contrast, PG neuron ablation or
misspecification eliminates induction of biosynthetic enzymes above basal levels.
However, in both situations, a pulse of E is produced, but with different kinetics. These
results suggest that the PG neurons produce other prothoraciotropic factors in addition to
PTTH. In collaboration with the labs of Kim Rewitz and Kirst King-Jones, we carried out a
genome wide RNAi screen to identify novel genes (including receptors and signal
transduction components) in the PG that affect developmental timing. This screen was
coupled with a temporal micro-array analysis of gene expression in the ring gland.
Together these methods enabled us to identify at least two new signaling pathways that
affect developmental timing: one involves Ca+2 signals and the other involves Tor
signaling and autophagy.
For the Ca+2 signaling pathway, we traced its origin back to a particular Gaq subunit
implying the involvement of an as yet uncharacterized GPCR. As part of this study, we
also discovered an inside-out ABC transporter that we show functions as an ecdysone
pump to fill novel synaptotagmin1 marked vesicles that fuse with the plasma membrane.
This transporter is related to the ABCA1/G1, transporters implicated in cholesterol efflux
from mammalian cells. On the basis of this data, we propose that trafficking of steroid
hormones out of endocrine cells in Drosophila is not through simple diffusion or facilitated
diffusion mechanisms as is presently thought, but instead involves a more complex
GPCR/Ca+2 signal-regulated vesicle release process.
For the Tor/autophagy pathway, we found that starvation before critical weight (CW), but
not after, markedly stimulates autophagy in PG tissue. We also found that activation of
Tor before attainment of CW suppresses starvation induced PG autophagy and leads to
pupariation without developmental delay. We hypothesize that differential activation of
autophagy is the function output of the CW checkpoint. Before CW, autophagy either
destroys or sequesters E biosynthetic enzymes, cholesterol or E itself thereby preventing
pupariation. After CW, starvation does not induce autophagy in the PG resulting in normal
developmental timing. We have also discovered that the Jeb/Alk signaling pathway
appears to regulate post-CW inhibition of autophagy induction since knockdown of Alk in
the PG enables robust autophagic induction after attainment of CW. These results will be
discussed with respect to current models for how developmental timing is regulated in
Drosophila.
49 POSTER ABSTRACTS
Characterization of a Drosophila melanogaster PTTH Null Mutant
MaryJane Shimell, Arpan C. Ghosh, and Michael B. O’Connor
Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, MN 55455 USA.
PTTH plays an important role in developmental timing in insects based on the
ecdysteroidogenic effect of the purified protein in Bombyx mori. These results were
further enforced in Drosophila melanogaster, where overexpression of PTTH results
in accelerated timing and genetic ablation of the PTTH-producing PG neurons leads
to a developmental timing delay. We wished to dissect loss of PTTH alone from loss
as a consequence of PG neuron ablation. However, in the absence of a PTTH null
mutation its singular role has remained elusive.
Here we report the isolation and characterization of a PTTH mutant in Drosophila
melanogaster. The PTTH mutant was assessed for developmental timing and was
found to have a delay of only one day, unlike the five-day delay described in the PG
neuron-ablated animal. The critical weight and terminal growth period of the mutant
was determined and found to be consistent with the delayed phenotype. Unlike the
ablated animals that showed relatively low levels of transcription of the Halloween
genes, we observed peaks of transcription in the PTTH mutant, in most cases at half
the level of wild type, the only difference being that those peaks of transcription
were delayed relative to the wild type. Additionally, the ecdysteroid titer results
were unexpected, with ecdysteroid levels in the whole body and hemolymph of the
mutant paralleling those of the wild type, the only difference being that the PTTH
mutant takes longer to mount the final ecdysteroid surge prior to pupariation. We
also show that PTTH is a tropic factor required for the correct size of the nuclei in PG
cells. These results have led us to re-consider how PTTH signaling through
Ras/Raf/Mek/Erk may affect developmental timing by controlling the amount of DNA
available as a transcriptional template for production of the ecdysone biosynthetic
components.
50 SDR positively regulates insulin/IGF signaling during neuroblast
reactivation in Drosophila
Takayuki Yamada, Naoki Okamoto, and Takashi Nishimura
Laboratory for Growth Control Signaling, RIKEN Center for Developmental Biology, Japan.
Members of the insulin/IGF family peptides are the central player regulating body
growth in a wide variety of metazoans. In the fruit fly Drosophila, nutritional signals
control systemic growth through regulation of the expression and secretion of
Drosophila insulin-like peptide (Dilps).
Secreted Dilps in circulating hemolymph are also subject to functional regulation
through their binding proteins. Despite the important role of Dilps in nutrientdependent growth control, the molecular mechanism that regulates the function of
Dilps remains largely unknown. We previously identified a new class of secreted Dilpbinding proteins that we named Secreted Decoy of InR (SDR). SDR negatively
regulates the function of Dilps and thereby suppresses body growth (Okamoto et al.,
Genes Dev, 2013). Because SDR is dominantly expressed in glial cells in the central
brain, we asked whether SDR plays a local function in brain development. Here we
found that SDR mutants significantly delayed the timing of neuroblast reactivation at
early larval stages, resulted in small brain phenotype. Glia-derived Dilps have been
reported to positively regulate neuroblast re-activation through IIS in neuroblasts.
The forced expression of Dilps and the activation of IIS in the SDR mutant
neuroblasts fully restored the delay of re-activation.
These results indicate that SDR positively regulates neuroblast re-activation in a noncell- autonomous manner, presumably through the interaction and stabilization of
glia-derived Dilps in the developing brain.
51 A search for the novel genes related to juvenile hormone
biosynthesis in Bombyx mori
Yuri Homma, Kazuei Mita, Yuki Nakamura, Toshiki Namiki, Hiroaki Noda, Tetsuro
Shinoda, Toru Togawa
College of Humanities and Sciences, Nihon University, Tokyo, Japan; National Institue of Agrobiological Sciences,
Tsukuba, Japan.
Juvenile hormones (JHs) are sesquiterpenoids functioning in various aspects
including development, reproduction, and polymorphism in insects. JHs are mainly
produced in the corpora allata (CA) from acetyl-CoA (and propyonyl-CoA in
lepidopteran insects) through two phases of biosynthetic pathway, the early
mevalonate pathway and the late JH-branching pathway. These two phases include
eight and five enzymatic steps, respectively. Although all the corresponding genes
have been identified in the mosquito, Aedes aegypti, the enzyme genes responsible
for the conversion of farnesyl pyrophosphate to farnesoic acid remain unknown in
other species including the silkworm, Bombyx mori. To identify the novel genes
related to JH biosynthesis, we searched for the genes whose expression is high and
biased in the complex of corpora cardiaca (CC) and CA (CC-CA) in B. mori.
We assembled the sequences from the CC-CA EST library and constructed their
contigs. In these contigs, we identified about 300 contigs that consist of the largest
numbers of ESTs; i.e. the corresponding genes are highly expressed in the CC-CA.
These contigs include more than 50% of all the ESTs, indicating that the
corresponding genes represent most of the function of CC-CA.
In order to analyze these genes for their tissue specificity, we designed a custommade DNA microarray using the CC-CA EST contigs as well as the predicted genes
from B. mori genome. Total RNA isolated from the CC-CA of day-2 fourth instar
larvae, that produce JH actively, and other tissues were examined with the
microarray. We analyzed the microarray data for the corresponding genes for the
about 300 contigs described above, and identified 20 genes whose expression is
prominent in the CC-CA. These genes included seven known JH-biosynthetic genes.
They also included three oxidoreductase genes, two protease genes, a gene for
carboxyl/choline esterase family (CCE), and a homolog of PTEN-like phosphatase
(Plip) of Drosophila melanogaster. Phylogenetic analyses showed that CCE and Plip
have one to one orthologs among insect species, suggesting their conserved function
probably in JH biosynthesis.
52 Evolution of SUMO function and chain formation in insects
Enric Ureña (1), Lucia Pirone (2), Coralia Pérez (2), James D. Sutherland (2), Valérie
Lang (3), Manuel S. Rodriguez (3), Fernando Lopitz-Otsoa (2), Francisco J. Blanco (2,
4), Rosa Barrio (2) and David Martín (1)
(1) Institute of Evolutionary Biology (CSIC-Universitat Pompeu Fabra), Passeig Marítim de la Barceloneta 37-49,
08003 Barcelona, Spain. (2) CIC bioGUNE, Bizkaia Technology Park, Building 801-A, 48160 Derio, Bizkaia, Spain.
(3) Cancer Unit. Inbiomed, Paseo Mikeletegi 81, 20009, San Sebastian, Gipuzkoa, Spain. (4) Ikerbasque, Basque
Foundation for Science, 4813 Bilbao, Spain.
SUMOylation, the covalent binding of Small Ubiquitin-like Modifier (SUMO) to target
proteins, is a posttranslational modification that regulates critical cellular processes
in eukaryotes. In insects, SUMOylation has been studied in holometabolous species,
particularly in the dipteran Drosophila melanogaster, which contains a single SUMO
gene (smt3). This has led to the assumption that insects contain a single SUMO
gene. However, the analysis of insect genomes shows that basal insects contain two
SUMO genes, orthologous to vertebrate SUMO1 and SUMO2/3. Our phylogenetical
analysis reveals that the SUMO gene has been duplicated giving rise to SUMO1 and
SUMO2/3 families early in Metazoan evolution, and that later in insect evolution the
SUMO1 gene has been lost after the Hymenoptera divergence. To explore the
consequences of this loss, we have examined the characteristics and different
biological functions of the two SUMO genes (SUMO1 and SUMO3) in the
hemimetabolous cockroach Blattella germanica and compared them with those of
Drosophila Smt3. Here, we show that the metamorphic role of the SUMO genes (cell
proliferation, ecdysone signalling response and proper molting) is evolutionary
conserved in insects, although there has been a regulatory switch from SUMO1 in
basal insects to SUMO3 in more derived ones. We also show that, unlike vertebrates,
insect SUMO3 proteins cannot form polySUMO chains due to the loss of critical lysine
residues within the N-terminal part of the protein. Furthermore, the formation of
polySUMO chains by expression of ectopic human SUMO3 has a deleterious effect in
Drosophila. These findings contribute to the understanding of the functional
consequences of the evolution of SUMO genes.
53 Intersection of the IP3 receptor calcium signalling and, InR and TOR
signalling pathways in dimm+ neuropeptide-producing cells during
Drosophila development.
Megha and Gaiti Hasan
National Centre For Biological Sciences - TIFR, GKVK Campus, Bellary Road, Bangalore 560065, India.
Depending on cell type and stimulus, diverse processes such as hormone release,
vesicle exocytosis, neurotransmission, fertilization etc., are controlled by transient
changes in cytosolic calcium. The release of ER calcium stores by the Inositol 1,4,5trisphosphate (IP3) receptor (itpr), downstream of G-protein coupled receptor
stimulation, is one route through which cytosolic calcium is elevated. Our laboratory
has generated a hypomorph mutant for itpr, termed itpr-ku, which is being utilized
to interrogate the functions of this receptor in Drosophila. This study has focused on
the requirement of itpr in dimm+ neuropeptide cells, during larval to pupal
transition. Functional itpr is required in these cells for larval development under
conditions of nutrient stress. As growth and development in larval stages is
coordinated by the InR and TOR signalling pathways, we investigated if these
pathways are compromised in itpr-ku. Genetic experiments suggest that these
pathways are indeed important in dimm+ neuropeptide cells for itpr-ku to survive
nutrient stress conditions. The current results suggests two possibilities: IP3mediated calcium release is required for neuropeptide-containing vesicle exocytosis,
whose components require a robust InR and TOR signalling pathway to be active or
IP3-mediated calcium release is required for InR and TOR signalling pathway to be
active during nutrient stress conditions in certain cell types. Additionally, we are
trying to identify the neuropeptide/(s) that require itpr to function in the nutrient
stress paradigm.
54 Live imaging the Drosophila salivary gland: Toward an understanding
of the role of steroid regulated cytoskeletal dynamic
Kathryn M. Lantz, Marwa K. Al-Karawi, and Andrew J. Andres
School of Life Sciences, University of Nevada, Las Vegas, NV, USA.
Regulated secretion in an exocrine tissue is well conserved throughout most
metazoans. The larval salivary gland of Drosophila melanogaster is well known for
its massive secretion of glue proteins that will cement puparia to a solid surface
during metamorphosis. This process is trigger by large pulse of 20-hydroxyecdysone
(20E) that occurs at the end of the third larval instar.
While it is understood that 20E is the cause of cargo exocytosis, there is little known
about the process at the molecular level. Using novel culturing techniques to image
live salivary gland ex vivo, we can faithfully reproduce the process of secretion
under controlled conditions in real-time using transgenic stocks that express
fluorescently tagged proteins. Furthermore, with the use of a dominant negative EcR
receptor transgene, we can investigate the process in live tissue when 20E signaling
is blocked. To find candidate genes working downstream of the receptor/hormone
complex, we used a combination of in house salivary gland transcriptome RNA-seq,
modENCODE anatomy RNA-seq analysis, and qRT-PCR to identify and confirm
targets that fit our criteria for induction. With these candidates in hand, we ordered
silencing stocks from the three major RNAi repositories and expressed them
specifically in larval salivary glands using two robust tissue specific drivers to test
function. Candidates necessary for secretion were monitored for their cellular
localization before, during, and after 20E exposure under live imaging conditions.
Here we present results of an analysis of 20E regulated cytoskeletal elements,
motors, and accessory proteins that are responsible for the dynamic reorganization
of the tissue during specialized, steroid-stimulated, regulated secretion.
55 Whole Phosphoproteome Analysis Reveals Rapid Non-Genomic Effects
Of The Steroid Hormone Ecdysone
Anne Færch Jørgensen (1), Lene Jakobsen (2), Maria Ibanez Vea (2), Janne Marie
Laursen (3), Martin Røssel Larsen (2), Kim Furbo Rewitz (1)
(1) Department of Biology, University of Copenhagen, Copenhagen, Denmark. (2)Department of Biochemistry and
Molecular Biology, University of Southern Denmark, Odense, Denmark. (3) Center for Biological Sequence Analysis,
Technical University of Denmark, Lyngby, Denmark.
Steroid hormones are major players in development and physiology. Although the
canonical steroid pathway is a genomic response elicited through interactions with
nuclear receptors (NRs), these hormones also induce rapid nongenomic effects
through mechanisms that are poorly understood. We have investigated the nature of
nongenomic steroid signaling using Drosophila melanogaster. To investigate the rapid
nongenomic pathways activated by steroids, we used quantitative phosphoproteomics
to examine changes in protein phosphorylation induced by the major active
ecdysteroid, 20-hydroxyecdysone (20E), after short term (5 min) stimulation. These
short term effects on protein phosphorylation were compared to long term (6 hour)
effects of 20E stimulation. GO term analysis revealed functions related to gene
expression and cytoskeletal reorganization among the short term effects of 20E.
Both cytoplasmic and membrane localized NRs and G-protein coupled receptors have
been proposed to mediate nongenomic steroid signaling. We provide evidence that the
ecdysone receptor (EcR), a member of the nuclear receptor family, undergoes
nucleocytoplasmic shuttling during the 3rd instar in the salivary gland and that
subcellular EcR localization is affected by the Ras-ERK mitogen activated kinase
protein (MAPK) pathway, making it possible that EcR may be involved in mediating
nongenomic 20E signaling.
56 A novel function of the hippo-pathway member, Warts, in controlling
organismal size of Drosophila
Morten E. Møller (1), Stephan Gerlach (2), E. Thomas Danielsen (1), Kim F. Rewitz
(1).
(1) Department of Biology, University of Copenhagen, Copenhagen, Denmark. (2) Department of Biology, University of
Münster, Germany.
In Drosophila, final body size is largely determined by the amount of growth during
the third and final larval stage (L3), in the period between the critical-weight
checkpoint in early L3 and the cessation of feeding in late L3, just prior to the
metamorphosis. The amount of growth is dependent on the availability of nutrients
and is reflected in the level of circulating insulin like peptides (ILPs). The binding of
ILPs to the insulin receptor (InR) promotes growth. On the other end, basal levels of
ecdysone, produced and released from the prothoracic gland (PG), has a negative
effect on systemic growth, as it inhibits insulin signaling. Thus, regulation of final body
size is fine-tuned by interplay between insulin signaling and basal levels of the
hormone ecdysone.
In a genome-wide RNAi screen targeting the PG, we found that PG-specific loss of the
tumor-suppressor gene warts (wts), a growth regulator in the hippo pathway, results
in an increase in final body size, but surprisingly with no delay in the onset of
pupariation. Thus, loss of wts in the PG accelerates growth during the larval stages
without affecting the duration of the growth period. Furthermore, we found that knock
down of wts in the PG affects the basal levels of ecdysone in an insulin dependent
manner, suggesting a mechanism whereby decreased wts expression in the PG leads
to an overall overgrowth. wts and the other members of the hippo pathway are keyregulators of tissue and organ size. Our data show a non-cell-autonomous function of
wts, via control of ecdysone biosynthesis in the PG, in regulating organismal size. In
conclusion, our data indicate a novel function of Wts in regulating final body size, in
addition to its known role in determining tissue and organ growth.
57 Coordination of ecdysone and heme production in Drosophila.
Qiuxiang Ou, Brian Phelps and Kirst King-Jones.
Department of Biological Sciences, University of Alberta, Edmonton, Canada.
Steroid hormones are signaling molecules that regulate developmental transitions
such as puberty in humans and metamorphosis in insects. While the actions of steroid
hormones are well understood, less is known about the regulatory pathways that
control steroid hormone production. Recently, our lab began investigating the role of
heme in the regulation of insect steroid hormone (= ecdysone) production, since
heme acts as a prosthetic group in most of the known steroid hormone-producing
enzymes. Steroid hormone-producing glands must anticipate the need for iron and
heme in order to synthesize adequate amounts of steroidogenic enzymes. In addition,
when cellular heme levels are too low, cells must be able to respond and upregulate
heme biosynthesis, suggesting the existence of a heme-sensing mechanism. We show
here that DHR51 (Drosophila hormone receptor 51), a nuclear receptor, is a likely
candidate for such a heme sensor in the steroid hormone-producing gland.
Specifically, RNA-Seq analysis of steroid hormone-producing tissues showed that lossof-DHR51 resulted in a similar transcriptional response when compared to disrupting a
heme enzyme via RNAi, indicating that interfering with DHR51 function impairs heme
biosynthesis. We also show that the induction of the rate-limiting enzyme of heme
production, ALAS, is dependent on DHR51. The RNA-Seq data also showed that key
genes associated with steroid hormone production were downregulated when DHR51
function was impaired. Feeding larvae ecdysone in their diet rescued the
developmental defects associated with loss-of-DHR51, suggesting that DHR51 was
necessary for steroid hormone production.
Taken together, DHR51 may coordinately regulate steroid hormone and heme
biosynthesis, allowing steroidogenic cells to adapt their iron and heme requirements
prior to the formation of steroid hormone pulse.
58 Gifts that keep on giving: Variable provisioning of ecdysteroids to
eggs has long-term consequences
Katherine C. Crocker, Elizabeth A. Tibbetts, Mark D. Hunter
830 N University Ave, Kraus Natural Sciences Building rm 2065, Ann Arbor, Michigan, USA 48109-1048.
Ecdysteroid hormones control early development, regulate molting and
metamorphosis, and are important for reproduction in insects. Although insects in
embryonic and larval stages are sensitive to minute changes in ecdysteroid
concentrations, we know little about the long-term consequences of such variability.
Preliminary data show that naturally occurring variation in the quantity of
ecdysteroids provided to eggs, while always sufficient to drive developmental
programs, may also play a role in generating phenotypic variability: we observed a
dose-dependent effect on growth rate (a common fitness proxy in insects) and
survival to maturity.
Natural variation in the quantity of ecdysteroids provided to eggs is known to occur in
several species; we chose house crickets (Acheta domesticus) to use in our
experiments.
Here we report (1) the ecdysteroid concentration in cricket eggs can be measured
using a newly validated Enzyme Immunoassay and (2) the results of an experiment in
which we varied maternal environment and genotype factorially across multiple
environments and generations.
Our results quantify the relative importance of genotype and environment in
determining the concentration of ecdysteroids provisioned to eggs and clarify whether
the consequences (for growth and survival rates) of variable ecdysteroid provisioning
persist across multiple generations of offspring, or are limited to the provisioned eggs.
We anticipate that this new knowledge of ecdysteroid effects in insects will inform
future work, particularly in light of growing general interest in transgenerational
environmental effects.
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