The golgi comprises a paired stack that is separated at G2 by modulation of the actin cytoskeleton through Abi and Scar/WAVE.

During the cell cycle, the Golgi, like other organelles, has to be duplicated in mass and number to ensure its correct segregation between the two daughter cells. It remains unclear, however, when and how this occurs. Here we show that in Drosophila S2 cells, the Golgi likely duplicates in mass to form a paired structure during G1/S phase and remains so until G2 when the two stacks separate, ready for entry into mitosis. We show that pairing requires an intact actin cytoskeleton which in turn depends on Abi/Scar but not WASP. This actin-dependent pairing is not limited to flies but also occurs in mammalian cells. We further show that preventing the Golgi stack separation at G2 blocks entry into mitosis, suggesting that this paired organization is part of the mitotic checkpoint, similar to what has been proposed in mammalian cells.

Control of antagonistic components of the hedgehog signaling pathway by microRNAs in Drosophila.

Hedgehog (Hh) signaling is critical for many developmental processes and for the genesis of diverse cancers. Hh signaling comprises a series of negative regulatory steps, from Hh reception to gene transcription output. We previously showed that stability of antagonistic regulatory proteins, including the coreceptor Smoothened (Smo), the kinesin-like Costal-2 (Cos2), and the kinase Fused (Fu), is affected by Hh signaling activation. Here, we show that the level of these three proteins is also regulated by a microRNA cluster. Indeed, the overexpression of this cluster and resulting microRNA regulation of the 3'-UTRs of smo, cos2, and fu mRNA decreases the levels of the three proteins and activates the pathway. Further, the loss of the microRNA cluster or of Dicer function modifies the 3'-UTR regulation of smo and cos2 mRNA, confirming that the mRNAs encoding the different Hh components are physiological targets of microRNAs. Nevertheless, an absence of neither the microRNA cluster nor of Dicer activity creates an hh-like phenotype, possibly due to dose compensation between the different antagonistic targets. This study reveals that a single signaling pathway can be targeted at multiple levels by the same microRNAs.

The cis-Golgi Drosophila GMAP has a role in anterograde transport and Golgi organization in vivo, similar to its mammalian ortholog in tissue culture cells.

The Golgi microtubule-associated protein of 210kDa (GMAP-210) has been shown to play a role in the assembly and maintenance of the Golgi apparatus in mammalian tissue culture cells. To develop a genetic model to study the function of GMAP-210 in vivo, we identified its closest relative in Drosophila melanogaster, dGMAP. We show that the dGMAP gene encodes two alternatively spliced transcripts, only one of which is translated into a protein product. To gain insight into the role of dGMAP, we generated a polyclonal antibody and investigated the protein distribution during development. This gene is ubiquitously expressed during embryonic and larval development with the highest level in polar cells, gut and salivary glands. We further show that dGMAP is present in the Golgi apparatus, and using electron microscopy of salivary glands, we observed a preferential localization at the cis side and at the rims of the Golgi stacks. Finally, we demonstrate that overexpression of dGMAP in salivary glands impairs Golgi architecture and function, whereas RNAi-mediated depletion of dGMAP does not induce any structural modification of the Golgi apparatus, and anterograde transport is seemingly unaffected. Altogether our results suggest that dGMAP is the ortholog of mammalian GMAP-210.

Dopamine and octopamine differentiate between aversive and appetitive olfactory memories in Drosophila.

The catecholamines play a major role in the regulation of behavior. Here we investigate, in the fly Drosophila melanogaster, the role of dopamine and octopamine (the presumed arthropod homolog of norepinephrine) during the formation of appetitive and aversive olfactory memories. We find that for the formation of both types of memories, cAMP signaling is necessary and sufficient within the same subpopulation of mushroom-body intrinsic neurons. On the other hand, memory formation can be distinguished by the requirement for different catecholamines, dopamine for aversive and octopamine for appetitive conditioning. Our results suggest that in associative conditioning, different memories are formed of the same odor under different circumstances, and that they are linked to the respective motivational systems by their specific modulatory pathways.

Cell-type-specific limitation on in vivo serotonin storage following ectopic expression of the Drosophila serotonin transporter, dSERT.

The synaptic machinery for neurotransmitter storage is cell-type specific. Although most elements of biosynthesis and transport have been identified, it remains unclear whether additional factors may be required to maintain this specificity. The Drosophila serotonin transporter (dSERT) is normally expressed exclusively in serotonin (5-HT) neurons in the CNS. Here we examine the effects of ectopic transcriptional expression of dSERT in the Drosophila larval CNS. We find a surprising limitation on 5-HT storage following ectopic expression of dSERT and green fluorescence protein-tagged dSERT (GFP-dSERT). When dSERT transcription is driven ectopically in the CNS, 5-HT is detectable only in 5-HT, dopamine (DA), and a very limited number of additional neurons. Addition of exogenous 5-HT does not dramatically broaden neuronal storage sites, so this limitation is only partly due to restricted intercellular diffusion of 5-HT. Furthermore, this limitation is not due to gross mislocalization of dSERT, because cells lacking or containing 5-HT show similar levels and subcellular distribution of GFP-dSERT protein, nor is it due to lack of the vesicular transporter, dVMAT. These data suggest that a small number of neurons selectively express factor(s) required for 5-HT storage, and potentially for function of dSERT.

Tissue-specific developmental requirements of Drosophila tyrosine hydroxylase isoforms.

Drosophila tyrosine hydroxylase (DTH) is a key enzyme in dopamine (DA) biosynthesis, which is expressed in neural and hypodermal DA-synthesizing cells. We previously reported that two DTH isoforms are produced in flies through tissue-specific alternative splicing that show distinct regulatory properties. We have now selectively expressed each DTH isoform in vivo in a pale (ple, i.e., DTH-deficient) mutant background. We show that the embryonic lethality of ple can be rescued by expression of the hypodermal, but not the neural, DTH isoform in all DA cells, indicating that the hypoderm- isoform is absolutely required for cuticle biosynthesis and survival in Drosophila. In addition, we report new observations on the consequences of DTH overexpression in the CNS and hypoderm. Our results provide evidence that tissue-specific alternative splicing of the DTH gene is a vital process in Drosophila development.

Tissue-specific developmental requirements of Drosophila tyrosine hydroxylase isoforms.

Drosophila tyrosine hydroxylase (DTH) is a key enzyme in dopamine (DA) biosynthesis, which is expressed in neural and hypodermal DA-synthesizing cells. We previously reported that two DTH isoforms are produced in flies through tissue-specific alternative splicing that show distinct regulatory properties. We have now selectively expressed each DTH isoform in vivo in a pale (ple, i.e., DTH-deficient) mutant background. We show that the embryonic lethality of ple can be rescued by expression of the hypodermal, but not the neural, DTH isoform in all DA cells, indicating that the hypoderm- isoform is absolutely required for cuticle biosynthesis and survival in Drosophila. In addition, we report new observations on the consequences of DTH overexpression in the CNS and hypoderm. Our results provide evidence that tissue-specific alternative splicing of the DTH gene is a vital process in Drosophila development.

Targeted gene expression in Drosophila dopaminergic cells using regulatory sequences from tyrosine hydroxylase.

Dopamine (DA) is the only catecholaminergic neurotransmitter in the fruit fly Drosophila melanogaster. Dopaminergic neurons have been identified in the larval and adult central nervous system (CNS) in Drosophila and other insects, but no specific genetic tool was available to study their development, function, and degeneration in vivo. In Drosophila as in vertebrates, the rate-limiting step in DA biosynthesis is catalyzed by the enzyme tyrosine hydroxylase (TH). The Drosophila TH gene (DTH) is specifically expressed in all dopaminergic cells and the corresponding mutant, pale (ple), is embryonic lethal. We have performed ple rescue experiments with modified DTH transgenes. Our results indicate that partially redundant regulatory elements located in DTH introns are required for proper expression of this gene in the CNS. Based on this study, we generated a GAL4 driver transgene, TH-GAL4, containing regulatory sequences from the DTH 5' flanking and downstream coding regions. TH-GAL4 specifically expresses in dopaminergic cells in embryos, larval CNS, and adult brain when introduced into the Drosophila genome. As a first application of this driver, we observed that in vivo inhibition of DA release induces a striking hyperexcitability behavior in adult flies. We propose that TH-GAL4 will be useful for studies of the role of DA in behavior and disease models in Drosophila.