Hedgehog (Hh) shape up development by playing important role in signaling, and thereby controlling growth and pattern formation. It is for this reason that their spatial distribution is tightly regulated. The 19kDa active form of Hh is modified with a palmitate at its N-terminal and with cholesterol at its C-terminal. This dually lipid modified form of Hh act as a morphogen, and is also referred to as HhNp (Mann and Beachy, 2004). In most cases, they are released from producing cells and spread into adjacent non-expressing cells within the tissue, where it activates target gene expression in a concentration-dependent manner. In Drosophila, Lipophorin (Lpp) particles carry these lipid-modified forms of Hh and play a role in long range signaling in the developing wing disc. Further, these particles circulate throughout the larvae in the hemolymph to distribute nutrients mostly in the form of lipids to different tissues of the animal. Thus, Lpp plays important role in metabolism and development. Hh as a morphogen plays a very important role in development and patterning of embryo and imaginal discs in Drosophila. We wanted to understand the role of Hh in overall development of Drosophila. In my thesis work, I discovered a new form of Hh that is systemically circulating in the 3rd instar larva of Drosophila. I show that imaginal tissues do not produce this form of circulating Hh. Our experiments strongly suggest that systemic Hh can travel from one tissue to another, a feature that was previously unknown. I also show that it could rescue the growth of the imaginal disc, implying its ability to influence cell proliferation. Since the concentration of systemic Hh is low it fails to up regulate the target genes. I characterized fat body as a target of systemically circulating Hh. I clearly demonstrate that fat body transcribes most of the components of Hh signaling pathway except Hh. Further, Hh accumulates in the fat body during late 3rd instar larvae. That makes the fat body an ideal target of systemic Hh. This could shed light in understanding the role of Hh in overall development of Drosophila melanogaster that includes tissue-based interaction.

Animals carry lipids such as hydrocarbons, fats, and sterols throughout their circulatory systems bound to a carrier protein known as lipophorin. The lipophorin receptor has been characterized in locusts, mosquitoes and cockroaches yet little is known about it in Drosophila melanogaster. An antibody against the eleven variants of the lipophorin receptor was developed and tested. Although this was the main feature of the work, several preliminary experiments using RNA interference were conducted to determine the effects of lipophorin receptor. Flies whose lipophorin receptor proteins were knocked down were found to have no major differences in locomotor activity in total darkness suggesting that their circadian rhythms are unaffected. The same flies were found to have extensive differences in their cuticular hydrocarbon profiles as compared with wild‐type flies. Whole‐mount tissue staining of Drosophila adult brains revealed that several cells in the central nervous system are immunoreactive with the anti‐Lipophorin receptor antibody.

Animals carry lipids such as hydrocarbons, fats, and sterols throughout their circulatory systems bound to a carrier protein known as lipophorin. The lipophorin receptor has been characterized in locusts, mosquitoes and cockroaches yet little is known about it in Drosophila melanogaster. An antibody against the eleven variants of the lipophorin receptor was developed and tested. Although this was the main feature of the work, several preliminary experiments using RNA interference were conducted to determine the effects of lipophorin receptor. Flies whose lipophorin receptor proteins were knocked down were found to have no major differences in locomotor activity in total darkness suggesting that their circadian rhythms are unaffected. The same flies were found to have extensive differences in their cuticular hydrocarbon profiles as compared with wild‐type flies. Whole‐mount tissue staining of Drosophila adult brains revealed that several cells in the central nervous system are immunoreactive with the anti‐Lipophorin receptor antibody.

Hedgehog (Hh) shape up development by playing important role in signaling, and thereby controlling growth and pattern formation. It is for this reason that their spatial distribution is tightly regulated. The 19kDa active form of Hh is modified with a palmitate at its N-terminal and with cholesterol at its C-terminal. This dually lipid modified form of Hh act as a morphogen, and is also referred to as HhNp (Mann and Beachy, 2004). In most cases, they are released from producing cells and spread into adjacent non-expressing cells within the tissue, where it activates target gene expression in a concentration-dependent manner. In Drosophila, Lipophorin (Lpp) particles carry these lipid-modified forms of Hh and play a role in long range signaling in the developing wing disc. Further, these particles circulate throughout the larvae in the hemolymph to distribute nutrients mostly in the form of lipids to different tissues of the animal. Thus, Lpp plays important role in metabolism and development. Hh as a morphogen plays a very important role in development and patterning of embryo and imaginal discs in Drosophila. We wanted to understand the role of Hh in overall development of Drosophila. In my thesis work, I discovered a new form of Hh that is systemically circulating in the 3rd instar larva of Drosophila. I show that imaginal tissues do not produce this form of circulating Hh. Our experiments strongly suggest that systemic Hh can travel from one tissue to another, a feature that was previously unknown. I also show that it could rescue the growth of the imaginal disc, implying its ability to influence cell proliferation. Since the concentration of systemic Hh is low it fails to up regulate the target genes. I characterized fat body as a target of systemically circulating Hh. I clearly demonstrate that fat body transcribes most of the components of Hh signaling pathway except Hh. Further, Hh accumulates in the fat body during late 3rd instar larvae. That makes the fat body an ideal target of systemic Hh. This could shed light in understanding the role of Hh in overall development of Drosophila melanogaster that includes tissue-based interaction.

The Drosophila Lipoprotein particles bear lipid-linked morphogens on their surface and are required for long-range signaling activity of Wingless and Hedgehog. They also bind a wide variety of gpi-linked proteins. Whether any of these proteins affect morphogen signaling is unknown. Here, I show that the gpi-linked heparan sulfate proteoglycan Dally is released from cell membranes and binds to lipoprotein particles both with and without its lipid anchor. Hedgehog signaling efficiency is reduced in Dally mutant discs, but can be rescued non-autonomously by expression of non-gpi-modified Dally. This Dally isoform colocalizes with Hedgehog, Patched and Lipophorin in endosomes and increases Hedgehog signaling efficiency without affecting Hedgehog distribution. These data show that Hedgehog signaling activity can be influenced by other Lipophorin-associated proteins, and suggest Lipoproteins provide a platform for regulation of morphogen signaling.

The Drosophila Lipoprotein particles bear lipid-linked morphogens on their surface and are required for long-range signaling activity of Wingless and Hedgehog. They also bind a wide variety of gpi-linked proteins. Whether any of these proteins affect morphogen signaling is unknown. Here, I show that the gpi-linked heparan sulfate proteoglycan Dally is released from cell membranes and binds to lipoprotein particles both with and without its lipid anchor. Hedgehog signaling efficiency is reduced in Dally mutant discs, but can be rescued non-autonomously by expression of non-gpi-modified Dally. This Dally isoform colocalizes with Hedgehog, Patched and Lipophorin in endosomes and increases Hedgehog signaling efficiency without affecting Hedgehog distribution. These data show that Hedgehog signaling activity can be influenced by other Lipophorin-associated proteins, and suggest Lipoproteins provide a platform for regulation of morphogen signaling.

Morphogens of the Wnt and Hedgehog families are secreted signaling molecules that coordinate growth and patterning of many different tissues. Both, Wingless and Hedgehog spread across long distances in developing wing of Drosophila melanogaster. However, both proteins are covalently modified with lipid moieties. The mechanisms that allow long-range movement of such hydrophobic molecules are unclear. Like Wingles and Hedgehog, glycosylphosphatidylinositol (gpi)-linked proteins also transfer between cells with their lipid anchor intact. It has been speculated that gpi-linked proteins and lipid-linked morphogens travel together on a membranous particle, which was termed an argosome. As yet however, no functional link between argosome production and dispersal of lipid-linked proteins has been established. The topic of this thesis is to understand the cell biological nature of the argosome and thus contribute to understanding of morphogen gradient formation. To address the question of argosome biosynthesis, at least two models have been proposed. One possibility is that argosomes are membranous exovesicles with a complete membrane bilayer. Alternatively, argosomes might resemble lipoprotein particles that comprise on of a family of apolipoproteins scaffolded around a phospholipid monolayer that surrounds a core of esterified cholesterol and triglyceride. Lipid-modified proteins of the exoplasmic face of the membrane (like GFPgpi, Wingless or Hedgehog) might fit well into the outer phospholipid monolayer of such a particle. Here, I utilize biochemical fractionation to determine the sort of particle that lipid-linked proteins associate with. I show that Wingless, Hedgehog and gpi-linked proteins bind Drosophila lipoprotein particles in vitro, and colocalize with them in wing imaginal discs. Next, I use genetic means to address the functional importance of this association. I demonstrate that reducing Lipophorin levels in Drosophila larvae perturbs long-range but not shor-range Wingless and Hedgehog signaling, and increases the sequestration of Hedgehog by Patched. I propose that Lipophorin particles are vehicles for the long-range movement of lipid-linked morphogens and gpi-linked proteins.

Morphogens of the Wnt and Hedgehog families are secreted signaling molecules that coordinate growth and patterning of many different tissues. Both, Wingless and Hedgehog spread across long distances in developing wing of Drosophila melanogaster. However, both proteins are covalently modified with lipid moieties. The mechanisms that allow long-range movement of such hydrophobic molecules are unclear. Like Wingles and Hedgehog, glycosylphosphatidylinositol (gpi)-linked proteins also transfer between cells with their lipid anchor intact. It has been speculated that gpi-linked proteins and lipid-linked morphogens travel together on a membranous particle, which was termed an argosome. As yet however, no functional link between argosome production and dispersal of lipid-linked proteins has been established. The topic of this thesis is to understand the cell biological nature of the argosome and thus contribute to understanding of morphogen gradient formation. To address the question of argosome biosynthesis, at least two models have been proposed. One possibility is that argosomes are membranous exovesicles with a complete membrane bilayer. Alternatively, argosomes might resemble lipoprotein particles that comprise on of a family of apolipoproteins scaffolded around a phospholipid monolayer that surrounds a core of esterified cholesterol and triglyceride. Lipid-modified proteins of the exoplasmic face of the membrane (like GFPgpi, Wingless or Hedgehog) might fit well into the outer phospholipid monolayer of such a particle. Here, I utilize biochemical fractionation to determine the sort of particle that lipid-linked proteins associate with. I show that Wingless, Hedgehog and gpi-linked proteins bind Drosophila lipoprotein particles in vitro, and colocalize with them in wing imaginal discs. Next, I use genetic means to address the functional importance of this association. I demonstrate that reducing Lipophorin levels in Drosophila larvae perturbs long-range but not shor-range Wingless and Hedgehog signaling, and increases the sequestration of Hedgehog by Patched. I propose that Lipophorin particles are vehicles for the long-range movement of lipid-linked morphogens and gpi-linked proteins.