In vitro differentiation of human retinoblastoma cells into neuronal phenotypes

Differentiation (1990) 45:25&257

Differentiation Ontogeny and Neoplasia 8 Springer-Verlag 1990

In vitro differentiation of human retinoblastoma cells into neuronal phenotypes Sabine Griegel, Kerstin Heise, Andrea Kindler-Rohrborn, and Manfred F. Rajewsky * Institute of Cell Biology (Cancer Research), West German Cancer Center Essen, University of Essen Medical School, Hufeland-Strasse 55, D-4300 Essen 1, Federal Republic of Germany Accepted in revised form September 27, 1990

Abstract. In order to characterize the cell type(s) of origin of human retinoblastoma cells by immunophenotyping, primary cells from seven retinoblastomas and of the corresponding cell lines (RBL lines), as well as four retinoblastoma (RB) lines established by other groups, were compared with rat and human retina cells, and with the adenovirus El A-transformed human retinoblast cell line HER-Xhol-CC2. Analyses using monoclonal antibodies (Mabs) RB13-2 and RB21-7, originally raised against prenatal rat brain cells and recognizing neural cell surface antigens expressed in a developmental-stage-dependent manner, and three cell-type-specific Mabs (421 1, M501, Mab directed against vimentin) developed by other groups, gave the following results: (i) Retinoblastomas consist of cells expressing differentiated neuronal phenotypes during cultivation in vitro; (ii) All of the newly established RBL lines express neuronal phenotypes; and (iii) Cell lines such as Y79, which have been propagated in vitro for extended periods, do not express antigens specific for the neuronal pathway and cannot, therefore, be considered phenotypically representative of retinoblastoma cells.

Introduction Retinoblastoma (RB), the most common intraocular neoplasm in early childhood, is of neuroectodermal origin. Predisposition to the development of RB is transmitted as an autosomal dominant trait [31], and the tumors occur in a hereditary (usually bilateral) or non-hereditary (unilateral) form. Tumorigenesis is assumed to be initiated by inactivation of both alleles of a regulatory (tumor-suppressor) gene (RB-1) located on chromosome 13q14 [4, 81. Contrary to its genetic background, the histogenesis of RB is poorly understood. It seems to be uncontested that RB originates from a proliferation-competent

* To whom offprint requests should be sent

retinoblast that fails to give rise to mature retina cells [23]; However, the lineage commitment and state of differentiation of the retinoblasts at the time of tumorigenic conversion remains a matter of dispute; in particular since expression of a malignant phenotype does not exclude that the tumorigenic cells may have retained some ability to differentiate [17]. It has been argued that “retinomas”, the benign type of manifestation of an inactivated RB-1 gene, may arise when the critical mutation of the RB-1 gene occurs in more-differentiated retinoblasts forced into only a few additional rounds of proliferation [15]. Inactivation of the RB-1 gene in terminally differentiated retina cells would have no effect because the cells’ proliferative machinery would have been shut off prior to the mutational event. The mammalian retina consists of six different neuronal cell types (ganglion cells, amacrine cells, bipolar cells, horizontal cells, rods and cones) and one glial cell type (Miiller cells), all of which are derived from an apparently homogeneous population of neuroepithelial precursor cells [3]. While some investigators have provided morphological, biochemical or electron-microscopic data, supporting neuronal differentiation of RB cells into photoreceptor cells [ 5 , 6, 13, 351, others have suggested that RB cells may be of glial origin [ l l , 361. Studies using the well-characterized but long-established RB cell lines Y79 and WERI have indicated that RB cells can express both neuronal and glial properties, pointing to their possible origin from a primitive pluripotential progenitor cell early in retina development [9, 21, 281. The present study was aimed at defining more precisely the phenotype(s) of RB cells on surgical removal of the tumors, as well as their potential for differentiation in vitro. Using a set of monoclonal antibodies (Mabs) specific for cell surface antigens of cell subpopulations of the developing rat brain [22, 241, we have analyzed 13 primary RB and 11 RB-derived cell lines in comparison to normal rat and human retina cells and to the adenovirus El A-transformed, human embryonal retinoblast line HER-Xhol-CC2 [38]. The data strongly

251

suggest that RB tumors consist of precursor cells that are committed to the neuronal lineage and can differentiate in vitro into mature neurons. The cells of seven newly established RB cell lines (RBL lines) exclusively express a neuronal phenotype. These results were further corroborated by the application of three Mabs directed against cell-type-specific antigens of the rat retina.

against embryonic chick retinal membranes and recognizes a cell surface antigen on neuronal cells (30). Mab M5Of binds to Miiller cells of the rat retina [39]. The Mab specific for human vimentin was obtained from Dakopatts, Glostrup, Denmark). Mab RB2f-7 and Mab RB 13-2 were established by immunization of mice with intact brain cells from BDIX rats at different stages of prenatal development [22]. Immunofluorescent cells were enumerated by microscope counting of labeled cells and from micrographs.

Methods

Results and discussion

RB tumors. Fresh tumor tissue was obtained from six cases of bilateral (RBL'IT, RBLl5T. RBLZOT, RBLZlT, RBL26T, and RBL27T) and seven cases of unilateral RB (RBL13T, RBL14T, RBLl8T. RBL25T. RBL28T, RBL30T, and RBL31T; the letter 'T symbolizes primary R B cells). After surgical removal and hemisection of the eyes, tumor tissue was transferred into cold phosphate-buffered saline (PBS) supplemented with 15% heat-inactivated fetal calf serum (FCS; Sebak, Aidenbach, Germany). The tumor tissue was dissociated by pipetting and cell debris was removed by three successive sedimentation steps at 1 g. The last cell pellet was either stored in liquid N, or used for immunocytochemistry immediately.

The expression of Mab-defined cell surface antigens was monitored as a function of retina development. Since specimens of normal human retina cannot be obtained routinely, for medical and ethical reasons, the retina of the BDIX-rat was used in the present study. This appears justified because the structure of the retina is very similar in higher mammals, in spite of somewhat varying proportions of the different cell types [3]. To ensure that the results obtained for rat retina are also representative of the human retina, the Mab binding pattern on the retina of a postmortem human embryo (19th week of gestation) was compared with the binding pattern on rat retina from a similar developmental stage (Pl-P7). No differences were observed.

Cell lines. Eileven RB-derived cell lines were included in this study. All cell lines were grown as suspension cultures in Dulbecco's modified Eagle's medium (DMEM), supplemented with 15% heat-inactivated FCS, penicillin (1 00 units/ml), streptomycin (100 pg/ml), D-glucose (4.5 mglml), insulin (1 0 pg/ml; Sigma, Deisenhofen, FRG, and 2-mercaptoethanol (5 x M ) [16]. Seven of the cell lines (RBL lines) were newly established [18]. Four of the RBL lines (RBL13, RBLl4, RBL18, and RBL30) were derived from unilateral, three RBL lines (RBL7, RBLlS, and RBL2O) from bilateral, tumors. The RB-derived lines RB383 and RB355 (both derived from unilateral tumors), and RB247C3 (originating from a bilateral tumor), were kindly provided by Dr. B. Gallie (Hospital for Sick Children, Toronto, Canada) [34]. The RB cell line Y79 [28] was obtained from the American Type Culture Collection (Rockville, MD, USA; ATCC HTB 18). The adenovirus 5-transformed cell line HER-Xhol-CC2 [38] was a gift from Dr. A.J. van der Eb (Department of Medical Biochemistry, The Sylvius Laboratories, State University of Leiden, The Netherlands).

Tissue samples and cryostat sections. Normal human retina was obtained from the eyes of a postmortem specimen (19th week of gestation). Eyes from rats of the strain BDIX [12] were used for analysis of Mab binding patterns during retina development at five distinct stages: prenatal day 16 (F16), and postnatal days 1 (Pi), 7 (P7), 12 (P12), and 96 (P96), the latter stage representing the mature retina. At stages F16, P1, P7, and P12, the appearance of differentiated cell types coincides with the formation of new cell layers. After enucleation and washing in PBS, whole eyes (unfixed) were mounted on copper pins in embedding solution (Tissue Tec I1 ; Lab-Tec Products, Naperville, 11, USA), immediately frozen on dry ice and stored at -80" C. Vertical sections (5-8 pm thick) were cut at - 20" C on a freezing microtome (SLEE, Mainz, FRG), spread on chrome alum/gelatine-coated coverslips, and dried at room temperature overnight. Some of the cryostat sections were processed for immunocytochemistry immediately, while the remaining sections were stored at - 80" C. Immunofluorescence analyses and antibodies. For immunocytochemistry, small RB cell aggregates or single RB cells were attached to glass coverslips coated with poly-L-ornithine (5 x lo5 cells/l2mm coverslip) and maintained under a humified atmosphere containing 10% C 0 2 for 24 h at 37" C. At this time the cells were firmly attached to the coated glass surface. Cells on coverslips and tissue sections were prepared for indirect immunofluorescence as described by Liepelt et al. [24]. Mab Q2f f was originally raised

Expression of the cell surface antigens defined by Mabs RB21-7 and RBl3-2 in the developing rat brain

In the developing rat brain, Mab RB21-7 recognizes a cell surface protein of 250 kDa expressed both on neuroectodermal precursor cells committed to the neuronal lineage and on differentiated neurons (as judged by neurofilament expression), and a small subpopulation of astrocytes (as judged by the expression of glial fibrillary acidic protein). Mab RB 21-7 also bound to the cells of eight out of nine human neuroblastoma cell lines tested [26]. Mab RB13-2, whose binding properties appear to be identical to Mab D1.l [lo] and partially identical to Mab Jones (which was originally raised against retina cells [25]), recognizes a set of 0-acetylated gangliosides [29] on neural precursor cells located in the germinal zones of rat brain during development [7]. The antigen is no longer expressed in adult rat brain. Cell culture studies have shown that Mab RBI3-2-binding cells, separated by fluorescence-activated cell sorting, lose the antigen while differentiating into neurons and astrocytes [24]. It is not yet clear whether some or all cells that bind Mab RB13-2 still possess the options for both differentiation pathways, or are already committed to one cell lineage when development is initiated in vivo. Expression of cell surface antigens recognized by Mabs RB21-7 and RBl3-2 in the rat retina

Characterization of retinal cells with respect to their cell type and stage of differentiation is comparatively easy. Retinal cells are organized in layers, and the retinoblasts differentiate in a defined order, beginning with the gan-

252

GcL[

IPL

[

.;[

OPL

[

oNL[

IPL

GC L

nn

OPL

[ Fig. 1 A-D. Immunoanalysis (indirect fluorescence) of frozen vertical sections of BDIX-rat retina. Mab RB21-7 (A, C: phase contrast; B, D: indirect immunofluorescence; A and B: P7; C and D: P98). N L , neuroblastic layer; GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer; IS, inner segments; OS, outer segments. Scale bar, 20 pm

glion cells and ending with the photoreceptor cells [3]. As in the brain, expression of the cell surface antigens recognized by Mab RB21-7 and Mab RB13-2 by retinal cells depends on their developmental/differentiation stage. Both Mabs can thus be used as markers for immature retina cells at discrete stages of development. Cellular differentiation in the retina occurs in the reverse direction from proliferation, progressing from the inner (future ganglion cell) layer to the outer (future photoreceptor) layer, so that the outer retina matures last [2]. On cryostat sections of the retina, Mab RB21-7 binds to cells of those retinal layers that are in the process of differentiation as well as to terminally differentiated cells of fully organized layers. Progenitor cells not yet associated with one of the retinal layers do not bind this Mab. At stage F16 of development, the rat retina is composed of a uniform layer of neural progenitor

cells [2]. At this time, the innermost cells of the retina have already begun to differentiate into mature ganglion cells, which thereafter form the ganglion cell layer [2]. These cells express the antigen defined by Mab RB21-7 (not shown). During further development, Mab RB21-7 immunostaining appears sequentially on each of the differentiated neuronal cell types of the retina. A minor subpopulation of astrocytes are also recognized by Mab RB21-7 [ S . Blass, A. Kindler-Rohrborn, and M.F. Rajewsky, in preparation] ; however, in the retina astrocytes are restricted to the nerve fiber layer, near blood vessls [32, 331. At stage P7, the cells of the ganglion cell, inner plexiform, and inner nuclear layer are terminally differentiated (and separated from the outer nuclear layer by the outer plexiform layer) [2]. These layers are stained by Mab RB21-7 (Fig. 1 A, B). The photoreceptor cell bodies, located mainly in the outer nuclear layer, become

253

GcL

[

NLp

IPL

[

GcLl IPL

OPL

IPL

r

L u u 0 PL

ONL

S

Fig. 2A-F. Immunoanalysis (indirect fluorescence) of frozen vertical sections of BDIX-rat retina. Mab RBf3-2 (A, C and E : phase contrast; B, D and F: indirect immunofluorescence; A and B: P1; C and D: P7; E and F: P12). Designation of rc4nal layers as in Fig. 1. Scale bar, 20 pm

weakly stained only in the mature retina (P98), while the inner and outer segments of these cells are not recognized by Mab RB21-7 at any developmental stage (Fig. 1 C, D). The composition of the segment membrane is thus different from that of the body of the photoreceptor cell. Contrasting with Mab RB21-7, Mab RB13-2 recognizes a cell surface antigen on neural progenitor cells that are not yet associated with defined layers. As in the developing rat brain [24], this antigen is briefly coexpressed with the antigen specified by Mab RB21-7 on more differentiated cells, which in the retina are organized in layers; however, the latter cells gradually lose the antigen defined by Mab RB13-2. From stage F16

to PI the antigen is expressed on all cells of the retina (Fig. 2A, B). At stage P7 the outer nuclear layer is formed, while the ganglion cell layer, the inner plexiform layer, and the inner region of the inner nuclear layer are already well organized [2] and no longer recognized by Mab RBl3-2 (Fig. 2C, D). At stage P12, the inner and outer segments of the photoreceptor cells become visible, and the morphology of the retina now resembles that of the mature retina [2]. Mab RB13-2 now binds only to the outer plexiform layer and to the cell bodies of the photoreceptor cells (Fig. 2E, F). In the mature retina (P98) the antigen is not expressed by any of the cells.

254

Fig. 3A-D. Immunoanalysis of RBLlST cells. A and C: phase contrast; B and D: indirect immunofluorescence; A and B: Mab RB21-7; C and D : Mab RBf 3-2, x 100

Expression of cell-type-specific antigens recognized by Mabs directed against neuronal or glial cells of the rat retina

To corroborate the specificity of Mab RB21-7 for neuronal cell types of the retina, the binding patterns of three other Mabs that recognize either Muller cells or neurons were compared with the expression of the Mab RB21-7defined antigen during retina development. Mab Q211, originally raised against embryonic chicken retinal membranes [20], binds to an antigen expressed by postmitotic neurons in embryonic chicken and rat brain. This Mab does not react with cells of nonneuronal origin. From F16 to PN7, the antigens defined by Mab Q211 and Mab RB21-7 were coexpressed on the same cell layers of the developing retina (results not shown). The Mab Q2ll-defined antigen was preferentially detected on cellular processes. Photoreceptor cells were not recognized by Mab Q211. Mab M501 [39] binds to a Muller-cell surface antigen. These cells are the primary supportive elements of the retina and the first of the mature retinal cells to appear, extending radially across the whole retina and reaching from the outer to the inner limiting membrane. Accordingly, the Mab M5Ol-defined antigen is detected in cryostat sections throughout the whole retina at all developmental stages (results not shown). This indicates

that the Mab M501-defined cell type, which is invisible under phase-contrast, is well represented in the retina. Vimentin is an intermediate filament protein, present in all cells of mesenchymal origin. In the retina vimentin is expressed only by Muller cells and horizontal cells [21]. In cryostat sections both cell types can be easily identified by the processes of the horizontal cells in the outer plexiform layer and the vimentin fibers of the Muller cells extending radially from the inner to the outer limiting membrane. By immunostaining with an antivimentin Mab, radially orientated vimentin fibers of Muller cells were detected across all cell layers at all developmental stages tested. The glial endfeet in the optic fiber layer and the processes extending to the outer plexiform layer and surrounding the photoreceptor segments were most intensely stained; however, numerous radially orientated fibers were also detected in the outer and inner nuclear layer. In contrast, the Mab RB21-7defined antigen only appeared on cells already organized in differentiated layers (results not shown). The results obtained with Mab Q211, Mab M501, and with the vimentin-specific Mab clearly show that Muller cells can be easily detected by indirect immunofluorescence in cryostat sections of the retina. The entirely different binding patterns of Mab M501 versus Mab RB21-7 confirms the selective binding of Mab RB21-7 to neuronal cell types of the retina.

255 Table 1. Monoclonal antibody (Mab) binding patterns on primary cells of 13 retinoblastoma (RB) tumors in comparison with 11 RBderived cell lines and the retinoblast cell line HER-Xhol-CC2 [38] RB tumors/ RB-derived lines

Percent of cells recognized by Mab R B f3-2

RBL7T RBL7 RBL13T RBL13 RBL14T RBL14 RBL15T RBLl5 RBL18T RBL18 RBL2OT RBL2O RBL2lT RBL25T RBL26T RBL27T RBL28T RBL3OT RBL3O RBL3lT RB247C3 RB355 RB383 Y79 HER-Xh01 -CC2

62 10 65 ND 50 ND 70 ND 20 ND 65 2 80 90 90 40 20 90 ND 40 3 7 21 ND ND

Mab RB2f-7 54 100 90 100 90 100 90 100 95 100 90 100 100 100 100 90 100 100 100 100

44 20 18 ND ND

T, Primary RB tumor cells; ND, no immunostaining detectable

Expression of the cell surface antigens specified by Mabs RB21-7 and RBl3-2 on primary cells of RB tumors and on cells of the RBL lines In primary cells of RB tumors, Mab RB21-7 is expressed on the majority of cells (90%-100% ; Fig. 3 A, B) while the antigen specified by Mab RB13-2 binds to subpopulations of different size (20%-90%) of the tumor cells; Fig. 3C, D) in 12 out of 13 RB tumors (Table 1). When cultured in vitro, the proportion of Mab RB21-7-positive cells increases to 100% in all cases (Table 2), and the antigen is recognized on all cells of the seven newly established RBL lines (tested for up to 18 months). Conversely, expression of the antigen specified by Mab RBl3-2 decreased rapidly under cell culture conditions (Table 2). In the RBL lines only a minor subpopulation of cells were recognized by Mab RB13-2, and in some cases, the antigen was not expressed at all (Table 1). These alterations in antigen expression during the initial period of in vitro cultivation occur in spite of the fact that the number of RB cells in the cultures remains constant [18]. It is, therefore, unlikely that these changes are due to selective overgrowth by cells recognized by Mab RB21-7. Primary RB cells are thus characterized by the expression of two cell surface antigens associated with suc-

Table 2. Binding of Mabs R B f 3 - 2 and RB21-7 to primary RB tumor cells and RB-derived cell lines Primary RB cells/ RB-derived lines

Time of cultivation (months)

RBL7T RBL7 RBL7 RBL13T RBLl3 RBL13 RBLl3 RBL14T RBL14 RBL14 RBL15T RBLl5 RBLl5 RBL18T RBLl8 RBL18 RBLZOT RBL20 RBL2O RBL30T RBL30 RBL30

ND

Mab RB13-2

Mab RB21-7

(% Mab-binding cells)

1

9 ND 1 2 18 ND 2 4 ND 8 12 ND 5

9 ND 7 11 ND 1 6

62 11 10 65 ND ND ND 50 3 ND 70 ND ND 20 ND ND 65 20 2 90 ND ND

54 67 100 90

too

100 100 90 100 100 90 100 100 95 100 100

90 100 100 100 100 100

T, Primary RB tumor cells; ND, no immunostaining detectable

cessive differentiation steps in the normal retina. The high proportion of Mab RB21-7-binding cells in RB tumors suggests that these are not composed of pluripotent progenitor cells [9,23,28], but rather consist of progenitor cells already committed to the neuronal lineage. This is further corroborated by the fact that primary RB cells are homogeneously recognized by Mab Q211 but not by Mab M501. Moreover, tumor induction does not seem to arrest the differentiation potential of RB cells completely. At least in vitro, RB cells follow to some extent the differentiation pathway characteristic of normal retinoblasts. Cells of the newly established RBL lines express neuronal properties without exception. The observation that RB-associated antigens are shared between RB-derived cell lines and primary RB tumor cells, suggests a common origin of these cells. Significantly, Y79 cells do not express the antigens specified by Mab RB21-7 and Mab13-2, nor by other Mabs of the same collection (RB15-11, RB21-13; results not shown; Table 1). The cell lines RB355, RB383, and RB247C3, which were established 6 years ago [34], exhibit an irregular binding pattern (Table 1). It has been argued [14, 151 that the expression of a less differentiated phenotype by Y79 cells could reflect tumor induction at an earlier stage of development. Contrary to this view, the present results show that cells of long-propagated RB lines lose at least some of their characteristic neuronal properties upon prolonged cultivation in vitro and that RB-derived cell lines

256

such as Y79, which have been propagated for long periods, are no longer representative of the RB phenotype. The cells of the established human retinoblast cell line HER-Xhol -CC2 [38] were recognized neither by Mab RB21-7 nor by Mab RB13-2. The present data strongly suggest that RB cells originate from progenitor cells committed to the neuronal pathway. However, it is not yet possible to decide whether the neuronal precursor cells giving rise to RB cells are already committed to one of the six distinct lineages of the retina. The retina contains seven different neural cell types derived from an apparently homogenous population of neuroepithelial precursor cells. Each differentiated phenotype is specified by the final position of a cell within the retina, and rods, bipolar cells, and Miiller cells may share the same lineage, until late in development [37]. The mechanisms determining the developmental pathway to be followed by a given precursor cell are unknown. Experiments with RB-derived cell lines in vitro may be misleading: photoreceptor cell differentiation could either be constitutive or a “faulty” pathway followed by retinal precursor cells in the absence of posi tion-dependent microenvironmental controls [ 11. Furthermore, phenotypic expression and transdifferentiation into other cell types are strongly influenced by the extracellular matrix in vitro or by the substrate on which neural progenitor cells are grown in culture. Either of these conditions can affect lineagespecific differentiation [19,27]. Expression of a photoreceptor phenotype by RB cells in vitro could, therefore, also be a cell-culture effect that would not occur in vivo. Further studies are, therefore, required to define more precisely the cells that give rise to RB cells among the different precursor cells of the developing retina. Acknowledgements. This work was supported by the Deutsche Forschungsgemeinschaft (Schwerpunktprogramm “Molekulare und klassische Tumorcytogenetik ”). The authors are indebted to Profs. W. Hopping and E.P. Messmer (Department of Ophthalmology, University of Essen Medical School) for providing the tumor material and to Dr. S. Henke-Fahle (Max-Planck-Institut fur Entwicklungsbiologie, Tubingen, FRG) and Prof. C.A.O. Stiirmer (Friedrich-Miescher-Laboratorium der Max-Planck-Gesellschaft, Tubingen, FRG) for providing Mab Q211 and M501, respectively. We are grcatful to Dipl.-Biol. S. Blass for valuable comments and to S. Dombrowski for preparing the manuscript.

References 1. Adler R, Hatlee M (1989) Plasticity and differentiation of em-

bryonic retinal cells after terminal mitosis. Science 243: 391-393 2. Araki M, Hanihara T, Saito T (1988) Histochemical observations on unique rod-like cells in the developing retina of the normal rat. J Neurocytol 17:179-188 3. Barnstable CJ (1987) Immunological studies of the diversity and development of the mammalian visual system. lmmunol Rev 100:47-78 4. Benedict WF, Murphree AL, Banejee A, Spina CA, Sparkes MC, Sparkes RS (1983) Patient with 1 3 chromosome deletion: evidence that the retinoblastoma gene is a recessive cancer gene. Science 21 9: 973-975 5. Bogenmann E, Mark C (1983) Routine growth and differentiation of primary retinoblastoma cells in culture. J Natl Cancer lnst 70:95-99

6. Bogenmann E, Lochrie MA, Simon MI (1988) Cone cell-specific genes expressed in retinoblastoma. Science 240: 76-78 7. Buse E, Kindler-Rohrborn A, Rajewsky MF (1989) In vitro experiments on neuronal and glial cell lineages among the ventricular cells of the mouse neural plate. Int J Dev Neurosci 7:103-113 8. Cavenee WK, Dryja TP, Phillips RA, Benedict WF, Godbout R, Gallie BL, Murphree AL, Strong LC, White RL (1983) Expression of recessive alleles by chromosomal mechanisms in retinoblastoma. Nature 305 :779-784 9. Chader GJ (1987) Multipotential differentiation of Y79 retinoblastoma cells in attachment culture. Cell Differ 20:209-216 10. Cheresh DA, Varki AP, Varki NM, Stallcup W, Levine J, Reisfeld RA (1984) Monoclonal antibody recognizes on O-acetylated sialic acid in a human melanoma-associated ganglioside. J Biol Chem 259: 7453-7459 1 1 . Detrick B, Chader GJ, Rodrigues M, Kyritsis AP, Chan C-C, Hooks JJ (1988) Coexpression of neuronal, glial, and major histocompability complex class I1 antigens on retinoblastoma cells. Cancer Res 48 : 1633-1 641 12. Druckrey H (1971) Genotypes and phenotypes of ten inbred strains of BD-rats. Arzneirnittel-Forsch 21 :1274-1278 13. Fong S-L, Balakier H, Bridges CDB, Gallie BL (1988) Retinoid binding proteins in retinoblastoma tumors. Cancer Res 48:11241128 14. Gallie BL, Albert DM, Wong JJW, Buyukmihic N, PuliaFato CA (1977) Heterotransplantation of retinoblastoma into the athymic “nude” mouse. Invest Ophthalmol Vis Sci 16: 256-259 15. Gallie BL, Ellsworth RM, Abramson DH, Phillips RA (1982) Retinoma : spontaneous regression of retinoblastoma or benign manifestation of the retinoblastoma gene. Brit J Cancer 45:513-521 16. Gallie BL, Holmes W, Phillips RA (1982) Reproducible growth in tissue culture of retinoblastoma tumor specimens. Cancer Res 42:301-305 17. Gallimore PH, Grand RJA, Byrd PJ (1986) Transformation of human embryo retinoblasts with simian virus 40, adenovirus and ras oncogenes. Anticancer Res 6 : 499-508 18. Griegel S, Chen H, Frotschl R, Hulser DF, Greger V, Horsthemke B, Rajewsky MF (1990) Newly established human retinoblastoma cell lines exhibit an “immortalized ” but not an invasive phenotype in vitro. Int J Cancer 46: 125132 19. Hall DE, Neugebauer KM, Reichard LF (1987) Embryonic neural retinal cell response to extracellular matrix proteins: developmental changes and effects of the cell substratum attachment antibody (CSAT). J Cell Biol 104:623-634 20. Henke-Fahle S (1983) Monoclonal antibodies recognize gangliosides in the chick brain. Neurosci Lett 14: 16&166 21. Huba R, Hofmann H-D (1988) Tetanus toxin binding to isolated and cultured rat retinal glial cells. Glia 1 :156-164 22. Kindler-Rohrborn A, Ahrens 0, Liepelt U, Rajewsky MR (1985) Expression of monoclonal antibody-defined cell surface antigens during rat brain development. Differentiation 30: 5360 23. Kyritsis A, Tsokos M, Triche L, Chader GJ (1984) Retinoblastoma: origin from a primitive neuroectodermal cell. Nature 307:471413 24. Liepelt U, Kindler-Rohrborn A, Lennartz K, Reinhardt-Maelicke S, Rajewsky MF (1990) Differentiation potential of a monoclonal antibody-defined neural progenitor cell population isolated from prenatal rat brain by fluorescence-activated cell sorting. Dev Brain Res 51 :267-278 25. Mendez-Otero R, Schlosshauer B, Barnstable CJ, ConstantinePaton M (1988) A developmentally regulated antigen associated with neural cell and process migration. J Neurosci 8:564-579 26. Moss TJ, Seeger RD, Kindler-Rohrborn A, Marangos PJ, Rajewsky MF, Reynolds CP (1985) Immunohistologic detection and phenotyping of neuroblastorna cells in bone marrow using cytoplasmic neuron specific enolase and cell surface antigens. In: Evans AE, D’Angio GJ (eds) Advances in Neuroblastoma Research. Prog Clin Biol Res 175 :365-378

257 27. Reh TA, Nagy T, Gretton H (1987) Retinal pigment epithelial cells induced to transdifferentiate to neurons by laminin. Nature 330:68-71 28. Reid TW, Albert DM, Rabson AS, Russel P, Craft J, Chu EW, Tralka TS, Wilcox JL (1974) Characteristics of an established cell line of retinoblastoma. J Natl Cancer Inst 53:347363 29. Reinhardt-Maelicke S, Cleeves V, Kindler-Rohrborn A, Rajewsky MF (1990) Differential recognition of a set of O-acetylated gangliosides by monoclonal antibodies RB13-2, D1 . l , and JONES during rat brain development. Dev Brain Res 51 :279282 30. Rosner H, Greis Ghr, Henke-Fahle S (1988) Developmental expression of embryonic rat and chicken brain of a polysialoganglioside-antigen reacting with the monoclonal antibody Q211. Dev Brain Res 42: 161-171 31. Schappert-Kimmijser J, Hemmes GD, Nijland R (1966) The heredity of retinoblastoma. Ophthalmol 151 :197-213 32. Scherer J, Schnitzer J (1989) The rabbit retina: a suitable mammalian tissue for obtaining astroglia-free Miiller cell cultures. Neurosci Letters 97: 51-56

33. Schnitzer J (1988) Astrocytes in the guinea pig, horse, and monkey retina : their occurrence coincides with the presence of blood vessels. Glia 1 : 74-89 34. Squire J, Gallie BL, Phillips RA (1985) A detailed analysis of chromosomal changes in hereditable and non-hereditable retinoblastoma. Hum Genet 70: 291-301 35. Ts’o MOM, Zimmermann LE, Fine BS (1970) The nature of retinoblastoma. 1. Photoreceptor differentiation : a clinical and histopathologic study. Am J Ophthalmol 69: 339-349 36. Ts’o MOM (1980) Clues to the cells of origin in retinoblastoma. Int Ophthalmol Clin 20: 191-210 37. Turner DL, Cepko CL (1987) A common progenitor for neurons and glia persists in rat retina late in development. Nature 328: 131-136 38. Vaessen RTMJ, Houweling A, Israel A, Kourilsky P, van der Eb AJ (1986) Adenovirus E1A-mediated regulation of class I MHC expression. EMBO J 5:33>341 39. Wehner K (1990) Identifizierung wachstumsdssoziierter Molekule im retinotectalen System des Goldfisches mit Hilfe neuer monoklonaler Antikorper. PhD Dissertation, Universitlt Stuttgart