Cells expressing Poxn in larval and adult brains are post-mitotic neurons.

<p>(<b>A–D</b>) Colocalization (white) in nuclei of Poxn (red) and Elav (green) proteins, visualized by immunofluorescent staining, is shown in wild-type (<i>Ore-R</i>) brain hemispheres of first (A), second (B), and late third instar larvae (C), and of adults (D). Staining of all <i>Poxn</i>-nuclei with Elav was corroborated by visual inspection in single layers of the Z-stacks. Panels show maximum intensity projections of CLSM sections of Z-stacks extending over 43 μm (A), 45 μm (B), and 50 μm (C) at 63x magnification, and of a Z-stack extending over 26 μm at 40x magnification (D). Scale bars: 10 μm (A–C) and 20 μm (D).</p

Most <i>Poxn</i>-neurons survive metamorphosis and continue to express Poxn in the adult brain.

<p>(<b>A–C</b>) The DC of <i>Poxn</i>-neurons, visualized by immunofluorescent staining for β-Gal (red) and GFP (green), is shown in the brain of a <i>w</i>^<i>1118</i> <i>UAS-Flp/</i>+; <i>tub-Gal80</i>^<i>ts</i><i>/+</i>; <i>Act5C>polyA>lacZ</i>.<i>nls1 Poxn-Gal4-13-1/Poxn-CD8</i>::<i>GFP</i> female. A maximum intensity projection of the same CLSM sections of an entire Z-stack is shown in the red channel (A), green channel (B), and in both channels (C) at 63x magnification. β-Gal-positive nuclei (red) belong to cells that expressed Poxn at the time of heat shock during the third larval instar (feeding stage). The membrane-associated CD8::GFP fusion protein labels Poxn-expressing cells at the time of fixation. (<b>D</b>–<b>F</b>) Maximum intensity projections of substacks at 0–5 μm (D), 5–10 μm (E), and 10–18 μm (F) of the Z-stack extending from 0 (anterior) to 31 μm (posterior) shown in (C), which includes all <i>Poxn</i>-nuclei. Virtually all β- Gal-labeled neurons also express GFP. Similar results were obtained for the VC of <i>Poxn</i>-neurons (data not shown). Scale bar: 20 μm.</p

Number of Poxn-expressing cells per brain hemisphere in embryos, larvae, and adults.

<p>Number of Poxn-expressing cells per brain hemisphere in embryos, larvae, and adults.</p

<i>w¹¹¹⁸</i> flies are not only optomotor blind but also dazzled by light.

<p>Courtship vigor indices were measured in single-choice courtship assays with mature males of indicated genotypes and receptive (<b>A</b>) or decapitated (<b>B</b>) <i>Ore-R</i> virgin females in daylight (yellow columns), under dim red light (red columns), or under low-intensity light conditions (orange columns). The numbers below the columns indicate the number of couples observed that initiated courtship, and error bars always represent double standard errors of the mean. Data for <i>ninaB^360d</i> males in panel A are from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077904#pone.0077904-Krstic1" target="_blank">[4]</a>.</p

Effect of <i>white</i> mutation on sexual arousal of males.

<p>Models explaining possible effects of extra-retinal lack of White function on general sexual arousal of males. Increased sexual arousal of <i>white</i> males over time <i>t</i> may be caused by (<b>A</b>) a change in the general alertness from a ground state in wild-type flies (solid blue line) to an enhanced state of alertness in <i>w¹¹¹⁸</i> mutants (broken blue line), resulting in a shortened, sensitized period of latency to sustained courtship, <i>t_s</i>, as compared to the control period of latency, <i>t_c</i>, or (<b>B</b>) a reduction of the threshold from its wild-type (solid red line) to a reduced <i>w¹¹¹⁸</i> level (broken red line), which relevant stimuli need to reach to elicit sustained courtship, also resulting in a shortened, sensitized period of latency, <i>t_s</i>. Since the processing of various sensory information by the CNS is not affected in either model, both changes can be taken to result from an enhanced sensitivity of the CNS to courtship stimuli. (<b>C</b>) Alternatively, <i>w¹¹¹⁸</i> males may reach the threshold level for sustained courtship faster (broken green line in <i>w¹¹¹⁸</i> mutants with slope greater than that of solid green line in wild-type flies) because of an altered processing or integration of courtship information by the CNS or an enhanced sensitivity to relevant stimuli, for example pheromones, of sensory neurons in the PNS. Also in this model, the augmented sexual arousal leads to a shortened, sensitized period of latency to sustained courtship, <i>t_s</i>, because of a steeper slope of the broken versus the solid green line.</p

Formation of ellipsoid body depends strongly on <i>Poxn</i> function.

<p>Brains of <i>w</i>^<i>1118</i>; <i>Poxn-CD8</i>::<i>GFP</i> (<b>A-H</b>) or <i>w</i>^<i>1118</i>; <i>Poxn</i>^{<i>ΔM22-B5</i>} <i>Poxn-Sbl-107</i>; <i>Poxn-CD8</i>::<i>GFP</i> (<b>A’-H’</b>) pupae, immunostained for GFP at 0 h APF (A,A’), 10 h APF (B,B’), 15 h APF (C,C’), 20 h APF (D,D’), 30 h APF (E, E’), 40 h APF (F,F’), 45 h APF (G,G’), and 50 h APF (H,H’), are shown as maximum intensity projections of confocal Z-stacks at 20x magnification (panels except E, G, and G’ show substacks that remove some of the cell bodies but improve the visibility of projections). Z-stacks extended over 89 μm (A), 67 μm (B), 82 μm (C), 52 μm (D), 72 μm (E), 54 μm (F), 79 μm (G), 51 μm (H), 44 μm (A’), 65 μm (B’), 90 μm (C’), 88 μm (D’), 56 μm (E’), 46 μm (F’), 102 μm (G’), and 66 μm (H’). Arrows point to the bilateral arc-like mlALTs visible at all pupal stages. Filled arrowheads point to the arborizations at the developing bulbs, while arrowheads point to the midline of the forming ellipsoid body neuropil. Note that arborizations at the lateral horn (LH) and antennal lobe (AL) become prominent around 40 h APF in the wild-type brain (F-H), while they are slightly delayed in mutant brains where they occur at the end of the stalled projections (G’,H’). Pupal stages, measured as hours APF, imply a developmental temporal variation corresponding to about 5%. Scale bars: 50 μm.</p

Projections of <i>Poxn</i>-neurons and axonal tracts labeled by FasII in wild-type and <i>Poxn</i> mutant brains of late third instar larvae.

<p><b>(A-D)</b><i>Poxn</i>-neurons, immunostained for the expression of <i>Poxn-CD8</i>::<i>GFP</i> (green), and neuropils, immunostained for the expression of FasII (red), are shown in brains of <i>w</i>^<i>1118</i>; <i>Poxn-CD8</i>::<i>GFP</i> <b>(A,D)</b> and <i>w</i>^<i>1118</i>; <i>Poxn</i>^{<i>ΔM22-B5</i>} <i>Poxn-Sbl-107</i>; <i>Poxn-CD8</i>::<i>GFP</i> <b>(B,C)</b> late third instar larvae in the red and green channel (A-C) and only in the green channel (D) at 40x magnification. The two <i>Poxn</i> mutant brains display the two types of mutant projection patterns commonly observed: projections from the VC seem to follow the mALT instead of the mlALT (B), or adopt an entirely different path, eventually running parallel to the projections from the DC before they stall (C). Scale bars: 50 μm. <b>(E-H')</b> Central parts of substacks of (A) are shown from posterior to anterior at 10–15 μm (E,E'), 23–28 μm (F,F'), 28–30 μm (G,G'), and 30–35 μm (H,H') as maximum intensity projections of CLSM sections of a Z-stack extending over 85 μm in both channels (E-H) and only in the green channel (E'-H') at 40x magnification. <i>Poxn</i>-neuron tracts that pass through the SEC and do or do not co-express FasII are indicated by arrowheads and arrows, respectively. DC, dorsal cluster of <i>Poxn</i>-neurons; vl, vertical lobe of mushroom bodies; mALT, middle antennal lobe tract; mlALT, mediolateral antennal lobe tract; ml, medial lobe of mushroom bodies; pBU, primordial bulb; VC, ventral cluster of <i>Poxn</i>-neurons.</p

Role of White protein in the biosynthesis of <i>Drosophila</i> eye pigments and the neurotransmitters serotonin and dopamine.

<p>The White protein is an ABC transporter that, in combination with the Scarlet protein, transports tryptophan and, in combination with the Brown protein, guanine across the cell membrane into the cytoplasm <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077904#pone.0077904-Ewart1" target="_blank">[7]</a>. Tryptophan is a precursor of the <i>Drosophila</i> Ommochrome pigment xanthommatin (brown) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077904#pone.0077904-Summers1" target="_blank">[6]</a>, but is also a precursor of the neurotransmitter serotonin <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077904#pone.0077904-Murch1" target="_blank">[13]</a>, as illustrated on the left. Guanine is a precursor of tetrahydrobiopterin (BH₄), which in turn is a precursor of most Drosopterins (red eye pigments of <i>Drosophila</i>) but also an essential cofactor in the conversion of tyrosine to dopamine, as indicated on the right, and of tryptophan to serotonin, as depicted on the left <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077904#pone.0077904-Visser1" target="_blank">[11]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077904#pone.0077904-Thny1" target="_blank">[12]</a>.</p

Lack of extra-retinal White function influences sexual orientation of males.

<p>Courtship vigor indices were measured in single-choice courtship assays with mature males of indicated genotypes and decapitated males (hatched columns) or decapitated females (filled columns) in the dark (red columns) or in daylight (yellow columns). The numbers below the columns indicate the number of males observed that initiated courtship. Data for <i>Ore-R</i> males are from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077904#pone.0077904-Krstic1" target="_blank">[4]</a>.</p

Projection patterns of <i>Poxn</i>-neurons in wild type and <i>Poxn</i> mutants during larval development.

<p>(<b>A–H</b>) <i>Poxn</i>-neurons are visualized by the expression of <i>Poxn-CD8</i>::<i>GFP</i> and immunofluorescent staining for GFP in first (A,E), late second (B,F), and late third instar (C,D,G,H) brains of <i>w</i>^<i>1118</i>; <i>Poxn-CD8</i>::<i>GFP</i> (A-C), <i>w</i>^<i>1118</i>; <i>Poxn</i>^{<i>ΔM22-B5</i>}; <i>Poxn-CD8</i>::<i>GFP</i> (E-G), <i>w</i>^<i>1118</i>; <i>Poxn</i>^{<i>ΔM22-B5</i>} <i>Poxn-SuperA-158</i>; <i>Poxn-CD8</i>::<i>GFP</i> (D), and <i>w</i>^<i>1118</i>; <i>Poxn</i>^{<i>ΔM22-B5</i>} <i>Poxn-Sbl-107</i>; <i>Poxn-CD8</i>::<i>GFP</i> (H) larvae. Note that the projection patterns of the latter two resemble those in wild-type (C) and <i>Poxn</i> mutant brains (G), respectively. Arrowheads in (A-C) point to arc-like projections of the VC and their targets in the lateral protocerebrum, and arrow in (C) points to tracts of the SEC, emanating from the DC of wild-type brains. Asterisks in (G) mark aberrant projections from both <i>Poxn</i> clusters in a <i>Poxn</i> mutant brain. Dashed lines indicate midlines of the flattened brains viewed along the anteroposterior axis. Panels show maximum intensity projections of CLSM sections of Z-stacks extending over 41 μm (A), 50 μm (B), 53 μm (C), 49 μm (D), 75 μm (E), 36 μm (F), 48 μm (G), and 77 μm (H) at 20x magnification. Panel H shows the same brain as <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0176002#pone.0176002.g006" target="_blank">Fig 6B</a>. DC, dorsal cluster of <i>Poxn</i>-neurons; LH, lateral horn; VC, ventral cluster of <i>Poxn</i>-neurons. Scale bars: 20 μm.</p