Patterns of cell proliferation and cell death in the developing retina and optic tectum of the brown trout

Developmental Brain Research 154 (2005) 101 – 119 www.elsevier.com/locate/devbrainres

Research report

Patterns of cell proliferation and cell death in the developing retina and optic tectum of the brown trout Eva Candal, Ramo´n Anado´n, Willem J. DeGrip, Isabel Rodrı´guez-Moldes* Department of Cell Biology and Ecology, Faculty of Biology, University of Santiago de Compostela, 15782-Santiago de Compostela, Spain Department of Biochemistry, Nijmegen Center for Molecular Life Sciences, University of Nijmegen, 6500 HB Nijmegen, The Netherlands Accepted 10 October 2004 Available online 6 November 2004

Abstract We have analyzed the patterns of cell proliferation and cell death in the retina and optic tectum of the brown trout (Salmo trutta fario) throughout embryonic and postembryonic stages. Cell proliferation was detected by immunohistochemistry with an antibody against the proliferating cell nuclear antigen (PCNA), and apoptosis by means of the TUNEL method. Haematoxylin and DAPI staining were also used to demonstrate apoptotic cells. Photoreceptor cell differentiation was assessed by immunohistochemistry with antibodies against opsins. Throughout embryonic development, PCNA-immunoreactive (PCNA-ir) cells become progressively restricted to the peripheral growth zone of the retina, which appears to be the principal source of new retinal cells from late embryos to adults. However, some PCNA-ir cells are observed secondarily in the differentiated retina, first in the inner nuclear layer of 15-mm alevins and later in the outer nuclear layer of 16-mm alevins, after differentiation of the first rods in the central retina, as demonstrated with opsin immunocytochemistry. Our observations also support the view that the PCNA-ir cells observed secondarily in the INL of the central retina of alevins are photoreceptor precursors. The number and distribution of apoptotic cells in the retina and optic tectum of the trout change throughout development, allowing distinction of several waves of apoptosis. Cell death is detected in proliferating areas at early stages, then in postmitotic or differentiating areas, and later concurring temporal and spatially with the establishment of visual circuits, thus indicating a relationship between apoptosis and proliferation, differentiation and synaptogenesis. D 2004 Elsevier B.V. All rights reserved. Theme: Development and regeneration Topic: Visual system Keywords: PCNA; TUNEL; Apoptosis; Opsin; Salmo trutta fario (Teleostei)

1. Introduction

Abbreviations: GCL, ganglion cell layer; H, hypothalamus; INL, inner nuclear layer; INLi, inner nuclear layer, inner part; INLo, inner nuclear layer, outer part; IPL, inner plexiform layer; IZ, intermediate zone; L, lens; MZ, marginal zone; on, optic nerve; ONL, outer nuclear layer; OPL, outer plexiform layer; OT, optic tectum; re, retinal epithelium; SGC, stratum griseum centrale; SGFS, stratum griseum et fibrosum superficiale; SGP, stratum griseum periventriculare; TgM, Mesencephalic tegmentum; TL, torus longitudinalis; VCb, Valvula cerebelli; VZ, ventricular zone * Corresponding author. Department of Fundamental Biology, Faculty of Biology, University of Santiago de Compostela, 15782-Santiago de Compostela, Spain. Tel.: +34 81 563100x13292; fax: +34 81 596904. E-mail address: [email protected] (I. Rodrı´guez-Moldes). 0165-3806/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.devbrainres.2004.10.008

Morphogenesis is a complex process that depends of the spatiotemporal control of cell proliferation, cell differentiation and migration of cells to their definitive positions. Equally important, programmed cell death acts as a specific surveillance mechanism to remove excess cells and cells failing to reach critical connections during morphogenesis. The retina and the optic tectum of teleosts appear especially suitable for studying morphogenesis, because during development these structures exhibit temporally and spatially ordered zones containing proliferating, early postmitotic or differentiated (mature) cells [14,24,32,54,56,62]. Much

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attention has focused on the time course of cell proliferation in the developing retina of teleosts [20,24,32,35,41,42,58]. In contrast, studies of cell death in the developing retina have been performed in only two teleost species, the zebrafish [7,10,26,32] and Haplochromis burtoni [23]. The low number of dying cells detected in the developing retina of these species has led to suggest that, in contrast with amniotes, either fish retinas never experience a significant cell death [32] or apoptosis is no longer used to remove excess of cells [7,23]. Since both zebrafish and Haplochromis are fast-developing species, it is possible that the observed time course of cell death was not representative for slow-developing teleosts. Comparative studies in fish appear necessary to know if there exists species where apoptosis may play an important role in the morphogenesis of the retina. Little is known about the relation between the spatiotemporal growing patterns of the retina and the optic tectum of teleosts, except for the autoradiographic studies made in goldfish [52,54] and trout [35]. In trout, available autoradiographic data are limited to three developmental stages [35] and cell proliferation has not been studied in the adult optic tectum. New methodological approaches are necessary to better know these spatiotemporal patterns in a slowdeveloping teleost species from embryo to adult, and the relationship between cell proliferation and cell death during the morphogenesis of the visual system. Trout is a suitable model for such a morphogenetic study because its slow embryonic development (about a month to hatching) allows a better estimation of the rates of cell death and cell proliferation than in species with fast development, as the zebrafish. Moreover, retinal morphogenesis extends to postembryonic stages [33], the cellular organization of the retina and the growth and maturation of retinotectal projections are well known [36,50,58], and the ontogeny of some types of photoreceptors [1] and of several neurochemically defined systems in the trout retina has been analyzed [3,5,9,62]. We present here evidence of a relationship between the patterns of cell proliferation and apoptosis during morphogenesis of the visual system of the developing brown trout, Salmo trutta fario. We have analyzed cell proliferation using the proliferating cell nuclear antigen (PCNA), which has been largely used as a proliferation marker both in the retina and in the brain ([13,30,42,44,65], among others). The immunohistochemical detection of PCNA allows distinguishing between highly proliferating areas and areas containing recent or early postmitotic cells, those with weakly PCNA-immunoreactive cells. Moreover, there has been reported a temporal coincidence between the absence of PCNA immunoreactivity and the detection of differentiated cells [42]. We have identified dying cells by means of the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) method, which allow detection of the controlled DNA cleavage switched on in apoptotic cells [18]. This method has been

successfully used for in situ visualization of apoptosis at single-cell level, while preserving cytoarchitecture. In this report, cell death was complementarily detected with haematoxylin and DAPI nucleic acid fluorescent staining, which has been widely used to identify chromatin condensation, nuclear shrinkage and formation of apoptotic bodies [25,69]; indeed, DAPI staining allows visualization of apoptotic cells even before DNA fragmentation can be detected by the TUNEL method. Moreover, we have studied early photoreceptor differentiation by immunohistochemistry with anti-rod and anti-cone opsin antibodies that label outer photoreceptor segments in the retina of a salmonid and other fish [17,39,49]. Knowledge of the patterns of cell proliferation, differentiation and dismissal in the trout retina and optic tectum will contribute to a more comprehensive understanding of morphogenesis of the vertebrate visual system.

2. Material and methods 2.1. Experimental animals Thirty-three embryos (total body lengths comprised between 5 and 14 mm; for the correspondence between lengths, days post-fertilization (dpf) and stages of Vernier [64], see Table 1), 14 alevins (between 15 and 26 mm), 6 juveniles (27, 30 and 35 mm, two of each), and 7 adults of the brown trout (S. trutta fario) were used. Animals were supplied by a fish farm (Centro Ictioxe´nico de Sobrado dos Monxes, A Corun˜a, Spain) and held in constantly aerated 5 l tanks at a mean water temperature of 10 8C and a photocycle of 12L:12D excepting embryos, which were maintained in darkness to avoid acceleration of development. All animals were deeply anaesthetized with a 0.05% solution of tricaine methane sulfonate (MS-222; Sigma, St. Louis, MO) in freshwater before fixation. All experiments were conducted in accordance with the European Community guidelines on animal care and experimentation.

Table 1 Correspondence between body length and days post-fertilization (dpf) of the brown trout (S. trutta fario) and the embryonic stages defined by Vernier [64] in the rainbow trout (O. mykiss=S. gairdneri) Early embryos

Late embryos

Hatching

Vernier stage

Length (mm)

dpf (at 10 8C)

19 20 21 22 24 25 27 28 29 30

4.5–5 6 7 7.5 8 9 11 11.5 13 14.5–15

11 12 15 16 17 21 26 27 31 35

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2.2. Immunohistochemistry 2.2.1. PCNA Animals were fixed by immersion in Bouin’s fluid for 4 h, and rinsed in 70% ethanol. Afterwards, samples were dehydrated, embedded in paraffin and cut on a rotary microtome (8 Am thickness) in transverse or sagittal planes, either into single series or into parallel series of sections. Before immunohistochemistry, sections were dewaxed and rehydrated. Endogenous peroxidase activity was blocked by incubation with 3% H2O2 in phosphatebuffered saline, pH 7.4 (PBS), for 30 min at room temperature (RT). The sections were then treated with 10% normal goat serum (NGS; Chemicon, Temecula, CA) in PBS with 0.2% TritonR X-100 (PBS–T) for 60 min, and incubated overnight with the monoclonal PCNA antibody (Sigma; dilution 1:1000) at 4 8C. After rinsing in PBS–T, the sections were incubated with goat antimouse IgG (Sigma; dilution 1:30) for 60 min at RT, rinsed in PBS–T and incubated with mouse peroxidase–antiperoxidase complex (Sigma; dilution 1:500) for 30 min. The immunoreaction was revealed with 0.5 mg/ml diaminobenzidine (DAB; Sigma) and 0.03% H2O2 for 15–30 min. Sections were dehydrated and mounted with Entellan (Merck, Darmstadt, Germany). Incubating sections as above but omitting either the primary antiserum or the goat anti-mouse IgG performed negative controls. In these controls, any staining was observed. 2.2.2. Opsins Series of transverse sections of 15- and 19-mm alevins were dewaxed, rehydrated and pretreated with H2O2 and NGS as described above. Afterwards, sections were incubated with rabbit polyclonal antibodies raised against cone and rod opsins (anti-chicken cone opsin, CERN-874: dilution 1:1000; anti-bovine rod opsin, CERN-922: dilution 1:1000) for 72 h at 4 8C. CERN-874 and CERN-922 antisera were prepared by one of the authors (W.J.G.G; for details see Ref. [15]). After three washes in PBS–T (10 min each), endogenous biotin was blocked (DAKOR biotin blocking system; DAKO, Glostrup, Denmark). After washes in PBS–T, sections were incubated with biotinylated goat anti-rabbit immunoglobulin (DAKO; dilution 1:500) for 30 min at RT, rinsed in PBS–T and incubated in StreptABComplex/HRP (DAKO) for 30 min. The immunoreaction was revealed as above. Previous studies have shown that these anti-opsin antibodies stain photoreceptors in a salmonid [49] and in other fish [17,39]. Negative controls were also performed, as indicated for PCNA. 2.3. Detection of apoptotic cells Animals were fixed by immersion in 4% paraformaldehyde in 0.1 M phosphate buffer (PB) at pH 7.4. The egg chorion of embryos was drilled before fixation in order to a better access of the fixative. Embryos, alevins and juveniles

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were immersed in cooled 30% sucrose in PB until they sank, and were then embedded in OCT Compound (Tissue Tek, Torrance, CA), frozen with liquid-nitrogen-cooled isopentane, and serially sectioned in transverse and sagittal planes on a cryostat. The sections (18 Am in thickness) were mounted on chrome alum-gelatin-coated slides. Sections were treated by the TUNEL method and/or stained with haematoxylin or DAPI. 2.3.1. TUNEL method The method was performed using the bIn situ Cell Detection, POD kitQ (Roche Molecular Biochemicals, Mannheim, Germany), based on labeling of DNA strand breaks. DNA breaks were recognized by the enzyme TdT (terminal deoxynucleotidyl transferase from calf thymus), which catalyses the polymerization of modified nucleotides to the free 3V-OH termini. Sections were rinsed in PBS, the endogenous peroxidase activity was blocked with 3% H2O2 in methanol for 10 min at room temperature, and cell permeabilization was done by incubation in ice-cooled 0.1% Triton X-100 in 0.1% sodium citrate for 2 min. Sections were then washed for 10 min in PBS and treated with the TUNEL reaction mixture (containing the TdT and the nucleotide mixture) in a humidified chamber for 60 min at 37 8C. After washing with PBS (15 min), they were incubated with the converter-POD (sheep anti-fluorescein Fab antibody fragment, conjugated with horseradish peroxidase) in a humidified chamber for 30 min at 37 8C. After incubation, samples were rinsed in PBS (15 min) and revealed with DAB and H2O2 as above for 10 min at RT. Sections were rinsed in PBS, dehydrated and mounted. In control sections in which the enzyme TdT was omitted from the reaction solution, no stained nuclei were observed. 2.3.2. Haematoxylin staining Haematoxylin staining provides optimal results for detecting fragments of dead cells [29,57]. This staining has been complementary used for demonstrate dying cells, since they allow visualization of apoptotic cells even before fragmentation of DNA can be detected by the TUNEL method. Serial transverse sections of Bouin’s fluid-fixed, paraffin-embedded embryos, alevins and adults were dewaxed, rehydrated and stained in Mayer’s haematoxylin for 3 min, rinsed in tap water for 10 min, dehydrated and mounted. 2.3.3. DAPI Chromatin condensation, nuclear shrinkage and formation of apoptotic bodies can be observed under fluorescence microscopy after staining of nuclei with DNA-specific fluorochromes. For nuclear staining, whole embryos were fixed, cryopreserved and serially sectioned in transverse planes on a cryostat as described above. Sections were equilibrated with PBS and covered with 300 nM DAPI for 3 min and then rinsed three times before observation. Samples

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were mounted with 1,2-Phenylendiamine (PDD)–glycerol (10% PDD in PBS, 90% glycerol). 2.4. Cell death quantification Since dying cells are labeled by the TUNEL method only during a relatively short period, this technique probably underestimates the actual number of apoptotic cells, especially in tissues with a low turnover rate. Therefore, for quantification we have considered as apoptotic cells those showing clear pyknotic nuclei, even if they were TUNEL-negative. The distribution of pyknotic nuclei was studied in haematoxylin or in DAPI-stained sections parallel to those stained with the TUNEL method. In some cases, samples treated with TUNEL were counterstained with haematoxylin for 3 min, washed in tap water for 10 min, dehydrated and mounted. Data were obtained from two different samples in each developmental stage. We analyzed the entire serial set in each sample and counted the apoptotic cells in alternate sections to avoid double cell counting, the number of sections depending on eye and tectum size. Counts in the retina were made in 38 (in embryos) and 56 (in alevins and juveniles) transverse sections, while in the tectum, 28 and 48 transverse sections were counted in embryos and in alevins and juveniles, respectively. Data were entered onto a spreadsheet (Microsoft Excel), and mean number of pyknotic nuclei and the standard errors were calculated. The mean retinal and tectal areas were calculated form 12 to 24 equidistant retinal sections and 11 to 21 tectal sections from each sample (the number of sections depending on eye size), which were measured on digital microphotographs using the Scion Image software (NIH, USA). The rate of apoptosis was expressed as apoptotic cells/mm2. Since the section thickness (18 Am) was much higher that the mean diameter of apoptotic bodies, and it was the same in all samples, no correction factor was introduced. Graphical representation of the data was done with Microsoft Graph 9; data of the rate of apoptosis in the whole retina and tectum (Fig. 6a) were represented in logarithmic scale due to the large differences between the earliest embryos and alevins. The statistical significances of the differences in the mean number of apoptotic cells were determined by Kruskal–Wallis test (performed with SPSS 11.5). 2.5. Imaging Most photomicrographs were made with an Olympus DP12 color digital camera and a Provis microscope (Olympus, Tokyo, Japan). DAPI fluorescence was observed in a Nikon E800 microscope (Nikon, Kanagawa, Japan) and photographs made with the NikonACT-1 software (LEAD technologies, Charlotte NC, USA). Contrast and brightness of photomicrographs were adjusted using Adobe Photoshop (Adobe Systems, San Jose, CA).

3. Results 3.1. PCNA and opsin expression in the retina and PCNA expression in the optic tectum PCNA immunohistochemistry allows distinguishing between highly proliferating areas, those containing intensely PCNA-immunoreactive (PCNA-ir) cells, and areas containing recent or early postmitotic cells, those with weakly PCNA-ir cells. The later are always adjacent to the intensely PCNA-ir areas since the PCNA level in cells leaving the cycle decreases only about 30% within 24 h [8]. Unless otherwise is indicated, the term PCNA-ir is here used to refer to intensely labeled areas or cells. The cells of PCNA-immunonegative areas are considered as differentiating because the expression of diverse neurochemical indicators of differentiation in the retina of teleosts reveals temporal coincidence between the absence of PCNA immunoreactivity and the detection of differentiation markers ([42], present results of opsin immunoreactivity). 3.2. Retina 3.2.1. PCNA expression We have characterized three periods in the trout retina development as regards the growing pattern. The first period, which comprises from 5- to 10-mm embryos, is characterized by the presence of a proliferating neuroepithelium (all cells, except mitotic figures, are PCNA-ir) (Fig. 1c). The central region of the optic cup is thicker than the peripheral region, but no regional differentiation or layering is still appreciable. Pigmentation of the retinal epithelium (outer layer of the optic cup) starts in the central region of 8-mm embryos, indicating the beginning of its cytodifferentiation. PCNA immunoreactivity disappears progressively from the retinal epithelium. The second period of the trout retinal growth, characterized by the progressive formation of layers in the central part of the retina, begins in embryos about 11-mm in length with the appearance of a differentiating central region where the inner plexiform layer (IPL) becomes recognizable (Figs. 1d,e and 2a). The end of the IPL defines the boundary between the differentiating central retina and the peripheral (proliferating) growth zone. In the central retina of 11-mm embryos, the primordial ganglion cell layer (GCL) is comprised of PCNA-negative cells and numerous PCNAnegative cells occupy the inner sublayer in the inner nuclear layer (INLi) (Fig. 1d,e), which extends as a band from temporal to nasal (Fig. 2). The peripheral growth zone and the retina neighbor to the optic fissure exhibit only PCNA-ir cells, retinal layering being no distinguishable (Fig. 1d). These growing borders remain intensely PCNA-ir throughout development. From 13-mm embryos onwards, the outer nuclear layer (ONL) is clearly distinguished in the most central part of the differentiating retina (Figs. 1f and 2b). Although PCNA-ir cells become mostly confined to the

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Fig. 1. Schematic drawings of dorsal (a) and lateral (b) views of the whole head of a trout alevin showing the nasal–temporal and dorsal–ventral axes considered in descriptions. Adapted from Vernier [64]. PCNA immunoreactivity in the retina of brown trout embryos (c–f) and alevins (g–i). (c) Vertical section of the central retina of a 5-mm embryo showing the optic cup formed by a PCNA-ir pseudostratified neuroepithelium. (d) Vertical section of the central retina of 11-mm embryo. Note that PCNA-ir cells are distributed throughout the retina except in the GCL and in the inner part of the INL (asterisk). (e) Detail of the area squared in d. (f) Section of the central retina of a 13-mm embryo. PCNA immunoreactivity becomes mostly confined to the peripheral growth zone of the retina although scattered PCNA-ir cells are also observed in the INLi (large arrows), outer sublayer of the INL (INLo, small arrows) and ONL (double arrow). Note that PCNA-ir cells extend to more central positions in the INLo than in the ONL and INLi. Lines indicate the limit between the peripheral growth zone and the central retina. (g) Photomicrograph of the retina of a 15-mm alevin. Note PCNA-ir cells in the INLo, either scattered or in small clusters (arrows), their absence from the GCL and the central ONL, and intense PCNA immunoreactivity in the peripheral growth zone retina (star). (h) Photomicrograph of the retina of a 16mm alevin. Note that PCNA-ir cells are distributed throughout the INL and in the central region of the GCL (small arrows) and ONL (large arrows). Inset: Detail of the squared area to show PCNA-ir cells in the ONL, INLo and between both layers (arrows). (i) Photomicrograph of the retina of a 24-mm alevin. Note that the temporoventral retina exhibits a PCNA-ir pattern similar to that found in the central retina of early alevins: a PCNA-ir peripheral growth zone (star), and a row of PCNA-ir cells in the INL (arrows). For abbreviations, see list. Scale bars: 100 Am.

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Fig. 2. Schematic drawings of vertical sections of the retina of embryos (a, b) and alevins (c, d) at temporal, intermediate and nasal levels to show the distribution of PCNA-immunoreactive and apoptotic cells. Dark gray areas represent high density of PCNA-immunoreactive cells (proliferating areas). Scattered proliferating cells are represented by dark gray dots while crosses represent apoptotic cells. The center (c) to peripheral (p) axis considered in descriptions is represented in c. Scale bar: 100 Am. For abbreviations, see list.

peripheral growth zone, in these late embryos scattered PCNA-ir cells are also observed extending some distance from this region in the INLi, outer sublayer of the inner nuclear layer (INLo) and ONL of the differentiating retina (Fig. 1f). Differences are observed regarding location of these cells since PCNA-ir cells in the INLo extend to more central positions than in the ONL and INLi (Figs. 1f and 2b). During this second period of retinal growth, which ends at hatching when the outer plexiform layer (OPL) becomes clearly distinguishable, PCNA-ir cells progressively dis-

appear from the central retina, their disappearance following a central-to-peripheral gradient. After hatching, the layers of the retina extend progressively following a central-toperipheral gradient, characterizing the third period of retinal growth. During this period, PCNA-ir cells progressively reappear in the central retina. In early alevins (15 mm), PCNA-ir cells are observed throughout the INLo, either scattered or in small clusters (Figs. 1g and 2c). The number of PCNA-ir cells observed in the INLi has also increased with respect to that of late embryos whereas no PCNA-ir

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cells are observed in the central part of the ONL. As development proceeds, the number of PCNA-ir cells in the INLo and INLi of the differentiated retina continues to increase following both temporal-to-nasal and peripheral-tocentral gradients (Fig. 2c,d), this number decreasing from 21-mm alevins. In the differentiated retina of 16-mm alevins, PCNA-ir cells are observed not only in the INL but also in the GCL, in the ONL and in between the INLo and the ONL (Fig. 1h). The number of PCNA-ir cells in the ONL continues to increase from central to peripheral regions until 19-mm alevins (Fig. 2d), while it decreases in the ONL of 24-mm alevins (Fig. 1i). In 24-mm alevins, the temporoventral retina presents a pattern of PCNA immunoreactivity similar to that observed in the remainder regions: a growing border consisting of PCNA-ir cells, and a differentiated, mostly PCNA-negative zone that contains a stripe of PCNA-ir cells in the INL (Fig. 1i). The distribution of PCNA-ir (proliferating) and PCNAnegative (postmitotic or differentiated) areas throughout the trout retina is asymmetrical. A dorsoventral asymmetry is noted regarding the appearance of PCNA-negative cells in the INLi (11-mm embryos) and in the ONL (13-mm embryos): from temporal-to-nasal, these negative cells appear first in the dorsal half, then in both the ventral and the dorsal halves, and then in the ventral half (Fig. 2a,b). Moreover, regarding the peripheral zone of the retina (the growing borders), the PCNA-ir cells occupy a larger extension in the ventral marginal zone than in the dorsal margin, especially at intermediate rostrocaudal levels (Fig. 2b). From 15-mm embryos, the dorsoventral asymmetry of the nasal region of the retina is lost, since the dorsal and ventral PCNA-ir margins occupy similar extensions in transverse sections (Fig. 2c,d). 3.2.2. Opsin expression The two antibodies we have used (CERN-922 and CERN-874) give similar staining patterns in the trout retina at the developmental stages studied. In that follows, we refer these immunoreactivities as opsin immunoreactivity. In early alevins (15 mm), some opsin-ir photoreceptors (putative rods, see below) have appeared in the central part of the ONL (i.e. the part that does not contain PCNA-ir cell, as described above) (Fig. 3a), where they form a horizontal band. Opsin immunoreactivity is observed in both the perikarya and photoreceptor segments (Fig. 3b,c). At this stage, the pear-shaped opsin-ir cells are associated in pairs rather close together (Fig. 3c). In 19-mm alevins, the number of opsin-ir photoreceptors increases from central to peripheral regions (coinciding with the increasing of PCNA-ir cells in this layer, see above) forming a broad equatorial band (Fig. 3d–g). The tallest opsin-ir cells are found in the temporal region of the band (Fig. 3e–h), and their length and maturation degree diminish in nasal, dorsal and ventral directions (Fig. 3d–f). In the most mature regions, the pear-shaped opsin-ir perikarya become elongated, being progressively separated by

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increasing numbers of immunonegative photoreceptor perikarya located in the outer ONL (Fig. 3h,i). Careful examination of parallel vertical sections of the retina of 19-mm alevins stained with the anti-cone opsin (CERN874) and the anti-rod opsin (CERN-922) antibodies reveals that both antibodies stain the same subset of photoreceptors (Fig. 3g–j), although the CERN-874 antibody yields a less intense immunoreaction. These cells are characterized by the position of their perikarya in the innermost ONL, the opsin immunoreactivity being observed in both the perikarya and photoreceptor segments (Fig. 3h,j). Analysis of tangential sections through the photoreceptor layer of a 19mm alevins allows studying the distribution of opsin immunoreactivity in the cone and rod mosaic, well defined in the central region of retina on these alevins. Groups of four opsin-ir cells are forming cartridges around the bcentral coneQ (immunonegative), the cartridges being surrounded by four immunonegative bdouble conesQ and four immunonegative baccessory conesQ (Fig. 3k–m). The inner position of perikarya in the ONL and the non-correspondence of cartridges with the previously reported cone mosaic (bcentral conesQ, bdouble conesQ and baccessoryQ or bcorner conesQ) indicate that the early-differentiated photoreceptors revealed in the trout retina by the CERN-874 and CERN922 antibodies are rods (for a detailed description of cone mosaics in trout see Ref. [33]). 3.3. Optic tectum 3.3.1. PCNA expression The PCNA distribution at different developmental stages of the trout optic tectum (OT) is schematized in Fig. 4a. The thin neuroepithelial alar plate of the midbrain that gives rise to the OT becomes distinguishable in 5-mm embryos. At this stage, the OT consists of a pseudostratified neuroepithelium with PCNA-ir cells radially oriented (Fig. 4b). From 6- to 8-mm embryos, the tectal neuroepithelium is formed of a ventricular zone (VZ) with densely packed cells, and a marginal zone (MZ) containing cell processes. All the cells bordering the tectal ventricle except those of the tectal midline are PCNA-ir. In 9-mm embryos, an intermediate zone (IZ) containing PCNA-immunonegative cells appears between the ventricular and the marginal zones (Fig. 4c), being more developed in lateroventral and rostral tectal regions than in mediodorsal and caudal regions. In these embryos, the tectal neuroepithelium becomes folded, and shows the most intensely PCNA-ir cells surrounding small infoldings. In 11.5-mm embryos, all the VZ cells are PCNA-ir in the caudal and rostral edges of the OT, whereas numerous PCNA-negative cells are seen in the VZ of the ventromedial OT (Fig. 4a). During development, the number of PCNA-negative cells in the tectal VZ increases from lateroventral to mediodorsal locations. Therefore, the PCNA-ir cells become progressively confined to the caudal and rostral borders of the OT and, in intermediate tectal regions, to the ventrolateral and dorsomedial VZ (Fig. 4a).

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Fig. 3. Photomicrographs showing the distribution of opsin-immunoreactive cells in the retina of 15- (a–c) and 19-mm (d–l) alevins, and a schematic representation of the photoreceptor mosaic in the trout retina (m). Panels a–i, k and l show the distribution of immunoreactivity to the CERN-922 (anti-rod opsin) antibody while panel j shows immunoreactivity to the CERN-874 antibody (anti-cone opsin). (a) Transverse section of the head of a 15-mm alevin passing through the eye at the level of the choroid fissure showing early opsin-expressing cells (arrows) in the central part of the ONL. (b) Detail of the central region with opsin-ir photoreceptors. (c) Detail of the area squared in b to show the immunoreactivity in the perikarya and photoreceptor segments. Note the paired distribution of immunoreactive photoreceptors. The line marks the outer limiting membrane. (d–f) Sagittal sections of the head of a 19-mm alevins showing opsin-ir cells in subequatorial eye sections from distal (d) to proximal (f). Note the choroid fissure (asterisk). Temporal is at the left and ventral is at the bottom. (d) At rather distal levels, opsin-ir cells are only located at the temporal retina (arrows). (e) At an intermediate level, opsin-ir cells are visible at the temporal (large arrows) and nasal poles (short arrows). (f) At a more proximal level, opsin-ir cell are visible throughout most the ONL, except ventrally. Note that the tallest opsin-ir cells are at temporal levels (large arrows). Short arrows indicate short opsin-ir cells. Details of the ONL at nasal (g) and temporal (h) levels showing the distribution of anti-rod opsin immunoreactivity. (i) High magnification of the area squared in h. The thin line marks the outer limiting membrane. (j) Detail of the temporal ONL of a section parallel to that of panel h to show that the CERN-874 antibody stains the same subset of photoreceptors that the CERN-922 antibody but with less intensity (compare with panel h). (k–l) Tangential sections through the photoreceptor layer of a 19-mm alevins stained with the CERN-922 antibody showing the position of cartridges (squared in k) of immunoreactive photoreceptors in relation to the cone mosaic. In panel l, the groups of four opsin-ir cells form a cartridge around a central immunonegative element (bcentral coneQ). Cones numbered as in panel m. (m) Schematic representation showing the position of CERN-922 and CERN-874 immunoreactive photoreceptors (black dots) in the photoreceptor mosaic. 1, bcentral coneQ; 2, bdouble coneQ; 3, baccessoryQ or bcorner coneQ. Scale bars: 100 Am (a, d–f); 25 Am (b, g, h, j); 15 Am (c, i, k); 10 Am (l).

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Fig. 4. Drawings (a) and photomicrographs (b–e) of transverse sections of the brown trout brain showing the distribution of PCNA immunoreactivity in the optic tectum of embryos and alevins. The distribution of apoptotic cells is also represented in a. (a) Schematic drawings of the optic tectum of embryos (11.5 and 13 mm) and alevins (15 and 17 mm) at rostral, intermediate and caudal levels. Dotted lines indicate the limit between the marginal and intermediate zones. Dark gray areas represent high density of PCNA-ir cells and crosses indicate apoptotic cells. (b) Cross section of the brain of a 5-mm embryo. Note that brain walls mostly consist of a PCNA-ir neuroepithelium. Areas with PCNA-negative cells are observed in the mesencephalic tegmentum (TgM) and hypothalamus (H). (c) Section of the optic tectum of a 9-mm embryo showing the thick PCNA-ir ventricular zone. The PCNA-negative intermediate zone (IZ) is appreciable between the ventricular (VZ) and the marginal (MZ) zones. (d, e) Transverse sections through the optic tectum of early (16.5 mm) and late (24 mm) alevins. Note the lateroventral (double arrow) and dorsomedial strongly PCNA-ir VZs of the tectum (arrow). For abbreviations, see list. Scale bars: 100 Am (a–c); 50 Am (d); 250 Am (e).

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The same pattern of PCNA immunoreactivity is observed in alevins (Fig. 4a,d,e). In the adult trout, only a small number of PCNA-ir cells is found in caudal, lateroventral and

mediodorsal regions of the OT. From hatching to adulthood, PCNA-ir cells are also seen in the VZ of the torus longitudinalis (TL) (Fig. 4a,d,e).

Fig. 5. Apoptotic cells in the retina of brown trout embryos (a–c) and alevins (d–g). (a) Cell death in the optic cup and lens primordium (L) of a 5-mm embryo revealed by the TUNEL method. The limits of the retinal neuroepithelium are outlined. Arrows point to some TUNEL-positive (+) cells. (b) Retina of a 7.5-mm embryo showing TUNEL+ nuclei (arrows) and TUNEL+ material appearing as remnant of a dead cell (double arrow). Note the relative low number of apoptotic cells with respect to that of the 5-mm embryo. (c) Vertical section through the central retina of an 11.5-mm embryo. Scarce apoptotic cells are found in the central part of the GCL, inner part of INL and ONL. TUNEL method. (d) Section through the temporal retina of a 15-mm alevin to show TUNEL+ cells (arrows). Note that they are more abundant in the INL than in the other layers. (e) Section of the temporal retina of a 17-mm alevin. Note the increased number of TUNEL+ cells in the GCL, with respect to previous stage. Section counterstained with haematoxylin. (f) Section of the retina of a 19-mm alevin stained with DAPI. Arrows point to some apoptotic nuclei. (g) Detail of the GCL of the retina of a 19-mm alevin stained with DAPI showing apoptotic cells (arrows). Note also a mitotic figure (double arrow). See list of abbreviations. Scale bars: 50 Am (a, b, e, f); 25 Am (c, d, g).

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3.4. Occurrence of apoptosis in the retina and optic tectum The location of TUNEL-positive (TUNEL+) cells within the proliferating, early postmitotic or differentiated areas has been determined by comparison with the distribution of PCNA immunoreactivity. TUNEL-labeled cells exhibit different appearances, either with a nucleus apparently normal, with a nucleus with rim-stained DNA, or with the fragmented nuclear morphology characteristic of the last stages of apoptosis. The labeling of these nuclei varies from weak to intense. TUNEL staining appears also in groups of fragments representing the remnants of a single dead cell, or in a halo of extracellular material (see discussion). For quantification, we have counted all these cells (with their distinct features), but not the halos of extracellular material. In all cases, the groups of small fragments resulting from a single dead cell have been counted as a unit. 3.4.1. Retina The distribution of apoptotic cells varies during the three growing periods of the retina defined above. During the first period, comprising early embryos from 5 to 10 mm whose retinal epithelium is massively proliferating (see PCNA results), TUNEL+ cells are clearly visible. After the formation of the optic cup (from 5- to 7-mm embryos), apoptotic cells are mostly observed throughout the borders of the inner layer of the optic cup, and in the lens primordium (Figs. 2 and 5a,b). When pigmentation of the retinal epithelium starts (8-mm embryos), apoptotic cells appear scattered at different retinal levels. During the second period (11- to 14-mm embryos), when different layers appear in the central retina, the distribution of apoptotic cells varies between proliferating and postmitotic areas and within the different cell layers. In 11-mm embryos, TUNEL+ cells are absent from both the GCL and the INLi (the areas containing postmitotic cells; see PCNA results), but they are found in the PCNA-ir areas: peripherally to the GCL and INLi, and throughout the prospective INLo and ONL (Fig. 2a). In 11.5-mm embryos, scarce TUNEL+ cells are seen in the central part of the GCL, INLi (the areas with postmitotic cells) and prospective ONL (Fig. 5c), while they are most abundant in areas peripheral to the GCL and INLi (Fig. 2). At temporal regions consisting only of proliferating cells, the pattern of apoptosis is similar to that observed in early embryos. From 12-mm embryos, more apoptotic cells appear first in the central part of the GCL and later in the INL but they progressively disappear in the ONL (containing now postmitotic cells), which does not contain apoptotic cells until hatching (Fig. 2b). During the third growing period of the retina (from hatchlings to 17-mm alevins), numerous apoptotic nuclei can be seen in all cell layers (Figs. 2c,d and 5d,e) throughout the retina (in temporal, intermediate and nasal regions). From 19-mm alevins, the number of apoptotic cells notably decreases in all layers (Fig. 5f,g). Scarce apoptotic cells are seen in the retina of

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21-mm alevins and they are almost absent from later alevins onwards. Although qualitative differences in the density of apoptotic cells in the different retinal layers or within each

Fig. 6. Rate of apoptosis in the trout retina (a–c) and optic tectum (a) at various developmental stages. (a) Rate of apoptosis in the whole retina (continuous line) and optic tectum (dashed line). Mean values are represented with standard errors. Rate of apoptosis in the different cell layers of the retina at intermediate (b) and temporal levels (c). The rate of apoptosis is averaged from data of two different specimens of each size (see Materials and methods). Data were expressed as number of apoptotic cells/ mm2. Mean values are represented with standard errors.

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layer throughout development can be noted by a simple comparison of the sections (see Fig. 5a–g), we have approached semiquantitatively these temporal and spatial variations by counting the number of apoptotic cells. In spite of the low number of specimens used for each developmental stage, the mean number of apoptotic cells in the retina among the different developmental stages was statistically different (Kruskal–Wallis test: chi-square= 20.25, df=8, P=0.009). Graphical representation of the semiquantitative results reveals temporal and spatial variations in the number of apoptotic cells that are compatible with the occurrence of several waves of cell dead throughout retinal development. The temporal variations in the number of apoptotic cells are represented in Fig. 6a: the number of apoptotic cells markedly decreases after the formation of the optic cup (from 5- to 7-mm embryos) while does not vary significantly between 7-mm embryos and hatchlings (14.5–15 mm). From hatchlings to

17-mm alevins, the relative number of apoptotic nuclei increases, and it decreases from 19-mm alevins onwards. Counts also evidence spatial (temporal-to-nasal) differences in the distribution of apoptotic cells within the different retinal layers (Fig. 6b,c). In 13-mm embryos, a wave of cell death has begun at intermediate levels of the retina (Fig. 6b): the rate of apoptosis increases first in the central part of the GCL and later in the INL but decreases in the recently postmitotic ONL. In the temporal retina of 13-mm embryos (Fig. 6c), the wave of apoptosis is delayed and appears to recapitulate that occurring in the central retina of 11.5 mm embryos: the number of apoptotic cells decreases first in the recently postmitotic GCL, INLi and then, in the ONL, and the wave of cell death begins later except for the ONL layer, as reported above. We have also observed that TUNEL+ cells exhibit different appearances throughout retinal development, as illustrated in Fig. 7. In the proliferating retina of early

Fig. 7. Photomicrographs showing different appearances exhibited by TUNEL-labeled cells. (a) Detail of the retina of a 5-mm embryo showing TUNEL+ nuclei (arrows) and scattered TUNEL+ remnants of a dead cell (double arrows). (b) Detail of the retina of 11-mm embryo showing TUNEL+ nuclei (arrows) and scattered TUNEL+ material surrounding apoptotic nuclei (double arrows). (c–e) Section of the retina of a 19-mm alevin. (c) Small arrows point to TUNEL+ material adhered to cell membranes along radial pathways from the GCL to the ONL. Section counter-stained with haematoxylin. (d) Detail of TUNEL-stained cells of the area squared in the GCL. The double arrow points to a TUNEL+ nucleus similar in size to neighboring normal nuclei, single arrows indicate rim-stained TUNEL+ nuclei, and small arrows point to small apoptotic fragments. (e) Detail of the area squared in the INL to show TUNEL+ material adhered to cell membranes. Scale bars: 25 Am (a, b); 50 Am (c); 15 Am (d); 5 Am (e).

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embryos (5- to 10-mm embryos), TUNEL+ material appears within cell bodies, and also as scattered remnants of dead cells (Fig. 7a). In postmitotic areas (as the GCL and INL) of late embryos (11- to 14.5-mm embryos), TUNEL positivity mostly appears as isolated pyknotic

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nuclei of different morphologies and size (Fig. 7b). In alevins, the TUNEL+ material appears as small apoptotic fragments, as well as within apparently intact nuclei and in nuclei with rim-stained chromatin (Fig. 7c–e). In 19-mm alevins, characterized by a marked decrease of the number

Fig. 8. Photomicrographs of transverse sections of the trout optic tectum showing cell death at different stages of development. (a) Section of a 7.5-mm embryo. Arrows indicate TUNEL+ cells located near the tectal margin (dashed line). (b) Caudal region of the tectum of 11.5-mm embryo. Apoptotic cells (arrows) are located in the proliferative area (as defined on the basis of PCNA immunoreactivity). Asterisk, ventricle. (c–e) 19-mm alevins. (c) Section of the intermediate tectum stained with DAPI. Arrows point to apoptotic nuclei in the intermediate zone (IZ). Note the absence of apoptotic cells in the ventricular (proliferating) zone (VZ). (d) Detail of a DAPI-stained section of the tectum at caudal level. Arrows point to apoptotic nuclei in the marginal (postmitotic) zone (MZ). (e) Detail of the intermediate zone of the tectum showing clusters of small pyknotic bodies (arrows), each of them probably representing the remnant of a single dead cell. Haematoxylin stain. (f, g) 24-mm alevins. (f) Most TUNEL+ cells are found in the stratum griseum periventriculare (SGP). (g) Detail of the area squared in f. Note different morphologies of TUNEL+ nuclei. Large arrows indicate rim-stained nuclei; small arrows indicate small apoptotic fragments. SGC: stratum griseum centrale; SGFS: stratum griseum et fibrosum superficiale; SGP: stratum griseum periventriculare. Scale bars: 50 Am (a–d, f); 25 Am (e, g).

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of apoptotic cells with respect to early alevins, TUNEL positivity also appears distributed in a radial fashion throughout the GCL to the ONL lining the cell membranes (Fig. 7c).

4. Discussion

3.4.2. Optic tectum As observed in the retina, the distribution of apoptotic cells with respect to proliferating and postmitotic areas in the tectum changes throughout development. In early embryos, apoptotic cells appear among proliferating cells located near the MZ (Fig. 8a). In late embryos (between 11.5-mm embryos and hatching), TUNEL+ cells also appear out of the proliferating zones (in the rostral and intermediate OT, see below) while in the caudal region, all the TUNEL+ cells appear within the proliferating areas: i.e. the pattern of apoptosis is similar to that observed in the rostral tectum of previous stages (Fig. 8b). The number of apoptotic cells in postmitotic areas increases at the time when different cell layers become distinguishable in the tectum, as observed with either the TUNEL method, DAPI (Fig. 8c,d) or haematoxylin staining (Fig. 8e). In 24-mm alevins, a few apoptotic cells are observed mainly in the postmitotic stratum griseum periventriculare SGP (Fig. 8f,g) and within other tectal layers as the stratum griseum centrale (SGC), the stratum griseum et fibrosum superficiale (SGFS) and the stratum opticum (SO). The distribution of apoptotic cells in proliferating and postmitotic areas changes throughout the rostral–caudal axis (Fig. 4a). In the rostral and intermediate tectum of 11.5-mm embryos, almost all TUNEL+ cells are located preferentially next to the outer fibrous layer, whereas in the caudal tectum, all the TUNEL+ cells still appear within the proliferating zone (Fig. 4a). In 13-mm embryos, TUNEL+ cells are almost exclusively located out of the proliferating areas, even in the caudal tectum. In 15-mm embryos, the relative number is higher in the rostral and intermediate tectum than in the caudal part, in which the cell layers are less developed (Fig. 4a). From 17-mm alevins onwards, the number of TUNEL+ cells also increases in the caudal tectum (Fig. 4a). As in the retina, the number of apoptotic cells in the OT is statistically different among different stages (Kruskal– Wallis test: chi-square=15.93, df=8, P=0.04). Graphical representation of these semiquantitative results of the trout OT reveals temporal and spatial variations that are compatible with the occurrence of two waves of cell dead throughout development. The highest proportion of TUNEL+ cells is detected at early embryonic stages (5 mm) (Fig. 6a). From this stage until 11-mm embryos, the relative number of apoptotic cells progressively decreases. In late embryos (embryos higher than 11 mm) and early alevins, the number of apoptotic cells in the OT slightly increases, peaking in 17-mm alevins to decrease from this stage onwards, showing a minimum in late alevins (24 mm) (Fig. 6a). After that stage, scarce variations occur in the number of apoptotic cells.

PCNA levels vary throughout the cell cycle: its expression begins in late G1 phase, is maximal during the S phase and decreases from the S/G2 transition towards mitosis. Therefore, a strong nuclear labeling is indicative of cells in the S phase, whereas a weaker labeling is indicative of cells in either the G1 or G2 phases [40,63]. The use of PCNA as a proliferation marker allows differentiating between highly proliferating brain areas consisting mainly of neuroepithelial stem cells and areas with weakly labeled cells that could give rise to differentiating postmitotic cells and to still pluripotent stem cells. Recent postmitotic cells may be PCNA-stained since the levels of PCNA in cells leaving the cell cycle decrease only about 30% within 24 h [8]. Studies of expression of diverse neurochemical indicators of development in the retina of fishes reveal temporal coincidence between the absence of PCNA immunoreactivity and the detection of differentiated cells [42]. Similar results have been obtained in our study, since the absence of PCNA immunoreactivity in cells in the central part of the ONL of the trout retina is closely associated with immunoreactivity to opsins and morphological differentiation of inner and outer segments (early photoreceptor differentiation) in photoreceptors of the most central ONL. Since our aim was to investigate the patterns of proliferation and the beginning of differentiation in the retina and OT, PCNA immunohistochemistry was useful to these purposes. The advantages of the use of PCNA with respect to other assays to detect cell proliferation, such us direct counting of mitotic figures and the use of DNA precursors such as tritiated (3H)-thymidine or nucleotide analogues such as bromodeoxyuridine (BrdU) have been already considered [47].

4.1. Relation between PCNA immunoreactivity and proliferation and cell-cycle exit

4.2. Cell addition in the developing trout retina As in other teleosts, it has long been recognized that the trout retina grows throughout life from the periphery and that the peripheral region at any particular time becomes more central as the eye grows [33]. We have observed that, in trout alevins, PCNA-ir cells are not restricted to the peripheral growth zone but also appear in central areas. Radioautography of larval goldfish retinas several days after injections of 3H-thymidine also reveals labeled cells in the differentiated retina [27]. Using PCNA immunohistochemistry, we have noted that in trout the apparition of proliferating cells in the differentiated retina comparatively occurs earlier than in goldfish, just at hatching, when the lamination of the trout retina is completed at central levels. As retinal differentiation follows its central-to-peripheral progression, more PCNA-ir cells extend from the center to the periphery.

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Our study also reveals that the appearance of PCNA-ir cells in the differentiated retina is spatiotemporally organized, they appearing first in the central INL of early alevins, and later in the most central part of the ONL and GCL, that happen following a central to peripheral gradient coinciding with the gradient of maturation/differentiation. Proliferating cells have been evidenced in the mature retina of trout [28], but our results report the spatiotemporal sequence of apparition of these cells. The sequence we observe in trout is rather similar to that reported in H. burtoni [20] in spite of the rapid growth of this species, thus revealing that the spatiotemporal pattern of cell proliferation in teleosts retinas is not depending on the rate of growth, in contrast with the pattern of cell death (see below). It has been proposed that in teleosts, a population of progenitor cells that divide throughout the ONL gives rise to new rods [20,27,53,55], the origin of these ONL proliferating cells remaining controversial. It has been argued that cells from clusters of proliferating cells located in the INL of the mature retina would migrate to the ONL, where they divide and originate new rods [20,27,28,48,55]. Other authors have considered that stem cells in the ONL that can remain mitotically active or quiescent are the main, if not the only, source of rod precursors [34]. Our PCNA observations in the developing trout retina support the hypothesis that proliferating cells of the INL are photoreceptor precursors. First, whereas in late embryos the central INL is devoid of PCNA-ir cells, PCNA-ir cells reappear in the central region of the INL of hatchlings. These INL proliferating cells appear to migrate centrally from the peripheral retina. By this stage, the central ONL presents opsin-ir cells (rods), whereas no PCNA-ir cells are observed. In this layer, PCNA-ir cells are observed in 16mm alevins, i.e. about 4 days after PCNA-ir cells reappear in the central region of the INL. Thus, proliferating cells reappear in the INL several days earlier than in the central ONL. Second, in alevins, some PCNA-ir cells are observed between the central INL and ONL, suggesting they are migrating from the INL to the ONL. In the retina of adults and postembryonic stages of goldfish, it has been noted that rod precursors migrate from the INL to the ONL, where they continue to divide and generate new rods continuously [27,48,55], which will be inserted in the adult mosaic [53]. Third, in larger trout alevins, the number of PCNA-ir cells in the ONL increases rapidly following a central-toperipheral gradient. Since in the trout ONL proliferating cells are observed at the same time as first postmitotic opsinir rods, before an increase in the number of cells in this layer, this suggests that most of them are photoreceptor progenitors. 4.3. Differentiation of the first rods in the trout retina Our results with the opsin antibodies CERN-922 and CERN-874 reveal new traits of rod differentiation and mosaic formation in salmonids. Most developmental studies

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of the salmonid retina have focused on generation of cones [1,46], while rod generation has received scarce attention. In trout, we have observed that both CERN-922 and CERN874 antibodies label groups of four opsin-ir cells that appear forming cartridges in the photoreceptor mosaic. Comparison of this cartridge photoreceptor mosaic with the photoreceptor cell mosaic reported in salmonids [1,4,33,46] indicates that the cells revealed by these antibodies in trout are rods. In this species, the CERN-874 (anti-cone opsin) does not stain any of the types of cones characterized to date in salmonids (i.e. bcentralQ, bdoubleQ and baccessoryQ or bcornerQ cones) [1,33,46] but stains perikarya located in the inner ONL. This antibody has been used in a study in salmon [49], and the cells stained by this antibody in the retina were referred to as cones, which is not supported by the present results. It has been reported that, at a low dilution, the antiserum CERN-874 shows some reaction with rod pigment upon immunoblot analysis [15]. In Xenopus, the CERN-874 antibody labels both cone and rod cells [2], while it is specific for cone opsins in salamander [60]. These results suggest that trout and Xenopus rod opsins share some immunological properties of cone opsins of chick that appear absent from trout cone opsins. Moreover, the CERN-874 and CERN-922 antibodies also recognized the opsin of the single photoreceptor type found in the retina of the larval sea lamprey [39]. At difference of most of other antibodies generated against vertebrate cone opsin, the CERN-874 antibody labels retinal neurons in a mollusk and, after immunoblot, it labels the same protein band that an antiserum generated against vertebrate rhodopsin [19]. The compact disposition of rod cartridges observed in early differentiating ONL regions is soon followed by an increasing separation between rod cartridges that is coincidental with the reappearance of PCNA-ir cells in the ONL. Our results suggest that the early photoreceptor mosaic grows in surface by intercalation and/or size growth of new cells between the rod cartridges that were immunonegative to the CERN-922 and CERN-874 antisera, which lead us to identify the immunonegative cells as cones. Our results thus suggest that in the trout retina rods are the first photoreceptors to differentiate. In goldfish, rod opsin is expressed before cone opsins although first cones are born first than rods [61]. Precedence of rod versus cone differentiation during early development has also been reported in zebrafish [56], although in this species the expression of rhodopsin can occur simultaneously with that of UV cone opsin thus revealing that the cell birth date and the onset of opsin expression are not closely tied [59]. Trout differs from zebrafish and goldfish in the locus of origin of the earliest opsin-expressing photoreceptors, a ventral patch of the nasal retina in the zebrafish and goldfish [56,61] and a central horizontal band in trout (present results), indicating that the spatial pattern of photoreceptor differentiation in trout is similar to that reported in amniotes.

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4.4. Proliferation pattern in the developing optic tectum Present results in the developing brown trout indicate that the optic tectum grows by the addition of cells from a crescent-shaped proliferative tectal zone that extends from the caudal pole to the ventrolateral and dorsomedial tectum. This pattern coincides with that reported previously in the developing rainbow trout by using 3H-thymidine [35,50] and a similar proliferation pattern has been observed in the tectum of the medaka, even in advanced periods of development [43,44]. Moreover, in postembryonic zebrafish cell proliferation in the optic tectum is found in a spherical cap at the periphery of the periventricular cell layer of each tectal hemisphere [66]. However, interspecies differences are noted in the abundance and location of proliferating cells in the adult tectum. Our results in the adult trout reveal a small number of PCNA-ir cells in the caudal, lateroventral and mediodorsal tectal regions. Using BrdU labeling, abundant proliferating cells were observed at caudal levels of the optic tectum of adult Apteronotus, being scarce in other tectal regions [70]. In adult Gasterosteus aculeatus, Ekstrfm et al. [13] observed a continuous proliferation zone (PZ) that extends throughout the dorsomedial, caudal and ventrolateral aspects of the tectum. The extension of the tectal PZ observed in adult Gasterosteus matches with that observed in alevins and juveniles of the brown trout (present results), and in postembryonic zebrafish and medaka [43,44,66]. All these results evidence that the teleosts optic tectum grows following a similar pattern although interspecies differences are noted in the timetable of tectal development in relation with the rate of growth. The asymmetrical growth pattern observed in the OT of teleosts is in contrast with that of the retina, where most new cells are added in concentric rings from the periphery. Tracttracing studies of the retinotectal projections in the trout show a rather specific topographic connection between retinal ganglion cells and their tectal targets [36]. The topological dissimilarity between the growth pattern of the retina and the tectum of trout supports the view that the retinotopic map must change continuously; maintenance of a correct retinotectal map requires that early-generated optic fiber terminals became shifted to more central portions in the tectum as it grows, either matching to neighboring cells or with new postmitotic cells [54]. Indeed, since in the trout the mature terminal field of retinotectal axons is established by pruning of primarily larger arbors [36], this change would allow accommodation of the maps between hatching and the stage 36 (14.5- to 23-mm alevins). 4.5. Some considerations on apoptosis in the developing retina and optic tectum TUNEL+ structures show different appearances depending on the stage of development. In proliferating areas, TUNEL+ material appears within cell bodies, but also scattered in the extracellular space. In postmitotic areas of

late embryos, most apoptotic cells do not show typical apoptotic bodies but rather intact TUNEL+ nuclei (either completely labeled or showing a stained nuclear rim), probably due to the ability of neighbor cells to remove dying cells at early stages, as proposed by other authors [6,18,45,68]. A predominance of small apoptotic bodies was noted in alevins. These fragmented TUNEL+ nuclei have also been reported in the chick retina at postnatal stages [12]. In the retina, TUNEL+ debris may extend throughout all layers, which suggests that radial cells (probably Mqller cells) could be involved in removal of apoptotic material. In the quail retina, Marı´n-Teva et al. [37] have also reported the presence of small apoptotic bodies, either singly or grouped in radial rows in the IPL. Their ultrastructural observations have shown than Mqller cells are able to phagocyte dead cell debris in the INL, IPL and the GCL by their radial processes. 4.6. Three waves of apoptosis occur in the developing trout retina In the trout retina, we have evidenced a reproducible distribution of TUNEL+ cells during development. Apoptosis follows specific spatial and temporal patterns, and three different periods or waves of cell death can be distinguished. The first wave occurs in early embryos (5mm embryos), coincidentally with the formation of the optic cup. After that, the rate of cell death progressively decreases throughout the neural retina, apoptotic cells appearing scattered among the proliferating cells. Although the global number of apoptotic cells in retina does not increase from 11-mm embryos to hatching (see Fig. 6a), a second wave of apoptosis is observed in the GCL and INL of the intermediate retina of 11- to 13-mm embryos (see Fig. 6b) at the time when the first postmitotic cells are observed at both sides of the primordial IPL. The number of apoptotic cells in these layers increases in a center-to-periphery gradient coinciding with the gradient of cell maturation. This second wave of apoptosis in the trout retina follows neurogenesis, as it has been evidenced in chick embryos [16]. The third wave of apoptosis is observed in all cell retinal layers from hatchlings to 17-mm alevins, coinciding with the third growing period of the retina of trout. In the ONL, this apoptotic wave is concurrent with differentiation of first photoreceptors (present results), and with the establishment of pathways between photoreceptors and GCL cells [58]. The number of apoptotic cells increases from hatchlings to 17-mm alevins, and then decreases, showing a minimum in 24-mm alevins. This period also coincides with the progressive invasion of the caudal pole of the tectum by retinal afferents [50,51], and the establishment of the global organization of retinotectal projections, which in Salmo gairdneri is completed in 23-mm alevins [36]. Julian et al. [28] have not found TUNEL+ cells in the mature retina of the rainbow trout.

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Only a few studies have reported apoptosis in developing teleost retinas (H. burtoni [23]; zebrafish [7,10,32]). Our results differ from those reported in H. burtoni, because no apoptotic waves were observed in this species [23]. In developing zebrafish, a wave of retinal cell death peaking at 36 h post-fertilization has been observed [10], coinciding with the initial outgrowth of retinal ganglion cell axons. The wave of apoptosis could be equivalent to the second wave of apoptosis we have observed in the GCL and INL of late embryos of trout, although in zebrafish, at difference of trout, the distribution of apoptotic cells does not reflect established and coordinated patterns of differentiation [10]. Biehlmaier et al. [7] have quantified the apoptosis in the retina of zebrafish larvae from 2 days post-fertilization (dpf), when the various retinal layers are histological identified in retinal sections (equivalent to just hatched trout alevins). They have observed two consecutive waves of apoptosis that peak at the same time (at 3 dpf) in the GCL and the INL, but peak clearly later in the ONL. Moreover, the second peak in the inner retina of zebrafish coincides with a peak of apoptosis in the ONL. Our results in the intermediate retina of trout also reveal a wave of apoptosis in early alevins but this wave peaks not only in GCL and INL but also in the ONL. However, in the temporal retina, apoptosis in the INL peaks early than in the GCL and ONL. The differences among teleosts in the pattern of apoptosis could be related with differences in the time course of retinal growth, since Haplochromis and zebrafish have a very fast growth compared with trout. The relative slow growth of the trout retina is rather similar to that of birds and some mammals, allowing a better discrimination of the cell death waves. It is remarkably that the pattern of cell death in the developing retina of trout is similar to that reported in chick and quail, where three well-defined periods of naturally occurring cell death have been reported coincident with the invagination of the optic vesicles, the birth of early ganglion cells and growth of their axons, and following tectal innervation [11,12,16,37,38]. Apoptosis is also closely related to proliferation, cell maturation and synaptogenesis during eye development in mammals (e.g. in mouse [31,67]). 4.7. Apoptosis in the developing optic tectum As indicated for the retina, the frequency and distribution of apoptotic cells in the developing trout OT vary following spatial and temporal patterns: in early developmental stages, cell death is detected in proliferative areas and in later stages in postmitotic areas following the arrival and arborization of retinal projections. In the trout OT, two spatial gradients of cell death can be defined: (a) a radial gradient (from ventricular to superficial), apoptosis occurring first in the VZ and later in more superficial, postmitotic areas; and (b) a rostrocaudal gradient, apoptosis occurring first in the rostral OT, and progressively in more caudal regions. Since the observed gradients of cell death

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appear to coincide with the gradients of differentiation in the OT, this suggests that apoptosis and differentiation are coordinated. A relative high number of apoptotic cells is observed in 5-mm embryos (first wave of apoptosis), when the OT appears as a single layer of cells formed by evagination and stretching of cells of the mesencephalic alar plate. Cell death at this time could be a consequence of the mechanical stress caused by morphogenetic movements (evagination and caudal extension), as it has been proposed to explain cell death during early development of Xenopus [22]. From 5- to 8-mm embryos, all the trout tectal cells are PCNA-ir (see PCNA results), the number of cells in the OT highly increasingly onwards and the OT becomes folded. The relative number of apoptotic cells in the tectum decreases throughout this period, although TUNEL+ cells are invariably found among proliferating cells. Since programmed cell death is topologically associated with proliferating regions, it could contribute to eliminate overproduced cells. From 11-mm embryos onward, apoptosis at intermediate OT levels is neither observed among PCNA-ir cells nor among recently postmitotic cells, i.e. those showing weak levels of PCNA immunoreactivity that are just bordering the VZ (see PCNA results). The second wave of apoptosis is also concurrent with these developmental processes. An increase of the rate of apoptosis (second wave) is observed in the tectum of early alevins, at the time when retinal axons invade the tectum [50]. Further increases in the SGP and other tectal layers follow the general stratification and maturation of retinotectal arborisation. Later in development, apoptosis is also detected in the SGC, SGFS and SO, i.e. in cells that have reached their corresponding strata. Our results in the OT do not reveal whether or not apoptosis was linked to particular cell lineages, but indicate that the variations in the number of TUNEL+ cells in superficial layers show a similar pattern to that observed in the SGP. The observed cell death in these tectal layers might reflect maturation of neuronal connections, as it has already been proposed in mammals [21]. Very few apoptotic cells are observed in trout from 24-mm onwards, when the retinal layering is completed and mature optic fiber arbors are established [36]. The occasional TUNEL+ cells observed in the OT of 27-mm juveniles and in adult trout appear to indicate that the processes that trigger cell death during early development continue at these stages, since the retina and the OT grows at low rate throughout life. In conclusion, our study presents evidence for a distinctive pattern of TUNEL labeling in both retina and optic tectum of the trout during different developmental stages: we have detected cell death in proliferating areas at early stages of development, then in postmitotic (differentiating areas), and later concurring temporal and spatially with the establishment of visual circuits. This clearly reflects a relationship between apoptosis and proliferation, differentiation and synaptogenesis. Subsequent studies will be

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required to demonstrate how control programs act to regulate proliferation, differentiation and apoptosis during development of the visual system in fish.

Acknowledgements The authors wish to thank Dr. Rube´n Retuerto of the University of Santiago for helping with the statistical analysis. The work was supported by Xunta de Galicia (PGIDT99BIO20002 and PGIDT01PXI20007PR) and Spanish Ministry of Science and Technology (BXX2000-0453-C02).

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