Gγ1, a Downstream Target for the hmgcr-Isoprenoid Biosynthetic Pathway, Is Required for Releasing the Hedgehog Ligand and Directing Germ Cell Migration

The isoprenoid biosynthetic pathway leading from the production of mevalonate by HMGCoA reductase (Hmgcr) to the geranylation of the G protein subunit, Gγ1, plays an important role in cardiac development in the fly. Hmgcr has also been implicated in the release of the signaling molecule Hedgehog (Hh) from hh expressing cells and in the production of an attractant that directs primordial germ cells to migrate to the somatic gonadal precursor cells (SGPs). The studies reported here indicate that this same hmgcr→Gγ1 pathway provides a novel post-translational mechanism for modulating the range and activity of the Hh signal produced by hh expressing cells. We show that, like hmgcr, gγ1 and quemao (which encodes the enzyme, geranylgeranyl diphosphate synthetase, that produces the substrate for geranylation of Gγ1) are components of the hh signaling pathway and are required for the efficient release of the Hh ligand from hh expressing cells. We also show that the hmgcr→Gγ1 pathway is linked to production of the germ cell attractant by the SGPs through its ability to enhance the potency of the Hh signal. We show that germ cell migration is disrupted by the loss or gain of gγ1 activity, by trans-heterozygous combinations between gγ1 and either hmgcr or hh mutations, and by ectopic expression of dominant negative Gγ1 proteins that cannot be geranylated

The DOCK Protein Sponge Binds to ELMO and Functions in Drosophila Embryonic CNS Development

Cell morphogenesis, which requires rearrangement of the actin cytoskeleton, is essential to coordinate the development of tissues such as the musculature and nervous system during normal embryonic development. One class of signaling proteins that regulate actin cytoskeletal rearrangement is the evolutionarily conserved CDM (C. elegans Ced-5, human DOCK180, Drosophila Myoblast city, or Mbc) family of proteins, which function as unconventional guanine nucleotide exchange factors for the small GTPase Rac. This CDM-Rac protein complex is sufficient for Rac activation, but is enhanced upon the association of CDM proteins with the ELMO/Ced-12 family of proteins. We identified and characterized the role of Drosophila Sponge (Spg), the vertebrate DOCK3/DOCK4 counterpart as an ELMO-interacting protein. Our analysis shows Spg mRNA and protein is expressed in the visceral musculature and developing nervous system, suggesting a role for Spg in later embryogenesis. As maternal null mutants of spg die early in development, we utilized genetic interaction analysis to uncover the role of Spg in central nervous system (CNS) development. Consistent with its role in ELMO-dependent pathways, we found genetic interactions with spg and elmo mutants exhibited aberrant axonal defects. In addition, our data suggests Ncad may be responsible for recruiting Spg to the membrane, possibly in CNS development. Our findings not only characterize the role of a new DOCK family member, but help to further understand the role of signaling downstream of N-cadherin in neuronal development

Interpretation of the UPD/JAK/STAT morphogen gradient in Drosophila follicle cells

We are using Drosophila follicle cells to study the mechanisms that promote cell motility. Using genetics we identified a gene regulatory network that controls the dynamic pattern of activation of JAK/STAT in anterior follicle cells. Under the influence of a graded signal, Unpaired (UPD), JAK/STAT becomes activated first in a graded fashion. STAT, in turn, locally activates its own repressor, Apontic (APT), a new feedback regulator of JAK/STAT signaling. High levels of JAK/STAT also activate Slow Border Cells (SLBO), which undermines APT-mediated repression. In this way, cells that achieve a high JAK/STAT level maintain SLBO expression and form border cells, which then migrate out of the cell layer. Cells with lower JAK/STAT activity express more APT than SLBO, ultimately lose STAT activity, and remain in the follicular epithelium. To better understand how the graded signal is converted to an all-or-none decision to move or stay, we developed a mathematical model. Simulations using the model reproduce the observed dynamics of JAK/STAT expression in the wild type and in several mutant situations. By combining biological experiments and mathematical modeling, we can achieve a more sophisticated understanding of how cells interpret molecular gradients

Fly and mammalian lipid phosphate phosphatase isoforms differ in activity both in vitro and in vivo

Wunen (Wun), a homologue of a lipid phosphate phosphatase (LPP), has a crucial function in the migration and survival of primordial germ cells (PGCs) during Drosophila embryogenesis. Past work has indicated that the LPP isoforms may show functional redundancy in certain systems, and that they have broad-range lipid phosphatase activities in vitro, with little apparent specificity between them. We show here that there are marked differences in biochemical activity between fly Wun and mammalian LPPs, with Wun having a narrower activity range than has been reported for the mammalian LPPs. Furthermore, although it is active on a range of substrates in vitro, mouse Lpp1 has no activity on an endogenous Drosophila germ-cell-specific factor in vivo. Conversely, human LPP3 is active, resulting in aberrant migration and PGC death. These results show an absolute difference in bioactivity among LPP isoforms for the first time in a model organism and may point towards an underlying signalling system that is conserved between flies and humans

Enhancing Undergraduate Teaching and Research with a Drosophila Virginizing System

Laboratory exercises using Drosophila crosses are an effective pedagogical method to complement traditional lecture and textbook presentations of genetics. Undergraduate thesis research is another common setting for using Drosophila. A significant barrier to using Drosophila for undergraduate teaching or research is the time and skill required to accurately collect virgins for use in controlled crosses. Erroneously collecting males or nonvirgin females contaminates crosses with unintended genotypes and confounds the results. Collecting adequate numbers of virgins requires large amounts of time, even for those skilled in virgin collection. I have adapted an effective method for virgin collection that eliminates these concerns and is straightforward to use in undergraduate settings. Using a heat-shock–induced, conditional lethal transgene specifically in males, male larvae can be eliminated from a culture before adults eclose. Females thus eclose in the absence of males and remain virgin, eliminating the need to laboriously score and segregate freshly eclosed females. This method is reliable, easily adaptable to any desired phenotypic marker, and readily scaleable to provide sufficient virgins for large laboratory classes or undergraduate research projects. In addition, it allows instructors lacking Drosophila expertise to use this organism as a pedagogical tool