Tryptophan derivatives rescue eye pigment in <i>tph</i> knockdowns.

<p>(A) Progress of pigment recovery after injection of 2.5 M 5-HTP is shown at hour-intervals. Eye pigment in <i>tph</i> knockdowns is largely recovered by 3 hr post-injection. (B) <i>tph(RNAi</i>) and control worms were injected with DMSO (n = 3), 100 mM tryptophan (n = 3), 100 mM 5-HTP (n = 5), 250 mM serotonin (n = 5), or 10 mM L-DOPA (n = 3). Images show phenotype at pre-injection and 24 hr time points. Insets highlight the increased pigmentation of control worms injected with 5-HTP vs. DMSO control. Scale bar is 200 μm. (C) Comparison of traditional and hypothesized melanin synthesis pathways.</p

Pigment cup and photoreceptors remain intact in <i>tph(RNAi)</i> animals.

<p>(A-B) The planarian visual system. Photoreceptors (magenta) are visualized by immunofluorescence with VC-1 antibody against arrestin, and pigment cup cells (green) are visualized by fluorescent <i>in situ</i> hybridization of <i>tyrosinase (tyr)</i>. (A) depicts a maximum intensity confocal projection, whereas (B) represents a single confocal section. (C) By 10 days post-amputation, control and <i>tph(RNAi)</i> animals regenerate both photoreceptors and pigment cup cells, as indicated by VC-1 staining and <i>tyr</i> mRNA expression. Scale bars 20 μm.</p

<i>Tryptophan hydroxylase</i> Is Required for Eye Melanogenesis in the Planarian <i>Schmidtea mediterranea</i>

<div><p>Melanins are ubiquitous and biologically important pigments, yet the molecular mechanisms that regulate their synthesis and biochemical composition are not fully understood. Here we present a study that supports a role for serotonin in melanin synthesis in the planarian <i>Schmidtea mediterranea</i>. We characterize the tryptophan hydroxylase (<i>tph</i>) gene, which encodes the rate-limiting enzyme in serotonin synthesis, and demonstrate by RNA interference that <i>tph</i> is essential for melanin production in the pigment cups of the planarian photoreceptors. We exploit this phenotype to investigate the biological function of pigment cups using a quantitative light-avoidance behavioral assay. Planarians lacking eye pigment remain phototactic, indicating that eye pigmentation is not essential for light avoidance in <i>S</i>. <i>mediterranea</i>, though it improves the efficiency of the photophobic response. Finally, we show that the eye pigmentation defect observed in <i>tph</i> knockdown animals can be rescued by injection of either the product of TPH, 5-hydroxytryptophan (5-HTP), or serotonin. Together, these results highlight a role for serotonin in melanogenesis, perhaps as a regulatory signal or as a pigment substrate. To our knowledge, this is the first example of this relationship to be reported outside of mammalian systems.</p></div

<i>tph(RNAi)</i> animals regenerate pigment cup cells largely devoid of mature melanosomes.

<p>All micrographs are transverse sections of the planarian eye. (A) Electron micrograph of the photoreceptor rhabdomes and the pigment cup cells in control RNAi animals. Panel (B) is a magnified view of the pigment cup cells highlighting the mature melanosomes. (C-D) Electron micrograph of the photoreceptors of <i>tph</i> knockdown animals. In <i>tph</i> knockdowns the photoreceptors and the pigment cups are intact, but the melanosomes appear immature and less electron dense compared to controls. Scale bars in A and C are 2000 nm; in B and D are 500 nm.</p

<i>tph(RNAi)</i> animals lacking eye pigment are slower to orient to light.

<p>Orientation dynamics of control and <i>tph(RNAi</i>) planarians under low (A-C), medium (D-F) and high (G-I) light gradient conditions. Both control and <i>tph(RNAi)</i> worms respond to all three gradients by turning away from the light source, but <i>tph(RNAi)</i> worms react less efficiently, especially in low light gradients. (A, D, G) Center-of-mass tracking results for both control and tph(RNAi) populations (n = 10). Triangles indicate the orientation of the light gradient, with the light source on the right. All points are color-coded for time, as shown by color bar legends. At t = 0 s, the worms are manually oriented toward the light source. (B, E, H) Orientational order parameters as a function of time for both populations (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127074#sec002" target="_blank">Methods</a>). The error bars show SEM at selected time points. The dashed y = 0 line is a guide for the eye showing the threshold between orientation biased toward the light souce (y > 0) and away from the light source (y < 0). Note the decrease in time scale as the gradient strength is increased, indicating a faster negative phototactic response of both populations at increased illumination. (C, F, I) Angular distributions of velocities at different time periods (corresponding to grayed regions in B, E and H) showing the reversal of polarity at different rates between control and <i>tph(RNAi</i>) worms. The size of the wedge indicates the proportion of worms traveling in the given direction in that time period, where 0° is towards the light source, and 180° is away from the light source. A two-way ANOVA test confirms statistical difference at the 1% level for TPH and control animals as well as for the three different gradient settings.</p

<i>tph</i> is essential for photoreceptor pigmentation after regeneration.

<p>(A) Structure and genomic organization of the <i>tph</i> gene. Top, gene structure of <i>tph</i>; the 5’-untranslated region, the coding region, and the 3’-untranslated region are depicted in yellow, red and blue, respectively. Below, organization of genomic regions (supercontigs) that encode <i>tph</i>. (B-C) Whole-mount <i>in situ</i> hybridization to localize <i>tph</i> expression. The <i>tph</i> gene was expressed in the pigment cups, the peripharyngeal secretory cells (dorsal) and cells within the central and peripheral nervous systems (ventral). (D-E) RNAi-mediated knockdown of <i>tph</i>. In comparison to controls (D), <i>tph</i> knockdowns (E) regenerate pigment cups that appear to lack pigment; 21-day regenerates shown. (F-G) <i>in situ</i> hybridizations to detect <i>tph</i> mRNA levels following RNAi treatment. Relative to controls (F), <i>tph</i> dsRNA-treated animals show dramatically reduced <i>tph</i> mRNA expression; 21-day regenerates shown. Scale bars 200 μm.</p