Translational repression determines a neuronal potential in Drosophila asymmetric cell division.

Asymmetric cell division is a fundamental strategy for generating cellular diversity during animal development. Daughter cells manifest asymmetry in their differential gene expression. Transcriptional regulation of this process has been the focus of many studies, whereas cell-type-specific 'translational' regulation has been considered to have a more minor role. During sensory organ development in Drosophila, Notch signalling directs the asymmetry between neuronal and non-neuronal lineages, and a zinc-finger transcriptional repressor Tramtrack69 (TTK69) acts downstream of Notch as a determinant of non-neuronal identity. Here we show that repression of TTK69 protein expression in the neuronal lineage occurs translationally rather than transcriptionally. This translational repression is achieved by a direct interaction between cis-acting sequences in the 3' untranslated region of ttk69 messenger RNA and its trans-acting repressor, the RNA-binding protein Musashi (MSI). Although msi can act downstream of Notch, Notch signalling does not affect MSI expression. Thus, Notch signalling is likely to regulate MSI activity rather than its expression. Our results define cell-type-specific translational control of ttk69 by MSI as a downstream event of Notch signalling in asymmetric cell division.

The Drosophila Netrin receptor Frazzled guides axons by controlling Netrin distribution.

Netrin is a secreted protein that can act as a chemotropic axon guidance cue. Two classes of Netrin receptor, DCC and UNC-5 (refs 6-9), are required for axon guidance and are thought to mediate Netrin signals in growth cones through their cytoplasmic domains. However, in the guidance of Drosophila photoreceptor axons, the DCC orthologue Frazzled is required not in the photoreceptor neurons but instead in their targets, indicating that Frazzled also has a non-cell-autonomous function. Here we show that Frazzled can capture Netrin and 'present' it for recognition by other receptors. Moreover, Frazzled itself is actively localized within the axon through its cytoplasmic domain, and thereby rearranges Netrin protein into a spatial pattern completely different from the pattern of Netrin gene expression. Frazzled-dependent guidance of one pioneer neuron in the central nervous system can be accounted for solely on the basis of this ability of Frazzled to control Netrin distribution, and not by Frazzled signalling. We propose a model of patterning mechanism in which a receptor rearranges secreted ligand molecules, thereby creating positional information for other receptors.

Sprouty: a common antagonist of FGF and EGF signaling pathways in Drosophila.

Extracellular factors such as FGF and EGF control various aspects of morphogenesis, patterning and cellular proliferation in both invertebrates and vertebrates. In most systems, it is primarily the distribution of these factors that controls the differential behavior of the responding cells. Here we describe the role of Sprouty in eye development. Sprouty is an extracellular protein that has been shown to antagonize FGF signaling during tracheal branching in Drosophila. It is a novel type of protein with a highly conserved cysteine-rich region. In addition to the embryonic tracheal system, sprouty is also expressed in other tissues including the developing eye imaginal disc, embryonic chordotonal organ precursors and the midline glia. In each of these tissues, EGF receptor signaling is known to participate in the control of the correct number of neurons or glia. We show that, in all three tissues, the loss of sprouty results in supernumerary neurons or glia, respectively. Furthermore, overexpression of sprouty in wing veins and ovarian follicle cells, two other tissues where EGF signaling is required for patterning, results in phenotypes that resemble the loss-of-function phenotypes of Egf receptor. These results suggest that Sprouty acts as an antagonist of EGF as well as FGF signaling pathways. These receptor tyrosine kinase-mediated pathways may share not only intracellular signaling components but also extracellular factors that modulate the strength of the signal.

sprouty encodes a novel antagonist of FGF signaling that patterns apical branching of the Drosophila airways.

Antagonists of several growth factor signaling pathways play important roles in developmental patterning by limiting the range of the cognate inducer. Here, we describe an antagonist of FGF signaling that patterns apical branching of the Drosophila airways. In wild-type embryos, the Branchless FGF induces secondary branching by activating the Breathless FGF receptor near the tips of growing primary branches. In sprouty mutants, the FGF pathway is overactive and ectopic branches are induced on the stalks of primary branches. We show that FGF signaling induces sprouty expression in the nearby tip cells, and sprouty acts nonautonomously and in a competitive fashion to block signaling to the more distant stalk cells. sprouty encodes a novel cysteine-rich protein that defines a new family of putative signaling molecules that may similarly function as FGF antagonists in vertebrate development.

The Drosophila mushroom body is a quadruple structure of clonal units each of which contains a virtually identical set of neurones and glial cells.

The mushroom body (MB) is an important centre for higher order sensory integration and learning in insects. To analyse the development and organisation of the MB neuropile in Drosophila, we performed cell lineage analysis in the adult brain with a new technique that combines the Flippase (flp)/FRT system and the GAL4/UAS system. We showed that the four mushroom body neuroblasts (MBNbs) give birth exclusively to the neurones and glial cells of the MB, and that each of the four MBNb clones contributes to the entire MB structure. The expression patterns of 19 GAL4 enhancer-trap strains that mark various subsets of MB cells revealed overlapping cell types in all four of the MBNb lineages. Partial ablation of MBNbs using hydroxyurea showed that each of the four neuroblasts autonomously generates the entire repertoire of the known MB substructures.

klingon, a novel member of the Drosophila immunoglobulin superfamily, is required for the development of the R7 photoreceptor neuron.

klingon is a member of the Immunoglobulin superfamily and is expressed in a restricted pattern of neurons during embryonic neurogenesis and in the R7 photoreceptor precursor throughout its development. Starting from the H214 enhancer trap line, we identified a transcription unit, klingon, that encodes a putative protein of 528 amino acids and contains three C2-type Immunoglobulin-like domains followed by one fibronectin type III repeat. When Klingon is expressed in S2 tissue culture cells, it is associated with the cell membrane by a glycosyl-phosphatidylinositol linkage and can mediate homophilic adhesion. Genetic analysis has revealed that klingon is an essential gene that participates in the development of the R7 neuron. Ectopic expression of klingon in all neurons in a sevenless background can alter the position of the R8 rhabdomere.

Biparous: a novel bHLH gene expressed in neuronal and glial precursors in Drosophila.

Genetic studies have uncovered many genes that are involved in the first steps of neuronal development in Drosophila. Less is known about the intermediate steps during which individual precursor cells follow either the neuronal pathway or the glial pathway. We report the identification of a novel bHLH gene, biparous, expressed in neuronal and glial precursors in Drosophila. Unlike most bHLH genes, biparous expression continues to the final stages of neurogenesis in the embryo. Expression of biparous is not observed in end stage postmitotic neurons and precedes the expression of repo, a gene activated in later stages of glial differentiation. The bHLH domain is sufficiently different from previously described bHLH domains to imply a novel function.

Cell fate control in the Drosophila retina by the orphan receptor seven-up: its role in the decisions mediated by the ras signaling pathway.

Drosophila seven-up is an orphan receptor of the steroid receptor family that is required to specify photoreceptor neuron subtypes in the developing compound eye. Expression of seven-up is confined to four of the eight photoreceptor precursors, R3/R4/R1/R6. We show that misexpression of seven-up in any of the other cell types within the developing ommatidium interferes with their differentiation. Each cell type responds differently to seven-up misexpression. For example, ectopic expression in the non-neuronal cone cells using the sevenless promoter/enhancer (sev-svp) causes the cone cells to take on a neuronal identity. Ectopic expression of seven-up in R2/R5 using the rough enhancer (ro-svp) causes these neurons to lose aspects of their photoreceptor subtype identity while remaining neuronal. Each cell type appears to have a different developmental time window that is sensitive to misexpressed seven-up. The temporal order of responsiveness of each cell type to misexpressed seven-up is similar but not identical to the order of neuronal differentiation. This suggests that there are processes of specification that are distinct from the specification to become a photoreceptor neuron. We have identified members of the ras signaling pathway as suppressors of the cone cell to R7 neuron transformation caused by sev-svp. Suppression of the sev-svp phenotype can be achieved by decreasing the gene-dosage of any of the members of the ras-pathway. This suggests that the function of seven-up in the cone cells requires ras signaling. However, a decrease in ras signaling results in enhancement of the phenotype caused by the ro-svp transgene. We discuss the relationship between decisions controlled by seven-up and those controlled by ras signaling.

Ectopic expression of seven-up causes cell fate changes during ommatidial assembly.

During Drosophila ommatidial development, a single cell is selected within the ommatidial cluster to become the R7 photoreceptor neuron. The seven-up gene has been shown to play a role in this process by preventing four other photoreceptor precursors, R3/R4/R1/R6, from adopting the R7 cell fate. The seven-up gene encodes a steroid receptor-like molecule that is expressed only in those four cells that require seven-up function in the developing Drosophila ommatidium. We have examined the functional significance of the spatially restricted expression of seven-up by misexpressing seven-up isoforms. As expected from the function that seven-up performs in R3/R4/R1/R6, ubiquitous expression of seven-up causes transformation of the R7 cell to an R1-R6 cell fate. In addition, depending on the timing and spatial pattern of expression, various other phenotypes are produced including the loss of the R7 cell and the formation of extra R7 cells. Ubiquitous expression of seven-up close to the morphogenetic furrow interferes with R8 differentiation resulting in failure to express the boss protein, the ligand for the sevenless receptor tyrosine kinase, and the R7 cell is lost consequently. Extra R7 cells are formed by recruiting non-neuronal cone cells as photoreceptor neurons in a sevenless and bride of sevenless independent way. Thus, the spatiotemporal pattern of seven-up expression plays an essential role in controlling the number and cellular origin of the R7 neuron in the ommatidium. Our results also suggest that seven-up controls decisions not only between photoreceptor subtypes, but also between neuronal and non-neuronal fates.

cactus, a gene involved in dorsoventral pattern formation of Drosophila, is related to the I kappa B gene family of vertebrates.

Among the maternally active genes of Drosophila, cactus is the only one whose loss of function mutations specifically produce ventralized embryos. Its product inhibits nuclear translocation of the dorsal morphogen in the dorsal region of the embryo. Here we report the cloning of cactus and the sequencing of its maternal transcript. The identity of our clones was verified by induction of phenocopies with antisense RNA and rescue of the mutant phenotype with sense RNA. cactus is predicted to encode an acidic, cytoplasmic protein with seven ankyrin repeats. The sequence has similarity to the I kappa B proteins that inhibit the vertebrate transcription factor NF-kappa B. In analogy to results obtained with I kappa B and NF-kappa B, bacterially expressed cactus protein can inhibit DNA binding of dorsal protein in vitro.

The presumptive R7 cell of the developing Drosophila eye receives positional information independent of sevenless, boss and sina.

Studies on the development of the R7 photoreceptor in the Drosophila eye thus far have identified three genes that specifically affect this cell: sevenless, boss and sina. In each of these mutants the R7 precursor develops instead as the equatorial cone cell (EQC). We have isolated an enhancer trap line, H214, in which beta-galactosidase is primarily expressed in the R7 cell throughout its development. In mutations of sevenless, boss and sina, expression in H214 is initially reduced although still present in the R7 precursor and persists in the EQC into which it develops. The EQC in wild type never expresses lacZ in H214. This result is in contrast to that seen with other enhancer trap lines that display expression in R7, and indicates that some aspect of R7 differentiation is independent of the genetic pathway(s) involving sevenless, boss and sina.

cactus, a maternal gene required for proper formation of the dorsoventral morphogen gradient in Drosophila embryos.

The dorsoventral pattern of the Drosophila embryo is mediated by a gradient of nuclear localization of the dorsal protein which acts as a morphogen. Establishment of the nuclear concentration gradient of dorsal protein requires the activities of the 10 maternal 'dorsal group' genes whose function results in the positive regulation of the nuclear uptake of the dorsal protein. Here we show that in contrast to the dorsal group genes, the maternal gene cactus acts as a negative regulator of the nuclear localization of the dorsal protein. While loss of function mutations of any of the dorsal group genes lead to dorsalized embryos, loss of cactus function results in a ventralization of the body pattern. Progressive loss of maternal cactus activity causes progressive loss of dorsal pattern elements accompanied by the expansion of ventrolateral and ventral anlagen. However, embryos still retain dorsoventral polarity, even if derived from germline clones using the strongest available, zygotic lethal cactus alleles. In contrast to the loss-of-function alleles, gain-of-function alleles of cactus cause a dorsalization of the embryonic pattern. Genetic studies indicate that they are not overproducers of normal activity, but rather synthesize products with altered function. Epistatic relationships of cactus with dorsal group genes were investigated by double mutant analysis. The dorsalized phenotype of the dorsal mutation is unchanged upon loss of cactus activity. This result implies that cactus acts via dorsal and has no independent morphogen function. In all other dorsal group mutant backgrounds, reduction of cactus function leads to embryos that express ventrolateral pattern elements and have increased nuclear uptake of the dorsal protein at all positions along the dorsoventral axis. Thus, the cactus gene product can prevent nuclear transport of dorsal protein in the absence of function of the dorsal group genes. Genetic and cytoplasmic transplantation studies suggest that the cactus product is evenly distributed along the dorsoventral axis. Thus the inhibitory function that cactus product exerts on the nuclear transport of the dorsal protein appears to be antagonized on the ventral side. We discuss models of how the action of the dorsal group genes might counteract the cactus function ventrally.

The Drosophila seven-up gene, a member of the steroid receptor gene superfamily, controls photoreceptor cell fates.

The Drosophila seven-up (svp) gene was isolated as a lethal insertion in an "enhancer trap" screen. It is expressed and required in photoreceptor cell precursors R1, R3, R4, and R6 during eye development. The absence of svp+ function causes a transformation of these cells toward an R7 cell fate, as judged by morphology and expression of an R7-specific marker. This transformation depends in part on the sevenless gene product. Our results show that svp is involved in control of cell fate during the generation of neuronal diversity. Molecular analysis of svp reveals that it is a member of the steroid receptor gene superfamily and is likely to be a Drosophila homolog of the human transcription factor COUP.

Lineage, migration, and morphogenesis of longitudinal glia in the Drosophila CNS as revealed by a molecular lineage marker.

Previous studies described three different classes of glial cells in the developing CNS of the early Drosophila embryo that prefigure and ensheath the major CNS axon tracts. Among these are 6 longitudinal glial cells on each side of each segment that overlie the longitudinal axon tracts. Here we use transformant lines carrying a P element containing a 130 bp sequence from the fushi tarazu gene in front of the lacZ reporter gene to direct beta-galactosidase expression in the longitudinal glia. Using this molecular lineage marker, we show that 1 of the "neuroblasts" in each hemisegment is actually a glioblast, which divides once symmetrically, in contrast to the typical asymmetric neuroblast divisions, producing 2 glial cells, which migrate medially and divide to generate the 6 longitudinal glial cells. As with neuroblasts, mutations in Notch and other neurogenic genes lead to supernumerary glioblasts. The results indicate that the glioblast is similar to other neuroblasts; however, the positionally specified fate of this blast cell is to generate a specific lineage of glia rather than a specific family of neurons.

Expression and function of the segmentation gene fushi tarazu during Drosophila neurogenesis.

Segmentation genes control cell identities during early pattern formation in Drosophila. One of these genes, fushi tarazu (ftz), is now shown also to control cell fate during neurogenesis. Early in development, ftz is expressed in a striped pattern at the blastoderm stage. Later, it is transiently expressed in a specific subset of neuronal precursor cells, neurons (such as aCC, pCC, RP1, and RP2), and glia in the developing central nervous system (CNS). The function of ftz in the CNS was determined by creating ftz mutant embryos that express ftz in the blastoderm stripes but not in the CNS. In the absence of ftz CNS expression, some neurons appear normal (for example, the aCC, pCC, and RP1), whereas the RP2 neuron extends its growth cone along an abnormal pathway, mimicking its sibling (RP1), suggesting a transformation in neuronal identity.

Regulation and function of the Drosophila segmentation gene fushi tarazu.

The Drosophila segmentation gene fushi tarazu (ftz) is expressed in a pattern of seven stripes at the blastoderm stage. Two cis-acting control elements are required for this expression: the zebra element, which confers the striped pattern by mediating the effects of a subset of segmentation genes; and the upstream element, an enhancer element requiring ftz+ activity for its action. Fusion of the upstream element to a basal promoter results in activation of the heterologous promoter in a ftz-dependent striped pattern, supporting the idea that ftz regulates itself by acting through its enhancer. The upstream element can also confer expression patterns similar to that of the homeotic gene Antennapedia, suggesting that a similar element may play a role in the activation of Antennapedia.

Molecular characterization of mutant actin genes which induce heat-shock proteins in Drosophila flight muscles.

Heat-shock proteins (hsps) are constitutively induced by the mutant actins in the Drosophila indirect flight muscles (IFM). We compared primary structures of the mutant actin genes (KM75 and HH5) which induce hsps and of the non-inducing alleles (KM129 and KM88). The KM75 actin has lost 20 amino acids at the C-terminus. The HH5 actin has only one amino acid substitution, from Gly-336 to Ser. In KM129, the C-terminal part of actin is replaced by novel amino acids. KM88 is a null allele, with an amber mutation early in the coding region of the mutated actin gene. Although all of the KM75, HH5 and KM129 actins have defects near the C-terminus, only hsp-inducing mutant actins cause enlargement of the IFM nuclei as well as a disruption of myofibrils even in the presence of two copies of the normal genes. We further consider the underlying mechanisms linking these features of the hsp-inducing alleles.

Germline transformation with Drosophila mutant actin genes induces constitutive expression of heat shock genes.

Two Drosophila mutants KM75 and HH5, which are mutated in the act88F actin gene specific for the indirect flight muscles (IFM), synthesize heat shock proteins (hsps) constitutively in a tissue-specific manner. We have introduced cloned mutant act88F genes into a strain containing the wild-type act88F allele by P-element-mediated transformation. Flies transformed with a 4.05 kb KM75 act88F gene fragment encoding the p42 actin variant express both p42 and hsps specifically in the IFM. Using normal/mutant chimeric genes, the mutation sites of KM75 and HH5 were mapped within the sequence encoding the last 72 amino acids of actin. An in vitro mutated gene encoding a protein that lacks the 72 carboxy-terminal amino acids also induces constitutive hsp synthesis.