egf and tgf-α stimulate retinal neuroepithelial cell proliferation in vitro

\euron, Vol. 6, 923-936, June, 1991, Copyright 0 1991 by Cell Press

EGF and TGF-a Stimulate Retinal Neuroepithelial Cell Proliferation In Vitro

Raymond M. Anchan, Thomas A. Reh, John Angello, Arthur Balliet, and Macie Walker Department of Biological Structure, SM-20 llniversity of Washington !ieattle, Washington 98195

Summary

Peptide growth factors have been shown to have diverse effects on cells of the CNS, such as promoting neuronal survival, neurite outgrowth, and several other aspects of neuronal differentiation. In addition, some of these factors have been shown to be mitogenic for particular classes of glial cells within the brain and optic nerve, and recently two peptide growth factors, fibroblast growth Factor and nerve growth factor, have been shown to have mitogenic activity on the CNS neuronal progenitors. We now report that two members of another peptide growth factor, epidermal growth factor and transform- kng growth factor-a, are mitogenic for retinal neuroepithelial cells in primary cultures and provide evidence for the presence of both of these factors in normal developing rat retina.

introduction

The multipotent progenitor ceils of the retinal germi- *ial neuroepithelium generate a large variety of differ- ?nt neuronal cell types, as well as Miiller glia, during the histogenesis of the retina (Sidman, 1961; Hinds and Hinds, 1978,1979; Turner and Cepko, 1988; Holt It al., 1988; Wetts and Fraser, 1988; Turner et al., 1990). 4lthough there is currently very little known about :he factors that regulate the mitotic activity of these rells, there is evidence from several sources that the ‘inal number of neurons produced by these cells is lot fixed and intrinsic to the cells, but rather under :he regulation of the developing microenvironment. :n the frog and fish, neurotoxic damage to the retina during larval or embryonic stages results in a consid- erable increase in the production of new neurons, to compensate for those destroyed by the lesion (Neg- :shi et al., 1982, 1985, 1987; Reh and Tully, 1986; Reh, 1987; see Reh, 1989, for review). Similarly, surgical re- moval of the retina in larval amphibians and embry- snic chicks induces adramatic regenerative response In the remaining marginal neuroepithelial cells (Stone, 1950a, 1950b, 1960; Coulombre and Coulombre, 1965; Reyer, 1977; Lopashov and Stroeva, 1964; Park and Hollenberg, 1989), as well as the pigmented epithelial cells, and again, these cells generate many more progeny than they otherwise would (see Reh, 1990, for review). A regenerative response is also present in the embryonic rat retina; X-irradiation studies have demonstrated that destruction of the newly postmitotic cells at early embryonic stages results in a com-

pensatory overproduction of cells so that the final number of cells in the retinaapproximates that found in the normal retina (Rugh and Wolff, 1955a, 1955b).

These studies indicate that the cells of the retinal neuroepithelium havethecapacityto respond todam- age by an up regulation in their production of neurons and glial cells, perhaps as a consequence of the re- lease of mitogenic factors from the damaged tissue. Since the neuroepithelial cells appear to be able to respond to signals in their microenvironment, it is possible that such signals are normally important in the regulation of neuronal and glial numbers during retinal histogenesis and that the cells of the neuroepithelium may respond to retinal damage as a consequence of the disruption of one of the normal mitotic regulators. We are therefore interested in determin- ing whether any known peptide growth factors are important in this response and have examined the actions of several peptide mitogenic factors for their actions on the multipotent progenitor cells of the retina. We havefound that epidermal growth factor (EGF) and transforming growth factor-a (TGF-a) are particularly effective mitogens for retinal cells in culture; we also provide evidence that message for both of these factors, as well as the EGF receptor, is present during much of the histogenesis of the retina and therefore conclude that either or both of these molecules may be important regulators of the numbers of neurons and glia produced during retinal neurogenesis.

Results

Retinal Neuroepithelial Cells Continue to Proliferate in Dissociated Cell Culture; EGF and TGF-a Stimulate the Mitotic Activity of These Cells We have previously reported (Reh and Kljavin, 1989) that small aggregates of embryonic and neonatal retinal cells will continue to undergo a degree of cell proliferation, while single cells will not. Therefore, in our initial experiments, retinal cells were dissociated in such a way as to produce many small aggregates, as well as a large number of single cells. The control wells, for both the postnatal and the prenatal cultures, show[3H]thymidinelabelingfor upto3days inculture (see Figures 2e and 2f), and the embryonic cells show an overall increase in cell number without the addition of EGF or TGF-a (see Figure 2a). When the radioac- tive nucleotide is present throughout the culture period of 6 days and the cells are subsequently processed for immunohistochemistrywith several neuron- specific antibodies, double-labeled cells of several neuronal and glial phenotypes are found in the cultures (see below), indicating that many of the cells that are actively dividing in the cultures are neuronal progenitors.

The initial response of the retinal cells to EGF or

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Figure 1. Effects of ECF and TGF-a on Primary Cell Cultures of Embryonic and Neonatal Retinal Cells

(A and B) After (12 hr) plating onto Matrigel substrates, EGF-treated cells (IO rig/ml) from P2 animals (B) have already begun to spread out more extensively onto the substratum than the cells in the control wells (A). This effect is even more pronounced after 2 days in culture (C and D) and occurs regardless of whether the cells are plated onto polylysine- or Matrigel-coated coverslips. After 3 days of EGF treatment in vitro, extensive cell spreading is evident in the phase micrograph of El8 retinal cells (E) while the same field, shown in bright field (0, shows that many of the cells in the culture are labeled with [3H]thymidine after a 6 hr exposure to the nucleotide. Bar, 80 pm.

TGF-a appears as a flattening of the cells on the sub- the control wells (Figure IA). This spreading becomes strate, regardless of whether the cells are plated di- even more pronounced on the second day in culture rectly on the plastic wells, or onto glass coverslips (Figure IC), and in addition to the flattening of the coated with polylysine or a combination of polylysine cells, there appear to be more cells than in the control and Matrigel (I:100 dilution). Figure IB shows this re- wells (Figure ID). By 3 days in vitro, there is extensive sponse for dissociated cells from a postnatal retina, flattening and apparent aggregation of the cells (Fig- treated with IO rig/ml EGF (Figure IB), compared with ures IE and IF), and in addition, the high degree of

EGF and TCF-a Are Mitogens for Neuroepithelial Cells 925

Figure 2. ECF and TGF-a Stimulate Cell Proliferation

(a and b) The number of cells was determined in cultures of El7 (a) or P3 (b) cells after 10 @ml EGF was added to the medium at the time of plating. The figure ex- presses the total cell number after 1,2, and 3 days in vitro. The initial number of cells plated was 100,000 per well. See text for details of the quantitation. Closed squares represent ECF-treated wells; open squares represent the control wells. Values represent means f SEM (c and d). The number of total cells and that of cells labeled with [‘Hlthymidine were quantified from more than six randomly selected fields from coverslips that had been processed for autoradiography after having been exposed to various concentrations of ECF for 3 days in culture, to show the dose-response rela- tionship of this factor. The number of [‘Hlthymidine-labeled cells after 3 days in culture for El8 (closed bars) and P3 (hatched bars) rat retinal cells is illustrated in (c) as a function of ECF concentration. (d) The total number of cells for El8 (closed bars) and P3 (hatched bars) as a function of EGF concentration, after 3 days in vitro. Initial plating density was 100,000 cells per well; however, a certain fraction of thecells is lost during the processing of the coverslips for immunohistochemistry and au-

I . . . . toradiography; therefore, these numbers cannot be readily compared with those of (a) and (b), in which the number ot total ilve cells in the wells was directly counted. (e and f) In a separate experiment the total number of [‘Hlthymidine-labeled cells in El8 (e) and P3 (f) cultures was determined after 3 days in vitro in cultures treated with ECF (IO nglml) and TCF-a (IO rig/ml). In both El8 and P3 cultures the number of [SH]thymidine-labeled cells is greater in EGF- and TGF-a-treated cultures when quantified either as the total number of labeled cells (closed bars) or as the number of labeled neuronal cells (hatched bars).

proliferative activity is demonstrated by the large number of [3H]thymidine-labeled cells following a 12 hr labeling period in the S-day cultures (Figure IF). These effects were essentially identical for all of the embryonic or neonatal ages of animal from which the cells were obtained.

Cell counts of sister wells after 1, 2, and 3 days in vitro confirmed that these growth factors were stimu- lating cell proliferation. Cellswere plated directly into wells of 24-well plates at a density of IO5 cells per well, and after each of the first 3 days in culture, the medium was removed and spun on a centrifuge to pellet any nonadherent cells (between 10% and 20% of the total cells plated). Since most of the cells in both the treated and control wells adhered to the plastic sur- faces of the wells, a trypsin-EDTA solution was added to each well. After a period of incubation, these cells were easily removed from the bottom of the wells by gentle trituration. The trypsin-EDTA-treated cells were then combined with the nonadherent cells (pel- leted earlier) and further incubated in trypsin-EDTA to dissociate completely any aggregates or clusters prior to counting on a hemacytometer. As can be seen in Figure 2, embryonic day 17 (E17) cells, plated at an initial density of 10scells per well, increased to almost 2.5 x IO5 cells per well after 2 days and 3.5 x IO5

cells per well after 3 days in vitro, while control wells contained less than 2 x IO5 cells per well at the end of this same period.

In cells from postnatal retinas, there is some cell addition in both the EGF-containing and the control wells during the first day in culture. Thereafter, the number of cells in the EGF-treated wells remains relatively constant, while the cell number in the control wells declines to approximately 80% of that initially plated. These data would seem to indicate that EGF is primarily acting to prevent cells from dying in the postnatal cultures; however, it is also possible that the rate of cell death is similar in both the control and EGF-treated cultures, but in the former the cell loss is balanced by an increase in the number of newly generated cells, while in the control wells, no such addition of new cells occurs to make up for the dying cells. In this scenario, the total number of cells per well declines in control cultures, but remainsconstant in the EGF-treated wells. To test this latter hypothesis, we labeled the actively proliferating cells in cells from El8 and postnatal day (P3) retinas with [3H]thymidine after 3 days in culture and calculated the number of radiolabeled cells and the total number of cells in the wells as a function of EGF concentration (Figures 2c and 2d). Figures 2c and 2d show a dosage response

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ECF and TCF-a Are Mitogens for Neuroepithelial Cells 927

for El8 (closed bars) and P3 (hatched bars) retinal cells. For both ages, the number of [3H]thymidine-labeled cells (Figure 2c) and the total number of cells (Figure 2d) increases with the addition of increasing amounts of ligand, up to 10 nglml. These data verify that the EGF is causing an increase in the number of [3H]thymidine-labeled cells, particularly at 1.0 and 10 rig/ml, for both prenatal and postnatal cultures, consistent with the stimulation of cell proliferation in these cultures.

small colonies consisting only of flat cells that would ultimately give rise to both neurons and additional flat cells. This can be seen in Figures 5A-5D, in which a small group of flat cells (2 or 3, pointed to by the small arrow in Figure 5A) generate over 50 flat and neuronal cells (Figure 5D).

ECF and TCF-a Stimulate the Proliferation of the Multipotent Progenitor Cells of the Retina There is no reliable way to label exclusively the multipotent progenitor cells of the retinal germinal neuroepithelium, and so our identification of thecells that have been stimulated to proliferate in response to the EGF and TGF-a is an indirect one; i.e., both neurons and glia are generated by these cells. The cells in our cultures can be morphologically classified intotwo basic types: round, neuronal cells with extensive processes and flat, glia-like cells. We have used two different strategies to determine whether the mitotically active cells in these cultures are producing neuronal and glial cells. In one set of experiments, the putative neuroepithelial cells were plated at low density (IO cells per well of a 24well plate) and then photographed each day, or the cells were plated at a somewhat higher density and continuously filmed with atime lapsevideo recorder. Figure 3 shows examples of two such colonies that were continuously recorded for the first 6 days in vitro. Both the flat cells and the round neuronal cells are increasing in number over the 6 days in culture, consistent with the possibility that neuroepithelial cells continue to proliferate and generate neurons in these cultures. In addition, in some cases, as their density increases, the neuroepithelial cells adopt a somewhat bipolar morphology and organize into rosette-like forma- tions, typical of their appearance in vivo in the developing retina. Extensive examination of the recordings showed two patterns of progeny following cell divi- sions. As they prepare to divide, the flat cells round up on the neighboring cells, and then when their mitosis is complete, the two progeny will either both become flat cells, or one of them will become a round neuronal cell and the other a flat cell. The round neuronal cells were never observed to undergo mitosis. The fact that only flat cells appear to divide in culture is consistentwith the immunohistochemical evidence of nestin immunoreactivity exclusively in these cells (see below). In addition, in some cases we observed

To characterize more fully the flat and neuronal cells produced in thesecultures, we used several anti- genie markers selective for different cell classes. In the rat retina, in vivo, the various types of neurons and glia are produced in a developmentally regulated manner, such that the majority of ganglion cells, cones, horizontal cells, and amacrine cells are generated prenatally, while the majority of rods, all bipolar cells, and Miller glia are produced after birth (Sid- man, 1961). Through the use of various specific antibody markers, several different types of neurons can now be labeled in the developing retina, as well as in cultures (Hicks and Barnstable, 1987; Barnstable and Drager, 1984; Shaw and Weber, 1983; Drager, 1983; Araki et al., 1987; Akagama and Barnstable, 1986; De- leeuw et al., 1990). We have found in a previous study that the development of the various classes of neurons and the Miller glia in culture parallels that observed in vivo (Reh and Kljavin, 1989; see also Watanabe and Raff, 1990; Adler and Hatlee, 1989). Therefore, in this study, we further characterized the cells stimulated to proliferate with EGF, using immunohistochemistry for antigens specific to particular types of retinal neurons and glia.

We found that the round neuronal cells generated in these cultures were immunoreactive for several different neuron-specific antigens, while the flat cells were not. The types of cells immunoreactive for the various antibodies that we used in this study are illustrated in Figures 4-7. Of the mitotically active cells, in cultures from both the prenatal and postnatal retina, neuroepithelial cells are labeled with antibodies against nestin (Cattaneo and McKay, 1990), as shown in Figures 4E and 4F, and vimentin (Bennett, 1987; Shaw and Weber, 1983; I. J. Kljavin and T. A. Reh, unpublished data). In the postnatal cuItures,vimentin also labels the Miiller cells (Shaw and Weber, 1983), but in addition, these cells are immunoreactive for cellular retinaldehyde-binding protein (CRALBP) (Fig- ures 7D-7F), the CRALBP found only in Mijller cells within the sensory retina (Deleeuw et al., 1990). All classes of postmitotic neurons express neural cell adhesion molecule (N-CAM) at high levels (Edelman et al., 1987), as shown in Figures 4A and 4B and Figure 6, and in addition, all classes are highly immunoreactive for neuron-specific enolase (Figures 4C and 4D) when well differentiated (Schilling et al., 1988; Reisert et al.,

Figure 3. Time Lapse Recording of ECF-Stimulated Cell Proliferation Micrographs of time lapse recordings from low density cultures of El4 retinal neuroepithelial cells cultured with EGF (IO rig/ml). Micrographs (A)-(D) and (E)-(H) represent separate experiments, both continuously recorded on days 2-6 in vitro. Over the period of examination, the clones increase in number of both phase-bright, round neuronal cells, as well as the flat neuroepithelial/glial cells. Note that the two flat cells in (A), indicated by small arrow, proliferate to a cluster of >50 cells (D), by 6 days in culture. Bar, 80, urn.

Figure 4. Antibodies to Specific Cell Classes Were Used to Characterize the Ceils Generated in the EGF-Treated Rat Retinal Cultures Cells were cultured from El8 (A and 6) or El5 (C-F) for 3 days and then labeled with antibodies raised against N-CAM (A and B), neuron-specific enolase (C and D), or nestin (RAT 401; [El and [F]). (A), (C), and (E) are fluorescence micrographs, and (61, (D), and (F) are the corresponding phase pictures. Note that virtually all of the round cells in (B) label positive for N-CAM (A). Many of these neuronal cells show immunoreactivity for neuron-specific enolase (0, although there is a less intense labeling of the underlying neuroepithelial cells. (E and F) Neuroepithelial cells labeled with RAT 401, an antibody against nestin, appear to have a primarily flat morphology in these cultures, though as the density increases they often adopt a more spindle or bipolar shape. Bar, 44 urn.

EGF and TCF-a Are Mitogens for Neuroepithelial Cells 929

Figure 5. Neurofilament-Containing Cells Are Produced in the Embryonic Cultures

Corresponding phase (A and C) and fluorescence (6 and D) micrographs show that neurofilament immunoreactivity, identified with RT97 monoclonal antibody is present in many of the neuronal cells and their processes. (A) and (B) show the labeled cells in El5 cultures, after 6 days in vitro with ECF; (C) and (D) show an example of a neurofilament-immunoreactive cell that was also labeled with [‘Hlthymidine (arrowhead) from an El7 culture after 3 days in vitro with EGF. Bar, 36 urn.

1982), although, as for N-CAM, a low level of immunoreactivity is also present in the flat cells in these cultures (see Schilling et al., 1988). Rods, the major class of photoreceptors in the rat retina, are immunoreactive for rhodopsin, and we have used a monoclonal antibody (4D2) to this molecule to identify the rods in these cultures (Figures 7A-7C). Ganglion cells or horizontal cells are the major retinal cell classes that express neurofilament proteins (Figure 5) and can be labeled with monoclonal RT97 (Drager, 1983).

It is apparent from the time lapse data that neuronal cells are generated in these cultures for at least the first 6 days, and the immunohistochemical characterization of these neuronal cells shows that they can be of various classes. However, a further series of experiments was carried out to corroborate this evidence. Both embryonic and postnatal retinal cells were plated onto glass coverslips that had been previously coated with either polylysine alone or in combination with a basement membrane extract (I:100 dilution of Matrigel in Hanks’ balanced salt solution).

Various concentrations of EGF or TGF-a were added to the wells at the time of plating, and the medium was not changed for the duration of the experiment. The cells were labeled with [3H]thymidine and then fixed after 1-6 days in culture. These coverslips were then labeled with particular neuron- or Miller glia- specific antibodies prior to processing for autoradiography. The results of these experiments are shown in Figures 6 and 7. Figure 6 shows several examples of N-CAM-immunoreactive cells that also have silver grains over their nuclei, indicating that theywere generated in culture. Similar examples of a double- labeled rod (opsin-immunoreactivecells)and aMi.iller glial cell (CRALBP-immunoreactive; Deleeuw et al., 1990) are shown in Figure 7, further supporting the multipotent character of the mitotically active cells in thesecultures. Both embryonic and neonatal cultures had examples of N-CAM-immunoreactive cells labeled with [3H]thymidine, but CRALBP and opsin- immunoreactive cells labeled with nucleotide were observed only in the postnatal retinal cultures within

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Figure 6. N-CAM-lmmunoreactive Cells Are Generated in the Embryonic Retinal Cultures Corresponding fluorescence (A and C) and phase (B and D) micrographs of El8 retinal ceils, after 3 days in vitro and continuous labeling with [‘Hlthymidine. Cells were fixed and labeled with an antibody directed against N-CAM, prior to autoradiography. Flat cells are only very dimly labeled with this antibody. Arrows point to N-CAM- immunoreactive cells that are also labeled with silver grains over their nuclei, directly demonstrating that some of the cells generated in these cultures are neurons. Bar, 33 pm.

6 days of plating. When we quantified the number of neuronal cells that were also labeled with [3H]thymidine after 3 days in culture, we found that both EGF and TGF-a increased the number of these cells in the cultures significantly over the control wells (Figures 2e and 2f) in both the embryonic and postnatal cultures. The antibodies and the types of cells in the cultures that are labeled with these antibodies are shown in diagramatic form in Figure 8. Neuroepithe- lial cells generate neurons and additional neuroepithelial cells in embryonic cultures, whereas neuroepithelial cells from postnatal retinas would be expected to give rise to both neurons and Miiller glia.

ECF and TGF-a mRNAs Are Present in Retinal Cells during Embryonic and Postnatal Development The mitogenic effects of EGF and TCF-a on the cells of the retinal germinal neuroepithelium suggested to us that these factors might normally play a role in the histogenesis of the retina. Although mRNA for both of these factors has been reported to be present in other areas of the fetal nervous system (Lee et al., 1985) and in the adult cow retina (Fassio et al., 1989), it is not known whether either of these factors or their receptors are present in the developing retina. There- fore, we used the polymerase chain reaction (PCR)

Figure 7. Rods and Mtiller Cells Are Generated in Postnatal Retinal Cultures

Corresponding fluorescence (A), bright-field (B), phase (C), fluorescence CD), bright-field (E), and phase (F) micrographs of P2 retinal ceils after 3 days in culture and continuous labeling with [jH]thymidine. Cells were fixed and labeled with Rho-4D2, a monoclonal

ECF and TGF-a Are Mitogens for Neuroepithelial Ceils 931

antibody directed against rhodopsin (an ant igen present only in the rod photoreceptor cells in the retina), prior to autoradiography or with anti-CRALBP (an ant igen present only in Miiller cells in the retina) prior to autoradiography. The cell that is labeled with the antibody as well as the silver grains in (A) and (6) is pointed out by the arrow and is a rod that was generated in culture. The cell that is labeled with the antibody as well as the silver grains in (D) and (E) is pointed out by the arrow and is a Miiller c:ell that was generated in culture. In (D)-(F), a pair of cells are labeled with [‘Hlthymidine (arrowhead), while only one cell in this pair is a Miiller glial cell, as demonstrated by the CRALBP immunoreactivity (arrow in [D]). Bar, 30 urn.

E15/E18 P2/P3

NE (nestin) I NE (nestin)

Nh IL1 N (NCAM) N (402) G KRALBP)

(RT97) 1

(NSE)

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Figure 8. Various Antibodies Were Employed to Identify the Dif- ferent Cell Phenotypes Generated in the Cultures with EGF after 3-6 Days In Vitro

The diagram illustrates the possible differentiation fates of retinal germinal neuroepithelial cells (NE). The neuroepithelial cells are immunoreactive for nestin and produce additional multipotent neuroepithelial cells or neuronal cells (N). The neuronal cells can be identified using antibodies to neuron-specific antigens such as N-CAM RT97, which labels the 200 kd neurofilament protein and anti-neuron-specific enolase (NSE). Postnatal retinal neuroepithelialcelIsgenerateneuronsorMiiIlerglia(G). lnaddi- tion to the neuronal antigens present in the round neuronal cells in the embryonic cultures, rod photoreceptors are also generated in postnatal cultures, as evidenced by the presence of cells immunoreactive to Rho-4D2 (a monoclonal antibody directed at rod rhodopsin). CRALBP is found only in Muller glia in rat retina and can be used to identify these cells reliably.

and subsequent Southern blotting and hybridization with probes to both EGF and TGF-a to determine whether either was present at the stages of retinal neurogenesis at which we found mitogenic effects for these factors. Figure 9a shows the results of an experiment in which total RNA was extracted from fetal and postnatal retina and subjected to PCR amplification of EGF message (upper blot) or TGF-a message (lower blot). Subsequent Southern blotting was per- formed prior to hybridization with probes to either EGF (upper blot) or TGF-a (lower blot). In Figure 9a, lanes 1,2, and 3, show the levels of the two messages in the fetal liver, whole brain, and cerebral cortex of an El8 rat, when equal amounts of RNA were amplified and loaded onto the gel, for comparison with the retinal message (lanes 4-8). Figure 9a, shows the relative amounts of message for EGF and TGF-a in the retina at E15, E18, PI, P7, and adult. Control samples that were treated in an identical manner, but were not reverse transcribed prior to amplification, showed no PCR products. It is interesting to note that TGF-a mRNA appears to be present at relatively higher levels in the embryonic retina and declines with increasing developmental- age, though it continues to be expressed in the adult, while EGF message is expressed at highest levels in the neonatal period, but appears not to be expressed in the adult or in the early embryonic retina.

a >

b

b

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116- 97-

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Figure 9. EGF and TCF-a, As Well As the EGF Receptor, Are Pres- ent in Rat Retina

(a) Southern blot of PCR products from total retinal RNA from various ages of rat retina. Between 4 and 6 ug of rat retinal total RNA was reverse transcribed with random hexamer primers and amplified 35 cycles. For the upper blot, primers were synthesized from the mouse EGF sequence; for the lower blot, oligonucleotide sequences in rat TGF-a were used to synthesize the primers. The PCR products were run on a 2% agarose gel prior to blotting and hybridization with a 12P-labeled mouse ECF probe (upper blot) or an end-labeled 32mer oligonucleotide probe comple- mentary to TGF-a (lower blot). (b) Western blot of neonatal rat retinal protein, showrng the presence of the EGF receptor as a band of approximately 165 kd in lane 1, incubated with an antiserum against the receptor, which is not present in lane 2, in which the primary antiserum was omitted from the incubation. Lane 3 shows an imido black stain of the total transferred protein.

Both TGF-a and EGF are thought to act via the same receptor, a 170 kd transmembrane glycoprotein with intrinsic tyrosine kinase activity (Cohen et al., 1982; Massague, 1983; Hunter and Cooper, 1985; Chandler et al., 1985). We therefore used an antibody directed against the extracellular domain of the human EGF receptor to determine whether this molecule is present in the retina at the stages in which we found these growth factors to exert mitogenic effects on the neuroepithelial cells. Total retinal protein was run on an SDS-PAGE from PI retinas and blotted onto nitrocellulose priorto incubation with a monoclonal antibody directed against the receptor (Figure 9b, lane 1) or control serum (Figure 9b, lane 2). The blot, shown in

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F!gure 9b, shows a band of approximately 165 kd that i! absent in the control (lane 2).

Discussion

W e have shown the following in this report: First, neuroepithelial cells continue to proliferate in culture. Second, several different types of retinal neurons, as well as Muller glial cells, are generated in these cultures. Third, EGF and TGF-a stimulate the neuronal and glial production by these cells. Fourth, the receptor and mRNA for these factors are present in the developing retina. On the basis of this evidence and that discussed below we propose that EGF and/or TGF-a are likely to be important regulatory molecules for the process of neurogenesis in the CNS.

The various types of neurons and glia present in the C-NS are derived from the multipotent neuroepithelial cells in the ventricular zone of the developing CNS. To characterize more fully factors that regulate the production of neurons and glia during development, several attempts have been made to recapitulate the process of neurogenesis in vitro. Cultures of embryonic rat cortex (Gensburger et al., 1987), striatum (Cattaneo and McKay, 1990), mesencephalon, or telencephalon (Murphyet al., 1990) have provided evi- dencethat neuroepithelial cells will continue to proliferate in vitro, and combined [3H]thymidine and immunohistochemistry show that some proportion of their progeny will differentiate into either neurons (neurofilament immunoreactive) or glia (glial fibrillary acidic protein immunoreactive). While it is clear from these reports that both neurons and glia are being generated in vitro, all of these studies used a culture density mat precluded the identification of individual neuroepithelial cells necessary to verify that a single neuroepithelial cell can give rise to both neurons and glia, as occurs in vivo. This was accomplished by Temple (1989) for another population of telencephalic progen- i:ors, the septal neuroepithelial cells, in which it was shown that single neuroepithelial cells remain multipotent in vitro and can give rise to large clones of r.eurons and glia.

The results of our studies of retinal neuroepithelial cells in culture are consistent with these earlier reports. The neuroepithelial cells adopt a flat, spread morphology in low density culture, but become more elongate and bipolar as their density increases. In the retina, as has been observed in other areas of the CNS, tne neuroepithelial cells give rise to both neurons and glia, both in vivo (Turner and Cepko, 1988) and in vitro. Since many of the cell c lasses in the retina can be identified with specific antigenic markers, we have been able to characterize the progeny of the neuroepithelial cells more fully than has been possible in clther areas of the CNS. Consequently, we have been able to show that the neuroepithelial cells can produce various types of neurons in culture and interest- ilgly continue to generate the classes of cells typical of their in vivo age. That is, neuroepithelial cells from

embryonic retina do not generate either rods or Mtiller cells for at least the first week in vitro, while both of these cell types are generated in postnatal cultures.

EGF and TGF-a are potent mitogens for a variety of different types of cells, including several classes of CNS glia (for review Simpson et al., 1982). W e have demonstrated in this report that these factors are also mitogenic for neuronal and glial progenitors in the rat retina in vitro. This raises the possibility that these molecules are involved in the regulation of neuronal numbersduring histogenesisoftheCNS. DuringCNS histogenesis, an enormous diversity of different neuronal cell types are generated from the multipotent neuroepithelial cells; perhaps mitogenic growth factors favor the production of particular classes of neurons or glia, as is thought to occur following cytokine activation of specific l ineages in the hemopoietic system (for review see Metcalf, 1989). W e do not think that EGF and TGF-a promote the differentiation of any particular cell type, since in our cultures, many different types of neurons, as well as Miiller glia, are produced by the neuroepithelial cells. In addition, these molecules appear to stimulate proliferation in thesecultures in both prenatal and postnatal cultures, when different classes of neurons are being generated. Instead, it is likelythat EGF and TGF-a play a role in regulating the overall numbers of cells during histogenesis and may therefore be important in the response of the germinal neuroepithelium to damage.

Previous investigators have demonstrated the presence of EGF and TGF-a in the CNS and have described several neurotrophic actions of these molecules. Mul- tiple species of EGF-immunoreact ive molecules have been reported to be present in the brain of adult rats (Schaudies et al., 1989; Werner et al., 1988; Gomez- Pinilla et al., 1988). In addition, message for EGF and TGF-a has also been found in adult and developing rat CNS (Lee et al., 1985; Fallon and Seeroogy, 1984; Wilcox and Derynek, 1988; Fassio et al., 1989). Several reports have also characterized the effects of EGF on various classes of CNS cells. Simpson et al. (1982) found that EGF is mitogenic for astrocytes and oligo- dendrocytes; however, no mitogenic effects were reported for neuronal progenitors. EGF has also been found to promote neurite outgrowth and cell survival in cultured cells from the CNS, particularly the sub- cortical telencephalon (Morrison et al., 1987). EGF, TGF-a, and amphiregulin all bind to and activate a 170 kd cell surface tyrosine kinase. In addition two related proteins, ERBB/HER2/neu and more recently HERY ERBB3, have also been shown to be expressed in the CNS (Kraus et al., 1989; Plowman et al., 1990), although the former has not been found to have any interaction with EGF or TGF-a. W e have found a 165 kd molecule in developing rat retina that is immunoreactive with antisera raised against the human EGF receptor, and so it is likely that the effects we observe are mediated through this receptor.

One of the most striking effects of EGF and TCF-a on neuroepithelial cells is the rapid and extensive flattening of the cells on the substratum. We have observed this flattening of the cells on tissue culture plastic, polylysine, and a basement membrane substrate. Could EGF and TGF-a be acting to enhance cell-substrate adhesion? A recent study of the interactions between Drosophila neurogenic loci Notch and Delta raised the possibility that the EGF repeats in these molecules might mediate a Ca*+-dependent adhesion between ceils expressing these molecules (Fehon et al., 1990). Although it is possible that cell membrane-associated EGF and TGF-a play such a role between retinal neuroepithelial cells, chick spinal neuroepithelial cells show a similar flattened mor- phologyuponexposureto basicfibroblastgrowthfac- tor in vitro (Heuer et al., 1990), suggesting that this response may have less to do with specific effects of EGF and instead represents a general response of neuroepithelial cells to mitogenic stimuli. Several studies have described a link between cell shape and proliferation; cell spreading generally correlates with an increasing probability of entering the S phase of the cell cycle (Folkman and Moscona, 1978; Schubert, 1986).

We have presented evidence that EGF and TGF-a have mitogenic effects on the multipotent neuroepithelial cells that give rise to retinal neurons and glial cells and are present at the appropriate stages of retinal histogenesis that would allow these factors to play a role normally in regulating their production. Other peptide growth factors as well as neurotransmitters have recently been described as having mitogenic effects on neuroblasts from other areas of the nervous system. Studies of in vitro DNA synthesis in neuroblasts from embryonic rat sympathetic ganglia dem- onstrate regulation by several factors, including insulin-like growth factors, insulin, EGF, and basic fibroblast growth factor (DiCicco-Bloom and Black, 1988; DiCicco-Bloom et al., 1990). In addition, the neu- rotransmitter vasoactive intestinal peptide is also mitogenic for these cells in culture (Pincus et al., 1990). Recent studies in the CNS have also shown that peptide growth factors can stimulate the proliferation of multipotent neuroepithelial cells. Temple (1989) found that clonal growth of striatal neuroepithelial cells was dependent on both serum-derived factors and conditioning factors from glial cells, while Mur- phy et al. (1990) and Cattaneo and McKay (1990) have described mitogenic effects of nerve growth factor and basic fibroblast growth factor on neuroepithelial cells from rat forebrain. Based on these findings and the results of this study, we propose that peptide growth factors normally play a role in the regulation of the numbers of neurons and glia produced by the neuroepithelium during histogenesis of the CNS, in addition to their actions on cell survival and differentiation.

Experimental Procedures

Cell Culture and lmmunofluorescence Neonatal or fetal rats were killed by decapitation following CO1 anesthesia, and the eyes were removed under sterile conditions. The neural retina was dissected away from the pigment epithelium and otheroculartissue. The retinawas then dissociated into a single-cell suspension by incubation in 0.25% trypsin solution (GIBCO, Grand Island, NY) in Ca2+, Mg*‘-free Hank’s balanced salt solution (GIBCO) at 37OC with gentle nutation for either 10 min (prenatal) or 20 min (postnatal). The cells were plated in a low serum medium consisting of DMEM-F12 (without glutamate or aspartate; CIBCO) with the following supplements: 25 &ml insulin, 100 kg/ml transferrin,60 PM putrescine, 30 nM selenium, progesterone, and either ECF (R& D Research, Minneapolis, MN) or recombinant TGF-a (generously supplied by Dr. T. Rose of Oncogen Seattle, WA), and 1% fetal bovine serum. Prior to plating, the cells were counted; postnatal retinas typically yield >2.5 x 106cells per retina. Cellswere grown at 37OC in an atmosphere of 5% CO>. In some experiments, retinal cells were plated at a final density of 103-IO5 cells per ml onto glass coverslips placed into 24well plates. Coverslips are coated with polylysine (50 pigi ml), laminin (IO pg/ml), or Matrigel (1:50-1:lOO) by incubating in the solution for morethan 1 hr. In another series of experiments, the retinal cells were plated directly into the 2Cwell plates.

To label the proliferating neuronal progenitors, [‘Hlthymidine (I pCi/ml of a 6.7 Cilmmol solution; New England Nuclear) was added to some of the cultures. In some experiments, the [‘Hlthymidine was added on the first day of culture; in other experiments, we added the radionucleotide as a pulse, 6-8 hr before the cells were fixed. Coverslips with cultured cells were then removed and fixed after periods of from l-6 days in vitro. Thecoverslipswerethen incubatedwithvarious neuron-specific antibodies to reveal the composition of the cultured cells. The rhodopsin monoclonal antibody Rho-4D2, which is specific to rod photoreceptor cells, was obtained from a hydridoma cell line (gift of Dr. Molday, University of British Columbia) and used at approximately IO pg/ml. Antiserum raised in rabbit against CRALBP and a monoclonal antibody raised against cellular reti- noic acid-binding protein, a specific marker for the majority of amacrine cells in the rat retina, were gifts from Dr. J. Saari (University of Washington, Seattle, WA), and a rabbit antiserum to N-CAM was a gift from Dr. A. Acheson (University of Alberta, Edmonton). A rabbit antiserum to neuron-specific enolase was purchased from Dakopatts (Denmark) and used at a dilution of 1:150; RT97 monoclonal antibody against neurofilament protein (200 kd subunit) was the generous gift of U. Drager (Harvard Medical School, Boston, Massachusetts) and was used as undi- luted hybridoma culture supernatant, as was RAT 401 (nestin) monoclonal antibody, which was obtained from S. Hockfield (Yale University School of Medicine, New Haven, CT). All antibodies were diluted to approximately 10 pgiml prior to use and visualized via a biotin-streptavidin-fluorochrome (Molecular Probes, Eugene, OR) complex. Following the immunohistochemistry, the coverslips were processed for autoradiography by stan- dard methods.

lmmunoblotting Samples for Western blotting were prepared by homogenizing P3 retinas in extraction buffer(4°C)consisting of 1% Triton X-100, 10 mM Tris, 5 mM EDTA, 30 mM sodium pyrophosphate, 50 mM sodium flouride, 100 mM sodium orthovanadate, 0.1% BSA, 50 mM NaCI, and 1 mM phenylmethylsulfonyl flouride (Sigma, St. Louis, MO). The samples were then diluted I:1 with SDS sample buffer and run on a 7.5% SDS-PAGE. The proteins were elec- troblotted on nitrocellulose, immunolabeled with an antibody raised in rabbits against a synthetic dodecapeptide derived from the extracellular domain of the human EGF receptor (CRB), and visualized with an alkaline phosphatase-conjugated goat anti- rabbit secondary antibody (Sigma).

ECF and TCF-a Are Mitogens for Neuroepithelial Cells 93!>

PCR and Southern Analysis Total RNA was extracted from whole retinas as follows. Tissue was rinsed in diethylpyrocarbonate-treated Hanks’balanced salt solution and frozen on dry ice. The tissue was then thawed and homogenized in lysis buffer containing 0.5% Nonidet P-40 in a 1.5 ml tube with a plastic pestle. Next, the lysate was underlaid with a25% sucrose solution and centrifuged to pellet the unlysed cells and nuclei. The supernatant was then treated with protein- ase K at 37OC for 30 min, followed by two successive phenol- chloroform extractions. An aliquot of the total RNA was then reverse transcribed with random hexamer primers for 1 hr at 42 ‘C in a 20 ~1 reaction containing the following: 4-6 wg of retinal RNA, 50 mM KCI, 20 mM Tris-HCI (pH 8.3), 2.5 mM MgCl?, 0.001% gelatin, 1 mM dNTPs (Pharmacia, Pleasant Hill, CA), 30 U of RNasin (Promega, Madison, WI), 100 pmol of random hexamers (Pharmacia), 200 U of reverse transcriptase from Maloney murine leukemia virus (BRL, Bethesda, MD), and 10 mM DTT. The cDNA was then amplified for 30-60 cycles on a Coy thermocycler in a IO!) ~1 reaction containing the following: 20 PI of cDNA, 500 ng of primers, 2.5 U of Taq polymerase (BRL), and final buffer concentrations of 50 mM KCI, 10 mM Tris-HCI (pH 8.3), and 1.9-2.3 mM MgCI,. The primer sequences used for TCF-a amplification were bases 280-297and 553-570 (Lee et al., 1985) for the upstream and downstream primers, respectively. The ECF primer sequences were bases 3265-3282 and 3613-3630 from the pub- lished sequence for mouse EGF RNA (Gray et al., 1983). After amplification, the PCR products were analyzed by electrophore- sis on a 2% agarose gel to verify that the appropriate predicted size product was produced, and subsequently the PCR products were blotted onto Hybond-N nylon membrane (Amersham) and hyhridized with either an end-labeled oligonucleotide probe wi: h the TGF-a sequence 409-431 or a 960 bp insert from pmECF- 261:12 (American Tissue Type Collection #37486).

Acknowledgments

The authors gratefully acknowledge support from the National Imtitutes of Health, grant ROI NS23808, and the Alfred E. Sloan Foundation to T. A. R. and acknowledge the critical comments on the manuscript of C. Jasoni.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 USC Sec- tion 1734 solely to indicate this fact.

Received December 5, 1990; revised February 27, 1991.

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egf and tgf-α stimulate retinal neuroepithelial cell proliferation in vitro

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