Interplay between Notch signaling and the homeoprotein Xiro1 is required for neural crest induction in Xenopus embryos

The neural crest is a population of cells that originates at the interface between the neural plate and non-neural ectoderm. Here, we have analyzed the role that Notch and the homeoprotein Xiro1 play in the specification of the neural crest. We show that Xiro1, Notch and the Notch target gene Hairy2A are all expressed in the neural crest territory, whereas the Notch ligands Delta1 and Serrate are expressed in the cells that surround the prospective crest cells. We have used inducible dominant-negative and activator constructs of both Notch signaling components and Xiro1 to analyze the role of these factors in neural crest specification without interfering with mesodermal or neural plate development. Activation of Xiro1 or Notch signaling led to an enlargement of the neural crest territory, whereas blocking their activity inhibited the expression of neural crest markers. It is known that BMPs are involved in the induction of the neural crest and, thus, we assessed whether these two elements might influence the expression of Bmp4. Activation of Xiro1 and of Notch signaling upregulated. Hairy2A and inhibited Bmp4 transcription during neural crest specification. These results, in conjunction with data from rescue experiments, allow us to propose a model wherein Xiro1 lies upstream of the cascade regulating Delta1 transcription. At the early gastrula stage, the coordinated action of Xiro1, as a positive regulator, and Snail, as a repressor, restricts the expression of Delta1 at the border of the neural crest territory. At the late gastrula stage, Delta1 interacts with Notch to activate Hairy2A in the region of the neural fold. Subsequently, Hairy2A acts as a repressor of Bmp4 transcription, ensuring that levels of Bmp4 optimal for the specification of the neural plate border are attained in this region. Finally, the activity of additional signals (WNTs, FGF and retinoic acid) in this newly defined domain induces the production of neural crest cells. These data also highlight the different roles played by BMP in neural crest specification in chick and Xenopus or zebrafish embryos.Fil: Glavic, Alvaro. Universidad de Chile; ChileFil: Silva, Francisca. Universidad de Chile; ChileFil: Aybar, Manuel Javier. Universidad de Chile; Chile. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; ArgentinaFil: Bastidas, Francisco. Universidad de Chile; ChileFil: Mayor, Roberto. Universidad de Chile; Chile. Colegio Universitario de Londres; Reino Unid

The Drosophila insulin-degrading enzyme restricts growth by modulating the PI3K pathway in a cell-autonomous manner

Mammalian insulin-degrading enzyme (IDE) cleaves insulin, among other peptidic substrates, but its function in insulin signaling is elusive. We use the Drosophila system to define the function of IDE in the regulation of growth and metabolism. We find that either loss or gain of function of Drosophila IDE (dIDE) can restrict growth in a cell-autonomous manner by affecting both cell size and cell number. dIDE can modulate Drosophila insulin-like peptide 2 levels, thereby restricting activation of the phosphatidylinositol-3-phosphate kinase pathway and promoting activation of Drosophila forkhead box, subgroup O transcription factor. Larvae reared in high sucrose exhibit delayed developmental timing due to insulin resistance. We find that dIDE loss of function exacerbates this phenotype and that mutants display increased levels of circulating sugar, along with augmented expression of a lipid biosynthesis marker. We propose that dIDE is a modulator of insulin signaling and that its loss of function favors insulin resistance, a hallmark of diabetes mellitus type II.Fil: Galagovsky, Diego. Fundación Instituto Leloir; ArgentinaFil: Katz, Maximiliano Javier. Fundación Instituto Leloir; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquimicas de Buenos Aires; ArgentinaFil: Acevedo, Julieta Maria. Fundación Instituto Leloir; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquimicas de Buenos Aires; ArgentinaFil: Sorianello, Eleonora Mariana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquimicas de Buenos Aires; Argentina. Fundación Instituto Leloir; ArgentinaFil: Glavic, Alvaro. Universidad de Chile. Facultad de Ciencias. Centro FONDAP de Regulación del Genoma; ChileFil: Wappner, Pablo. Fundación Instituto Leloir; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquimicas de Buenos Aires; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Fisiología, Biología Molecular y Celular; Argentin

Modulation of the Proteostasis Machinery to Overcome Stress Caused by Diminished Levels of t6A‐Modified tRNAs in Drosophila

Transfer RNAs (tRNAs) harbor a subset of post‐transcriptional modifications required for structural stability or decoding function. N6‐threonylcarbamoyladenosine (t6A) is a universally conserved modification found at position 37 in tRNA that pair A‐starting codons (ANN) and is required for proper translation initiation and to prevent frame shift during elongation. In its absence, the synthesis of aberrant proteins is likely, evidenced by the formation of protein aggregates. In this work, our aim was to study the relationship between t6A‐modified tRNAs and protein synthesis homeostasis machinery using Drosophila melanogaster. We used the Gal4/UAS system to knockdown genes required for t6A synthesis in a tissue and time specific manner and in vivo reporters of unfolded protein response (UPR) activation. Our results suggest that t6A‐modified tRNAs, synthetized by the threonyl‐carbamoyl transferase complex (TCTC), are required for organismal growth and imaginal cell survival, and is most likely to support proper protein synthesis

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

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

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.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.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

The homeoprotein Xiro1 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.This research was supported by an International Research Scholar Award from the Howard Hughes Medical Institute to R. M. and by grants from Fondecyt (#2990122 to A. G., #1990570 to R. M.), the Millennium Program (P99-137F) to R. M.; the Dirección General de Investigación Científica y Técnica (BMC2001-2122) to J. L. G.-S.; and the Human Frontier Science Program (RG0042-98) to R. M. and Juan Modolell. A. G. is a fellow from Fundación Andes (Chile).Peer reviewe

Xiro‐1 controls mesoderm patterning by repressing bmp‐4 expression in the spemann organizer

The Iroquois genes code for homeodomain proteins that have been implicated in the neural development of Drosophila and vertebrates. We show here for the first time that Xiro‐1, one of the Xenopus Iroquois genes, is expressed in the Spemann organizer from the start of gastrulation and that its overexpression induces a secondary axis as well as the ectopic expression of several organizer genes, such as chordin, goosecoid, and Xlim‐1. Our results also indicate that Xiro‐1 normally functions as a transcriptional repressor in the mesoderm. Overexpression of Xiro‐1 or a chimeric form fused to the repressor domain of Engrailed cause similar phenotypes while overexpression of functional derivatives of Xiro‐1 fused with transactivation domains (VP16 or E1A) produce the opposite effects. Finally, we show that Xiro‐1 works as a repressor of bmp‐4 transcription and that its effect on organizer development is dependent on BMP‐4 activity. We propose that the previously observed down regulation of bmp‐4 in the dorsal mesoderm during gastrulation can be explained by the repressor activity of Xiro‐1 described here. Thus, Xiro‐1 seems to have at least two different functions: control of neural plate and organizer development, both of which could be mediated by repression of bmp‐4 transcription.A.G is a fellow from Fundación Andes.Peer reviewe

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-mannose-deshydratase (GMD), the first enzyme in the synthesis of GDP-fucose. Our results show that GMD activity, and likely the levels of GDP-fucose 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

A Gain-of-Function Screen Identifying Genes Required for Growth and Pattern Formation of the Drosophila melanogaster Wing

The Drosophila melanogaster wing is a model system for analyzing the genetic control of organ size, shape, and pattern formation. The formation of the wing involves a variety of processes, such as cell growth, proliferation, pattern formation, and differentiation. These developmental processes are under genetic control, and many genes participating in specific aspects of wing development have already being characterized. In this work, we aim to identify novel genes regulating wing growth and patterning. To this end, we have carried out a gain-of-function screen generating novel P-UAS (upstream activating sequences) insertions allowing forced gene expression. We produced 3340 novel P-UAS insertions and isolated 300 that cause a variety of wing phenotypes in combination with a Gal4 driver expressed exclusively in the central domain of the presumptive wing blade. The mapping of these P-UAS insertion sites allowed us to identify the gene that causes the gain-of-function phenotypes. We show that a fraction of these phenotypes are related to the induction of cell death in the domain of ectopic gene expression. Finally, we present a preliminary characterization of a gene identified in the screen, the function of which is required for the development of the L5 longitudinal vein