The balance between GMD and OFUT1 regulates notch signaling pathway activity by modulating notch stability

The Notch signaling pathway plays an important role in development and physiology. In Drosophila, Notch is activated by its Delta or Serrate ligands, depending in part on the sugar modifications present in its extracellular domain. O-fucosyltransferase-1 (OFUT1) performs the first glycosylation step in this process, O-fucosylating various EGF repeats at the Notch extracellular domain. Besides its O-fucosyltransferase activity, OFUT1 also behaves as a chaperone during Notch synthesis and is able to down regulate Notch by enhancing its endocytosis and degradation. We have reevaluated the roles that O-fucosylation and the synthesis of GDP-fucose play in the regulation of Notch protein stability. Using mutants and the UAS/Gal4 system, we modified in developing tissues the amount of GDP-mannosedeshydratase (GMD), the first enzyme in the synthesis of GDP-fucose. Our results show that GMD activity, and likely the levels of GDPfucose and O-fucosylation, are essential to stabilize the Notch protein. Notch degradation observed under low GMD expression is absolutely dependent on OFUT1 and this is also observed in Notch Abruptex mutants, which have mutations in some potential O-fucosylated EGF domains. We propose that the GDP-fucose/OFUT1 balance determines the ability of OFUT1 to endocytose and degrade Notch in a manner that is independent of the residues affected by Abruptex mutations in Notch EGF domains.This work was funded by ICM P06-039F grant to A.G. and by a BFU2009-09403 grant of the M.E.C. to J.F.dC. An institutional grant from the Ramón Areces Foundation to the CBMSO is also acknowledged.Peer Reviewe

The Serine Protease Homolog, Scarface, Is Sensitive to Nutrient Availability and Modulates the Development of the <i>Drosophila</i> Blood-Brain Barrier.

The adaptable transcriptional response to changes in food availability not only ensures animal survival but also lets embryonic development progress. Interestingly, the CNS is preferentially protected from periods of malnutrition, a phenomenon known as "brain sparing." However, the mechanisms that mediate this response remain poorly understood. To get a better understanding of this, we used Drosophila melanogaster as a model, analyzing the transcriptional response of neural stem cells (neuroblasts) and glia of the blood-brain barrier (BBB) from larvae of both sexes during nutrient restriction using targeted DamID. We found differentially expressed genes in both neuroblasts and glia of the BBB, although the effect of nutrient deficiency was primarily observed in the BBB. We characterized the function of a nutritional sensitive gene expressed in the BBB, the serine protease homolog, scarface (scaf). Scaf is expressed in subperineurial glia in the BBB in response to nutrition. Tissue-specific knockdown of scaf increases subperineurial glia endoreplication and proliferation of perineurial glia in the blood-brain barrier. Furthermore, neuroblast proliferation is diminished on scaf knockdown in subperineurial glia. Interestingly, reexpression of Scaf in subperineurial glia is able to enhance neuroblast proliferation and brain growth of animals in starvation. Finally, we show that loss of scaf in the blood-brain barrier increases sensitivity to drugs in adulthood, suggesting a physiological impairment. We propose that Scaf integrates the nutrient status to modulate the balance between neurogenesis and growth of the BBB, preserving the proper equilibrium between the size of the barrier and the brain.SIGNIFICANCE STATEMENT The Drosophila BBB separates the CNS from the open circulatory system. The BBB glia are not only acting as a physical segregation of tissues but participate in the regulation of the metabolism and neurogenesis during development. Here we analyze the transcriptional response of the BBB glia to nutrient deprivation during larval development, a condition in which protective mechanisms are switched on in the brain. Our findings show that the gene scarface reduces growth in the BBB while promoting the proliferation of neural stem, assuring the balanced growth of the larval brain. Thus, Scarface would link animal nutrition with brain development, coordinating neurogenesis with the growth of the BBB

p53 Related Protein Kinase is Required for Arp2/3-Dependent Actin Dynamics of Hemocytes in Drosophila melanogaster.

Cells extend membrane protrusions like lamellipodia and filopodia from the leading edge to sense, to move and to form new contacts. The Arp2/3 complex sustains lamellipodia formation, and in conjunction with the actomyosin contractile system, provides mechanical strength to the cell. Drosophila p53-related protein kinase (Prpk), a Tsc5p ortholog, has been described as essential for cell growth and proliferation. In addition, Prpk interacts with proteins associated to actin filament dynamics such as α-spectrin and the Arp2/3 complex subunit Arpc4. Here, we investigated the role of Prpk in cell shape changes, specifically regarding actin filament dynamics and membrane protrusion formation. We found that reductions in Prpk alter cell shape and the structure of lamellipodia, mimicking the phenotypes evoked by Arp2/3 complex deficiencies. Prpk co-localize and co-immunoprecipitates with the Arp2/3 complex subunit Arpc1 and with the small GTPase Rab35. Importantly, expression of Rab35, known by its ability to recruit upstream regulators of the Arp2/3 complex, could rescue the Prpk knockdown phenotypes. Finally, we evaluated the requirement of Prpk in different developmental contexts, where it was shown to be essential for correct Arp2/3 complex distribution and actin dynamics required for hemocytes migration, recruitment, and phagocytosis during immune response

Intertissue mechanical stress affects frizzled-mediated planar cell polarity in the drosophila notum epidermis

Frizzled/planar cell polarity (Fz/PCP) signaling controls the orientation of sensory bristles and cellular hairs (trichomes) along the anteroposterior axis of the Drosophila thorax (notum) [1-4]. A subset of the trichome-producing notum cells differentiate as "tendon cells," serving as attachment sites for the indirect flight muscles (IFMs) to the exoskeleton [5]. Through the analysis of chascon (chas), a gene identified by its ability to disrupt Fz/PCP signaling under overexpression conditions, and jitterbug (jbug)/filamin [6], we show that maintenance of anteroposterior planar polarization requires the notum epithelia to balance mechanical stress generated by the attachment of the IFMs. chas is expressed in notum tendon cells, and its loss of function disturbs cellular orientation at and near the regions where IFMs attach to the epidermis. This effect is independent of the Fz/PCP and fat/dachsous systems [7]. The chas phenotype arises during normal shortening of the IFMs [8] and is

Drosophila p115 is required for Cdk1 activation and G2/M cell cycle transition

Golgi complex inheritance and its relationship with the cell cycle are central in cell biology. Golgi matrix proteins, known as golgins, are one of the components that underlie the shape and functionality of this organelle. In mammalian cells, golgins are phosphorylated during mitosis to allow fragmentation of the Golgi ribbon and they also participate in spindle dynamics; both processes are required for cell cycle progression. Little is known about the function of golgins during mitosis in metazoans in vivo. This is particularly significant in Drosophila, in which the Golgi architecture is distributed in numerous units scattered throughout the cytoplasm, in contrast with mammalian cells. We examined the function of the ER/cis-Golgi golgin p115 during the proliferative phase of the Drosophila wing imaginal disc. Knockdown of p115 decreased tissue size. This phenotype was not caused by programmed cell death or cell size reductions, but by a reduction in the final cell number due to an accumulation of cells at the G2/M transition. This phenomenon frequently allows mitotic bypass and re-replication of DNA. These outcomes are similar to those observed following the partial loss of function of positive regulators of Cdk1 in Drosophila. In agreement with this, Cdk1 activation was reduced upon p115 knockdown. Interestingly, these phenotypes were fully rescued by Cdk1 overexpression and partially rescued by Myt1 depletion, but not by String (also known as Cdc25) overexpression. Additionally, we confirmed the physical interaction between p115 and Cdk1, suggesting that the formation of a complex where both proteins are present is essential for the full activation of Cdk1 and thus the correct progression of mitosis in proliferating tissues.FONDAP 15090007 PIA ACT1401 FONDECYT 1140522 CONICYT Ph.D. Fellowshi

p53 is required for brain growth but is dispensable for resistance to nutrient restriction during Drosophila larval development

Background Animal growth is influenced by the genetic background and the environmental circumstances. How genes promote growth and coordinate adaptation to nutrient availability is still an open question. p53 is a transcription factor that commands the cellular response to different types of stresses. In adult Drosophila melanogaster, p53 regulates the metabolic adaptation to nutrient restriction that supports fly viability. Furthermore, the larval brain is protected from nutrient restriction in a phenomenon called 'brain sparing'. Therefore, we hypothesised that p53 may regulate brain growth and show a protective role over brain development under nutrient restriction. Results Here, we studied the function of p53 during brain growth in normal conditions and in animals subjected to developmental nutrient restriction. We showed that p53 loss of function reduced animal growth and larval brain size. Endogenous p53 was expressed in larval neural stem cells, but its levels and activity were not affected by nutritional stress. Interestingly, p53 knockdown only in neural stem cells was sufficient to decrease larval brain growth. Finally, we showed that in p53 mutant larvae under nutrient restriction, the energy storage levels were not altered, and these larvae generated adults with brains of similar size than wild-type animals. Conclusions Using genetic approaches, we demonstrate that p53 is required for proper growth of the larval brain. This developmental role of p53 does not have an impact on animal resistance to nutritional stress since brain growth in p53 mutants under nutrient restriction is similar to control animals.Fondo Nacional de Desarrollo Cientifico y Tecnologico FONDECYT 3160412 1140522 1171800 Anillo de Investigacion en Ciencia y Tecnologia DRiDANS ACT1401 Institute Milenio de Neurociencia Biomedica (BNI) ICM P09015-F Fondo de Financiamiento de Centros de Investigacion en Areas Prioritarias (Fondap) 1509000

Extracellular signals, cell interactions and transcription factors involved in the induction of the neural crest cells

The neural crest is induced at the border between the neural plate and the epidermis. A complex set of signals is required for the specification of the crest cells between the epidermis and the neural plate. Here we discuss evidence supporting a model for neural crest induction in which different signals contribute in a sequential order. First, a gradient of bone morphogenic proteins (BMPs) is established in the ectoderm that results in segreggation into neural plate, neural folds and epidermis at increasing levels of BMP activity. Thus, the neural folds are induced at a precise threshold concentration of BMP, but this neural fold has an anterior character. In a second step, these anterior neural folds are transformed into prospective neural crest by posteriorizing signals due to fibroblast growth factor, Wnts and retinoic acid. Finally, the induced cells interact to complete neural crest induction by a process that requires Notch/Delta signaling. Once neural crest formation has been induced by this combination of extracellular and intracellular signals. a cascade of transcription factors is activated in these cells that culminates in the ultimate steps of neural crest differentiatio

The homeoprotein Xiro 1 is required for midbrain-hindbrain boundary formation

The isthmic organizer, which patterns the anterior hindbrain and midbrain, is one of the most studied secondary organizers. In recent years, new insights have been reported on the molecular nature of its morphogenetic activity. Studies in chick, mouse and zebrafish have converged to show that mutually repressive interactions between the homeoproteins encoded by Otx and Gbx genes position this organizer in the neural primordia. We present evidence that equivalent, in addition to novel, interactions between these and other genes operate in Xenopus embryos to position the isthmic organizer. We made use of fusion proteins in which we combined Otx2 or Gbx2 homeodomains with the E1A activation domain or the EnR repressor element which were then injected into embryos. Our results show that Otx2 and Gbx2 are likely to be transcriptional repressors, and that these two proteins repress each other transcription. Our experiments show that the interaction between these two proteins is required for the positioning of the isthmic organizer genes Fgf8, Pax2 and En2. In this study we also developed a novel in vitro assay for the study of the formation of this organizer. We show that conjugating animal caps previously injected with Otx2 and Gbx2 mRNAs recreate the interactions required for the induction of the isthmic organizer. We have used this assay to determine which cells produce and which cells receive the Fgf signal. Finally, we have added a novel genetic element to this process, Xiro1, which encode another homeoprotein. We show that the Xiro1 expression domain overlaps with territories expressing Otx2, Gbx2 and Fgf8. By expressing wild-type or dominant negative forms of Xiro1, we show that this gene activates the expression of Gbx2 in the hindbrain. In addition, Xiro1 is required in the Otx2 territory to allow cells within this region to respond to the signals produced by adjacent Gbx2 cells. Moreover, Xiro1 is absolutely required for Fgf8 expression at the isthmic organizer. We discuss a model where Xiro1 plays different roles in regulating the genetic cascade of interactions between Otx2 and Gbx2 that are necessary for the specification of the isthmic organizer

Posteriorization by FGF, Wnt, and retinoic acid is required for neural crest induction

The neural crest is a unique cell population induced at the lateral border of the neural plate. Neural crest is not produced at the anterior border of the neural plate, which is fated to become forebrain. Here, the roles of BMPs, FGFs, Wnts, and retinoic acid signaling in neural crest induction were analyzed by using an assay developed for investigating the posteriorization of the neural plate. Using specific markers for the anterior neural plate border and the neural crest, the posterior end of early neurula embryos was shown to be able to transform the anterior neural plate border into neural crest cells. In addition, tissue expressing anterior neural plate markers, induced by an intermediate level of BMP activity, was transformed into neural crest by posteriorizing signals. This transformation was mimicked by bFGF, Wnt-8, or retinoic acid treatment and was also inhibited by expression of the dominant negative forms of the FGF receptor, the retinoic acid receptor, and Wnt signaling