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The embryogenesis of rod photoreceptors in the teleost fish retina, Haplochromis burtoni
Developmental Brain Research 108 Ž1998. 217–227
Research report
The embryogenesis of rod photoreceptors in the teleost fish retina, Haplochromis burtoni Mary Hagedorn ) , Andreas F. Mack 1, Barbara Evans 2 , Russell D. Fernald
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Institute of Neuroscience, UniÕersity of Oregon, Eugene, OR 97403, USA Accepted 3 March 1998
Abstract Development of the retina, like that of other tissues, occurs via an orderly sequence of cell division and differentiation, producing the functional retina. In teleost fish, however, cell division and differentiation in the retina continue throughout the life of the animal in two distinct ways. Stem cells in a circumferential germinal zone at the periphery of the retina give rise to all retinal cell types and progenitor cells located throughout the retina in the outer nuclear layer ŽONL. produce new rod photoreceptors. These processes in adult retina recapitulate in space the embryonic events responsible for forming the retina. Analysis of these events in an African cichlid fish, Haplochromis burtoni, confirmed that cone photoreceptors differentiate first, followed by rod photoreceptors. Correspondingly, at the margin of the eye, cone photoreceptors differentiate nearer to the margin than do rods. Control of photoreceptor production is not understood. Here we present the time of appearance and distribution pattern of GABA and vimentin which are candidates for the control of retinal cell division and differentiation. Antibody staining reveals that both GABA and vimentin exhibit unique patterns of expression during embryonic retinal development. Vimentin immunoreactivity is evident throughout the retina in a spoke-like pattern between developmental Days 4 and 7, as both cone and rod photoreceptors are being formed. GABA is expressed in horizontal cells between Days 5 and 7, corresponding to the onset of rod differentiation in time and in position within the retina. Moreover, the wave of GABAergic staining in the horizontal cells parallels the wave of rod differentiation across the embryonic retina of H. burtoni. Thus, GABA may play a role in the development of rod photoreceptors. q 1998 Elsevier Science B.V. All rights reserved. Keywords: Retina; Development; Teleost; Cone photoreceptor; Rod photoreceptor; Muller cell; GABA; Vimentin ¨
1. Introduction Unlike the retinas of mammals, the teleost retina continues to add new neurons throughout the life of the animal. Stem cells arranged circumferentially at the periphery of the retina give rise to all retinal cell types except rods and progenitor cells located throughout the retina in the outer nuclear layer ŽONL. produce new rod photoreceptors. These processes in adult retina recapitulate in space the
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Corresponding author. Dept. of Reproductive Physiology, Smithsonian Institution, National Zoological Park, 3001 Conn. Ave, NW, Washington, DC 20008, USA. Fax: q 1-202-673-4733; E-mail: [email protected] 1 Current Address: Anatomisches Institut, Universitaet Tuebingen, Oesterbergstr.3, 72074 Tuebingen, Germany. 2 Current Address: Dept. Biol., Lake Superior State University, Sault St. Marie, MI 49783, USA. 3 Current Address: Neuroscience Program, Stanford University, Stanford, CA 94305, USA. 0165-3806r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 1 6 5 - 3 8 0 6 Ž 9 8 . 0 0 0 5 1 - 0
embryonic events in time responsible for forming the retina. How these events are controlled, either in the embryo or adult, is not understood. Here we present the time of appearance and distribution pattern of GABA and vimentin which are candidates for the control of retinal cell division and differentiation, because of their time of expression. In embryogenesis, the teleost retina begins as an invaginated optic cup consisting of spindle-shaped neuroepithelial cells which is transformed through development into a highly organized, laminated structure with several distinct types of retinal neurons w24,18x. In Haplochromis burtoni, an African cichlid fish, neurogenesis begins in the central, vitread portion of the retina, radiates toward the photoreceptor layer and spreads peripherally towards the margin of the eye w11x. In most vertebrate retinas, rod photoreceptors are the last cells to differentiate w7,30,14x, which is the case in H. burtoni. Differentiation of cone photoreceptors begins on Day 3 while the onset of rod differentiation is delayed until Day 4.5 w11x. This late onset of rod differen-
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tiation in fish can sometimes be quite extreme. For example, Sandy and Blaxter w36x observed in the retinas of flounder and sole that the rod differentiation was delayed for months until the onset of metamorphosis. Moreover, in the larval winter flounder, photoreceptors are uniformly
single cones with no double cones or rods appearing before metamorphosis w5,6x. In contrast, it has been reported in the zebrafish that rod opsin positive cells appear shortly before or around the time of cone opsins in a ventral patch of the retina w31,34x.
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In this paper, we have used immuno-histochemical tracers to identify cellular events which surround the late onset of rod neurogenesis in the teleost, H. burtoni. We have examined the neurogenesis of the photoreceptors, Muller ¨ cells, as well as the onset of putative guidance substances responsible for triggering the differentiation in the developing teleost retina. Specifically, we have stained the developing retina with vimentin and g-aminobutyric acid ŽGABA. to determine whether their timing or pattern of appearance might correlate with rod neurogenesis. The cytoskeletal protein, vimentin, is one of the key structural elements in the Muller ¨ cell, a major type of glial cell present in the retina w4,38,39x. It is not clear what the function of vimentin is, but Wylie w42x speculates that it is responsible for the movement of cells during early development. Wylie has shown that blocking vimentin caused improper cleavage patterns in Xenopus embryos. Moreover, Johns w15x, Raymond w28x, and Raymond and Rivlin w29x have implicated Muller ¨ cells as ‘guidance’ cells which assist rod progenitors in migrating from the inner nuclear layer to the outer nuclear layer during early development and growth. If so, during embryogenesis, the origin of Muller ¨ cells andror vimentin must precede the differentiation of rod photoreceptors. In order to test this hypothesis, we examined the onset of vimentin immunoreactivity and compared it to the onset time of rod neurogenesis. The inhibitory neurotransmitter GABA has also been implicated as a pioneering or trophic substance both in the developing central nervous system and in the developing retina w37,26,32,30x. In the developing rat brain, Lauder et al. w16x observed GABAergic fibers running from the cortex to the telencephalon at the onset of neurogenesis in this area. Early in the development of certain mammals, such as the mouse and the rabbit, type A horizontal cells mature precociously and transiently produce GABA w37,32,41x. Destruction of these GABAergic horizontal cells with kainic acid resulted in a destruction of the horizontal cells, and a deregulation resulting in an overproduction of rod photoreceptors in the outer nuclear layer and a disruption of the synaptic connections in the outer plexiform layer w22x. Such transient production of GABA by the horizontal cells suggests that GABA is important for neuronal development, in general, and may play a role
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in the regulation of photoreceptor differentiation in the retina. Additionally, Mitchell and Redburn w23x suggested a role for GABA during cone synaptogenesis in the rabbit. If a similar function occurs in fish, this would imply that GABA immunoreactivity must precede or co-occur with the onset of photoreceptor differentiation. In order to test this hypothesis, we examined the onset of GABA immunoreactivity in horizontal cells and compared it to the onset of rod neurogenesis. In contrast to the transitory embryonic pattern of GABAergic staining in the horizontal cells of mammals, in most lower vertebrates, Žsuch as fish, frogs and birds., GABAergic staining is retained in H 1 horizontal cells, a sub-type of cone horizontal cells, across the entire adult retina w20,27,2,40,14x. Little is known about the embryonic onset of GABAergic staining in the horizontal cells of lower vertebrates; however, chicken horizontal cells first express GABA-like immunoreactivity as early as Day 8 w14x. Negishi and Wagner w25x report GABAergic staining in a cichlid retina Ž Aequidens pulcher . around the time of synaptogenesis but do not specify which horizontal cells. Additionally, transient expression of GABA was found in ganglion cells but not in horizontal cells of the developing zebrafish retina w35x. To understand the late onset of rod neurogenesis in the teleost retina, we used immunocytochemistry to determine the onset and pattern of GABA and vimentin expression at the time of rod neurogenesis. We identified the onset of rod neurogenesis with a rod-specific opsin antibody w1x and compared the adult pattern Žthe central retina of a 2month-old fish. of immunohistochemical staining with the embryonic pattern between Days 4 and 7. We used six separate antibodies to delineate separate aspects of the developing retina for three reasons. First, we wanted to describe the onset of photoreceptor maturation and distinguish between rod and cone maturation. Second, we were interested in how the spatial patterns of embryonic neurogenesis related to the continuing patterns of neurogenesis in the adult retina. Third, we wished to examine the correlation of reputed guidance and pioneering substances in the retina Žwhich may function in the guidance and insertion of rod photoreceptors into the ONL. with the onset of photoreceptor maturation.
Fig. 1. Immunostaining of the photoreceptors in adult H. burtoni. The retinas are viewed in cross-section on the left and in tangential section on the level of the inner segments of the photoreceptors on the right. The retinas were dark adapted to retract the pigmented epithelium and reveal the morphology of the photoreceptors. Bars s 20 m m. ŽA. The 9C1 antibody stains both double and single cones including inner segments, nuclei, myoid processes and pedicles. The tangential section on the right shows the positively stained surrounding octet of double cones Žwhite arrows. and the central cones Žopen white arrows.. ŽB. The 12H10 antibody stains only one member of the double cones. On the left, the cross-sections show the tips of the outer segments, the inner segments, the myoid processes and a very dark staining body above the cone nuclei. The tangential section on the right shows a dark, cross-shaped pattern surrounding a single member of the double cone inner segments and their nearest neighbor. The inset shows a drawing of this pattern. ŽC. The cross-section is stained with a specific rod photoreceptor antibody ŽK16-107. which illustrates that rod photoreceptors heavily dominate the H. burtoni retina. In adults, only the outer segments of rods show positive staining. On the right, the tangential section of the positively stained punctate rods Žarrows. are interspersed among the much larger unstained cones. Abbreviations: inner segments i; margins m; myoid processs mp; nucleuss n; outer segments o; pedicles p.
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2. Materials and methods 2.1. Animal care The fish were maintained in an environmentally controlled facility with a 12:12 lightrdark cycle intended to mimic natural conditions w8,9x. Small social groups were kept in 200-l aquaria and fed twice a day with either cichlid flakes ŽTetra-min. or cichlid pellets ŽWardley.. Approximately 10% of the water was changed daily and replaced with water treated with cichlid salts ŽAqua Stock, Bayonne, NJ.. Most commonly, H. burtoni breeds in the morning hours and the females brood their young for approximately 2 weeks in their mouth. Following spawning, we removed embryos from the mother’s mouth and staged them according to the external morphology in Hagedorn and Fernald w11x. If the animals were removed and raised outside the
mother’s mouth during the first day after fertilization, the mortality of the brood was high and often developmental abnormalities appeared. However, on Day 2, animals from a single brood could be removed from the mother’s mouth and maintained in a 60-l aquarium at 308C in a buoyant plastic chamber with a mesh bottom to permit aerated water flow over the embryos. This apparatus allowed the eggs to roll gently, mimicking the action of the eggs in the mother’s mouth. We compared the developmental progress of these animals raised in vitro with that of the animals raised in the mother’s mouth. The mortality of the animals raised in vitro was very low and their developmental rate matched the rate of those animals raised in the mother’s mouth. 2.2. Histology Prior to fixation, the animals were sacrificed by placing them in ice-cold aquarium water. Adult animals were
Fig. 2. Immunostaining of the adult H. burtoni retina against vimentin and GABA. Sections on the left show the overall staining pattern of the antibodies across the retina while the figures on the right show enlarged detail of particular cells. Bars s 40 m m. ŽA. Muller ¨ cells show positive immunostaining for vimentin in the adult retina. The radial processes and the Muller cell bodies Žarrows. are shown in greater detail on the right. Their processes surround ¨ nuclei in the ganglion cell layer Žopen arrows.. ŽB. GABA-like immunoreactivity stains horizontal, amacrine and ganglion cells; however, only the horizontal cells Žarrows. close to the margin show positive staining. The horizontal cells in the central retina Žto the right of the arrows. do not show GABA-like immunoreactivity. A tangential section of the margin, reveals large GABA-positive horizontal cells in a grid-like pattern Žwhite rectangle..
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sacrificed by spinal section and then whole embryos or eyes removed from the adults were placed in the appropriate fixative. In adult eyes, the corneas were pierced to ensure proper fixation of the retina. Following fixation, the retinas were rinsed in phosphate buffered saline ŽPBS., embedded in a 1.5% agar, 5% sucrose and 0.1 M phosphate buffer solution, cryoprotected in 30% sucrose in 0.1 M phosphate buffer solution overnight, and then sectioned at 10 m m with a cryostat onto gelatinized slides. To characterize the onset of the immunoreactivity to specific antibodies, serial sections were made through the retinas of the embryos from Day 4 through Day 7 Ž N s 2–5 at each developmental time point.. Only sections from the center of the adult retina were used to characterize the adult staining pattern. Because it is often difficult to determine the immunostaining pattern from cross-sections of the retina, we made both cross-sectional Žwhich shows layers as concentric circles. and tangential sections Žwhich viewed a single layer at a time. of the areas of interest. 2.3. Immunohistochemical procedures Since techniques varied amongst antibodies, methods for each one will be described separately. For all antibodies, a dilution series was performed for both adult and embryonic tissue to discover the dilution needed for dark staining of individual cells or fibers with little or no staining of the surrounding tissue. Four antibodies were used to analyze the onset of rods and cones in the developing retina. Three cone monoclonal antibodies ŽT7, 9C1 and 12H10. were developed in collaboration with our laboratory by the Monoclonal Facility at the University of Oregon using the techniques of Marusich w21x. The fourth antibody was a rod specific antibody ŽK16-107. which recognizes eight amino acids Ž340–348. of bovine rhodopsin w1x. For these four antibodies, the
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animals were fixed overnight at 48C in 4% paraformaldehyde in 0.1 M phosphate buffer. The dilution, blocking and incubation for each antibody is as follows. Two cone antibodies ŽT7 and 12H10. were diluted 1:800 and 1:3 with PBS, respectively. The sections were blocked for 20 min in a solution of 10% bovine serum albumin and 0.1% horse serum and then incubated with the primary antibody for 1 hour at room temperature. The cone and rod antibodies Ž9C1 and K16-107., were undiluted, and diluted 1:20 with PBS, respectively. The sections were blocked for 20 min in a solution of 10% bovine serum albumin, 0.1% horse serum and 0.1% Triton-X and then incubated with the primary antibody overnight at 48C. The dilutions used for all of the photoreceptor antibodies were equivalent for both adult and embryonic tissue. To localize Muller cells, we used a monoclonal anti¨ body against the cytoskeletal protein, vimentin ŽSigma., and glial fibrillary acidic protein ŽGFAP. w3x. Animals were fixed in 4% paraformaldehyde in 0.1 M phosphate buffer overnight at 48C. Vimentin-antibody dilutions of 1:5 for the embryos and 1:20 for the adult yielded good results. The vimentin antibody was blocked in a 0.1% horse serum in PBS for 20 min, and the primary antibody was incubated overnight at 48C. To examine GABAergic staining, we used a monoclonal antibody for GABA–BSA. GABA-antibody dilutions of approximately 1:5000 for the embryos and of 1:15,000 for adults yielded good results. Animals were fixed overnight in 2–4% glutaraldehyde in 0.1 M phosphate buffer at 48C. We followed the immunohistochemical procedures of Glasener et al. w10x for the staining of ¨ GABA and used 10% bovine serum albumin with 0.1% Triton-X in PBS as 20 min block and in diluting solution with the antibody. The GABA antibody was incubated overnight at 48C.
Fig. 3. A summary of the onset of immunoreactivity of six antibodies in the central portion of the developing retina of H. burtoni from Day 4 to Day 7. The most distinctive feature of this pattern is the transient production of GABA in the central retina from Day 5 to Day 6.
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Fig. 4. Immunoreactivity of the developing cone ŽA. and rod ŽB. photoreceptors on embryonic Day 6 in the H. burtoni retina. A developmental gradient in the maturation of photoreceptors exists. The central retina Žleft. exhibits darker stained, more mature cells than in the peripheral retina Žright.. Bar s 50 m m. ŽA. In the central retina Žleft., the cone-specific antibody, 9C1, reveals positively stained inner segments Ži., nuclei Žn., and pedicles Žp.. In the periphery Žright., cones show a lighter staining and a rectangular shape near the margin Žm.. ŽB. In the central part of the retina Žleft., the rod opsin-specific antibody, K16-107, stains developing rod photoreceptors. Cells show positive staining for the embryonic inner segment Ži., the dark-staining nucleus Žn., and the myoid process Žmp. leading out of the nucleus. This pattern is different than observed in the adult pattern which only stains outer segments. Although there is a developmental gradient in the intensity of the opsin-stain at the periphery of the retina Žright., it does not reveal the developing morphology of the rods near the margin Žm..
In addition, we examined a cone horizontal cell antibody ŽIII F7.1. w43x diluted 1:10 with PBS and processed the same as the 9C1 cone antibody. All the antibodies were visualized with the avidin–biotin peroxidase method ŽVectastain Mouse IgG Kit, Vector Laboratories, Burlingame, CA.. The primary antibody was removed, the secondary biotinylated anti-mouse IgG and the avidin–biotin horseradish peroxidase complex were prepared and used according to the standard procedure given with the kit. The peroxidase was developed using a
0.1% nickel chloride for intensification and 3,3X-diaminobenzidine tetrahydrochloride Ž0.5 mgrml; DAB., peroxide Ž0.01%. and PBS solution for 15 min. 3. Results 3.1. Adult pattern of retinal immunostaining In the outer nuclear layer ŽONL., we used three antibodies specific for cone photoreceptors and a single anti-
Fig. 5. GABA-like immunoreactivity in the horizontal cells parallels the onset of rod neurogenesis in the embryonic H. burtoni retina. Sections sampled from the central retina on Day 5 to 7. On the left is the developmental pattern of rod-specific opsin ŽK16-107. immunoreactivity present in the outer nuclear layer illustrating the pattern of rod neurogenesis across the developing retina. On the right is the GABA-like immunoreactivity across the developing retina. Bar s 50 m m. Day 5: At the onset of rod neurogenesis Žleft. only a small portion of the retina is immunopositive Žarea between the black arrows.. The onset of GABA-like immunoreactivity in horizontal cells is shown Žwhite box, right.. An enlargement of this area indicates the immunopositive horizontal cells Žwhite arrows, right.. In addition, the amacrine cells, inner plexiform layer and ganglion cells show immunopositive staining for GABA, as well. The onset of the staining of these cells is Day 4. Day 6: The retina has enlarged and rod neurogenesis Žleft. is expanding across the retina Žblack arrows, left.. This is paralleled by an expansion of GABA production in horizontal cells across the retina which now show GABA-like immunoreactivity Žin the area between the open arrows, right.. Day 7: Rod neurogenesis is continuing in the areas towards the margin Žleft.; however, all rods are labeled, as indicated by the black arrows. The GABA-like immunoreactivity of the horizontal cells is now absent from the central retina, only the areas between the open arrows Žright. are immunopositive.
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body specific for rod opsin ŽFig. 1.. Two antibodies ŽT7 and 9C1. label most of the double and single cone photoreceptor Žinner segment, nucleus, myoid process and pedicle; Fig. 1A.. Because T7 and 9C1 showed similar staining patterns in the adult, only 9C1 was shown in Fig. 1A; however, we utilized both of these antibodies here since they differ in their onset time during development. The 12H10 antibody also labels cones Žinner and outer segment, myoid process and nucleus; Fig. 1B.. Tangential sections revealed that the antibody stained only a single member of the double cones and their nearest neighbor in the adjacent double cone, forming an x-shaped pattern ŽFig. 1B, inset.. Moreover, 12H10 seems to be membrane associated, as can be seen in the tangential section where the heavy staining pattern surrounds the outside of the cell. The rod-opsin specific antibody ŽK16-107. was used to show the pattern of rods in the adult retina. Rod photoreceptors dominate the adult retina ŽFig. 1C. and only rod outer segments are heavily stained. The selectivity of this antibody is demonstrated in the tangential sections where rod outer segments appear as dark dots that fill the spaces between the larger, unstained cones ŽFig. 1C.. We also examined the staining pattern revealed by antibodies to vimentin and GABA in adult retinal tissue. Both revealed staining in the inner nuclear layer ŽINL. and the ganglion cell layer ŽGCL.. The cytoskeletal protein, vimentin, is broadly distributed throughout these areas of the retina, but Muller cells and their processes are most ¨ clearly filled with this protein in the areas surrounding ganglion cell bodies ŽFig. 2A.. GABA stains three distinct types of cells within the adult H. burtoni retina, large amacrine cells, ganglion cells and horizontal cells. Large amacrine cells close to the inner plexiform layer and some ganglion cells were observed to stain uniformly across the whole retina; however, horizontal cells show GABA-like immunoreactivity only near the margin ŽFig. 2B.. This restriction of GABAergic staining to only those horizontal cells near the edge of the retina is unique to teleosts. A tangential section of the margin area revealed large GABAergic horizontal cells in a grid-like pattern typical of the embryonic form of horizontal cell w11x. Additionally, it appears that GABA immunoreactivity is quite intense in the inner plexiform layer across the whole retina ŽFig. 2B arrow.. Two antibodies, GFAP w3x and a cone horizontal cell antibody ŽIII F7.1; w43x. which react with the goldfish, either do not cross-react or only cross-react weakly with the H. burtoni retina Ždata not shown.. 3.2. Embryonic pattern of retinal immunostaining A summary of the onset of immunostaining in the central retina of the embryonic H. burtoni retina is shown
in Fig. 3. On Day 4, cone photoreceptors ŽT7., and amacrine and ganglion cells ŽGABA. show positive staining. At this time, vimentin reactivity is widely distributed throughout the retina in radial fibers that cross through most layers of the retina in a spoke-like pattern, but the vimentin antibody does not stain any cell bodies. At Day 5, a variety of antibodies show positive immunostaining. Specifically, two cone antibodies Ž9C1. and Ž12H10., and rod photoreceptors ŽK16-107. and horizontal cells ŽGABA. first show immunoreactivity in the central part of the retina. By Day 7, the horizontal cells in the center of the retina are no longer GABAergic, the spoke-like vimentin pattern is no longer as pronounced and Muller cell bodies ¨ become vimentin-positive. To understand the potential importance of this sequence of staining in a developmental context, we focused on the differentiation of cells in three areas of the retina: first, photoreceptors within the ONL; second, horizontal cells near the ONL; and third, cells in other parts of the retina. Within the ONL, rod and cone photoreceptors do not differentiate at the same time. In H. burtoni, the onset of cone photoreceptor labeling ŽT7, Day 4; 9C1, Day 5. precedes rod photoreceptors ŽK16-107; Day 5. by 1 day. Although the cone antibodies were first visualized at slightly different times, the stained cells showed similar cellular morphologies, whereas from the onset of differentiation, rods and cones show divergent morphological patterns ŽFig. 4.. On Day 5, the 9C1 antibody begins to stain cuboidally-shaped cells in the central ONL Žthat are presumably the cone nuclei.. On the following day, inner and outer segments, cone nuclei, pedicles and myoid processes are evident in the central ONL, with a developmental gradient in cell morphology continuing towards the margin ŽFig. 4A.. Rods differentiate on Day 5 and rod-specific opsin immunoreactivity originates in the small round nuclei. Some nuclei also have a slender darkly staining process emerging sclerad ŽFig. 4B.. Within 1 day ŽDay 6., these cells appear to be morphologically mature rod photoreceptors. The onset of rod differentiation is correlated with a transient production of GABA immunoreactivity by the horizontal cells located in the inner plexiform layer ŽIPL; Fig. 5.. Rod differentiation is observed in the central portion of the ONL on Day 5 and spreads tangentially towards the peripheral margin by Day 7 ŽFig. 5.. At this time, the rods in the central retina resemble the adult phenotype whereas the rods toward the edge of the retina are actively differentiating. By Day 5, horizontal cells have formed a grid-like sheet over the entire embryonic retina w11x; however, GABAergic staining is only observed in horizontal cells in the central part of this pattern. By Day 6, this GABAergic staining extends toward the peripheral
Fig. 6. Immunoreactivity of vimentin in the central portion of the embryonic H. burtoni retina. Bars s 50 m m. ŽA. On Day 4, vimentin-positive fibers radiate from the darkly-staining inner plexiform layer. ŽB. By Day 7, much of the radiating immunoreactive fibers are now found in the periphery and less distinct in the center, indicating a more elaborate branching of emerging Muller cell processes. ŽC. Muller cells can be identified on Day 7 and are ¨ ¨ characterized as large cell bodies darkly-outlined with vimentin Žarrows..
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margin in a continuous row of cells; however, this pattern becomes discontinuous on Day 7 when the cells in the central portion of the retina Ždark arrows. are no longer GABA positive ŽFig. 5.. Horizontal cells in the retina of H. burtoni transiently produce GABA for approximately 2 days. The embryonic retina of H. burtoni is strongly immunopositive for the intermediate filament protein vimentin. On Day 4, vimentin fibers radiate in a spoke-like pattern from a densely stained IPL ŽFig. 6A.. This radial pattern continues until Day 7 where vimentin is deposited around the cells and most strongly around large cell bodies near the IPL, putative Muller ¨ cells ŽFig. 6B.. Thus, Muller ¨ cells cannot be identified in the embryonic retina until Day 7 which is 2 days past neurogenesis and differentiation of the first rods within the retina ŽFigs. 5 and 6C.. 4. Discussion What controls the development of rod photoreceptors in the teleost retina? Evidence suggests that stem cells in the teleost retina left behind in the INL produce rod precursors that migrate to the ONL w15,28,29,11x. In the zebrafish retina, some rhodopsin expressing cells emerge around the same time as cone opsin expressing cells w31,34x; yet, it is not clear whether they are produced by the same mechanism as the rod precursors which arise later in development. In H. burtoni, cone differentiation clearly precedes rod differentiation and thus follows the cell-type gradients of histogenesis found in most vertebrates w12x. Rod precursors have migratory ability even in adult fish w19x and presumably must be guided to their appropriate location. Muller ¨ cells have been suggested as possibly playing a role in the guidance of these migrating rod progenitors exiting from the INL into the ONL w15,28,29x. If Muller cells are ¨ involved in the guidance of rod progenitors, then these cells must be present in the developing retina prior to the onset of rod differentiation. The cytoskeletal protein, vimentin is one of the key structural elements in Muller ¨ cells w4,38,39x. In the H. burtoni retina, vimentin-positive radial fibers exist on Day 4, yet they cannot be identified as Muller cells. It is known that vimentin is expressed in ¨ retinal neuroblasts during development and is later restricted to Muller ¨ glial cells w17x. Vimentin-positive Muller ¨ cells cannot be identified in the embryonic retina until Day 7 which is 2 days after the onset of rod differentiation. Although vimentin is often taken as an indicator of early neural or glial radial fibers, which supposedly play a role in guiding migrating neuroblasts, to test this hypothesis directly, it will be important to characterize surface marker molecules which are in direct contact with the migrating cells. If vimentin-positive cells have a guidance function for migrating cells w42x then this function might be performed by neuroblasts in early development and taken over by Muller cells at later stages. ¨ GABA is considered to be a signaling substance in the
retina w33x. GABAergic staining in embryonic horizontal cells is a common developmental feature of most vertebrate retinas. In all cases, the embryonic sequence includes horizontal cells staining positively for GABA, across the entire retina. In some groups, such as mammals, this embryonic pattern of GABA is transient w41,26x, whereas most lower vertebrates maintain their embryonic staining pattern throughout life w2x. The horizontal cells of H. burtoni combine these two patterns. Specifically, in the central retina, transient activity is observed in horizontal cells while horizontal cells next to the developing margin maintain GABAergic staining throughout life. The pattern of GABAergic staining in horizontal cells of H. burtoni is unlike the reported patterns in the goldfish, where GABAergic horizontal cells have been reported w20x. On the other hand, it is similar to the distribution of GABA-positive horizontal cells in zebrafish where they are found only in the retinal periphery w35x. The zebrafish study, however, did not reveal transient GABA in central horizontal cells as observed in H. burtoni. This may reflect differences in the fishes’ visual processing and acuity, or different developmental strategies. In the fast developing zebrafish retina patterning signals such as GABA might be compressed or omitted, whereas in the somewhat slower developing, mouth brooding H. burtoni transient GABA might establish precisely patterned synaptic connections. There are several lines of evidence which suggest that GABA may be acting to control the differentiation of photoreceptors. Messersmith and Redburn w22x measured an increase in the rod to cone ratio when they selectively destroyed type A horizontal cells early in development. Although other aspects of the retina were disrupted by the kainic acid treatment, rods developed with normal morphology and synaptic connections. This suggests GABA may regulate the production of photoreceptors. More recently, Mitchell and Redburn w23x showed that GABA A receptors are expressed in the cone photoreceptor terminals around the time of synaptogenesis. In summary, the transient GABAergic staining in developing horizontal cells correlates spatially and temporally with the traveling wave of rod neurogenesis in the embryonic H. burtoni retina. This transient pulse of GABA may play a role both in the initiation and regulation of rod neurogenesis in the central retina during embryogenesis, and directly or indirectly in the developing margin. Acknowledgements We would like to thank the following people for their technical help and insightful discussions: R. Bremiller, G. Glasener-Cippillone, S. Kroger, M. McDowell, N. Tublitz, ¨ ¨ and S. Vollmer. We received several gifts of monoclonal antibodies from several laboratories: a GABA monoclonal antibody from C. Matute and P. Streit, University of Zurich; a rod-specific opsin monoclonal antibody ŽK16-
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107. from G. Adamus, University of Florida; a GFAP monoclonal antibody ŽA2D4. from A. Bignami, Harvard Medical School, and; a horizontal cell monoclonal antibody ŽIII F7.1. from L. Young, Harvard University. We thank the two anonymous reviewers for their careful reading of the manuscript. This grant was supported by NIH grant EY 05051 to RDF.
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Research report
The embryogenesis of rod photoreceptors in the teleost fish retina, Haplochromis burtoni Mary Hagedorn ) , Andreas F. Mack 1, Barbara Evans 2 , Russell D. Fernald
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Institute of Neuroscience, UniÕersity of Oregon, Eugene, OR 97403, USA Accepted 3 March 1998
Abstract Development of the retina, like that of other tissues, occurs via an orderly sequence of cell division and differentiation, producing the functional retina. In teleost fish, however, cell division and differentiation in the retina continue throughout the life of the animal in two distinct ways. Stem cells in a circumferential germinal zone at the periphery of the retina give rise to all retinal cell types and progenitor cells located throughout the retina in the outer nuclear layer ŽONL. produce new rod photoreceptors. These processes in adult retina recapitulate in space the embryonic events responsible for forming the retina. Analysis of these events in an African cichlid fish, Haplochromis burtoni, confirmed that cone photoreceptors differentiate first, followed by rod photoreceptors. Correspondingly, at the margin of the eye, cone photoreceptors differentiate nearer to the margin than do rods. Control of photoreceptor production is not understood. Here we present the time of appearance and distribution pattern of GABA and vimentin which are candidates for the control of retinal cell division and differentiation. Antibody staining reveals that both GABA and vimentin exhibit unique patterns of expression during embryonic retinal development. Vimentin immunoreactivity is evident throughout the retina in a spoke-like pattern between developmental Days 4 and 7, as both cone and rod photoreceptors are being formed. GABA is expressed in horizontal cells between Days 5 and 7, corresponding to the onset of rod differentiation in time and in position within the retina. Moreover, the wave of GABAergic staining in the horizontal cells parallels the wave of rod differentiation across the embryonic retina of H. burtoni. Thus, GABA may play a role in the development of rod photoreceptors. q 1998 Elsevier Science B.V. All rights reserved. Keywords: Retina; Development; Teleost; Cone photoreceptor; Rod photoreceptor; Muller cell; GABA; Vimentin ¨
1. Introduction Unlike the retinas of mammals, the teleost retina continues to add new neurons throughout the life of the animal. Stem cells arranged circumferentially at the periphery of the retina give rise to all retinal cell types except rods and progenitor cells located throughout the retina in the outer nuclear layer ŽONL. produce new rod photoreceptors. These processes in adult retina recapitulate in space the
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Corresponding author. Dept. of Reproductive Physiology, Smithsonian Institution, National Zoological Park, 3001 Conn. Ave, NW, Washington, DC 20008, USA. Fax: q 1-202-673-4733; E-mail: [email protected] 1 Current Address: Anatomisches Institut, Universitaet Tuebingen, Oesterbergstr.3, 72074 Tuebingen, Germany. 2 Current Address: Dept. Biol., Lake Superior State University, Sault St. Marie, MI 49783, USA. 3 Current Address: Neuroscience Program, Stanford University, Stanford, CA 94305, USA. 0165-3806r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 1 6 5 - 3 8 0 6 Ž 9 8 . 0 0 0 5 1 - 0
embryonic events in time responsible for forming the retina. How these events are controlled, either in the embryo or adult, is not understood. Here we present the time of appearance and distribution pattern of GABA and vimentin which are candidates for the control of retinal cell division and differentiation, because of their time of expression. In embryogenesis, the teleost retina begins as an invaginated optic cup consisting of spindle-shaped neuroepithelial cells which is transformed through development into a highly organized, laminated structure with several distinct types of retinal neurons w24,18x. In Haplochromis burtoni, an African cichlid fish, neurogenesis begins in the central, vitread portion of the retina, radiates toward the photoreceptor layer and spreads peripherally towards the margin of the eye w11x. In most vertebrate retinas, rod photoreceptors are the last cells to differentiate w7,30,14x, which is the case in H. burtoni. Differentiation of cone photoreceptors begins on Day 3 while the onset of rod differentiation is delayed until Day 4.5 w11x. This late onset of rod differen-
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tiation in fish can sometimes be quite extreme. For example, Sandy and Blaxter w36x observed in the retinas of flounder and sole that the rod differentiation was delayed for months until the onset of metamorphosis. Moreover, in the larval winter flounder, photoreceptors are uniformly
single cones with no double cones or rods appearing before metamorphosis w5,6x. In contrast, it has been reported in the zebrafish that rod opsin positive cells appear shortly before or around the time of cone opsins in a ventral patch of the retina w31,34x.
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In this paper, we have used immuno-histochemical tracers to identify cellular events which surround the late onset of rod neurogenesis in the teleost, H. burtoni. We have examined the neurogenesis of the photoreceptors, Muller ¨ cells, as well as the onset of putative guidance substances responsible for triggering the differentiation in the developing teleost retina. Specifically, we have stained the developing retina with vimentin and g-aminobutyric acid ŽGABA. to determine whether their timing or pattern of appearance might correlate with rod neurogenesis. The cytoskeletal protein, vimentin, is one of the key structural elements in the Muller ¨ cell, a major type of glial cell present in the retina w4,38,39x. It is not clear what the function of vimentin is, but Wylie w42x speculates that it is responsible for the movement of cells during early development. Wylie has shown that blocking vimentin caused improper cleavage patterns in Xenopus embryos. Moreover, Johns w15x, Raymond w28x, and Raymond and Rivlin w29x have implicated Muller ¨ cells as ‘guidance’ cells which assist rod progenitors in migrating from the inner nuclear layer to the outer nuclear layer during early development and growth. If so, during embryogenesis, the origin of Muller ¨ cells andror vimentin must precede the differentiation of rod photoreceptors. In order to test this hypothesis, we examined the onset of vimentin immunoreactivity and compared it to the onset time of rod neurogenesis. The inhibitory neurotransmitter GABA has also been implicated as a pioneering or trophic substance both in the developing central nervous system and in the developing retina w37,26,32,30x. In the developing rat brain, Lauder et al. w16x observed GABAergic fibers running from the cortex to the telencephalon at the onset of neurogenesis in this area. Early in the development of certain mammals, such as the mouse and the rabbit, type A horizontal cells mature precociously and transiently produce GABA w37,32,41x. Destruction of these GABAergic horizontal cells with kainic acid resulted in a destruction of the horizontal cells, and a deregulation resulting in an overproduction of rod photoreceptors in the outer nuclear layer and a disruption of the synaptic connections in the outer plexiform layer w22x. Such transient production of GABA by the horizontal cells suggests that GABA is important for neuronal development, in general, and may play a role
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in the regulation of photoreceptor differentiation in the retina. Additionally, Mitchell and Redburn w23x suggested a role for GABA during cone synaptogenesis in the rabbit. If a similar function occurs in fish, this would imply that GABA immunoreactivity must precede or co-occur with the onset of photoreceptor differentiation. In order to test this hypothesis, we examined the onset of GABA immunoreactivity in horizontal cells and compared it to the onset of rod neurogenesis. In contrast to the transitory embryonic pattern of GABAergic staining in the horizontal cells of mammals, in most lower vertebrates, Žsuch as fish, frogs and birds., GABAergic staining is retained in H 1 horizontal cells, a sub-type of cone horizontal cells, across the entire adult retina w20,27,2,40,14x. Little is known about the embryonic onset of GABAergic staining in the horizontal cells of lower vertebrates; however, chicken horizontal cells first express GABA-like immunoreactivity as early as Day 8 w14x. Negishi and Wagner w25x report GABAergic staining in a cichlid retina Ž Aequidens pulcher . around the time of synaptogenesis but do not specify which horizontal cells. Additionally, transient expression of GABA was found in ganglion cells but not in horizontal cells of the developing zebrafish retina w35x. To understand the late onset of rod neurogenesis in the teleost retina, we used immunocytochemistry to determine the onset and pattern of GABA and vimentin expression at the time of rod neurogenesis. We identified the onset of rod neurogenesis with a rod-specific opsin antibody w1x and compared the adult pattern Žthe central retina of a 2month-old fish. of immunohistochemical staining with the embryonic pattern between Days 4 and 7. We used six separate antibodies to delineate separate aspects of the developing retina for three reasons. First, we wanted to describe the onset of photoreceptor maturation and distinguish between rod and cone maturation. Second, we were interested in how the spatial patterns of embryonic neurogenesis related to the continuing patterns of neurogenesis in the adult retina. Third, we wished to examine the correlation of reputed guidance and pioneering substances in the retina Žwhich may function in the guidance and insertion of rod photoreceptors into the ONL. with the onset of photoreceptor maturation.
Fig. 1. Immunostaining of the photoreceptors in adult H. burtoni. The retinas are viewed in cross-section on the left and in tangential section on the level of the inner segments of the photoreceptors on the right. The retinas were dark adapted to retract the pigmented epithelium and reveal the morphology of the photoreceptors. Bars s 20 m m. ŽA. The 9C1 antibody stains both double and single cones including inner segments, nuclei, myoid processes and pedicles. The tangential section on the right shows the positively stained surrounding octet of double cones Žwhite arrows. and the central cones Žopen white arrows.. ŽB. The 12H10 antibody stains only one member of the double cones. On the left, the cross-sections show the tips of the outer segments, the inner segments, the myoid processes and a very dark staining body above the cone nuclei. The tangential section on the right shows a dark, cross-shaped pattern surrounding a single member of the double cone inner segments and their nearest neighbor. The inset shows a drawing of this pattern. ŽC. The cross-section is stained with a specific rod photoreceptor antibody ŽK16-107. which illustrates that rod photoreceptors heavily dominate the H. burtoni retina. In adults, only the outer segments of rods show positive staining. On the right, the tangential section of the positively stained punctate rods Žarrows. are interspersed among the much larger unstained cones. Abbreviations: inner segments i; margins m; myoid processs mp; nucleuss n; outer segments o; pedicles p.
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2. Materials and methods 2.1. Animal care The fish were maintained in an environmentally controlled facility with a 12:12 lightrdark cycle intended to mimic natural conditions w8,9x. Small social groups were kept in 200-l aquaria and fed twice a day with either cichlid flakes ŽTetra-min. or cichlid pellets ŽWardley.. Approximately 10% of the water was changed daily and replaced with water treated with cichlid salts ŽAqua Stock, Bayonne, NJ.. Most commonly, H. burtoni breeds in the morning hours and the females brood their young for approximately 2 weeks in their mouth. Following spawning, we removed embryos from the mother’s mouth and staged them according to the external morphology in Hagedorn and Fernald w11x. If the animals were removed and raised outside the
mother’s mouth during the first day after fertilization, the mortality of the brood was high and often developmental abnormalities appeared. However, on Day 2, animals from a single brood could be removed from the mother’s mouth and maintained in a 60-l aquarium at 308C in a buoyant plastic chamber with a mesh bottom to permit aerated water flow over the embryos. This apparatus allowed the eggs to roll gently, mimicking the action of the eggs in the mother’s mouth. We compared the developmental progress of these animals raised in vitro with that of the animals raised in the mother’s mouth. The mortality of the animals raised in vitro was very low and their developmental rate matched the rate of those animals raised in the mother’s mouth. 2.2. Histology Prior to fixation, the animals were sacrificed by placing them in ice-cold aquarium water. Adult animals were
Fig. 2. Immunostaining of the adult H. burtoni retina against vimentin and GABA. Sections on the left show the overall staining pattern of the antibodies across the retina while the figures on the right show enlarged detail of particular cells. Bars s 40 m m. ŽA. Muller ¨ cells show positive immunostaining for vimentin in the adult retina. The radial processes and the Muller cell bodies Žarrows. are shown in greater detail on the right. Their processes surround ¨ nuclei in the ganglion cell layer Žopen arrows.. ŽB. GABA-like immunoreactivity stains horizontal, amacrine and ganglion cells; however, only the horizontal cells Žarrows. close to the margin show positive staining. The horizontal cells in the central retina Žto the right of the arrows. do not show GABA-like immunoreactivity. A tangential section of the margin, reveals large GABA-positive horizontal cells in a grid-like pattern Žwhite rectangle..
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sacrificed by spinal section and then whole embryos or eyes removed from the adults were placed in the appropriate fixative. In adult eyes, the corneas were pierced to ensure proper fixation of the retina. Following fixation, the retinas were rinsed in phosphate buffered saline ŽPBS., embedded in a 1.5% agar, 5% sucrose and 0.1 M phosphate buffer solution, cryoprotected in 30% sucrose in 0.1 M phosphate buffer solution overnight, and then sectioned at 10 m m with a cryostat onto gelatinized slides. To characterize the onset of the immunoreactivity to specific antibodies, serial sections were made through the retinas of the embryos from Day 4 through Day 7 Ž N s 2–5 at each developmental time point.. Only sections from the center of the adult retina were used to characterize the adult staining pattern. Because it is often difficult to determine the immunostaining pattern from cross-sections of the retina, we made both cross-sectional Žwhich shows layers as concentric circles. and tangential sections Žwhich viewed a single layer at a time. of the areas of interest. 2.3. Immunohistochemical procedures Since techniques varied amongst antibodies, methods for each one will be described separately. For all antibodies, a dilution series was performed for both adult and embryonic tissue to discover the dilution needed for dark staining of individual cells or fibers with little or no staining of the surrounding tissue. Four antibodies were used to analyze the onset of rods and cones in the developing retina. Three cone monoclonal antibodies ŽT7, 9C1 and 12H10. were developed in collaboration with our laboratory by the Monoclonal Facility at the University of Oregon using the techniques of Marusich w21x. The fourth antibody was a rod specific antibody ŽK16-107. which recognizes eight amino acids Ž340–348. of bovine rhodopsin w1x. For these four antibodies, the
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animals were fixed overnight at 48C in 4% paraformaldehyde in 0.1 M phosphate buffer. The dilution, blocking and incubation for each antibody is as follows. Two cone antibodies ŽT7 and 12H10. were diluted 1:800 and 1:3 with PBS, respectively. The sections were blocked for 20 min in a solution of 10% bovine serum albumin and 0.1% horse serum and then incubated with the primary antibody for 1 hour at room temperature. The cone and rod antibodies Ž9C1 and K16-107., were undiluted, and diluted 1:20 with PBS, respectively. The sections were blocked for 20 min in a solution of 10% bovine serum albumin, 0.1% horse serum and 0.1% Triton-X and then incubated with the primary antibody overnight at 48C. The dilutions used for all of the photoreceptor antibodies were equivalent for both adult and embryonic tissue. To localize Muller cells, we used a monoclonal anti¨ body against the cytoskeletal protein, vimentin ŽSigma., and glial fibrillary acidic protein ŽGFAP. w3x. Animals were fixed in 4% paraformaldehyde in 0.1 M phosphate buffer overnight at 48C. Vimentin-antibody dilutions of 1:5 for the embryos and 1:20 for the adult yielded good results. The vimentin antibody was blocked in a 0.1% horse serum in PBS for 20 min, and the primary antibody was incubated overnight at 48C. To examine GABAergic staining, we used a monoclonal antibody for GABA–BSA. GABA-antibody dilutions of approximately 1:5000 for the embryos and of 1:15,000 for adults yielded good results. Animals were fixed overnight in 2–4% glutaraldehyde in 0.1 M phosphate buffer at 48C. We followed the immunohistochemical procedures of Glasener et al. w10x for the staining of ¨ GABA and used 10% bovine serum albumin with 0.1% Triton-X in PBS as 20 min block and in diluting solution with the antibody. The GABA antibody was incubated overnight at 48C.
Fig. 3. A summary of the onset of immunoreactivity of six antibodies in the central portion of the developing retina of H. burtoni from Day 4 to Day 7. The most distinctive feature of this pattern is the transient production of GABA in the central retina from Day 5 to Day 6.
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Fig. 4. Immunoreactivity of the developing cone ŽA. and rod ŽB. photoreceptors on embryonic Day 6 in the H. burtoni retina. A developmental gradient in the maturation of photoreceptors exists. The central retina Žleft. exhibits darker stained, more mature cells than in the peripheral retina Žright.. Bar s 50 m m. ŽA. In the central retina Žleft., the cone-specific antibody, 9C1, reveals positively stained inner segments Ži., nuclei Žn., and pedicles Žp.. In the periphery Žright., cones show a lighter staining and a rectangular shape near the margin Žm.. ŽB. In the central part of the retina Žleft., the rod opsin-specific antibody, K16-107, stains developing rod photoreceptors. Cells show positive staining for the embryonic inner segment Ži., the dark-staining nucleus Žn., and the myoid process Žmp. leading out of the nucleus. This pattern is different than observed in the adult pattern which only stains outer segments. Although there is a developmental gradient in the intensity of the opsin-stain at the periphery of the retina Žright., it does not reveal the developing morphology of the rods near the margin Žm..
In addition, we examined a cone horizontal cell antibody ŽIII F7.1. w43x diluted 1:10 with PBS and processed the same as the 9C1 cone antibody. All the antibodies were visualized with the avidin–biotin peroxidase method ŽVectastain Mouse IgG Kit, Vector Laboratories, Burlingame, CA.. The primary antibody was removed, the secondary biotinylated anti-mouse IgG and the avidin–biotin horseradish peroxidase complex were prepared and used according to the standard procedure given with the kit. The peroxidase was developed using a
0.1% nickel chloride for intensification and 3,3X-diaminobenzidine tetrahydrochloride Ž0.5 mgrml; DAB., peroxide Ž0.01%. and PBS solution for 15 min. 3. Results 3.1. Adult pattern of retinal immunostaining In the outer nuclear layer ŽONL., we used three antibodies specific for cone photoreceptors and a single anti-
Fig. 5. GABA-like immunoreactivity in the horizontal cells parallels the onset of rod neurogenesis in the embryonic H. burtoni retina. Sections sampled from the central retina on Day 5 to 7. On the left is the developmental pattern of rod-specific opsin ŽK16-107. immunoreactivity present in the outer nuclear layer illustrating the pattern of rod neurogenesis across the developing retina. On the right is the GABA-like immunoreactivity across the developing retina. Bar s 50 m m. Day 5: At the onset of rod neurogenesis Žleft. only a small portion of the retina is immunopositive Žarea between the black arrows.. The onset of GABA-like immunoreactivity in horizontal cells is shown Žwhite box, right.. An enlargement of this area indicates the immunopositive horizontal cells Žwhite arrows, right.. In addition, the amacrine cells, inner plexiform layer and ganglion cells show immunopositive staining for GABA, as well. The onset of the staining of these cells is Day 4. Day 6: The retina has enlarged and rod neurogenesis Žleft. is expanding across the retina Žblack arrows, left.. This is paralleled by an expansion of GABA production in horizontal cells across the retina which now show GABA-like immunoreactivity Žin the area between the open arrows, right.. Day 7: Rod neurogenesis is continuing in the areas towards the margin Žleft.; however, all rods are labeled, as indicated by the black arrows. The GABA-like immunoreactivity of the horizontal cells is now absent from the central retina, only the areas between the open arrows Žright. are immunopositive.
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body specific for rod opsin ŽFig. 1.. Two antibodies ŽT7 and 9C1. label most of the double and single cone photoreceptor Žinner segment, nucleus, myoid process and pedicle; Fig. 1A.. Because T7 and 9C1 showed similar staining patterns in the adult, only 9C1 was shown in Fig. 1A; however, we utilized both of these antibodies here since they differ in their onset time during development. The 12H10 antibody also labels cones Žinner and outer segment, myoid process and nucleus; Fig. 1B.. Tangential sections revealed that the antibody stained only a single member of the double cones and their nearest neighbor in the adjacent double cone, forming an x-shaped pattern ŽFig. 1B, inset.. Moreover, 12H10 seems to be membrane associated, as can be seen in the tangential section where the heavy staining pattern surrounds the outside of the cell. The rod-opsin specific antibody ŽK16-107. was used to show the pattern of rods in the adult retina. Rod photoreceptors dominate the adult retina ŽFig. 1C. and only rod outer segments are heavily stained. The selectivity of this antibody is demonstrated in the tangential sections where rod outer segments appear as dark dots that fill the spaces between the larger, unstained cones ŽFig. 1C.. We also examined the staining pattern revealed by antibodies to vimentin and GABA in adult retinal tissue. Both revealed staining in the inner nuclear layer ŽINL. and the ganglion cell layer ŽGCL.. The cytoskeletal protein, vimentin, is broadly distributed throughout these areas of the retina, but Muller cells and their processes are most ¨ clearly filled with this protein in the areas surrounding ganglion cell bodies ŽFig. 2A.. GABA stains three distinct types of cells within the adult H. burtoni retina, large amacrine cells, ganglion cells and horizontal cells. Large amacrine cells close to the inner plexiform layer and some ganglion cells were observed to stain uniformly across the whole retina; however, horizontal cells show GABA-like immunoreactivity only near the margin ŽFig. 2B.. This restriction of GABAergic staining to only those horizontal cells near the edge of the retina is unique to teleosts. A tangential section of the margin area revealed large GABAergic horizontal cells in a grid-like pattern typical of the embryonic form of horizontal cell w11x. Additionally, it appears that GABA immunoreactivity is quite intense in the inner plexiform layer across the whole retina ŽFig. 2B arrow.. Two antibodies, GFAP w3x and a cone horizontal cell antibody ŽIII F7.1; w43x. which react with the goldfish, either do not cross-react or only cross-react weakly with the H. burtoni retina Ždata not shown.. 3.2. Embryonic pattern of retinal immunostaining A summary of the onset of immunostaining in the central retina of the embryonic H. burtoni retina is shown
in Fig. 3. On Day 4, cone photoreceptors ŽT7., and amacrine and ganglion cells ŽGABA. show positive staining. At this time, vimentin reactivity is widely distributed throughout the retina in radial fibers that cross through most layers of the retina in a spoke-like pattern, but the vimentin antibody does not stain any cell bodies. At Day 5, a variety of antibodies show positive immunostaining. Specifically, two cone antibodies Ž9C1. and Ž12H10., and rod photoreceptors ŽK16-107. and horizontal cells ŽGABA. first show immunoreactivity in the central part of the retina. By Day 7, the horizontal cells in the center of the retina are no longer GABAergic, the spoke-like vimentin pattern is no longer as pronounced and Muller cell bodies ¨ become vimentin-positive. To understand the potential importance of this sequence of staining in a developmental context, we focused on the differentiation of cells in three areas of the retina: first, photoreceptors within the ONL; second, horizontal cells near the ONL; and third, cells in other parts of the retina. Within the ONL, rod and cone photoreceptors do not differentiate at the same time. In H. burtoni, the onset of cone photoreceptor labeling ŽT7, Day 4; 9C1, Day 5. precedes rod photoreceptors ŽK16-107; Day 5. by 1 day. Although the cone antibodies were first visualized at slightly different times, the stained cells showed similar cellular morphologies, whereas from the onset of differentiation, rods and cones show divergent morphological patterns ŽFig. 4.. On Day 5, the 9C1 antibody begins to stain cuboidally-shaped cells in the central ONL Žthat are presumably the cone nuclei.. On the following day, inner and outer segments, cone nuclei, pedicles and myoid processes are evident in the central ONL, with a developmental gradient in cell morphology continuing towards the margin ŽFig. 4A.. Rods differentiate on Day 5 and rod-specific opsin immunoreactivity originates in the small round nuclei. Some nuclei also have a slender darkly staining process emerging sclerad ŽFig. 4B.. Within 1 day ŽDay 6., these cells appear to be morphologically mature rod photoreceptors. The onset of rod differentiation is correlated with a transient production of GABA immunoreactivity by the horizontal cells located in the inner plexiform layer ŽIPL; Fig. 5.. Rod differentiation is observed in the central portion of the ONL on Day 5 and spreads tangentially towards the peripheral margin by Day 7 ŽFig. 5.. At this time, the rods in the central retina resemble the adult phenotype whereas the rods toward the edge of the retina are actively differentiating. By Day 5, horizontal cells have formed a grid-like sheet over the entire embryonic retina w11x; however, GABAergic staining is only observed in horizontal cells in the central part of this pattern. By Day 6, this GABAergic staining extends toward the peripheral
Fig. 6. Immunoreactivity of vimentin in the central portion of the embryonic H. burtoni retina. Bars s 50 m m. ŽA. On Day 4, vimentin-positive fibers radiate from the darkly-staining inner plexiform layer. ŽB. By Day 7, much of the radiating immunoreactive fibers are now found in the periphery and less distinct in the center, indicating a more elaborate branching of emerging Muller cell processes. ŽC. Muller cells can be identified on Day 7 and are ¨ ¨ characterized as large cell bodies darkly-outlined with vimentin Žarrows..
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margin in a continuous row of cells; however, this pattern becomes discontinuous on Day 7 when the cells in the central portion of the retina Ždark arrows. are no longer GABA positive ŽFig. 5.. Horizontal cells in the retina of H. burtoni transiently produce GABA for approximately 2 days. The embryonic retina of H. burtoni is strongly immunopositive for the intermediate filament protein vimentin. On Day 4, vimentin fibers radiate in a spoke-like pattern from a densely stained IPL ŽFig. 6A.. This radial pattern continues until Day 7 where vimentin is deposited around the cells and most strongly around large cell bodies near the IPL, putative Muller ¨ cells ŽFig. 6B.. Thus, Muller ¨ cells cannot be identified in the embryonic retina until Day 7 which is 2 days past neurogenesis and differentiation of the first rods within the retina ŽFigs. 5 and 6C.. 4. Discussion What controls the development of rod photoreceptors in the teleost retina? Evidence suggests that stem cells in the teleost retina left behind in the INL produce rod precursors that migrate to the ONL w15,28,29,11x. In the zebrafish retina, some rhodopsin expressing cells emerge around the same time as cone opsin expressing cells w31,34x; yet, it is not clear whether they are produced by the same mechanism as the rod precursors which arise later in development. In H. burtoni, cone differentiation clearly precedes rod differentiation and thus follows the cell-type gradients of histogenesis found in most vertebrates w12x. Rod precursors have migratory ability even in adult fish w19x and presumably must be guided to their appropriate location. Muller ¨ cells have been suggested as possibly playing a role in the guidance of these migrating rod progenitors exiting from the INL into the ONL w15,28,29x. If Muller cells are ¨ involved in the guidance of rod progenitors, then these cells must be present in the developing retina prior to the onset of rod differentiation. The cytoskeletal protein, vimentin is one of the key structural elements in Muller ¨ cells w4,38,39x. In the H. burtoni retina, vimentin-positive radial fibers exist on Day 4, yet they cannot be identified as Muller cells. It is known that vimentin is expressed in ¨ retinal neuroblasts during development and is later restricted to Muller ¨ glial cells w17x. Vimentin-positive Muller ¨ cells cannot be identified in the embryonic retina until Day 7 which is 2 days after the onset of rod differentiation. Although vimentin is often taken as an indicator of early neural or glial radial fibers, which supposedly play a role in guiding migrating neuroblasts, to test this hypothesis directly, it will be important to characterize surface marker molecules which are in direct contact with the migrating cells. If vimentin-positive cells have a guidance function for migrating cells w42x then this function might be performed by neuroblasts in early development and taken over by Muller cells at later stages. ¨ GABA is considered to be a signaling substance in the
retina w33x. GABAergic staining in embryonic horizontal cells is a common developmental feature of most vertebrate retinas. In all cases, the embryonic sequence includes horizontal cells staining positively for GABA, across the entire retina. In some groups, such as mammals, this embryonic pattern of GABA is transient w41,26x, whereas most lower vertebrates maintain their embryonic staining pattern throughout life w2x. The horizontal cells of H. burtoni combine these two patterns. Specifically, in the central retina, transient activity is observed in horizontal cells while horizontal cells next to the developing margin maintain GABAergic staining throughout life. The pattern of GABAergic staining in horizontal cells of H. burtoni is unlike the reported patterns in the goldfish, where GABAergic horizontal cells have been reported w20x. On the other hand, it is similar to the distribution of GABA-positive horizontal cells in zebrafish where they are found only in the retinal periphery w35x. The zebrafish study, however, did not reveal transient GABA in central horizontal cells as observed in H. burtoni. This may reflect differences in the fishes’ visual processing and acuity, or different developmental strategies. In the fast developing zebrafish retina patterning signals such as GABA might be compressed or omitted, whereas in the somewhat slower developing, mouth brooding H. burtoni transient GABA might establish precisely patterned synaptic connections. There are several lines of evidence which suggest that GABA may be acting to control the differentiation of photoreceptors. Messersmith and Redburn w22x measured an increase in the rod to cone ratio when they selectively destroyed type A horizontal cells early in development. Although other aspects of the retina were disrupted by the kainic acid treatment, rods developed with normal morphology and synaptic connections. This suggests GABA may regulate the production of photoreceptors. More recently, Mitchell and Redburn w23x showed that GABA A receptors are expressed in the cone photoreceptor terminals around the time of synaptogenesis. In summary, the transient GABAergic staining in developing horizontal cells correlates spatially and temporally with the traveling wave of rod neurogenesis in the embryonic H. burtoni retina. This transient pulse of GABA may play a role both in the initiation and regulation of rod neurogenesis in the central retina during embryogenesis, and directly or indirectly in the developing margin. Acknowledgements We would like to thank the following people for their technical help and insightful discussions: R. Bremiller, G. Glasener-Cippillone, S. Kroger, M. McDowell, N. Tublitz, ¨ ¨ and S. Vollmer. We received several gifts of monoclonal antibodies from several laboratories: a GABA monoclonal antibody from C. Matute and P. Streit, University of Zurich; a rod-specific opsin monoclonal antibody ŽK16-
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107. from G. Adamus, University of Florida; a GFAP monoclonal antibody ŽA2D4. from A. Bignami, Harvard Medical School, and; a horizontal cell monoclonal antibody ŽIII F7.1. from L. Young, Harvard University. We thank the two anonymous reviewers for their careful reading of the manuscript. This grant was supported by NIH grant EY 05051 to RDF.
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