Research Article

Micchelli CA, The I, Selva E, Mogila V, Perrimon N. Rasp, a putative transmembrane acyltransferase, is required for Hedgehog signaling. Development. 2002;129 (4) :843-51. Abstract

Members of the Hedgehog (Hh) family encode secreted molecules that act as potent organizers during vertebrate and invertebrate development. Post-translational modification regulates both the range and efficacy of Hh protein. One such modification is the acylation of the N-terminal cysteine of Hh. In a screen for zygotic lethal mutations associated with maternal effects, we have identified rasp, a novel Drosophila segment polarity gene. Analysis of the rasp mutant phenotype, in both the embryo and wing imaginal disc demonstrates that rasp does not disrupt Wnt/Wingless signaling but is specifically required for Hh signaling. The requirement of rasp is restricted only to those cells that produce Hh; hh transcription, protein levels and distribution are not affected by the loss of rasp. Molecular analysis reveals that rasp encodes a multipass transmembrane protein that has homology to a family of membrane bound O-acyl transferases. Our results suggest that Rasp-dependent acylation is necessary to generate a fully active Hh protein.

Boutros M, Agaisse H, Perrimon N. Sequential activation of signaling pathways during innate immune responses in Drosophila. Dev Cell. 2002;3 (5) :711-22. Abstract

Innate immunity is essential for metazoans to fight microbial infections. Genome-wide expression profiling was used to analyze the outcome of impairing specific signaling pathways after microbial challenge. We found that these transcriptional patterns can be dissected into distinct groups. We demonstrate that, in addition to signaling through the Toll and Imd pathways, signaling through the JNK and JAK/STAT pathways controls distinct subsets of targets induced by microbial agents. Each pathway shows a specific temporal pattern of activation and targets different functional groups, suggesting that innate immune responses are modular and recruit distinct physiological programs. In particular, our results may imply a close link between the control of tissue repair and antimicrobial processes.

2002_Dev Cell_Boutros.pdf Supplemental
Stronach B, Perrimon N. Activation of the JNK pathway during dorsal closure in Drosophila requires the mixed lineage kinase, slipper. Genes Dev. 2002;16 (3) :377-87. Abstract

The Jun kinase (JNK) pathway has been characterized for its role in stimulating AP-1 activity and for modulating the balance between cell growth and death during development, inflammation, and cancer. Six families of mammalian kinases acting at the level of JNKKK have emerged as upstream regulators of JNK activity (MLK, LZK, TAK, ASK, MEKK, and TPL); however, the specificity underlying which kinase is utilized for transducing a distinct signal is poorly understood. In Drosophila, JNK signaling plays a central role in dorsal closure, controlling cell fate and cell sheet morphogenesis during embryogenesis. Notably, in the fly genome, there are single homologs of each of the mammalian JNKKK families. Here, we identify mutations in one of those, a mixed lineage kinase, named slipper (slpr), and show that it is required for JNK activation during dorsal closure. Furthermore, our results show that other putative JNKKKs cannot compensate for the loss of slpr function and, thus, may regulate other JNK or MAPK-dependent processes.

2002_Genes Dev_Stronach.pdf
Chen H-W, Marinissen MJ, Oh S-W, Chen X, Melnick M, Perrimon N, et al. CKA, a novel multidomain protein, regulates the JUN N-terminal kinase signal transduction pathway in Drosophila. Mol Cell Biol. 2002;22 (6) :1792-803. Abstract

The Drosophila melanogaster JUN N-terminal kinase (DJNK) and DPP (decapentaplegic) signal transduction pathways coordinately regulate epithelial cell sheet movement during the process of dorsal closure in the embryo. By a genetic screen of mutations affecting dorsal closure in Drosophila, we have now identified a multidomain protein, connector of kinase to AP-1 (cka), that functions in the DJNK pathway and controls the localized expression of dpp in the leading-edge cells. We have also investigated how CKA acts. This unique molecule forms a complex with HEP (DJNKK), BSK (DJNK), DJUN, and DFOS. Complex formation activates BSK kinase, which in turn phosphorylates and activates DJUN and DFOS. These data suggest that CKA represents a novel molecule regulating AP-1 activity by organizing a molecular complex of kinases and transcription factors, thus coordinating the spatial-temporal expression of AP-1-regulated genes.

2002_Mol Cell Bio_Chen.pdf
Li WX, Agaisse H, Mathey-Prevot B, Perrimon N. Differential requirement for STAT by gain-of-function and wild-type receptor tyrosine kinase Torso in Drosophila. Development. 2002;129 (18) :4241-8. Abstract

Malignant transformation frequently involves aberrant signaling from receptor tyrosine kinases (RTKs). These receptors commonly activate Ras/Raf/MEK/MAPK signaling but when overactivated can also induce the JAK/STAT pathway, originally identified as the signaling cascade downstream of cytokine receptors. Inappropriate activation of STAT has been found in many human cancers. However, the contribution of the JAK/STAT pathway in RTK signaling remains unclear. We have investigated the requirement of the JAK/STAT pathway for signaling by wild-type and mutant forms of the RTK Torso (Tor) using a genetic approach in Drosophila. Our results indicate that the JAK/STAT pathway plays little or no role in signaling by wild-type Tor. In contrast, we find that STAT, encoded by marelle (mrl; DStat92E), is essential for the gain-of-function mutant Tor (Tor(GOF)) to activate ectopic gene expression. Our findings indicate that the Ras/Raf/MEK/MAPK signaling pathway is sufficient to mediate the normal functions of wild-type RTK, whereas the effects of gain-of-function mutant RTK additionally require STAT activation.

Song H-J, Billeter J-C, Reynaud E, Carlo T, Spana EP, Perrimon N, et al. The fruitless gene is required for the proper formation of axonal tracts in the embryonic central nervous system of Drosophila. Genetics. 2002;162 (4) :1703-24. Abstract

The fruitless (fru) gene in Drosophila melanogaster is a multifunctional gene that has sex-specific functions in the regulation of male sexual behavior and sex-nonspecific functions affecting adult viability and external morphology. While much attention has focused on fru's sex-specific roles, less is known about its sex-nonspecific functions. We have examined fru's sex-nonspecific role in embryonic neural development. fru transcripts from sex-nonspecific promoters are expressed beginning at the earliest stages of neurogenesis, and Fru proteins are present in both neurons and glia. In embryos that lack most or all fru function, FasII- and BP102-positive axons have defasciculation defects and grow along abnormal pathways in the CNS. These defects in axonal projections in fru mutants were rescued by the expression of specific UAS-fru transgenes under the control of a pan-neuronal scabrous-GAL4 driver. Our results suggest that one of fru's sex-nonspecific roles is to regulate the pathfinding ability of axons in the embryonic CNS.

Mathew D, Gramates SL, Packard M, Thomas U, Bilder D, Perrimon N, et al. Recruitment of scribble to the synaptic scaffolding complex requires GUK-holder, a novel DLG binding protein. Curr Biol. 2002;12 (7) :531-9. Abstract

BACKGROUND: Membrane-associated guanylate kinases (MAGUKs), such as Discs-Large (DLG), play critical roles in synapse maturation by regulating the assembly of synaptic multiprotein complexes. Previous studies have revealed a genetic interaction between DLG and another PDZ scaffolding protein, SCRIBBLE (SCRIB), during the establishment of cell polarity in developing epithelia. A possible interaction between DLG and SCRIB at synaptic junctions has not yet been addressed. Likewise, the biochemical nature of this interaction remains elusive, raising questions regarding the mechanisms by which the actions of both proteins are coordinated. RESULTS: Here we report the isolation of a new DLG-interacting protein, GUK-holder, that interacts with the GUK domain of DLG and which is dynamically expressed during synaptic bouton budding. We also show that at Drosophila synapses DLG colocalizes with SCRIB and that this colocalization is likely to be mediated by direct interactions between GUKH and the PDZ2 domain of SCRIB. We show that DLG, GUKH, and SCRIB form a tripartite complex at synapses, in which DLG and GUKH are required for the proper synaptic localization of SCRIB. CONCLUSIONS: Our results provide a mechanism by which developmentally important PDZ-mediated complexes are associated at the synapse.

2002_Curr Bio_Mathew.pdf Supplement.pdf
Bai J, Chiu W, Wang J, Tzeng T, Perrimon N, Hsu J. The cell adhesion molecule Echinoid defines a new pathway that antagonizes the Drosophila EGF receptor signaling pathway. Development. 2001;128 (4) :591-601. Abstract

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.

Suzanne M, Perrimon N, Noselli S. The Drosophila JNK pathway controls the morphogenesis of the egg dorsal appendages and micropyle. Dev Biol. 2001;237 (2) :282-94. Abstract

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.

2001_Dev Bio_Suzanne.pdf
Hong Y, Stronach B, Perrimon N, Jan LY, Jan YN. Drosophila Stardust interacts with Crumbs to control polarity of epithelia but not neuroblasts. Nature. 2001;414 (6864) :634-8. Abstract

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.

2001_Nat_Hong.pdf Supplement.pdf
Selva EM, Hong K, Baeg GH, Beverley SM, Turco SJ, Perrimon N, et al. Dual role of the fringe connection gene in both heparan sulphate and fringe-dependent signalling events. Nat Cell Biol. 2001;3 (9) :809-15. Abstract

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.

2001_Nat Cell Bio_Selva.pdf
Baeg GH, Lin X, Khare N, Baumgartner S, Perrimon N. Heparan sulfate proteoglycans are critical for the organization of the extracellular distribution of Wingless. Development. 2001;128 (1) :87-94. Abstract

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.

Stronach BE, Perrimon N. Investigation of leading edge formation at the interface of amnioserosa and dorsal ectoderm in the Drosophila embryo. Development. 2001;128 (15) :2905-13. Abstract

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.

Baum B, Perrimon N. Spatial control of the actin cytoskeleton in Drosophila epithelial cells. Nat Cell Biol. 2001;3 (10) :883-90. Abstract

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.

2001_Nat Cell Bio_Baum.pdf
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