Identification of Vangl2 and Scrb1 as planar polarity genes in mammals

In mammals, an example of planar cell polarity (PCP) is the uniform orientation of the hair cell stereociliary bundles within the cochlea. The PCP pathway of Drosophila refers to a conserved signalling pathway that regulates the coordinated orientation of cells or structures within the plane of an epithelium. Here we show that a mutation in Vangl2, a mammalian homologue of the Drosophila PCP gene Strabismus/Van Gogh, results in significant disruptions in the polarization of stereociliary bundles in mouse cochlea as a result of defects in the direction of movement and/or anchoring of the kinocilium within each hair cell. Similar, but less severe, defects are observed in animals containing a mutation in the LAP protein family gene Scrb1 (homologous with Drosophila scribble). Polarization defects in animals heterozygous for Vangl2 and Scrb1 are comparable with Vangl2 homozygotes, demonstrating genetic interactions between these genes in the regulation of PCP in mammals. These results demonstrate a role for the PCP pathway in planar polarization in mammals, and identify Scrb1 as a PCP gene.     

A subset of dopamine neurons signals reward for odour memory in Drosophila

Animals approach stimuli that predict a pleasant outcome. After the paired presentation of an odour and a reward, Drosophila melanogaster can develop a conditioned approach towards that odour. Despite recent advances in understanding the neural circuits for associative memory and appetitive motivation, the cellular mechanisms for reward processing in the fly brain are unknown. Here we show that a group of dopamine neurons in the protocerebral anterior medial (PAM) cluster signals sugar reward by transient activation and inactivation of target neurons in intact behaving flies. These dopamine neurons are selectively required for the reinforcing property of, but not a reflexive response to, the sugar stimulus. In vivo calcium imaging revealed that these neurons are activated by sugar ingestion and the activation is increased on starvation. The output sites of the PAM neurons are mainly localized to the medial lobes of the mushroom bodies (MBs), where appetitive olfactory associative memory is formed. We therefore propose that the PAM cluster neurons endow a positive predictive value to the odour in the MBs. Dopamine in insects is known to mediate aversive reinforcement signals. Our results highlight the cellular specificity underlying the various roles of dopamine and the importance of spatially segregated local circuits within the MBs.     

Paracrine Wingless signalling controls self-renewal of Drosophila intestinal stem cells

In the Drosophila midgut, multipotent intestinal stem cells (ISCs) that are scattered along the epithelial basement membrane maintain tissue homeostasis by their ability to steadily produce daughters that differentiate into either enterocytes or enteroendocrine cells, depending on the levels of Notch activity. However, the mechanisms controlling ISC self-renewal remain elusive. Here we show that a canonical Wnt signalling pathway controls ISC self-renewal. The ligand Wingless (Wg) is specifically expressed in the circular muscles next to ISCs, separated by a thin layer of basement membrane. Reduced function of wg causes ISC quiescence and differentiation, whereas wg overexpression produces excessive ISC-like cells that express high levels of the Notch ligand, Delta. Clonal analysis shows that the main downstream components of the Wg pathway, including Frizzled, Dishevelled and Armadillo, are autonomously required for ISC self-renewal. Furthermore, epistatic analysis suggests that Notch acts downstream of the Wg pathway and a hierarchy of Wg/Notch signalling pathways controls the balance between self-renewal and differentiation of ISCs. These data suggest that the underlying circular muscle constitutes the ISC niche, which produce Wg signals that act directly on ISCs to promote ISC self-renewal. This study demonstrates markedly conserved mechanisms regulating ISCs from Drosophila to mammals. The identification of the Drosophila ISC niche and the principal self-renewal signal will facilitate further understanding of intestinal homeostasis control and tumorigenesis.     

Shaggy/GSK3 antagonizes Hedgehog signalling by regulating Cubitus interruptus

The Drosophila protein Shaggy (Sgg, also known as Zeste-white3, Zw3) and its vertebrate orthologue glycogen synthase kinase 3 (GSK3) are inhibitory components of the Wingless (Wg) and Wnt pathways. Here we show that Sgg is also a negative regulator in the Hedgehog (Hh) pathway. In Drosophila, Hh acts both by blocking the proteolytic processing of full-length Cubitus interruptus, Ci (Ci155), to generate a truncated repressor form (Ci75), and by stimulating the activity of accumulated Ci155 (refs 2-6). Loss of sgg gene function results in a cell-autonomous accumulation of high levels of Ci155 and the ectopic expression of Hh-responsive genes including decapentaplegic (dpp) and wg. Simultaneous removal of sgg and Suppressor of fused, Su(fu), results in wing duplications similar to those caused by ectopic Hh signalling. Ci is phosphorylated by GSK3 after a primed phosphorylation by protein kinase A (PKA), and mutating GSK3-phosphorylation sites in Ci blocks its processing and prevents the production of the repressor form. We propose that Sgg/GSK3 acts in conjunction with PKA to cause hyperphosphorylation of Ci, which targets it for proteolytic processing, and that Hh opposes Ci proteolysis by promoting its dephosphorylation.     

Sleep in Drosophila is regulated by adult mushroom bodies

Sleep is one of the few major whole-organ phenomena for which no function and no underlying mechanism have been conclusively demonstrated. Sleep could result from global changes in the brain during wakefulness or it could be regulated by specific loci that recruit the rest of the brain into the electrical and metabolic states characteristic of sleep. Here we address this issue by exploiting the genetic tractability of the fruitfly, Drosophila melanogaster, which exhibits the hallmarks of vertebrate sleep. We show that large changes in sleep are achieved by spatial and temporal enhancement of cyclic-AMP-dependent protein kinase (PKA) activity specifically in the adult mushroom bodies of Drosophila. Other manipulations of the mushroom bodies, such as electrical silencing, increasing excitation or ablation, also alter sleep. These results link sleep regulation to an anatomical locus known to be involved in learning and memory.     

A product of the prune locus of Drosophila is similar to mammalian GTPase-activating protein.

The X-linked prune (pn) eye-colour mutation of Drosophila melanogaster has a highly specific, complementary lethal interaction with the conditional dominant Killer of prune (awdK-pn) mutation. Although awdK-pn flies have no apparent phenotype on their own, pn awdK-pn double mutants die as second or third larval instars. The awd locus encodes a nucleoside diphosphate kinase, an enzyme that catalyses the transfer of high-energy phosphate bonds between nucleoside diphosphates and nucleoside triphosphates, which is essential for the normal development of Drosophila. Analysis of the pn locus has suggested that the complementary DNA, TcD37, encodes a putative pn+ product. Here we report the nucleotide sequence of TcD37 and the similarity of its deduced protein product to the catalytic domain of mammalian GTPase-activating proteins (GAPs); GAPs stimulate the GTPase activity of Ras (ref. 6), which are plasma membrane-bound proteins involved in the regulation of cell proliferation and differentiation. These results suggest that the Drosophila TcD37 protein participates in a biochemical pathway similar to that of Ras and GAPs in mammals and yeast. We propose that the interaction between pn and awd is due to a neomorphic mutation that enhances the ability of AwdK-pn nucleoside diphosphate kinase to induce a regulatory GTPase into a GTP-bound 'on' state, whereas Pn modulates the activity of this GTPase either by switching it to a GDP-bound 'off' state or by interfering with its effector function.     

Hedgehog signalling activity of Smoothened requires phosphorylation by protein kinase A and casein kinase I

The Hedgehog (Hh) family of secreted proteins governs cell growth and patterning in animal development. The Hh signal is transduced by the seven-transmembrane protein Smoothened (Smo); however, the mechanism by which Smo is regulated remains largely unknown. Here we show that protein kinase A (PKA) and casein kinase I (CKI) regulate Smo cell-surface accumulation and activity in response to Hh. Blocking PKA or CKI activity in the Drosophila wing disc prevents Hh-induced Smo accumulation and attenuates pathway activity, whereas increasing PKA activity promotes Smo accumulation and pathway activation. We show that PKA and CKI phosphorylate Smo at several sites, and that phosphorylation-deficient forms of Smo fail to accumulate on the cell surface and are unable to transduce the Hh signal. Conversely, phosphorylation-mimicking Smo variants show constitutive cell-surface expression and signalling activity. Furthermore, we find that the levels of Smo cell-surface expression and activity correlate with its levels of phosphorylation. Our data indicate that Hh induces progressive Smo phosphorylation by PKA and CKI, leading to elevation of Smo cell-surface levels and signalling activity.     

Axon routing across the midline controlled by the Drosophila Derailed receptor

In nervous systems with symmetry about the midline, many neurons project axons from one side to the other. Although several of the components controlling midline crossing have been identified, little is known about how axons choose the appropriate pathway when crossing. For example, in the Drosophila embryo axons cross the midline in one of two distinct tracts, the anterior or posterior commissure (AC or PC, respec tively). Here we show that the Derailed (Drl) receptor tyrosine kinase is expressed by neurons that project in the AC, and that in the absence of Drl such neurons often project abnormally into the PC. Conversely, misexpression of Drl in PC neurons forces them to cross in the AC. The behaviour of Drl-misexpressing neurons and the in vivo binding pattern of a soluble Drl receptor probe indicate that Drl acts as a guidance receptor for a repellent ligand present in the PC. Our results show that Drl is a novel component in the control of midline crossing.     

Mitochondrial dysfunction in Drosophila PINK1 mutants is complemented by parkin

Autosomal recessive juvenile parkinsonism (AR-JP) is an early-onset form of Parkinson's disease characterized by motor disturbances and dopaminergic neurodegeneration. To address its underlying molecular pathogenesis, we generated and characterized loss-of-function mutants of Drosophila PTEN-induced putative kinase 1 (PINK1), a novel AR-JP-linked gene. Here, we show that PINK1 mutants exhibit indirect flight muscle and dopaminergic neuronal degeneration accompanied by locomotive defects. Furthermore, transmission electron microscopy analysis and a rescue experiment with Drosophila Bcl-2 demonstrated that mitochondrial dysfunction accounts for the degenerative changes in all phenotypes of PINK1 mutants. Notably, we also found that PINK1 mutants share marked phenotypic similarities with parkin mutants. Transgenic expression of Parkin markedly ameliorated all PINK1 loss-of-function phenotypes, but not vice versa, suggesting that Parkin functions downstream of PINK1. Taken together, our genetic evidence clearly establishes that Parkin and PINK1 act in a common pathway in maintaining mitochondrial integrity and function in both muscles and dopaminergic neurons.     

Coupled oscillators control morning and evening locomotor behaviour of Drosophila

Daily rhythms of physiology and behaviour are precisely timed by an endogenous circadian clock. These include separate bouts of morning and evening activity, characteristic of Drosophila melanogaster and many other taxa, including mammals. Whereas multiple oscillators have long been proposed to orchestrate such complex behavioural programmes, their nature and interplay have remained elusive. By using cell-specific ablation, we show that the timing of morning and evening activity in Drosophila derives from two distinct groups of circadian neurons: morning activity from the ventral lateral neurons that express the neuropeptide PDF, and evening activity from another group of cells, including the dorsal lateral neurons. Although the two oscillators can function autonomously, cell-specific rescue experiments with circadian clock mutants indicate that they are functionally coupled.     

The hangover gene defines a stress pathway required for ethanol tolerance development

Repeated alcohol consumption leads to the development of tolerance, simply defined as an acquired resistance to the physiological and behavioural effects of the drug. This tolerance allows increased alcohol consumption, which over time leads to physical dependence and possibly addiction. Previous studies have shown that Drosophila develop ethanol tolerance, with kinetics of acquisition and dissipation that mimic those seen in mammals. This tolerance requires the catecholamine octopamine, the functional analogue of mammalian noradrenaline. Here we describe a new gene, hangover, which is required for normal development of ethanol tolerance. hangover flies are also defective in responses to environmental stressors, such as heat and the free-radical-generating agent paraquat. Using genetic epistasis tests, we show that ethanol tolerance in Drosophila relies on two distinct molecular pathways: a cellular stress pathway defined by hangover, and a parallel pathway requiring octopamine. hangover encodes a large nuclear zinc-finger protein, suggesting a role in nucleic acid binding. There is growing recognition that stress, at both the cellular and systemic levels, contributes to drug- and addiction-related behaviours in mammals. Our studies suggest that this role may be conserved across evolution.     

Visual place learning in Drosophila melanogaster

The ability of insects to learn and navigate to specific locations in the environment has fascinated naturalists for decades. The impressive navigational abilities of ants, bees, wasps and other insects demonstrate that insects are capable of visual place learning, but little is known about the underlying neural circuits that mediate these behaviours. Drosophila melanogaster (common fruit fly) is a powerful model organism for dissecting the neural circuitry underlying complex behaviours, from sensory perception to learning and memory. Drosophila can identify and remember visual features such as size, colour and contour orientation. However, the extent to which they use vision to recall specific locations remains unclear. Here we describe a visual place learning platform and demonstrate that Drosophila are capable of forming and retaining visual place memories to guide selective navigation. By targeted genetic silencing of small subsets of cells in the Drosophila brain, we show that neurons in the ellipsoid body, but not in the mushroom bodies, are necessary for visual place learning. Together, these studies reveal distinct neuroanatomical substrates for spatial versus non-spatial learning, and establish Drosophila as a powerful model for the study of spatial memories.     

Drosophila pink1 is required for mitochondrial function and interacts genetically with parkin

Parkinson's disease is the second most common neurodegenerative disorder and is characterized by the degeneration of dopaminergic neurons in the substantia nigra. Mitochondrial dysfunction has been implicated as an important trigger for Parkinson's disease-like pathogenesis because exposure to environmental mitochondrial toxins leads to Parkinson's disease-like pathology. Recently, multiple genes mediating familial forms of Parkinson's disease have been identified, including PTEN-induced kinase 1 (PINK1; PARK6) and parkin (PARK2), which are also associated with sporadic forms of Parkinson's disease. PINK1 encodes a putative serine/threonine kinase with a mitochondrial targeting sequence. So far, no in vivo studies have been reported for pink1 in any model system. Here we show that removal of Drosophila PINK1 homologue (CG4523; hereafter called pink1) function results in male sterility, apoptotic muscle degeneration, defects in mitochondrial morphology and increased sensitivity to multiple stresses including oxidative stress. Pink1 localizes to mitochondria, and mitochondrial cristae are fragmented in pink1 mutants. Expression of human PINK1 in the Drosophila testes restores male fertility and normal mitochondrial morphology in a portion of pink1 mutants, demonstrating functional conservation between human and Drosophila Pink1. Loss of Drosophila parkin shows phenotypes similar to loss of pink1 function. Notably, overexpression of parkin rescues the male sterility and mitochondrial morphology defects of pink1 mutants, whereas double mutants removing both pink1 and parkin function show muscle phenotypes identical to those observed in either mutant alone. These observations suggest that pink1 and parkin function, at least in part, in the same pathway, with pink1 functioning upstream of parkin. The role of the pink1-parkin pathway in regulating mitochondrial function underscores the importance of mitochondrial dysfunction as a central mechanism of Parkinson's disease pathogenesis.     

Control of neuronal fate by the Drosophila segmentation gene even-skipped

The central nervous system (CNS) contains a remarkable diversity of cell types. The molecular basis for generating this neuronal diversity is poorly understood. Much is known, however, about the regulatory genes which control segmentation and segment identity during early Drosophila embryogenesis. Interestingly, most of the segmentation and homoeotic genes in Drosophila, as well as many of their vertebrate homologues, are expressed during the development of the nervous system (for example, ref. 3). Are these genes involved in specifying the identity of individual neurons during neurogenesis, just as they specify the identity of cells during segmentation? We previously described the CNS expression of the segmentation gene fushi tarazu (ftz) and showed that ftz CNS expression is involved in the determination of an identified neuron. Here we show that another segmentation gene, even-skipped (eve), is expressed in a different but overlapping subset of neurons. Temperature-sensitive inactivation of the eve protein during neurogenesis alters the fate of two of these neurons. Our results indicate that the nuclear protein products of the eve and ftz segmentation genes are components of the mechanism controlling cell fate during neuronal development.     

PTK7/CCK-4 is a novel regulator of planar cell polarity in vertebrates

In addition to the apical-basal polarity pathway operating in epithelial cells, a planar cell polarity (PCP) pathway establishes polarity within the plane of epithelial tissues and is conserved from Drosophila to mammals. In Drosophila, a 'core' group of PCP genes including frizzled (fz), flamingo/starry night, dishevelled (dsh), Van Gogh/strabismus and prickle, function to regulate wing hair, bristle and ommatidial polarity. In vertebrates, the PCP pathway regulates convergent extension movements and neural tube closure, as well as the orientation of stereociliary bundles of sensory hair cells in the inner ear. Here we show that a mutation in the mouse protein tyrosine kinase 7 (PTK7) gene, which encodes an evolutionarily conserved transmembrane protein with tyrosine kinase homology, disrupts neural tube closure and stereociliary bundle orientation, and shows genetic interactions with a mutation in the mouse Van Gogh homologue vangl2. We also show that PTK7 is dynamically localized during hair cell polarization, and that the Xenopus homologue of PTK7 is required for neural convergent extension and neural tube closure. These results identify PTK7 as a novel regulator of PCP in vertebrates.