The differentiation of retinal photoreceptors and neurons in vitro

CHAPTER 1

The Differentiation of Retinal Photoreceptors and Neurons in vitro RUBEN ADLER

The Michael M. Wynn Center for the Study of Retinal Degenerations, The Wilmer Ophthalmological Institute, The Johns Hopkins University, School of Medicine, Baltimore, Maryland 21205, USA

CONTENTS 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1

2. Studies with Complex Monolayer Cultures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Development of the Cultures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Complex Cultures as an Experimental System to Study Retinal Cell Differentiation . . . . . . . . .

3 3 5

3. Studies with Retinal Neurons in Low Density, Glia-free Monolayer Cultures . . . . . . . . . . . . . . . . . . . 3.1. Methodological Considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Predetermination of Neuronal Development: A First Approximation . . . . . . . . . . . . . . . . . . . . . .

7 7 8

4. The Problem of Cell Identification in Retinal Cultures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

12

5. Studies with Cultured Photoreceptor Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1. Culture Methods for Photoreceptor Ceils . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2. Are Precursor Cells Predetermined to Differentiate as Photoreceptors? . . . . . . . . . . . . . . . . . . .

13 13 18

6. Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

24

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

24

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

24

I. INTRODUCTION

The mature retina is characterized by the presence of different cell types, orderly arranged in layers and interconnected with each other. In contrast, the embryonic rudiment that gives rise to the retina is a simple epithelial structure containing only one recognizable cell type, the neuroepithelial cell. When and how are these neuroepithelial cells induced to differentiate as photoreceptors, glia, retinal ganglion cells, etc? Figure 1 shows in graphic f o r m two hypothetical scenarios that can be proposed to answer this

question. In the first scenario, cells require a separate inductive signal for each of the developmental transformations necessary for the transition from the undifferentiated state to the mature phenotype. In the alternative scenario, cells become committed early in embryogenesis to the development of a 'master plan', that they will carry out without further instructive signals f r o m the microenvironment. Intermediate developmental strategies could be proposed in which m a n y phenotypic properties would be predetermined early in development as part of a 'master plan', while others would be induced by separate signals acting later in

R. ADLER Instructive Signals

_

Precurs°rNalve

'

2

_

J

i i T T 1~::;,erer'tiated

[ ,ns,r°c,,ve S,gna,s I FIG. 1. Two alternative hypotheses concerning the regulation of the events leading to photoreceptor cell differentiation are presented graphically in this figure, and explained in the text in more detail.

development. These questions are important beyond the limits of developmental biology. We know, for example, that repair and regeneration of the injured retina occur in adult individuals in lower vertebrates such as fish, but fail in higher species including man. There is a double link between development and regeneration. First, regenerative events to a large extent represent a recapitulation of developmental mechanisms by post-embryonic (i.e. 'adult') cells. Second, even in species in which regeneration does not occur in the adult, that capacity is present in the early embryo and is lost as differentiation advances (Coulombre and Coulombre, 1965; Grafstein, 1986). Thus, it is probably warranted to propose that our capacity to intervene upon the injured retina and stimulate its regeneration will be largely dependent upon our understanding of the mechanisms controlling the development and maintenance of the differentiated state of retinal cells. The intact animal, as it exists in the in vivo situation, is much too complex to allow the type of experimentation necessary to manipulate the microenvironment surrounding a cell and study its responses in a dynamic fashion. From this point of view, the use of in vitro techniques to study questions such as those summarized in the preceding paragraphs is justified by the impossibility to carry out similar studies in vivo. It is fair to ask, however, how relevant are these in vitro studies with respect to the in vivo organism (or, as the question is occasionally phrased, to 'real life')? What is the value of tissue culture

experiments in which in vitro and in vivo conditions are not identical? Should researchers make every possible effort to find in vitro systems which mimick to perfection the conditions present in the intact organism? As illustrated in Fig. 2, there is very limited gain in experimental and analytical possibilities if, after the complex series of manipulations involved in tissue culture techniques, the final product is an in vitro organism identical to the one which exists in 'real life'. In vivo and in vitro conditions are nonidentical, by definition. Moreover, comparisons between different in vitro procedures regarding their similarity with the intact organism frequently are only a matter of personal preference and semantics. The approach followed in our laboratory is based on the assumption that in vivo and in vitro conditions necessarily are (and should be) different (Fig. 2). In this alternative approach, specific cell types are dissociated away from the organism, and grown in vitro under defined conditions. This approach allows adequate control of the environment surrounding the cultured cells, which at the same time become amenable to direct inspection with high resolution techniques. While observations made with these purified cultures cannot be automatically considered representative of phenomena occurring in the intact organism, they are likely to disclose the repertoire of cellular responses to specific sets of microenvironmental conditions, as well as the cell machineries involved in these responses. These cultures also allow investigating how the differentiation of the cultured ceils is affected by molecules isolated from the original organism, thus potentially disclosing the existence and mechanism of action of regulatory factors present in vivo. The relevance of these in vitro methods, then, derives from the comparison between in vivo and in vitro behaviors, comparison which in turn assumes that both situations are indeed different. This chapter will deal with some of the questions presented here, as well as with the methods which allow their investigation. This double set of objectives will require alternating the emphasis of the discussion between both areas, hopefully without creating excessive confusion in the process. As is the norm in the Progress in Retinal Research series, this monograph will be

3

RETINAL PHOTORECEPTORS AND NEURONS

mainly devoted to recent work from the author's laboratory. Important contributions from other laboratories, which would have a protagonistic role in a more comprehensive review, will be discussed only as background information. The presentation will be biased towards experiments using chick embryo retinal monolayer cultures, the system most frequently used in this laboratory. Evidence will be presented suggesting that retinal precursor cells become committed rather early in embryogenesis to the development of specific phenotypes. At the same time, the experiments will emphasize the critical role played by microenvironmental factors in regulating the differentiation of retinal neurons and photoreceptors. Given the impact that progressive developments in tissue culture methods have had upon our capacity to investigate these questions, it appears advisable to follow a historical approach, centered around the different monolayer culture systems which became available over the years to study retina ceils. 2. STUDIES W I T H C O M P L E X M O N O L A Y E R CULTURES 2.1. Development of the Cultures

The chick embryo neural retina was extensively used for explant and reaggregation cultures in the 1950s and 60s (Adler et al., 1976; Hild and Callas,

1967; LaVail and Hild, 1971; Moscona, 1965; Stefanelli et al., 1967; Steinberg, 1963; Tamai et al., 1978 reviewed in Adler, 1986a). However, the first report of a monolayer culture of dissociated retina cells seems to be that by Barr-Nea and Barishak (1970). Their procedure, and similar ones used later on in different laboratories (Crisanti-Combes et al., 1977; Dutt and ReifLehrer, 1981; Gremo et al., 1984; Itoh, 1976; Kaplowitz and Moscona, 1976; Okada, 1980; Pritchard et al., 1978; Puro et al., 1977; Redfern et al., 1976; Thompson and Pelto, 1982) involved: (i) the careful dissection of the neural retina from other ocular tissues, including the retinal pigment epithelium; (ii) the use of trypsin to dissociate the retina into a single cell suspension; (iii) the use of fetal calf serum-supplemented medium; and (iv) the use of cell culture substrata such as uncoated glass, and later on plastic, without any special 'attachment factors'. Although other stages have also been used, the eight-day-old chick embryo is probably the most frequent source of cells for these studies, and the description that follows will refer to this stage of development. A very important early event in these cultures is the occurrence of spontaneous cell reaggregation, favored by the poor adhesiveness of untreated plastic for retinal cells. Multicellular clumps thus become the predominant element in the cultures during the first two or three days in vitro (Fig. 3).

llg

"in ovo" chicken (real life)

II

Tissue Culture Black Box

rest of chicken

FIG. 2. This cartoon illustrates two alternative strategies that can be followedin the design of tissue culture studies. A detailed explanation is presented in the text (Section 1).

R. ADLER ' F l a t ' cells b e g i n to e m e r g e f r o m t h e m u l t i c e l l u l a r c l u m p s in a r a t h e r s y n c h r o n o u s f a s h i o n t o w a r d s t h e e n d o f this p e r i o d (Fig. 3). T h i s is a m o s t s t r i k i n g p h e n o m e n o n , a n d o n e w h i c h has n o t r e c e i v e d t h e a t t e n t i o n it p r o b a b l y d e s e r v e d .

A l t h o u g h t h e s i t u a t i o n m i g h t be d i f f e r e n t in t h e case o f o l d e r e m b r y o s ( M o y e r a n d S h e f f i e l d , 1985), ' f l a t ' cells are n o t p r e s e n t as s u c h in eightd a y r e t i n a cell s u s p e n s i o n o r e a r l y c u l t u r e s . M o r e o v e r , t h e y fail t o d e v e l o p i f t h e f o r m a t i o n o f

FIG. 3. Clump-containing cultures are generated when neural retina cell suspensions are seeded on poorly adhesive substrata. (a) During the first three days in vitro, cultures supported by fetal calf serum (shown here) or horse serum are similar, and consist essentially of multicellular clumps which occasionally show neuritic processes. (b) six days in vitro, pb = Processbearing cells. (c) six days in vitro. When fetal calf serum is present in the medium, flat cells (fc) start migrating out of the clumps towards the fourth day in culture, and give rise to a confluent monolayer, cl = clumps; pb = process-bearing cells. (d) ten days in vitro. The multicellular clumps and most single cells attached onto the flat cell carpet can be mechanically removed using a jet of fluid, giving rise to a purified flat cell monolayer. Magnification bar: (a) 100/am; (b)- (d) 25/am. Reproduced from Adler et al., 1982, with permission from the publisher.

RETINAL PHOTORECEPTORS AND NEURONS

multicellular clumps is prevented (Adler et al., 1979, 1982). Analysis of this phenomenon led these authors to propose that 'flat cells' will only develop after appropriate (but still undetermined) cell-cell interactions take place within the multicellular clumps. It is of interest that differentiated biochemical properties of retinal glial cells, such as the inducibility of the enzyme glutamine synthetase, are also regulated by intercellular interactions (Moscona and Linser, 1983). After a period of about a week, which varies according to the initial seeding density, practically 100°/0 of the dish surface becomes covered with flat cells. This population expands by cell proliferation (Kaplowitz and Moscona, 1976; Adler et al., 1982) as well as cell migration (Li and Sheffield, 1986b). At the same time, cell processes with the microscopic appearance of 'neurites' (i.e. axons and dendrites) can be seen to emerge from multicellular clumps and from some isolated cells. By the end of the first week in vitro, then, the cultures show a very complex structure, with a network of cell clusters and single cells interconnected by neurite processes, sitting on top of a confluent monolayer of flat cells (see Fig. 3). This configuration, however, will soon change through two different processes. First, there is progressive loss of multicellular clumps and neurons. The starting time of these changes has not be accurately determined, but the attrition process is fairly obvious during the third week in vitro, and eventually leads to cultures made up exclusively of 'flat cells'. Second, some of the flat cells undergo very profound changes that lead to the appearance of new cellular phenotypes through the 'transdifferentiation' process discussed below (Moscona and Linser, 1983; Okada, 1980; see also Pritchard, 1983).

2.2. Complex Cultures as an Experimental System to Study Retinal Cell Differentiation 2.2.1. FLAT CELLS AND THE 'TRANSDIFFERENTIATION' PHENOMENON

Several properties of the flat cells suggest their glial identity, including the ultrastructural

presence of abundant intermediate filaments and junctional complexes (Araki et al., 1982; CrisantiCombes et al., 1977; Li and Sheffield, 1984) and the presence of immunocytochemically detectable vimentin and GFAP (glial fibrillary acidic protein) (Li and Sheffield, 1984). Vimentin is not usually considered a glial 'marker', because it is present in cells such as fibroblasts and vascular endothelium. However, the absence of these cells from the chick retina enhances the relevance of vimentin cytochemistry as a useful (although not definitive) criterium for Muller cell identification (Lemmon and Rieser, 1983). The interpretation of GFAP immunoreactivity in retinal flat cells is somewhat more complicated, because this protein can not be found in chick Mfiller cells in vivo. Li and Sheffield (1984) have suggested that its presence in cultured flat cells represents a response to the in vitro situation, mimicking reactive responses observed in the injured retina in other animal species (Bignami and Dahl, 1979). The presence of the enzymes carbonic anhydrase and glutamine synthetase in flat cells is also consistent with their glial identity (Dutt and Reif-Lehrer, 1981; Linser and Moscona, 1983; Moscona and Linser, 1983) as is their capacity to take up some putative neurotransmitters by means of high affinity mechanisms (dePomerai and Carr, 1982; dePomerai et al., 1983; Hyndman and Adler, 1982a,b). Taking into consideration all these properties, as well as the absence of fibroblasts and endothelial cells from the chick retina, the generally accepted identification of flat cells as 'glial-like' cells seems warranted (their 'flat' morphology, although different from the configuration shown by glial cells in vivo, resembles the appearance adopted by other CNS glial cells in culture). It is less clear, however, whether they are actually derived from the Miiller cells present in the retina in vivo. The preceding description of the flat cells is only valid for relatively young cultures, because these cells tend to 'transdifferentiate' in vitro. The transdifferentiation phenomenon has been extensively studied by several laboratories, and there are comprehensive reviews which should be consulted for further information (Moscona and Linser, 1983; Okada, 1980). In essence, flat cells undergo a change in gene expression, with loss of

6

R. A D L E R

proteins typical of glial cells accompanied by new synthesis of large amounts of other proteins characteristic of either lens cells or pigmented epithelial cells. This is a fascinating system for the investigation of gene regulation in vertebrate cells, but unfortunately it also limits the usefulness of flat cells as a model system to study 'retinal glia'. Given that the changes in gene expression seem to occur in increasing numbers of cells, the cultures can be expected to contain different mixtures of glial, lens and pigmented epithelial cells at different in vitro stages. This variability can be further increased by the sensitivity of the transdifferentiation process to relatively subtle changes in culture conditions, including the volume, composition and bicarbonate concentration of the culture medium, the type of serum used as supplement, etc. (Gall and dePomerai, 1984; Okada, 1980; Pritchard et al., 1978).

2.2.2. N E U R O N S AND P H O T O R E C E P T O R S

Analysis of the differentiation of neurons and photoreceptor in complex monolayer cultures has been limited by the presence of flat cells and multicellular clumps in the cultures. The neuronal identity of most of the process-bearing, isolated cells detectable by phase contrast microscopy is supported by their positive staining by tetanus toxin cytochemistry (Adler et al., 1982), a property consistent with a neuronal identity (Mirsky et al., 1978). Some of these cells also show high-affinity uptake mechanisms (dePomerai et al., 1983; dePomerai and Carr, 1982; Guerinot and Pessac, 1979; Hyndman and Adler, 1982a,b), catecholamine fluorescence (Araki et al., 1984) and can be stained for acetylcholinesterase (Araki et al., 1982). Moreover, transmission electron microscopical studies have shown that multicellular clumps contain cells with phenotypic properties typical of differentiated neurons and photoreceptors, including neuritic processes and typical synaptic terminals, although photoreceptor outer segments have not been observed (Araki et al., 1982; Crisanti-Combes et al., 1977). These findings, together with the 'rosette-like' configuration seen in the multicellular clumps by the same authors, suggest that the 'clumps'

present in complex 'monolayer' cultures are actually equivalent to multicellular reaggregates of retinal cells described by Sheffield and Moscona (1970). 2.2.3. A N A L Y T I C A L POSSIBILITIES OFFERED BY COMPLEX RETINAL CULTURES

The presence of multicellular clumps in the cultures is a limiting factor for the analysis of cell differentiation. Individual cells can not be resolved within multicellular clumps with the microscopical techniques available to study live monolayer cultures (i.e. phase contrast, Nomarski). Similar lack of resolution is found when otherwise powerful analytical techniques, such as autoradiography and immunocytochemistry, are applied to clump-containing cultures. In the autoradiogram of a thymidinelabelled culture shown in Fig. 4, for example, it is frequently impossible to determine which are the individual cells responsible for the presence of silver grains because of the overlapping between neurons and the adjacent flat cells. The interpretation of in vitro experiments can be frequently complicated by 'hidden' interactions between constituents of a culture system. These complications are even more likely to occur in the case of complex cultures with high cell density and heterogeneous composition. The example presented below to illustrate this point is by no means unique (see, for example, Dutt and ReifLehrer, 1981; Linser and Moscona, 1983). Our example derives from a study with monosodium glutamate (MSG) a substance known to destroy many retinal neurons in vivo. During an in vitro investigations of the responses of cultured retinal cells to MSG, Hyndman and Adler (1982a) observed that this substance caused the loss of many neurons in complex retinal cultures, but failed to kill the neurons when they were grown in glial-free, low density cultures (these cultures will be described in Section 3). A possible explanation for this discrepancy was that the flat cells present in complex cultures were somehow involved in the neuronotoxic effects of MSG. This was shown to be the case by experiments in which MSG-containing medium, exposed briefly to a monolayer

RETINAL PHOTORECEPTORS AND NEURONS

of retinal flat cells, acquired the capacity to kill retinal neurons even when they were grown in gliafree cultures. Thus, the availability o f separate, purified cultures of retinal neurons and glial cells made it possible to avoid the erroneous conclusion that would have been drawn if MSG had only been tested using complex cultures. Interestingly, the in vitro results obtained with the purified populations supported the suggestion by Casper et al. (1982), based on EM and autoradiographic observations in vivo, that glial cells mediate the toxicity of MSG for retinal neurons. Different treatments have been successfully used in several laboratories to generate purified flat cell populations through the elimination of multicellular clumps and neurons (Adler et al., 1982; dePomerai and Cart, 1982; Koh et al., 1984; Li and Sheffield, 1984; Linser and Moscona, 1983; See Fig. 3). The usefulness o f these purified monolayers of non-neuronal cells as a model system to study the biology of retinal glia-like cells is obvious, although the transdifferentiation phenomenon represents a complicating factor that must be taken into consideration (see above). These populations can also be of use to study

molecules released by glial-like cells into 'conditioned media' potentially capable of modulating neuronal development (cf. Adler et al., 1981). 3. S T U D I E S W I T H R E T I N A L N E U R O N S IN LOW D E N S I T Y , G L I A - F R E E M O N O L A Y E R CULTURES 3.1. Methodological Considerations Methodological changes allowing growth of retinal neurons in glial-free cultures (Adler et al 1982) were actually derived f r o m previous experience with chick embryo optic lobe cells (Adler et al., 1979). Observations made with both visual organs showed that the formation of multicellular clumps by suspensions of dissociated cells could be prevented by increasing the adhesiveness of the substrata used for cell attachment. Lack of flat cell development in the cultures was seen as a consequence of the absence of multicellular clumps. Highly adhesive substrata such as polyornithine or a special collagen preparation could be used for this purpose (Adler

FIG. 4. Complex retinal monolayer cultures grown on substrata of low adhesiveness were exposed to 3H-thymidine-labelled cells. Flat cells do not show any detectable tetanus toxin immunofluorescence, but appear labelled with 3H-thymidine(short arrows). Multicellular clumps show tetanus toxin-positive cells (long arrows). No thymidine-labelledcells can be seen in the clumps at six days, although they are present at three days in vitro (not shown). Magnification bar = 25/am. Reproduction from Adler et al., 1982, with permission from the publisher.

8

R. ADLER

et al., 1979, 1982; see also Betz and Mtiller, 1982). Autoradiographic studies using [3H] thymidine as a precursor showed lack of cell proliferation in low density cultures on these adhesive substrata, in contrast to the abundant proliferation seen in clumped cultures (Adler et al., 1982). The predominant element in the low density, clumpfree cultures are non-mitotic, process-bearing, tetanus toxin-positive cells, which could therefore be identified as neurons (Adler et al., 1982). It is not clear what happens in these cultures to the 'flat cell precursors' (i.e. the cells which would give rise to flat cells if clump formation had been allowed). Some process-free, round cells present in retinal cultures on highly adhesive substrata could well represent these undifferentiated precursor cells, but no markers are available to identify them more conclusively, or to document their eventual disappearance. In any event, it is clear that retinal cell suspensions will originate either flat cell-free or flat cell-containing cultures depending on the substratum used, even if the same (fetal calf serum-containing) medium is used in both cases. Flat cell-free neuronal cultures can also be supported by horse serum-containing medium, or by serum-free, chemically defined medium supplemented with the ' N I ' formulation developed by Bottenstein and Sato (1979). The possibility to grow CNS neurons in this medium was first demonstrated by Skaper et al. (1979) and extended to retinal neurons by Betz and Miiller (1982) and Hyndman and Adler (1982c). Specific requirements for culturing retinal cells in chemically defined medium are discussed in more detail below (Section 5.2).

3.2. Predetermination of Neuronal Development: A First Approximation

Low density, purified neuronal cultures offer better possibilities than complex, glia-containing cultures for the analysis of factors involved in the regulation of neuronal differentiation. For example, the absence of a confluent flat cell monolayer allows direct contact between neurons and the substratum, thus making it possible to investigate the effects of individual molecules acting from a substratum-bound situation. The

lower~ cell density of the cultures reduces significantly the extent of intercellular contacts and, concomitantly, the likelihood of 'hidden' cell - cell interactions. Finally, neuronal behaviors can be analyzed with high resolution both in live cultures (i.e. by phase contrast or Normaski microscopy) or in fixed preparations processed for techniques such as autoradiography or immunocytochemistry. These features allowed the investigation of retinal cell responses to environmental factors, disclosing at the same time that the expression of some aspects of the neuronal phenotype appear to be predetermined early in development.

3 . 2 . 1 . EFFECTS OF EXTRACELLULAR MATRIX MOLECULES

(ECM) ON

RETINAL CELL DEVELOPMENT

There is some confusion in the literature regarding the presence (and possible functions) of ECMs in the central nervous system. This confusion is probably derived from the classic perception of ECMs as exclusive components of connective tissues, a concept rendered obsolete by more recent observations showing the broad distribution of these materials. In fact, the avascular, connective tissue-free, purely 'neural' retina of the chick is a very good example, since it has been demonstrated to synthesize collagen (Smith et al., 1976) as well as proteoglycans (Morris et al., 1977; Morris and Ting, 1981). Moreoever, its inner limiting membrane contains immunocytochemically detectable laminin (Adler et al., 1985), and the photoreceptor cells are in close contact with an 'interphotoreceptor matrix', located between the neural retina and the pigment epithelium (Hewitt, 1986). Thus, it appears that ECM molecules are not only present but are probably also functionally important in the retina. In recent studies we tested the effects of ECM molecules upon retinal neurons by allowing these molecules to bind to tissue culture plastic dishes precoated with polyornithine (PORN) (Fig. 5 and Table 1). The coated dishes were washed to remove unbound materials, and seeded with freshly dissociated retinal cells suspended in serum-free medium (Adler et al., 1985). Similar numbers of cells attached to untreated PORN, to

RETINAL PHOTORECEPTORS AND NEURONS P O R N c o a t e d w i t h f e t a l c a l f s e r u m (a c o m m o n source of 'attachment factors'), or to PORN coated with the ECM glycoproteins fibronectin, laminin or chondronectin. However, cell attachment to these different substrata appeared to b e m e d i a t e d b y at least t w o d i f f e r e n t

(a)

m e c h a n i s m s , since it w a s t e m p e r a t u r e - d e p e n d e n t in t h e cases o f l a m i n i n a n d f e t a l c a l f s e r u m , a n d temperature-independent for fibronectin and untreated polyornithine substrata. Further d e v e l o p m e n t o f t h e cells was a l s o d e p e n d e n t u p o n t h e n a t u r e o f t h e s u b s t r a t a ( T a b l e 1). T h u s , r e t i n a l

(b)

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FIG. 5. Effects of extraceUular matrix molecules on retinal cell behavior. Dissociated cells from eight-day chick embryo neural retina were cultured for 6 hr in serum-free medium on different substrata. (a) Untreated polyornithine; most cells are well spread out and several cells show extensive vacuolization (arrows). Neuritic processes are essentially absent. (b) Fetal calf serum-treated polyornithine; cells appear phase bright and are considerably less spread out than on untreated polyornithine. Neurite development is very limited. (c) Fibronectin-treated polyornithine; note considerable cell spreading. Neuritic development is minimal. Similar behavior was observed on chondronectin-treated polyornithine. (d) Laminin-treated polyornithine; cells are less spread out than on untreated polyornithine but are not as phase bright as on fetal calf serumtreated polyornithine. Note extensive neurite development and the presence of growth cones on some neurite processes (arrows). Hematoxylin-eosin stained, phase contrast microscopy. Magnification bar = 25 ~,m. Reproduced from Adler et al., 1985, with permission from the publisher.

10

R. ADLER TABLE 1. Behavior o f Retinal Cells Cultured in Serum-free Medium on Different Substrata: A Summary Cell behavior

Substratum

Attachment

Polyornithine (untreated) + fibronectin Very good + chondronectin

Extensively spread cells

Cell degeneration

Neurite development 6 hr

72 hr

Abundant

Extensive

Essentially none

Essentially none

+ FCS

Very good

Minimal to intermediate

Minimal

Minimal

Moderate

+ PNPF + laminin

Very good

Intermediate

Minimal

Extensive

Extensive

T. culture plastic (TCP) Very good T C P + laminin Poor

Intermediate Absent

Not studied Not studied

None None

None

cells attached to fibronectin-coated P O R N spread very extensively, displayed very little if any neurite development and showed conspicuous signs of degeneration (Fig. 5). In contrast, cell spreading was much less extensive and cell survival much better when the cultures were grown on P O R N coated with either fetal calf serum or laminin, although other differences were in turn found between these last two substrata (Fig. 5). These differences were particularly obvious regarding their capacity to stimulate neurite development: In a short term bioassay, some 45 - 50°70 of the cells showed lavish neurite development on laminin, while only 7°7o of the cells did so on fetal calf serum-treated dishes. It is of interest that the neurite-promoting effects of laminin were in turn dependent upon the nature of the substratum to which laminin itself was bound (Fig. 6). Laminin bound directly to tissue culture plastic (in the absence of polyornithine) failed to stimulate neurite development while showing a concentrationdependent inhibitory effect upon cell attachment. This behavior was completely different from the powerful neurite stimulation seen with P O R N bound laminin. Studies with iodinated laminin showed that similar amounts of this glycoprotein bound to untreated plastic as it did to P O R N treated plastic. It is possible that the complex laminin molecule undergoes different conformational changes when bound to positively charged polyornithine or to negatively charged tissue culture plastic. It is an intriguing (and still unexplored) possibility that laminin could also

affect neuronal behavior in the intact retina, and that its properties could be regulated by lamininassociated molecules also in the in vivo situation. The effects of laminin upon retinal neurons were remarkably similar to those of ' P N P F ' , a neurite-promoting activity present in Schwannoma conditioned medium (Adler, 1982). Antilaminin antibodies failed to block neurite stimulation by P N P F , although they inhibit the neurite promoting effect of purified laminin (Adler et al., 1985). A similar difference has been described using other neuronal systems (Manthorpe et al., 1983; Lander et al., 1985a,b). Recent purification work has shown that the neurite-promoting activity present in conditioned medium is actually associated with a laminincontaining complex (Davis et al., 1985; Lander et al., 1985a,b). Surprisingly, the same antibodies that fail to block the biological activity of the conditioned medium neurite-promoting factor do bind to the molecules apparently responsible for that activity. The reason for this discrepancy is unknown. Together with related studies from other laboratories, these findings suggest the possibility that ECMs could play a role in the regulation of neuronal behaviors in CNS organs such as the retina. We now know that these effects are selective, since the stimulatory effects of laminin are not reproduced by fibronectin or chondronectin (Adler, 1985) or by several classes of collagen and proteoglycans (Hewitt and Adler, in preparation). Given that several of these ECM molecules (including laminin) are present in the

RETINAL PHOTORECEPTORS AND NEURONS

retina, their possible in vivo function should be tested. These substances could be involved in the control of axonal growth not only in embryonic development, but also in the regeneration of retinal ganglion cell axons after optic nerve lesions

D,,P,, Ce,= 6

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FIG. 6. Substratum dependency of laminin effects on retinal cells. "SI-labelled laminin was used to coat either untreated or polyornithine-treated tissue culture plastic dishes. (a) Effects of PORN-bound laminin on cell attachment ( e ) and neurite development (©). PORN-bound laminin did not modify to any large extent the total number of cells bound to the substratum, but had a dose-dependent stimulatory effect on neurite formation by retinal neurons. (b) Effects of tissue culture plastic-bound laminin on cell attachment ( e ) and neurite development (©). Plasticbound laminin showed a dose-dependent inhibitory effect on the attachment of retina cells to the substratum. No neurite formation was observed in these cultures. (c) The amount of radioactive laminin bound to the dishes was similar on both polyornithine-treated ( I ) or untreated (D) tissue culture plastic dishes. From Adler et al., 1985, with permission from the publisher.

11

- - a phenomenon which normally occurs in lower animals such as amphibians and fish, but fails in birds and mammals. 3.2.2. CELL DIVERSITY IN RETINAL CULTURES

Is neurite development controlled exclusively by microenvironmental factors such as those described in the preceding section, or is it also regulated by intracellular determinants? This second alternative is supported by the finding that, while many of the cultured retinal cells develop neurites in a permissive environment, not all the process-bearing neurons produce the same pattern of neurite outgrowth. Quantitative analysis has shown that neurons with different numbers of neurites occur in the cultures with different frequencies (Hyndman and Adler, 1982c; and Fig. 7). These findings suggest that, although microenvironmental factors can determine whether retinal neurons will grow neurites, different retinal neurons seem to be predetermined to produce a specific number of nerve fibers. Predetermination is also apparent in the fact that, even when the cultures are grown under conditions that stimulate neurite development, only s o m e of the neurons respond with the production of very long neurites (See Fig. 7). A similar selectivity was detected in a study of the responses of retinal neurons to extracts from the optic lobe, the organ normally innervated by retinal ganglion cells (Hyndman and Adler, 1982c). These extracts determined the appearance in the cultures of some cells characterized by an extremely long neurite, in the absence of detectable changes in overall neurite development in the cultures. In summary, then, the starting cell population gives rise to a highly heterogenous (but predictable) set of neuronal phenotypes, even when developing in an apparently homogeneous microenvironment. The logical conclusion suggested by these observations (i.e. that cells are predetermined to follow a specific pattern of development) can only be considered tentative in the absence of clear correlations between each particular phenotype observed in vitro, and the different classes of neurons known to exist in the retina in viva. The next two sections will address this problem.

12

R. ADLER

",

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i FIG. 7. Flat cell-free, clump-free, low density monolayer culture from eight-day chick embryo retina neurons grown on a very/tdhesive substratum (polyornithine) coated with a Schwanoma conditioned medium which contains the neurite promoting factor ' P N P F ' . Besides emphasizing the absence of glial cells from the cultures, the photomicrograph shows the diversity of neuronal phenotypes present in the cultures. Note the presence of one neuron which gives rise to a very long neurite (arrow), which is m a n y times longer than the neurites from other neurons• Magnification bar = 20/am. Reproduced from Adler, 1982, with permission from the publisher•

4. THE PROBLEM OF CELL I D E N T I F I C A T I O N IN R E T I N A L CULTURES Accurate cell identification is not always easy in the in vivo situation, and is even more difficult in

cell culture where normal tissue architecture is absent. The state of the art of cell identification in vitro can be summarized in the following two principles (rev. in Adler, 1986b): (i) cell morphology is a necessary, but not sufficient criterion for unambiguous cell identification; and (ii) the presence or absence of a molecular 'marker' is seldom conclusive in the absence of other typical phenotypic features. The two principles emphasize the need to base cell identification on the investigation of as many components of each specific phenotype as possible. This comprehensive approach excludes old fashioned attempts to classify cells on the basis of subjective morphological comparisons, as well as more contemporary procedures based exclusively on the investigation of a single 'marker' such as the binding of a monoclonal antibody. Fortunately, there are now many procedures that can be directly applied to low density monolayer cultures and are useful for the identification and analysis of retinal neurons and photoreceptors, including: (i) immunocytochemical detection of cell surface antigens, (i.e. Barnstable et al., 1983; Lemmon and Gottlieb, 1982); (ii) cytochemical investigation of lectin receptors (Blanks and Johnson, 1983, 1984; Bee, 1982; Adler et al., 1984); (iii) autoradiographic and immunocytochemical analysis of neurotransmitter-related cellular machineries, including synthetic enzymes and uptake mechanisms (Adler, 1983; dePomerai and Cart, 1982; dePomerai et al., 1983; Hyndman and Adler, 1982b,c; Pessin and Adler, 1985); (iv) neuronal 'backfilling' with fluorescent dyes or horseradish peroxidase injected in target territories and retrogradely transported to the neuronal soma (cf. Armson and Bennett, 1983; Sarthy et al., 1983); (v) identification by immunocytochemistry of cellspecific products, such as opsin in photoreceptors (Adler, 1985, 1986c); and (vi) techniques for morphological analysis, including phase contrast, Nomarski and scanning and transmission electron microscopy. While retinal neurons can be adequately identified as such with most of these techniques, it is much more difficult to recognize in culture neuronal subpopulations such as amacrine, horizontal or bipolar cells. In contrast, photoreceptor cells show unique features when

13

RETINAL PHOTORECEPTORS AND NEURONS

studied with most o f these analytical methods, and, as discussed in the next section, can be readily identified from other neuronal cells.

(a)

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5. STUDIES W I T H C U L T U R E D P H O T O R E C E P T O R CELLS Photoreceptor cells were not apparent in gliafree, retinal neuronal cultures such as those described in Section 3. More recently, conditions have been found that support the development of identifiable photoreceptors in similar cultures by using medium 199 supplemented with linoleic acid and fetal calf serum (Adler et ai., 1984). The cultured photoreceptors show an elongated, polarized configuration, with a single, short neurite emerging from the nucleus-containing cell body (Fig. 8). More distally there is an inner segment with a phase-dark accumulation o f organelles and a refractile vacuole resembling the 'lipid droplet' characteristic o f avian cones. These pigmented lipid droplets are not present in other cultured neurons. The identity of these cultured photoreceptors was corroborated by peanut lectin cytochemistry, by the immunocytochemical demonstration o f opsin, and by scanning and transmission electron microscopy (see Section 5.2). Methodological aspects of this work will be summarized first, before discussing in more detail the differentiation o f cultured photoreceptor cells.

5.1. Culture Methods for Photoreceptor Cells 5.1.1. SERUM-CONTAINING AND SERUM-FREE DEFINED MEDIA

As mentioned above, photoreceptor cells were first detected in cultures supported by serumsupplemented medium 199. Because o f its highly complex and variable composition, serum supplementation of culture media is likely to pose some difficulties for the interpretation of experimental results. Taking as a starting point available information about serum-free, chemically defined media (rev. Bottenstein and Sato, 1985), a systemic screening was undertaken to identify supplements that would substitute for

F1G. 8. Retinal cells grown under conditions which allow the expression of the photoreceptor phenotype (see text). (a) Phase contrast microscopy image of a cone photoreceptor (C) and two multipolar retinal neurons (N). Six days in vitro. (b) Enlarged phase-contrast microscopy image of a cone like cell iUustrating the characteristiclipid droplet (arrow). Six day culture. (c) Bright field microscopy image illustrating the pigmented lipid droplet (arrow). Sevenday culture. From Adler et al., 1984, with permission from the publishers.

14

R. A D L E R

serum to support growth of retinal neurons and photoreceptors (Lindsey and Adler, 1985; and manuscript in preparation). Pilot experiments showed that the N1 supplement (Bottenstein and Sato, 1979) which consists of insulin, selenium, putrescine, progesterone and transferrin failed to support the survival of retinal cells in serum-free medium 199. This was a surprising observation since previous work had shown that N1 did support the survival of retinal neurons when added to DME (Dulbecco's modified Eagle's medium). Pyruvate, which is present in DME and not in 199, appeared as a logical candidate to explain this difference. This metabolite is essential for the survival of various chick neuronal populations in serum-free media (Selak et al., 1985). Retinal cells appeared to have this same requirement, since they did survive when medium 199 was supplemented with pyruvic acid in addition to the N1 cocktail. Another interesting observation was that insulin was the only N1 ingredient that was essential for photoreceptor survival. Insulin supported the same number of photoreceptors as fetal calf serum when tested in the presence of medium 199, BSA, linoleic acid and pyruvate. Conversely, no photoreceptors were present when the medium contained the other four N1 ingredients in the absence of insulin. It must be emphasized that both pyruvate and insulin are necessary for photoreceptor survival. Transferrin and progesterone did not affect the number of photoreceptors in the cultures, although qualitative observations suggested that they had some beneficial effects. Both BSA and linoleic acid caused concentration-dependent increases in the number of photoreceptors detectable in the cultures when tested in medium 199 supplemented with pyruvate, insulin, transferrin and progesterone. It still remains to be investigated whether BSA effects are not due to other molecules frequently found as contaminants even in the most highly purified commercial preparations available. In summary, it is possible to grow photoreceptor-containing cultures for as long as 14 days in a chemically defined medium containing medium 199 supplemented with pyruvate, insulin, transferrin, progesterone, BSA and linoleic acid. Although photoreceptor survival

is similar in serum-free and serum-supplemented cultures, there are differences between these cultures regarding the expression of some features of the photoreceptor phenotype. For example, opsin immunoreactive materials, which normally appear in serum-supported photoreceptors, are not detectable in ceils grown in serum-free medium (Lindsey and Adler, 1986; and in preparation). Interestingly, opsin does appear in photoreceptors when the cultures are switched from serum-free to serum-containing medium after a few days in vitro. The nature of the serum substances regulating opsin development in photoreceptor cells is now under investigation.

5.1.2. G E N E R A T I O N O F S E P A R A T E , ENRICHED POPULATIONS OF P H O T O R E C E P T O R S AND NEURONS

The coexistence of photoreceptors and neurons within one same culture is advantageous for experiments comparing both cell types with techniques such as light and electron microscopy, immunocytochemistry, or autoradiography. On the other hand, the availability of separate populations of neurons or photoreceptors would be much more suitable for biochemical measurements carried out in homogenates or extracts of the cultures, which otherwise would reflect the sum of the properties of different cell types. Recent work has shown that it is possible to generate enriched populations of either photoreceptor cells or neurons. 5.1.2.1. Kainic acid (KA) and 13-bungarotoxin (BT) as tools f o r photoreceptor purification. Work from several laboratories has shown that selective cell destruction is a useful method for the generation of purified populations in vitro (rev. Adler, 1986b; Schaffner and Schnaar, 1983; Varon and Manthorpe, 1980). The selective toxicity of kainic acid (KA) and /3-bungarotoxin (BT) suggested their potential usefulness as tools for cell separation in retinal cultures. Through mechanisms not yet fully elucidated, kainic acid causes the selective destruction of most of the amacrine cells in the chick embryo retina, and, at higher concentrations, of some bipolar and horizontal neurons as well. Retinal cells not

RETINAL PHOTORECEPTORS AND NEURONS

affected by kainic acid include ganglion cells, Miiller cells and, of particular relevance in the context o f this review, photoreceptor cells (rev. by Morgan, 1983). Beta-bungarotoxin (BT) is another selective toxin which causes a concentration-dependent destruction of the ganglion cells and some amacrine neurons in the chick retina (Hirokawa, 1978; Rehm et al., 1982). The remaining retinal cells, including the photoreceptors, are not affected by this toxin. As shown in Fig. 9 and 10 both KA and BT are able to destroy substantial numbers of cultured retinal neurons without affecting the photoreceptor cells (Politi and Adler, 1985, 1986a). In vitro neuronotoxic effects are specific and concentration-dependent. A maximum degeneration of 60°7o o f the neurons present in the cultures is achieved with 2 mM KA, while no changes in photoreceptor numbers were observed at any of the concentrations tested (Fig. 9). BT-induced neuronal degeneration is more extensive (70°7o) and is achieved at lower concentrations (1 nM) than that caused by KA. On the other hand, BT

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also lacks detectable effects upon cultured photoreceptor ceils. Some neuronal sensitivity to KA and BT is already present in two-day cultures, and reaches a maximum towards the e n d . o f the first week in vitro. At least 8 hr of exposure are necessary to achieve a full response at this stage. The effects o f KA and BT are only marginally additive, suggesting that a same subpopulation of neurons (probably amacrine cells) is affected by both toxins through different mechanisms. However, the maximum number of neurons which can be destroyed by BT is approximately 10°70 higher than the maximum number affected by KA. It would be of interest to determine whether these neurons are retinal ganglion cells, the only cells known to be sensitive to BT and resistant to KA in the chick retina in vivo (Hirokawa, 1978; Morgan, 1983). While transmission electron microscopy shows no signs of damage in photoreceptor cells in cultures treated with either KA or BT, these toxins cause extensive losses of neuronal 'markers' such as choline acetyltransferase or the high-affinity uptake

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FIG. 9. Effects of kainic acid and fl-bungarotoxin on retinal neurons. The results are expressed as percent of the n u m b e r s present in control cultures (26,000 neurons, 26,000 process-free cells and 7000 photoreceptors). Left-side graph: kainic acid. Note that pbotoreceptor destruction is reached at approximately 2 mM and does not increase with concentrations as high as 4 mM. A slight increase in process-free round ceils at low kainic acid concentrations probably represents neurite loss, which appears as an early sign of neurotoxicity. Right-side graph:/J-bungarotoxin. Photoreceptor cells are also resistant to this toxin, as are the process-free cells. There is a concentration-dependent neuronal loss which reaches m a x i m u m values at approximately 1 - 2 riM. The maximal neuronal loss caused by/3-bungarotoxin was slightly (but consistently) higher than that caused by kainic acid. Reproduced from Politi et al., 1986a, with permission from the publisher.

16

R. ADLER

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(a)

FIG. 10. Generation of enriched populations of photoreceptor ceils in vitro. Chick embryo neural retina ceils cultured for seven days in glial-free monolayers. (a) Untreated cultures. Note the presence of multipolar neurons (short arrows), photoreceptors (long arrow) and process-free cells. (b) Cultured treated for 24 hr with 2 mM kainic acid. Note extensive neuronal degeneration. Photoreceptor cells appear unaffected, as are the process-free cells. (c) Culture treated for 24 hr with 1 nM/3-bungarotoxin, showing extensive neuronal degeneration while photoreceptors in process-free cells appear unaffected. (d) Higher magnification picture of a KA-treated culture illustrating the cytological changes shown by degenerating cells (arrows). A photoreceptor cell (PhR) appears unaffected by the treatment. Magnifications: (a) 375 x ; (b) 460 × ; (c) 410 x ; (d) 1100 ×. Reproduction from Politi and Adler, 1986a, with permission from the publisher.

RETINAL PHOTORECEPTORS AND NEURONS

mechanism for GABA. Other cellular activities known to occur in both neurons and photoreceptor cells (such as high-affinity uptakes for aspartate or glutamate) show much smaller decreases in the treated cultures. In summary, then, selective neuronal destruction with KA and/ or BT generates a highly enriched (though not yet pure) population of photoreceptors (Fig. 10), which apparently are not affected by the treatments. 5.1.2.2. Photoreceptor-free neuronal cultures: selective photoreceptor lysis by antiopsin antiserum. The capacity of some immunoglobulins to produce cell lysis when they bind to plasma membrane antigens in the presence of complement has been exploited to purify subpopulations of cultured cells. For example, highly homogeneous Schwann cell preparations have been obtained by antibody-mediated elimination of fibroblastic contaminants (Brockes et al., 1979). As described in more detail in Section 5.2, opsin immunoreactive materials are present in the plasma membrane of cultured photoreceptor cells. This observation led to the development of a method that allows generating photoreceptor-free, purified populations of retinal neurons by complement-mediated lysis using an antiopsin antiserum (Adler and Politi, 1986; Politi and Adler, 1986b). A concentration-dependent lysis of photoreceptor cells can be observed when sevenday cultures are exposed to antiopsin antiserum in the presence of complement. The treatment causes little change in the number of neurons present in the cultures, or in neuronal markers such as choline acetyltransferase or high affinity GABA uptake. Photoreceptor sensitivity to complement mediated lysis by antiopsin antibodies develops in vitro in parallel to the appearance of immunocytochemically-detectable opsin in these cells. Thus, practically no photoreceptors could be lysed in two-day cultures, and the percentage of sensitive photoreceptors increased with time in culture to reach a maximum by the end of the first week. 'Blocking' experiments in which the antiserum was preincubated with purified rhodopsin showed that cell lysis is actually mediated by antiopsin antibodies present in the serum. From an experimental point of view, these

17

photoreceptor-free cultures should offer a useful counterpart to the neuron-depleted photoreceptor preparations described in the preceding section. It should now be possible to compare biochemically the properties of mixed cultures (containing neurons and photoreceptors) with other cultures in which either photoreceptors are eliminated using antibodies, or the majority of the neurons are destroyed with kainic acid and fJbungarotoxin. The cultures also appear as a useful bioassay to investigate the presence of 'antineuron' and 'antiphotoreceptor' antibodies in serum samples from patients or experimental animals affected by retinal degenerations. The bioassay is fast ( 1 - 2 hr) and allows discriminating between activities affecting both cell types; immune and nonimmune toxic mechanisms can be distinguished by testing serum in the presence or absence of complement.

5.1.3. PHOTORECEPTOR-CONTAINING CULTURES AS AN EXPERIMENTAL SYSTEM

5.1.3.1. Control o f experimental conditions. In this, like in any other in vitro technique, the investigator has control of the composition of the culture microenvironment only until the moment when the cells are introduced in the system. Thereafter, the metabolic activities of the cells will almost always change the composition of culture media and substrata. It is very important to minimize and/or detect these 'self-conditioning' effects. Some features of the retinal cultures which are useful in this context include their low density, the absence of glial cells and the feasibility of using chemically defined media. Serum-free cultures, for example, allow the detection of metabolic requirements that would remain unrecognized under other circumstances. The absolute photoreceptor dependence on pyruvate and insulin, for example, was not recognized while the cells were grown in serum-containing medium. It is similarly advantageous to grow the cells on chemically defined substrata, rather than on the surface of non-neuronal cells. Intercellular contacts can also be regulated to some degree by altering the seeding density and the composition of the culture substratum to control clump

18

R. A D L E R

formation and neurite development. The absence of substantial cell proliferation is another useful feature in this context. Limited cell contacts do occur in these cultures, however, largely through cell migration and the growth of nerve fibers. This allows comparisons between cells developing in complete isolation and those that have contacts with other cells - - a comparison largely impossible in complex, fiat cell-containing, high density cultures. 5.1.3.2. Accessibility to analytical techniques. One of the most useful features of low-density retinal cultures is their amenability to m a n y different high-resolution analytical techniques (see Section 4), which can frequently be applied in sequence to a same cell. Thus, the maturation of a cell can be monitored by video recording and computer-assisted image analysis, allowing dynamic as well as quantitative descriptions of developmental events. The cell can then be double (or even triple) labeled using techniques such as autoradiography and immunocytochemistry that can be applied to the cultures without histological sectioning, and the sparsity of the cultures usually allows unambiguous localization of immunoreactive products or silver grains. Alternatively, the cultures can be readily examined by scanning electron microscopy, and are amenable to transmission EM (the latter technique is only practical for studies requiring a small number of samples). Photoreceptors and neurons are also amenable to biophysical techniques, including electrophysiology with intracellular electrodes or patch clamp methods.

5.2. Are Precursor Cells Predetermined to Differentiate as Photoreceptors?

The properties of these low-density cultures of retinal neurons and photoreceptors appear very well suited for the investigation of the differentiation of these cells. Ongoing studies of this process are summarized in this section.

5.2.1. T H E EIGHT-DAY EMBRYONIC RETINA AS A REFERENCE POINT

The chick embryo neural retina contains no

differentiated photoreceptors and relatively few recognizable neurons at eight days of development (rev. Grun, 1982). Many of the cells that will develop as neurons or photoreceptors at later stages are already post mitotic, but still maintain a neuroepithelial appearance at this stage. The neuroepithelial cells closer to the retinal pigment epithelium in an eight-day retina are the ones that will presumably differentiate as photoreceptors both in vivo and in the dissociated cell cultures described below.

5.2.2.

T H E EMERGENCE OF CELL DIVERSITY ill v i t r o

Dissociated eight-day embryonic retinal cells grown in vitro for only one hr appear as a morphologically homogeneous population of process-free, round cells [Fig. ll(a)]. This homogeneous appearance is in marked contrast with the heterogeneity detectable in cultures grown for a few additional days, which show a variety of multipolar neurons as well as cells that can be identified as photoreceptors [Fig. l l(b)]. Which are the determining factors that guide some of the cells present in early cultures to express a neuronal phenotype while others are directed to develop as photoreceptors? It is unlikely that the in vitro microenvironment is inducing these different developmental pathways, because cells can be seen to differentiate as neurons or photoreceptors in the absence of contacts with other cells. Given that the culture medium must be assumed to be homogeneous, it appears that the eight-day chick embryo retina contains some cells that are already predetermined to express either the photoreceptor or the neuronal phenotypes.

5.2.3. T H E P H O T O R E C E P T O R P H E N O T Y P E 'itl vitro'

It appears pertinent to compare photoreceptor cells developing in vivo and in vitro in some detail, including in the comparison the expression of cellspecific molecules as well as the acquisition of a characteristic pattern of structural organisation.

RETINAL PHOTORECEPTORSAND NEURONS

5.2.3.1. The polarized, compartmentalized photoreceptor phenotype. As their in vivo counterparts, cultured photoreceptors are highly polarized cells showing a series of subcellular c o m p a r t m e n t s organized in a defined apico-basal sequence along their longitudinal axis (Fig. 8, 1 1 - 1 5 ) . These c o m p a r t m e n t s include a small outer segment, an inner segment, the nucleuscontaining cell body and a very short neurite. The possible presence of a fifth c o m p a r t m e n t (a synaptic terminal) has not yet been documented. The development of this highly polarized phenotype involves changes in cell shape as well as the segregation of the nucleus f r o m the organelles that accumulate in the inner segment region (Fig. 13). There is no information about the molecular mechanisms determining these and other morphogenetic events, including the characteristic localization of the neurite and the outer segment at opposite ends of the cell. The polarization of cultured photoreceptors can also be recognized at the molecular level. As their in vivo counterparts (Blanks and Johnson, 1983,

1984), cultured cones show selective and polarized binding of peanut lectin, circumscribed to the region of the inner and outer segments (Adler et al., 1984). Opsin immunoreactive materials are also selectively expressed and asymmetrically distributed in cultured photoreceptors (Araki, 1984; Adler, 1985, 1986c), again reproducing the well known polarized distribution of this molecule in vivo (rev. Papermaster and Schneider, 1982; Besharse, 1986). Opsin is not yet present in the eight-day chick embryo retina (Araki, 1984; Adler and Lindsey, 1985), or during early phases of photoreceptor development in vitro. Cultured photoreceptors first show diffuse and 'spotty' immunostaining towards in vitro days 4 - 5 (Adler, 1986c; see Fig. 15). The distribution of immunoreactive materials seems to change with time: by the end of the first week in culture m a n y photoreceptors show a polarized staining pattern, with a negative cell body and a brightly stained outer segment process (Fig. 15). Similar patterns can be seen regardless of the permeation of the cells with detergents, suggesting that at least some

(a ) 4

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19

6

FIG. 11. Developmentof photoreceptors and neurons in vitro. Isolated eight-day chick embryo neuralretinas were dissociated and cultured as described in Section 5. (a) Cell-substratum attachment is complete 1 hr after seeding, when the cultures appear as a morphologically homogeneous population of process-free, round ceils. Phase contrast: 400 ×. (b) Similar culture grown for seven days. Some cells have retained a circular outline and failed to develop processes. Some 80070of the procdss-bearing cells show a typical multipolar neuronal phenotype (long arrow), and the remaining 20070differentiate as photoreceptors (short arrow). Phase contrast: 300×. (c) Photoreceptors are elongated, highly asymmetric, polarized cells. A single, characteristically short neurite emerges from one of the cell poles. The adjacent cell body is almost completelyoccupied by the nucleus (N). The organelle-rich inner segment region contains a conspicuous lipid droplet (arrow). Phase contrast: 700x. Reproduced from Adler, 1986, with permission from the publishers.

20

R. ADLER

FIG. 12. Scanning electron micrograph illustrating the morphology of a photoreceptor cell. Note the apical membranous expansion of the cone-like cell (arrowheads). Sometimes a stout process suggestive of an outer segment arises from the apical region (arrowhead in inset). Reproduced from Adler et al., 1984, with permission from the publisher.

opsin molecules are present (in a polarized fashion) in the plasma membrane. A similar asymmetry can be seen in the distribution of cytoskeletal elements such as filamentous actin (Fig. 15). Contact-mediated mechanisms which have been suggested to explain plasma membrane asymmetries in other cells (rev. Rodriguez Boulan, 1983) are not likely to be important in cultured photoreceptors lacking intercellular contacts and cell junctions. It is also unlikely that photoreceptor polarization in vitro could be determined exclusively by hypothetical environmental cues, since the culture environment appears homogeneous and neighboring neurons do not show similar polarization (see Figs 11, 15). Photoreceptor polarization occurs in vitro along a plane parallel to the bottom of the dish, suggesting that the culture substratum does not play a role similar to that suggested in the case of epithelial cells that become polarized perpendicularly to the bottom of the dish (Rodriguez Boulan, 1983). At the stages studied in our experiment, in summary, the in vitro microenvironment appears to play a permissive rather than a determining role regarding photoreceptor polarization. 5.2.3.2. Neurite development. In contrast to the multiple neurites shown by most neurons

(Hyndman and Adler, 1982c), cultured photoreceptors develop a single neurite which originates from the cell body region more distal with respect to the inner and outer segments. Cultured photoreceptors resemble their in vivo counterparts in this behavior as well as in the fact that their ,neurite also remains characteristically short. After three days in vitro, for example, the majority of the photoreceptor neurites measure between 15 and 30/~m, with a maximum of 45/~m, while 80070 of the neurites from other neurons measure between 60 and 270/~m (Adler, 1986c). Similar differences can be observed at later stages. Given that these measurements included only those neurites devoid of contacts with other cells, these observations suggest that intracellular determinants are responsible for at least some aspects of the specific pattern of neurite outgrowth shown by cultured photoreceptor cells and neurons. This is consistent with concepts proposed by Solomon (1984) on the bases of studies of neurite formation by neuroblastoma cells. The existence of intracellular determinants of neurite development does not exclude a complementary regulatory role for the microenvironment as demonstrated by the overall stimulation of neurite development by retinal cells

RETINAL PHOTORECEPTORS AND NEURONS

21

V

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FIG. 13. Transmission electron micrographs o f six-day cone photoreceptors illustrating the polarized position of the nuclei (N) and mitochondria (M) as well as the large vacuole (V) corresponding to the lipid droplet. Some cells (B) also contain golgi apparatus (G) and a parabolloid (P). Reproduced from Adler et al., 1984, with permission from the publishers.

22

R. ADLER

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FIG. 14. Transmission electron micrographs of a photoreceptor cell. A short, outer segment-like process [enlarged in (b) from box in (a)] contains abundant smooth tubulo-vesicular elements (tve) and some compacted m e m b r a n o u s profiles reminiscent of the discs present in photoreceptor outer segments in vivo. One of these discs appears continuous with the plasma m e m b r a n e (arrow). A connecting cilium cannot be seen in this section, but has been observed in other specimens. N = nucleus; L = lipid droplet. (a) 6000x ; (b) 69,000 x . Reproduced from Adler, 1986, with permission from the publisher.

observed with some extracellular matrix molecules (Adler, 1982; Adler et al., 1985; Manthorpe et al., 1983; Rogers et al., 1983; see Section 3). 5.2.3.3. Development o f inner and outer segments. Cultured photoreceptors also resemble their in vivo counterparts regarding the accumulation of organelles such as mitochondria, golgi apparatus and endoplasmic reticulum in a specialized inner segment region, which also contains a pigmented lipid droplet (Figs 8 and 13). They also develop a rudimentary outer segment, which can be observed with different techniques. This short, finger-shaped process can be seen in the apical region of the cells with both scanning (Fig. 12) and transmission electron microscopy (Fig. 14). The latter technique shows that this process contains tubulovesicular elements, as well as some compacted membranous elements reminiscent of outer segment discs (Fig. 14). Moreoever, light microscopical immunocytochemistry demonstrates an accumulation of opsin immunoreactive materials in the apical finger-shaped process (Fig. 15). Thus, three complementary sets of observations indicate that

this apical process represents an immature outer segment process. It is of interest that the development of the outer segment process does not seem to continue beyond the stage reached by the end of the first week in culture even when the cells are grown for an additional 14 days in vitro. This arrested outer segment growth could be due to the absence of some regulatory agent capable of stimulating its further maturation. Two likely candidates for this role are Vitamin A, as well as molecules derived from the pigment epithelium. Vitamin A is necessary for the synthesis of light-sensitive visual pigments, and also seems to have a 'trophic' role for photoreceptors, which undergo degenerative changes in its absence (rev. Bridges et al., 1983). The hypothetical role of pigment epithelium-derived materials stems from the known anatomical and functional interdependence between photoreceptors and pigment epithelium as well as from the finding that outer segment development becomes abortive in retinas deprived of pigment epithelium (Hollyfield and Witkovsky, 1974; see also McLoon and McLoon,

23

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FIG. 15. Cytochemical analysis of cultured photoreceptor cells. ( a ) - (g) Opsin immunocytochemistry. The cultures were fixed in 4% formaldehyde in phosphate buffered saline (PBS) for 45 min, rinsed, incubated overnight in 2% rabbit serum, and then incubated 30 min with 1:100 dilution of a sheep antibovine rhodopsin antiserum, and for 30 min with a 1:40 dilution of secondary antibody (rabbit antisheep lgG, labelled with flourescein isothiocyanate). In some cases the cultures were treated before antibody incubations with ethanol and 0.25% Triton X-100 in PBS to facilitate antibody penetration into the cells. No staining was observed with any of the following controls: (i) omission of the primary antibody; (ii) substitution of non-immune sheep serum for the primary antibody; and (iii) preincubation of antiopsin antibody with bovine rhopsin for 3 hr at 4°C. (a), (b) Retinal culture grown for four days; (a) phase microscopy; (b) fluorescence microscopy. The photoreceptor shows a diffuse pattern of immunoreactive materials in the cell body as well as the neurite. Ethanol-Triton X-pretreated culture (825 x ). (c) Five-day culture stained without prior permeation. A similar diffuse pattern in seen (105 x ). (d), (e) Culture grown for seven days; (d) phase contrast; (e) fluorescence microscopy. Note negative neurons (N), a positive photoreceptor (P), and the polarized distribution of opsin immunoreactivity (750 x ). (f), (g) Photoreceptor cells after seven days in vitro. The neurite and cell body appear negative. Immunoreactive materials accumulate in the region occupied by the outer segment process. Similar patterns could be seen in unpermeated (f) and permeated preparations (g) (1200 × ). (h) Paraformaldehyde-fixed, seven-day cultures were permeated as described above and incubated for 20 min with Rhodamine-labelled phalloidin (Molecular Probes, Oregon) diluted 1:10 with PBS. Phalloidin is known to bind selectively to filamentous actin. Bright fluorescence could be observed only in the region from which outer segment process can be seen to emerge (arrows) and in the neurite (700×). Reproduced from Adler, 1986c, with permission from the publisher.

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R. ADLER

1984). Cultured photoreceptors should be useful as a bioassay for the investigation of these putative regulatory factors.

6. CONCLUDING REMARKS

The magnitude of our ignorance regarding the molecular mechanisms controlling the differentiation of retinal neurons and photoreceptors continues to have humbling dimensions. There are, however, reasons for optimism with respect to the prospects for furthering our knowledge in this area. Many of the methodological problems limiting our capacity to investigate these mechanisms have been recently overcome. It is now possible to monitor dynamically the development of individual, identifiable cells, manipulate their microenvironment and analyze with powerful techniques their responses to putative regulatory agents. Questions about cell differentiation which were already recognized and lucidly formulated many years ago (i.e. Coulombre, 1965) can now be reinvestigated with new methods and new approaches. One of the main conclusions of the present monograph is that some aspects of photoreceptor differentiation are predetermined at early stages of embryonic development while others are apparently determined later on. Another conclusion is that microenvironmental factors play a critical role in controlling the development of many cellular properties - - including those for which the cell is already 'predetermined'. These are essentially reformulations of classic concepts in developmental biology that the reported cell culture studies allow to test for the specific case of retinal neurons and photoreceptors. These culture systems should serve as bioassays to search for, and eventually purify molecules acting as 'instructive' and/or 'permissive' factors in the development of retinal cells. They should also contribute to the analysis of the molecular mechanisms used by undifferentiated precursor cells to store and express the information that they acquire as they become programmed to follow specific developmental pathways. We have now

many of the tools necessary to exploit these possibilities.

Acknowledgements - - Research from the author's laboratory was supported by USPHS grants EY02854 and EY04859. Ruben Adler is a William and Mary Greeve International Research Scholar from Research to Prevent Blindness. The author is grateful to James D. Lindsey, Luis Politi, Steve Madreperla and Cindy Winter for their comments on the manuscript, and to Doris Golembieski for her patient and efficient secretarial help.

REFERENCES ADLER, R. (1982) Regulation of neurite growth in purified retina cultures. Effects of PNPF, a substratum-bound neurite-promoting factor. J. Neurosci. Res. 8: 165 - 177. ADLER, R. (1983) Taurine uptake by chick embryo retinal neurons and glial cells in purified culture. J. Neurosci. Res. 10: 3 6 9 - 379. ADLER, R. (1985) Photoreceptor morphogenesis and polarization in low density retinal cell cultures. A m . Soc. Cell Biol. 101: 222a. ADLER, R. (1986a) Trophic interactions and cell death in retina development and pathology. In: The Retina: A Model f o r Cell Biology, (Adv. cell. Neurobiol. Vol. 7) (R. Adler and D. Farber, eds) Academic Press, New York, Part 1, pp. 112 150. ADLER, R. (1986b) In vitro techniques for the investigation of trophic factors active on CNS neurons. In: Handbook o f Nervous System and Muscle Factors (J. R. PerezPolo, ed.) C.R.C. Press, Florida (in press). ADLER, R. (1986c) Developmental predetermination of the structural and molecular polarization of photoreceptor cells. Devl Biol. (in press). ADLER, R. and LlrqDSEV, J. D. (1985) Photoreceptor cells in vivo and in monolayer culture: development of opsinlike immunoreactivity. Invest. Ophthalmol. Vis. Sci. (Suppl.) 26: 335. ADLER, R. and POEm, L. (1986) Selective photoreceptor degeneration by antiopsin antisera: in vitro studies. Invest. Ophthalmol. Vis. Sci. (Suppl.) 27: 57. ADLER, R., TEITEEMAN, G. and StmuRo, A. M. (1976) Cell interactions and the regulation of cholinergic enzymes during neural differentiation "'in vRro". Devl Biol. 50: 48 - 58. ADLER, R., MANTHORPE,M. and VARON, S. (1979) Separation of neuronal and nonneuronal cells in monolayer cultures from chick embryo optic lobe. Devl Biol. 69: 424 - 435. ADLER, R., MANTHORPE, M., SKAPER, S. D. and VARON, S. (1981) Polyornit hine-attached neurite-promoting factors (PNPFs). Culture sources and responsive neurons. Brain Res. 206: 129- 144. ADLER, R., MAGISTRETTI, P. J., HYNDMAN, A. G. and SHOEMAKER, W. J. (1982) Purification and cytochemical identification of neuronal and nonneuronal cells in chick embryo retina cultures. Devl Neurosci. 5: 2 7 - 39.

RETINAL PHOTORECEPTORS AND NEURONS ADLER, R., MANTHORPE, M. and VARON, S. (1983) Lectin reactivity of PNPF, a polyornithine-binding neuritepromoting factor. Devl Brain Res. 6: 6 9 - 75. ADLER, R., LINDSEY, J. D. and ELSNER, C. L. (1984) Expression of cone-like properties by chick embryo neural retina cells in glial-free monolayer cultures. J. CelIBiol. 99: 1173- 1178. ADLER, R., JERDAN, J. and HEWITT, A. T. (1985) Responses of cultured retinal cells to substratum-bound laminin and other extracellular matrix molecules. Devl Biol. 112: 100-114. ARAKI, M. (1984) Immunocytochemical study on photoreceptor cell differentiation in the cultured retina of the chick. Devl Biol. 103: 313-318. ARAKI, M., IDE, C., SAITO, F. and SATO, F. (1982) Ultracytochemical study on in vitro differentiation of neural retina cells of chick embryos. Devl BioL 94: 51-61. ARAKI, M., SAITO, T., TAKEUCHI, Y. and KIMURA, H. (1984) Differentiation of monoamine accumulating neuron systems in cultured chick retina: an immunohistochemical and fluorescence histochemical study. Brain Res. 317: 229-237. ARMSON, P. F. and BENNETT, M. R. (1983) Neonatal retinal ganglion cell cultures of high purity: effect of superior colliculus on their survival. Neurosci. Lett. 38: 181 - 186. BARNSTABLE, C. J., AKAGAWA, K., HOFSTEIN, R. and HORN, J. P. (1983) Monoclonal antibodies that label discrete cell types in the mammalian nervous system. Cold Spring Harb. Syrup. quant. Biol. 48: 863- 876. BARR-NEA, L. and BARISHAK, R. Y. (1970) Tissue culture studies of the embryonal chick retina. Invest. Ophthalmol. 9: 447- 457. BEE, J. A. (1982) A cytochemical study of lectin receptors on isolated chick neural retina neurons in vitro. J. Cell Sci. 53: l - 20. BESHARSE, J. (1986) Photosensitive membrane turnover: differentiated membrane domains and cell-cell interaction. In: The Retina, A Model f o r Cell Biology Studies (R. Adler and D. Farber, eds) Part I, pp. 297- 352. Academic Press, Florida. BETZ, H. and MOLLER, U. (1982) Culture of chick embryo neural retina in serum-free medium. Expression of cholinergic proteins. Expl Cell Res. 138:297 - 302. BIGNAMI, A. and DAHL, D. (1979) The radial glia of Miiller in the rat retina and their response to injury. An immunoflourescence study with antibodies to glial fibrillary acidic (GFA) protein. Expl Eye Res. 28: 63 - 69. BLANKS, J. C. and JOHNSON, L. V. (1983) Selective lectin binding of the developing mouse retina. J. comp. Neurol. 221:31-41. BLANKS, J. C. and JOHNSON, L. V. (1984) Specific binding of peanut lectin to a class of retinal photoreceptor cells. Invest. Ophthalmol. Vis. Sci. 25: 546-557. BOTTENSTEIN, J. and SATO, G. (1979) Growth of a rat neuroblastoma cell line in serum-free supplemented medium. Proc. natn. Acad. Sci. U.S.A. 76: 514-517. BOTTENSTEIN, J. E. and SATO, G. (1985) Cell Culture in the Neurosciences. Plenum Press, New York. BRIDGES, C. D. B., FONG, S.-L., LIOU, G. I., ALVAREZ, R. A. and LANDERS, R. A. (1983) Transport, utilization and metabolism of visual cycle retinoids in the retina and pigment epithelium. Prog. Ret. Res. 2: 137- 162. PRR V O L 6 - C

25

BROCKES, J. P., FIELDS, K. L. and RAFF, M. C. (1979) Studies on cultured rat Schwann cells. I. Establishment of purified populations from cultures of peripheral nerve. Brain Res. 165: 105- 118. CASPER, D. S., TRELSTAD, R. L. and RE1E-LEHRER, L. (1982) Glutamate-induced cellular injury in isolated chick embryo retina: Miiller cell localization of initial effects. J. comp. Neurol. 209: 7 9 - 90. COULOMBRE, A. J. (1965) The eye. In: Organogenesis (H. Ursprung and R. DeHaan, eds) pp. 219-257. Holt, Rinehart and Winston, New York. COULOMBRE, J. L. and COULOMBRE,A. J. (1965) Regeneration of neural retina from the pigmented epithelium in the chick embryo. Devl Biol. 12: 7 9 - 92. CRISANTI-COMBES, P., PRIVAT, A., PESSAC, B. and CALOTHY, G. (1977) Differentiation of chick embryo neuroretina cells in monolayer cultures. An ultrastructural study: I. Seven-day retina. Cell Tiss. Res. 185: 159- 173. DAVIS, G. E., MANTHORPE, M., ENGVALL, E. and VARON, S. (1985) Isolation and characterization of rat Schwannoma neurite promoting factor: evidence that the factor contains laminin. J. Neurosci. 5:2662 - 2671. DEPOMERAI, D. I. and CARR, A. (1982) GABA and choline accumulation by cultures of chick embryo neuroretinal cells. Expl Eye Res. 34:553 - 563. DEPOMERAI, D. 1., KOTECHA, B., FLOR-HENRY, M., FULLICK, C., YOUNG, A. and GALI, M. A. (1983) Expression of differentiation markers by chick embryo neuroretinal cells in vivo and in culture. J. Embryol. exp. Morphol. 77:201 - 220. DUTT, K. and REIE-LEIHRER, L. (1981) Glutamine synthetase induction in chick embryo retina monolayers. Cell Biophys. 3 : 1 - 17. GALl, M. and DEPOMERAI, D. (1984) Differential effects of culture media on normal and foreign differentiation pathways followed by chick embryo neuroretinal cells in vitro. Differentiation 25: 238- 246. GRAFSTEIN, B. (1986) The retina as a regenerating organ. In: The Retina as a Model f o r Cell Biology Studies (R. Adler and D. B. Farber, eds) Part II, pp. 275-335. Academic Press, New York. GREMO, F., PORRU, S. and VERNADAKIS, A. (1984) Effects of corticosterone on chick embryonic retinal cells in culture. Brain Res. 317: 4 5 - 52. GRUN, G. (1982) The development of the vertebrate retina: a comparative study. Adv. Anat. Embryol. Cell Biok 78: 7 - 85. GUERINOT, F. and PESSA¢, B. (1979) Uptake of y-aminobutyric acid and glutamic acid decarboxylase activity in chick embryo neuroretinas in monolayer cultures. Brain Res. 162: 179- 183. HEWlTT, A. T. (1986) Extracellular matrix molecules: their importance in the structure and function of the retina. In: The Retina as a Model f o r Cell Biology Studies (R. Adler and D. B. Farber, eds) Part I1, pp. 170-214. Academic Press, New York. HILD, W. and CALLAS,G. (1967) The behavior of retinal tissue in vitro. Light and electron microscopic observations. Z. Zellforsch. 8 0 : 1 - 2 1 . HIROKAWA, N. (1978) Characterization of various nervous tissues of the chick embryo through responses to chronic application and immunocytochemistry of /3bungarotoxin. J. comp. Neurol. 180:'449-466. HOLLYFIELD, J. G. and WITKOVSKY, P. (1974) Pigmented retinal epithelium involvement in photoreceptor

26

R. ADLER

development and function. J. expl Zool. 189:357 - 378. HYNDMAN, A. G. and ADLER, R. (1982a)Analysis of glutamate uptake and m o n o s o d i u m glutamate toxicity in neural retina monolayer cultures. DevIBrain Res. 2:303 314. HYNDMAN, A. G. and ADLER, R. (1982b) G A B A uptake and release in purified neuronal and nonneuronal cultures from chick embryo retina. Devl Brain Res. 3:167 - 180. HYNDMAN, A. G. and ADLER, R. (1982c) Neural retina development in vitro. Effects of tissue extracts on survival and neuritic development in purified neuronal cultures. Devl Neurosci. 5: 4 0 - 5 3 . IrOH, Y. (1976) Enhancement of differentiation of lens and pigment cells by ascorbic acid in cultures of neural retinal cells of chick embryos. Devl Biol. 54:157 162. KAPLOWITZ,P. B. and MOSCONA,A. A. (1976a) Stimulation of DNA synthesis by ouabain and concanavalin A in cultures of embryonic neural retina cells. Cell Diff. 5: 109 119. KAPLOWITZ, P. B. and MOSCONA, A. A. (1976b) Lectinmediated stimulation of D N A synthesis in cultures of embryonic neural retina cells. Expl Cell Res. 100: 177 - 189. KOH, S. W. M., KYRITSIS, A. and CHADER, G. (1984) Interaction of neuropeptides and cultured glial (Mfiller) cells of the chick retina: elevation of intracellular cyclic A M P by vasoactive intestinal peptide and glucagon. J. Neurochem. 43: 1 9 9 - 203. LANDER, A. D., FUJII, D. K. and REICHARDT, L. F. (1985a) Laminin is associated with the "neurite outgrowth promoting factors" found in conditioned media. Proc. Natn. Acad. Sci. UIS.A. 82:2183 2187. LANDER, A. D., FuJII, D. K. and REICHERT, L. F. (1985b) Purification of a factor that promotes neurite outgrowth: isolation of laminin and associated molecules. J. Cell Biol. 101: 8 9 8 - 9 1 3 . LAVAIL, M. and HILD, W. (1971) Histotypic organization of the rat retina in vitro. Z. Zellforsch. 114: 5 5 7 - 5 7 9 . LEMMON, V. and GOTTLIEB, D. I. (1982) Monoclonal antibodies selective for the inner portion of the chick retina. J. Neurosci. 2: 5 3 1 - 535. LEMMON, V. and RIESER, G. (1983) The development and distribution of vimentin in the chick retina. Brain Res. 313:191 - 197. LI, H. P. and SHEFFIELD, J. B. (1984) Isolation and characterization of flat cells, a subpopulation of the embryonic chick retina. Tiss. Cell 16:843 857. LI, H. P. and SHEFFIELD, J. B. (1986a) Retinal flat cells are a substrate that facilitates retinal neuron growth and fiber formation. Invest. Ophthalmol. Vis. Sei. 27: 2 9 6 - 306. LI, H. P. and SHEFFIELD, J. B. (1986b) Retinal flat cells participate in the formation of fibers by retinal neuroblasts in vitro. Time lapse studies. Invest. Ophthalmol. Vis. Sci. 27: 3 0 7 - 3 1 5 . LINDSE¥, J. D. and ADLER, R. (1985) A chemically defined medium that support photoreceptor differentiation in vitro. Invest. Ophthalmol. Vis. Sci. 26: 335. LINDSEY, J. D. and ADLER, R. (1986) Comparative effects of serum-containing and serum-free media on the survival and maturation of retinal neurons and photoreceptors. Invest. Ophthalmol. Vis. Sci. (Suppl.) 27: 329. LINSER, P. and MOSCONA, A. A. (1983) Hormonal induction of glutamine synthetase in cultures of embryonic retina cells: requirement for neuron-gila cell contacts. Devl Biol. 96: 5 2 9 - 5 3 4 . MANTHORPE, M., ENGVALL, E., RUOSLAHTI, E., LONGO, F.

M., DAVIS, G. E. and VARON, S. (1983) Laminin promotes neuritic regeneration from cultured peripheral and central neurons. J. Cell Biol. 97: 1882- 1890. McLooN, S. C. and McLooN, L. K. (1984) Transplantation of the developing m a m m a l i a n visual system. In: Neural Transplants (J. R. Sladek, Jr. and D. M. Gash, eds) pp. 9 9 - 124. Plenum Press, New York. MIRSKY, R., WENDON, L. M. B., BLACK, P., STOLKIN, C. and BRAY, D. (1978) Tetanus toxin: a cell surface maker for neurones in culture. Brain Res. 148: 2 5 1 - 259. MORGAN, 1. G. (1983) Kainic acid as a tool in retinal research. Prog. Ret. Res. 2: 2 4 9 - 266. MORRIS, J. E. and TING, J.-P. (1981) Comparison of proteoglycans extracted by saline and guanidinium chloride from cultured chick retinas. J. Neurochem. 37: 1 5 9 4 - 1602. MORRIS, J. E., HOPWOOD, J. J. and DORFMAN, A. (1977) Biosynthesis of glycosaminoglycans in the developing retina. Devl Biol. 58:313 327. MOSCONA, A. A. (1965) Recombination of dissociated cells and the development of cell aggregates, in: Cells and Tissues in Culture (E. Wilmer, ed.) pp. 4 8 9 - 5 2 9 . Academic Press, New York. MOSCONA, A. A. and LINSER, P. J. (1983) Developmental and experimental changes in retinal gila cells: cell interactions and control of phenotype expression and stability. Curr. Top. devl Biol. 18: 1 5 5 - 1 8 8 . MOYER, M. and SHEFFIEI.D, J. B. (1985) The emergence of flat cells in stationary cultures of embryonic chick neural retina. J. Cell Biol. 101: 223a. OKADA, T. S. (1980) Cellular metaplasia or transdifferentiation as a model for retinal cell differentiation. Curr. Top. devl Biol. 16: 3 4 9 - 380. PAPERMASTER, D. S. and SCHNEIDER, B. G. (1982) Biosynthesis and morphogenesis of outer segment membranes in vertebrate photoreceptor cells. In: Cell Biology o f the Eye (D. McDevitt, ed.) pp. 4 7 5 - 5 3 1 . PESSIN, M. and ADLER, R. (1985) Coexistence of high affinity uptake mechanisms for putative neurotransmitter molecules in chick embryo retinal neurons in purified culture. J. Neurosci. Res. 14: 3 1 7 - 3 2 8 . POLITl, L. E. and ADLER, R. (1985) Generation of enriched populations of cultured photoreceptor cells by selective neuronal destruction. Soc. Neurosci. l l : 628. POLITI, L. E. and ADLER, R. (1986a) Generation of enriched populations of cultured photoreceptor cells. Invest. Ophthalmol. Vis. Sci. 27:656 665. POILTI, L. E. and ADLER, R. (1986b) Selective destruction of photoreceptor cells by antiopsin antibodies. Invest. Ophthalmol. Vis. Sei. (in press). PRITCHARD, D. J. (1983) The effects of light on transdifferentiation and survival of chicken neural retina cells. Expl Eye Res. 37:315 - 3 2 6 . PRITCHARD, D. J., CI.AYTON, R. M. and DEPOMERAI, D. 1. (1978) Transdifferentiation of chicken neural retina into lens and pigment epithelium in culture: controlling influences. J. Embryol. exp. Morphol. 48:1 21. PURO, D. G., DEMELLO, F.G. and NIRENBERG, M. (1977) Synapse turnover and termination of transient synapses. Proc. natn. Acad. Sci. U.S.A. 74: 4 9 7 7 - 4 9 8 1 . REDFERN, N., ISRAEt., P., BERGSMA, D., ROBISON, W. G., WHIKEHART, D. and CHADER, G. (1976) Neural retina and pigment epithelial cells in culture: patterns of differentiation and effects of prostaglandins and cyclicA M P on pigmentation. Expl Eye Res. 22:559 568.

RETINAL PHOTORECEPTORS AND NEURONS REHM, H., SCHAFER, T. and BETZ, H. (1982) Betabungarotoxin-induced cell-death of neurons in chick retina. Brain Res. 250: 309-319. RODRIGUEZ BOULAN, E. (1983) Membrane biogenesis, enveloped RNA viruses, and epithelial polarity. Mud. Cell Biol. 1 : 1 1 9 - 170. ROGERS, S. L., LETOURNEAU,P. C., PALM, S. L., MCCARTHY, J. and FURCHT, L. T. (1983) Neurite extension by peripheral and central nervous system neurons in response to substratum-bound fibronectin and laminin. Devl Biol. 98:212 - 220. SARTHY, P. V., CURTIS, B. M. and CATTERALL, W. A. (1984) Retrograde labeling, enrichment, and characterization of retinal ganglion cells from the neonatal rat. J. Neurosci. 3: 2532- 2544. SCHAFFNER, A. E. and SCHNAAR, R. L. (1983) The isolation and purification of neurons from the vertebrate central nervous system, ln: Current Methods in Cellular Neurobiology (J. Barker and J. McKeivy, eds) Wiley, New York. SELAK, I., SKAPER, S. D. and VARON, S. (1985) Pyruvate participation in the low molecular weight trophic activity for CNS neurons in gila-conditioned media. J. Neurosci. 5: 2 3 - 28. SHEFFIELD, J. B. and MOSCONA, A. A. (1970) Electron microscopic analysis of aggregation of embryonic cells: the structure and differentiation of aggregates of neural

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retinal cells. Devl Biol. 23:36 - 61. SKAPER, S. D., ADLER, R. and VARON, S. (1979) Generalized procedure for purifying neuron-like cells in cultures from central nervous tissues with a defined medium. Devl Neurosci. 2: 233- 237. SMITH, G. N., LINSENMEYER,T. F. and NEWSOME,D. A. (1976) Synthesis of the type II collagen in vitro by embryonic chick neural retina tissue. Proc. natn. Acad. Sci. U.S.A. 73:4420 - 4423. SOLOMON, F. (1984) Determinants of neuronal form. Trends Neurosci. 7:17 - 20. STEFANELLI, A. A., ZACCHEI, A. M., CARSVITA, S., CATALDI, A. and TERALDI, L. A. (1967) New forming retinal synapses in vitro. Experientia 23:199 - 200. STEINBERG, M. S. (1963) Reconstitution of tissues by dissociated cells. Science 141: 401- 408. TAMAI, M., TAKAHASHI,J., NOHI, T. and MIZUNO, K. (1978) Development of photoreceptor cells in vitro: influence and phagocytic activity of homo- and heterogenic pigment epithelium. Expl Eye Res. 26: 581- 590. THOMPSON, J. M. and PELTO, D. J. (1982) Attachment, survival and neurite extension of chick embryo retinal neurons on various culture substrates. Devl Neurosci. 5: 447 - 457. VARON,S. and MANTHORPE,M. (1980) Separation of neurons and glial cells by affinity methods. Adv. Cell Neurobiol. 1: 405.