Adult zebrafish outer retina; structure and probe specificity.

<p>The schematic diagram of the outer retina to the left is based on Raymond and Barthel, 2004 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0121330#pone.0121330.ref036" target="_blank">36</a>] and a Nomarski contrast photomicrograph from a radial section of an adult AB zebrafish retina (<b>A</b>). All photoreceptors have a terminal in the outer plexiform layer (OPL). The outer nuclear layer (ONL) is subdivided by the outer limiting membrane (olm) into a sublayer where the rods have their nuclei (black asterisk: rONL) and a more external sublayer where the cones have their nuclei (empty asterisks: cONL). Note that the short single cones (SSC) have their nuclei in the rONL while their oil droplets (black arrows) and outer segments (OS) are in the cONL. The rods’ myoids (empty arrows) are embedded in the cONL. The long cone inner segment sublayer (lCIS) includes the oil droplets and myoids of the double cones (DC) and the long single cones (LSC) and part of the LSC OS. The outermost part of the retina (top) is covered by the pigmentary epithelium where the DC OS (dCOS) and the rods’ ellipsoid (arrowhead) and outer segments (ROS) are embedded. <b>B</b> and <b>C</b> are confocal photomicrographs of <i>in situ</i> hybridisation experiments performed using Tg(<i>gnat2</i>:EGFP) zebrafish, which expresses EGFP in all cones. Specific Fast Red staining in rods for <i>gnb1a</i> (<b>B</b>) and cones for <i>gnat2</i> (<b>C</b>) can be observed. Arrows in <b>B</b> point to the rod´s myoids that in some cases could be mistaken as SSC. In <b>C</b>, the arrowhead points to a DC, the asterisk to a LSC and the star to a SSC. Scale bar in <b>A</b> represents 25 μm.</p

Co-expression of <i>gnb1a</i> and <i>gnb1b</i> genes in the adult zebrafish retina.

<p>Photomicrographs from an adult retina showing combination of <i>gnb1a</i> and <i>gnb1b</i> antisense riboprobes in a double ISH experiment. The upper row displays radial sections of the entire adult retina (dorsal side to the left), while the middle row shows higher magnification of the area marked within the square in the upper pictures (outermost side at the top). <b>A</b> and <b>C</b> are fluorescent pictures showing Fast Red staining for <i>gnb1a</i>. <b>D</b> is a bright-field picture showing mainly NBT/BCIP staining for <i>gnb1b</i> in purple but also a minimal red from the Fast Red. <b>B</b> and <b>E</b> pictures combine bright-field and fluorescence to show the co-expression of both mRNAs in the same rods. Empty arrows in panels <b>C-E</b> point at single rods and arrows mark the oil droplets region that gives autofluorescence in panel <b>C</b>. Scale bars are 200 μm for panels <b>A-B</b> and 50 μm for panels <b>C-E</b>. The bottom of the figure displays a graph showing the expression levels of the two genes at different Zeitgeber time points during the day (0 to 20 in X-axis) in relation to β-actin as housekeeping gene (Y-axis), using the 2^-ΔCt method (<b>F</b>). Note that the expression level of <i>gnb1a</i> is significantly higher than <i>gnb1b</i> at all time points. Moreover, <i>gnb1a</i> expression oscillates, with significantly higher peaks of expression (p<0,05) at ZT8 and ZT16, while <i>gnb1b</i> does not oscillate.</p

Summary of the transducin subunit gene ontogeny in the zebrafish pineal complex.

<p>The photomicrographs show the presence of mRNAs for the transducin genes in the pineal complex of zebrafish. In adults, transversal sections from the dorsal diencephalon show expression of <i>gnat1</i> (<b>D</b>), <i>gnat2</i> (<b>H</b>), <i>gnb1a</i> (<b>L</b>), <i>gngt1</i> (<b>V</b>) and <i>gngt2a</i> (<b>Z</b>) in the pineal complex (asterisks) but not in the dorsal diencephalon (d). Observe the complete lack of mRNA for the <i>gnb3a</i> gene (<b>P</b>). The ontogenetic analysis shows a synchronised onset and development of expression starting at around 26 hpf for <i>gnat1</i> (<b>A-C</b>), <i>gnat2</i> (<b>E-G</b>), <i>gnb1a</i> (<b>I-K</b>), <i>gnb3a</i> (<b>M-O</b>), <i>gngt1</i> (<b>S-U</b>) and <i>gngt2a</i> (<b>W-Y</b>). In addition, there is a transient expression of <i>gnb3b</i> (<b>Q</b>, <b>R</b>) and <i>gnbt2b</i> (<b>A´, B´</b>) in the pineal complex prior to hatching. Note the background-like expression found in the early stages for <i>gnb1a</i> (<b>I-K</b>), <i>gnb3a</i> (<b>M</b>, <b>N</b>) and <i>gngt2a</i> (<b>W-Y</b>). In all cases, arrows point to the location of the pineal complex, and “e” mark the location of the eyes. The photomicrographs in panels <b>C</b>, <b>G</b>, <b>K</b>, <b>O</b>, <b>U</b> and <b>Y</b> show a dorsal view of the larvae, while the others show lateral views. Scale bars: 50 μm in larvae and 30 μm in adults. For abbreviations see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0121330#pone.0121330.g002" target="_blank">Fig. 2</a> legend.</p

Phylogenetic cladogram summarising the evolution of the transducin subunit genes within vertebrates.

<p>The three transducin subunit genes present in the vertebrate ancestor cone-like photoreceptors (“proto-cones”) (<i>GNAT</i>, <i>GNB</i> and <i>GNGT</i>) gave rise to two paralogs for each gene represented in extant tetrapods after 1R and 2R: <i>GNAT1</i>-<i>GNAT2</i>, <i>GNB1</i>-<i>GNB3</i> and <i>GNGT1</i>-<i>GNGT2</i>. This facilitated the appearance of rods. In teleost fishes the 3R tetraploidisation resulted in three pairs of paralogs that have been retained in zebrafish: <i>gnb1a</i>/<i>gnb1b</i>, <i>gnb3a</i>/<i>gnb3b and gngt2a</i>/<i>gngt2b</i>.</p

Expression patterns of each transducin subunit gene in the adult zebrafish retina.

<p>Nomarski contrast photomicrographs from radial sections of adult zebrafish retina showing the expression of all the transducin mRNAs. Panels <b>A, C, D</b> and <b>G</b> show the rod-specific expression of <i>gnat1</i>, <i>gnb1a</i>, <i>gnb1b</i> and <i>gngt1</i>, respectively. Their expression is observed in the rod nuclei of the rONL, but the strongest staining is observed in their myoids, which are embedded in the cONL sublayer (thin arrows). Panels <b>B</b>, <b>F</b>, <b>H</b> and <b>I</b> show the expression of <i>gnat2</i>, <i>gnb3b</i>, <i>gngt2a</i> and <i>gngt2b</i>, respectively, in all cones: DC (arrowheads), LSC (asterisks) and SSC (stars). The brown retinal pigment epithelium is shown the uppermost part for each picture. The stratification of the outer retina is evident in all panels except <b>H</b>, due to the exclusive ventral expression of <i>gngt2a</i>, where the stratification becomes unclear. Panel <b>E</b> shows the lack of staining in the adult retina for the <i>gnb3a</i> gene. Note that the weaker stained band into the ONLc for the cone-specific transducin subunits (thick arrows) corresponds to the rods’ myoid position (thin arrows in rods photomicrographs). Scale bar in panel <b>A</b>: 50 μm. For abbreviations see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0121330#pone.0121330.g002" target="_blank">Fig. 2</a> legend.</p

Expression of the <i>gnb3</i> paralogs retained in zebrafish after 3R.

<p>The upper part of the figure shows two photomicrographs from radial sections of adult zebrafish eyes. The inner side is to the right and the dorsal side to the top. No mRNA could be detected for the <i>gnb3a</i> gene (<b>A</b>). Expression of the <i>gnb3b</i> gene (in purple) was observed in the dorsal and medial retina (<b>B</b>), while the ventral retina lacks mRNA (asterisk). The black-brown tissue surrounding the retina corresponds to the pigmentary epithelium. ON; optic nerve. Scale bars: 200 μm. At the bottom of the figure there is a graph comparing the cone-specific <i>gnat2</i>, <i>gnb3a</i> and <i>gnb3b</i> expression levels between 1 dpf, 2 dpf, 3 dpf zebrafish and eyes of 1 year old individuals in relation to the β-actin housekeeping gene using the 2^-ΔCt method (<b>C</b>). Note the increment in the expression of <i>gnat2</i> and <i>gnb3b</i> in the adult stage while the similar amount of mRNA for the <i>gnb3a</i> gene present in the 3 dpf embryos and an adult eye.</p

Compartmentalisation of the <i>gngt2a</i> and <i>gngt2b</i> cone-specific paralogs in the adult retina.

<p>The upper part shows two photomicrographs from radial sections of adult zebrafish eyes. The inner side is to the right and the dorsal side to the top. In them, expression patterns (asterisks) can be observed in the ventral retina for the <i>gngt2a</i> gene (<b>A</b>) and in the dorsal and medial retina for the <i>gngt2b</i> gene (<b>F</b>). At the bottom are two drawings of a zebrafish adult head in a lateral view (<b>B</b>, <b>G</b>), pointing out the level where the sections were taken for the schematic drawings in panels <b>C</b>, <b>D</b> and <b>E</b> for <i>gngt2a</i>, and panels <b>H</b>, <b>I</b> and <b>J</b> for <i>gngt2b</i>. L; lens, ON; optic nerve. Scale bars: 200 μm.</p

Photomontage that highlights gene specialisations of the zebrafish ventral retina.

<p>These photomicrographs show radial sections of adult zebrafish retinae. They demonstrate specific expression of <i>rh2–4</i> (<b>A</b>), <i>lws-1</i> (<b>B</b>) and <i>gngt2a</i> (<b>C</b>) in the ventral retina (arrowheads), while <i>gnb3b</i> (<b>D</b>) shows expression in the dorsal and medial retina (black arrow). Note that <i>gnb3b</i> expression is absent in the ventral retina (asterisk). The pictures in panels <b>A</b> and <b>B</b> are used with permission from Takechi and Kawamura (2005) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0121330#pone.0121330.ref051" target="_blank">51</a>] and Tsujimura <i>et al</i>. (2010) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0121330#pone.0121330.ref050" target="_blank">50</a>], respectively. The pictures in panels <b>C</b> and <b>D</b> are extracted from the present results. Scale bars: 200 μm.</p

Opsin-transducin model suggested for the retina and pineal complex photoreceptors.

<p>Drawing of an adult zebrafish head showing the opsin and transducin genes expressed in rods and cones of the retina, as well as a suggested model for the rod- and cone-like photoreceptors of the pineal complex. Due to the low expression of <i>gnb3a</i> in the adult retina, and the fact that its expression could not be demonstrated in the adult pineal complex, it is shown without a box.</p

Timetable comparing the ontogeny of retina and pineal complex expression for each transducin gene.

<p>The expression in the retina (blue bars) starts either right before 48 hpf for <i>gnat1</i>, <i>gnb3a</i>, <i>gngt1</i> or slightly after 48 hpf for <i>gnat2</i>, <i>gnb1a</i>, <i>gnb1b</i>, <i>gnb3b</i>, <i>gngt2a</i> and <i>gngt2b</i>. The onset of expression in the pineal complex (red bars) is synchronised at 26 hpf for all the genes except <i>gnb1b</i>, which is not expressed at all, and <i>gngt2b</i>, which is only transiently expressed right before hatching. Furthermore, <i>gnb3b</i> also shows a transient expression, and <i>gnb3a</i> could not be detected by ISH in the adult stage in the retina or the pineal complex. Dpf; days post-fertilisation, hpf; hours post-fertilisation.</p