The I, Perrimon N. Morphogen diffusion: the case of the wingless protein. Nat Cell Biol. 2000;2 (5) :E79-82. 2000_Nat Cell Bio_The.pdf
Lin X, Perrimon N. Role of heparan sulfate proteoglycans in cell-cell signaling in Drosophila. Matrix Biol. 2000;19 (4) :303-7. Abstract

Heparan sulfate proteoglycans (HSPGs) are abundant molecules associated with the cell surface and extracellular matrix, and consist of a protein core to which heparan sulfate (HS) glycosaminoglycan (GAG) chains are attached. Although these molecules have been the focus of intense biochemical studies in vitro, their biological functions in vivo were unclear until recently. We have undertaken an in vivo functional study of HSPGs in Drosophila. Our studies, as well as others, demonstrate the critical roles of HSPGs in several major signaling pathways, including ibroblast growth factor (FGF), Wnt, Hedgehog (Hh) and TGF-beta. Our results also suggest that specific HS GAG chain modifications, as well as specific HSPG protein cores, are involved in specific signaling pathways.

2000_Matrix Bio_Lin.pdf
Zeidler MP, Bach EA, Perrimon N. The roles of the Drosophila JAK/STAT pathway. Oncogene. 2000;19 (21) :2598-606. Abstract

The JAK/STAT signal transduction pathway has been conserved throughout evolution such that true structural and functional homologues of components originally identified in vertebrate systems are also present in the model genetic system Drosophila melanogaster. In addition to roles during larval hematopoiesis reminiscent of the requirement for this pathway in mammalian systems, the JAK/STAT pathway in Drosophila is also involved in a number of other developmental events. Recent data has demonstrated further roles for the JAK/STAT pathway in the establishment of sexual identity via the early embryonic expression of Sex lethal, the segmentation of the embryo via the control of pair rule genes including even skipped and the establishment of polarity within the adult compound eye via a mechanism that includes the four jointed gene. Use of the powerful genetics in the model organism Drosophila may identify new components of the JAK/STAT pathway, define new roles for this pathway, and provide insights into the function of this signal transduction system. Here we review the roles of STAT and its associated signaling pathway during both embryonic and adult stages of Drosophila development and discuss future prospects for the identification and characterization of novel pathway components and targets. Oncogene (2000).

Zeidler MP, Perrimon N. Sex determination: co-opted signals determine gender. Curr Biol. 2000;10 (18) :R682-4. Abstract

The Drosophila JAK-STAT pathway and its ligand Unpaired are required for a wide range of developmental processes. Recent results have identified Unpaired as an activator of sex-lethal and revealed a new role for the JAK-STAT pathway in sex determination.

2000_Curr Bio_Zeidler.pdf
Noselli S, Perrimon N. Signal transduction. Are there close encounters between signaling pathways?. Science. 2000;290 (5489) :68-9. Abstract

Do different signaling pathways inside the same cell talk to each other? Evidence suggests that in the worm and fly, signaling pathways exist as separate linear cassettes, whereas in mammalian cells there does appear to be cross talk between signaling pathways. However, as Noselli and Perrimon argue in their Perspective, most of the evidence in mammalian cells comes from tumor cells and overexpression assays. They suggest that true cross talk may not actually exist in mammalian cells under normal circumstances.   

Perrimon N, Bernfield M. Specificities of heparan sulphate proteoglycans in developmental processes. Nature. 2000;404 (6779) :725-8. Abstract

Heparan sulphate proteoglycans are abundant cell-surface molecules that consist of a protein core to which heparan sulphate glycosaminoglycan chains are attached. The functions of these molecules have remained mostly underappreciated by developmental biologists; however, the actions of important signalling molecules, for example Wnt and Hedgehog, depend on them. To understand both the mechanisms by which ligands involved in development interact with their receptors and how morphogens pattern tissues, biologists need to consider the functions of heparan sulphate proteoglycans in signalling and developmental patterning.

Bilder D, Li M, Perrimon N. Cooperative regulation of cell polarity and growth by Drosophila tumor suppressors. Science. 2000;289 (5476) :113-6. Abstract

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.

Baum B, Li W, Perrimon N. A cyclase-associated protein regulates actin and cell polarity during Drosophila oogenesis and in yeast. Curr Biol. 2000;10 (16) :964-73. Abstract

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.

2000_Curr Bio_Baum.pdf
Tanaka K, Okabayashi K, Asashima M, Perrimon N, Kadowaki T. The evolutionarily conserved porcupine gene family is involved in the processing of the Wnt family. Eur J Biochem. 2000;267 (13) :4300-11. Abstract

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.

2000_Eur J Biochem_Tanaka.pdf
Bilder D, Perrimon N. Localization of apical epithelial determinants by the basolateral PDZ protein Scribble. Nature. 2000;403 (6770) :676-80. Abstract

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.

Zeidler MP, Perrimon N, Strutt DI. Multiple roles for four-jointed in planar polarity and limb patterning. Dev Biol. 2000;228 (2) :181-96. Abstract

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.

2000_Dev Bio_Zeidler.pdf
Noll E, Medina M, Hartley D, Zhou J, Perrimon N, Kosik KS. Presenilin affects arm/beta-catenin localization and function in Drosophila. Dev Biol. 2000;227 (2) :450-64. Abstract

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.

2000_Dev Bio_Noll.pdf
Bilder D, Birnbaum D, Borg JP, Bryant P, Huigbretse J, Jansen E, et al. Collective nomenclature for LAP proteins. Nat Cell Biol. 2000;2 (7) :E114. 2000_Nat Cell Bio_Bilder.pdf
Li W, Noll E, Perrimon N. Identification of autosomal regions involved in Drosophila Raf function. Genetics. 2000;156 (2) :763-74. Abstract

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.

Lin X, Perrimon N. Dally cooperates with Drosophila Frizzled 2 to transduce Wingless signalling. Nature. 1999;400 (6741) :281-4. Abstract

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.

Zeidler MP, Perrimon N, Strutt DI. The four-jointed gene is required in the Drosophila eye for ommatidial polarity specification. Curr Biol. 1999;9 (23) :1363-72. Abstract

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.

1999_Curr Bio_Zeidler.pdf
The I, Bellaiche Y, Perrimon N. Hedgehog movement is regulated through tout velu-dependent synthesis of a heparan sulfate proteoglycan. Mol Cell. 1999;4 (4) :633-9. Abstract

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.

1999_Mol Cell_The.pdf
Lin X, Buff EM, Perrimon N, Michelson AM. Heparan sulfate proteoglycans are essential for FGF receptor signaling during Drosophila embryonic development. Development. 1999;126 (17) :3715-23. Abstract

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.

Ghiglione C, Perrimon N, Perkins LA. Quantitative variations in the level of MAPK activity control patterning of the embryonic termini in Drosophila. Dev Biol. 1999;205 (1) :181-93. Abstract

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.

1999_Dev Bio_Ghiglione.pdf
Gayko U, Cleghon V, Copeland T, Morrison DK, Perrimon N. Synergistic activities of multiple phosphotyrosine residues mediate full signaling from the Drosophila Torso receptor tyrosine kinase. Proc Natl Acad Sci U S A. 1999;96 (2) :523-8. Abstract

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.