Herder, Rachel Jeanne2012-10-252012-10-252012-08https://hdl.handle.net/11299/137106University of Minnesota Ph.D dissertation. August 2012. Advisor: Dr. Michael B. O’Connor. 1 computer file (PDF); ix, 127 pages, appendices A-D.Animals use steroid hormones to regulate the timing of development and growth. In insects, the developmental processes of hatching, molting, metamorphosis and eclosion are all regulated by a steroid hormone, 20-hydroxyecdysone (20E). Biosynthesis of ecdysone (E), the immediate precursor to 20E, is thought to be regulated, in part, by a small neuropeptide called prothoracicotropic hormone (PTTH ). PTTH is produced by neurons that innervate the prothoracic gland (PG) and signals to this tissue to up-regulate biosynthesis of E in insects, including Drosophila melanogaster (fruit fly) and Bombyx mori (silk worm). When PTTH signaling is disrupted, developmental timing is altered. This thesis focuses on further elucidating the role of PTTH signaling in development through three separate but related aims. First, to better understand the global effects of PTTH signaling on transcriptional regulation in the PG, we used Illumina Next Generation sequencing to compare the transcriptome of PTTH-stimulated and –unstimulated PGs. This was used as an unbiased approach to determine what genes are up and down regulated in response to PTTH stimulation. At the time of this thesis writing, the sequencing reactions have been completed, however, the bioinformatics analysis is still underway. Second, to better understand how PTTH acts to up-regulate gene expression, we analyzed regulatory elements in target genes using a promoter-bashing approach. Using this approach, we uncovered three minimal enhancer regions from phantom, spookier and disembodied, members of the Halloween family of E biosynthetic genes that are expressed in the PG. Additionally, we have discovered several small, highly conserved, sequence motifs that are necessary for reporter gene expression in the PG. Third, we examined a single PTTH-responsive gene, called Membrane Steroid Binding Protein (MSBP), with the goal of elucidating its role in ecdysone biosynthesis. We have confirmed that MSBP is expressed in ecdysone producing tissues and that MSBP expression changes in response to PTTH. However, MSBP-/- animals show no obvious phenotype, suggesting either redundancy or no requirement of MSBP in regulating developmental timing.en-USBombyx moriDrosophila melanogasterEcdysoneProthoracic glandProthoracicotropic hormoneThesis or Dissertation