Axon guidance of Drosophila SNb motoneurons depends on the cooperative action of muscular Krüppel and neuronal capricious activities.

The body wall musculature of the Drosophila larva consists of a stereotyped pattern of 30 muscles per abdominal hemisegment which are innervated by about 40 distinct motoneurons. Proper innervation by motoneurons is established during late embryogenesis. Guidance of motor axons to specific muscles requires appropriate pathfinding decisions as they follow their pathways within the central nervous system and on the surface of muscles. Once the appropriate targets are reached, stable synaptic contacts between motoneurons and muscles are formed. Recent studies revealed a number of molecular components required for proper motor axon pathfinding and demonstrated specific roles in fasciculation/defasciculation events, a key process in the formation of discrete motoneuron pathways. The gene capricious (caps), which encodes a cell-surface protein, functions as a recognition molecule in motor axon guidance, regulating the formation of the selective connections between the SNb-derived motoneuron RP5 and muscle 12. Here we show that Krüppel (Kr), best known as a segmentation gene of the gap class, functionally interacts with caps in establishing the proper axonal pathway of SNb including the RP5 axons. The results suggest that the transcription factor Krüppel participates in proper control of cell-surface molecules which are necessary for the SNb neurons to navigate in a caps-dependent manner within the array of the ventral longitudinal target muscles.

Cooperative action of antioxidant defense systems in Drosophila.

Molecular oxygen is key to aerobic life but is also converted into cytotoxic byproducts referred to as reactive oxygen species (ROS). Intracellular defense systems that protect cells from ROS-induced damage include glutathione reductase (GR), thioredoxin reductase (TrxR), superoxide dismutase (Sod), and catalase (Cat). Sod and Cat constitute an evolutionary conserved ROS defense system against superoxide; Sod converts superoxide anions to H(2)O(2), and Cat prevents free hydroxyl radical formation by breaking down H(2)O(2) into oxygen and water. As a consequence, they are important effectors in the life span determination of the fly Drosophila. ROS defense by TrxR and GR is more indirect. They transfer reducing equivalents from NADPH to thioredoxin (Trx) and glutathione disulfide (GSSG), respectively, resulting in Trx(SH)(2) and glutathione (GSH), which act as effective intracellular antioxidants. TrxR and GR were found to be molecularly conserved. However, the single GR homolog of Drosophila specifies TrxR activity, which compensates for the absence of a true GR system for recycling GSH. We show that TrxR null mutations reduce the capacity to adequately protect cells from cytotoxic damage, resulting in larval death, whereas mutations causing reduced TrxR activity affect pupal eclosion and cause a severe reduction of the adult life span. We also provide genetic evidence for a functional interaction between TrxR, Sod1, and Cat, indicating that the burden of ROS metabolism in Drosophila is shared by the two defense systems.

From first base: the sequence of the tip of the X chromosome of Drosophila melanogaster, a comparison of two sequencing strategies.

We present the sequence of a contiguous 2.63 Mb of DNA extending from the tip of the X chromosome of Drosophila melanogaster. Within this sequence, we predict 277 protein coding genes, of which 94 had been sequenced already in the course of studying the biology of their gene products, and examples of 12 different transposable elements. We show that an interval between bands 3A2 and 3C2, believed in the 1970s to show a correlation between the number of bands on the polytene chromosomes and the 20 genes identified by conventional genetics, is predicted to contain 45 genes from its DNA sequence. We have determined the insertion sites of P-elements from 111 mutant lines, about half of which are in a position likely to affect the expression of novel predicted genes, thus representing a resource for subsequent functional genomic analysis. We compare the European Drosophila Genome Project sequence with the corresponding part of the independently assembled and annotated Joint Sequence determined through "shotgun" sequencing. Discounting differences in the distribution of known transposable elements between the strains sequenced in the two projects, we detected three major sequence differences, two of which are probably explained by errors in assembly; the origin of the third major difference is unclear. In addition there are eight sequence gaps within the Joint Sequence. At least six of these eight gaps are likely to be sites of transposable elements; the other two are complex. Of the 275 genes in common to both projects, 60% are identical within 1% of their predicted amino-acid sequence and 31% show minor differences such as in choice of translation initiation or termination codons; the remaining 9% show major differences in interpretation.

The Drosophila homolog of the human AF10 is an HP1-interacting suppressor of position effect variegation.

In chromosomal rearrangements of acute myeloid leukaemia patients the mixed lineage leukaemia (MLL) gene, a human homolog of the Drosophila gene trithorax, is frequently fused to AF10. Here we describe the identification and a functional characterization of the Drosophila homolog dAF10. We show that dAF10 functions in heterochromatin-dependent genomic silencing of position effect variegation, a phenomenon associated with chromosomal rearrangements that cause mosaic expression of euchromatic genes when relocated next to heterochromatin. We also demonstrate that dAF10 can associate with the heterochromatin protein 1 (HP1) in vitro and in vivo. The results indicate that dAF10 is an HP1-interacting component of the heterochromatin-dependent gene silencing pathway, which either contributes to the stability of the heterochromatin complex or to its function.

Regulation of Drosophila wing vein patterning: net encodes a bHLH protein repressing rhomboid and is repressed by rhomboid-dependent Egfr signalling.

The stereotyped pattern of veins in the Drosophila wing is generated in response to local EGF signalling. Mutations in the rhomboid (rho) gene, which encodes a sevenpass membrane protein required to enhance signalling transmitted by the EGF receptor (Egfr), inhibit vein development and disrupt the vein pattern. By contrast, net mutations produce ectopic veins in intervein regions. We have cloned the net gene and show that it encodes a basic HLH protein that probably acts as a transcriptional repressor. net and rho are expressed in mutually exclusive patterns during the development of the wing imaginal disc. Lack of net activity causes rho expression to expand, and vice versa. Furthermore, ectopic expression of net or rho results in their mutual repression and thus suppresses vein formation or generates tube-like wings composed of vein-like tissue. Egfr signalling and net exert mutually antagonising activities during the specification of vein versus intervein fate. While Egfr signalling represses net transcription, net exhibits a two-tiered control by repressing rho transcription and interfering with Egfr signalling downstream of Rho. Our results further suggest that net is required to maintain intervein development by restricting Egfr signalling, which promotes vein development, to the Net-free vein regions of the wing disc.

A modifier screen of ectopic Krüppel activity identifies autosomal Drosophila chromosomal sites and genes required for normal eye development.

Irregular facets (If) is a dominant gain-of-function allele of the Drosophila segmentation gene Krüppel (Kr) that interferes with eye development. In a search for genes that interact with Kr activity, we recently performed a systematic genetic screen to identify dominant enhancers and suppressors of the If eye phenotype that are located on the third chromosome. Here we describe locations and candidate genes of the second chromosome that act as dominant modifiers of ectopic Kr activity during eye development. The collection of more than 40 modifiers of Kr activity located on the second and third chromosomes, from which a total of 16 genes were identified, includes genes encoding transcription factors and components of signal transduction pathways that may regulate or be regulated by Kr activity. We also identified genes coding for more general cellular factors that could interfere with the intracellular transport or the half-life of the Kr protein. The data demonstrate that the If mutation provides a means to screen the Drosophila genome for functional components of developmental pathways that depend on or can be modified by Kr activity. Owing to the bias of the screening system applied, these modifier genes will be expressed and are likely to be required during Drosophila wild-type eye development.

Drosophila segment borders result from unilateral repression of hedgehog activity by wingless signaling.

Body structures of Drosophila develop through transient developmental units, termed parasegments, with boundaries lying between the adjacent expression domains of wingless and engrailed. Parasegments are transformed into the morphologically distinct segments that remain fixed. Segment borders are established adjacent and posterior to each engrailed domain. They are marked by single rows of stripe expressing cells that develop into epidermal muscle attachment sites. We show that the positioning of these cells is achieved through repression of Hedgehog signal transduction by Wingless signaling at the parasegment boundary. The nuclear mediators of the two signaling pathways, Cubitus interruptus and Pangolin, function as activator and symmetry-breaking repressor of stripe expression, respectively.

Homeodomain position 54 specifies transcriptional versus translational control by Bicoid.

Bicoid (BCD), the anterior determinant of Drosophila, controls embryonic gene expression by transcriptional activation and translational repression. Both functions require the homeodomain (HD), which recognizes DNA motifs at target gene enhancers and a specific sequence interval in the 3' untranslated region of caudal (cad) mRNA. Here we show that the BCD HD is a nucleic acid-binding unit. Its helix III contains an arginine-rich motif (ARM), similar to the RNA-binding domain of the HIV-1 protein REV, needed for both RNA and DNA recognition. Replacement of arginine 54, within this motif, alters the RNA but not the DNA binding properties of the HD. Corresponding BCD mutants fail to repress cad mRNA translation, whereas the transcriptional target genes are still activated.

Phenotypic suppression of empty spiracles is prevented by buttonhead.

Unlike the trunk segments, the anterior head segments of Drosophila are formed in the absence of pair-rule and HOX-cluster gene expression, by the activities of the gap-like genes orthodenticle (otd), empty spiracles (ems) and buttonhead (btd). The products of these genes are transcription factors, but only EMS has a HOX-like homeodomain. Indeed, ems can confer identity to trunk segments when other HOX-cluster gene activities are absent. In trunk segments of wild-type embryos, however, ems activity is prevented by phenotypic suppression, in which more posterior HOX-cluster genes inactivate the more anterior without affecting transcription or translation. ems is suppressed by all other Hox-cluster genes and so is placed at the bottom of their hierarchy. Here we show that misexpression of EMS in the head transforms segment identity in a btd-dependent manner, that misexpression of BTD in the trunk causes ems-dependent structures to develop, and that EMS and BTD interact in vitro. The data indicate that this interaction may allow ems to escape from the bottom of the HOX-cluster gene hierarchy and cause a dominant switch of homeotic prevalence in the anterior-posterior direction.

VASA mediates translation through interaction with a Drosophila yIF2 homolog.

The Drosophila gene vasa (vas) encodes an RNA-binding protein required for embryonic patterning and germ cell specification. In vas mutants, translation of several germline mRNAs is reduced. Here we show that VAS interacts directly with the Drosophila homolog of yeast translation initiation factor 2, encoded by a novel gene, dIF2. Embryos produced by vas/+; dIF2/+ females have pattern defects and fewer germline progenitor cells, indicating a functional interaction between endogenous vas and dIF2 activities. Mutations in other translation initiation factors do not enhance the vas phenotype, suggesting that dIF2 has a particular role in germ plasm function. We conclude that VAS regulates translation of germline mRNAs by specific interaction with dIF2, an essential factor conserved from bacteria to humans.

From sequence to chromosome: the tip of the X chromosome of D. melanogaster.

One of the rewards of having a Drosophila melanogaster whole-genome sequence will be the potential to understand the molecular bases for structural features of chromosomes that have been a long-standing puzzle. Analysis of 2.6 megabases of sequence from the tip of the X chromosome of Drosophila identifies 273 genes. Cloned DNAs from the characteristic bulbous structure at the tip of the X chromosome in the region of the broad complex display an unusual pattern of in situ hybridization. Sequence analysis revealed that this region comprises 154 kilobases of DNA flanked by 1.2-kilobases of inverted repeats, each composed of a 350-base pair satellite related element. Thus, some aspects of chromosome structure appear to be revealed directly within the DNA sequence itself.

The Drosophila gene abstrakt, required for visual system development, encodes a putative RNA helicase of the DEAD box protein family.

The molecular mechanisms underlying axonal pathfinding are not well understood. In a genetic screen for mutations affecting the projection of the larval optic nerve we isolated the abstrakt locus. abstrakt is required for pathfinding of the larval optic nerve, and it also affects development in both the adult visual system and the embryonic CNS. Here we report the molecular characterization of abstrakt. It encodes a putative ATP-dependent RNA helicase of the DEAD box protein family, with two rare substitutions in the PTRELA and the RG-D motifs, thought to be involved in oligonucleotide binding: serine for threonine, and lysine for arginine, respectively. Two mutant alleles of abstrakt show amino acid exchanges in highly conserved positions. A glycine to serine exchange in the HRIGR motif, which is involved in RNA binding and ATP hydrolysis, results in a complete loss of protein function; and a proline to leucine exchange located between the highly conserved ATPase A and PTRELA motifs results in temperature-sensitive protein function. Both the broad requirement for abstrakt gene function and its ubiquitous expression are consistent with a molecular function of the abstrakt protein in mRNA splicing or translational control.

Receptor tyrosine kinase signaling regulates different modes of Groucho-dependent control of Dorsal.

Transcriptional control of the Drosophila terminal gap gene huckebein (hkb) depends on Torso (Tor) receptor tyrosine kinase (RTK) signaling and the Rel/NFkappaB homolog Dorsal (DI). DI acts as an intrinsic transcriptional activator in the ventral region of the embryo, but under certain conditions, such as when it is associated with the non-DNA-binding co-repressor Groucho (Gro), it is converted into a repressor. Gro is recruited to the enhancer element in the vicinity of DI by sequence-specific transcription factors such as Dead Ringer (Dri). We examined the interplay between DI, Gro and Dri on the hkb enhancer and show that when acting over a distance, Gro abolishes rather than converts DI activator function. Reducing the distance between DI- and Dri-binding sites, however, switches DI into a Gro-dependent repressor that overrides activation of transcription. Both of the distance-dependent regulatory options of Gro - quenching and silencing of transcription - are inhibited by RTK signaling. These data describe a newly identified mode of function for Gro when acting in concert with DI. RTK signaling provides a way of modulating DI function by interfering either with Gro activity or with Dri-dependent recruitment of Gro to the enhancer.

Restricted expression and subnuclear localization of the Drosophila gene Dnop5, a member of the Nop/Sik family of the conserved rRNA processing factors.

Members of the conserved nop5/sik1 gene family encode components of small nucleolar ribonucleoprotein (snoRNP) complexes, which have an essential function in rRNA-processing. We describe a novel Drosophila member of this family, termed Dnop5. The gene is expressed in nurse cells during oogenesis and transcripts are deposited into the growing oocyte. Maternal transcripts become evenly distributed in the egg and remain in a ubiquitous pattern during early embryogenesis. Zygotic Dnop5 expression is initiated during the extended germband stage. Transcripts accumulate in mesoderm and midgut primordia, and in the developing imaginal discs of the larvae. Consistent with a function in rRNA processing, Dnop5 protein (DNop5) accumulates in a nuclear substructure, likely to be the nucleolus. Maternal protein accumulates in the nucleolus of all cells in the early embryo, whereas DNop5 that is derived from zygotic mRNA, is restricted to the nuclei of muscles and midgut.

Localized expression of the Drosophila gene Dxl6, a novel member of the serine/arginine rich (SR) family of splicing factors.

Members of the highly conserved family of serine/arginine rich (SR) splicing factors play an essential role in the recognition of the exonic splicing enhancers that control the choice of splice sites in primary transcripts. Here, we report the cloning and the expression pattern of Dxl6, a novel Drosophila member of this protein family. Dxl6 is located on the second chromosome in a position next to hrp48 and Dwee1. Its intron contains Dnop5, a small nucleolar ribonucleoprotein (snoRNP) which is essential for rRNA-processing. During oogenesis, Dxl6 transcripts are expressed in nurse cells. Transcripts are transported into the oocyte and maintained in a ubiquitous pattern in the egg and early embryo. Zygotic Dxl6 transcripts accumulate in the neuroectodermal region of the gastrulating embryo and become highly enriched in the central nervous system (CNS) and brain of embryos. During larval stages, Dxl6 transcripts are detected in distinct patterns in the developing imaginal discs.

The class 2 selenophosphate synthetase gene of Drosophila contains a functional mammalian-type SECIS.

Synthesis of monoselenophosphate, the selenium donor required for the synthesis of selenocysteine (Sec) is catalyzed by the enzyme selenophosphate synthetase (SPS), first described in Escherichia coli. SPS homologs were identified in archaea, mammals and Drosophila. In the latter, however, an amino acid replacement is present within the catalytic domain and lacks selenide-dependent SPS activity. We describe the identification of a novel Drosophila homolog, Dsps2. The open reading frame of Dsps2 mRNA is interrupted by an UGA stop codon. The 3'UTR contains a mammalian-like Sec insertion sequence which causes translational readthrough in both transfected Drosophila cells and transgenic embryos. Thus, like vertebrates, Drosophila contains two SPS enzymes one with and one without Sec in its catalytic domain. Our data indicate further that the selenoprotein biosynthesis machinery is conserved between mammals and fly, promoting the use of Drosophila as a genetic tool to identify components and mechanistic features of the synthesis pathway.

Tip cell-derived RTK signaling initiates cell movements in the Drosophila stomatogastric nervous system anlage.

The stomatogastric nervous system (SNS) of Drosophila is a simply organized neural circuitry that innervates the anterior enteric system. Unlike the central and the peripheral nervous systems, the SNS derives from a compact epithelial anlage in which three invagination centers, each giving rise to an invagination fold headed by a tip cell, are generated. Tip cell selection involves lateral inhibition, a process in which Wingless (Wg) activity adjusts the range of Notch signaling. Here we show that RTK signaling mediated by the Drosophila homolog of the epidermal growth factor receptor, DER, plays a key role in two consecutive steps during early SNS development. Like Wg, DER signaling participates in adjusting the range of Notch-dependent lateral inhibition during tip cell selection. Subsequently, tip cells secrete the DER ligand Spitz and trigger local RTK signaling, which initiates morphogenetic movements resulting in the tip cell-directed invaginations within the SNS anlage.

Common and diverged functions of the Drosophila gene pair D-Sp1 and buttonhead.

The Drosophila gene buttonhead (btd) is required for the formation of the mandibular, the intercalary and the antennal head segments of the embryo. The btd protein (BTD) is functionally and structurally related to the human C(2)H(2) zinc finger transcription factor Sp1. A second Sp1-like Drosophila gene, termed Drosophila Sp1 (D-Sp1), had been identified on the basis of a partial sequence showing that the gene encodes a characteristic zinc finger domain, composed of three finger motifs similar to both Sp1 and btd. D-Sp1 is located in the same cytological location as btd in chromosome band 9A on the X-chromosome. It had been proposed that D-Sp1 and btd are likely to act as a gene pair and function in a at least partially redundant manner. Here we report the molecular analysis of D-Sp1 and its expression pattern during embryonic and larval development. We show that D-Sp1 acts as a transcriptional regulator. Lack-of-function analysis combined with rescue and gain-of-function studies indicates that btd and D-Sp1 play essential and redundant roles for mechanosensory organ development. However, D-Sp1 lacks the specific features of BTD required for embryonic intercalary and antennal segment formation.

hairy stripe 7 element mediates activation and repression in response to different domains and levels of Krüppel in the Drosophila embryo.

The Drosophila gap gene Krüppel (Kr) encodes a zinc finger-type transcription factor required for controlling the spatial expression of other segmentation genes during early blastoderm stage. Here we show that two independent and transferable repressor domains of Krüppel act to control expression of the pair-rule gene hairy, and that the minimal cis-acting element of hairy stripe7 (h7) mediates either Krüppel-dependent activation or repression in different regions of the blastoderm embryo. The C-terminal region of Krüppel which encompasses the predominant repressor domain is not essential for activation, but is required to fully suppress h7-mediated transcription in response to high levels of Krüppel activity. This domain contains an interaction motif for dCtBP, a homologue of the human co-repressor CtBP. dCtBP activity is, however, dispensable for Krüppel-mediated repression in the embryo since Krüppel-mediated repression functions in the absence of dCtBP. Possible modes of h7-mediated gene regulation in response to the different domains and levels of Krüppel are discussed.

The range of spalt-activating Dpp signalling is reduced in endocytosis-defective Drosophila wing discs.

Pattern formation along the anterior-posterior (A/P) axis of the developing Drosophila wing depends on Decapentaplegic (Dpp), a member of the conserved transforming growth factor beta (TGFbeta) family of secreted proteins. Dpp is expressed in a stripe along the A/P compartment boundary of the wing imaginal disc and forms a long-range concentration gradient with morphogen-like properties which generates distinct cell fates along the A/P axis. We have monitored Dpp expression and Dpp signalling in endocytosis-mutant wing imaginal discs which develop severe pattern defects specifically along the A/P wing axis. The results show that the size of the Dpp expression domain is expanded in endocytosis-mutant wing discs. However, this expansion did not result in a concomitant expansion of the functional range of Dpp activity but rather its reduction as indicated by the reduced expression domain of the Dpp target gene spalt. The data suggest that clathrin-mediated endocytosis, a cellular process necessary for membrane recycling and vesicular trafficking, participates in Dpp action during wing development. Genetic interaction studies suggest a link between the Dpp receptors and clathrin. Impaired endocytosis does not interfere with the reception of the Dpp signal or the intracellular processing of the mediation of the signal in the responder cells, but rather affects the secretion and/or the distribution of Dpp in the developing wing cells.