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The physiological response to environmental cues involves complex interorgan communication via endocrine factors and hormones, but the underlying mechanisms are poorly understood. In particular, little is known about how animals coordinate systemic growth and developmental timing in response to environmental changes. The morphogen Hedgehog (Hh), which is well studied in tissue patterning and homeostasis, has only recently been implicated in the regulation of lipid and sugar metabolism. Interestingly, Hh is present in systemic circulation in both, ies and mammals. Here, we demonstrate that systemic Hh is produced in the midgut and secreted in association with the lipoprotein particle lipophorin (Lpp) into the hemolymph to mediate the interorgan communication between the midgut and two tissues, the fat body and the prothoracic gland (PG). We show that midgut hh expression is regulated by dietary sugar and amino acid levels, and RNAi-mediated knock-down of circulating Hh leads to starvation sensitivity. We demonstrate that circulating Hh is required to inhibit systemic growth and developmental progression. In insects, developmental transitions are regulated by steroid hormones, which are produced by the PG. Nutritional regulation of growth is, in part, mediated by the Drosophila fat body. Strikingly, canonical Hh pathway components are present in both tissues, the fat body and the PG. To understand the Hh-mediated function during nutritional stress, we ectopically activated or inhibited the Hh signaling pathway specifically in the fat body and the PG. Our results show that systemic Hh exerts its function through these two target tissues. Hh signaling in the fat body is required for survival during periods of nutrient deprivation, and ectopic activation of fat body Hh signaling causes an inhibition of systemic growth. Hh signaling in the PG slows down developmental progression by inhibiting steroid hormone biosynthesis. In conclusion, we propose that the midgut senses the uptake of dietary sugar and amino acids and secrets Hh in association with Lpp particles into circulation to relay information about the feeding status to the developing animal. Therefore, circulating Hh functions as a hormone and signals in an endocrine manner to the fat body and the prothoracic gland to coordinate systemic growth and developmental timing in response to changes in nutrient availability.

Cell cycle checkpoints are signal transduction pathways that control the order and timing of cell cycle transitions, ensuring that critical events are completed before the cell cycle proceeds. The Chk2 family of kinases plays a central role in mediating responses to DNA damage or DNA replication blocks in various organisms. My functional analysis of the Drosophila serine/threonine kinase Loki/Chk2 shows that fly chk2 monitors double-strand breaks caused by irradiation during S and G2 phases and induces cell cycle arrest in embryonic cells around cellularization. / loki is also required for the normal number of germ line cells to form in the embryo, and for normal modification of Vasa, a crucial factor in germ cell formation. However, during normal oogenesis loki expression is suppressed by orb. Another group described the involvement of Drosophila loki/chk2 in the meiotic pachytene checkpoint. Using our loki·null mutant, I obtained the opposite result: loki/chk2 does not have an essential function in this process. / The second part of my thesis deals with the question of how cells are instructed about their identity in a developing organism. (Abstract shortened by UMI.)

The clk-1 gene was found in the screen for the maternal effect viable mutants in Caenorhabditis elegans. It is believed to be involved in controlling timing of various biological processes in the nematode including lengthening it's life span, and, as such, it belongs to the Clock group of genes. The CLK-1 homologue in yeast, COQ7, was shown to be involved in ubiquinone biosynthesis and gluconeogenic gene activation. The Drosophila clk-1 gene has been cloned. As a means to assay probable correlation between the timing mechanism of clk-1 and the circadian clocks, well studied in Drosophila, the clk-1 circadian mRNA expression has been monitored. At the middle of the dark phase, the level of clk-1 transcript decreases by approximately 30%. The Drosophila clk-1 mRNA expression during developmental stages was also analyzed. The amount of clk-1 mRNA is doubled during the larvae stage, the most metabolically active stage in the early Drosophila development.

Drosophila Lasp contains a LIM domain, two nebulin repeats, and a SH3 domain, and exhibits high homology with mammalian Lasp family proteins. Vertebrate Lasp localizes to focal adhesions and to the leading edge of migrating cells and binds filamentous actin. To investigate Drosophila Lasp in vivo, we generated a Lasp null mutant, named Laspl, and showed that Laspl is male sterile. We observed two major functions of Lasp during Drosophila spermatogenesis. First, in the stem cell niche, hub cells fail to localize to the apical end of Drosophila testis in Laspl mutant. Hub cell anchoring is dependent on cell adhesion between cells and extracellular matrix (ECM), which is mediated by integrins. Lasp genetically interacts with betaPS integrin showing complete hub cell mislocalization. This indicates that Lasp is involved in an integrin-dependent process. However, hub cell anchoring is not required for fertility or stem cell maintenance. Secondly, we observe that actin cones, a unique actin structure during spermatid individualization, are perturbed in Laspl. Our data for Lasp expression in actin cones and incomplete individualization indicate that Lasp may play a role in tethering actin to the plasma membrane.

This thesis reports the results of genetic and molecular analysis of polytene region 38 of D. melanogaster. / A detailed genetic map of the region has been constructed through the complementation analysis of 22 genes with 37 deficiency stocks, providing 48 breakpoints. 44 of the breakpoints have been precisely mapped and separate the complementation groups into 16 distinct intervals. / Molecular characterisation was achieved using a combination of computational sequence analysis and experimental techniques. In this manner, 46 new transcription units and their developmental expression patterns were identified and the physical location of 7 deficiency breakpoints revealed. Links have been established between the genetic and the physical maps using STSs and sequence information from previously cloned genes. This allowed the possible correlation of 4 previously genetically defined genes to their transcription units.

We report the cloning and characterization of valois (vls), a posterior group gene of Drosophila melanogaster , which was initially identified in a screen for female steriles. Three EMS alleles of vls contain premature stop codons in the open reading frame. Sequence analyses show the presence of WD domains in Vls and find significant similarity with the human MEP50 protein which is involved in the assembly of the splicing machinery. We did not find evidence that this function is conserved in flies yet. / We created a null mutant for vls, which shows a maternal effect lethal phenotype accompanied by posterior polarity defects in the embryos. Hemizygous vlsEMS females show a weaker, partially maternal-effect lethal and a fully penetrant grandchildless phenotype. The posterior localization of Vasa is disrupted in vlsnull ovaries, but the initial distribution of Oskar protein and mRNA appear normal. However, levels of the Short Oskar isoform responsible for pole plasm assembly are greatly reduced and Vasa appears to be differently modified post-translationally. Furthermore, a Vls::GFP fusion protein is detected all throughout oogenesis in the nurse cell and oocyte cytoplasm. Taken together, these data suggest that Vls is a cytoplasmic protein involved in the transport or activation of Vasa at the posterior of the oocyte essential for the accumulation of Short Osk.

Thesis (Ph. D. in Neuroscience)--Vanderbilt University, May 2008. / Title from title screen. Includes bibliographical references.

Thesis (Ph. D.)--University of Washington, 2002. / Vita. Includes bibliographical references (leaves 100-111).

Undergraduate honors paper--Mount Holyoke College, 2005. Dept. of Biological Sciences. / Includes bibliographical references (leaves 74-79).