Molecular Genetic Analyses of Development of the Mushroom Bodies, Centers for Learning and Memory in Drosophila

ショウジョウバエの脳は，ヒトの脳の百万分の一の神経細胞から構成されるにすぎないが，学習，記憶，空間認識等，多様な高次脳機能が存在する。これらショウジョウバエの脳機能の中枢として機能しているのが，キノコ体と呼ばれる大規模な神経構造である。本研究では，胚期，及び幼虫期のキノコ体の分子解剖学的解析によりその詳細な形成過程と内部構造を明らかにすると共に，キノコ体形成を支配する転写制御因子群の同定をおこなった。　まず，これまで報告のほとんどなかったキノコ体の初期発生過程の詳細な解析を行い，胚発生期におけるキノコ体の初期神経軸索の伸長様式を明らかにすると共に，幼虫期キノコ体の構造を詳細に記載した。この解析により，胚発生過程の脳におけるキノコ体前駆細胞の正確な位置と，初期神経軸索の走行路を明らかにした。さらに，幼虫期におけるキノコ体が，均質な神経構造ではなく，遺伝子発現の異なる同心円状の層構造から成り立つことを発見した。次に，キノコ体神経細胞のモザイク解析を行い，新規に分裂供給された神経細胞がキノコ体層構造の中心領域に軸索を付加していくことを明らかにした。さらに，遺伝学的解析から，層構造の形成に細胞接着因子Fasciclin IIが必要であることを明らかにした。　つぎに，キノコ体発生を制御する遺伝子群を明らかにすることを目的に，以上の解剖学的解析によって同定したキノコ体前駆細胞の位置を手がかりに，胚発生過程のキノコ体でショウジョウバエPax-6相同遺伝子であるeyeless，及び，複眼形成遺伝子dachshundが高レベルに発現していることを見いだした。さらに，遺伝学的解析の結果，これらの遺伝子の変異体では，キノコ体の形成が著しく阻害されることを明らかにした。eyelessは，複眼形成においてdachshund，eyes absent，sine oculis等の転写制御遺伝子と協調的に機能する。下流のsine oculis，eyes absent，dachshundは，eyelessの発現をフィードバック増強し，最終的にこれらの制御網がさらに下流の数千の複眼形成遺伝子の発現を誘導する。しかしながら，キノコ体形成においては，同じくeyelessを中核としつつも，eyes absentもsine oculisも発現せず，さらにeyelessとdachshundの発現は，独立に制御されていることを明らかにした。これらの結果は，キノコ体発生では複眼形成とは異なる新規遺伝子群と制御ネットワークが関与することを示唆するものである。 The mushroom bodies (MBs) are uniquely indefinable brain structures present in the brains of arthropods. Functional studies have showed that the MBs are involved in higher-order behaviors such as olfactory learning and cognition. Despite wealth of knowledge on the anatomy and functions of the adult brain, ...Thesis (Ph. D. in Science)--University of Tsukuba, (A), no. 2771, 2002.3.25Includes bibliographical referencesTITLE,CONTENTS,ABBREVIATIONS -- ABSTRACT -- INTRODUCTION -- MATERIALS AND METHODS -- RESULTS -- DISCISSION -- ACKNOWLEDGEMENTS -- REFERENCES -- TABLES -- FIGURE

Developmental changes in expression, subcellular distribution, and function of Drosophila N-cadherin, guided by a cell-intrinsic program during neuronal differentiation

Cell adhesion molecules (CAMs) perform numerous functions during neural development. An individual CAM can play different roles during each stage of neuronal differentiation; however, little is known about how such functional switching is accomplished. Here we show that Drosophila N-cadherin (CadN) is required at multiple developmental stages within the same neuronal population and that its sub-cellular expression pattern changes between the different stages. During development of mushroom body neurons and motoneurons, CadN is expressed at high levels on growing axons, whereas expression becomes downregulated and restricted to synaptic sites in mature neurons. Phenotypic analysis of CadN mutants reveals that developing axons require CadN for axon guidance and fasciculation, whereas mature neurons for terminal growth and receptor clustering. Furthermore, we demonstrate that CadN downregulation can be achieved in cultured neurons without synaptic contact with other cells. Neuronal silencing experiments using Kir_2.1 indicate that neuronal excitability is also dispensable for CadN downregulation in vivo. Interestingly, downregulation of CadN can be prematurely induced by ectopic expression of a nonselective cation channel, dTRPA1, in developing neurons. Together, we suggest that switching of CadN expression during neuronal differentiation involves regulated cation influx within neurons

A screen of cell-surface molecules identifies leucine-rich repeat proteins as key mediators of synaptic target selection

In Drosophila embryos and larvae, a small number of identified motor neurons innervate body wall muscles in a highly stereotyped pattern. Although genetic screens have identified many proteins that are required for axon guidance and synaptogenesis in this system, little is known about the mechanisms by which muscle fibers are defined as targets for specific motor axons. To identify potential target labels, we screened 410 genes encoding cell-surface and secreted proteins, searching for those whose overexpression on all muscle fibers causes motor axons to make targeting errors. Thirty such genes were identified, and a number of these were members of a large gene family encoding proteins whose extracellular domains contain leucine-rich repeat (LRR) sequences, which are protein interaction modules. By manipulating gene expression in muscle 12, we showed that four LRR proteins participate in the selection of this muscle as the appropriate synaptic target for the RP5 motor neuron

Leucine-rich repeat transmembrane proteins instruct discrete dendrite targeting in an olfactory map

Olfactory systems utilize discrete neural pathways to process and integrate odorant information. In Drosophila, axons of first-order olfactory receptor neurons (ORNs) and dendrites of second-order projection neurons (PNs) form class-specific synaptic connections at ~50 glomeruli. The mechanisms underlying PN dendrite targeting to distinct glomeruli in a three-dimensional discrete neural map are unclear. We found that the leucine-rich repeat (LRR) transmembrane protein Capricious (Caps) was differentially expressed in different classes of PNs. Loss-of-function and gain-of-function studies indicated that Caps instructs the segregation of Caps-positive and Caps-negative PN dendrites to discrete glomerular targets. Moreover, Caps-mediated PN dendrite targeting was independent of presynaptic ORNs and did not involve homophilic interactions. The closely related protein Tartan was partially redundant with Caps. These LRR proteins are probably part of a combinatorial cell-surface code that instructs discrete olfactory map formation

Unc-51/ATG1 Controls Axonal and Dendritic Development via Kinesin-Mediated Vesicle Transport in the Drosophila Brain

Background:Members of the evolutionary conserved Ser/Thr kinase Unc-51 family are key regulatory proteins that control neural development in both vertebrates and invertebrates. Previous studies have suggested diverse functions for the Unc-51 protein, including axonal elongation, growth cone guidance, and synaptic vesicle transport.Methodology/Principal Findings:In this work, we have investigated the functional significance of Unc-51-mediated vesicle transport in the development of complex brain structures in Drosophila. We show that Unc-51 preferentially accumulates in newly elongating axons of the mushroom body, a center of olfactory learning in flies. Mutations in unc-51 cause disintegration of the core of the developing mushroom body, with mislocalization of Fasciclin II (Fas II), an IgG-family cell adhesion molecule important for axonal guidance and fasciculation. In unc-51 mutants, Fas II accumulates in the cell bodies, calyx, and the proximal peduncle. Furthermore, we show that mutations in unc-51 cause aberrant overshooting of dendrites in the mushroom body and the antennal lobe. Loss of unc-51 function leads to marked accumulation of Rab5 and Golgi components, whereas the localization of dendrite-specific proteins, such as Down syndrome cell adhesion molecule (DSCAM) and No distributive disjunction (Nod), remains unaltered. Genetic analyses of kinesin light chain (Klc) and unc-51 double heterozygotes suggest the importance of kinesin-mediated membrane transport for axonal and dendritic development. Moreover, our data demonstrate that loss of Klc activity causes similar axonal and dendritic defects in mushroom body neurons, recapitulating the salient feature of the developmental abnormalities caused by unc-51 mutations.Conclusions/Significance:Unc-51 plays pivotal roles in the axonal and dendritic development of the Drosophila brain. Unc-51-mediated membrane vesicle transport is important in targeted localization of guidance molecules and organelles that regulate elongation and compartmentalization of developing neurons