Department of Biological Structure, University of Washington, Seattle, WA 98195, USA
*Author for correspondence (e-mail: tomreh{at}u.washington.edu)
Accepted 31 January 2002
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SUMMARY |
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Key words: Growth factors, Chick, FGF2, Insulin
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INTRODUCTION |
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Despite these clear demonstrations of the remarkable plasticity of neural stem cells, evidence from in vivo studies has demonstrated restrictions in the types of neurons generated by neural stem cells. In the brain of the adult songbird, for example, neurons that project from the higher vocal center (HVC) to the robust nucleus of the archistriatum (RA) are produced throughout life, while other types of HVC neurons are not (reviewed by Goldman, 1998; Scharff et al., 2000
). Similarly, in the subventricular zone of the adult rodent, stem cells produce only glia and granule neurons in the olfactory bulbs (Luskin, 1993
; Luskin, 1998
; Goldman, 1995
; Lois and Alvarez-Buylla, 1993
; Lois et al., 1996
). Other types of neurons present in the olfactory bulbs are not generated in adult animals. Similarly, hippocampal stem cells in the subgranular zone of the dentate gyrus give rise to only interneurons of the granule cell layer, not other types of hippocampal neurons (Altman, 1965
; Altman, 1969
; Altman and Das, 1966
; Bayer, 1982
; Kaplan and Bell, 1984
; Kuhn et al., 1996
).
Taken together, in vivo studies suggest that neural stem cells are restricted to producing specific cell types, but when placed into an appropriate in vitro environment, this restriction can be over-ridden (reviewed by Anderson, 2001). It remains unknown what factors in the microenvironment limit or promote cell fate decisions of adult neural stem cells. It is also not clear whether the same factors that regulate neuronal phenotype in the developing CNS will continue to be active in controlling the cell fate decisions of the stem cells in the mature CNS.
Recent studies have demonstrated that the eyes of birds and mammals contain neural stem cells (Fischer and Reh, 2000; Tropepe et al., 2000
; Ahmad et al., 2000
). In the retinal margin of the post-hatch chicken, there is a population of neural stem cells that proliferate and generate neurons that are integrated into the peripheral edge of the retina (Fischer and Reh, 2000
). These cells are reminiscent of the well-described stem cells of the ciliary marginal zone (CMZ) of fish and amphibians (reviewed by Reh and Levine, 1998
). However, under normal conditions, the retinal CMZ cells of the chick produce only amacrine and bipolar neurons, while all types of retinal neurons are produced by stem cells in the CMZ of fish and amphibians. Amacrine and bipolar cells are generated late relative to other retinal cell types during embryonic histogenesis (Prada et al., 1991
), and it has been proposed that retinal progenitors become progressively restricted to producing specific cell types as development proceeds (Jasoni et al., 1994
). Retinal stem cells in the adult rodent eye may be similar to late-stage embryonic progenitors and the CMZ cells of the post-hatch chick eye in that they are restricted to producing late-generated cell types in vitro (Tropepe et al., 2000
). The projection neurons of the retina, the ganglion cells, are not generated in vitro or in vivo by retinal stem cells of mature birds or mammals.
Neural stem cells in the eyes of adult birds and mammals appear to be restricted in the types of neurons that they are capable of producing. The purpose of this study was to test whether exogenous growth factors are capable of inducing the production of ganglion cells from the CMZ cells at the retinal margin of the chicken. We show that intraocular injections of insulin and fibroblast growth factor 2 (FGF2) stimulated the proliferation of CMZ cells and production of ganglion cells at the retinal margin. These results show that the same microenvironmental factors that are known to control the cell fate choices in the developing retina continue to act in the mature retina, and further suggest that the potential of neural stem cells in the mature CNS can be expanded by experimental manipulations.
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MATERIALS AND METHODS |
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Injections
Chicks were anesthetized and injected as described elsewhere (Fischer et al., 1998; Fischer et al., 1999
; Fischer and Reh, 2000
). The left eye (control) was injected with 20 µl of vehicle (sterile saline plus 0.1 mg/ml bovine serum albumin) and the right eye (treated) was injected with growth factors. Growth factors used in these experiments included purified bovine insulin (2 µg per injection) and purified bovine fibroblast growth factor 2 (FGF2; 100 ng per injection). All growth factors were obtained from R & D Systems and were dissolved in saline plus 0.1 mg/ml BSA and 100 µg/ml 5-bromo-2-deoxyuridine (BrdU; Sigma). We made two or three consecutive daily injections of BrdU plus growth factors starting at post-hatch day 8 (P8) and harvested tissues at 1, 4 or 10 days after the final injection
Fixation and sectioning
Dissection, fixation and sectioned where performed as described elsewhere (Fischer et al., 1998; Fischer et al., 1999
; Fischer and Reh, 2000
).
Immunocytochemistry
Standard immunocytochemical techniques were applied to sections as described elsewhere (Fischer et al., 1998; Fischer et al., 1999
; Fischer and Reh, 2000
). To obtain adequate immunolabeling in wholemounts of the peripheral retina, the following procedures were used to overcome barriers to antibody penetration. Twenty-four hours before dissection, eyes were injected with 300 units of hyaluronidase (Sigma). Eyes were enucleated, transected equatorially and the anterior hemi-segment immersed in fixative (4% paraformaldehyde in 0.1 M dibasic phosphate buffer (PB) plus 3% sucrose pH 7.4) for 30 minutes at room temperature. All washes and incubations were done on free-floating samples at room temperature and on a nutator, with washes lasting 15 minutes and incubations lasting 24 hours. Tissues were washed three times in phosphate-buffered saline (PBS), equilibrated in 20% sucrose in PBS and subjected to three freeze/thaw cycles. The retina, pars plana and adherent pigmented epithelium was dissected away from the choroid, sclera and lens, cut into four radial quadrants, and each piece of tissue placed individually into the wells of a 24-well plate. This was followed by consecutive washes in PBS, deionized H2O, 30% dimethylsulfoxide (DMSO) in deionized H2O, 70% DMSO, 30% DMSO, deionized H2O and PBS. Tissues were incubated in 250 µl of antibody solution (antibody diluted in PBS added with 5% goat serum, 0.3% Triton X-100 and 0.01% NaN3). Tissues were washed three times in PBS and incubated with secondary antibody. After incubation in the secondary, tissues were washes three times in PBS, fixed for 30 minutes in 2% paraformaldehyde in 0.1M PB plus 3% sucrose. This was followed by a series of washes in PBS, 4 N HCl for 7 minutes, PBS, and incubation with BrdU antibodies. After incubation with antibodies to BrdU, tissues were washed twice in PBS, incubated with secondary antibodies, and washed twice in PBS. Tissues were mounted in 4:1 glycerol to water for observation under an epifluoresence microscope.
Working dilutions and sources of antibodies used in this study included the following: mouse anti-Pax6 at 1:50 (Developmental Studies Hybridoma Bank; DSHB); rabbit anti-Chx-10 at 1:4000 (Dr T. Jessell, Colombia University); mouse anti-Islet-1 at 1:50 (39d.45; DSHB); mouse anti-Hu at 1:200 (Monoclonal Antibody Facility, University of Oregon); rabbit anti-Brn3.0 at 1:1000 (Dr E. Turner, University of California, San Diego); mouse anti-neurofilament at 1:2000 (recognizes the 160 kDa subunit of neurofilament; RMO270; Zymed); rabbit anti-neurofilament at 1:1000 (recognizes the 145 kDa subunit of neurofilament; Chemicon); rabbit anti-glutamine synthetase at 1:2000 (Dr P. Linser, University of Florida); rat anti-BrdU at 1:80 (Accurate Chemicals); and mouse anti-BrdU at 1:80 (G3B4; DSHB). Secondary antibodies included goat-anti-rabbit-Alexa568, goat-anti-mouse-Alexa568, goat-anti-mouse-Alexa488 and goat-anti-rat-Alexa488 (Molecular Probes, Eugene, OR) diluted to 1:500 in PBS plus 0.3% Triton X-100.
In situ hybridization
Tissues were dissected and immediately embedded and frozen in OCT medium (Tissue-Tek). Sections (14 µm) were cut in the naso-temporal plane, thaw mounted onto Super-FrostTM slides (Fischer Scientific), and stored dessicated at 80°C until use. Upon thawing, slides were immediately fixed for 10 minutes in 4% paraformaldehyde in DEPC-treated PBS, followed by two 15 minutes washes in 0.1% active DEPC in PBS, and a 15 minute wash in DEPC-treated 5xSSC. Sections were prehybridized for 2 hours at 60°C in 50% formamide, 5x SSC, 5x Denhardts, 250 µg/ml yeast RNA and 500 µg/ml herring sperm DNA. This solution was replaced with fresh hybridization buffer with 1 µg/ml DIG-labeled riboprobe and sections were incubated over night at 60°C in a humidified chamber. Sections were rinsed with 2x SSC at 65°C and washed for 1 hour in 0.2x SSC at 72°C. Sections were processed for DIG-immunolabeling as described elsewhere (Jasoni et al., 1994). Riboprobes to Cath5 were made from base pairs 11-501 by using an in vitro transcription kit (New England Biolabs).
Measurements, cell counts, and statistical analyses
Errors were calculated as the standard deviation of each sample that was comprised of at least five individuals per group. To compare data from treated and control eyes, statistical significance was assessed by using a two-tailed Student t-test. All measurements were made from digital micrographs of the retinal margin, while all cell-counts were made under the microscope on at least eight different sections per individual.
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RESULTS |
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Insulin and FGF2 induce the production of ganglion cells at the retinal margin
To determine whether the intraocular injections of insulin and FGF2 caused a change in the types of neurons generated by the cells of the CMZ, we used double-labeling with BrdU and several markers of retinal neurons. We concentrated our analysis on retinal ganglion cells, as they are not normally generated by the CMZ cells (Fischer and Reh, 2000). Neurofilament protein expression is normally confined to orthotopic and ectopic ganglion cells in the chick retina (Fig. 2A). Thus, we labeled sections of the retinal margin with antibodies to the 160 kDa subunit of neurofilament. At the retinal margin of the untreated post-hatch chick eye, we found a few neurofilament immunoreactive cells (Fig. 2B). These neurofilament-positive cells in the CMZ at the retinal margin were vertically oriented and appeared to produce processes that spanned the depth of the retinal margin. The morphology of these cells is similar to that of differentiating ganglion cells in the embryonic retina (McLoon and Barnes, 1989
; Brittis et al., 1995
; McCabe et al., 1999
). Three consecutive daily injections of FGF2 alone did not cause an increase in the number of neurofilament-positive cells in the CMZ (Fig. 2F), while three injections of insulin alone caused an increase in the number of neurofilament-positive cells in the CMZ (Fig. 2C,F). This effect was further increased when FGF2 was applied with insulin (Fig. 2D,F). The effect of these factors on neurofilament expression in the CMZ was transient because at 4 days after the final dose either factor, the number of neurofilament-positive cells in the CMZ at the retinal margin was reduced to levels seen in saline-treated eyes (Fig. 2E,F). We found many neurofilament-immunoreactive cells in the CMZ that were labeled for BrdU (Fig. 3A-C), indicating that these cells were newly generated.
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As noted above, intraocular injections of insulin and FGF caused many of the BrdU-labeled cells from the CMZ to migrate to the ganglion cell layer. To test whether BrdU-labeled nuclei in the GCL were those of ganglion cells, we double labeled sections for BrdU and either Islet1 or Brn3.0. One day after the final injection of three consecutive daily injections of insulin alone, we found few cells that were double-labeled for Islet1 and BrdU (Fig. 4A). By comparison, 4 days after the final injection of insulin alone we found increased numbers of cells that were double labeled for Islet1 and BrdU, and most of the these cells were in the INL (Fig. 4A). Four days after the final injection of insulin and FGF2, we found many cells in the GCL that were labeled for BrdU and Islet1 (Fig. 4B-E). In eyes treated with saline, we did not find any cells in the GCL that were labeled for BrdU and Islet1 (Fig. 4B). By comparison, we did find a few cells in the GCL (<1 cell per section of the retinal margin) that were labeled for BrdU and Islet1 in retinas that were treated with insulin alone (Fig. 4B). Compared with retinas treated with insulin alone, retinas treated with insulin and FGF2 had about 10 times as many cells in the GCL that were double labeled for BrdU and Islet1 in the far peripheral retina (Fig. 4B).
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DISCUSSION |
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The expression of the ganglion cell marker neurofilament in the CMZ was stimulated by insulin alone, but insulin alone did not induce the production of ganglion cells. This finding suggests that the expression of neurofilament may not represent a commitment to a ganglion cell phenotype. We found that numbers of neurofilament-expressing cells in the CMZ were related to the proliferation of CMZ cells, but not the production of ganglion cells. For example, we found that insulin alone increased the number BrdU-labeled cells added to the retina, increased the number of Pax6/Chx10-expressing cells within the retinal margin and increased the number of neurofilament-expressing cells in the CMZ, but did not increase the number ganglion cells that were produced (i.e. cells that were labeled for BrdU and Islet1, Brn3.0 or neurofilament at 4 days after the final injection of growth factor). By contrast, the production of ganglion cells was induced when FGF2 was applied with insulin without further stimulating the proliferation of CMZ cells. The fate of neurofilament-expressing cells observed in the CMZ of eyes treated with insulin alone remains uncertain. It is possible that the expression of neurofilament is transient in some CMZ cells or in some types of differentiating neurons. Consistent with this hypothesis, Bennett and DiLullo (Bennett and DiLullo, 1985a; Bennett and DiLullo, 1985b
) reported that neuroepithelial and neuroblastic cells of the chick central nervous system express neurofilament proteins and that some of this expression is transient. In addition, others have shown that neurofilament expression is transient during the differentiation of hair cells in the mouse cochlea (Hasko et al., 1990
), cerebellar granule cells in the rat (Cambray-Deakin and Burgoyne, 1986
) and cholinergic amacrine cells in the chick retina (A. J. F. and T. A. R., unpublished).
Our data suggest that the differentiation of neurons at the retinal margin begins in sclerad layers of the CMZ. Neurofilament-positive cells in the CMZ expressed Islet1 or Hu, and many of these cells were located towards the sclerad surface of the CMZ. Coincident with the location of these cells, we found cells that expressed Cath5 or Brn3.0. In the embryonic chick retina, both Brn3 and Cath5 are expressed by cells distal to the developing GCL; the expression of these factors ectopic to the GCL is presumed to be produced by differentiating ganglion cells that are migrating toward the GCL (Liu et al., 2000; Matter-Sadzinski et al., 2001
). By comparison, others have found that the differentiation of ganglion cells, as detected by the expression of RA4, neurofilament or Islet1, in the embryonic retina begins near the ventricular surface (McLoon and Barnes, 1989
; Brittis et al., 1995
; McCabe et al., 1999
; Zhang and Yang, 2001
), which is equivalent to the sclerad surface of the post-hatch chick CMZ.
The notion of FGF-induced production of ganglion cells is consistent with observations made in the developing retina. Previous studies have shown that exogenous FGFs can promote the production of ganglion cells in cultures of embryonic retinas from chicks and rodents (Pittack et al., 1991; Pittack et al., 1997
; Guillemot and Cepko, 1992
; Zhao and Barnstable, 1996
; McCabe et al., 1999
). In addition, overexpression of FGF2 in Xenopus retina promotes the production of ganglion cell (Patel and McFarlane, 2000
). Consistent with the findings of these gain-of-function studies, the differentiation of ganglion cells can be inhibited by antisense-mediated suppression of FGF expression in embryonic chick retinas in vitro and in vivo (Desire et al., 1998
), and by the FGF-receptor inhibitor SU5402 in explant cultures of the embryonic chick retina (McCabe et al., 1999
). Thus, it appears that CMZ cells at the margin of the chicken eye resemble embryonic chick retinal precursors in their response to FGFs.
Our results suggest that adult neural stem cells may be restricted in the types of neurons they generate because of limitations imposed by the secreted factors in the local microenvironment. As noted in the Introduction, a variety of different approaches have been used to address this question. In vitro studies have generally emphasized the plasticity of neural stem cells, and some amount of cell culture may even induce this plasticity. For example, studies showing that the neural stem cells can generate diverse cell types appear to require that neural stem cells proliferate in culture prior to differentiation (for a review, see Anderson, 2001). It is possible that the growth factors used to stimulate the proliferation of neural stem cells and, subsequently, to induce differentiation into different cell types are required to bestow plasticity upon these cells. The data from in vivo studies has generally shown that neural stem cells have a more limited potential. For example, targeted photolysis of projections neurons in the HVC of adult songbirds results in a compensatory replacement of neurons that are normally turned over, but replacement of neuronal types that are not normally generated does not occur (Scharff et al., 2000
). Despite the evidence for some intrinsic limits to the types of progeny derived from neural stem cells in vivo, recent studies by Magavi and colleagues (Magavi et al., 2000
), indicate that selective ablation of projection neurons stimulates the regeneration of the ablated cell type in the cerebral cortex of adult mice. It is possible that the highly selective loss of a cell type via photolysis induces changes in the microenvironment that allow or promote the differentiation of cells derived from adult neural stem cells.
In conclusion, the findings presented demonstrate that the same factors that control neuronal cell fate decisions in the embryonic CNS are also active in stem cell zones such as the CMZ to direct precursors to specific fates. Moreover, our results are consistent with the hypothesis that part of the restriction in cell fates generated by the cells of the CMZ in the post-hatch chicken is due to limiting amounts of FGF in the microenvironment. Thus, these findings suggest that exogenous growth factors can be used in vivo to influence the types of neurons produced by stem cells and possibly stimulate the replacement of particular types of neurons.
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ACKNOWLEDGMENTS |
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