Chihiro Hama

The Drosophila Trio Plays an Essential Role in Patterning of Axons by Regulating Their Directional Extension

We identified the Drosophila trio gene, which encodes a Dbl family protein carrying two Dbl homology (DH) domains, each of which potentially activates Rho family GTPases. Trio was distributed along axons in the central nervous system (CNS) of embryos and was strongly expressed in subsets of brain regions, including the mushroom body (MB). Loss-of-function trio mutations resulted in the misdirection or stall of axons in embryos and also caused malformation of the MB. The MB phenotypes were attributed to alteration in the intrinsic nature of neurites, as revealed by clonal analyses. Thus, Trio is essential in order for neurites to faithfully extend on the correct pathways. In addition, the localization of Trio in the adult brain suggests its postdevelopmental role in neurite terminals.

Microglial α7 nicotinic acetylcholine receptors drive a phospholipase C/IP3 pathway and modulate the cell activation toward a neuroprotective role

Microglia perform both neuroprotective and neurotoxic functions in the brain, with this depending on their state of activation and their release of mediators. Upon P2X7 receptor stimulation, for example, microglia release small amounts of TNF, which protect neurons, whereas LPS causes massive TNF release leading to neuroinflammation. Here we report that, in rat primary cultured microglia, nicotine enhances P2X7 receptor‐mediated TNF release, whilst suppressing LPS‐induced TNF release but without affecting TNF mRNA expression via activation of α7 nicotinic acetylcholine receptors (α7 nAChRs). In microglia, nicotine elicited a transient increase in intracellular Ca2+ levels, which was abolished by specific blockers of α7 nAChRs. However, this response was independent of extracellular Ca2+ and blocked by U73122, an inhibitor of phospholipase C (PLC), and xestospongin C, a blocker of the IP3 receptor. Repeated experiments showed that currents were not detected in nicotine‐stimulated microglia. Moreover, nicotine modulation of LPS‐induced TNF release was also blocked by xestospongin C. Upon LPS stimulation, inhibition of TNF release by nicotine was associated with the suppression of JNK and p38 MAP kinase activation, which regulate the post‐transcriptional steps of TNF synthesis. In contrast, nicotine did not alter any MAP kinase activation, but enhanced Ca2+ response in P2X7 receptor‐activated microglia. In conclusion, microglial α7 nAChRs might drive a signaling process involving the activation of PLC and Ca2+ release from intracellular Ca2+ stores, rather than function as conventional ion channels. This novel α7 nAChR signal may be involved in the nicotine modification of microglia activation towards a neuroprotective role by suppressing the inflammatory state and strengthening the protective function.

Cloning of the Drosophila prospero gene and its expression in ganglion mother cells.

The Drosophila central nervous system comprises an enormous diversity of neurons that are originated from neuronal stem cells, neuroblasts. They generate a specific series of ganglion mother cells, each of which is once cleaved into a pair of neurons. Among genes known to control neurogenesis, prospero (pros) was recently identified as a gene required for gene expression specifying properties of some identified neurons. Here we report that pros encodes a nuclear protein containing a homeodomain-like sequence. In neuronal lineages of the central nervous system, pros protein is specifically detected in ganglion mother cells, although their parental neuroblasts have begun expressing a significant level of pros transcripts, suggesting a post-transcriptional control of pros expression. Our results provoke that in neuronal cell differentiation ganglion mother cells might play a pivotal role associating with the pros function.

Identification of the stef Gene That Encodes a Novel Guanine Nucleotide Exchange Factor Specific for Rac1

The Rho family GTPases are involved in a variety of cellular events by changing the organization of actin cytoskeletal networks in response to extracellular signals. However, it is not clearly known how their activities are spatially and temporally regulated. Here we report the identification of a novel guanine nucleotide exchange factor for Rac1, STEF, which is related in overall amino acid sequence and modular structure to mouse Tiam1 andDrosophila SIF proteins. STEF protein contains two pleckstrin homology domains, a PDZ domain and a Dbl homology domain. The in vitro assay showed that STEF protein specifically enhanced the dissociation of GDP from Rac1 but not that from either RhoA or Cdc42. Expression of a truncated STEF protein in culture cells induced membrane ruffling with altered actin localization, which implies that this protein also activates Rac1 in vivo. Thestef transcript was observed in restricted parts of mice, including cartilaginous tissues and the cortical plate of the central nervous system during embryogenesis. These findings suggested that STEF protein participates in the control of cellular events in several developing tissues, possibly changing the actin cytoskeletal network by activating Rac1.

Notch signal organizes the Drosophila olfactory circuitry by diversifying the sensory neuronal lineages

An essential feature of the organization and function of the vertebrate and insect olfactory systems is the generation of a variety of olfactory receptor neurons (ORNs) that have different specificities in regard to both odorant receptor expression and axonal targeting. Yet the underlying mechanisms that generate this neuronal diversity remain elusive. Here we demonstrate that the Notch signal is involved in the diversification of ORNs in Drosophila melanogaster. A systematic clonal analysis showed that a cluster of ORNs housed in each sensillum were differentiated into two classes, depending on the level of Notch activity in their sibling precursors. Notably, ORNs of different classes segregated their axonal projections into distinct domains in the antennal lobes. In addition, both the odorant receptor expression and the axonal targeting of ORNs were specified according to their Notch-mediated identities. Thus, Notch signaling contributes to the diversification of ORNs, thereby regulating multiple developmental events that establish the olfactory map in Drosophila.

Fruitless Recruits Two Antagonistic Chromatin Factors to Establish Single-Neuron Sexual Dimorphism

The Drosophila fruitless (fru) gene encodes a set of putative transcription factors that promote male sexual behavior by controlling the development of sexually dimorphic neuronal circuitry. However, the mechanism whereby fru establishes the sexual fate of neurons remains enigmatic. Here, we show that Fru forms a complex with the transcriptional cofactor Bonus (Bon), which, in turn, recruits either of two chromatin regulators, Histone deacetylase 1 (HDAC1), which masculinizes individual sexually dimorphic neurons, or Heterochromatin protein 1a (HP1a), which demasculinizes them. Manipulations of HDAC1 or HP1a expression change the proportion of male-typical neurons and female-typical neurons rather than producing neurons with intersexual characteristics, indicating that on a single neuron level, this sexual switch operates in an all-or-none manner.

GAL4/UAS-WGA system as a powerful tool for tracing Drosophila transsynaptic neural pathways.

Visualization of specific transsynaptic neural pathways is an indispensable technique for understanding the relationship between structure and function in the nervous system. Here, we demonstrate the application of the wheat germ agglutinin (WGA) transgene technique for tracing transsynaptic neural pathways in Drosophila. The intracellular localization of WGA was examined by immunoelectron microscopy. WGA signals were detected in granule‐like structures in both the outer photoreceptor cells expressing WGA and the second‐order laminar neurons. Misexpression of tetanus toxin (TNT), which inactivates N‐synaptobrevin, in the outer photoreceptor cells resulted in the elimination of on/off transients in electroretinogram (ERG) recordings and in a great reduction in WGA transfer into laminar neurons, suggesting that anterograde WGA transsynaptic transfer is dependent mainly on synaptic transmission. Retrograde WGA transfer was also detected upon its forced expression in muscle cells. WGA primarily expressed in muscle cells was taken up by motoneuron axons and transported to their cell bodies in the ventral nerve cord, suggesting that WGA can trace motoneuronal pathways in combination with the muscle‐specific GAL4 driver. Thus, the GAL4/UAS‐WGA system should facilitate the dissection of the Drosophila neural circuit formation and/or synaptic activity in various regions and at various developmental stages. J. Neurosci. Res. 59:94–99, 2000

hikaru genki, a CNS-specific gene identified by abnormal locomotion in drosophila, encodes a novel type of protein

We have identified a gene, hikaru genki (hig), whose mutant phenotype includes abnormal locomotor behavior. Mutant first instar larvae have uncoordinated movements, and both larvae and adults have reduced locomotion. Sequence analyses revealed that this gene encodes a novel type of protein with a signal sequence, but without transmembrane regions. One of its domains has similarities with immunoglobulin domains; three or four regions are similar to a complement-binding domain found in complement-related proteins and selectins. In situ hybridization to embryos revealed that accumulation of the hig transcripts is restricted to subsets of cells in the CNS. Our data suggest that hig has a role in the development of CNS functions involved in locomotor activity.

Differentially Expressed Drl and Drl-2 Play Opposing Roles in Wnt5 Signaling during Drosophila Olfactory System Development

In Drosophila, odor information received by olfactory receptor neurons (ORNs) is processed by glomeruli, which are organized in a stereotypic manner in the antennal lobe (AL). This glomerular organization is regulated by Wnt5 signaling. In the embryonic CNS, Wnt5 signaling is transduced by the Drl receptor, a member of the Ryk family. During development of the olfactory system, however, it is antagonized by Drl. Here, we identify Drl-2 as a receptor mediating Wnt5 signaling. Drl is found in the neurites of brain cells in the AL and specific glia, whereas Drl-2 is predominantly found in subsets of growing ORN axons. A drl-2 mutation produces only mild deficits in glomerular patterning, but when it is combined with a drl mutation, the phenotype is exacerbated and more closely resembles the Wnt5 phenotype. Wnt5 overexpression in ORNs induces aberrant glomeruli positioning. This phenotype is ameliorated in the drl-2 mutant background, indicating that Drl-2 mediates Wnt5 signaling. In contrast, forced expression of Drl-2 in the glia of drl mutants rescues the glomerular phenotype caused by the loss of antagonistic Drl function. Therefore, Drl-2 can also antagonize Wnt5 signaling. Additionally, our genetic data suggest that Drl localized to developing glomeruli mediates Wnt5 signaling. Thus, these two members of the Ryk family are capable of carrying out a similar molecular function, but they can play opposing roles in Wnt5 signaling, depending on the type of cells in which they are expressed. These molecules work cooperatively to establish the olfactory circuitry in Drosophila.

Neural expression of hikaru genki protein during embryonic and larval development of Drosophila melanogaster

Abstract Hikaru genki (HIG) is a putative secreted protein of Drosophila that belongs to immunoglobulin and complement-binding protein superfamilies. Previous studies reported that, during pupal and adult stages, HIG protein is synthesized in subsets of neurons and appears to be secreted to the synaptic clefts of neuron-neuron synapses in the central nervous system (CNS). Here we report the analyses of distribution patterns of HIG protein at embryonic and larval stages. In embryos, HIG was mainly observed in subsets of neurons of the CNS that include pCC interneurons and RP5 motorneurons. At third instar larval stage, this protein was detected in a limited number of cells in the brain and ventral nerve cord. Among them are the motorneurons that extend their axons to make neuromuscular junctions on body wall muscle 8. Immunoelectron microscopy showed that these axonal processes as well as the neuromuscular terminals contain numerous vesicles with HIG staining, suggesting that HIG is in a pathway of secretion at this stage. Some neurosecretory cells were also found to express this protein. These data suggest that HIG functions in the nervous system through most developmental stages and may serve as a secreted signalling molecule to modulate the property of synapses or the physiology of the postsynaptic cells.