Fig 1.
tph is essential for photoreceptor pigmentation after regeneration.
(A) Structure and genomic organization of the tph gene. Top, gene structure of tph; 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 tph. (B-C) Whole-mount in situ hybridization to localize tph expression. The tph 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 tph. In comparison to controls (D), tph knockdowns (E) regenerate pigment cups that appear to lack pigment; 21-day regenerates shown. (F-G) in situ hybridizations to detect tph mRNA levels following RNAi treatment. Relative to controls (F), tph dsRNA-treated animals show dramatically reduced tph mRNA expression; 21-day regenerates shown. Scale bars 200 μm.
Fig 2.
Pigment cup and photoreceptors remain intact in tph(RNAi) animals.
(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 in situ hybridization of tyrosinase (tyr). (A) depicts a maximum intensity confocal projection, whereas (B) represents a single confocal section. (C) By 10 days post-amputation, control and tph(RNAi) animals regenerate both photoreceptors and pigment cup cells, as indicated by VC-1 staining and tyr mRNA expression. Scale bars 20 μm.
Fig 3.
tph(RNAi) animals regenerate pigment cup cells largely devoid of mature melanosomes.
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 tph knockdown animals. In tph 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.
Fig 4.
tph(RNAi) animals lacking eye pigment are slower to orient to light.
Orientation dynamics of control and tph(RNAi) planarians under low (A-C), medium (D-F) and high (G-I) light gradient conditions. Both control and tph(RNAi) worms respond to all three gradients by turning away from the light source, but tph(RNAi) 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 Methods). 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 tph(RNAi) 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.
Fig 5.
Tryptophan derivatives rescue eye pigment in tph knockdowns.
(A) Progress of pigment recovery after injection of 2.5 M 5-HTP is shown at hour-intervals. Eye pigment in tph knockdowns is largely recovered by 3 hr post-injection. (B) tph(RNAi) 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.