New reproductive anomalies in fruitless‐mutant Drosophila males: Extreme lengthening of mating durations and infertility correlated with defective serotonergic innervation of reproductive organs

Several features of male reproductive behavior are under the neural control of fruitless (fru) in Drosophila melanogaster. This gene is known to influence courtship steps prior to mating, due to the absence of attempted copulation in the behavioral repertoire of most types of fru‐mutant males. However, certain combinations of fru mutations allow for fertility. By analyzing such matings and their consequences, we uncovered two striking defects: mating times up to four times the normal average duration of copulation; and frequent infertility, regardless of the time of mating by a given transheterozygous fru‐mutant male. The lengthened copulation times may be connected with fru‐induced defects in the formation of a male‐specific abdominal muscle. Production of sperm and certain seminal fluid proteins are normal in these fru mutants. However, analysis of postmating qualities of females that copulated with transheterozygous mutants strongly implied defects in the ability of these males to transfer sperm and seminal fluids. Such abnormalities may be associated with certain serotonergic neurons in the abdominal ganglion in which production of 5HT is regulated by fru. These cells send processes to contractile muscles of the male's internal sex organs; such projection patterns are aberrant in the semifertile fru mutants. Therefore, the reproductive functions regulated by fruitless are expanded in their scope, encompassing not only the earliest stages of courtship behavior along with almost all subsequent steps in the behavioral sequence, but also more than one component of the culminating events. © 2001 John Wiley & Sons, Inc. J Neurobiol 47: 121–149, 2001     

Multiple mechanisms mediate motor neuron migration in the zebrafish hindbrain

The transmembrane protein Van gogh‐like 2 (Vangl2) is a component of the noncanonical Wnt/Planar Cell Polarity (PCP) signaling pathway, and is required for tangential migration of facial branchiomotor neurons (FBMNs) from rhombomere 4 (r4) to r5‐r7 in the vertebrate hindbrain. Since vangl2 is expressed throughout the zebrafish hindbrain, it might also regulate motor neuron migration in other rhombomeres. We tested this hypothesis by examining whether migration of motor neurons out of r2 following ectopic hoxb1b expression was affected in vangl2^? (trilobite) mutants. Hoxb1b specifies r4 identity, and when ectopically expressed transforms r2 to an “r4‐like” compartment. Using time‐lapse imaging, we show that GFP‐expressing motor neurons in the r2/r3 region of a hoxb1b‐overexpressing wild‐type embryo migrate along the anterior‐posterior (AP) axis. Furthermore, these cells express prickle1b (pk1b), a Wnt/PCP gene that is specifically expressed in FBMNs and is essential for their migration. Importantly, GFP‐expressing motor neurons in the r2/r3 region of hoxb1b‐overexpressing trilobite mutants and pk1b morphants often migrate, even though FBMNs in r4 of the same embryos fail to migrate longitudinally (tangentially) into r6 and r7. These observations suggest that tangentially migrating motor neurons in the anterior hindbrain (r1‐r3) can use mechanisms that are independent of vangl2 and pk1b functions. Interestingly, analysis of tri; val double mutants also suggests a role for vangl2‐independent factors in neuronal migration, since the valentino mutation partially suppresses the trilobite mutant migration defect. Together, the hoxb1b and val experiments suggest that multiple mechanisms regulate motor neuron migration along the AP axis of the zebrafish hindbrain. © 2009 Wiley Periodicals, Inc. Develop Neurobiol, 2010     

Regulated vnd expression is required for both neural and glial specification in Drosophila

The Drosophila embryonic CNS arises from the neuroectoderm, which is divided along the dorsal‐ventral axis into two halves by specialized mesectodermal cells at the ventral midline. The neuroectoderm is in turn divided into three longitudinal stripes—ventral, intermediate, and lateral. The ventral nervous system defective, or vnd, homeobox gene is expressed from cellularization throughout early neural development in ventral neuroectodermal cells, neuroblasts, and ganglion mother cells, and later in an unrelated pattern in neurons. Here, in the context of the dorsal‐ventral location of precursor cells, we reassess the vnd loss‐ and gain‐of‐function CNS phenotypes using cell specific markers. We find that over expression of vnd causes significantly more profound effects on CNS cell specification than vnd loss. The CNS defects seen in vnd mutants are partly caused by loss of progeny of ventral neuroblasts—the commissures are fused and the longitudinal connectives are aberrantly positioned close to the ventral midline. The commissural vnd phenotype is associated with defects in cells that arise from the mesectoderm, where the VUM neurons have pathfinding defects, the MP1 neurons are mis‐specified, and the midline glia are reduced in number. vnd over expression results in the mis‐specification of progeny arising from all regions of the neuroectoderm, including the ventral neuroblasts that normally express the gene. The CNS of embryos that over express vnd is highly disrupted, with weak longitudinal connectives that are placed too far from the ventral midline and severely reduced commissural formation. The commissural defects seen in vnd gain‐of‐function mutants correlate with midline glial defects, whereas the mislocalization of interneurons coincides with longitudinal glial mis‐specification. Thus, Drosophila neural and glial specification requires that vnd expression by tightly regulated. © 2002 Wiley Periodicals, Inc. J Neurobiol 50: 118–136, 2002; DOI 10.1002/neu.10022     

Nucleoside diphosphate kinase Nm23-M1 involves in oligodendroglial versus neuronal cell fate decision in vitro

The adult glial progenitor cells were recently shown to be able to produce neurons in central nervous system (CNS) and to become multipotent in vitro. Although the fate decision of glial progenitors was studied extensively, the signals and factors which regulate the timing of neuronal differentiation still remain unknown. To elucidate the mechanisms underlying the neuronal differentiation from glial progenitors, we modified the gene expression profile in NG2⁺ glial progenitor cells using enhanced retroviral mutagen (ERM) technique followed by phenotype screening to identify possible gene(s) responsible for glial-neuronal cell fate determination. Among the identified molecules, we found the gene named non-metastatic cell 1 which encodes a nucleoside diphosphate kinase protein A (Nm23-M1 or NME1). So far, the Nm23 members have been shown to be involved in various molecular processes including tumor metastasis, cell proliferation, differentiation and cell fate determination. In the present study, we provide evidence suggesting the role of NME1 in glial-neuronal cell fate determination in vitro. We showed that NME1 is widely expressed in neuronal structures throughout adult mouse CNS. Our immunohistochemical results revealed that NME1 is strongly colocalized with NF200 through white matter of spinal cord and brain. Interestingly, NME1 overexpression in oligodendrocyte progenitor OLN-93 cells potently induced the acquisition of neuronal fate, while its silencing was shown to promote oligodendrocyte differentiation. Furthermore, we demonstrated that dual-functional role of NME1 is achieved through cAMP-dependent protein kinase (PKA). Our data therefore suggested that NME1 acts as a switcher or reprogramming factor which involves in oligodentrocyte versus neuron cell fate specification in vitro.     

Selective degeneration of dopaminergic neurons by MPP+ and its rescue by D2 autoreceptors in Drosophila primary culture

Drosophila melanogaster is widely used to study genetic factors causing Parkinson's disease (PD) largely because of the use of sophisticated genetic approaches and the presence of a high conservation of gene sequence/function between Drosophila and mammals. However, in Drosophila, little has been done to study the environmental factors which cause over 90% of PD cases. We used Drosophila primary neuronal culture to study degenerative effects of a well‐known PD toxin MPP⁺. Dopaminergic (DA) neurons were selectively degenerated by MPP⁺, whereas cholinergic and GABAergic neurons were not affected. This DA neuronal loss was because of post‐mitotic degeneration, not by inhibition of DA neuronal differentiation. We also found that MPP⁺‐mediated neurodegeneration was rescued by D2 agonists quinpirole and bromocriptine. This rescue was through activation of Drosophila D2 receptor DD2R, as D2 agonists failed to rescue MPP⁺‐toxicity in neuronal cultures prepared from both a DD2R deficiency line and a transgenic line pan‐neuronally expressing DD2R RNAi. Furthermore, DD2R autoreceptors in DA neurons played a critical role in the rescue. When DD2R RNAi was expressed only in DA neurons, MPP⁺ toxicity was not rescued by D2 agonists. Our study also showed that rescue of DA neurodegeneration by Drosophila DD2R activation was mediated through suppression of action potentials in DA neurons.     

A Small Subset of Fruitless Subesophageal Neurons Modulate Early Courtship in Drosophila

We show that a small subset of two to six subesophageal neurons, expressing the male products of the male courtship master regulator gene products fruitless^Male (fru^M), are required in the early stages of the Drosophila melanogaster male courtship behavioral program. Loss of fru^M expression or inhibition of synaptic transmission in these fru^M(+) neurons results in delayed courtship initiation and a failure to progress to copulation primarily under visually-deficient conditions. We identify a fru^M-dependent sexually dimorphic arborization in the tritocerebrum made by two of these neurons. Furthermore, these SOG neurons extend descending projections to the thorax and abdominal ganglia. These anatomical and functional characteristics place these neurons in the position to integrate gustatory and higher-order signals in order to properly initiate and progress through early courtship.     

Gatekeeper role of brain antigen‐presenting CD11c+ cells in neuroinflammation

Multiple sclerosis is the most frequent chronic inflammatory disease of the CNS. The entry and survival of pathogenic T cells in the CNS are crucial for the initiation and persistence of autoimmune neuroinflammation. In this respect, contradictory evidence exists on the role of the most potent type of antigen‐presenting cells, dendritic cells. Applying intravital two‐photon microscopy, we demonstrate the gatekeeper function of CNS professional antigen‐presenting CD11c⁺ cells, which preferentially interact with Th17 cells. IL‐17 expression correlates with expression of GM‐CSF by T cells and with accumulation of CNS CD11c⁺ cells. These CD11c⁺ cells are organized in perivascular clusters, targeted by T cells, and strongly express the inflammatory chemokines Ccl5, Cxcl9, and Cxcl10. Our findings demonstrate a fundamental role of CNS CD11c⁺ cells in the attraction of pathogenic T cells into and their survival within the CNS. Depletion of CD11c⁺ cells markedly reduced disease severity due to impaired enrichment of pathogenic T cells within the CNS.     

Altered outward K+ currents in Drosophila larval neurons of memory mutants rutabaga and amnesiac

K⁺ currents in cultured Drosophila larval neurons have been classified into four categories according to their inactivation time constants, relative amplitude, and response to K⁺ channel blockers 4‐AP and triethylammonium. The percentage (65%) of neurons displaying K⁺ currents which were reduced to 30% in amplitude by 5 mM cyclic adenosine monophosphate (cAMP) analog 8‐bromo‐cAMP in both Drosophila memory mutants rutabaga (rut) and amnesiac (amn) was significantly larger than that (50%) in wild type. This initial characterization provides evidence for altered K⁺ currents in both rut and amn mutants. Arachidonic acid, a specifical inhibitor of Kv4 family (shal) K⁺ channels, was found to inhibit K⁺ currents in cultured Drosophila neurons, suggesting the presence of shal channels in these neurons. © 1999 John Wiley & Sons, Inc. J Neurobiol 40: 158–170, 1999     

A glial K+/Cl− cotransporter modifies temperature‐evoked dynamics in Caenorhabditis elegans sensory neurons

K⁺/Cl^? cotransporters (KCCs) are known to be crucial in the control of neuronal electrochemical Cl^? gradient. However, the role of these proteins in glial cells remains largely unexplored despite a number of studies showing expression of KCC proteins in glial cells of many species. Here, we show that the Caenorhabditis elegans K⁺/Cl^? cotransporter KCC‐3 is expressed in glial‐like cells and regulates the thermosensory behavior through modifying temperature‐evoked activity of a thermosensory neuron. Mutations in the kcc‐3 gene were isolated from a genetic screen for mutants defective in thermotaxis. KCC‐3 is expressed and functions in the amphid sheath glia that ensheathes the AFD neuron, a major thermosensory neuron known to be required for thermotaxis. A genetic analysis indicated that the regulation of the thermosensory behavior by KCC‐3 is mediated through AFD, and we further show that KCC‐3 in the amphid sheath glia regulates the dynamics of the AFD activity. Our results show a novel mechanism by which the glial KCC‐3 protein non‐cell autonomously modifies the stimulus‐evoked activity of a sensory neuron and highlights the functional importance of glial KCC proteins in modulating the dynamics of a neural circuitry to control an animal behavior.     

Conditional modification of behavior in Drosophila by targeted expression of a temperature‐sensitive shibire allele in defined neurons

Behavior is a manifestation of temporally and spatially defined neuronal activities. To understand how behavior is controlled by the nervous system, it is important to identify the neuronal substrates responsible for these activities, and to elucidate how they are integrated into a functional circuit. I introduce a novel and general method to conditionally perturb anatomically defined neurons in intact Drosophila. In this method, a temperature‐sensitive allele of shibire (shi^ts1) is overexpressed in neuronal subsets using the GAL4/UAS system. Because the shi gene product is essential for synaptic vesicle recycling, and shi^ts1 is semidominant, a simple temperature shift should lead to fast and reversible effects on synaptic transmission of shi^ts1 expressing neurons. When shi^ts1 expression was directed to cholinergic neurons, adult flies showed a dramatic response to the restrictive temperature, becoming motionless within 2 min at 30°C. This temperature‐induced paralysis was reversible. After being shifted back to the permissive temperature, they readily regained their activity and started to walk in 1 min. When shi^ts1 was expressed in photoreceptor cells, adults and larvae exhibited temperature‐dependent blindness. These observations show that the GAL4/UAS system can be used to express shi^ts1 in a specific subset of neurons to cause temperature‐dependent changes in behavior. Because this method allows perturbation of the neuronal activities rapidly and reversibly in a spatially and temporally restricted manner, it will be useful to study the functional significance of particular neuronal subsets in the behavior of intact animals. © 2001 John Wiley & Sons, Inc. J Neurobiol 47: 81–92, 2001     

NaV1.6a is required for normal activation of motor circuits normally excited by tactile stimulation

A screen for zebrafish motor mutants identified two noncomplementing alleles of a recessive mutation that were named non‐active (nav^mi89 and nav^mi130). nav embryos displayed diminished spontaneous and touch‐evoked escape behaviors during the first 3 days of development. Genetic mapping identified the gene encoding Na_V1.6a (scn8aa) as a potential candidate for nav. Subsequent cloning of scn8aa from the two alleles of nav uncovered two missense mutations in Na_V1.6a that eliminated channel activity when assayed heterologously. Furthermore, the injection of RNA encoding wild‐type scn8aa rescued the nav mutant phenotype indicating that scn8aa was the causative gene of nav. In‐vivo electrophysiological analysis of the touch‐evoked escape circuit indicated that voltage‐dependent inward current was decreased in mechanosensory neurons in mutants, but they were able to fire action potentials. Furthermore, tactile stimulation of mutants activated some neurons downstream of mechanosensory neurons but failed to activate the swim locomotor circuit in accord with the behavioral response of initial escape contractions but no swimming. Thus, mutant mechanosensory neurons appeared to respond to tactile stimulation but failed to initiate swimming. Interestingly fictive swimming could be initiated pharmacologically suggesting that a swim circuit was present in mutants. These results suggested that Na_V1.6a was required for touch‐induced activation of the swim locomotor network. © 2010 Wiley Periodicals, Inc. Develop Neurobiol 70:508–522, 2010     

Deletion of the Drosophila neuronal gene found in neurons disrupts brain anatomy and male courtship

The fne (found‐in‐neurons) locus encodes one of the three paralogs of the ELAV gene family of Drosophila melanogaster. Members of this family are found throughout metazoans and encode RNA‐binding proteins with primarily neuronal localization, but with remarkably diverse functions given their high level of amino acid sequence conservation. The first identified member of the family, elav of Drosophila is a vital gene. Mutations in the second Drosophila elav paralog, rbp9, are viable but female sterile. No alleles of fne were previously available. FNE protein is normally present in the cytoplasm of all neurons throughout development. Here we describe the generation and characterization of fne^null mutations by homologous recombination. In contrast to elav and similar to rbp9, fne^null mutants are viable, but exhibit a specific and fully penetrant fusion of the β‐lobes in their mushroom bodies (MB), a paired neuropil of the central brain involved in a variety of complex behaviors. Mutant males have reduced courtship indices, but normal short‐ and long‐term courtship memory. Our data show that fne has specific functions which are non‐overlapping with the other two family members, namely in courtship behavior and in the development of the adult MB. The data further show that courtship memory does not require intact β‐lobes in the MB.     

Effects of amyloid peptides on A‐type K+ currents of Drosophila larval cholinergic neurons

Accumulation of amyloid (Aβ) peptides has been suggested to be the primary event in Alzheimer's disease. In neurons, K⁺ channels regulate a number of processes, including setting the resting potential, keeping action potentials short, timing interspike intervals, synaptic plasticity, and cell death. In particular, A‐type K⁺ channels have been implicated in the onset of LTP in mammalian neurons, which is thought to underlie learning and memory. A number of studies have shown that Aβ peptides alter the properties of K⁺ currents in mammalian neurons. We set out to determine the effects of Aβ peptides on the neuronal A‐type K⁺ channels of Drosophila. Treatment of cells for 18 h with 1 μM Aβ1‐42 altered the kinetics of the A‐type K⁺ current, shifting steady‐state inactivation to more depolarized potentials and increasing the rate of recovery from inactivation. It also caused a decrease in neuronal viability. Thus it seems that alteration in the properties of the A‐type K⁺ current is a prelude to the amyloid‐induced death of neurons. This alteration in the properties of the A‐type K⁺ current may provide a basis for the early memory impairment that was observed prior to neurodegeneration in a recent study of a transgenic Drosophila melanogaster line over‐expressing the human Aβ1‐42 peptide. © 2006 Wiley Periodicals, Inc. J Neurobiol, 2006