Guidance of cell migration by the Drosophila PDGF/VEGF receptor.

Directed cell migrations are important for development, but the signaling pathways and mechanisms responsible for guiding cell migration in vivo are poorly understood. Migration of border cells during Drosophila oogenesis is a simple and attractive model system in which to address these questions. We demonstrate that PVR, a receptor tyrosine kinase related to mammalian PDGF and VEGF receptors, acts in border cells to guide them to the oocyte. The oocyte is the source of a ligand for PVR, PDGF/VEGF factor 1 (PVF1). Intriguingly, the guidance function of PVR is largely redundant with that of EGFR. We present evidence implicating Rac and the Rac activator Mbc/DOCK180/CED-5 as mediators of the guidance signal.

Cloning and expression of sprint, a Drosophila homologue of RIN1.

The small GTPase Ras is critical for regulation of growth and differentiation during development. The mammalian protein RIN1 is a potential Ras effector protein, which can also interact with the Abelson tyrosine kinase. However, its biological function is unknown. We have identified the Drosophila homologue of RIN1, called sprint, for SH2, poly-proline containing Ras interactor. The sprint locus is very large and contains at least two differentially expressed isoforms (sprint-a and sprint-b). Both isoforms are expressed in the ovary and maternal mRNA is deposited into embryos. In addition, sprint is zygotically expressed in the developing midgut, amnioserosa and in a specific subset of CNS neurons. The expression patterns of the two sprint isoforms are temporally distinct suggesting that the isoforms may have unique functions.

Guidance of cell migration by EGF receptor signaling during Drosophila oogenesis.

Directed cell migration is important for many aspects of normal animal development, but little is known about how cell migrations are guided or the mechanisms by which guidance cues are translated into directed cell movement. Here we present evidence that signaling mediated by the epidermal growth factor receptor (EGFR) guides dorsal migration of border cells during Drosophila oogenesis. The transforming growth factor-alpha (TGF-alpha)-like ligand Gurken appears to serve as the guidance cue. To mediate this guidance function, EGFR signals via a pathway that is independent of Raf-MAP kinase and receptor-specific.

The level of C/EBP protein is critical for cell migration during Drosophila oogenesis and is tightly controlled by regulated degradation.

The C/EBP transcription factor, Slbo, is required for migration of border cells during Drosophila oogenesis. Unexpectedly, we find that neither increase nor decrease of Slbo activity is tolerated in border cells. Correct protein level is in part ensured by cell type-specific regulated turnover of Slbo protein. Through genetic screening, we identify two genes that are involved in this regulation. The Ubp64 ubiquitin hydrolase acts as a stabilizer of Slbo protein. A novel gene, tribbles, is a negative regulator of slbo in vivo. Tribbles acts by specifically targeting Slbo for rapid degradation via ubiquitination.

Tribbles coordinates mitosis and morphogenesis in Drosophila by regulating string/CDC25 proteolysis.

Morphogenesis and cell differentiation in multicellular organisms often require accurate control of cell divisions. We show that a novel cell cycle regulator, tribbles, is critical for this control during Drosophila development. During oogenesis, the level of tribbles affects the number of germ cell divisions as well as oocyte determination. The mesoderm anlage enters mitosis prematurely in tribbles mutant embryos, leading to gastrulation defects. We show that Tribbles acts by specifically inducing degradation of the CDC25 mitotic activators String and Twine via the proteosome pathway. By regulating CDC25, Tribbles serves to coordinate entry into mitosis with morphogenesis and cell fate determination.

Gal4 in the Drosophila female germline.

The modular Gal4 system has proven to be an extremely useful tool for conditional gene expression in Drosophila. One limitation has been the inability of the system to work in the female germline. A modified Gal4 system that works throughout oogenesis is presented here. To achieve germline expression, it was critical to change the basal promoter and 3'-UTR in the Gal4-responsive expression vector (generating UASp). Basal promoters and heterologous 3'-UTRs are often considered neutral, but as shown here, can endow qualitative tissue-specificity to a chimeric transcript. The modified Gal4 system was used to investigate the role of the Drosophila FGF homologue branchless, ligand for the FGF receptor breathless, in border cell migration. FGF signaling guides tracheal cell migration in the embryo. However, misexpression of branchless in the ovary had no effect on border cell migration. Thus border cells and tracheal cells appear to be guided differently.

Systematic gain-of-function genetics in Drosophila.

A modular misexpression system was used to carry out systematic gain-of-function genetic screens in Drosophila. The system is based on inducible expression of genes tagged by insertion of a P-element vector carrying a GAL4-regulated promoter oriented to transcribe flanking genomic sequences. To identify genes involved in eye and wing development, the 2300 independent lines were screened for dominant phenotypes. Among many novel genes, the screen identified known genes, including hedgehog and decapentaplegic, implicated in these processes. A genetic interaction screen for suppressors of a cell migration defect in a hypomorphic slow border cells mutant identified known genes with likely roles in tyrosine kinase signaling and control of actin cytoskeleton, among many novel genes. These studies demonstrate the ability of the modular misexpression system to identify developmentally important genes and suggest that it will be generally useful for genetic interaction screens.

A modular misexpression screen in Drosophila detecting tissue-specific phenotypes.

Genetic screens in Drosophila have lead to the discovery of many genes important for patterning and signal transduction in diverse organisms. Traditionally, the phenotypic effects of loss-of-function mutations are analyzed. As an alternative way to link genes and function, I have developed a versatile misexpression screen in Drosophila, the first such screen in higher eukaryotes. The screen identifies genes that, when over- or misexpressed in a pattern of interest, give a specific phenotype or modulate an existing mutant phenotype. It is based on Gal4 transactivation of a mobile enhancer and promoter that "targets" random endogenous genes for expression. The modular design of the screen allows directed expression in any temporal or spatial pattern. When activated in the developing eye, 4% of target inserts gave dominant phenotypes. One insertion was in the gene encoding Ras GTPase-activating protein; its overexpression phenotype was strongly enhanced by a mutation in Ras1. Thus, biologically relevant phenotypes and genetic interactions are identified using this method. The screen is a powerful new tool for developmental genetics; similar approaches can also be developed for other organisms.

Specification of C/EBP function during Drosophila development by the bZIP basic region.

The biologically relevant interactions of a transcription factor are those that are important for function in the organism. Here, a transgenic rescue assay was used to determine which molecular functions of Drosophila CCAAT/enhancer binding protein (C/EBP), a basic region-leucine zipper transcription factor, are required for it to fulfill its essential role during development. Chimeric proteins that contain the Drosophila C/EBP (DmC/EBP) basic region, a heterologous zipper, and a heterologous activation domain could functionally substitute for DmC/EBP. Mammalian C/EBPs were also functional in Drosophila. In contrast, 9 of 25 single amino acid substitutions in the basic region disrupted biological function. Thus, the conserved basic region specifies DmC/EBP activity in the organism.

Drosophila C/EBP: a tissue-specific DNA-binding protein required for embryonic development.

Recently, we reported the cloning of the Drosophila melanogaster homolog of the vertebrate CCAAT/enhancer-binding protein (C/EBP). Here, we describe studies of the DNA-binding and dimerization properties of Drosophila C/EBP (DmC/EBP), as well as its tissue distribution, developmental regulation, and essential role in embryonic development and conclude that it bears functional as well as structural similarity to mammalian C/EBP. DmC/EBP contains a basic region/leucine zipper (bZIP) DNA-binding domain very similar to that of mammalian C/EBP and the purified C/EBPs bound to DNA with the same sequence specificity. Among the DNA sequences that DmC/EBP bound with high affinity was a conserved site within the promoter of the DmC/EBP gene itself. In vitro, DmC/EBP and mammalian C/EBP specifically formed functional heterodimers; however, as we found no evidence for a family of DmC/EBPs, DmC/EBP may function as a homodimer in vivo. The DmC/EBP protein was expressed predominantly during late embryogenesis in the nuclei of a restricted set of differentiating cell types, such as the lining of the gut and epidermis, similar to the mammalian tissues that express C/EBP. We have characterized mutations in the DmC/EBP gene and found that deleting the gene caused late embryonic lethality. Embryos that lack C/EBP die just before or just upon hatching. The lethal phenotype of C/EBP mutants can be rescued with the cloned C/EBP gene introduced by P-element-mediated germ-line transformation. The strict requirement for C/EBP during Drosophila embryogenesis, coupled with its structural and functional similarities to mammalian C/EBP, provides a useful genetic system in which to study the role of C/EBP in development.

A novel temporal expression pattern of three C/EBP family members in differentiating myelomonocytic cells.

Members of the CCAAT/enhancer binding protein (C/EBP) family have been shown to regulate the terminal differentiation of adipocytes and hepatocytes. In these cell lineages, high levels of C/EBP alpha are found only in mature, nondividing cells. Using Western blotting and immunohistochemical staining, we have determined the temporal order of expression for C/EBP alpha, C/EBP beta, and C/EBP delta in differentiating myelomonocytic marrow cells. These studies show a unique temporal pattern of C/EBP isoform expression in the myeloid lineage. In particular, C/EBP alpha expression is very high in proliferative myelomonocytic cells, and diminishes during phenotypic maturation. While we have detected C/EBP alpha, C/EBP beta, and C/EBP delta in multiple myeloid leukemia cell lines, and C/EBP alpha in normal myeloid cells and in de novo human myeloid leukemias, we have not detected these C/EBP isoforms in either erythroid or lymphoid cells. Finally, we show that C/EBP alpha, C/EBP beta, and C/EBP delta protein and messenger RNA levels correlate in maturing granulocytic cells. The formation of tissue-specific combinations of C/EBP homodimers and heterodimers may allow this family of transcription factors to regulate different sets of genes in adipocytes, hepatocytes, and myelomonocytes.

slow border cells, a locus required for a developmentally regulated cell migration during oogenesis, encodes Drosophila C/EBP.

During Drosophila oogenesis six to ten follicle cells, the border cells, undergo a dramatic and stereotypic migration through the developing egg chamber. We identified four independent P element insertion mutations that specifically blocked border cell migration. They defined a single, novel locus that was named slow border cells (slbo), because hypomorphic alleles caused delayed onset of the migration. Laser ablation of the border cells, or failure of their migration, caused improper morphogenesis of the micropyle, the egg-shell structure through which the sperm enters at fertilization. The slbo locus was found to encode a product homologous to the CCAAT/enhancer-binding protein (C/EBP), a basic region-leucine zipper transcription factor. Drosophila C/EBP may be required for the expression of gene products mediating border cell migration.

Essential AP-1 and PEA3 binding elements in the human urokinase enhancer display cell type-specific activity.

We have characterized a transcriptional enhancer of the human urokinase-type plasminogen activator (uPA) gene in three transformed human cell lines: HeLa, HepG2 and HT1080. The enhancer is located approximately 2 kbp upstream of the mRNA cap site and is active in all three cell lines. By footprinting and gel retardation analysis we found that it contained two binding sites for transcription factor AP-1, encoded by the fos and jun proto-oncogene families. The most upstream of these sites was juxtaposed to a binding site for PEA3, a product of the ets/Spi proto-oncogene family. By transient transfection analysis of deletions, point mutations and subcloned fragments, we found these sites to be crucial for enhancer activity. However, the sites displayed differences in activity in the three different cell lines. The downstream AP-1 site was almost exclusively responsible for enhancer activity in HeLa cells, whereas the AP-1/PEA3 site played a major role in HT1080 and HepG2 cells. The implications of our findings for the known regulation of uPA expression by transforming oncogenes, adenovirus E1A protein and glucocorticoids are discussed.

Transcription factor PEA3 participates in the induction of urokinase plasminogen activator transcription in murine keratinocytes stimulated with epidermal growth factor or phorbol-ester.

Keratinocytes in culture represent cells which exhibit continued and controlled growth in the organism. We have investigated the synthesis of urokinase plasminogen activator mRNA in exponentially growing cultures of primary murine keratinocytes and the keratinocyte cell line BALB/MK. The tumor promotor 12-O-tetradecanoyl phorbol-13-acetate (TPA) and epidermal growth factor (EGF) induced urokinase mRNA synthesis. We made a series of progressive 5' deletions as well as internal deletions in the region upstream of the murine uPA gene. These were joined to the cat reporter gene, and used to map the TPA and EGF responsive regions of the promoter. We found both responsive sequences within a 90 base pair Hae III fragment, located 2.4 kb. upstream of the mRNA cap site. This DNA fragment conferred TPA inducibility on reporter gene expression independent of its distance and orientation to the transcription initiation site. Footprinting and gel retardation studies identified the responsible sequence to be a binding site for PEA3 juxtaposed to an octameric TRE-element. Transfections with point mutants showed that these target sequences were necessary for TPA and EGF induction of transcription.