Specific Aims Body size profoundly affects many aspects of animal biology, yet the mechanisms that determine body size remain one of the fundamental unsolved problems of developmental biology. Holometabolous insects the primary model for the study of size regulation in animals do not grow as adults, so the size at which larvae initiate metamorphosis determines their adult size. In holometabolous insect larvae, the developmental decision to stop growing and metamorphose is attained at a particular weight, called the critical weight. Attainment of critical weight initiates a hormonal cascade that ultimately results in the synthesis and release of ecdysone, the hormone that coordinates the developmental events necessary for a larva to molt and metamorphose. Recent research has elucidated the signaling pathways that regulate the synthesis of ecdysteroids. However, the mechanisms that a larva uses to sense its size and activate these signaling pathways are largely unknown. The result is a conspicuous gap in our understanding of the mechanisms that regulate body size. Insects routinely double their mass within an instar, while tracheal structures remain largely fixed, leading oxygen demand to outstrip supply capacity. What remains unclear is whether oxygen sensing is a proximate mechanism for insects to sense their size during the instar and regulate molting. I hypothesize that oxygen regulates body size in holometabolous insects in part by regulating the timing of molting, and that this effect is mediated by direct or indirect effects of hypoxia on the molecular pathways that regulate the synthesis and secretion of ecdysone. My proposed research will address three specific research aims based on this hypothesis, using Drosophila as a model organism. Aim 1: To determine how oxygen supply affects the size at which Drosophila larvae commit to metamorphosis. Rationale. My data demonstrate that critical weight is reduced in hypoxia in Manduca and Drosophila, and that hypoxia extends the duration of the last larval instar. This suggests that oxygen influences the moleculargenetic mechanisms that regulate the degree and timing of ecdysone signaling. Methods. I will use hormone titer assays and qPCR to measure the changes in the underlying hormonal physiology reflected by the shift in critical weight. Aim 2: To determine how oxygen interacts with the molecular-genetic pathways known to control ecdysteroidogenesis. Rationale. Insulin, Target of Rapamycin (TOR), and Prothoracicotropic Hormone (PTTH)/Ras/Raf/MAP kinase signaling are key regulators of ecdysteroidogenesis, with similar but non-overlapping effects on critical weight, growth rate, and final body size. I hypothesize that the effects of hypoxia on critical weight are due to the effects of hypoxia on these pathways. By directly manipulating these pathways in the prothoracic glands of hypoxic and normoxic larvae, I can use epistatic analysis to test whether any of these pathways are essential for the effects of oxygen on critical weight. Methods. I will up- and down-regulate the activity of different components of the insulin/IGF-, TOR- and PTTH/Ras/Raf/MAPK signaling pathways in the PG of developing larvae. I will then rear these larvae in both normoxic (21% oxygen) and hypoxic (10% and 5% oxygen) conditions and assay critical weight, the timing of metamorphosis and body size. Using epistatic analysis, I will determine whether the effects of oxygen on critical weight act up- or down-stream of insulin, TOR and PTTH/Ras/Raf/MAPK. Aim 3: To determine whether HIF mediates the effects of oxygen on critical weight. Rationale. HIF-1 (Hypoxia Inducible Factor-1) is a central mediator of the response of growth and development to changes in oxygen levels in Drosophila. There is considerable cross talk between HIFsignaling and the signaling pathways that have been shown to regulate ecdysteroidgenesis (insulin/IGF-, TORand PTTH/Ras/Raf/MAPK- signaling), although the nature of this crosstalk is only partially elucidated. I hypothesize that the effects of hypoxia on critical weight, development time, and final size are mediated by interactions of HIF with insulin and TOR signaling. Methods. I will first up- and down-regulate HIF activity in specific tissues and systemically in developing larvae, rear these larvae in both normoxic (21% oxygen) and hypoxic (10% oxygen) conditions, and measure the critical weight in these larvae. I will measure changes in HIF activity through the instar under normal physiological conditions, using western blotting and a GFP-reporter line that expresses GFP when hypoxic.
|Effective start/end date||7/1/13 → 8/31/13|
- HHS-NIH: National Institute of General Medical Sciences (NIGMS): $10,819.00
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