Research ArticleORGANISMAL BIOLOGY

Pushing the limits of photoreception in twilight conditions: The rod-like cone retina of the deep-sea pearlsides

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Science Advances  08 Nov 2017:
Vol. 3, no. 11, eaao4709
DOI: 10.1126/sciadv.aao4709
  • Fig. 1 Vertebrate opsin gene phylogeny and pearlside opsin gene expression.

    (A) The pearlside retinal transcriptomes (n = 5 per species) contained three opsin genes: one rod opsin (rh1) and two rhodopsin-like 2 (rh2) cone opsins. Black spheres indicate Bayesian posterior probabilities >0.8. Asterisk indicates that this opsin gene class was not present in the pearlside transcriptome. lws, long-wavelength sensitive; sws1 and sws2, short-wavelength sensitive 1 and 2; va, vertebrate ancient opsin (outgroup). A detailed phylogeny and GenBank accession numbers are shown in fig. S1. Pearlside-specific accession numbers are given in table S4. (B) The per-species mean of the proportional opsin gene expression shows the almost exclusive use of cone opsins in pearlside vision.

  • Fig. 2 Vertebrate phylogenies of phototransduction cascade genes and phototransduction cascade gene expression.

    (A and B) Vertebrate Gα transducin (A) and vertebrate arrestin (B) gene phylogenies. Black spheres indicate Bayesian posterior probabilities >0.8. Detailed phylogenies and GenBank accession numbers are shown in figs. S3 and S4. Pearlside-specific accession numbers are given in table S4. gnat2, G protein subunit α transducin 2; gnat1, G protein subunit α transducin 1; “taste,” G protein subunit α transducin 3; arrb2, arrestin β2; arr3, arrestin 3; sag, s-antigen arrestin [saga is present in the outer segment, and sagb is present in the synapses (16)]; arrb1, arrestin β1. (C and D) The per-species mean of the proportional transducin (C) and arrestin (D) gene expression shows the almost exclusive use of cone transducin (gnat2) and cone arrestin (arr3) in pearlside vision.

  • Fig. 3 Absorbance spectra of photopigments expressed in two representative Maurolicus spp.

    (A) Experimentally determined absorbance spectra of M. muelleri and human (Homo sapiens; control) rod opsin photopigments, reconstituted with 11-cis-retinal. For all pigments, representative dark (filled circles) and light-bleached (open circles) spectra are shown, with difference spectra (open diamonds) that have been fitted with a Govardovskii rhodopsin/vitamin A1 template (63) (black line) in the inset to determine the λmax. (B) Predicted spectral sensitivities of rh1 and the two rh2 opsins found in the two pearlside species, M. muelleri (black) and M. mucronatus (gray).

  • Fig. 4 Opsin R-ISH and the distribution of the two photoreceptor types in the retina of M. muelleri.

    (A and C) Retinal cryosections showing the expression of rh1 (A) and rh2 (C) opsin genes in cryosections. Arrowheads highlight labeled cells. Note that both genes are expressed in rod-like photoreceptor cells. (B) Distribution of rh1 photoreceptors labeled with anti-rhodopsin antibodies. Each dot represents one labeled photoreceptor. Black arrows indicate the orientation of the retina. N, nasal; V, ventral. (D) Topographic map of rh2 photoreceptor densities (cells × 104 mm−2). Percentages indicate the proportion of each cell type. Scale bars, 50 μm (A and C) and 1 mm (B and D).

  • Fig. 5 Morphology of the two photoreceptor types in M. muelleri.

    (A) Schematic of the rod-like cone (yellow; left) and rod (blue; right) drawn from the 3D reconstruction using 3View. OS, outer segment; IS, inner segment; SE, synaptic ending; Di, discs; Mt, mitochondria; ILM, inner limiting membrane; Nc, nucleus. Note the displaced nucleus and synaptic ending in the rod. (B) Immunofluorescence labeling of transverse retinal cryosections. Rod outer segments were labeled with anti-rhodopsin antibodies (red), inner segments with NeuN (white), cell nuclei with 4′,6-diamidino-2-phenylindole (DAPI) (blue), and synaptic connections with synapsin (green). Note that NeuN does not usually stain photoreceptor inner segments, but in M. muelleri, the inner segments of the rods were strongly labeled compared to the rod-like cones. PRL, photoreceptor layer; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; c, rod-like cone; r, rod; cse, rod-like cone synaptic ending; rce, rod synaptic ending. Scale bar, 5 μm. (C to F) TEM of transverse retinal sections showing the two photoreceptor types (C), the polysynaptic ending of the rod-like cone (D), the oligosynaptic ending of the rod (E), and the sealed discs and incisures of the outer segments (F). The white arrowheads in (D) and (E) show the synaptic ribbons, and the black arrowheads in (F) show the incisures present in the rod-like cone. Scale bars, 2 μm (C) and 1 μm (D to F).

  • Fig. 6 Ambient light environment and pearlside visual capabilities.

    (A) Light levels associated with different photoreceptor functionalities. M. muelleri is only active during mesopic and low-level photopic light intensities (39). R, rod; C, cone. Scotopic vision is defined by the use of rods. Mesopic vision is defined by the use of both rods and cones limited by cone threshold and rod saturation. Photopic vision is defined by the use of cones and ends when light intensities start to be damaging (75). Environmental light sources (from left to right) are as follows: starlight, full moon, civil twilight, sunset/sunrise, and sunlight (76). Figure partially redrawn from Hood and Finkelstein (75). (B) Spectral sensitivity curves of the pearlside M. muelleri rh2 (a, black line; predicted λmax = 441 nm; fig. S7), the deep-sea myctophid Symbolophorus evermanni rh1 [b, dark gray line; λmax = 476 (23)], and the nocturnal squirrelfish Neoniphon sammara rh1 [c, light gray line; λmax = 502 nm (77)] along with the relative downwelling vector irradiance spectra (courtesy of S. Johnsen) of their respective light environments: twilight (−6.5° solar elevation) at the surface (d, black dashed line), downwelling light at 500 m (e, dark gray dashed line), and moonlight (full moon at 70° elevation above horizon) at the surface (f, light gray dashed line). Note how the spectral sensitivity of each species is tuned to the light spectra of their respective habitat.

  • Table 1 Characteristics summary (morphology, opsin, phototransduction cascade, and electrophysiology) of the transmuted photoreceptors of different species compared to true rods and true cones.

    Lizard data are for the genus Anolis. Lamprey is the sea lamprey Petromyzon marinus. Salamander is the tiger salamander Ambystoma tigrinum. Pearlside is the Mueller’s pearlside M. muelleri. The snake with the cone-like rods is the diurnal garter snake Thamnophis proximus. The snake with the rod-like cones data are for the nocturnal genus Hypsiglena. The gecko is the nocturnal Tokay gecko Gekko gekko. The skate is the genus Raja. R, true rod; C, true cone; n.a., not available; poly, polysynaptic.

    Photoreceptor
    characteristics
    True
    rod (1, 19)
    True
    cone (1, 19)
    Cone-like rodRod-like cone
    Snake
    (7, 78)
    Skate
    (10, 45, 79)
    Lizard
    (5, 80, 81)
    Lamprey
    (9, 82, 83)
    Gecko
    (6, 8486)
    Snake
    (4, 35)
    Salamander
    (8, 87)
    Pearlside
    (this study)
    Outer segment
    shape
    Long, rod-
    shaped
    (cylindrical)
    Short, cone-
    shaped (distally
    tapering)
    CRCRRRRR
    Outer segment
    discs
    Individual sealed
    disc, separated
    from the plasma
    membrane
    Discs continuous
    with the plasma
    membrane (open)
    RRn.a.R CR Cn.a.n.a.R
    IncisurePresentAbsentn.a.n.a.CCRn.a.RR
    ParaboloidAbsentPresentCRRR
    Oil dropletAbsentSometimes presentRn.a.Cn.a.RRRR
    Synaptic endingSmall, spherical,
    oligosynaptic
    Large, conical,
    flat-end base,
    polysynaptic
    n.a.Cn.a.R C
    Small poly
    Cn.a.R C
    Small poly
    R C
    Small poly
    Opsinrh1sws1, sws2, lws, rh2RRRRC
    rh2
    C
    sws1
    lws
    C
    sws2
    C
    rh2-1
    rh2-2
    Spectral
    sensitivity
    (nm)
    480–510rh2, 450–530
    sws1, 360–440
    sws2, 400–450
    m/lws, 510–560
    R
    482
    R
    500
    R
    491
    n.a.C
    521
    C
    358, 536
    C
    432
    C
    441 (both pigments)
    Phototransduction
    cascade
    Rod-likeCone-likeRn.a.n.a.n.a.C(R)n.a.RC
    Cell physiologyRod properties
    (high sensitivity)
    Cone properties
    (fast, never
    saturate)
    n.a.R Cn.a.RRn.a.n.a.n.a.
  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/3/11/eaao4709/DC1

    fig. S1. Gene coding region (CDS) inferred vertebrate opsin gene phylogeny.

    fig. S2. Reconstruction of amino acid changes at known key spectral tuning sites of pearlside opsins.

    fig. S3. Vertebrate Gα transducin gene phylogeny.

    fig. S4. Vertebrate arrestin gene phylogeny.

    fig. S5. rh2 opsin gene conversion phylogeny.

    fig. S6. Topographic distribution of rod-like cone photoreceptors in three individuals of M. muelleri.

    fig. S7. Topographic distribution of ganglion cells (excluding amacrine cells and glial cells) in three individuals of M. muelleri.

    fig. S8. Expression of rh2 and rh1 opsin genes from sectional RNA in situ hybridization analyses of the eye of M. muelleri.

    fig. S9. Location of the ”true“ rod photoreceptors and their distribution across the retina in M. muelleri.

    fig. S10. Manual approach to extract genes from back-mapped reads.

    table S1. Summary of transcriptomes, GenBank accession numbers, opsin mapping (including base pair coverage), and proportional opsin gene expression.

    table S2. Summary of transcriptomes, GenBank accession numbers, transducin mapping (including base pair coverage), and proportional transducin gene expression.

    table S3. Summary of transcriptomes, GenBank accession numbers, arrestin mapping (including base pair coverage), and proportional arrestin gene expression.

    table S4. Single-gene GenBank accession numbers of gene sequences produced during this study.

    table S5. Summary of the stereology parameters used for the analysis of the rod-like cone photoreceptors and ganglion cell distribution along with the quantitative results obtained using the optical fractionator methods in six retinas of M. muelleri.

    movie S1. 3D reconstruction of the two photoreceptor types in M. muelleri.

    movie S2. Close-up 3D reconstruction of the nucleus and synaptic terminal of the two photoreceptor types in M. muelleri.

  • Supplementary Materials

    This PDF file includes:

    • fig. S1. Gene coding region (CDS) inferred vertebrate opsin gene phylogeny.
    • fig. S2. Reconstruction of amino acid changes at known key spectral tuning sites of pearlside opsins.
    • fig. S3. Vertebrate Gα transducin gene phylogeny.
    • fig. S4. Vertebrate arrestin gene phylogeny.
    • fig. S5. rh2 opsin gene conversion phylogeny.
    • fig. S6. Topographic distribution of rod-like cone photoreceptors in three individuals of M. muelleri.
    • fig. S7. Topographic distribution of ganglion cells (excluding amacrine cells and glial cells) in three individuals of M. muelleri.
    • fig. S8. Expression of rh2 and rh1 opsin genes from sectional RNA in situ hybridization analyses of the eye of M. muelleri.
    • fig. S9. Location of the “true” rod photoreceptors and their distribution across the retina in M. muelleri.
    • fig. S10. Manual approach to extract genes from back-mapped reads.
    • table S1. Summary of transcriptomes, GenBank accession numbers, opsin mapping (including base pair coverage), and proportional opsin gene expression.
    • table S2. Summary of transcriptomes, GenBank accession numbers, transducin mapping (including base pair coverage), and proportional transducin gene expression.
    • table S3. Summary of transcriptomes, GenBank accession numbers, arrestin mapping (including base pair coverage), and proportional arrestin gene expression.
    • table S4. Single-gene GenBank accession numbers of gene sequences produced during this study.
    • table S5. Summary of the stereology parameters used for the analysis of the rod-like cone photoreceptors and ganglion cell distribution along with the quantitative results obtained using the optical fractionator methods in six retinas of M. muelleri.
    • Legends for movies S1 and S2

    Download PDF

    Other Supplementary Material for this manuscript includes the following:

    • movie S1 (.mp4 format). 3D reconstruction of the two photoreceptor types in M. muelleri.
    • movie S2 (.mp4 format). Close-up 3D reconstruction of the nucleus and synaptic terminal of the two photoreceptor types in M. muelleri.

    Files in this Data Supplement: