A function of spalt major as a sequence-specific DNA binding transcription factor mediates repression of knirps in the Drosophila wing imaginal disc.

The Spalt transcriptional regulators participate in a variety of cell fate decisions during multicellular development. Vertebrate Spalt proteins have been mostly associated to the organization of heterochromatic regions, but they also contribute regulatory functions through binding to A/T rich motives present in their target genes. The developmental processes in which the Drosophila spalt genes participate are well known through genetic analysis, but the mechanism by which the Spalt proteins regulate transcription are still unknown. Furthermore, despite the prominent changes in gene expression associated to mutations in the spalt genes, the specific DNA sequences they bind are unknow. Here, we analyze a DNA fragment present in the regulatory region of the knirps gene. Spalt proteins are candidate repressors of knirps expression during the formation of the venation pattern in the wing disc, and we identified a minimal conserved 30bp sequence that binds to Spalt major both in vivo and in vitro. This sequence mediates transcriptional repression in the central region of the wing blade, constituting the first confirmed case of a direct regulatory interaction between Spalt major and its target DNA in Drosophila. Interestingly, we also find similar sequences in a set of eight novel candidate Spalt target genes, pointing to a common mechanism of transcriptional repression mediated by Spalt proteins.

RNAi screen in the Drosophila wing of genes encoding proteins related to cytoskeleton organization and cell division.

Cell division and cytoskeleton organization are fundamental processes participating in the development of Drosophila imaginal discs. In this manuscript we describe the phenotypes in the adult fly wing generated by knockdowns of 85% of Drosophila genes encoding proteins likely related to the regulation of cell division and cytoskeleton organization. We also compile a molecular classification of these proteins into classes that describe their expected or known main biochemical characteristics, as well as mRNA expression in the wing disc and likely protein subcellular localization for a subset of these genes. Finally, we analyze in more detail one protein family of cytoskeleton genes (Arp2/3 complex), and define the consequences of interfering with cell division for wing growth and patterning.

Functional requirements of protein kinases and phosphatases in the development of the Drosophila melanogaster wing.

Protein kinases and phosphatases constitute a large family of conserved enzymes that control a variety of biological processes by regulating the phosphorylation state of target proteins. They play fundamental regulatory roles during cell cycle progression and signaling, among other key aspects of multicellular development. The complement of protein kinases and phosphatases includes approximately 326 members in Drosophila, and they have been the subject of several functional screens searching for novel components of signaling pathways and regulators of cell division and survival. These approaches have been carried out mostly in cell cultures using RNA interference to evaluate the contribution of each protein in different functional assays and have contributed significantly to assign specific roles to the corresponding genes. In this work, we describe the results of an evaluation of the Drosophila complement of kinases and phosphatases using the wing as a system to identify their functional requirements in vivo. We also describe the results of several modifying screens aiming to identify among the set of protein kinases and phosphatases additional components or regulators of the activities of the epidermal growth factor and insulin receptors signaling pathways.

Genome-wide phenotypic RNAi screen in the Drosophila wing: phenotypic description of functional classes.

The Drosophila genome contains approximately 14,000 protein-coding genes encoding all the necessary information to sustain cellular physiology, tissue organization, organism development, and behavior. In this manuscript, we describe in some detail the phenotypes in the adult fly wing generated after knockdown of approximately 80% of Drosophila genes. We combined this phenotypic description with a comprehensive molecular classification of the Drosophila proteins into classes that summarize the main expected or known biochemical/functional aspect of each protein. This information, combined with mRNA expression levels and in situ expression patterns, provides a simplified atlas of the Drosophila genome, from housekeeping proteins to the components of the signaling pathways directing wing development, that might help to further understand the contribution of each gene group to wing formation.

Genome-wide phenotypic RNAi screen in the Drosophila wing: global parameters.

We have screened a collection of UAS-RNAi lines targeting 10,920 Drosophila protein-coding genes for phenotypes in the adult wing. We identified 3653 genes (33%) whose knockdown causes either larval/pupal lethality or a mutant phenotype affecting the formation of a normal wing. The most frequent phenotypes consist of changes in wing size, vein differentiation, and patterning, defects in the wing margin and in the apposition of the dorsal and ventral wing surfaces. We also defined 16 functional categories encompassing the most relevant aspect of each protein function and assigned each Drosophila gene to one of these functional groups. This allowed us to identify which mutant phenotypes are enriched within each functional group. Finally, we used previously published gene expression datasets to determine which genes are or are not expressed in the wing disc. Integrating expression, phenotypic and molecular information offers considerable precision to identify the relevant genes affecting wing formation and the biological processes regulated by them.

EGFRAP encodes a new negative regulator of the EGFR acting in both normal and oncogenic EGFR/Ras-driven tissue morphogenesis.

Activation of Ras signaling occurs in ~30% of human cancers. However, activated Ras alone is insufficient to produce malignancy. Thus, it is imperative to identify those genes cooperating with activated Ras in driving tumoral growth. In this work, we have identified a novel EGFR inhibitor, which we have named EGFRAP, for EGFR adaptor protein. Elimination of EGFRAP potentiates activated Ras-induced overgrowth in the Drosophila wing imaginal disc. We show that EGFRAP interacts physically with the phosphorylated form of EGFR via its SH2 domain. EGFRAP is expressed at high levels in regions of maximal EGFR/Ras pathway activity, such as at the presumptive wing margin. In addition, EGFRAP expression is up-regulated in conditions of oncogenic EGFR/Ras activation. Normal and oncogenic EGFR/Ras-mediated upregulation of EGRAP levels depend on the Notch pathway. We also find that elimination of EGFRAP does not affect overall organogenesis or viability. However, simultaneous downregulation of EGFRAP and its ortholog PVRAP results in defects associated with increased EGFR function. Based on these results, we propose that EGFRAP is a new negative regulator of the EGFR/Ras pathway, which, while being required redundantly for normal morphogenesis, behaves as an important modulator of EGFR/Ras-driven tissue hyperplasia. We suggest that the ability of EGFRAP to functionally inhibit the EGFR pathway in oncogenic cells results from the activation of a feedback loop leading to increase EGFRAP expression. This could act as a surveillance mechanism to prevent excessive EGFR activity and uncontrolled cell growth.

Ras2, the TC21/R-Ras2 Drosophila homologue, contributes to insulin signalling but is not required for organism viability.

Ras1 (Ras85D) and Ras2 (Ras64B) are the Drosophila orthologs of human H-Ras/N-Ras/K-Ras and R-Ras1-3 genes, respectively. The function of Ras1 has been thoroughly characterised during Drosophila embryonic and imaginal development, and it is associated with coupling activated trans-membrane receptors with tyrosine kinase activity to their downstream effectors. In this capacity, Ras1 binds and is required for the activation of Raf. Ras1 can also interact with PI3K, and it is needed to achieve maximal levels of PI3K signalling in specific cellular settings. In contrast, the function of the unique Drosophila R-Ras member (Ras2/Ras64B), which is more closely related to vertebrate R-Ras2/TC21, has been only studied through the use of constitutively activated forms of the protein. This pioneering work identified a variety of phenotypes that were related to those displayed by Ras1, suggesting that Ras1 and Ras2 might have overlapping activities. Here we find that Ras2 can interact with PI3K and Raf and activate their downstream effectors Akt and Erk. However, and in contrast to mutants in Ras1, which are lethal, null alleles of Ras2 are viable in homozygosis and only show a phenotype of reduced wing size and extended life span that might be related to reduced Insulin receptor signalling.

Patterning of the Drosophila L2 vein is driven by regulatory interactions between region-specific transcription factors expressed in response to Dpp signalling.

Pattern formation relies on the generation of transcriptional landscapes regulated by signalling pathways. A paradigm of epithelial patterning is the distribution of vein territories in the Drosophila wing disc. In this tissue, Decapentaplegic signalling regulates its target genes at different distances from the source of the ligand. The transformation of signalling into coherent territories of gene expression requires regulatory cross-interactions between these target genes. Here, we analyse the mechanisms generating the domain of knirps expression in the presumptive L2 vein of the wing imaginal disc. We find that knirps is regulated by four Decapentaplegic target genes encoding the transcription factors aristaless, spalt major, spalt-related and optix The expression of optix is activated by Dpp and repressed by the Spalt proteins, becoming restricted to the most anterior region of the wing blade. In turn, the expression of knirps is activated by Aristaless and repressed by Optix and the Spalt proteins. In this manner, the expression of knirps becomes restricted to those cells where Spalt levels are sufficient to repress optix, but not sufficient to repress knirps.

A Search for Genes Mediating the Growth-Promoting Function of TGFß in the Drosophila melanogaster Wing Disc.

Transforming Growth Factor ß (TGFß) signaling has a complex influence on cell proliferation, acting to stop cell division in differentiating cells, but also promoting cell division in immature cells. The activity of the pathway in Drosophila is mostly required to stimulate the proliferation of neural and epithelial tissues. Most interestingly, this function is not absolutely required for cell division, but it is needed for these tissues to reach their correct size. It is not known how TGFß signaling promotes cell division in imaginal discs, or what the interactions between TGFß activity and other signaling pathways regulating cell proliferation are. In this work, we have explored the disc autonomous function of TGFß that promotes wing imaginal disc growth. We have studied the genetic interactions between TGFß signaling and other pathways regulating wing disc growth, such as the Insulin and Hippo/Salvador/Warts pathways, as well as cell cycle regulators. We have also identified a collection of TGFß candidate target genes affecting imaginal growth using expression profiles. These candidates correspond to genes participating in the regulation of a variety of biochemical processes, including different aspects of cell metabolism, suggesting that TGFß could affect cell proliferation by regulating the metabolic fitness of imaginal cells.

Structure of developmental gene regulatory networks from the perspective of cell fate-determining genes.

The core of gene regulatory networks (GRNs) is formed by transcription factors (TF) and cis-regulatory modules (CRMs) present in their downstream genes. GRNs have a modular structure in which complex circuitries link TFs to CRMs to generate specific transcriptional outputs. (1) Of particular interest are those GRNs including cell fate-determining genes, as they constitute developmental switches which activity is necessary and sufficient to promote particular cellular fates. Most of the genetic analysis of developmental processes deals with the composition and structure of GRNs acting upstream of cell fate-determining genes, as they are best suited for genetic analysis and molecular deconstruction. More recently, the application of a variety of in vivo, computational and genome-wide approaches is allowing the identification and functional analysis of GRNs acting downstream of cell fate-determining genes. In this review we discuss several examples of GRNs acting upstream and downstream of cell fate-determining genes, including other TFs which activity pervade across both regulatory networks.

The Spalt Transcription Factors Generate the Transcriptional Landscape of the Drosophila melanogaster Wing Pouch Central Region.

The Drosophila genes spalt major (salm) and spalt-related (salr) encode Zn-finger transcription factors regulated by the Decapentaplegic (Dpp) signalling pathway in the wing imaginal disc. The function of these genes is required for cell survival and proliferation in the central region of the wing disc, and also for vein patterning in the lateral regions. The identification of direct Salm and Salr target genes, and the analysis of their functions, are critical steps towards understanding the genetic control of growth and patterning of the Drosophila wing imaginal disc by the Dpp pathway. To identify candidate Salm/Salr target genes, we have compared the expression profile of salm/salr knockdown wing discs with control discs in microarray experiments. We studied by in situ hybridization the expression pattern of the genes whose mRNA levels varied significantly, and uncovered a complex transcription landscape regulated by the Spalt proteins in the wing disc. Interestingly, candidate Salm/Salr targets include genes which expression is turned off and genes which expression is positively regulated by Salm/Salr. Furthermore, loss-of-function phenotypic analysis of these genes indicates, for a fraction of them, a requirement for wing growth and patterning. The identification and analysis of candidate Salm/Salr target genes opens a new avenue to reconstruct the genetic structure of the wing, linking the activity of the Dpp pathway to the development of this epithelial tissue.

Tay bridge is a negative regulator of EGFR signalling and interacts with Erk and Mkp3 in the Drosophila melanogaster wing.

The regulation of Extracellular regulated kinase (Erk) activity is a key aspect of signalling by pathways activated by extracellular ligands acting through tyrosine kinase transmembrane receptors. In this process, participate proteins with kinase activity that phosphorylate and activate Erk, as well as different phosphatases that inactivate Erk by de-phosphorylation. The state of Erk phosphorylation affects not only its activity, but also its subcellular localization, defining the repertoire of Erk target proteins, and consequently, the cellular response to Erk. In this work, we characterise Tay bridge as a novel component of the EGFR/Erk signalling pathway. Tay bridge is a large nuclear protein with a domain of homology with human AUTS2, and was previously identified due to the neuronal phenotypes displayed by loss-of-function mutations. We show that Tay bridge antagonizes EGFR signalling in the Drosophila melanogaster wing disc and other tissues, and that the protein interacts with both Erk and Mkp3. We suggest that Tay bridge constitutes a novel element involved in the regulation of Erk activity, acting as a nuclear docking for Erk that retains this protein in an inactive form in the nucleus.

Understanding the determinants of notch interactions with its ligands.

The Notch signaling pathway involves ligand-activated cleavage of the receptor Notch and the interaction of the intracellular fragment with the transcriptional regulators CSL and Mastermind. Additional complexity in the system arises through the differential interaction of Notch with its ligands of the Delta and Serrate families. Glycosylation of the extracellular portion of Notch by Fringe proteins contributes to receptor selectivity toward its ligands. Recent research suggests that a glycosylation-independent function of the Notch epidermal growth factor repeats also plays an important role in specifying activation of Notch by Ser.

Activation and function of TGFß signalling during Drosophila wing development and its interactions with the BMP pathway.

The development of the Drosophila wing disc requires the activities of the BMP and TGFß signalling pathways. BMP signalling is critical for the correct growth and patterning of the disc, whereas the related TGFß pathway is mostly required for growth. The BMP and TGFß pathways share a common co-receptor (Punt) and a nuclear effector (Medea), and consequently it is likely that these pathways can interfere with each other during normal development. In this work we focus on the spatial activation domains and requirements for TGFß signalling during wing disc development. We found that the phosphorylation of Smad2, the specific transducer for TGFß signalling, occurs in a generalised manner in the wing disc. It appears that the expression of the four candidate TGFß ligands (Activinß, Dawdle, Maverick and Myoglianin) in the wing disc is required to obtain normal levels of TGFß signalling in this tissue. We show that Baboon, the specific receptor of the TGFß pathway, can phosphorylate Mad, the specific transducer of the BMP pathway, in vivo. However, this activation only occurs in the wing disc when the receptor is constitutively activated in a background of reduced expression of Smad2. In the presence of Smad2, the normal situation during wing disc development, high levels of activated Baboon lead to a depletion in Mad phosphorylation and to BMP loss-of-function phenotypes. Although loss of either babo or Smad2 expression reduce growth in the wing blade in a similar manner, loss of Smad2 can also cause phenotypes related to ectopic BMP signalling, suggesting a physiological role for this transducer in the regulation of Mad spatial activation.

The Spalt transcription factors regulate cell proliferation, survival and epithelial integrity downstream of the Decapentaplegic signalling pathway.

The expression of the spalt genes is regulated by the Decapentaplegic signalling pathway in the Drosophila wing. These genes participate in the patterning of the longitudinal wing veins by regulating the expression of vein-specific genes, and in the establishment of cellular affinities in the central region of the wing blade epithelium. The Spalt proteins act as transcription factors, most likely regulating gene expression by repression, but the identity of their target genes in the wing is still unknown. As a preliminary step to unravel the genetic hierarchy controlled by the Spalt proteins, we have analysed their requirements during wing development, and addressed to what extent they mediate all the functions of the Decapentaplegic pathway in this developmental system. We identify additional functions for Spalt in cell division, survival, and maintenance of epithelial integrity. Thus, Spalt activity is required to promote cell proliferation, acting in the G2/M transition of the cell cycle. The contribution of Spalt to cell division is limited to the central region of the wing blade, as they do not mediate the extra growth triggered by Decapentaplegic signalling in the peripheral regions of the wing disc. In addition, Spalt function is required to maintain cell viability in cells exposed to high levels of Decapentaplegic signalling. This aspect of Spalt function is related to the repression of JNK signalling in the spalt domain of expression. Finally, we further characterise the requirements of Spalt to maintain epithelial integrity by regulating cellular affinities between cells located in the central wing region. Our results indicate that Spalt function mediates most of the requirements identified for Decapentaplegic signalling, contributing to establish the cellular qualities that differentiate central versus peripheral territories in the wing blade.

Genetic annotation of gain-of-function screens using RNA interference and in situ hybridization of candidate genes in the Drosophila wing.

Gain-of-function screens in Drosophila are an effective method with which to identify genes that affect the development of particular structures or cell types. It has been found that a fraction of 2-10% of the genes tested, depending on the particularities of the screen, results in a discernible phenotype when overexpressed. However, it is not clear to what extent a gain-of-function phenotype generated by overexpression is informative about the normal function of the gene. Thus, very few reports attempt to correlate the loss- and overexpression phenotype for collections of genes identified in gain-of-function screens. In this work we use RNA interference and in situ hybridization to annotate a collection of 123 P-GS insertions that in combination with different Gal4 drivers affect the size and/or patterning of the wing. We identify the gene causing the overexpression phenotype by expressing, in a background of overexpression, RNA interference for the genes affected by each P-GS insertion. Then, we compare the loss and gain-of-function phenotypes obtained for each gene and relate them to its expression pattern in the wing disc. We find that 52% of genes identified by their overexpression phenotype are required during normal development. However, only in 9% of the cases analyzed was there some complementarity between the gain- and loss-of-function phenotype, suggesting that, in general, the overexpression phenotypes would not be indicative of the normal requirements of the gene.

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.

Role of the Drosophila non-visual ß-arrestin kurtz in hedgehog signalling.

The non-visual ß-arrestins are cytosolic proteins highly conserved across species that participate in a variety of signalling events, including plasma membrane receptor degradation, recycling, and signalling, and that can also act as scaffolding for kinases such as MAPK and Akt/PI3K. In Drosophila melanogaster, there is only a single non-visual ß-arrestin, encoded by kurtz, whose function is essential for neuronal activity. We have addressed the participation of Kurtz in signalling during the development of the imaginal discs, epithelial tissues requiring the activity of the Hedgehog, Wingless, EGFR, Notch, Insulin, and TGFß pathways. Surprisingly, we found that the complete elimination of kurtz by genetic techniques has no major consequences in imaginal cells. In contrast, the over-expression of Kurtz in the wing disc causes a phenotype identical to the loss of Hedgehog signalling and prevents the expression of Hedgehog targets in the corresponding wing discs. The mechanism by which Kurtz antagonises Hedgehog signalling is to promote Smoothened internalization and degradation in a clathrin- and proteosomal-dependent manner. Intriguingly, the effects of Kurtz on Smoothened are independent of Gprk2 activity and of the activation state of the receptor. Our results suggest fundamental differences in the molecular mechanisms regulating receptor turnover and signalling in vertebrates and invertebrates, and they could provide important insights into divergent evolution of Hedgehog signalling in these organisms.

MAP4K3 is a component of the TORC1 signalling complex that modulates cell growth and viability in Drosophila melanogaster.

BACKGROUND: MAP4K3 is a conserved Ser/Thr kinase that has being found in connection with several signalling pathways, including the Imd, EGFR, TORC1 and JNK modules, in different organisms and experimental assays. We have analyzed the consequences of changing the levels of MAP4K3 expression in the development of the Drosophila wing, a convenient model system to characterize gene function during epithelial development. METHODOLOGY AND PRINCIPAL FINDINGS: Using loss-of-function mutants and over-expression conditions we find that MAP4K3 activity affects cell growth and viability in the Drosophila wing. These requirements are related to the modulation of the TORC1 and JNK signalling pathways, and are best detected when the larvae grow in a medium with low protein concentration (TORC1) or are exposed to irradiation (JNK). We also show that MAP4K3 display strong genetic interactions with different components of the InR/Tor signalling pathway, and can interact directly with the GTPases RagA and RagC and with the multi-domain kinase Tor. CONCLUSIONS AND SIGNIFICANCE: We suggest that MAP4K3 has two independent functions during wing development, one related to the activation of the JNK pathway in response to stress and other in the assembling or activation of the TORC1 complex, being critical to modulate cellular responses to changes in nutrient availability.

A conserved function of the chromatin ATPase Kismet in the regulation of hedgehog expression.

The development of the Drosophila melanogaster wing depends on its subdivision into anterior and posterior compartments, which constitute two independent cell lineages since their origin in the embryonic ectoderm. The anterior-posterior compartment boundary is the place where signaling by the Hedgehog pathway takes place, and this requires pathway activation in anterior cells by ligand expressed exclusively in posterior cells. Several mechanisms ensure the confinement of hedgehog expression to posterior cells, including repression by Cubitus interruptus, the co-repressor Groucho and Master of thick veins. In this work we identified Kismet, a chromodomain-containing protein of the SNF2-like family of ATPases, as a novel component of the hedgehog transcriptional repression mechanism in anterior compartment cells. In kismet mutants, hedgehog is ectopically expressed in a domain of anterior cells close to the anterior-posterior compartment boundary, causing inappropriate activation of the pathway and changes in the development of the central region of the wing. The contribution of Kismet to the silencing of hedgehog expression is limited to anterior cells with low levels of the repressor form of Cubitus interruptus. We also show that knockdown of CHD8, the kismet homolog in Xenopus tropicalis, is also associated with ectopic sonic hedgehog expression and up-regulation of one of its target genes in the eye, Pax2, indicating the evolutionary conservation of Kismet/CHD8 function in negatively controlling hedgehog expression.