<i>amn</i> mutants show defects in egg-laying decisions.

<p>(A) In sucrose/caffeine chamber, <i>amn</i>^<i>1</i>, <i>amn</i>^<i>28A</i>, <i>amn</i>^<i>c651</i>, and <i>amn</i>^<i>X8</i> flies showed significant difference in egg-laying decisions compared to wild-type flies. <i>amn</i>^<i>X8</i> showed significant difference compared to the other <i>amn</i> mutants. Each value represents mean ± SEM (n = 34–35, ***P < 0.001). (B) In sucrose/plain chamber, <i>amn</i>^<i>1</i>, <i>amn</i>^<i>28A</i>, <i>amn</i>^<i>c651</i>, and <i>amn</i>^<i>X8</i> flies showed significant difference in egg-laying decisions compared to wild-type flies. <i>amn</i>^<i>X8</i> showed significant difference compared to the other <i>amn</i> mutants. Each value represents mean ± SEM (n = 20, ***P < 0.001, **P < 0.01). (C) In caffeine/plain chamber, <i>amn</i>^<i>1</i>, <i>amn</i>^<i>28A</i>, <i>amn</i>^<i>c651</i>, and <i>amn</i>^<i>X8</i> flies showed significant difference in egg-laying decisions compared to wild-type flies. Each value represents mean ± SEM (n = 23–27, ***P < 0.001).</p

Aging does not alter <i>Drosophila</i> egg-laying decisions.

<p>(A) Schematic representation of the chamber for egg-laying decisions assays. Two 1% agarose media, containing either 100 mM sucrose (orange) or 100 mM caffeine (blue), were loaded above the 3% hard agarose medium (grey). A gap (0.1 cm) was made on the 3% agarose medium in the middle of the chamber. (B) Egg-laying decisions of wild-type flies at different ages (5-day and 21-day). Each value represents mean ± SEM (N = 36–42, n.s., not statistically significant).</p

Neural activity in mushroom body αβ neurons is required for normal egg-laying decisions.

<p>(A) Preferential expression of <i>R16A06-GAL4</i> in mushroom body γ neurons (green). The brain was immunostained with DLG antibody (red). The scale bar represents 50 μm. Genotype was as follow: <i>UAS-mCD8</i>::<i>GFP/+; R16A06-GAL4/UAS-mCD8</i>::<i>GFP</i>. (B) Preferential expression of <i>VT30604-GAL4</i> in mushroom body α´β´ neurons (green). The brain was immunostained with DLG antibody (red). The scale bar represents 50 μm. Genotype was as follow: <i>UAS-mCD8</i>::<i>GFP/+; VT30604-GAL4/UAS-mCD8</i>::<i>GFP</i>. (C) Preferential expression of <i>VT49246-GAL4</i> in mushroom body αβ neurons (green). The brain was immunostained with DLG antibody (red). The scale bar represents 50 μm. Genotype was as follow: <i>UAS-mCD8</i>::<i>GFP/+; VT49246-GAL4/UAS-mCD8</i>::<i>GFP</i>. (D) Effects of acute silencing of neuronal activity in different mushroom body neuron subsets on egg-laying decisions. The temperature shift protocols are shown schematically above each graph. Each value represents mean ± SEM (n = 10–25, ***P < 0.001. n.s., not statistically significant). Genotypes were as follows: (1) <i>tubP-GAL80</i>^<i>ts</i><i>/+; +/UAS-Kir2</i>.<i>1</i>, (2) <i>+/+; R16A06-GAL4/+</i>, (3) <i>+/+; VT30604-GAL4/+</i>, (4) <i>+/+; VT49246-GAL4/+</i>, (5) <i>tubP-GAL80</i>^<i>ts</i><i>/+;R16A06-GAL4/UAS-Kir2</i>.<i>1</i>, (6) <i>tubP-GAL80</i>^<i>ts</i><i>/+;VT30604-GAL4/UAS-Kir2</i>.<i>1</i>, and (7) <i>tubP-GAL80</i>^<i>ts</i><i>/+;VT49246-GAL4/UAS-Kir2</i>.<i>1</i>.</p

Expression of <i>amn</i> transgene in DPM neurons reverses the defects of egg-laying decisions in <i>amn</i> mutants.

<p>(A) The expression pattern of <i>C316-GAL4</i> (green). The brain was immunostained with DLG antibody (red). Arrowheads indicate the somata of DPM neurons. The scale bar represents 50 μm. Genotype was as follow: <i>UAS-mCD8</i>::<i>GFP/+; C316-GAL4/UAS-mCD8</i>::<i>GFP</i>. (B) The expression pattern of <i>VT6412-GAL4</i> (green). The brain was immunostained with DLG antibody (red). Arrowheads indicate the somata of DPM neurons. The scale bar represents 50 μm. Genotype was as follow: <i>UAS-mCD8</i>::<i>GFP/+; VT64246-GAL4/UAS-mCD8</i>::<i>GFP</i>. (C) The expression pattern of <i>VT64246-GAL4</i> (green). The brain was immunostained with DLG antibody (red). Arrowheads indicate the somata of DPM neurons. The scale bar represents 50 μm. Genotype was as follow: <i>UAS-mCD8</i>::<i>GFP/+; VT64246-GAL4/UAS-mCD8</i>::<i>GFP</i>. (D) Overexpression of the <i>amn</i> transgene (<i>amn</i>^<i>+</i>) in DPM neurons reversed the deficiency of egg-laying decisions in <i>amn</i>^<i>1</i> background. Each value represents mean ± SEM (n = 22–24, ***P < 0.001, n.s., not statistically significant). Genotypes were as follows: (1) +/+, (2) <i>amn</i>^<i>1</i><i>/amn</i>^<i>1</i>, (3) <i>amn</i>^<i>1</i><i>/amn</i>^<i>1</i><i>; +/+; +/UAS-amn</i>^<i>+</i>, (4) <i>amn</i>^<i>1</i><i>/amn</i>^<i>1</i><i>; +/+; C316-GAL4/+</i>, (5) <i>amn</i>^<i>1</i><i>/amn</i>^<i>1</i><i>; +/+; VT6412-GAL4/+</i>, (6) <i>amn</i>^<i>1</i><i>/amn</i>^<i>1</i><i>; +/+; VT64246-GAL4/+</i>, (7) <i>amn</i>^<i>1</i><i>/amn</i>^<i>1</i><i>; +/+; C316-GAL4/UAS-amn</i>^<i>+</i>, (8) <i>amn</i>^<i>1</i><i>/amn</i>^<i>1</i><i>; +/+; VT6412-GAL4/UAS-amn</i>^<i>+</i>, and (9) <i>amn</i>^<i>1</i><i>/amn</i>^<i>1</i><i>; +/+; VT64246-GAL4/UAS-amn</i>^<i>+</i>. (E) Effects of acute silencing of neuronal activity in DPM neurons on egg-laying decisions. The schematics of the temperature shift protocols are shown above each graph. Each value represents mean ± SEM (n = 17–26, ***P < 0.001. n.s., not statistically significant). Genotypes were as follows: (1) <i>+/+; VT6412-GAL4/+</i>, (2) <i>tubP-GAL80</i>^<i>ts</i><i>/+; UAS-Kir2</i>.<i>1/+</i>, and (3) <i>tubP-GAL80</i>^<i>ts</i><i>/+; VT6412-GAL4/UAS-Kir2</i>.<i>1</i>.</p