Doctor of Philosophy / Genetics Interdepartmental Program / Susan J. Brown / In Drosophila, all segments form in the blastoderm where morphogen gradients spanning the entire anterior-posterior axis of the embryo provide positional information. However, in the beetle Tribolium castaneum and most other insects, a number of anterior segments form in the blastoderm, and the remaining segments form sequentially from a posterior growth zone during germband elongation. In this work, I show that segmentation at both blastoderm and germband stages of Tribolium is based on a segmentation clock. Specifically, I show that the Tribolium primary pair-rule gene, Tc-even-skipped (Tc-eve), is expressed in waves propagating from the posterior pole and progressively slowing until they freeze into stripes; such dynamics are a hallmark of clock-based segmentation. Phase shifts between Tc-eve transcripts and protein confirm that these waves are due to expression dynamics. Such waves, like their counterparts in vertebrates, are assumed to arise due to the modulation of a molecular clock by a posterior-to-anterior frequency gradient. I provide evidence that the posterior gradient of Tc-caudal (Tc-cad) expression regulates the oscillation frequency of pair-rule gene expression in Tribolium. I show this by correlating the gradient of Tc-cad expression to the spatiotemporal dynamics of Tc-even-skipped expression in WT as well as in different knockdowns of Tc-cad regulators. Specifically, the spatial extent, frequency, and width of Tc-eve waves correlate with the spatial extent, expression level, and slope of Tc-cad gradient, respectively, as predicted by computer modeling. These results pose intriguing evolutionary questions, since Drosophila and Tribolium segment their blastoderms using the same genes but different mechanisms, and highlight the role of frequency gradients in pattern formation.

Doctor of Philosophy / Department of Division of Biology / Rollie J. Clem / Mosquitoes are carriers of a variety of harmful human pathogens, including viruses. In order to be successfully transmitted, a virus must evade mosquito immune responses. In this work, the innate immune role of apoptosis in mosquito-virus interactions was examined utilizing the disease vector Aedes aegypti and Sindbis virus. Ae. aegypti is the main vector for yellow fever and dengue virus, which result in over 100 million infections per year. Sindbis virus (Togaviridae) can be transmitted to vertebrates by Ae. aegypti in the laboratory. Sindbis is also well characterized molecularly, making it a good model system for understanding virus-vector interactions. Sindbis MRE-16 recombinant virus clones were utilized to express either an antiapoptotic or pro-apoptotic gene during virus replication. Mosquitoes were infected with recombinant virus clones during a blood meal or by intrathoracic injection. Midgut tissue and whole body samples were analyzed for virus infection and dissemination. Virus was also quantified in saliva and mosquito survival was assayed. Decreased infection in the midgut and delayed virus replication were observed in mosquitoes that were infected with virus expressing a pro-apoptotic gene. Infection with this virus clone also resulted in less virus in the saliva and reduced survival of infected mosquitoes. In addition, negative selection against pro-apoptotic gene expression during virus replication was observed. Collectively, these data suggest that apoptosis can serve as an antiviral defense in Ae. aegypti and may potentially be exploited to control virus transmission. An additional study included in this dissertation focused on zebrafish development and migration of somitic precursors from the tailbud. The tailbud consists of a population of stem cells at the posterior tip of the embryonic tail. The exit of these stem cells from the tailbud is required for the formation of tail somites. A novel double mutant was identified that lacked the t-box transcription factor spadetail and the BMP inhibitor chordin. Double mutants completely lacked somites and had an enlarged tailbud due to accumulation of stem cells that were unable to exit the tailbud. This study indicates the importance of BMP inhibition and spadetail expression in the proper exit of muscle precursors from the tailbud.

Master of Science / Department of Biology / Tonia L. Von Ohlen / The Drosophila embryonic nervous system develops from an array of neural precursor cells called, neuroblasts. These neuroblasts give rise to all the cell types that populate the mature central nervous system (CNS). The CNS originates from a bilateraly symmetric neurectoderm that is subdivided into three domains along the dorso-ventral (DV) axis. One of these domains is defined by the expression of the Homeodomain protein ventral nervous system defective (vnd). Regulation of neuroblast designation is very precise and controlled. Extensive research has been done on neuroblast formation along the anteroposterior axis, most of which indicates that neuroblast selection within a cluster of neurectodermal cells is controlled by segmentation genes. However, much more research is required to elucidate the function of genes along the DV axis. Early studies indicate that vnd is required for neuroblast formation in the ventral column. Here, we show that vnd function, but not expression, is dependent on MAPK activity downstream of Drosophila EGF-R (DER). Specifically, we show that vnd activity is eliminated in EGF-R mutant embryos in a stage specific manner by evaluating vnd’s ability to inhibit intermediate neuroblast defective (ind), muscle segment homeobox (msh), and the newly identified neural tube development player, neu3. Finally, we show that DER functionality in the ventral column is entirely dependent on the processing protein rhomboid (rho) in later stage embryos.

Doctor of Philosophy / Department of Biochemistry / Subbaratnam Muthukrishnan / In order to grow and develop, insects must undergo a process of molting, wherein the old cuticle is replaced with a new one. A thin envelope layer has been predicted to act as a physical barrier between molting fluid chitinases and the site of new chitin synthesis ensuring selective protection of newly synthesized chitin. The factors that help the new exoskeleton withstand the deleterious effects of chitinolytic enzymes remain poorly understood. In the current study a mechanistic role for two proteins, Knickkopf (Knk) and Retroactive (Rtv), was explored in organization and protection of the newly synthesized procuticular chitin. Our study demonstrated colocalization of molting fluid chitinases (chitinase-5) with chitin in T. castaneum pharate adult elytral cuticle. Presence of chitinases in the new cuticle, disproved the old theory of the envelope being a protective barrier against chitinases. Confocal and transmission electron microscopic imaging of T. castaneum pharate adult elytral cuticle suggested that Knk protein selectively colocalizes with chitin in the new procuticle, organizes chitin into laminae and protects it from the activity of molting fluid chitinases. Down-regulation of Knk expression resulted in reduction of procuticular chitin, disruption of the laminar architecture of the procuticle and severe molting defects that are ultimately lethal at all stages of insect growth. The presence and activity of Rtv protein ensures the trafficking of Knk into the procuticle. Down regulation of Rtv transcripts showed molting defects and a significant decrease in chitin content similar to those following Knk dsRNA treatment. Confocal microscopic analysis revealed an essential role for Rtv in proper trafficking of Knk from epithelial cells to within the newly synthesized procuticule. Once released into the procuticle, Knk organizes and protects chitin from chitinases. The conservation of Knk and Rtv in all insect species suggests a critical role for these proteins in maintenance and protection of chitin in the insect exoskeleton.