The JAK/STAT pathway is a non-receptor tyrosine kinase signaling pathway that is well conserved and highly re-utilized in many mammalian and Drosophila developmental processes. Compared to dozens of ligands and receptors in mammalian JAK/STAT, Drosophila JAK/STAT pathway is simpler with one receptor and three ligands, Upd, Upd2 and Upd3, which have similar amino acid sequences. Previous literature shows that upd and upd2 exhibit the same dynamic striped expression pattern in embryos and have semi-redundant functions during embryogenesis. Do Upd and Upd3 also have redundant functions? To answer this question, the functions of Upd3 in Drosophila development were investigated in this dissertation. In addition, the coordinate expression mechanism of upd and upd3 in eye discs was also analyzed. To study the functions of Upd3 in development, the expression pattern of upd3 was examined and detected in larval eye discs, wing discs, haltere discs, lymph glands and adult ovaries with in situ hybridization to upd3 mRNA and an upd3 reporter line. Consistent with the expression pattern, the loss of function mutants of upd3 exhibit small eyes, outstretched wings, downward extended halteres and reduced circulating blood cell concentration, demonstrating the roles of Upd3 in these tissues’ development. However, functions of Upd3 in other aspects of immune response were not detected. To investigate the mechanism of the coordinate expression of upd and upd3, the genetic and molecular relationship of upd, upd3 and os was dissected. The os alleles, oso, oss and os1, are a group of classical alleles which display outstretched wings, small eyes, or both, respectively. The genetic complementation tests of upd, upd3 and os showed that both upd and upd3 failed to complement os while upd complemented upd3, suggesting functions of both upd and upd3 are affected in os alleles. Consistent with the genetic tests, the expression of upd and upd3 in eye discs is lost in os allele. Molecularly, putative enhancer regions are deleted at the 5’ end of upd3 in os alleles. Hence, a transcriptional co-regulation model of upd and upd3 is proposed in which upd and upd3 share a common cis-regulatory region, lesions of which cause the os phenotype.

Janus Kinase (JAK) activity specifies the cell fates of the follicular epithelium during Drosophila oogenesis by establishing a gradient of JAK activity with highest levels at the A/P poles. Unpaired (Upd), a ligand for the pathway, is expressed and secreted exclusively from the polar cells potentially establishing the JAK activity gradient. This project proposed that Upd acts as a morphogen to directly establish the JAK activity gradient, specifying the fates of the follicular epithelium. The aims of this work were to investigate the extracellular distribution of Upd and, in addition, factors that may be involved. Furthermore, upd3, a gene encoding a protein with sequence similarity to Upd, is also co-expressed with upd in the polar cells. An additional aim of this project was to determine what role, if any, Upd3 plays in follicular development. Immunostaining was used to reveal Upd distribution during oogenesis. The data revealed an Upd gradient on the apical membrane of the follicular epithelium. By virtue of the extracellular gradient, Upd fulfills the requirements necessary to be classified as a morphogen. Some morphogens are dependent on heparan sulphate proteoglycans (HSPGs) for distribution. Using mitotic recombination to make mosaics, this work reveals that Dally, a glypican, is essential for the distribution of Upd and establishment of the JAK gradient during oogenesis. The data suggests Dally is involved with stability of extracellular Upd. Mosaic analysis of an additional HSPGs revealed that they are not essential for the Upd gradient or JAK activity during oogenesis. upd3 mutant flies have small eyes and outstretched wings, a phenotype consistent reduced JAK activity. In upd3 mutant ovaries it is shown that there is a higher frequency of deteriorating egg chambers, a higher frequency of egg chamber fusions, and a decrease in border cells per egg chamber compared to wildtype controls; all of which support a reduction of JAK activity. Furthermore, ovarian phenotypes of upd3 get worse as the fly ages suggesting that upd3 is required over time. The data presented suggests that Upd3 does act to maintain JAK activity in the ovary as the fly ages.