Deborah K. Hoshizaki

Serpent regulates Drosophila immunity genes in the larval fat body through an essential GATA motif

Insects possess a powerful immune system, which in response to infection leads to a vast production of different antimicrobial peptides. The regulatory regions of many immunity genes contain a GATA motif in proximity to a κB motif. Upon infection, Rel proteins enter the nucleus and activate transcription of the immunity genes. High levels of Rel protein‐mediated Cecropin A1 expression previously have been shown to require the GATA site along with the κB site. We provide evidence demonstrating that the GATA motif is needed for expression of the Cecropin A1 gene in larval fat body, but is dispensable in adult fat body. A nuclear DNA‐binding activity interacts with the Cecropin A1 GATA motif with the same properties as the Drosophila GATA factor Serpent. The GATA‐binding activity is recognized by Serpent‐specific antibodies, demonstrating their identity. We show that Serpent is nuclear in larval fat body cells and haemocytes both before and after infection. After overexpression, Serpent increases Cecropin A1 transcription in a GATA‐dependent manner. We propose that Serpent plays a key role in tissue‐specific expression of immunity genes, by priming them for inducible activation by Rel proteins in response to infection.

The role of larval fat cells in adult Drosophila melanogaster.

SUMMARY In the life history of holometabolous insects, distinct developmental stages are tightly linked to feeding and non-feeding periods. The larval stage is characterized by extensive feeding, which supports the rapid growth of the animal and allows accumulation of energy stores, primarily in the larval fat body. In Drosophila melanogaster access to these stores during pupal development is possible because the larval fat body is preserved in the pupa as individual fat cells. These larval fat cells are refractive to autophagic cell death that removes most of the larval cells during metamorphosis. The larval fat cells are thought to persist into the adult stage and thus might also have a nutritional role in the young adult. We used cell markers to demonstrate that the fat cells in the young adult are in fact dissociated larval fat body cells, and we present evidence that these cells are eventually removed in the adult by a caspase cascade that leads to cell death. By genetically manipulating the lifespan of the larval fat cells, we demonstrate that these cells are nutritionally important during the early, non-feeding stage of adulthood. We experimentally blocked cell death of larval fat cells using the GAL4/UAS system and found that in newly eclosed adults starvation resistance increased from 58 h to 72 h. Starvation survival was highly correlated with the number of remaining larval fat cells. We discuss the implications of these results in terms of the overall nutritional status of the larva as an important factor in adult survival in environmental stresses such as starvation.

THE SERPENT GENE IS NECESSARY FOR PROGRESSION THROUGH THE EARLY STAGES OF FAT-BODY DEVELOPMENT

The serpent (srp) gene, also known as ABF, codes for a GATA-like transcription factor and is involved in the transcription activation of Adh in the larval fat body or adipose tissue. Here, we describe the tissue-specific distribution of SRP protein in various stages of embryonic development and describe srps role in early fat-cell development. SRP protein was detected in the progenitor fat-body cells and is present in the developing fat-body cells and in the mature embryonic fat body. An analysis of srp embryos revealed a gradual loss of precursor fat cells that is likely due to apoptosis. Within the fat-cell lineage, srp is necessary for progression through early stages of fat-cell development and may be involved in the transactivation of genes necessary for fat-cell differentiation.

Energetics of metamorphosis in Drosophila melanogaster

We measured the energetic cost of metamorphosis in the fruitfly, Drosophila melanogaster. Metabolic rates decreased rapidly in the first 24h and remained low until shortly before eclosion, when the rates increased rapidly, thus creating a U-shaped metabolic curve. The primary fuel used during metamorphosis was lipid, which accounted for >80% of total metabolism. The total energy consumed during metamorphosis was lowest at 25°C, compared to 18 and 29°C, due to differences in metabolic rates and the length of pupal development. Temperature differentially affected metabolic rates during different stages of metamorphosis. Prepupal and late pupal stages exhibited typical increases in metabolic rate at high temperatures, whereas metabolic rates were independent of temperature during the first 2/3 of pupal development. We tested two hypotheses for the underlying cause of the U-shaped metabolic curve. The first hypothesis was that pupae become oxygen restricted as a result of remodeling of the larval tracheal system. We tested this hypothesis by exposing pupae to hypoxic and hyperoxic atmospheres, and by measuring lactic acid production during normoxic development. No evidence for oxygen limitation was observed. We also tested the hypothesis that the U-shaped metabolic curve follows changes in metabolically active tissue, such that the early decrease in metabolic rates reflects the histolysis of larval tissues, and the later increase in metabolic rates is associated with organogenesis and terminal differentiation of adult tissues. We assayed the activity of a mitochondrial indicator enzyme, citrate synthase, and correlated it with tissue-specific developmental events during metamorphosis. Citrate synthase activity exhibited a U-shaped curve, suggesting that the pattern of metabolic activity is related to changes in the amount of potentially active aerobic tissue.

ßFTZ-F1 and Matrix metalloproteinase 2 are required for fat-body remodeling in Drosophila

During metamorphosis, holometabolous insects eliminate obsolete larval tissues via programmed cell death. In contrast, tissues required for further development are retained and often remodeled to meet the needs of the adult fly. The larval fat body is involved in fueling metamorphosis, and thus it escapes cell death and is instead remodeled during prepupal development. The molecular mechanisms by which the fat body escapes programmed cell death have not yet been described, but it has been established that fat-body remodeling requires 20-hydroxyecdysone (20E) signaling. We have determined that 20E signaling is required within the fat body for the cell-shape changes and cell detachment that are characteristic of fat-body remodeling. We demonstrate that the nuclear hormone receptor ßFTZ-F1 is a key modulator of 20E hormonal induction of fat body remodeling and Matrix metalloproteinase 2 (MMP2) expression in the fat body. We show that induction of MMP2 expression in the fat body requires 20E signaling, and that MMP2 is necessary and sufficient to induce fat-body remodeling.

Homeotic selector genes control the patterning of seven-up expressing cells in the Drosophila dorsal vessel

The linear cardiac tube of Drosophila, the dorsal vessel, is an important model organ for the study of cardiac specification and patterning in vertebrates. In Drosophila, the Hox segmentation gene abdominal-A (abd-A) is required for the specification of a functionally distinct heart region at the posterior of the dorsal vessel, from which blood is pumped anteriorly through a tube termed the aorta. Since we have previously shown that the posterior part of the aorta is specified during embryogenesis to form the adult heart during metamorphosis, we determined if the embryonic aorta is also patterned by the function of Hox segmentation genes. Using gain- and loss-of-function experiments, we demonstrate that the three Hox genes expressed in the posterior aorta and heart are sufficient to confer heart or posterior aorta fate throughout the dorsal vessel. Additionally, we demonstrate that Ultrabithorax and abd-A, but not Antennapedia, function to control cell number in the dorsal vessel. These studies add robustness to the model that homeotic selector genes pattern the Drosophila dorsal vessel, and further extend our understanding of how the cardiac tube is patterned in animal models.

Toll Mediated Infection Response Is Altered by Gravity and Spaceflight in Drosophila

Space travel presents unlimited opportunities for exploration and discovery, but requires better understanding of the biological consequences of long-term exposure to spaceflight. Immune function in particular is relevant for space travel. Human immune responses are weakened in space, with increased vulnerability to opportunistic infections and immune-related conditions. In addition, microorganisms can become more virulent in space, causing further challenges to health. To understand these issues better and to contribute to design of effective countermeasures, we used the Drosophila model of innate immunity to study immune responses in both hypergravity and spaceflight. Focusing on infections mediated through the conserved Toll and Imd signaling pathways, we found that hypergravity improves resistance to Toll-mediated fungal infections except in a known gravitaxis mutant of the yuri gagarin gene. These results led to the first spaceflight project on Drosophila immunity, in which flies that developed to adulthood in microgravity were assessed for immune responses by transcription profiling on return to Earth. Spaceflight alone altered transcription, producing activation of the heat shock stress system. Space flies subsequently infected by fungus failed to activate the Toll pathway. In contrast, bacterial infection produced normal activation of the Imd pathway. We speculate on possible linkage between functional Toll signaling and the heat shock chaperone system. Our major findings are that hypergravity and spaceflight have opposing effects, and that spaceflight produces stress-related transcriptional responses and results in a specific inability to mount a Toll-mediated infection response.

The role of 20-hydroxyecdysone signaling in Drosophila pupal metabolism.

In holometabolous insects, the steroid hormone 20-hydroxyecdysone (20E), in coordination with juvenile hormone, regulates the major developmental events that promote larval development and the transition from the larval to the pupal stage. Intimately entwined with the hormonal control of development is the control of larval growth and the acquisition of energy stores necessary for the development of the non-feeding pupa and immature adult. Studies of the coordination of insect development and growth have suggested that the larval fat body plays a central role in monitoring animal size and nutritional status by integrating 20E signaling with the insulin signaling pathway. Previous studies have shown that tissue-specific loss of 20E signaling in the fat body causes pupal lethality (Cherbas et al., 2003). Because the fat body is the major organ responsible for nutrient homeostasis, we hypothesized that the observed pupal mortality is due to a metabolic defect. In this paper we show that disruption of 20E signaling in the fat body does not disrupt nutrient storage, animal size at pupariation, or nutrient utilization. We conclude that 20E signaling in the fat body is not necessary for normal pupal metabolism.

Drosophila windpipe codes for a leucine-rich repeat protein expressed in the developing trachea.

The embryonic tracheal system of Drosophila provides an important model for understanding the process of epithelial branching morphogenesis. Here we report the sequence and expression analysis of a novel tracheal gene, named windpipe (wdp). wdp is identical to the predicted gene CG3413 and encodes a transmembrane, leucine-rich repeat family member. wdp transcripts appear abruptly at stage 15 and are restricted to primary tracheal branches that give rise to secondary branches.

Life is a gas.

Meeting report from 49th Annual Drosophila Research Conference, April 2008 in San Diego, CA