Novel Middle-Type Kenyon Cells in the Honeybee Brain Revealed by Area-Preferential Gene Expression Analysis

<div><p>The mushroom bodies (a higher center) of the honeybee (<i>Apis mellifera</i> L) brain were considered to comprise three types of intrinsic neurons, including large- and small-type Kenyon cells that have distinct gene expression profiles. Although previous neural activity mapping using the immediate early gene <i>kakusei</i> suggested that small-type Kenyon cells are mainly active in forager brains, the precise Kenyon cell types that are active in the forager brain remain to be elucidated. We searched for novel gene(s) that are expressed in an area-preferential manner in the honeybee brain. By identifying and analyzing expression of a gene that we termed <i>mKast</i> (<i>middle-type Kenyon cell-preferential arrestin-related protein</i>), we discovered novel ‘middle-type Kenyon cells’ that are sandwiched between large- and small-type Kenyon cells and have a gene expression profile almost complementary to those of large– and small-type Kenyon cells. Expression analysis of <i>kakusei</i> revealed that both small-type Kenyon cells and some middle-type Kenyon cells are active in the forager brains, suggesting their possible involvement in information processing during the foraging flight. <i>mKast</i> expression began after the differentiation of small- and large-type Kenyon cells during metamorphosis, suggesting that middle-type Kenyon cells differentiate by modifying some characteristics of large– and/or small-type Kenyon cells. Interestingly, <i>CaMKII</i> and <i>mKast</i>, marker genes for large– and middle-type Kenyon cells, respectively, were preferentially expressed in a distinct set of optic lobe (a visual center) neurons. Our findings suggested that it is not simply the Kenyon cell-preferential gene expression profiles, rather, a ‘clustering’ of neurons with similar gene expression profiles as particular Kenyon cell types that characterize the honeybee mushroom body structure.</p></div

Identification of mKCs that are characterized by <i>mKast</i> expression.

<p>(<b>A–D</b>) The same double <i>in situ</i> hybridization result with <i>CaMKII</i> and <i>mKast</i> antisense probes shown in Fig. 3 (D). (<b>A</b>) <i>mKast-</i>signals detected by HNPP/FastRed, (<b>B</b>) <i>mblk-1-</i>signals detected by fluorescein, (<b>C</b>) nuclear signals detected with DAPI, and (<b>D</b>) merged images of the three. Dashed lines indicate borders between cells expressing <i>mKast</i> mKCs, magenta) and cells expressing <i>mblk-1</i> (lKCs, green), and cells expressing <i>mKast</i> (mKCs) and cells stained with DAPI (sKCs, blue). The bar indicates 100 µm.</p

Double fluorescent <i>in situ</i> hybridization of <i>mKast</i> and <i>CaMKII</i> or <i>mblk1</i> in the worker brain.

<p>(<b>A–D</b>) Double <i>in situ</i> hybridization with <i>CaMKII</i> and <i>mKast</i> antisense probes. Nuclear signals detected by DAPI (A), <i>mKast</i> signals detected by HNPP/FastRed (B), <i>CaMKII-</i>signals detected by fluorescein (C) and merged images of the two (D) are shown. In the merged image (D), DAPI signals, <i>mKast</i> signals and <i>CaMKII</i> signals are colored in blue, magenta, and green, respectively. White arrowheads indicate regions with <i>mKast</i> signals. Bars indicate 100 µm. (<b>E–H</b>) Double <i>in situ</i> hybridization with <i>mblk-1</i>and <i>mKast</i> antisense probes. Nuclear signals detected by DAPI (E), <i>mKast</i> signals detected by HNPP/FastRed (F), <i>mblk-1</i> signals detected by fluorescein (G) and merged images of the two (H) are shown. In the merged image (H), DAPI signals, <i>mKast</i> signals and <i>mblk-1</i> signals are colored blue, magenta, and green, respectively. White arrowheads indicate regions with <i>mKast-</i>signals. (<b>I–L</b>) Schematic drawings of the signal pattern corresponding to the above two panels, respectively. Signals of DAPI, <i>mKast</i>, <i>CaMKII</i> or <i>mblk-1</i> are colored blue, magenta, and green, respectively. lKCs, large-type Kenyon cells; sKCs, small-type Kenyon cells.</p

Double fluorescent <i>in situ</i> hybridization of <i>mKast</i> and <i>jhdk</i> or <i>trp</i> in the worker brain.

<p>(<b>A–D</b>) Double <i>in situ</i> hybridization with <i>jhdk</i> and <i>mKast</i> antisense probes. Nuclear signals detected by DAPI (A), <i>mKast</i> signals detected by HNPP/FastRed (B), <i>jhdk-</i>signals detected by fluorescein (C) and merged images of the three (D) are shown. In the merged image (D), DAPI signals, <i>mKast</i> signals and <i>jhdk</i> signals are colored blue, magenta and green, respectively. White arrowheads indicate regions with <i>mKast</i> signals. Bars indicate 100 µm. (<b>E–H</b>) Double <i>in situ</i> hybridization with <i>trp</i> and <i>mKast</i> antisense probes. Nuclear signals detected by DAPI (E), <i>mKast</i> signals detected by HNPP/FastRed (F), <i>trp</i> signals detected by fluorescein (G) and merged images of the two (H) are shown. In the merged image (H), DAPI signals, <i>mKast</i> signals and <i>trp</i> signals were colored blue, magenta, and green, respectively. White arrowheads indicate regions with <i>mKast-</i>signals. (<b>I–L</b>) Schematic drawings of the signal pattern corresponding to the above two panels, respectively. Signals of DAPI, <i>mKast</i>, and <i>jhdk</i> or <i>trp</i> are colored blue, magenta and green, respectively. lKCs, large-type Kenyon cells; sKCs, small-type Kenyon cells.</p

Phylogenetic tree analysis of genes containing arrestin-like domains.

<p>A phylogenetic tree was constructed using the neighbor-joining method <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071732#pone.0071732-DeWire1" target="_blank">[51]</a>. Known arrestin genes and other genes that encode proteins with arrestin-like domains of <i>Apis mellifera</i> (Bee-) <i>Drosophila melanogaster</i> (Fly-), <i>Caenorhabditis elegans</i>, (c. elegans-)and <i>Homo sapiens</i> (human-) origin are indicated by the pink and orange background color, respectively. The scale bar corresponds to 0.1 substitutions/site.</p

Expression analysis of <i>mKast</i> and <i>CaMKII</i> revealed a novel ‘module-like structure’ in the honeybee brain.

<p>(<b>A</b>) Double <i>in situ</i> hybridization of <i>CaMKII</i> and <i>mKast</i> in the worker OL area. Nuclear signals detected by DAPI, <i>mKast</i> signals detected by HNPP/FastRed and <i>CaMKII</i>-signals detected by fluorescein are colored blue, magenta, and green, respectively. (<b>B</b>) Schematic drawing of the distribution of <i>mKast</i>-expressing cells (red dots) in a left hemisphere section of the worker brain. (<b>C</b>) Double <i>in situ</i> hybridization of <i>CaMKII</i> and <i>mKast</i> in the worker MB area. Nuclear signals detected by DAPI, <i>mKast</i> signals detected by HNPP/FastRed, and <i>CaMKII</i> signals detected by fluorescein are colored blue, magenta and green, respectively. (<b>D–E</b>) Magnified views of the box shown in panel (A). (<b>D</b>) Nuclear signals detected by DAPI, (<b>E</b>) <i>mKast</i> signals detected by HNPP/FastRed, (<b>F</b>) <i>CaMKII</i> signals detected by fluorescein, and (<b>G</b>) a merged image of three. Note that there are three types of cells expressing either <i>mKast</i> (indicated by red arrowheads), <i>CaMKII</i> (indicated by green arrowheads) or neither (indicated by white arrowheads) in panels (D–G). Bars indicate 100 µm in panels (A and C) and 20 µm in panels (D–G).</p

Activity mapping of mKCs and sKCs in the forager brain.

<p>(A) <i>In situ</i> hybridization of <i>kakusei</i>, an immediate early gene, to map active KCs in the forager brain. Note that <i>kakusei</i> signals (purple dots) were detected in the inner region of the MB calyx. (B) <i>In situ</i> hybridization of <i>mKast</i> to identify mKCs in the forager brain. Note that <i>mKast</i>-signals (purple dots) represent the localization of mKCs. (C) Schematic drawing of the distribution area of mKCs expressing <i>mKast</i> (orange) and active KCs expressing <i>kakusei</i> (blue). Note that not only sKCs (blue area) but also some mKCs (merged red area) are active in the forager brain. Bars indicate 100 µm.</p

Gene structure and primary structure of <i>mKast</i> (GB18367/NC_007082.3).

<p>Gene structure of <i>mKast</i> (GB18367/NC_007082.3). The exon (blue closed boxes)/intron (blue horizontal lines) structure of <i>mKast</i> is presented below the corresponding part of Chromosome LG13. Note that <i>mKast</i> is located in the reverse strand. Numbers indicate the positions of the start and end nucleotides of <i>mKast</i> in Chromosome LG13. XM_00121375.2 indicates the mRNA structure of <i>mKast</i>, which corresponds to all of the exons (blue boxes), whereas XP_00121375.2 indicates the coding regions of <i>mKast</i>, which correspond to parts of the first and eighth exons and all of the second to seventh exons (red boxes). Clone #3 (magenta box) corresponds to almost the entire 3–UTR of the eighth exon. (B) Domain structure of mKast. Arrestin-like_N and _C domains are indicated in the primary structure (gray box) of mKast. Numbers indicate amino acid positions.</p

<i>mKast</i> expression begins after the proliferation of lKCs and sKCs ceases.

<p>(Upper arrow) Staging of the honeybee metamorphosis. P1–P9 in the arrow colored in light purple indicate the pupal stages. P5–P8 stages analyzed by in situ hybridization of <i>mKast</i> are boxed with red lines. (Middle panels labeled with P5–P6) <i>In situ</i> hybridization with <i>mKast</i>-antisense probe to identify mKCs in the developing pupal worker brain. Areas corresponding to lKCs and sKCs, that had already stopped proliferating, are colored light green and purple, respectively. (Lower panels) <i>In situ</i> hybridization with <i>mKast</i> sense probe (control experiments) corresponding to the above sections. Note that <i>mKast</i> signals indicated by red arrowheads are detectable only in the P7 and P8 stages in the middle panels.</p

Single fluorescent <i>in situ</i> hybridization of <i>mKast</i> in the worker brain.

<p>(<b>A</b>) Schematic drawing of the distribution of the <i>mKast</i>-expressing cells in a sagittal left hemisphere section of a worker brain. <i>mKast</i>-expressing cells (black dots) were scattered between the lamina (la) and medulla (me), whereas almost all of the cells located between the medulla (me) and lobula (lo) expressed <i>mKast</i>. (<b>B</b>) Results of single <i>in situ</i> hybridization with <i>mKast</i> antisense probe in a worker brain area corresponding to box (B) shown in panel (A). <i>mKast</i> expression is indicated in magenta, whereas nuclei were stained with DAPI (blue). (<b>C–H</b>) Magnified views of boxes (C–E) and (F–H) in panel (B). (<b>I–K</b>) Magnified views of box (I–K) in panel (A). Signals of <i>mKast</i> (C, F, I) and DAPI (D, G, J), and merged images of signals of <i>mKast</i> and DAPI are shown in independent panels, respectively. la, lamina; lo, lobula; me, medulla; OL, optic lobe; MB, mushroom body; Re, retina. Bars represent 100 µm in panel (B) and 20 µm in panels (C–H), respectively.</p