Co-expression of <i>tmt-opsins</i> with <i>choline acetyltransferase</i> in distinct inter- and motorneurons.

<p>Two-color ISH of <i>tmtops1b</i> (blue) and <i>chat1/2</i> (red/red fluorescence) on coronal adult medaka brain sections. Transversal planes of (A), (D), and (G) correspond to planes in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001585#pbio-1001585-g003" target="_blank">Figure 3H, D, and F</a>, respectively. (B, E, H) Magnification of boxed areas. (C, F, I) Fluorescent images of <i>chat1</i>/<i>2</i> staining. Arrowheads, co-expressing cells. Scale bars, 50 µm. Co-staining in facial nerve motorneurons (A–C), in interneurons of the periventricular grey zone of the tectum (D–F), and in interneurons of the rostral tegmental nucleus (G–I). Note that fluorescent signal of <i>chat1/2</i> expression can be quenched in areas of strong <i>tmt-opsin</i> staining (for higher magnification see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001585#pbio.1001585.s010" target="_blank">Figure S10</a>).</p

<i>Tmt</i>- and <i>val-opsin</i> co-expression domains in inter- and motorneuron cluster are conserved in vertebrates.

<p>Two-color ISH of <i>tmt-opsin1b</i> (blue) and <i>val-opsins</i> (red/red fluorescence) on coronal medaka (A–F) and zebrafish (G–L) sections. Magnifications are indicated as boxes. Scale bars, 50 µm. <i>Tmtops1b</i> and <i>valopb</i> co-expression in the central posterior thalamic nucleus of medaka (A–B) and zebrafish (G–H), in the dorsal tegmental nucleus in medaka (C) and zebrafish (I) and in the facial nerve nucleus in medaka (D–F) and zebrafish (J–L).</p

Co-Expression of VAL- and TMT-Opsins Uncovers Ancient Photosensory Interneurons and Motorneurons in the Vertebrate Brain

<div><p>The functional principle of the vertebrate brain is often paralleled to a computer: information collected by dedicated devices is processed and integrated by interneuron circuits and leads to output. However, inter- and motorneurons present in today's vertebrate brains are thought to derive from neurons that combined sensory, integration, and motor function. Consistently, sensory inter­motorneurons have been found in the simple nerve nets of cnidarians, animals at the base of the evolutionary lineage. We show that light-sensory motorneurons and light-sensory interneurons are also present in the brains of vertebrates, challenging the paradigm that information processing and output circuitry in the central brain is shielded from direct environmental influences. We investigated two groups of nonvisual photopigments, VAL- and TMT-Opsins, in zebrafish and medaka fish; two teleost species from distinct habitats separated by over 300 million years of evolution. TMT-Opsin subclasses are specifically expressed not only in hypothalamic and thalamic deep brain photoreceptors, but also in interneurons and motorneurons with no known photoreceptive function, such as the typeXIV interneurons of the fish optic tectum. We further show that TMT-Opsins and Encephalopsin render neuronal cells light-sensitive. TMT-Opsins preferentially respond to blue light relative to rhodopsin, with subclass-specific response kinetics. We discovered that <i>tmt-opsins</i> co-express with <i>val-opsins</i>, known green light receptors, in distinct inter- and motorneurons. Finally, we show by electrophysiological recordings on isolated adult tectal slices that interneurons in the position of typeXIV neurons respond to light. Our work supports “sensory-inter-motorneurons” as ancient units for brain evolution. It also reveals that vertebrate inter- and motorneurons are endowed with an evolutionarily ancient, complex light-sensory ability that could be used to detect changes in ambient light spectra, possibly providing the endogenous equivalent to an optogenetic machinery.</p></div

Phylogenetic and sequence analyses of TMT-Opsins and Encephalopsins.

<p>(A) Maximum likelihood (ML) and neighbor joining (NJ) trees group TMT-Opsins into three distinct subclasses conserved across vertebrates with high branch support. Encephalopsins form a fourth, closely related group. The topology of the NJ tree is shown, and support values are given as NJ/ML next to critical branches. Grey box, ciliary-type opsins; yellow, TMT-Opsins; blue, Encephalopsins. (B, C, D, F) Conserved sequence stretches in the c-terminus of the indicated opsin subfamilies. Numbers on <i>x</i>-axis refer to the amino acid positions in bovine rhodopsin (B, C, D, F) or human Encephalopsin (G). (E) Comparative analysis of characteristic opsin sequence features critical for photopigment function. Bovine rhodopsin is used as reference for counterion position. (G) Conserved sequence stretch in the c-terminus of Encephalopsins.</p

NASCAR workflow.

<p>(1) Seeds perfectly matching between query (i.e. enhancer) and target (e.g. genomic window) sequence (small black segments) are extended up- and downstream (red segments) using a match/mismatch scoring scheme to generate a raw motif profile. Motifs that overlap the predefined window boundaries are also taken into account and virtually extend the window (grey areas). (2) As a next step, overlapping regions of the extracted raw motifs in the target sequence are determined (grey areas) and the smaller motif truncated whenever it overlaps a larger one (2 to 3). Motifs smaller than the initial seed size after truncation are discarded in this step. (3) Same filtering procedure is repeated in the query sequence for the processed profile (3 to 4). (4) Motifs below the noise threshold (bright blue segment) are discarded and the basic similarity (“PURE”) score calculated from the fully filtered motif profile (dark blue). (5) In addition, a pattern detection method searches for co-linear arrangements in the profile (grey area). Panel shows the same motif composition as (4) but in a co-linear configuration. This time, the motif below the noise threshold (bright pink) is kept as it is contained in a pattern. The score of the full pattern (all pink motifs) is subsequently added to the previously calculated basic score, resulting in the “COMB” score. For a given enhancer, the whole process is repeated window by window until the last window in the target sequence is reached.</p

Deletion of conserved motifs (grey area) from the predicted fish regions results in change of enhancer activity in both tested constructs.

<p><b>Schematic on the right shows the motif configuration in the human and medaka locus for hs1344 and hs865, respectively. The full grey area is deleted from the medaka enhancer and the remaining sequence tested for reporter expression. Images on the left show the reporter activity of the medaka constructs prior to and after the deletion.</b> Hs1344 ol2-1delta gains two symmetrical domains in the midbrain (red arrowheads), while hs865 ol2-1delta shows a loss of expression in the central part of the original domain.</p

<i>TMT-Opsin</i> expression in inter- and motorneuron nuclei is maintained from larvae to adult stages.

<p>(Left topmost panel) Dorsal view of a schematized medaka larva; red boxes indicate positions of sections displayed below. (Right topmost panel) lateral view of an adult medaka brain, transversal planes corresponding to sections below. ISH on 7 dpf larvae (A, C, E, G) and coronal sections of the adult brain (B, D, F, H). Magnifications of boxed areas on the right; corresponding expression domains in larvae are indicated with arrowheads. Scale bars, 50 µm. Expression domains: <i>tmtops1b</i>, granular layer of the olfactory bulb (A, B); <i>tmtops3a</i>, semicircular torus (C, D); <i>tmtops2</i>, dorsal tegmental nucleus (E, F), facial nerve nucleus of the hindbrain (G, H).</p

TMT-Opsins and Encephalopsin are functional light receptors.

<p>(A) Example traces of Neuro2A (N2A) cell stimulation by two consecutive light pulses of 12 min and 10 min. Data are presented as means (<i>N</i> = 4). (B–D) Traces of N2A cells transfected with medaka <i>tmt-opsins</i> (red) versus mutated (L294A) <i>tmt-opsins</i> (black), 10 min light stimulation. Data are presented as mean ± SEM (grey lines) (<i>N</i> = 4). (E) Different kinetics of TMT-2 (blue) compared to TMT-1B and TMT-3A in N2A cells (see also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001585#pbio.1001585.s003" target="_blank">Figure S3</a>). Baseline normalized CI values were normalized to the maximum and data are presented as means (<i>N</i> = 4). (F) Quantification of opsin-dependent N2A cell responses to light. Relative light responses are displayed as mean ± SEM (<i>N</i> = 72–136). (G) Quantification of opsin-dependent N2A cell responses to different spectra compared to human rhodopsin. Data represent mean ± SEM (<i>N</i> = 12–36). (H) HEK cells transfected with medaka <i>encephalopsin</i> (red) versus Schiff base mutant version (black). Data represent mean ± SEM (grey lines) (<i>N</i> = 32). (I) Quantification of Encephalopsin-dependent cell responses in HEK and N2A cells. Data represent mean ± SEM (<i>N</i> = 44–120); **** <i>p</i><0.0001; *** <i>p</i><0.0005; ns, not significant; yellow background box, light stimulation. See Figures S4 and S13 for analyses details.</p

<i>TMT-Opsin 1B</i> is expressed in inter- and motorneuron nuclei on mRNA and protein level.

<p>ISH (A, C) and immunohistochemistry (B, D, E, F) of TMT-Opsin 1b on coronal adult medaka brain sections. Magnification of black boxes in insets. Scale bars, 50 µm. (A) mRNA expression of <i>tmtops1b</i> in the dorsal tegmental nucleus. (B) Protein expression of TMTopsin1b in cells of the dorsal tegmental nucleus, the same area as in (A). Arrowheads indicate TMTopsin1b+ cells. (C) Multiple domains of mRNA expression of <i>tmtops1b</i> in the hindbrain. (D) TMTopsin1b+ cells localize to sites of mRNA expression indicated by a yellow box in (C). (E) Overview of TMTopsin1b protein expression in the hindbrain. Arrows and asterisks indicate protein expression domains that correspond to mRNA expression in (C). (F) Magnification of box in (E), <i>z</i>-stack: 13.87 µm. Note the projections (arrowheads) extending from a TMTopsin1b+ cell verifying its neuronal nature.</p

Handling Permutation in Sequence Comparison: Genome-Wide Enhancer Prediction in Vertebrates by a Novel Non-Linear Alignment Scoring Principle

<div><p>Enhancers have been described to evolve by permutation without changing function. This has posed the problem of how to predict enhancer elements that are hidden from alignment-based approaches due to the loss of co-linearity. Alignment-free algorithms have been proposed as one possible solution. However, this approach is hampered by several problems inherent to its underlying working principle. Here we present a new approach, which combines the power of alignment and alignment-free techniques into one algorithm. It allows the prediction of enhancers based on the query and target sequence only, no matter whether the regulatory logic is co-linear or reshuffled. To test our novel approach, we employ it for the prediction of enhancers across the evolutionary distance of ~450Myr between human and medaka. We demonstrate its efficacy by subsequent <i>in vivo</i> validation resulting in 82% (9/11) of the predicted medaka regions showing reporter activity. These include five candidates with partially co-linear and four with reshuffled motif patterns. Orthology in flanking genes and conservation of the detected co-linear motifs indicates that those candidates are likely functionally equivalent enhancers. In sum, our results demonstrate that the proposed principle successfully predicts mutated as well as permuted enhancer regions at an encouragingly high rate.</p></div