ミツバチ脳キノコ体で選択的に発現する遺伝子の変態期での発現とPLCの記憶学習における機能の解析

学位の種別: 課程博士審査委員会委員 : （主査）東京大学教授 岡 良隆, 東京大学教授 多羽田 哲也, 東京大学教授 久保 健雄, 東京大学准教授 近藤 真理子, 東京大学准教授 國友 博文University of Tokyo(東京大学

Kenyon Cell Subtypes/Populations in the Honeybee Mushroom Bodies: Possible Function Based on Their Gene Expression Profiles, Differentiation, Possible Evolution, and Application of Genome Editing

Mushroom bodies (MBs), a higher-order center in the honeybee brain, comprise some subtypes/populations of interneurons termed as Kenyon cells (KCs), which are distinguished by their cell body size and location in the MBs, as well as their gene expression profiles. Although the role of MBs in learning ability has been studied extensively in the honeybee, the roles of each KC subtype and their evolution in hymenopteran insects remain mostly unknown. This mini-review describes recent progress in the analysis of gene/protein expression profiles and possible functions of KC subtypes/populations in the honeybee. Especially, the discovery of novel KC subtypes/populations, the “middle-type KCs” and “KC population expressing FoxP,” necessitated a redefinition of the KC subtype/population. Analysis of the effects of inhibiting gene function in a KC subtype-preferential manner revealed the function of the gene product as well as of the KC subtype where it is expressed. Genes expressed in a KC subtype/population-preferential manner can be used to trace the differentiation of KC subtypes during the honeybee ontogeny and the possible evolution of KC subtypes in hymenopteran insects. Current findings suggest that the three KC subtypes are unique characteristics to the aculeate hymenopteran insects. Finally, prospects regarding future application of genome editing for the study of KC subtype functions in the honeybee are described. Genes expressed in a KC subtype-preferential manner can be good candidate target genes for genome editing, because they are likely related to highly advanced brain functions and some of them are dispensable for normal development and sexual maturation in honeybees

Pharmacologic inhibition of phospholipase C in the brain attenuates early memory formation in the honeybee (Apis mellifera L.)

Although the molecular mechanisms involved in learning and memory in insects have been studied intensively, the intracellular signaling mechanisms involved in early memory formation are not fully understood. We previously demonstrated that phospholipase C epsilon (PLCe), whose product is involved in calcium signaling, is almost selectively expressed in the mushroom bodies, a brain structure important for learning and memory in the honeybee. Here, we pharmacologically examined the role of phospholipase C (PLC) in learning and memory in the honeybee. First, we identified four genes for PLC subtypes in the honeybee genome database. Quantitative reverse transcription-polymerase chain reaction revealed that, among these four genes, three, including PLCe, were expressed higher in the brain than in sensory organs in worker honeybees, suggesting their main roles in the brain. Edelfosine and neomycin, pan-PLC inhibitors, significantly decreased PLC activities in homogenates of the brain tissues. These drugs injected into the head of foragers significantly attenuated memory acquisition in comparison with the control groups, whereas memory retention was not affected. These findings suggest that PLC in the brain is involved in early memory formation in the honeybee. To our knowledge, this is the first report of a role for PLC in learning and memory in an insect

Analysis of the Differentiation of Kenyon Cell Subtypes Using Three Mushroom Body-Preferential Genes during Metamorphosis in the Honeybee (Apis mellifera L.).

The adult honeybee (Apis mellifera L.) mushroom bodies (MBs, a higher center in the insect brain) comprise four subtypes of intrinsic neurons: the class-I large-, middle-, and small-type Kenyon cells (lKCs, mKCs, and sKCs, respectively), and class-II KCs. Analysis of the differentiation of KC subtypes during metamorphosis is important for the better understanding of the roles of KC subtypes related to the honeybee behaviors. In the present study, aiming at identifying marker genes for KC subtypes, we used a cDNA microarray to comprehensively search for genes expressed in an MB-preferential manner in the honeybee brain. Among the 18 genes identified, we further analyzed three genes whose expression was enriched in the MBs: phospholipase C epsilon (PLCe), synaptotagmin 14 (Syt14), and discs large homolog 5 (dlg5). Quantitative reverse transcription-polymerase chain reaction analysis revealed that expression of PLCe, Syt14, and dlg5 was more enriched in the MBs than in the other brain regions by approximately 31-, 6.8-, and 5.6-fold, respectively. In situ hybridization revealed that expression of both Syt14 and dlg5 was enriched in the lKCs but not in the mKCs and sKCs, whereas expression of PLCe was similar in all KC subtypes (the entire MBs) in the honeybee brain, suggesting that Syt14 and dlg5, and PLCe are available as marker genes for the lKCs, and all KC subtypes, respectively. In situ hybridization revealed that expression of PLCe is already detectable in the class-II KCs at the larval fifth instar feeding stage, indicating that PLCe expression is a characteristic common to the larval and adult MBs. In contrast, expression of both Syt14 and dlg5 became detectable at the day three pupa, indicating that Syt14 and dlg5 expressions are characteristic to the late pupal and adult MBs and the lKC specific molecular characteristics are established during the late pupal stages

Summary of stage-specific gene expression profiles in the MBs during metamorphosis.

<p>Changes in the MB organization and gene expression profiles during metamorphosis are schematized. Expression level of each gene is represented by red intensity. NA, not analyzed in this study. (1) <i>Mblk-1</i> expression in the adult MBs reported previously [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0157841#pone.0157841.ref040" target="_blank">40</a>] is also included in this figure. Ca, calyces; Nb, MB neuroblasts; Ped, peduncles.</p

<i>In situ</i> hybridization of <i>dlg5</i> in the larval (L5F) and pupal (P3) brains.

<p>(A-D) Frontal sections of the brain right hemispheres hybridized with antisense (A and C) and sense (B and D) probes. (A, B) The larval (L5F) brain. (C, D) The pupal (P3) brain. Arrowheads in (C) indicate prominent signals in the MBs. MB, mushroom body; OLp, optic lobe primordium; OL, optic lobe; AL, antennal lobe. (E-H) Magnified views of the MBs shown in (A) and (C) and their schematic views. (E and F) The larval MBs. (G and H) The pupal MBs. Black and white arrowheads indicate moderate signals in the outer halves of the lKCs and weak signals in the remaining lKCs and class-II KCs, respectively. White arrows indicate weak signals in the MB neuroblast clusters. The lKCs, class-II KCs, MB neuroblast clusters, and regions where somata of the other cells exist are colored by purple, green, grey, and light grey, respectively. Ca, calyces; Nb, MB neuroblasts; Ped, peduncles. Bars indicate 200μm. The probes were prepared with the second set of primers mentioned in Materials and Methods.</p

<i>In situ</i> hybridization of <i>Syt14</i> in the larval (L5F) and pupal (P3) brains.

<p>(A-D) Frontal sections of the brain right hemispheres hybridized with antisense (A, C) and sense (B, D) probes. (A, B) The larval (L5F) brain. (C, D) The pupal (P3) brain. Arrowheads in (C) indicate prominent signals in the MBs. MB, mushroom body; OLp, optic lobe primordium; OL, optic lobe; AL, antennal lobe. (E-H) Magnified views of the MBs shown in (A) and (C) and their schematic views. (E and F) The larval MBs. (G and H) The pupal MBs. Arrowheads indicate prominent signals in the lKCs and class-II KCs, while white arrows indicate weak signals in the MB neuroblast clusters. The lKCs, class-II KCs, MB neuroblast clusters, and regions where somata of the other cells exist are colored by purple, green, grey, and light grey, respectively. Ca, calyces; Nb, MB neuroblasts; Ped, peduncles. Bars indicate 200μm.</p

Expressions of <i>Syt14</i> and <i>dlg5</i> do not overlap with that of <i>mKast</i> in the worker MB.

<p>(A-C) <i>In situ</i> hybridization of <i>Syt14</i> (A), <i>mKast</i> (B) and <i>dlg5</i> (C) using serial worker MB sections. <i>mKast</i> signals represent the localization of mKC somata. (D) Schematic drawings of the expression of each gene in (A-C). Left illustration represents regions strongly expressing <i>Syt14</i> (blue) and <i>mKast</i> (yellow), respectively. Right illustration represents regions strongly expressing <i>dlg5</i> (magenta) and <i>mKast</i>, respectively. Note that regions strongly expressing <i>Syt14</i> and <i>dlg5</i> do not overlap with that strongly expressing <i>mKast</i>. Bars indicate 100μm. The probe for <i>dlg5</i> was prepared with the second set of primers mentioned in Materials and Methods.</p

<i>In situ</i> hybridization of <i>Syt14</i> in the worker brain.

<p>(A, B) Frontal section of the whole brain hybridized with antisense (A) and sense (B) probes. (C) Magnified view of the right MB shown in (A). Filled triangles, open triangles and arrows indicate the lKCs, mKCs and sKCs, and class-II KCs, respectively. The lKCs strongly expressed <i>Syt14</i>. Note that regions where the somata of mKCs are localized are not discriminated in this experiment. (D) Schematic view of (C). Boundary between the lKCs (purple), and mKCs and sKCs (orange) are indicated by dotted lines. Brain regions where the somata of the class-II KCs and other neurons exist are colored by green and light grey, respectively. (E, F) Anterior section, which contained the ALs, and posterior section, which contained the SOG, of (A) hybridized with antisense probes, respectively. MB, mushroom body; OL, optic lobe; mCa, medial calyx; lCa, lateral calyx; AL, antennal lobe; SOG, suboesophageal ganglion. Bars indicate 100 μm.</p