<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

<i>In situ</i> hybridization of <i>Mblk-1</i> in the pupal (P3) brain.

<p>(A, B) Frontal sections of the right brain hemispheres hybridized with antisense (A) and sense (B) probes. Arrowheads and arrows indicate prominent signals in the MBs, OLs and ALs, respectively. MB, mushroom body; OL, optic lobe; AL, antennal lobe; R, retina. (C) Magnified view of the MB shown by a square in (A). Black and white arrowheads represent strong signals in the lKCs and weak signals in the class-II KCs, respectively. White arrows represent signals in the MB neuroblasts. (D) Schematic drawing of the brain structure seen in (C). 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

<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>dlg5</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>dlg5</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. The probes were prepared with the first set of primers mentioned in Materials and Methods.</p

Quantitative RT-PCR analysis of <i>PLCe</i>, <i>Syt14</i>, and <i>dlg5</i>.

<p>(A) <i>PLCe</i> (B) <i>Syt14</i> (C) <i>dlg5</i>. Relative expression of each gene was normalized to <i>RpL32</i> and compared between the MBs and other brain parts, taking expression in the other brain region as 1. One lot contained two workers, and four lots were used for the experiment. Bars indicate standard deviation. * indicates <i>P</i> < 0.01 (Welch’s t-test).</p

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

Characteristics of identified honeybee genes showing preferential MB expression.

<p>The gene and protein structures of (A) <i>PLCe</i>, (B) <i>Syt14</i>, and (C) <i>dlg5</i>. In each panel, the upper line indicates linkage group where the corresponding gene is mapped. The second line indicates the predicted gene structure. Closed boxes indicate coding regions, while open boxes indicate 5’ and 3’ UTRs. Positions of differential display products (clone Nos. 231, 116, and 314, respectively) and probes used for <i>in situ</i> hybridization are shown below the gene structure. The lower bar indicates protein structure. Numbers indicate amino acid positions. Colored boxes on the protein structure show functional domains.</p

Alignment of amino acid sequences of PLCe, Syt14, and dlg5.

<p>Alignments of domains showing the highest similarity in each protein with other known protein orthologues. (A) PLCX catalytic domain of PLCe, (B) C2 of Syt14 and Syt16, (C) The third PDZ domain of dlg5. Amino acid sequence of honeybee PLCe was deduced from the nucleotide sequence of GB42984. Alignments were performed using ClustalW. Identical amino acids in the same position are labelled by asterisks under the alignments. Abbreviations of species are follows: Am, <i>Apis mellifera</i>; Dm, <i>Drosophila melanogaster</i>; Tc, <i>Tribolium castaneum</i>; Aa, <i>Aedes aegypti</i>; Cq, <i>Culex quinquefasciatus</i>; Ce, <i>Caenorhabditis elegans</i>; Mm, <i>Mus musculus</i>; Hs, <i>Homo sapiens</i>. Some species are not used in each alignment because their proteins or domains are not annotated.</p

<i>In situ</i> hybridization of <i>RpL32</i> in the larval (L5F) brain.

<p>(A, B) Frontal sections of the left brain hemispheres hybridized with antisense (A) or sense (B) probes. Arrows indicate prominent signals in the MB neuroblasts and primordium of OL. (C) Magnified view of the MB shown by a square in (A). Arrows indicate prominent signals in the MB. (D) Schematic drawing of the brain structure seen in (C). Regions for the MB neuroblasts (Nb) and for the other cells are colored by grey and light grey, respectively. MB, mushroom body; OLp, optic lobe primordium; AL, antennal lobe; Ped, peduncles. Bars indicate 200μm.</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