Project summary Intellectual merit. Body size profoundly affects many aspects of animal biology, yet it remains 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 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. The phenomenon of the critical weight has been observed for decades, and more 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. We hypothesize that, as larvae grow through an instar, the growth of tissues relative to supply structures creates internal hypoxia, and internal hypoxia is a physiological cue that initiates the hormonal cascade for molting and metamorphosis. Further, we hypothesize that oxygen effects on critical weight are mediated by hypoxias interaction with the known pathways (insulin and TOR) that regulate ecdysone secretion. We will investigate this question using physiological, morphological and molecular-genetic methods. First we will test whether hypoxia causes a reduction in the critical weight at which commitment to molting occurs. Next, we will test whether oxygen demand actually outstrips supply during the latter part of the final larval instar by assessing tracheal supply using quantitative x-ray 3D tomography and confocal microscopy, and by measuring the metabolic rates of larvae throughout the instar. We will test whether larvae become hypoxic later in the instar by measuring HIF-1 (hypoxia inducible factor) proteins using western blotting on whole flies, and by assessing HIF-1 signaling in different cell types using flies with GFP-reporter constructs. We will test whether the effects of hypoxia on size are mediated by insulin and TOR signaling, which are key regulators of ecdysone secretion. We will test whether HIF-1 signaling is necessary for effects of hypoxia on molting and size by reducing HIF- 1 signaling in a cell-type specific manner using GAL4 drivers and RNAi lines. We will test whether HIF- 1 signaling is sufficient for hypoxia effects on molting and size by increasing HIF-1 signaling using fatiga mutants that produce constitutive HIF-1 signaling, again, using cell-type-specific GAL4 drivers. Together, these experiments will test whether internal hypoxia is a critical cue in the sensing and regulation of molting and body size in Drosophila. This study has the potential to reveal a fundamental process for the regulation of body size in animals. Because body size is such a critical factor in ecology and physiology, and we do not understand the mechanisms by which any animal decides the appropriate developmental stage to transition from juvenile to adult, this study will be of wide interest in biology and medicine. Broader impacts. Understanding the physiological and genetic mechanisms that regulate body size is fundamentally important for understanding biological phenomena in development, physiology, ecology and evolution. Our data will contribute to an integrated, multi-scale mechanism for size regulation. Educationally, this research will fund the training and recruitment of an outstanding young female postdoc, Dr. Viviane Callier. Dr. Callier will receive a broad training in molecular biology and physiology. We will also train a variety of undergraduate students in both the Shingleton and Harrison labs, with monthly joint lab meetings via videoconference. We will link our labs to local high schools with a high fraction of students from groups underrepresented in science. Funds will be used to support high school teachers and students to conduct research in the Harrison and Shingleton labs. These outreach efforts will function to help recruit minority high school students into university science programs, enhance high school science curricula, and improve public understanding and acceptance of evolution. We will also partner with the ASU Ask-A-Biologist web site and the Arizona Science Center to produce a new K-12 teaching resource. In collaboration with teachers, graphic artists, and web designers we will develop a lab exercise that focuses on the physiology and genetics of growth and size.
|Effective start/end date||9/1/13 → 8/31/18|
- NSF: Directorate for Biological Sciences (BIO): $638,855.00