Regulation of cell division and rod differentiation in the teleost retina

Developmental Brain Research, 76 (1993) 183-187

183

© 1993 Elsevier Science Publishers B.V. All rights reserved 0165-3806/93/$06.00 BRESD 51715

Research Report

Regulation of cell division and rod differentiation in the teleost retina Andreas F. Mack *, Russell D. Fernald Neuroscience Program, Psychology Department, Building 420, Stanford University. Stanford. CA 94305, USA

(Accepted 29 June 1993)

Key words." Retina; Teleost; Insulin-like growth factor; Fibroblast growth factor; Proliferation; Differentiation

We tested the effects of several growth factors on the proliferation and differentiation of cells in the teleost retina which typicallybecome rod photoreceptors to understand their regulation. Using organotypicslice cultures of differentiated teleost fish retinal tissue, we found that insulin and insulin-like growth factor I (IGF-I) stimulate proliferation of rod precursor cells whereas basic fibroblast growth factor (bFGF) does not. In the presence of bFGF, however, a greater proportion of the cells that had divided expressed a rod photoreceptor-specific phenotype than did control slices. This suggests insulin and the related IGF-I can influence the regulation of neuronal cell division whereas bFGF promotes the differentiation of neuronal stem cells into rod photoreceptors in retinal slice culture. These results support the idea that cell division and differentiation are differentially regulated and diffusible factors play a role in this process.

INTRODUCTION Cell addition to the neural retina of teleost fish occurs throughout life in two distinct ways 7. First, new retinal cells divide and differentiate in a circumferential growth zone at the margin of the eye, contiguous with extant retina. Second, in the outer nuclear layer, individual cells divide and differentiate into rod photoreceptors 22. Thus, both cell production and phenotype specification are ongoing processes in the fish retina. During embryogenesis in vertebrates, retinal cell fate appears to be largely independent of cell lineage 24'12, suggesting it is determined late, at the time of the last cell division, although the likelihood of a particular cell phenotype being produced changes as a function of developmental time 23"28. Thus, lineage-independent mechanisms such as extracellular cues (hormones, growth factors and neurotransmitters) as well as cell-cell contact could regulate proliferation a n d / o r differentiation in the developing vertebrate retina. Similar and possibly related mechanisms may also influence continued specification of rod progenitors in the developing and adult teleost retina. To discover if growth factors influence the production of new rods in

*Corresponding author. Fax: (1) (415) 725-5699.

teleosts, we have developed a method for growing slices of fish retina in vitro tS. These thin retinal sections grown in culture mimic important properties of intact teleost retina, including the location of cell division and differentiation ~4. We tested two types of growth factors which have been implicated in control of retinal development and maintenance: basic fibroblast growth factor (bFGF) jl, and insulin and insulin-like growth factor I (IGF-I) 17'2~'. Receptors for these growth factors occur in the adult and developing retina 25'3 and b F G F has previously been reported to slow retinal degeneration 6, stimulate photoreceptor differentiation ~ and retinal regeneration 2°. We measured the effect of these growth factors on retinal cell proliferation by using [3H]thymidine to visualize cell division and their effect on differentiation by using rod-specific antibodies. We assayed the effect of these growth factors on the rod progenitor cells located in the outer nuclear layer (ONL) which makes them easy to identify. Moreover, the proliferation rate of these cells in the O N L has been shown to be regulated in intact animals 9. Hence this stem cell population is a likely target for control in the differentiated teleost retina. Since no other cell type undergoes cell division in the O N L after embryogenesis, we can exclude proliferating non-neuronal cells in the slice cultures as a possible source of error in our analysis.

184

Fig. la, lb, lc (top), Fig. 3a (middle), Fig. 3b (bottom). For legends, see next page.

185 MATERIALS

Increase of cell divisions stimulated by growth factors

AND METHODS

Retinal slices were prepared from eyes of 12- to 15-day-old, juvenile cichlid fish, Haplochrornis burtoni. The fish were anesthetized on ice, quickly decapitated, and 150-/zm-thick slices prepared as described previously 15. At this age, the retina of H. burtoni is fully developed and grows in a pattern characteristic of adults s. Slices were cultured in L-15 medium, supplemented with 10 mM glucose, 5 ~zg/ml transferrin, 5 n g / m l selenite, 25 mM taurine, 5 mM HEPES, and 10 g / m l gentamicin (all Sigma) at 28°C. [3H]thymidine was added to the culture medium to label dividing cells (2.5/xCi/ml medium, spec. activity 68 Ci/mmol). To measure the effects of growth factors on cell division, slices were cultured for 24 h and in the final 12 h, [3H]thymidine was added to the culture medium to label dividing cells (2.5 /xCi/ml medium, spec. activity 68 Ci/mmol). Growth factors were added to the culturing medium as follows: 50 n g / m l bFGF (bovine recombinant), 20 n g / m l IGF-I (human recombinant), and 5 /~g/ml insulin (bovine pancreas; all from Boehringer). To minimize variation among slices, only slices from the center of the eye with approximately the same total amount of tissue were used for this experiment. Experimental and control slices for each experiment were from animals of the same brood raised together. After culturing, the slices were fixed in 4% paraformaldehyde in PBS for 1-2 h at room temperature, dehydrated and embedded in plastic (Immunobed, Polysciences). The blocks were then sectioned serially at 3 p~m approximately parallel to the slice surface and the sections processed for autoradiography and stained with Cresyl violet. All the cells in the ONE labeled by silver grains were counted. Since the number of sections obtained per slice varied somewhat, depending on the angle between the plane of sectioning and the surface of the slice, all sections were counted. Thus, it is possible that a cell could be counted twice if there were significant label associate with it in adjacent sections. However, since the ONL cell nuclei are significantly smaller than the section thickness, this is a rare event. To test the effects of growth factors on photoreceptor differentiation, retinal slices were cultured for a total of 3.5 days. [3H]thymidine was added to the culture medium during the first 12 h. Then, slices were rinsed several times in unsupplemented medium. Afterwards, fresh supplemented medium was added with the appropriate growth factor (50 n g / m l bFGF; 5 ~ g / m l insulin) and replaced daily. After additional 3 days in culture the slices were fixed as above, washed in PBS and incubated in primary antibody with 0.1% triton X and 1% DMSO for 5-8 days. We used the monoclonal antibody K16 155C developed against mouse rbodopsin by Dr. G. Adamus, which binds exclusively to rod photoreceptors in the teleost retina s. This step was followed by incubation with secondary anti-mouse antibody and peroxidase-anti-peroxidase complex (Jackson Immuno Res. Lab., Inc.) for ca. 24 h each. Antibody binding was visualized by diaminobenzidine reaction. Immediately after the antibody staining, the slices were dehydrated and embedded in plastic and sectioned at 3 ~m. Sections were then processed for autoradiography and developed 3 days later. The fraction of rod photoreceptor cells in the slice cultures in each condition were compared.

Ceils labeled with 3H-thymidine in the ONL in [%] above control

160" I

140 • 120 100 8O

4O 20 0 IGF-I

Insulin

bFGF

Fig. 2. Effect of bFGF, insulin and IGF-I on the proliferation of stem cells in the ONL of retinal slices. Insulin increased the number of labeled cells in the ONL on average by 110% and IGF-I by 79%. This increase in the number of labeled cells was statistically significant (t-test, for insulin, n = 7, P < 0.001, t = 4.3; for IGF-I, P < 0.01, t = 2.9, n = 7). The difference to control slices was calculated in percent for each experimental slice. Error bars = S.E.M.

RESULTS

First we asked whether either of the two growth factors affected the amount of cell proliferation in the ONL and second whether either growth factor affected the fate of newly divided cells. In the retinal slice culture, as in vivo, dividing ceils labeled by [3H]thymidine are found at the ciliary margin and in the ONL as revealed by autoradiography (Fig. 1). Retinal slices cultured either in the presence of insulin or IGF-I had a significantly greater number of ONL cells which divided (Fig. 2). No significant difference in pattern or number of dividing cells was observed comparing insulin and IGF-I treated cultures. In contrast to insulin and IGF-I, b F G F did not increase cell proliferation in the ONL of cultured slices significantly but did affect the fate of dividing cells, as shown in the experiment described below.

Fig. 1. Photomicrograph of teleost retinas in vivo and in vitro, indicating the locations of cell divisions. (a) Autoradiograph of dividing neuronal progenitor cells labeled by a single injection with [3Hlthymidine in a 3-month-old fish (30 ~ C i / g body weight). This 3-/~m plastic section was counterstained with Cresyl violet. Labeled cells are found in the peripheral germinal zone and in the outer nuclear layer (ONL; arrows) as indicated by silver grains above the cell bodies. (b) The edge of a freshly sectioned retinal slice. The arrows point to area of proliferation in the germinal zone. (c) A 3-/xm section through a retinal slice showing cell divisions of neuronal stem cells in vitro. The slice was cultured for 24 h in defined culture medium containing insulin (5 /xg/ml). Dividing cells were labeled with [3H]thymidine. The slice was subsequently fixed, processed histologically for autoradiography and the sections stained with cresyl violet. Many cells in the germinal zone and in the ONL are labeled by silver grains (arrows). Scale bars = 25/xm. Fig. 3. Photomicrographs of a 3-/xm section through the inner (INL) and outer (ONL) nuclear layers of a retinal slice. (a) focus on the cell bodies with Nomarski optics; (b) focus on the silver grains in bright field. Cells in the ONL are stained positively with an antibody specific for rod photoreceptors (arrow heads). One cell is double-labeled with the antibody and silver grains (large arrows) indicating [3H]thymidine uptake. A cell next to it is labeled by silver grains only (small arrow in b). Scale bar = 25 ~zm.

186 To discover whether any of these factors affect cell fate in the ONL, retinal slice cultures were first labeled with [3H]thymidine to detect newly divided cells. Then after a total of 3.5 days in culture cells were stained with a rhodopsin-specific antibody, expressed only in rods in teleost fish Is. Cells labeled both with [3H] thymidine and the specific-antibody must have undergone both cell division and a subsequent differentiation step toward becoming a rod photoreceptor in the slice culture. Examples of such double-labeled cells are shown in Fig. 3. Considering only cells in which there were clear autoradiographic silver grains and immunocytochemical staining, we counted and compared the fraction of new cells which had differentiated after application of each growth factor. When b F G F was added to retinal slices cultured in insulin-containing medium to test for a possible synergistic effect of the growth factors on cell proliferation the total number of newly divided cells was not different from insulin treated control cultures (Fig 4a). In slices cultured with just insulin, only a small fraction (6.2%) of these cells express rhodopsin after 3.5 days in vitro. When b F G F is added in addition to insulin the percentage of newly divided cells which express rhodopsin increases dramatically (19.7%; Fig. 4b).

Effect of bFGF on proliferation

Number of dividing cells (3H-thymidine positive} / retinal slice

250 200 150 1O0 50

Exact roles for insulin and I G F - I in the retina have not been identified but they have been associated with synaptogenesis in the developing retina 17, and in the maintenance of the pigment epithelium in the adult retina 26'5'19. Both insulin and I G F - I have distinct receptors and both have been shown to stimulate the proliferation of cultured sympathetic neuroblasts 4 implying a possible regulatory role in the cell cycle of neuronal precursors. In the amphibian retina, insulin and I G F - I bind to a single receptor with equal affinity 29, consistent with our data that insulin and 1GF-1 have similar stimulating effects on the proliferation of stem cells in the outer nuclear layer of teleost retinal cultures. It is tempting to speculate that the lack of the distinction between the effects of insulin and I G F - I are related to the continued growth of the teleost nervous system since even the mature fish continues to add neurons to brain and retina. Perhaps the regulation of developmental and metabolic events by insulin-like molecules might have been coupled early in evolution, and this might still be the case in growing teleosts. The specific effect produced by b F G F suggests that it plays an important role in normal rod photoreceptor differentiation. Although we attempted to test this

0

a

Y///////~ /////////A ~///////A I////////A Y//d////A bFGF + Insulin

Insulin

Effect of bFGF on differentiation Fraction of newly divided cells which express rhodopsin

30

[%]

25 20

T

15 10 5 0

b

DISCUSSION

300

)

i

bFGF + Insulin

Insulin

Fig. 4. Effect of b F O F on the proliferation and differentiation of

progenitor cells in the ONL of a retinal slice in the presence or absence of insulin. (a) To test for a possible synergistic effect of insulin and bFGF, we repeated the experiment described in Fig. 2 but did not find any significant difference in proliferating cells in bFGF and insulin treated cultures (n = 6) compared to cultures containing only insulin (n = 3). (b) Slices were cultured as described in Fig. 3. The number of cells which divided in culture and stained positively with rhodopsin-specific antibody is expressed as percentage of total [3H]thymidine labeled cells in the ONL. Cultures treated with bFGF had a significantly higher rate of double-labeled cells (t-test, P < 0.01, t = 4.06, n = 6 for both, control and experimental slices). Experimental as well as the control culture medium contained insulin because without the addition of insulin, there were not enough [3H]thymidine labeled cells in the ONL tO allow a reasonable analysis. Error bars = S.E.M.

effect without the addition of insulin to the culture medium there were not enough cells labeled with [3H]thymidine after 3.5 days in vitro. This might be due to cell death or detachment of proliferating cells from the slice tissue. Since loss of labeled cells occurred in both, insulin-treated and untreated slices it does not affect our analysis. The best known members of the fibroblast growth factor family, a F G F and bFGF; have both been isolated from retinal tissue 2J6'22 and their m R N A s are also localized in the retina a3JS. Thus it seems reasonable that b F G F could play a role in regulating retinal cell fate, particularly since F G F s can

187 induce regeneration of the embryonic chick retina in vivo 2°. A recent study on dissociated rat retinal neuroblast provides evidence that b F G F stimulates photoreceptor differentiation in embryogenesis II. Our results suggest a similar role for b F G F in the mature teleost retina in an organotypic preparation. This notion remains to be shown in vivo. Two other reports, using two different culture systems also suggest that diffusible factors rather than direct cell-cell contact promote rod cell differentiation in the developing rat

retina 1,27. HOW could the effects of b F G F be regulated? It may be present in sufficient quantities such that newly divided cells have ready access to it making regulation of cell phenotype dependent on cell division. Conversely, local release of b F G F could be the regulatory step. Experiments are underway to distinguish these between possibilities. The teleost retina provides unique opportunities to study neuronal cell production. The neuronal stem cells in the outer nuclear layer studied here are added in substantial numbers to the differentiated fish retina 7, and these cells must respond to permissive or inductive environmental cues in order to differentiate into rods. Our data suggest that b F G F may be partially responsible for the differentiation of newly divided cells into rod photoreceptors. How these new retinal cells adapt their phenotype in response to extracellular signals remains unknown. Acknowledgements. We thank B. Evans, L. Goodrich, M. NadalVicens and S. White for reviewing the manuscript. Supported by NIH T32 GM 07257 to A.F.M. and E.Y. 05051 to R.D.F.

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