The Drosophila tumor suppressor gene lethal(2)giant larvae is required for the emission of the Decapentaplegic signal.

The Drosophila tumor suppressor gene lethal(2) giant larvae (lgl) encodes a cytoskeletal protein required for the change in shape and polarity acquisition of epithelial cells, and also for asymmetric division of neuroblasts. We show here that lgl participates in the emission of Decapentaplegic (Dpp), a member of the transforming growth factor beta (TGFbeta) family, in various developmental processes. During embryogenesis, lgl is required for the dpp-dependent transcriptional activation of zipper (zip), which encodes the non-muscle myosin heavy chain (NMHC), in the dorsalmost ectodermal cells - the leading edge cells. The embryonic expression of known targets of the dpp signaling pathway, such as labial or tinman was abolished or strongly reduced in lgl mutants. lgl mutant cuticles exhibited phenotypes resembling those observed in mutated partners of the dpp signaling pathway. In addition, lgl was required downstream of dpp and upstream of its receptor Thickveins (Tkv) for the dorsoventral patterning of the ectoderm. During larval development, the expression of spalt, a dpp target, was abolished in mutant wing discs, while it was restored by a constitutively activated form of Tkv (Tkv(Q253D)). Taking into account that the activation of dpp expression was unaffected in the mutant, this suggests that lgl function is not required downstream of the Dpp receptor. Finally, the function of lgl responsible for the activation of Spalt expression appeared to be required only in the cells that produce Dpp, and lgl mutant somatic clones behaved non autonomously. We therefore position the activity of lgl in the cells that produce Dpp, and not in those that respond to the Dpp signal. These results are consistent with a same role for lgl in exocytosis and secretion as that proposed for its yeast ortholog sro7/77 and lgl might function in parallel or independently of its well-documented role in the control of epithelial cell polarity.

The heterotrimeric protein Go is required for the formation of heart epithelium in Drosophila.

The gene encoding the alpha subunit of the Drosophila Go protein is expressed early in embryogenesis in the precursor cells of the heart tube, of the visceral muscles, and of the nervous system. This early expression coincides with the onset of the mesenchymal-epithelial transition to which are subjected the cardial cells and the precursor cells of the visceral musculature. This gene constitutes an appropriate marker to follow this transition. In addition, a detailed analysis of its expression suggests that the cardioblasts originate from two subpopulations of cells in each parasegment of the dorsal mesoderm that might depend on the wingless and hedgehog signaling pathways for both their determination and specification. In the nervous system, the expression of Goalpha shortly precedes the beginning of axonogenesis. Mutants produced in the Goalpha gene harbor abnormalities in the three tissues in which the gene is expressed. In particular, the heart does not form properly and interruptions in the heart epithelium are repeatedly observed, henceforth the brokenheart (bkh) name. Furthermore, in the bkh mutant embryos, the epithelial polarity of cardial cells was not acquired (or maintained) in various places of the cardiac tube. We predict that bkh might be involved in vesicular traffic of membrane proteins that is responsible for the acquisition of polarity.

A Drosophila RNA helicase gene, pitchoune, is required for cell growth and proliferation and is a potential target of d-Myc.

This article describes the characterization of a new Drosophila gene that we have called pitchoune (pit) (meaning small in Provence) because mutations in this gene produce larvae that cannot grow beyond the first instar larval stage although they can live as long as 7-10 days. All the tissues are equally affected and the perfectly shaped larvae are indistinguishable from first instar wild-type animals. Analysis of mutant somatic clones suggests a function in cell growth and proliferation, which is supported by the fact that cell proliferation is promoted by pit overexpression. Tagged-Pit, when transfected in S2 cells, localizes mainly to the nucleolus, pointing towards a possible role in ribosome biogenesis and, consequently, in protein biosynthesis. pit encodes a DEAD-box RNA helicase, a family of proteins involved in the control of RNA structure in many cellular processes and its closest homologue is a human DEAD-box RNA helicase, MrDb, whose corresponding gene transcription is directly activated by Myc-Max heterodimers (Grandori, C., Mac, J., Siëbelt, F., Ayer, D. E. and Eisenman, R. N. (1996) EMBO J. 15, 4344-4357). The patterns of expression of d-myc and pit are superimposable. Ectopic expression of myc in the nervous system drives an ectopic expression of pit in this tissue indicating that in Drosophila as well, pit is a potential target of d-Myc. These results suggest that myc might promote cell proliferation by activating genes that are required in protein biosynthesis, thus linking cell growth and cell proliferation.

The held out wings (how) Drosophila gene encodes a putative RNA-binding protein involved in the control of muscular and cardiac activity.

In an attempt to identify genes that are involved in Drosophila embryonic cardiac development, we have cloned and characterized a gene whose function is required late in embryogenesis to control heart rate and muscular activity. This gene has been named held out wings (how) because hypomorphic mutant alleles produce adult animals that have lost their ability to fly and that keep their wings horizontal at a 90 degree angle from the body axis. In contrast to the late phenotype observed in null mutants, the How protein is expressed early in the invaginating mesoderm and this expression is apparently under the control of twist. When the different mesodermal lineages segregate, the expression of How becomes restricted to the myogenic lineage, including the cardioblasts and probably all the myoblasts. Antibodies directed against the protein demonstrate that How is localized to the nucleus. how encodes a protein containing one KH-domain which has been implicated in binding RNA. how is highly related to the mouse quaking gene which plays a role at least in myelination and that could serve to link a signal transduction pathway to the control of mRNA metabolism. The properties of the how gene described herein suggest that this gene participates in the control of expression of as yet unidentified target mRNAs coding for proteins essential to cardiac and muscular activity.

Evolution of the aminoacyl-tRNA synthetase family and the organization of the Drosophila glutamyl-prolyl-tRNA synthetase gene. Intron/exon structure of the gene, control of expression of the two mRNAs, selective advantage of the multienzyme complex.

In Drosophila, glutamyl-prolyl-tRNA synthetase is a multifunctional synthetase encoded by a unique gene and composed of three domains: the amino- and carboxy-terminal domains catalyze the aminoacylation of glutamic acid and proline tRNA species, respectively, and the central domain is made of 75 amino acids repeated six times amongst which 46 are highly conserved and constitute the repeated motifs [Cerini, C., Kerjan, P., Astier, M., Gratecos, D., Mirande, M. & Sémériva, M. (1991) EMBO J. 10, 4267-4277]. The intron/exon organization of the Drosophila gene reveals the presence of six exons among which four are in the 5'-end encoding glutamic acid activity. Only one exon encodes the repeated motifs. A comparison of introns positions, intron classes and intron/exon boundaries in the Drosophila gene and in its human counterpart is compatible with the intron-early hypothesis presiding, at least in part, to the evolution of the synthetases. The full-length fly protein is encoded by a 6.1-kb mRNA which is expressed throughout development. In addition, a shorter transcript encompasses the 3'-end of the cDNA and it is especially abundant in 5-10-h embryos until the first larval stage. Expression of these two mRNAs seems to be controlled by two independent promoters. The 6.1-kb mRNA promoter is probably localized in the 5'-end of the gene. The small mRNA promoter resides in the 4th intron and evidence is provided that the mRNA encodes only the domain corresponding to prolyl-tRNA synthetase and is functional in vivo. Finally, transgenic flies have been established by using constructs corresponding to the three domains of the protein. Overexpression of the repeated motifs leads to a sterility of the flies that suggests a role of these motifs in linking the multienzyme complex to an, as yet, unknown structure of the protein synthesis apparatus.

The tumor suppressor gene, lethal(2)giant larvae (1(2)g1), is required for cell shape change of epithelial cells during Drosophila development.

Inactivation of the lethal(2)giant larvae (l(2)gl) gene results in malignant transformation of imaginal disc cells and neuroblasts of the larval brain in Drosophila. Subcellular localization of the l(2)gl gene product, P127, and its biochemical characterization have indicated that it participates in the formation of the cytoskeletal network. In this paper, genetic and phenotypic analyses of a temperature-sensitive mutation (l(2)glts3) that behaves as a hypomorphic allele at restrictive temperature are presented. In experimentally overaged larvae obtained by using mutants in the production of ecdysone, the l(2)glts3 mutation displays a tumorous potential. This temperature-sensitive allele of the l(2)gl gene has been used to describe the primary function of the gene before tumor progression. A reduced contribution of both maternal and zygotic activities in l(2)glts3 homozygous mutant embryos blocks embryogenesis at the end of germ-band retraction. The mutant embryos are consequently affected in dorsal closure and head involution and show a hypertrophy of the midgut. These phenotypes are accompanied by an arrest of the cell shape changes normally occurring in lateral epidermis and in epithelial midgut cells. l(2)gl activity is also necessary for larval fife and the critical period falls within the third instar larval stage. Finally, l(2)gl activity is required during oogenesis and mutations in the gene disorganize egg chambers and cause abnormalities in the shape of follicle cells, which are eventually internalized within the egg chamber. These results together with the tumoral phenotype of epithelial imaginal disc cells strongly suggest that the l(2)gl product is required in vivo in different types of epithelial cells to control their shape during development.

Cellular interactions during heart morphogenesis in the Drosophila embryo.

The formation of the dorsal vessel or heart in a Drosophila melanogaster embryo can be divided into three main steps: i) the determination step allows individualization of heart precursor cells from the dorsal mesoderm. They are arranged in clusters of seven to nine cells, located in each of the eleven segments of the trunk. Preliminary observations suggest that the gene Notch could participate in the choice of fate that the cardioblasts and the pericardial cells will adopt within the cardiogenic region. In the same line, a new gene, whose expression, as revealed by a P-lacZ insertion, is initiated at gastrulation in the developing mesoderm and becomes restricted within the mesoderm to the myogenic lineages, could participate in the determination of the cardioblasts identity; ii) once the cardioblasts have separated from the dorsal mesoderm, they reorganize to form an epithelial monolayer. The gene coding for the alpha-subunit of the transduction protein Go, which is expressed in the cardioblasts shortly before this step, could be involved in this process. Indeed, mutants in the Go alpha gene are affected in the formation of the cardiac endothelium; and iii) the last step consists of the migration of the cardiac epithelium towards the dorsal midline of the embryo to form the dorsal vessel by apposition of the two layers of cardioblasts. We show that an extracellular matrix component is specifically expressed at the surface of the dorsal vessel and could participate in the interaction between the dorsalmost ectodermal cells and the heart during this migration step.

The multienzyme complex containing nine aminoacyl-tRNA synthetases is ubiquitous from Drosophila to mammals.

In all mammalian cells studied so far, a multienzyme complex containing the nine aminoacyl-tRNA synthetases specific for the amino acids Glu, Pro, Ile, Leu, Met, Gln, Lys, Arg and Asp was characterized. The complexes purified from various sources display very similar polypeptide compositions; they are composed of 11 polypeptides with molecular masses ranging from 18 to 150 kDa. By contrast, the corresponding enzymes from prokaryotes and lower eukaryotes behave as free enzymes. In order to test for the ubiquity of the multisynthetase complex in all metazoan species, we have searched for a similar complex in Drosophila. We have purified to homogeneity, from Schneider cells, a high molecular weight complex comprising the same nine synthetase activities. Its polypeptide composition resembles that of the complexes isolated from mammalian sources. By using the Western blotting procedure, some of the constituent polypeptides of the Drosophila complex were assigned to specific aminoacyl-tRNA synthetases. These findings support the proposal according to which the multisynthetase complex is an idiosyncratic feature of all higher eukaryotic cells.

A component of the multisynthetase complex is a multifunctional aminoacyl-tRNA synthetase.

In higher eukaryotes, nine aminoacyl-tRNA synthetases are associated within a multienzyme complex which is composed of 11 polypeptides with molecular masses ranging from 18 to 150 kDa. We have cloned and sequenced a cDNA from Drosophila encoding the largest polypeptide of this complex. We demonstrate here that the corresponding protein is a multifunctional aminoacyl-tRNA synthetase. It is composed of three major domains, two of them specifying distinct synthetase activities. The amino and carboxy-terminal domains were expressed separately in Escherichia coli, and were found to catalyse the aminoacylation of glutamic acid and proline tRNA species, respectively. The central domain is made of six 46 amino acid repeats. In prokaryotes, these two aminoacyl-tRNA synthetases are encoded by distinct genes. The emergence of a multifunctional synthetase by a gene fusion event seems to be a specific, but general attribute of all higher eukaryotic cells. This type of structural organization, in relation to the occurrence of multisynthetase complexes, could be a mechanism to integrate several catalytic domains within the same particle. The involvement of the internal repeats in mediating complex assembly is discussed.

The transduction signalling protein Go during embryonic development of Drosophila melanogaster.

G proteins are heterotrimeric proteins that play a key role in signalling transduction conveying signals from cell surface receptors to intracellular effector proteins. In particulate preparations from Drosophila melanogaster embryos, only one substrate of 39,000-40,000 molecular weight could be ADP-ribosylated with pertussis toxin. This substrate reacted in immunoblotting and immunoprecipitation experiments with a polyclonal antibody directed against the carboxy-terminal sequence of the alpha subunit of the mammalian Go protein. The Drosophila Go alpha protein was present at all stages of embryonic development; however, its expression markedly increased after 10 h embryogenesis, a period of time during which there is an active development of axonal tracts. Immunolocalization on whole mount embryos has indicated that this protein is principally localized in the CNS and is mainly restricted to the neuropil without any labelling of the cell bodies. In contrast, all the axon tracts of the CNS appeared to be highly labelled. The distribution of the Go alpha protein was also examined in several neurogenic mutants. The Go alpha protein expression was not altered in any of them but the pattern of labelling was disorganized as was the neuronal network. These results suggest a possible role for the Go protein during axonogenesis.

Towards the molecular biology of cell adhesion in Drosophila.

The characterization of extracellular matrix molecules and their putative receptors is rapidly evolving in Drosophila. Where corresponding vertebrate and Drosophila extracellular proteins have been identified they are very similar with respect to their structural properties, suggesting a high degree of conservation during evolution. By contrast, indications for components homologous to vertebrate cell-cell adhesion molecules are still very sparse. Studies on the regulation of the Drosophila genes encoding cell adhesion molecules that are involved in general basic functions during morphogenesis, together with a knowledge of the function of the genes responsible for pattern formation, should lead towards a more complete understanding of the organism's developmental program.

Drosophila fibronectin: a protein that shares properties similar to those of its mammalian homologue.

This is the first report on the existence in Drosophila of a protein with properties similar to those of vertebrate fibronectin that we shall refer to as Drosophila fibronectin. Rabbit antibodies against human plasma fibronectin have allowed the detection of this molecule in Drosophila haemolymph; common epitopes are shared by the two proteins. Drosophila fibronectin with a subunit mol. wt of approximately 230 kd is a glycoprotein which binds to denatured mammalian collagen. It is present throughout development and is as abundant in embryos as in larvae and adult flies. Drosophila fibronectin is differentially expressed during embryogenesis, a small amount being present before the blastoderm stage. Its concentration increases at gastrulation and reaches a steady-state value at the end of organogenesis. Drosophila fibronectin is predominantly detected by immunofluorescence on frozen sections of 16 h embryos in the extracellular spaces lying between the different tissues and organs. In mature third instar larvae, most of the staining is concentrated in fat body and imaginal discs, and the pattern strongly supports an extracellular localization of the protein. In addition, it is shown that Drosophila embryonic cells can functionally utilize vertebrate fibronectin for their spreading and differentiation. Finally, injection of antihuman plasma fibronectin antibodies in early embryos leads to the same phenotype as injection of Arg-Gly-Asp-containing peptides. This result suggests that one of the Arg-Gly-Asp-bearing protein(s) involved in gastrulation might be fibronectin.

A new approach to monoclonal antibody production. In vitro immunization with antigens on nitrocellulose using Drosophila myosin heavy chain as an example.

A methodology of broad applicability is described for the production of monoclonal antibodies to antigens with interesting developmental properties using Drosophila myosin heavy chain to exemplify the procedure. The technique consists of two parts. (1) Identification of the antigen with any tool which allows its detection on a Western blot. In the present case, this was achieved by monospecific polyclonal antibodies, prepared as described by Smith and Fisher (J. Cell Biol. (1984) 99, 20), which usefully define the characteristic properties of the antigen. (2) In vitro immunization of splenocytes with antigen on nitrocellulose whose position on Western blots was detected using monospecific polyclonal antibodies, followed by generation of monoclonal antibodies. A total of 19 hybridomas were selected by immunohistochemistry screening and at least six of them were directed against antigens possessing the specific characteristic properties of myosin heavy chain.

Peptides containing the cell-attachment recognition signal Arg-Gly-Asp prevent gastrulation in Drosophila embryos.

It has recently been suggested that the Arg-Gly-Asp sequence (RGD) forms part of a widespread cell-extracellular matrix recognition system. Analysis of the cell binding sites of vertebrate fibronectin and other extracellular proteins that interact with cell surfaces implicate the same amino acid triplet. Peptides containing this sequence inhibit certain developmental events such as cell-matrix adhesion or cellular migration in vitro and in vivo. The RGD-sequence is also part of the cellular recognition site of the aggregation protein discoidin I in Dictyostelium suggesting that the RGD-recognition system could be universally used. In Drosophila, despite its advanced genetics, very little is known about the extracellular components that are involved in cell movements and morphogenesis. We report here that peptides containing the RGD-sequence prevent gastrulation of Drosophila embryos. The phenotypic effect is similar to that observed in the dorsal-group mutants: no ventral furrow is formed and the embryos lack dorsal-ventral polarity. The specificity of the inhibiting action suggests that the RGD-sequence may also be used by invertebrates to mediate cell-attachment phenomena.

Photoaffinity labeling of membrane-bound porcine aminopeptidase N.

To investigate the possible role of aminopeptidase N (alpha-aminoacyl-peptide hydrolase (microsomal), EC 3.4.11.2) in the transport of amino acids from oligopeptides, the modified amino acids Phe(N3) and Phe(N3, I) and the tetrapeptides Phe(N3) or Phe(N3, I)-L-or-DAla-Gly-Gly have been synthesized. The azido-amino acids were radioactively labeled by tritium or 125I before their coupling with the tripeptides. Their utilization as photoaffinity labels for aminopeptidase N has been studied. The modification imposed at the N-terminal residue of the tetrapeptides has not impaired their hydrolysis by porcine aminopeptidase N (same kinetic parameters as unmodified peptides). In addition, evidence is presented for a specific and reversible interaction in the dark of the azido-derivatives at the substrate recognition site of the enzyme. Upon photolysis, irreversible inactivation of aminopeptidase N and covalent attachment of Phe(N3, I) have been demonstrated. Soluble and membrane-bound aminopeptidases are both labeled to the same extent indicating that the free azido-amino acid preferentially reacts with the external part of the enzyme. Although the linkage of the azido-derivative is not strictly restricted to the region of the active site, the values obtained strongly suggest that 1 mol probe has been covalently attached per mol monomer of inhibited aminopeptidase.

Studies on transport of amino acids from peptides by rat small intestine in vitro. Synthesis, properties and uptake of a photosensitive tetrapeptide.

By comparison with what is known of disaccharides transport, it has been suggested that intestinal aminopeptidase N could, hydrolyze, on the surface of the microvillus membrane, oligopeptides longer than tripeptides and itself subserve the translocation function for the amino acids released from these peptides. This article describes the synthesis of the tritiated azido-tetrapeptides p-azido[3H]phenylalanyl-alanyl-glycyl-glycine containing L or D-alanine. The synthesized products possess a function which displays all the characteristics of an aryl-azide. The photosensitive tetrapeptide formed with LAla-Gly-Gly is as good a substrate for porcine and rat aminopeptidases N as unmodified peptides while the tetrapeptide formed with DLa-Gly-Gly is not hydrolyzed at all. In addition a pattern of stepwise hydrolysis could be demonstrated and aminopeptidase N is the only exopeptidase present in the mucosal cells capable of utilizing the modified tetrapeptide as substrate. Uptake assays performed on everted rings of jejunum with the azido-tetrapeptide as substrate have shown that: (a) the azido-tetrapeptide is not transported intact but must be hydrolyzed first; (b) p-azido-phenylalanine is not released in the external medium and therefore its observed uptake is not from the bulk medium and (c) the azido-D-tetrapeptide is only accumulated by passive diffusion. These observations suggest the presence on the brush border membrane of an aminopeptidase-related transport system.

Effect of taurodeoxycholate, colipase and temperature on the interfacial inactivation of porcine pancreatic lipase.

Beside their inhibitory effect upon lipase adsorption, bile salts at low concentration (around 0.2 mM) have repeatedly been shown to enhance lipolysis slightly. From the data reported in this paper, this activation has been attributed to a stabilization of the adsorbed lipase brought about by low concentrations of bile salts. This hypothesis relies on the following observations. (a) For a given temperature, the activation by bile salts depends on the substrate. It is maximum for trihexanoin (trihexanoylglycerol) and does not exist for tripropionin (tripropionylglycerol). (b) In the absence of bile salts, the optimal activities are obtained for different temperatures depending on the substrate. (c) For a given substrate, the activation by a low concentration of bile salts depends on the temperature. It increases when the temperature is raised (up to 35-40 degrees C) and completely disappears at a sufficiently low temperature (around 10 degrees C). (d) This temperature effect does not seem to be due to a modification of the physical parameters of the interface as measured by the interfacial tension. (r) Colipase, like bile salts, increases the lipase activity on short-chain triglycerides but only at high temperature when lipase denaturation occurs. It has no influence upon the activity when the temperature is sufficiently low (around 10 degrees C).