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The larval visual system of Drosophila melanogaster consists of two bilateral clusters of 12 photoreceptors, which express Rhodopsin 5 and 6 (Rh5 and Rh6) in a non-overlapping manner. These neurons send their axons in a fascicle, the larval optic nerve (LON), which terminates in the larval optic neuropil. The LON is required for the development of a serotonergic arborization originating in the central brain and for the development of the dendritic tree of the circadian pacemakers, the small ventral lateral neurons (LNv) [Malpel, S., Klarsfeld, A., Rouyer, F., 2002. Larval optic nerve and adult extra-retinal photoreceptors sequentially associate with clock neurons during Drosophila brain development. Development 129, 1443–1453; Mukhopadhyay, M., Campos, A.R., 1995. The larval optic nerve is required for the development of an identified serotonergic arborization in Drosophila melanogaster . Dev. Biol., 169, 629–643]. Here, we show that both Rh5- and Rh6-expressing fibers overlap equally with the 5-HT arborization and that it, in turn, also contacts the dendritic tree of the LNv. The experiments described here aimed at determining whether Rh5- or Rh6-expressing fibers, as well as the LNv, influence the development of this serotonergic arborization. We conclude that Rh6-expressing fibers play a unique role in providing a signal required for the outgrowth and branching of the serotonergic arborization. Moreover, the innervation of the larval optic neuropil by the 5-HT arborization depends on intact Rac function. A possible role for these serotonergic processes in modulating the larval circadian rhythmicity and photoreceptor function is discussed.

We report the identification of the Disco Interacting Protein 1 (DIP1) gene isolated in a yeast interaction trap screen using the zinc finger protein disconnected (disco) as a bait. DIP1 encodes a protein containing two double-stranded RNA binding domains (dsRBD). Consistent with the presence of dsRBD, DIP1 binds dsRNA or structured RNAs in Northwestern assays. DIP1 is found in nuclear subdomains resembling speckles known to accumulate transcription and splicing factors. In early embryos, nuclear localization of DIP1 protein coincides with the onset of zygotic gene expression. Later in development DIP1 expression is decreased in dividing cells in different tissues. Overexpression of DIP1 in the eye-antennal imaginal disc, early in embryonic and larval development, causes the formation of supernumerary structures in the head capsule. A role for DIP1 in epigenetic mechanisms that lead to the establishment and/or maintenance of cell fate specification is discussed.

The Drosophila disconnected (disco) gene encodes a transcription factor containing two C2H2 zinc-fingers that functions in the development of both embryonic and adult structures. The disco gene was initially identified from a mutation in which adult photoreceptor axons fail to connect with their target cells in the larval optic lobe (Steller et al., 1987). The optic lobes of disco mutants are poorly developed and lack optic lobe neurons, including the lateral neurons. Abnormal circadian rhythmicity has been attributed to the absence or aberrant differentiation of these lateral neurons (Hardin et al., 1992; Helfrich-Forster et al., 1998). Additionally, disco mutants show defects in thoracic and abdominal sensory axon development (Glossop and Shepherd, 1998). More recently, disco was found to have a broader role in embryonic pattern formation, functioning as a cofactor for the Hox proteins Deformed (Dfd) and Sex Combs Reduced (Scr) in the gnathocephalic segments (mandibular, maxillary, and labial); this more extensive role was masked by the partially redundant gene, disco-related (disco-r) (Mahaffey et al., 2001). Lack of both Disco and Disco-r causes embryonic lethality and the absence of some larval mouth structures arising from the gnathocephalic segments. The disco gene is also expressed in the antennal and leg imaginal discs and likely has a role in the development of these structures (Lee et al., 1991). The disco gene is located in region 14B of the X chromosome and consists of two exons separated by a single intron. The first exon is untranslated and comprises the majority of the disco 5 UTR. The second exon contains the complete open reading frame (ORF) encoding the 568 amino acid protein. The two C2H2 zinc finger regions, defining the DNA binding domain, are located between amino acids 89 and 149 (Heilig et al., 1991). As tools for further study of Disco function, we generated three UAS-disco constructs—two encoding wildtype proteins and one producing an altered protein like that encoded by the disco null allele. The UAS-disco wild-type construct was generated by the sequential insertion of two disco fragments into the Drosophila transformation vector pUAST (Brand and Perrimon, 1993). A 1,270-bp BglII-XbaI fragment representing the majority of the disco ORF (a subclone from 542.2; Surdej et al., 1990) was inserted into BglII-XbaI digested pUAST. Next, a 473-bp fragment, with an artificial EcoRI site immediately upstream of the initiation codon, was obtained by an EcoRI-BglII digestion of a 1,276-bp PCR product generated with the following primers: Forward primer, 5 GGA TCC GAATTC ATG GAG CAC ATA ATG 3 ; Reverse Primer, 5 ATG TAG GCA CTG TCG CTA TCC 3 . This fragment was inserted into the EcoRI-BglII sites of the initial BglII-XbaI pUAST construct. The final construct includes a 1,743-bp EcoRI-XbaI insert encoding the entire disco ORF with some 3 UTR. The second wild-type construct (further referred to as UAS-disco) and the mutant construct (further referred to as UAS-disco) were generated by PCR amplification of genomic DNA. A 1,704-bp fragment containing the complete disco ORF was generated using primers containing EcoRI sites: Forward primer, 5 CCC ACC ACA GAATTC ATG GAG CAC 3 ; Reverse Primer, 5 CCA TGG ATC GAATTC TGG ACA TTA 3 . After EcoRI digestion, the resulting PCR product was inserted into an EcoRI digested pUAST. For the UAS-disco construct, Oregon R genomic DNA was used as a template, while homozygous disco genomic DNA was used as a template for the mutant UAS-disco construct. This form of the protein contains a single amino acid change— Cysteine 127 is substituted with a Serine—which disrupts the first zinc finger. Prior analysis indicated that this mutant form of the Disco protein is nonfunctional (Heilig et al., 1991; Mahaffey et al., 2001). The sequence and reading frame of all constructs were verified by DNA sequence analysis. Integration of UAS-disco constructs into the Drosophila genome was carried out using P-element-mediated germ-line transformation essentially as described by Rubin and Spradling (1982). Two UAS-disco lines were obtained—a heterozygous line carrying UAS-disco on the second chromosome balanced over CyO (UAS-disco(II)), and a homozygous line with the insert on the

DNA polymerase γ, Pol γ, is the key replicative enzyme in animal mitochondria. The Drosophila enzyme is a heterodimer comprising catalytic and accessory subunits of 125 kDa and 35 kDa, respectively. Both subunits have been cloned and characterized in a variety of model systems, and genetic mutants of the catalytic subunit were first identified in Drosophila , as chemically induced mutations that disrupt larval behavior ( tamas ) . Mutations in the gene encoding the accessory subunit have not yet been described in any organism. Here, we report the consequences of null mutations upon mitochondrial DNA (mtDNA) replication and morphology , cell proliferation, and organismal viability. Mutations in the accessory subunit cause lethality during early pupation, concomitant with loss of mtDNA and mitochondrial mass, and reduced cell proliferation in the central nervous system. Surprisingly, the function of the central nervous system and muscle, as assessed in a locomotion assay, are only marginally affected. This finding is in contrast to our previous findings that disruption in the function of the catalytic subunit causes severe reduction in larval locomotion. We discuss our results in the context of current hypotheses for the function of the accessory subunit in mtDNA replication.

Clathrin-mediated endocytosis is required to recycle synaptic vesicles for fast and efficient neurotransmission. Amphiphysins are thought to be multiprotein adaptors that may contribute to this process by bringing together many of the proteins required for endocytosis. Their in vivo function, however, has yet to be determined. Here, we show that the Drosophila genome encodes a single amphiphysin gene that is broadly expressed during development. We also show that, unlike its vertebrate counterparts, Drosophila Amphiphysin is enriched postsynaptically at the larval neuromuscular junction. To determine the role of Drosophila Amphiphysin, we also generated null mutants which are viable but give rise to larvae and adults with pronounced locomotory defects. Surprisingly, the locomotory defects cannot be accounted for by alterations in the morphology or physiology of the neuromuscular junction. Moreover, using stimulus protocols designed to test endocytosis under moderate and extreme vesicle cycling, we could not detect any defect in the neuromuscular junction of the amphiphysin mutant. Taken together, our findings suggest that Amphiphysin is not required for viability, nor is it absolutely required for clathrin-mediated endocytosis. However, Drosophila Amphiphysin function is required in both larvae and adults for normal locomotion.

The Drosophila disconnected (disco) gene is required for the formation of appropriate connections between the larval optic nerve and its target cells in the brain. The disco gene encodes a nuclear protein with two zinc fingers, which suggests that the gene product is a transcription factor. Here, we present data supporting this notion. We find that disco expression in the optic lobe primordium, a group of cells contacted by the developing optic nerve, depends on an autoregulatory feedback loop. We show that wild-type disco function is required for maintenance of disco mRNA and protein expression in the developing optic lobe. In addition, we demonstrate that ubiquitous Disco activity supplied by a heat-inducible gene construct activates expression from the endogenous disco gene specifically in the optic lobe primordium. Consistent with a role of Disco as a transcriptional regulatory protein, we show that portions of the Disco protein are capable of activating the transcription of reporter constructs in a heterologous system. Moreover, we find that the zinc finger portion of Disco binds in vitro to sequences located near the disco transcription unit, suggesting that Disco autoregulates its transcription in the optic lobe primordium by direct binding to a regulatory element in its own promoter.

SUMMARYInsects excrete a wide variety of toxins via the Malpighian (renal) tubules. Previous studies have implicated three transporters in the secretion of the organic anion (OA) methotrexate (MTX) by the Drosophila Malpighian tubule: Drosophila multidrug resistance-associated protein (dMRP, CG6214), a multidrug efflux transporter (MET, CG30344), and an organic anion transporting polypeptide 58Dc (OATP58Dc, CG3380). RNA interference (RNAi) knockdown and P-element insertion mutation of single OA transporter genes were used to evaluate the importance of these three putative transporters in the secretion of MTX by the Malpighian tubules of Drosophila melanogaster. A major finding is that genetic knockdown of a single OA transporter gene leads to reductions in the expression of at least one other OA transporter gene and in secretion of MTX by Malpighian tubules isolated from flies reared on a standard diet. The pattern of changes indicates that decreases in MTX secretion do not correspond to decreases in dMRP expression in all of the RNAi lines. Genetic knockdown of a single OA transporter gene also alters the extent of upregulation of multiple OA transporter genes in the tubules in response to dietary MTX. Knockdown of dMRP is associated with a decrease in MET expression but an increase in OATP expression when flies are reared on MTX-enriched diet. Our results indicate that dMRP and MET are not the dominant MTX transporters in the tubules when flies are reared on MTX-enriched diets. At least one additional transporter, and possibly OATP, are required for MTX secretion. The implications of our results for studies using genetic knockdown techniques to identify OA transporters in whole tissues such as Malpighian tubules are discussed.

Mutations in the disco (disconnected) gene prevent the establishment of stable connections between the larval optic nerves, the Bolwig's nerves, and their target cells in the brain during embryonic development. The failure of this initial connection is associated with aberrant development of the optic lobes which are largely degenerate in the mutant adult fly. In order to understand the role of disco in establishing this connection, we isolated and characterized the disco gene. A 22 kb DNA fragment can completely rescue the mutant phenotype. A single transcript, 2.9 kb in length, is found in this region and is expressed throughout development of the fly. We determined the nucleotide sequence of the disco gene to be unique when compared with sequences in a number of databases. The predicted amino acid sequence contains a region with similarity to the consensus established for the zinc finger motif. Mobilization of a P-element inserted near the gene resulted in the deletion of the 5' end of the gene and produced flies indistinguishable from those carrying the disco allele.

Menin, the product of the multiple endocrine neoplasia type I gene, has been implicated in several biological processes, including the control of gene expression and apoptosis, the modulation of mitogen-activated protein kinase pathways, and DNA damage sensing or repair. In this study, we have investigated the function of menin in the model organism Drosophila melanogaster. We show that Drosophila lines overexpressing menin or an RNA interference for this gene develop normally but are impaired in their response to several stresses, including heat shock, hypoxia, hyperosmolarity and oxidative stress. In the embryo subjected to heat shock, this impairment was characterized by a high degree of developmental arrest and lethality. The overexpression of menin enhanced the expression of HSP70 in embryos and interfered with its down-regulation during recovery at the normal temperature. In contrast, the inhibition of menin with RNA interference reduced the induction of HSP70 and blocked the activation of HSP23 upon heat shock, Menin was recruited to the Hsp70 promoter upon heat shock and menin overexpression stimulated the activity of this promoter in embryos. A 70-kDa inducible form of menin was expressed in response to heat shock, indicating that menin is also regulated in conditions of stress. The induction of HSP70 and HSP23 was markedly reduced or absent in mutant embryos harboring a deletion of the menin gene. These embryos, which did not express the heat shock-inducible form of menin, were also hypersensitive to various conditions of stress. These results suggest a novel role for menin in the control of the stress response and in processes associated with the maintenance of protein integrity.

From a screen of pupal lethal lines of Drosophila melanogaster we identified a mutant strain that displayed a reproducible reduction in the larval response to light. Moreover, this mutant strain showed defects in the development of the adult visual system and failure to undergo behavioral changes characteristic of the wandering stage. The foraging third instar larvae remained in the food substrate for a prolonged period and died at or just before pupariation. Using a new assay for individual larval photobehavior we determined that the lack of response to light in these mutants was due to a primary deficit in locomotion. The mutation responsible for these phenotypes was mapped to the lethal complementation group l(2)34Dc, which we renamed tamas (translated from Sanskrit as “dark inertia”). Sequencing of mutant alleles demonstrated that tamas codes for the mitochondrial DNA polymerase catalytic subunit (DNApol-γ125).

The embryonic lethal abnormal visual system (elav) locus in Drosophila melanogaster, a vital gene mapping within the 1B5-1B9 region of the X-chromosome has been cloned and analysed. Previous developmental analyses have shown that in addition to the embryonic requirement there is a post-embryonic requirement for elav function in the cells of the visual system. A DNA segment containing elav+ function was defined through germ line transformation experiments. This region encodes three embryonic poly(A)+ RNAs and two adult transcripts which are preferentially expressed in the head. In situ hybridization experiments clearly demonstrate that the embryonic expression of elav is restricted to the nervous system.

The larval visual system in the fruitfly Drosophila melanogaster consists of two bilateral clusters of 12 photoreceptor cells. These neurons send their axons in a fascicle, the Bolwig's nerve, toward the target area in the ventral lateral region of the brain hemispheres. We describe the development of a serotonergic arborization originating in the central brain found in the larval optic center in association with the larval optic nerve. This arborization is formed by processes from larval neurons born during embryogenesis. However, these neuronal processes do not reach their final destination, the larval optic center, until late in larval development. Using mutations that disrupt the connectivity and/or development of the larval photoreceptor cells, as well as mosaic analysis, we demonstrate that the innervation of the larval optic center by this serotonergic arborization depends upon contact with the larval optic nerve.