Photoreceptor and cone cells in the Drosophila eye are recruited following activation of the epidermal growth factor receptor (EGFR) pathway. We have identified echinoid (ed) as a novel putative cell adhesion molecule that negatively regulates EGFR signaling. The ed mutant phenotype is associated with extra photoreceptor and cone cells. Conversely, ectopic expression of ed in the eye leads to a reduction in the number of photoreceptor cells. ed expression is independent of EGFR signaling and ED is localized to the plasma membrane of every cells throughout the eye disc. We present evidence that ed acts nonautonomously to generate extra R7 cells by a mechanism that is sina-independent but upstream of Tramtrack (TTK88). Together, our results support a model whereby ED defines an independent pathway that antagonizes EGFR signaling by regulating the activity, but not the level, of the TTK88 transcriptional repressor.

During Drosophila oogenesis, the formation of the egg respiratory appendages and the micropyle require the shaping of anterior and dorsal follicle cells. Prior to their morphogenesis, cells of the presumptive appendages are determined by integrating dorsal-ventral and anterior-posterior positional information provided by the epidermal growth factor receptor (EGFR) and Decapentaplegic (Dpp) pathways, respectively. We show here that another signaling pathway, the Drosophila Jun-N-terminal kinase (JNK) cascade, is essential for the correct morphogenesis of the dorsal appendages and the micropyle during oogenesis. Mutant follicle cell clones of members of the JNK pathway, including DJNKK/hemipterous (hep), DJNK/basket (bsk), and Djun, block dorsal appendage formation and affect the micropyle shape and size, suggesting a late requirement for the JNK pathway in anterior chorion morphogenesis. In support of this view, hep does not affect early follicle cell patterning as indicated by the normal expression of kekkon (kek) and Broad-Complex (BR-C), two of the targets of the EGFR pathway in dorsal follicle cells. Furthermore, the expression of the TGF-beta homolog dpp, which is under the control of hep in embryos, is not coupled to JNK activity during oogenesis. We show that hep controls the expression of puckered (puc) in the follicular epithelium in a cell-autonomous manner. Since puc overexpression in the egg follicular epithelium mimics JNK appendages and micropyle phenotypes, it indicates a negative role of puc in their morphogenesis. The role of the JNK pathway in the morphogenesis of follicle cells and other epithelia during development is discussed.

Establishing cellular polarity is critical for tissue organization and function. Initially discovered in the landmark genetic screen for Drosophila developmental mutants, bazooka, crumbs, shotgun and stardust mutants exhibit severe disruption in apicobasal polarity in embryonic epithelia, resulting in multilayered epithelia, tissue disintegration, and defects in cuticle formation. Here we report that stardust encodes single PDZ domain MAGUK (membrane-associated guanylate kinase) proteins that are expressed in all primary embryonic epithelia from the onset of gastrulation. Stardust colocalizes with Crumbs at the apicolateral boundary, although their expression patterns in sensory organs differ. Stardust binds to the carboxy terminus of Crumbs in vitro, and Stardust and Crumbs are mutually dependent in their stability, localization and function in controlling the apicobasal polarity of epithelial cells. However, for the subset of ectodermal cells that delaminate and form neuroblasts, their polarity requires the function of Bazooka, but not of Stardust or Crumbs.

The precise regulation of growth factor signalling is crucial to the molecular control of development in Drosophila. Post-translational modification of signalling molecules is one of the mechanisms that modulate developmental signalling specificity. We describe a new gene, fringe connection (frc), that encodes a nucleotide-sugar transporter that transfers UDP-glucuronic acid, UDP-N-acetylglucosamine and possibly UDP-xylose from the cytoplasm into the lumen of the endoplasmic reticulum/Golgi. Embryos with the frc mutation display defects in Wingless, Hedgehog and fibroblast growth factor signalling. Clonal analysis shows that fringe-dependent Notch signalling is disrupted in frc mutant tissue.

Recent studies in Drosophila have shown that heparan sulfate proteoglycans (HSPGs) are required for Wingless (Wg/Wnt) signaling. In addition, genetic and phenotypic analyses have implicated the glypican gene dally in this process. Here, we report the identification of another Drosophila glypican gene, dally-like (dly) and show that it is also involved in Wg signaling. Inhibition of dly gene activity implicates a function for DLY in Wg reception and we show that overexpression of DLY leads to an accumulation of extracellular Wg. We propose that DLY plays a role in the extracellular distribution of Wg. Consistent with this model, a dramatic decrease of extracellular Wg was detected in clones of cells that are deficient in proper glycosaminoglycan biosynthesis. We conclude that HSPGs play an important role in organizing the extracellular distribution of Wg.

The leading edge (LE) is a single row of cells in the Drosophila embryonic epidermis that marks the boundary between two fields of cells: the amnioserosa and the dorsal ectoderm. LE cells play a crucial role in the morphogenetic process of dorsal closure and eventually form the dorsal midline of the embryo. Mutations that block LE differentiation result in a failure of dorsal closure and embryonic lethality. How LE cells are specified remains unclear. To explore whether LE cells are specified in response to early dorsoventral patterning information or whether they arise secondarily, we have altered the extent of amnioserosa and dorsal ectoderm genetically, and assayed LE cell fate. We did not observe an expansion of LE fate in dorsalized or ventralized mutants. Furthermore, we observed that the LE fate arises as a single row of cells, wherever amnioserosa tissue and dorsal epidermis are physically juxtaposed. Taken together our data indicate that LE formation is a secondary consequence of early zygotic dorsal patterning signals. In particular, proper LE specification requires the function of genes such as u-shaped and hindsight, which are direct transcriptional targets of the early Decapentaplegic/Screw patterning gradient, to establish a competency zone from which LE arises. We propose that subsequent inductive signaling between amnioserosa and dorsal ectoderm restricts the formation of LE to a single row of cells.

The actin cytoskeleton orders cellular space and transduces many of the forces required for morphogenesis. Here we combine genetics and cell biology to identify genes that control the polarized distribution of actin filaments within the Drosophila follicular epithelium. We find that profilin and cofilin regulate actin-filament formation throughout the cell cortex. In contrast, CAP-a Drosophila homologue of Adenylyl Cyclase Associated Proteins-functions specifically to limit actin-filament formation catalysed by Ena at apical cell junctions. The Abl tyrosine kinase also collaborates in this process. We therefore propose that CAP, Ena and Abl act in concert to modulate the subcellular distribution of actin filaments in Drosophila.

Loss of cell polarity and tissue architecture are characteristics of malignant cancers derived from epithelial tissues. We provide evidence from Drosophila that a group of membrane-associated proteins act in concert to regulate both epithelial structure and cell proliferation. Scribble (Scrib) is a cell junction-localized protein required for polarization of embryonic and, as demonstrated here, imaginal disc and follicular epithelia. We show that the tumor suppressors lethal giant larvae (lgl) and discs-large (dlg) have identical effects on all three epithelia, and that scrib also acts as a tumor suppressor. Scrib and Dlg colocalize and overlap with Lgl in epithelia; activity of all three genes is required for cortical localization of Lgl and junctional localization of Scrib and Dlg. scrib, dlg, and lgl show strong genetic interactions. Our data indicate that the three tumor suppressors act together in a common pathway to regulate cell polarity and growth control.

BACKGROUND: A polarised cytoskeleton is required to pattern cellular space, and for many aspects of cell behaviour. While the mechanisms ordering the actin cytoskeleton have been extensively studied in yeast, little is known about the analogous processes in other organisms. We have used Drosophila oogenesis as a model genetic system in which to investigate control of cytoskeletal organisation and cell polarity in multicellular eukaryotes. RESULTS: In a screen to identify genes required for Drosophila oocyte polarity, we isolated a Drosophila homologue of the yeast cyclase-associated protein, CAP. Here we show that CAP preferentially accumulates in the oocyte, where it inhibits actin polymerisation. CAP also has a role in oocyte polarity, as cap mutants fail to establish the proper, asymmetric distribution of mRNA determinants within the oocyte. Similarly in yeast, loss of CAP causes analogous polarity defects, altering the distribution of actin filaments and mRNA determinants. CONCLUSIONS: This study identifies CAP as a new effector of actin dynamics in Drosophila. As CAP controls the spatial distribution of actin filaments and mRNA determinants in both yeast and Drosophila, we conclude that CAP has an evolutionarily conserved function in the genesis of eukaryotic cell polarity.

The Drosophila segment polarity gene product Porcupine (Porc) was first identified as being necessary for processing Wingless (Wg), a Drosophila Wnt (Wnt) family member. Mouse and Xenopus homologs of porc (Mporc and Xporc) were identified and found to encode endoplasmic reticulum (ER) proteins with multiple transmembrane domains. In contrast with porc, four different types of Mporc and Xporc mRNA (A-D) are generated from a single gene by alternative splicing. Mporc mRNA is differentially expressed during embryogenesis and in various adult tissues, demonstrating that the alternative splicing is regulated to synthesize the specific types of Mporc. In transfected mammalian cells, all Mporc types affect the processing of mouse Wnt 1, 3A, 4, 6, and 7B but not 5A. Furthermore, all Mporc types are co-immunoprecipitated with various Wnt proteins. These results suggest that Mporc may function as a chaperone-like molecule for Wnt. Interestingly, all Mporc types can substitute for Porc, as they are able to rescue the phenotypes of Drosophila porc embryos. Consistent with this observation, Mporc, like Porc, modifies the processing of Wg expressed in mammalian cells. These results demonstrate that the porc gene family encodes the multitransmembrane ER proteins, which are evolutionarily well conserved and involved in processing the Wnt family.

The generation of membrane domains with distinct protein constituents is a hallmark of cell polarization. In epithelia, segregation of membrane proteins into apical and basolateral compartments is critical for cell morphology, tissue physiology and cell signalling. Drosophila proteins that confer apical membrane identity have been found, but the mechanisms that restrict these determinants to the apical cell surface are unknown. Here we show that a laterally localized protein is required for the apical confinement of polarity determinants. Mutations in Drosophila scribble (scrib), which encodes a multi-PDZ (PSD-95, Discs-large and ZO-1) and leucine-rich-repeat protein, cause aberrant cell shapes and loss of the monolayer organization of embryonic epithelia. Scrib is localized to the epithelial septate junction, the analogue of the vertebrate tight junction, at the boundary of the apical and basolateral cell surfaces. Loss of scrib function results in the misdistribution of apical proteins and adherens junctions to the basolateral cell surface, but basolateral protein localization remains intact. These phenotypes can be accounted for by mislocalization of the apical determinant Crumbs. Our results show that the lateral domain of epithelia, particularly the septate junction, functions in restricting apical membrane identity and correctly placing adherens junctions.

Insect cuticles have been a model system for the study of planar polarity for many years and a number of genes required for this process have been identified. These genes organise the polarised arrangement of hairs on the legs, wings, thorax, and abdomen of adult Drosophila. It has previously been shown that four-jointed is involved in planar polarity decisions in the eye as well as proximal distal leg and wing development. We now present evidence that four-jointed is expressed in a gradient through the developing wing and show that it is required for planar polarity determination in both the wing and the abdomen. Clones of cells either lacking or ectopically expressing four-jointed cause both autonomous and nonautonomous repolarisation of hairs in these tissues. We propose that the inferred four-jointed expression gradient is important for planar polarity establishment and that local inversions of the gradient by the clones are the probable cause of the observed polarity phenotypes. In addition we observe defects in wing vein development. The subtle phenotypes of mutant flies, and the diverse patterning processes in which it is involved, suggest that four-jointed may act as a modifier of the activity of multiple other signalling factors.

Presenilin is an essential gene for development that when disrupted leads to a neurogenic phenotype that closely resembles Notch loss of function in Drosophila. In humans, many naturally occurring mutations in Presenilin 1 or 2 cause early onset Alzheimer's disease. Both loss of expression and overexpression of Presenilin suggested a role for this protein in the localization of Armadillo/beta-catenin. In blastoderm stage Presenilin mutants, Arm is aberrantly distributed, often in Ubiquitin-immunoreactive cytoplasmic inclusions predominantly located basally in the cell. These inclusions were not observed in loss of function Notch mutants, suggesting that failure to process Notch is not the only consequence of the loss of Presenilin function. Human presenilin 1 expressed in Drosophila produces embryonic phenotypes resembling those associated with mutations in Armadillo and exhibited reduced Armadillo at the plasma membrane that is likely due to retention of Armadillo in a complex with Presenilin. The interaction between Armadillo/beta-catenin and Presenilin 1 requires a third protein which may be delta-catenin. Our results suggest that Presenilin may regulate the delivery of a multiprotein complex that regulates Armadillo trafficking between the adherens junction and the proteasome.

Raf is an essential downstream effector of activated p21(Ras) (Ras) in transducing proliferation or differentiation signals. Following binding to Ras, Raf is translocated to the plasma membrane, where it is activated by a yet unidentified "Raf activator." In an attempt to identify the Raf activator or additional molecules involved in the Raf signaling pathway, we conducted a genetic screen to identify genomic regions that are required for the biological function of Drosophila Raf (Draf). We tested a collection of chromosomal deficiencies representing approximately 70% of the autosomal euchromatic genomic regions for their abilities to enhance the lethality associated with a hypomorphic viable allele of Draf, Draf(Su2). Of the 148 autosomal deficiencies tested, 23 behaved as dominant enhancers of Draf(Su2), causing lethality in Draf(Su2) hemizygous males. Four of these deficiencies identified genes known to be involved in the Drosophila Ras/Raf (Ras1/Draf) pathway: Ras1, rolled (rl, encoding a MAPK), 14-3-3epsilon, and bowel (bowl). Two additional deficiencies removed the Drosophila Tec and Src homologs, Tec29A and Src64B. We demonstrate that Src64B interacts genetically with Draf and that an activated form of Src64B, when overexpressed in early embryos, causes ectopic expression of the Torso (Tor) receptor tyrosine kinase-target gene tailless. In addition, we show that a mutation in Tec29A partially suppresses a gain-of-function mutation in tor. These results suggest that Tec29A and Src64B are involved in Tor signaling, raising the possibility that they function to activate Draf. Finally, we discovered a genetic interaction between Draf(Su2) and Df(3L)vin5 that revealed a novel role of Draf in limb development. We find that loss of Draf activity causes limb defects, including pattern duplications, consistent with a role for Draf in regulation of engrailed (en) expression in imaginal discs.

The Drosophila wingless gene (wg) encodes a protein of the Wnt family and is a critical regulator in many developmental processes. Biochemical studies have indicated that heparan sulphate proteoglycans, consisting of a protein core to which heparan sulphate glycosaminoglycans are attached, are important for Wg function. Here we show that, consistent with these findings, the Drosophila gene sulfateless (sfl), which encodes a homologue of vertebrate heparan sulphate N-deacetylase/N-sulphotransferase (an enzyme needed for the modification of heparan sulphate) is essential for Wg signalling. We have identified the product of division abnormally delayed (dally), a glycosyl-phosphatidyl inositol (GPI)-linked glypican, as a heparan sulphate proteoglycan molecule involved in Wg signalling. Our results indicate that Dally may act as a co-receptor for Wg, and that Dally, together with Drosophila Frizzled 2, modulates both short- and long-range activities of Wg.

BACKGROUND: The Drosophila eye is composed of about 800 ommatidia, each of which becomes dorsoventrally polarised in a process requiring signalling through the Notch, JAK/STAT and Wingless pathways. These three pathways are thought to act by setting up a gradient of a signalling molecule (or molecules) often referred to as the 'second signal'. Thus far, no candidate for a second signal has been identified. RESULTS: The four-jointed locus encodes a type II transmembrane protein that is expressed in a dorsoventral gradient in the developing eye disc. We have analysed the function and regulation of four-jointed during eye patterning. Loss-of-function clones or ectopic expression of four-jointed resulted in strong non-autonomous defects in ommatidial polarity on the dorsoventral axis. Ectopic expression experiments indicated that localised four-jointed expression was required at the time during development when ommatidial polarity was being determined. In contrast, complete removal of four-jointed function resulted in only a mild ommatidial polarity defect. Finally, we found that four-jointed expression was regulated by the Notch, JAK/STAT and Wingless pathways, consistent with it mediating their effects on ommatidial polarity. CONCLUSIONS: The clonal phenotypes, time of requirement and regulation of four-jointed are consistent with it acting in ommatidial polarity determination as a second signal downstream of Notch, JAK/STAT and Wingless. Interestingly, it appears to act redundantly with unknown factors in this process, providing an explanation for the previous failure to identify a second signal.

Hedgehog (Hh) molecules play critical roles during development as a morphogen, and therefore their distribution must be regulated. Hh proteins undergo several modifications that tether them to the membrane. We have previously identified tout velu (ttv), a homolog of the mammalian EXT tumor suppressor gene family, as a gene required for movement of Hh. In this paper, we present in vivo evidence that ttv is involved in heparan sulfate proteoglycan (HSPG) biosynthesis, suggesting that HSPGs control Hh distribution. In contrast to mutants in other HSPG biosynthesis genes, the activity of the HSPG-dependent FGF and Wingless signaling pathways are not affected in ttv mutants. This demonstrates an unexpected level of specificity in the regulation of the distribution of extracellular signals by HSPGs.

The Drosophila sugarless and sulfateless genes encode enzymes required for the biosynthesis of heparan sulfate glycosaminoglycans. Biochemical studies have shown that heparan sulfate glycosaminoglycans are involved in signaling by fibroblast growth factor receptors, but evidence for such a requirement in an intact organism has not been available. We now demonstrate that sugarless and sulfateless mutant embryos have phenotypes similar to those lacking the functions of two Drosophila fibroblast growth factor receptors, Heartless and Breathless. Moreover, both Heartless- and Breathless-dependent MAPK activation is significantly reduced in embryos which fail to synthesize heparan sulfate glycosaminoglycans. Consistent with an involvement of Sulfateless and Sugarless in fibroblast growth factor receptor signaling, a constitutively activated form of Heartless partially rescues sugarless and sulfateless mutants, and dosage-sensitive interactions occur between heartless and the heparan sulfate glycosaminoglycan biosynthetic enzyme genes. We also find that overexpression of Branchless, the Breathless ligand, can partially overcome the requirement of Sugarless and Sulfateless for Breathless activity. These results provide the first genetic evidence that heparan sulfate glycosaminoglycans are essential for fibroblast growth factor receptor signaling in a well defined developmental context, and support a model in which heparan sulfate glycosaminoglycans facilitate fibroblast growth factor ligand and/or ligand-receptor oligomerization.

We have examined the role in patterning of quantitative variations of MAPK activity in signaling from the Drosophila Torso (Tor) receptor tyrosine kinase (RTK). Activation of Tor at the embryonic termini leads to differential expression of the genes tailless and huckebein. We demonstrate, using a series of mutations in the signal transducers Corkscrew/SHP-2 and D-Raf, that quantitative variations in the magnitude of MAPK activity trigger both qualitatively and quantitatively distinct transcriptional responses. We also demonstrate that two chimeric receptors, Torextracellular-Egfrcytoplasmic and Torextracellular-Sevcytoplasmic, cannot fully functionally replace the wild-type Tor receptor, revealing that the precise activation of MAPK involves not only the number of activated RTK molecules but also the magnitude of the signal generated by the RTK cytoplasmic domain. Altogether, our results illustrate how a gradient of MAPK activity controls differential gene expression and, thus, the establishment of various cell fates. We discuss the roles of quantitative mechanisms in defining RTK specificity.

Here, we identify four tyrosine residues (Y644, Y698, Y767, and Y772) that become phosphorylated after activation of the Torso (Tor) receptor tyrosine kinase. Previously, we characterized phosphotyrosine sites (P-Y630 and P-Y918). Of the six P-Y sites identified, three (Y630, Y644, and Y698) are located in the kinase domain insert region, one (Y918) is located in the C-terminal tail region, and two (Y767 and Y772) are located in the activation loop of the kinase domain. To investigate the function of each P-Y residue in Tor signaling, we have generated transgenic Drosophila embryos expressing mutant Tor receptors containing either single or multiple tyrosine to phenylalanine substitutions. Single P-Y mutations were found to have either positive, negative, or no effect on the signaling activity of the receptor. Elimination of all P-Y sites within the kinase insert region resulted in the complete loss of receptor function, indicating that some combination of these sites is necessary for Tor signaling. Mutation of the C-terminal P-Y918 site revealed that this site is responsible for negative signaling or down-regulation of receptor activity. Mutation of the P-Y sites in the kinase domain activation loop demonstrated that these sites are essential for enzymatic activity. Our analysis provides a detailed in vivo example of the extent of cooperativity between P-Y residues in transducing the signal received by a receptor tyrosine kinase and in vivo data demonstrating the function of P-Y residues in the activation loop of the kinase domain.