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Ann Anat 187 (2005) 43—50

KEYWORDMuller celZebrafish;Retina;PhotoreceElectron m

0940-9602/$ - sdoi:10.1016/j.

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An ultramicroscopic study on the distribution ofMuller cell processes in the outer retinal layers ofthe zebrafish

Eunju Leea, Byung Soo Changb, Ga-Hee Munc, Yoon Hee Chungb,Jaehyup Kimc, Dong Hoon Shinc,�

aDepartment of Internal Medicine, Ulsan University College of Medicine, 28 Yongon-Dong, Chongno-Gu, Seoul 110-799,Republic of KoreabDepartment of Clinical Pathology, Dongnam Health College, Suwon, Republic of KoreacDepartment of Anatomy, Seoul National University College of Medicine, 28 Yongon-Dong, Chongno-Gu, Seoul 110-799,Republic of Korea

Received 3 March 2004; accepted 31 August 2004

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AbstractThe zebrafish, Brachydanio rerio, is a good model for studying the development ofvarious organs. We have assayed the distribution pattern of Muller cell processes inzebrafish retinas by electron microscopy. In the outer nuclear (ON) layer, multiplelayers of Muller cell processes were present along both sides of the large pyramidalendings of the synaptic terminals. We found that the inner segments (ISs) of thezebrafish photoreceptors (PRs), including the cones, double cones and rods, werearranged in different planes, and that the Muller cell processes formed multilayeredsheaths around virtually all PR compartments except their outer segments. Thus,Muller cell processes beyond the outer limiting membrane (OLM) are more easilyobserved in zebrafish retina than in the retinas of other species. To our knowledge,this is the first study to show the exact ultrastructural distribution of Muller cellprocesses around the OLM and the PR layer in zebrafish retina.& 2005 Elsevier GmbH. All rights reserved.

Introduction

Some structures in the outer layers of the teleostretina have been reported to differ from those of

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740 8203; fax: +82 2 745 9528..net (D. Hoon Shin).

other species. For example, cones in the zebrafishphotoreceptor (PR) layer are arranged in a two-dimensional crystalline pattern, whereas the var-ious types of cones are located at specific positions

rved.

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(Engstrom, 1960; Engstrom, 1963; Branchek andBremiller, 1984; Nawrocki et al., 1985; Raymond etal., 1993, 1995). These results indicate that thereare several morphological subtypes of cones in thezebrafish, including double cone pairs and longsingle and short single cones; the same retinalstructures have also been observed in somenocturnal land vertebrate species and deep-seadwelling fishes. In addition, in the retina of a fewspecies, the Muller cell processes, called microvilli,extend beyond the outer limiting membrane (OLM)(Mack et al., 1988; Reichenbach et al., 1988, 1995;Prada et al., 1989; Peterson et al., 2001). Thus,while Muller cells of virtually all vertebrates extendmicrovilli beyond the OLM, the number, shape andlength of microvilli varies among species (Uga andSmelser, 1973).

The zebrafish, Brachydanio rerio, is a commonlyused model in studies of organ development.Although the distribution of Muller cells aroundthe OLM has been studied in the retinas of variousvertebrate species, an exact ultrastructural studyhas not been performed on zebrafish retina. Wetherefore examined the zebrafish retina by trans-mission electron microscopy in order to determinethe ultrastructural distribution of Muller cellprocesses around the OLM and in the PR layer.

Materials and methods

Adult zebrafish were maintained in 45-l aquariaat 28.5 1C. Experiments were performed in accor-dance with the ‘‘Guide for the care and use oflaboratory animals’’ (NIH publication No. 86-23,1985 edition). Retinas were harvested during thedaytime and fixed in freshly prepared 4% parafor-maldehyde and 0.1% glutaraldehyde in neutral0.1M phosphate buffer. Tissues were postfixed for1 h in PBS containing 1% (W/V) osmic acid,dehydrated in graded ethanol, and embedded inEpon 812 (EMS, Fort Washington, PA). Ultrathinsections were cut and mounted onto nickel gridscoated with Formvar film. The sections wereexamined under a JEOL 1200 EX-II electron micro-scope (Tokyo, Japan), with or without uranyl-leadcounter staining.

Zebrafish retinas were immunostained withmouse antibody against intermediate filamentprotein vimentin (Immunotech, 1:500), a glialmarker in the central nervous system. Briefly, afterremoving a retina, the tissue was fixed in freshlyprepared 4% paraformaldehyde in neutral 0.05Mphosphate buffer. Sections were incubated over-night in primary antibody, and staining was visua-

lized using the diaminobenzidine reaction (Shin etal., 2000). The distribution of nuclei in the outerretinal layers of the zebrafish was assayed by DAPIstaining (1:5000, Sigma, St. Louis, MO), in accor-dance with the manufacturer’s protocol.

Results

In most vertebrate retinas, the Muller cellprocesses are located in the space between thecell bodies, which are present in the outer nuclear(ON) layer (Yamada et al., 1968). Although thispattern is similar in the zebrafish retina, Muller cellprocesses in this species show unique patterns inthe ON, with multiple layers present along bothsides of the large pyramidal endings of the synapticterminals (Fig. 1A). These processes were observedto terminate in small round pockets in the transi-tional region between the base and the other sidesof the pyramidal endings (Fig. 1B). This patternwas observed not only in the synaptic region ofthe outer plexiform (OP) and the ON, but also inthe other intercellular spaces of the ON (Figs. 1Cand 1D).

This unique ultrastructure of the Muller cellprocesses was also observed in the PR layer. Thatis, the inner segments (ISs) of the PRs, whichinclude the cones, double cones and rods, werearranged in different planes (Fig. 2A). This multi-layered distribution of PRs also causes their nucleito be differentially located. While the nuclei ofcones and rods were observed in the ON layer(Branchek and Bremiller, 1984; Peterson et al.,2001), the nuclei of the double cones were locatedbeyond the OLM (Fig. 2A). This also affected thedistribution of a dense staining line between the ONand the PR layer, termed the OLM. Although thetraditional OLM was also detected in the zebrafishretina, we observed an electron-dense linear linebetween the intercellular spaces beyond the OLM(Fig. 2A).

A previous electron microscopic study showedthat the OLM, which is apparent by light micro-scopy, corresponds to a row of zonulae adherentesformed by the PRs and Muller cells. In otherspecies, these cell junctions were found to form asingle membrane, which defines the boundaries ofthe ON and PR layers, as all PRs are located in thesame plane. Similarly, we found that both thecytoplasmic membrane of the ISs of the cones andthe processes of the rods were involved in theformation of an OLM (Fig. 2B). In addition to thisclassical OLM, we observed continuous electrondense lines between the intercellular spaces from

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Figure 1. Electron microscopy of the zebrafish retina. (A) Multi-layered Muller cell processes (arrows) were observedbetween the cells in the ON layer. ST, synaptic terminal. (B) Magnification of the image in (A), showing that the multiplelayers terminate in small round pockets (Rectangle). (C) and (D) ON layer and Muller cell processes. Multiple layers(arrows) were observed between the cells in the ON. Scale bars, 200 nm.

An ultramicroscopic study on the distribution of Muller cell 45

the OLM to the IS of the double cones or to the ISof the rod PRs (Fig. 2C). At higher magnification,we found that these electron dense structureswere composed of multiple parallel lines, whichresembled the Muller cell processes in the ON(Fig. 2D).

The relationship between the Muller cell pro-cesses and the various PRs in the zebrafish retina,including the cones, double cones and rods, isgraphically summarized in Fig. 3. The presence ofMuller cell processes in the region beyond the OLMis likely due to the presence of the ISs of the

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Figure 2. Electron microscopy of the PR layer of zebrafish retinas. (A) Structures belonging to the cones and doublecones are marked in red and green, respectively. The nucleus (N) in the ON layer is continuous with the IS of the cone(IC). Arrows indicate the electron dense line extending from the outer limiting membrane to the IS of the PRs. Someprocesses (diamond shaped symbols) from the double cones were observed to join the formation of the outer limitingmembrane. A long slender nucleus (asterisk) of a rod is shown between the PRs. (B) Magnified image of the outerlimiting membrane of (A). The junctional complexes are present between the ISs of the double cones and the Muller cellprocesses (*). (C) Micrograph showing the continuity of the electron dense lines (arrows) from the outer limitingmembrane. IS, inner segment. (D) Magnified image of the electron dense lines (arrows) present in the intercellularspaces between the double cones. Scale bars, (A) and (C) 4 mm; (B) 2 mm; (D) 200 nm.

E. Lee et al.46

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Figure 3. Schematic representation of the relationshipof the Muller cell processes with the photoreceptors (PRs)in (A) mammalian and (B) zebrafish retinas. The dottedred line represents the outer limiting membrane. (A) Inmammalian retinas, the cones and rods are arranged in aplane. The terminals of the Muller cell processes (browncolored structures), which make contact with the innersegment (IS) of the PRs, do not extend far from the OLM.(B) In the zebrafish retina, the ISs of the double cone androd PRs are present far beyond the ONL. Thus, in thisspecies, the Muller cell processes are much longer than inmammals.

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zebrafish PRs far beyond the OLM. Similar to thecones and rods in mammalian retinas, which arearranged in a plane, the terminals of Muller cellprocesses, which make contact with the IS of PRs,did not extend far from the OLM (Fig. 3A). The ISsof the double cones and rods within zebrafishretinas are present far beyond the ONL, indicatingthat the Muller cell processes in the zebrafish retinaare much longer than those in mammalian retinas(Fig. 3B).

DAPI staining showed that nuclei of some PRsbeyond the OLM were arranged with regular spacing(Fig. 4A). In the horizontal section parallel to theretinal layers (as seen in the yellow line in Fig. 4A),the nuclei of the rod PRs were arranged with ablank region between them (Fig. 4B). Theseregions, in which nuclei were not observed(Fig. 3B), apparently result from the presence ofthe nuclei of the cones and rods within the ON. Thepresence of nuclei in the region outside the OLMwas confirmed by electron microscopy, whichshowed that the nuclei of the cone PRs were

localized in the ON, in a manner similar to thatobserved in mammalian retinas (Fig. 4C). We alsoobserved electron dense lines localized in theregion adjacent to the IS of the rods (Fig. 4C).Magnification of this image showed that theelectron dense lines were multilayered sheaths,which may belong to Muller cells (Fig. 4D).

To show that Muller cell processes extended be-yond the OLM in zebrafish retina, we immunohisto-chemically stained these tissues with anti-vimentinantibody. Vimentin immunoreactivity was localizedin the intercellular spaces between the ISs of thePRs (Fig. 5A). By electron microscopy, we foundthat vimentin immunoreactivity was present in aline from the OLM to the IS of the PRs, which werelocalized far beyond the OLM (Fig. 5B).

Discussion

Using electron microscopy, we observed severalinteresting structures related to the distribution ofMuller cell processes in the PR layer. Muller cellsare supporting elements with a neuroglial char-acter. In most vertebrate retinas, the oval nuclei ofMuller cells are observed in the middle zone of theinner nuclear layer, and their processes extendradially from the outer to the inner limitingmembrane. At the limit, between the ON layerand the layer containing rods and cones, Mullercells have prominent zonulae adherentes, whichproduce the linear density interpreted as an OLM(Fawcett, 1986). In the majority of vertebratespecies, the cones and rods of PRs are arranged in asingle plane, and the OLM is the outer boundary ofMuller cells. Thus, in most of these species, Mullercell processes do not extend far beyond the OLM.

We have shown here that the structural patternformed by Muller cell processes in zebrafish retinais different from that in other vertebrates, in thatthe former involves multi-layered processes termi-nating in small round pockets in the ON. Similarmultiple layers were also present in the PR layerand in the intercellular space of the IS of PRs. Thesemultiple layers may be due to Muller cells, asobserved in the ON. Since Muller cell processes inmost vertebrates terminate in the OLM to form thezonula adherens and do not extend beyond theOLM, our results obtained by electron microscopyand immunohistochemistry using an antibody spe-cific for Muller cells, show unusual patterns.Specifically, in the zebrafish retina, Muller cellprocesses are present in the intercellular spacesbetween the cells in the ON and the regionsbetween the ISs of PRs.

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Figure 4. (A) DAPI staining of zebrafish retinas, showing the presence of nuclei of some PRs beyond the OLM (blue).These nuclei (red dotted line) were arranged with regular spacing (*). (B) DAPI staining of the horizontal section parallelto the retinal layers (as seen in the yellow line in A), showing that the nuclei of the rod PRs were arranged with a blankregion (*) between them. (C) Electron microscopy confirming the presence of nuclei in the region beyond the OLM (reddotted line). The nucleus (black asterisk) of the cone PR (red) was localized in the ON. An electron dense line wasobserved to extend into the region adjacent to the IS of the rods (brown). (D) Magnification of the image in (C), showingthat the electron dense lines were multi-layered sheaths (arrows). Scale bars, (A) and (B) 30 mm; (C) 4 mm; (D) 200 nm.

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The presence of Muller cell processes in the PRmay be explained by the structure of the zebrafishretina. In this species, PRs are present in different

parallel planes (Raymond et al., 1995), and theelectron-dense bands of multilayered Muller cellprocesses begin at the OLM and are arranged

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Figure 5. (A) Immunohistochemical staining of zebrafish retinas with anti-vimentin antibody. Vimentin immunor-eactivity was localized in the intercellular spaces (*) between the cells in the ON layer. Black arrows indicate the OLM,and white arrows indicate vimentin immunoreactivity localized in the region beyond the OLM. IS, inner segment of a PR;OP, outer plexiform layer; PR, PR layer. (B) Electron microscopic evaluation of vimentin immunoreactivity in thezebrafish retina. Vimentin immunoreactivity (white arrows) was observed in a line from the OLM (black arrows) to the ISof the PRs, which are localized far beyond the OLM. Scale bars, 4 mm.

An ultramicroscopic study on the distribution of Muller cell 49

orthogonally to it within the PR. Therefore, ifMuller cell processes are in contact with all PR cellsin different planes, these processes should bepresent in regions extending far beyond the OLM.

Even though the presence of Muller cell processesin those regions beyond the OLM has been reportedpreviously, the current data could provide theunique ultrastructural patterns of Muller cellprocesses in zebrafish retina, which has not beenrevealed in the other studies.

Acknowledgements

This study was supported by a grant (01-PJ1-PG3-20700-0041) from the Korea Health 21 R&D Project,Ministry of Health and Welfare, Republic of Korea.

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