Drosophila mushroom body mutants are deficient in olfactory learning

Two Drosophila mutants are described in which the connections between the input to and the output from the mushroom bodies is largely interrupted. In all forms of the flies (larva, imago, male, female) showing the structural defect, olfactory conditioning is impaired. Learning is completely abolished when electroshock is used as reinforcement and partially suppressed in reward learning with sucrose. No influence of the mushroom body defect on the perception of the conditioning stimuli or on spontaneous olfactory behavior is observed. The defect seems not to impair learning of color discrimination tasks or operant learning involving visual cues.     

EGFR and wingless signaling pathways interact to specify the ocellar pattern in Drosophila

This report shows that the Wingless signaling pathway regulates the expression of epidermal growth factor receptor (EGFR) in Drosophila. This pathway specifies cell fate during head development. Blocking wingless signaling upregulates Drosophila epidermal growth factor receptor (DER) activity and expands its expression domains in the eye primordium. Moreover, ectopic expression of wingless inhibits DER signaling and dramatically restricts its expression domain. This study suggests a novel role of wingless in specifying the Drosophila dorsal head via blocking vein expression in this region.     

Cell identity and sexual development in Cryptococcus neoformans are controlled by the mating-type-specific homeodomain protein Sxi1alpha

Virulence in the human fungal pathogen Cryptococcus neoformans is associated with the alpha mating type. Studies to identify the properties of alpha cells that enhance pathogenesis have led to the identification of a mating-type locus of unusually large size and distinct architecture. Here, we demonstrate that the previously identified MATalpha components are insufficient to regulate sexual differentiation, and we identify a novel alpha-specific regulator, SXI1alpha. Our data show that SXI1alpha establishes alpha cell identity and controls progression through the sexual cycle, and we discover that ectopic expression of SXI1alpha in a cells is sufficient to drive a/alpha sexual development. SXI1alpha is the first example of a key regulator of cell identity and sexual differentiation in C. neoformans, and its identification and characterization lead to a new model of how cell fate and the sexual cycle are controlled in C. neoformans.     

Cytoskeleton proteins are modulators of mutant tau-induced neurodegeneration in Drosophila

Tauopathies, including Alzheimer's disease and fronto-temporal dementia and parkinsonism linked to chromosome 17 (FTDP-17), are a group of neurodegenerative disorders characterized by the presence of intraneuronal filamentous inclusions of aberrantly phosphorylated-tau. Tau is a neuronal microtubule-associated protein involved in microtubule assembly and stabilization. Currently, the molecular mechanisms underlying tau-mediated cellular toxicity remain elusive. To address the determinants of tau neurotoxicity, we first characterized the cellular alterations resulting from the over-expression of a mutant form of human tau associated with FTDP-17 (tau V337M) in Drosophila. We found that the over-expression of tau V337M, in Drosophila larval motor neurons, induced disruption of the microtubular network at presynaptic nerve terminals and changes in neuromuscular junctions morphological features. Secondly, we performed a misexpression screen to identify genetic modifiers of the tau V337M-mediated rough eye phenotype. The screening of 1250 mutant Drosophila lines allowed us to identify several components of the cytoskeleton, and particularly from the actin network, as specific modifiers of tau V337M-induced neurodegeneration. Furthermore, we found that numerous tau modulators identified in our screen were involved in the maintenance of synaptic function. Taken together, these findings suggest that disruption of the microtubule network in presynaptic nerve terminals could constitute early events in the pathological process leading to synaptic dysfunction in tau V337M pathology.     

The embryology of body wall closure: relevance to gastroschisis and other ventral body wall defects

During the 3rd and 4th weeks post-fertilization (5 and 6 weeks from the last normal menstrual period [LNMP]), the human embryo is transformed from a flat disc-shaped organism into the classic shape of an embryo in the "fetal" position. This change is effected by simultaneously rolling the top layer of the disc, the ectoderm, into the neural tube and the bottom layers of the disc, the endoderm and mesoderm, into the gut tube and body wall, respectively. In this manner, the flat disc is transformed into two tubes, one dorsal to the other, surrounded by supporting structures in the body wall. If closure of the neural tube fails, then neural tube defects (NTDs), such as anencephaly and spina bifida, occur; if closure of the ventral body wall fails, then ventral body wall defects, such as ectopia cordis, gastroschisis, and bladder and cloacal exstrophy, occur. Interestingly, no known closure defects have been described for the gut tube. Note, however, that all of the closure defects that do occur have their origins early in gestation during the third and fourth weeks of development.     

Organ identity specification factor WGE localizes to the histone locus body and regulates histone expression to ensure genomic stability in Drosophila

Over‐expression of Winged‐Eye (WGE) in the Drosophila eye imaginal disc induces an eye‐to‐wing transformation. Endogenous WGE is required for organ development, and wge‐deficient mutants exhibit growth arrest at the larval stage, suggesting that WGE is critical for normal growth. The function of WGE, however, remains unclear. Here, we analyzed the subcellular localization of WGE to gain insight into its endogenous function. Immunostaining showed that WGE localized to specific nuclear foci called the histone locus body (HLB), an evolutionarily conserved nuclear body required for S phase‐specific histone mRNA production. Histone mRNA levels and protein levels in cytosolic fractions were aberrantly up‐regulated in wge mutant larva, suggesting a role for WGE in regulating histone gene expression. Genetic analyses showed that wge suppresses position‐effect variegation, and that WGE and a HLB component Mute appears to be synergistically involved in heterochromatin formation. Further supporting a role in chromatin regulation, wge‐deficient mutants showed derepression of retrotransposons and increased γH2Av signals, a DNA damage marker. These findings suggest that WGE is a component of HLB in Drosophila with a role in heterochromatin formation and transposon silencing. We propose that WGE at HLB contributes to genomic stability and development by regulating heterochromatin structure via histone gene regulation.     

Neisseria meningitidis RTX proteins are not required for virulence in infant rats

RTX cytotoxins play an important role in virulence of numerous gram-negative pathogens. Unexpectedly, however, we show here that the RTX proteins of Neisseria meningitidis are dispensable for virulence in the infant rat model of infection.     

The translocated Salmonella effector proteins SseF and SseG interact and are required to establish an intracellular replication niche

The facultative intracellular pathogen Salmonella enterica causes a variety of diseases, including gastroenteritis and typhoid fever. Inside epithelial cells, Salmonella replicates in vacuoles, which localize in the perinuclear area in close proximity to the Golgi apparatus. Among the effector proteins translocated by the Salmonella pathogenicity island 2-encoded type III secretion system, SifA and SseG have been shown necessary but not sufficient to ensure the intracellular positioning of Salmonella vacuoles. Hence, we have investigated the involvement of other secreted effector proteins in this process. Here we show that SseF interacts functionally and physically with SseG but not SifA and is also required for the perinuclear localization of Salmonella vacuoles. The observations show that the intracellular positioning of Salmonella vacuoles is a complex phenomenon resulting from the combined action of several effector proteins.     

Xenopus BTBD6 and its Drosophila homologue lute are required for neuronal development

BBP proteins constitute a subclass of CUL3 interacting BTB proteins whose in vivo function remains unknown. Here, we show that the Xenopus BBP gene BTBD6 and the single Drosophila homologue of mammalian BBP genes lute are strongly expressed in the developing nervous system. In Xenopus, BTBD6 expression responds positively to proneural and negatively to neurogenic gene overexpression. Knockdown of BTBD6 in Xenopus or loss of Drosophila lute result in embryos with strong defects in late neuronal markers and strongly reduced and disorganized axons while early neural development is unaffected. XBTBD6 knockdown in Xenopus also affects muscle development. Together, these data indicate that BTBD6/lute is required for proper embryogenesis and plays an essential evolutionary conserved role during neuronal development.     

Mutations in the Drosophila gene extradenticle affect the way specific homeo domain proteins regulate segmental identity

We characterized a gene, extradenticle, which seems to interact with a specific subset of Drosophila homeo domain proteins, possibly affecting their target specificity. This interpretation is based on an examination of the zygotic and maternal effect phenotypes of extradenticle mutations. In embryos with reduced levels of extradenticle gene product, anterior and posterior segmental transformations occur. Segmental identity in Drosophila is mediated by the products of the Antennapedia and bithorax complexes. These homeo domain proteins are thought to regulate different target genes specifically in each segment, resulting in different morphologies. extradenticle alters segmental identity without affecting the pattern of expression of homeotic genes. Genetic tests demonstrate that in extradenticle mutants, the homeotic proteins are functional and act in their normal segmental domains, yet segmental identities are altered. Even when homeotic proteins are ectopically expressed under the control of a heterologous promoter, extradenticle mutations affect their consequences. Thus, in the absence of sufficient extradenticle product, altered segmental morphology results from alteration of the functional consequences of specific homeo domain proteins, possibly through alterations in their target gene specificity. extradenticle is also expressed maternally. Complete removal of extradenticle, maternally and zygotically, leads to specific alterations in segmentation, many of which result from failure to maintain the expression of the homeo domain protein engrailed.     

Endocytic Rab proteins are required for hepatitis C virus replication complex formation

During infection, hepatitis C virus (HCV) NS4B protein remodels host membranes to form HCV replication complexes (RC) which appear as foci under fluorescence microscopy (FM). To understand the role of Rab proteins in forming NS4B foci, cells expressing the HCV replicon were examined biochemically and via FM. First, we show that an isolated NS4B-bound subcellular fraction is competent for HCV RNA synthesis. Further, this fraction is differentially enriched in Rab1, 2, 5, 6 and 7. However, when examined via FM, NS4B foci appear to be selectively associated with Rab5 and Rab7 proteins. Additionally, dominant negative (DN) Rab6 expression impairs Rab5 recruitment into NS4B foci. Further, silencing of Rab5 or Rab7 resulted in a significant decrease in HCV genome replication. Finally, expression of DN Rab5 or Rab7 led to a reticular NS4B subcellular distribution, suggesting that endocytic proteins Rab5 and Rab7, but not Rab11, may facilitate NS4B foci formation.     

Changes in protein turnover after heat shock are related to accumulation of abnormal proteins in aging Drosophila melanogaster

Adult Drosophila melanogaster kept at 24 degrees C show a progressive decline in the synthesis and degradation of proteins with age. After exposure of young, 7-10 days old flies to 20 min of heat shock at 37 degrees C, the incorporation of [35S]-methionine into trichloroacetic acid precipitable proteins decreases to more than 60% of that observed in non-stressed flies. This decrease is also accompanied by a lower protein degradation rate. In contrast, the same stress in old, 49 days old insects results in a 3-fold increase in protein synthesis as compared to either non-heat shocked senescent flies or to young heat-shocked flies. The older flies also have faster protein turnover than unshocked controls. An effect similar to that observed in senescent Drosophila also occurs in young flies that have been fed canavanine, an arginine analogue, before and during heat shock. These results suggest that an age dependent accumulation of abnormal proteins may be responsible for the changes in protein turnover observed in the heat-shocked old flies.     

In search of proteins that are important for synaptic functions in Drosophila visual system

This is the second of two reviews that include some of the studies we, members of the Pak laboratory and collaborators, did from 2000 to 2010 on the mutants that affect synaptic transmission in the Drosophila visual system. Of the five mutants we discuss, two turned out to also play roles in the larval neuromuscular junction. This review complements the one on phototransduction to give a fairly complete account of what we focused on during the 10-year period, although we also did some studies on photoreceptor degeneration in the early part of the decade. Besides showing the power of using a genetic approach to the study of synaptic transmission, the review contains some unexpected results that illustrate the serendipitous nature of research.