Research ArticleLIFE SCIENCES

Hierarchies in light sensing and dynamic interactions between ocular and extraocular sensory networks in a flatworm

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Science Advances  28 Jul 2017:
Vol. 3, no. 7, e1603025
DOI: 10.1126/sciadv.1603025
  • Fig. 1 Planarians show the ability to resolve light inputs of distinct wavelengths.

    (A) Schematic of the binary phototactic choice assay performed. Two simultaneous light inputs of specific wavelengths were provided, and the movement of planarians was measured to determine the choice made—either movement away from λ2 (R1 localization), away from λ1 (R3 localization), or no choice (R2 localization). (B) DI shown here for binary choice assays performed with either of the following light pairs: blue and green (450 and 545 nm), green and red (545 and 625 nm), or blue and red (450 and 625 nm). Results of measurements on 10 worms, n = 6. Error bars indicate SEM. DI as indicated here and detailed in Materials and Methods. (C) Data from binary phototactic choice assay between a 500 nm light input and a second wavelength (525/545/590 nm). DI = (NR3 − NR1)/total number of worms, where NR is the number of worms in a region. A DI value of 1 or −1 indicates a complete aversion or preference for the 500 nm light relative to the second wavelength. A value of 0 indicates no preference. All choice measurements were performed by providing constant light intensity of 316 μW/cm2 (± 10) for both inputs. Results of measurements on 30 worms, n = 5. (D) Wavelength discrimination assay performed with wavelengths from 425 to 625 nm with ~25 nm spectral separation. Results of measurements on 10 worms, n = 4. (B to D) Error bars indicate SEM.

  • Fig. 2 Light intensity–based neutralization of wavelength choice yields an “action” spectrum that resembles an opsin absorption curve.

    (A) Overturning of choice through light intensity dosage. Binary choice experiments were initially performed with equal amounts of photon flux (photon fluence rate − X = 1.59 × 1014/s per cm2). Photon flux of less-aversive input was then increased as indicated. Results of measurements on 10 worms, n = 4. (B) Putative action spectrum determined by measuring the amount of light required for choice neutralization in binary choice assays with respect to 500 nm (near peak). Plotted here is a ratio of photon fluence rates (F500/Fλ) versus wavelength (λ) in nanometers at the point of choice neutralization. The photon fluence rate at 500 nm was kept constant (1.59 × 1014/s per cm2), whereas the amount of light for other wavelengths (Fλ) was modulated to determine minimum light required for choice neutralization in binary assays. For all wavelengths other than 450 nm, an increase in photon fluence rate was required to neutralize choice with 500 nm. Results of measurements on 10 worms, n = 4. This obtained profile was matched with the predicted spectrum of the alpha band of an opsin with a λmax of 475 nm, based on Govardovskii’s template (34).

  • Fig. 3 Smed eye opsin expression is restricted only to eye and is expressed in all PRNs.

    (A) Confocal Z-projected image of FISH of Smed eye opsin (red) and 4′,6-diamidino-2-phenylindole (DAPI) (blue) highlighting cephalic ganglion in the worms. Scale bar, 100 μm. (B) Representative stack of laser scanning confocal image of Smed eye opsin (red) FISH showing individual photoreceptor cells. Scale bar, 10 μm. (C) VC-1 (green) antibody staining along with Smed eye opsin FISH (red) merged with the transmitted image showing eye pigmentation. Dorsal, middle, and ventral stacks (top to bottom) showing coexpression of arrestin and Smed eye opsin. Scale bars, 10 μm.

  • Fig. 4 Mapping recovery of planarian phototactic abilities during regeneration.

    (A) Images showing head regeneration in S. mediterranea from days 1 to 8. (B) Wavelength discrimination assay performed on regenerating worms, with blue and green (450 and 545 nm), green and red (545 and 625 nm), and blue and red (450 and 625 nm) light inputs. Measurements with 10 worms each, n = 6. Gray dashed line indicates the return of single-wavelength input (500 nm) negative phototactic ability (see also fig. S4). (C) Comparative recovery of finer wavelength discrimination ability in regeneration. Binary choice assays with the light wavelength pairs 500/590, 500/545, and 500/590 nm over regeneration. Measurements on 10 worms each, n = 4. (B and C) Error bars indicate SEM. ns, not significant. (D) Immunostaining of planarian cephalic ganglion using SYNORF1 antibody during the course of regeneration from days 3 to 7. Scale bar, 100 μm. *P < 0.05.

  • Fig. 5 Planarian extraocular photoresponse and hierarchical relationship with ocular light response.

    (A) Snapshots of amputated worms showing light avoidance under UV light (350 to 400 nm) and no light avoidance under white light (W). (B) Dose dependence of extraocular photoresponse. Time taken for a headless tail piece (24 hours after amputation) to move out of a circular spot of the 365 or 395 nm light (n = 30; see details in the Supplementary Materials). (C) Interplay between ocular and extraocular light response in regeneration. DI for choice assays between 395 and 500 nm light in anterior and posterior regenerates during time course of regeneration. DI for intact worms is also shown (right). Error bars indicate SEM. (D) Conceptual model indicates multiple facets and diversity in planarian light sensing. The ocular response mediated through the cerebral eye is a “processive” response, including the capacity for wavelength discrimination. This discrimination appears dependent on comparative processing of input signals through the visual network. On the other hand, the extraocular response to UV light appears to be a more rudimentary response with the ventral nerve cord and peripheral network likely functioning as an integrator, collecting and mediating a whole-body response leading to coordinated phototaxis. Both ocular and extraocular light sensing modalities engage the same motor machinery.

  • Fig. 6 Hierarchical light sensing and processing in planarians revealed through recovery of function with regeneration.

    (A) Schematic showing a timeline of return of different phototactic abilities during head regeneration in S. mediterranea. By day 5 after amputation, worms sense light but have no ability to finer intensity discrimination (assayed through wavelength choice), which is acquired gradually significantly later. See (B) for ocular control. (B) Model for hierarchical relationship between networks regulating ocular and extraocular sensing, including switching during regeneration. During days 1 to 4 after amputation, worms show only extraocular response anchored in ventral nerve cord (VNC) and peripheral neurons. By about day 5, ocular response recovers in worms, but signaling flux from cerebral visual networks is unable to override signals resulting from extraocular photosensing. Ocular and extraocular photosensing coexist, but extraocular sensing is dominant in competition assays. Day 7 onward, flux from visual network strengthens, allowing the ocular response to be dominant, as in intact worms. Black arrow indicates visible light input, whereas violet arrow indicates long UV light input.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/3/7/e1603025/DC1

    fig. S1. Planarian light sensing apparatus.

    fig. S2. Planarians are aversive to light across a broad wavelength range.

    fig. S3. Planarian eye opsin knockdown attenuates light sensing across the color spectrum.

    fig. S4. Recovery of single-input light sensing and response during head regeneration.

    fig. S5. Wavelength dependence of extraocular photoreception.

    fig. S6. Extraocular response attenuates in visible light (~405 nm and longer).

    fig. S7. Aggregate time taken for eye-mediated phototaxis is insensitive to light intensity.

    fig. S8. Lack of wavelength discrimination in extraocular photoresponse.

    fig. S9. Phylogenetic tree (maximum likelihood) of planarian opsin sequence.

    fig. S10. Phylogenetic analysis of Smed TRP channels along with known UV-sensitive TRPA1 channels (human and Drosophila).

    fig. S11. Light setup for phototaxis experiments and recording.

    movie S1. Planarian worm shows wavelength discrimination.

    movie S2. Planarians show robust wavelength discrimination (group response).

    movie S3. Imaging Smed eye opsin expression.

    movie S4. Smed eye opsin is expressed in all photoreceptor cells.

    movie S5. Regenerating cephalic ganglion (day 3).

    movie S6. Regenerating cephalic ganglion (day 4).

    movie S7. Regenerating cephalic ganglion (day 5).

    movie S8. Regenerating cephalic ganglion (day 6).

    movie S9. Regenerating cephalic ganglion (day 7).

    movie S10. Extraocular photoreception and phototaxis in planarians.

    movie S11. Light avoidance (extraocular) response to long UV is directional.

  • Supplementary Materials

    This PDF file includes:

    • fig. S1. Planarian light sensing apparatus.
    • fig. S2. Planarians are aversive to light across a broad wavelength range.
    • fig. S3. Planarian eye opsin knockdown attenuates light sensing across the color spectrum.
    • fig. S4. Recovery of single-input light sensing and response during head regeneration.
    • fig. S5. Wavelength dependence of extraocular photoreception.
    • fig. S6. Extraocular response attenuates in visible light (~405 nm and longer).
    • fig. S7. Aggregate time taken for eye-mediated phototaxis is insensitive to light intensity.
    • fig. S8. Lack of wavelength discrimination in extraocular photoresponse.
    • fig. S9. Phylogenetic tree (maximum likelihood) of planarian opsin sequence.
    • fig. S10. Phylogenetic analysis of Smed TRP channels along with known UV-sensitive TRPA1 channels (human and Drosophila).
    • fig. S11. Light setup for phototaxis experiments and recording.
    • Legends for movies S1 to S11

    Download PDF

    Other Supplementary Material for this manuscript includes the following:

    • movie S1 (.avi format). Planarian worm shows wavelength discrimination.
    • movie S2 (.avi format). Planarians show robust wavelength discrimination (group response).
    • movie S3 (.avi format). Imaging Smed eye opsin expression.
    • movie S4 (.avi format). Smed eye opsin is expressed in all photoreceptor cells.
    • movie S5 (.avi format). Regenerating cephalic ganglion (day 3).
    • movie S6 (.avi format). Regenerating cephalic ganglion (day 4).
    • movie S7 (.avi format). Regenerating cephalic ganglion (day 5).
    • movie S8 (.avi format). Regenerating cephalic ganglion (day 6).
    • movie S9 (.avi format). Regenerating cephalic ganglion (day 7).
    • movie S10 (.avi format). Extraocular photoreception and phototaxis in planarians.
    • movie S11 (.avi format). Light avoidance (extraocular) response to long UV is directional.

    Download Movies S1 to S11

    Files in this Data Supplement: