Exp Brain Res (1995) 105:59-66 �9 Springer-Verlag 1995

Magdalene J. Seiler �9 Robert B. Aramant

Transplantation of embryonic retinal donor cells labelled with BrdU or carrying a genetic marker to adult retina

Received: 10 November 1994 / Accepted: 15 February 1995

Abstract After transplantation of embryonic retinal cells to injured adult retina, it is often difficult to distin- guish donor from host cells. To overcome this problem, two methods were applied: labelling donor cells with the nuclear marker bromodeoxyuridine (BrdU) and use of transgenic donor tissue. BrdU was injected into timed- pregnant rats on 2 or 3 consecutive days. The donor em- bryos were taken 1 4 days later for transplantation. The BrdU-labelled donor tissue was examined in transplants sampled up to 1 year after grafting. Labelled donor cells were specifically identified in the transplants and in the interface with the adjacent host retina. The varying inten- sities of cell labelling indicated differences in the initial uptake of BrdU in the S-phase, or the dilution of the la- bel by cell divisions after BrdU injection. The best la- belled cells were presumably the ones that stopped divid- ing shortly after injection of BrdU. As controls, the nor- mal development of BrdU-labelled retinas from the off- spring of females that had been BrdU-injected at E l6 and E l7 and not used for transplantation was studied. Near the time of birth, clones of labelled cells were radi- ally distributed. In the mature retina, labelled cells were seen in all retinal layers. Embryonic retina derived from transgenic (NSE-lacZ) mice was transplanted to 'nude' , immunodeficient rats (xenografts). These transgenic mice contain the Escherichia coli [~-galactosidase gene, coupled to the promoter for neuron-specific enolase (NSE). Thus, all retinal donor cells that contain NSE could be identified by histochemistry or immunohisto- chemistry. The donor cells expressing the transgene could be detected several months after transplantation.

Key words Retinal transplantation �9 Donor cell label - E. coli [3-galactosidase �9 Bromodeoxyuridine �9 Rat - Mouse

M. J. Seller (~ ) �9 R. B. Aramant Department of Ophthalmology and Visual Sciences and Department of Anatomical Sciences and Neurobiology, University of Louisville Medical School, Louisville, KY 40292, USA; Tel. no: +1-502-852-7442, Fax no.: +1-502-852-0128

Introduction

Our experimental model consists of transplanting em- bryonic retina to injured adult host retina (review in Ar- amant et al. 1991; Seiler et al. 1991; Aramant and Seiler 1993). The immature neuroblastic donor cells differenti- ate into the different cell types and layers found in nor- mal retina (Aramant et al. 1990a; Seiler and Aramant 1994). The transplants can integrate with the host retina without the formation of a glial barrier (Seiler and Turn- er 1988; Aramant and Seller 1994). Distinguishing graft from host cells is often difficult, and it is therefore nec- essary to find a reliable marker for the transplanted cells. Preincubation of donor cells with a cytoplasmic label before transplantation is not reliable because, if some of the transplant cells degenerate, the label can diffuse out and be taken up by host cells (Aramant and Seiler, unpublished observations). To overcome this problem, we have applied two strategies: (1) transplan- tation of bromodeoxyuridine-labelled cells (Miller and Nowakowski 1988; Sieradzan and Vrbovfi 1989), and (2) transplantation of transgenic mouse cells, carrying the Escherichia coli ~-galactosidase gene coupled to the promoter for neuron-specific enolase (Forss-Petter et al. 1990). This work has been presented previously in ab- stract form (Aramant and Seiler 1992; Seiler and Aram- ant 1992).

Material and methods

Animals

As embryo donors, a total of nine timed-pregnant Long-Evans rats and two timed-pregnant NSE-lacZ mice were used. Thirty-four fe- male Long-Evans and eight homozygous athymic (nu/nu) rats were used as host animals.

Bromodeoxyuridine labelling of rat embryos

Four timed-pregnant rats were obtained from Harlan Spragne- Dawley, Indianapolis. Bromodeoxyuridine (BrdU; 20-30 mg/ml)

60

was dissolved in 0.07 N NaOH by sonication and sterile filtered. Three of the timed-pregnant rats were injected intraperitoneally with BrdU at a dose of 40 mg/kg on embryonic days 16 and 17 (El6 and El7) (vaginal plug=E0). From two of the BrdU-injected rats, El8 or E21 donor retinas were dissected and used for trans- plantation. As controls, the third BrdU-injected pregnant rat and the fourth unlabelled pregnant rat were allowed to bear and raise their pups. Pups were killed at El8, the day of birth (P1), P6, P14, P21, and P74. BrdU-labelled pups were smaller than unlabelled control pups; however, the BrdU-labelled litter consisted of 13 and the control litter of six pups.

Later, five more timed-pregnant rats were labelled with BrdU and used as donors for transplantation. Two of these rats were in- jected with BrdU (40 mg/kg) at El4 and El5, and their embryos were used for transplantation at El6. Two other rats were injected at El3, 14, and 15. One of these two rats absorbed the fetuses after the second injection; the embryos of the other rat were used for transplantation at El5. The fifth rat was injected with BrdU at El7, and the fetuses taken for transplantation 1 h later.

Host animals for BrdU-labelled transplants

In total, 34 female Long-Evans host rats (weight at surgery 130-241 g, average 181_+27 g) were used. Four host rats received El8 retinal transplants (BrdU-treated at E16, 17) in one eye. Ten rats received E21 transplants (BrdU at El6, 17) in both eyes. Ten rats received El6 transplants (BrdU at El4, 15) in both eyes. Four rats received El5 transplants (BrdU at E13,14,15) in one eye. Six rats received El7 transplants (BrdU at El7) in both eyes.

NSE-lacZ mouse donor tissue

A breeding colony of transgenic NSE-lacZ mice was obtained from Drs. Sutcliffe and Forss-Petter, La Jolla, Calif. These mice contain the E. coli [~-galactosidase gene, coupled to the neuron- specific enolase (NSE) promoter (Forss-Petter et al. 1990). Timed- pregnant mice were obtained by placing males overnight together with females (one male+three females). Two timed-pregnant fe- males (gestational stage El3 and El8) were injected with a sodi- um pentobarbital overdose, and their uteri were removed immedi- ately and stored on ice in Dulbecco's phosphate-buffered saline (D-PBS, Gibco) containing 1 mM glucose.

Fig. l a - c Development of BrdU-labelled cells in normal retina. Different developmental stages of rat retina labelled by BrdU injec- tions at embryonic day (E) 16 and El7. All pictures are oriented with the inner (vitreous) side of the retina up and the outer (photore- ceptor) side down. a Central retina of a rat El8 embryo. Intensely labelled nuclei can be seen in the ganglion cell layer (GC) and in the outer neuroblastic layer (NB). Other faint punctate labelling is seen in between. Paraffin section (8 gm). IP Inner plexiform layer, b Cen- tral region of rat E21 retina. Most intensely labelled cells are found in the outer neuroblastic layer. Cells in the inner neuroblastic layer close to the inner plexiform layer and in the ganglion cell layer are more faintly stained. Note that the fine punctate labelled nuclei in between are arranged in columns. Paraffin section (8 gm). c Central region of rat postnatal day 21 (P21) rat retina. BrdU-immunoreactive nuclei can be seen in the ganglion cell layer, inner nuclear layer (IN) and outer nuclear layer (ON). The staining intensities vary from complete filling to fine punctate labelling. The best-labelled cells are presumably the ones that stopped dividing shortly after injection of BrdU. Paraffin section (8 gm). IL Inner limiting membrane, OP out- er plexiform layer, OL outer limiting membrane. Bars 50 gm

Host animals for transgenic mouse tissue

Eight homozygous nu/nu athymic rats were obtained from Harlan Sprague-Dawley, Indianapolis. Six animals received NSE-lacZ mouse embryonic day 18 (El8) donor retina. Their weight at sur- gery ranged from 160 to 181 g. The remaining two animals re- ceived NSE-lacZ El3 donor retina later, at weights of 265 and 273 g.

Dissection of donor tissue

Embryos were collected in D-PBS containing 1 mM glucose. Em- bryonic retinas were dissected free of surrounding tissue and stored in ice-cold D-PBS/glucose until used. In some cases, em- bryos were stored overnight in hibernation medium (Kawamoto and Barrett 1986).

Transplantation procedure

Lesions were created in the retinas of host animals by making a dorsal incision approximately 0.5 mm long in the eyeball through sclera, choroid, and retina, using a sterile 15 ~ microblade (George Tiemann, Hauppauge, N.Y.). This incision was closed with 10-0 sutures. The donor retina was taken up in custom-made glass nee-

61

Fig. 2a -d Development of BrdU-labelled cells in transplants, a Transplant (Z) of BrdU-labelled rat E 21 retina, 9 weeks after transplantation. Donor cells can be found both on top of the host retina (H), inside the host retina at the lesion site (arrows), and in the subretinal space (box). Paraffin section (8 gin). b Enlargement of a. Darkly and faintly labelled nuclei of donor cells. Note the close apposition of transplant and host cells, c Epiretinal trans- plant of BrdU-labelled rat E 21 retina, 4 months after transplanta- tion, at lesion site in host retina. Vibratome section (80 gm). d En- largement of c. Darkly and faintly labelled donor cell nuclei. Note that there is no glial barrier between host and transplant. Bars 50 btm

dies, thus breaking the tissue up into cell aggregates, and then in- jected into the vitreous through a smaller, self-closing incision, covering the lesion site of the host retina. The transplantation pro- cedure has been described in detail elsewhere (Aramant et al. 1990b).

Att animals were treated according to N[H and ARVO guide- lines. The surgical procedures with the nude rats were performed under aseptic conditions: the surgery room and operation micro- scopes were fogged with disinfectant, and instruments, solutions, surgery gowns, masks, gloves were sterilized. The animals were kept in microisolator cages and handled under a laminar flow

hood. The animals were weighed weekly to monitor their condi- tion. The Long-Evans rats were kept in standard cages.

Tissue processing

Long-Evans rats with BrdU-labelled rat transplants were killed 3 weeks, 2 months, 3 months, 4 months, 7 months, and 1 year after surgery. After injection with an overdose of sodium pentobarbital, most of the animals were perfused through the ascending aorta with saline at 37~ followed by ice-cold paraformaldehyde 4% in

62

0.1 N Na-phosphate buffer, pH 7,2. Forty-five of the eyecups con- taining grafts were fixed in paraformaldehyde 4%, embedded in gelatin and sectioned on a vibratome. Eight eyes were embedded in plastic. Five eyes were immersion fixed in Bouin's fixative (Sigma) and embedded in low-melting-point paraffin.

Athymic nude rats with NSE-lacZ mouse transplants were sac- rificed 6 and 13 weeks after transplantation. The animals were perfusion fixed as described above. Eyecups containing grafts were fixed in paraformaldehyde 4%, cryoprotected in phosphate- buffered saline (PBS; 0.05 M sodium phosphate buffer, 0.1 M NaC1, pH 7.2) with sucrose 30%, embedded in Tissue-Tek, and frozen on dry ice. Sections 8 gm thick were cut in a Hacker-Bright cryostat.

Immunohistochemistry for BrdU

Deparaffinized paraffin sections or vibratome sections of 2, 3, 4, and 7 months and 1 year-old transplants were preincubated for 30 min in 2 N HC1 at 30~ to expose the nuclear antigen accord- ing to Sieradzan and Vrbovfi (1989). The sections were incubated with PBS containing 1% BSA and 0.3% Triton X-100 for at least 20 rain, then blocked in 20% horse serum for 30 rain. The sections were incubated in 1:100-1:500 dilution of a monoclonal mouse antibody against BrdU (DAKO) from overnight to several days at 4~ A monoclonal BrdU antibody from another supplier did not give satisfactory staining. An Elite-ABC kit against mouse IgG (Vector Labs) was used for the detection of the primary antibody. To reduce nonspecific staining, the secondary antibody was dilut- ed in PBS with 1% BSA containing blocking serum 10-20%; the incubation time was 15-30 rain at room temperature. Incubation time in the ABC-complex was usually 30 min. The substrate solu- tion for the HRP reaction was either 3,3'-diaminobenzidine (Sig- ma) 0.03%, 0.01 M imidazole and H202 0.01% in 0.1 M Tris buff- er, pH 7.2, resulting in brown staining, or Vector-SG (Vector Labs), which gave a blue reaction product.

Histochemistry for E. coli [~-galactosidase

Cryostat sections with retinal transplants were incubated at 37~ for 22 h with a reaction mixture for E. coli ~-galactosidase (Sanes et al. 1986) consisting of 5 mM K3Fe(CN) 3, 5 mM K4Fe(CN)6, 1 mg/ml X-gal (Sigma), 2 mM MgC12, sodium deoxycholate 0.01% and NP-40 0.02% in PBS (0.1 N sodium phosphate buffer pH 7.5, 0.15 M NaC1), rinsed three times with DMSO 3% in PBS and coverslipped in 1:1 PBS:glycerol. The reaction product was turquoise blue.

Immunohistochemistry for E. coli ~-galactosidase

The immunohistochemical procedures for cryostat sections con- taining NSE-lacZ mouse transplants were similar to those de- scribed for BrdU-immunohistochemistry. No HC1 preincubation was used, and the detection kit for the primary antibody was the Vector Elite kit against rabbit IgG, with goat serum as the block- ing serum. The sections were incubated with a 1:20 000 dilution of the primary antibody (rabbit antiserum against E. coli ~-galac- tosidase; 5 Prime - 3 Prime, Boulder, Colo.).

Results

Development of BrdU-labelled cells in normal retina

The development of BrdU staining was studied in retinas of animals that had been labelled by injecting the mother with BrdU at El6 and El7. One day after the last injec-

tion of the tracer (El8), the embryonic retina contained many intensely labelled cells and, in addition, some punctate label (Fig. la). Three days later (at E21), short- ly before birth, the label was less intense, and the la- belled cells were arranged in radial columns (Fig. lb). The label was further diluted at P6 and P14. In the fully mature retina, at P 21, labelled nuclei were seen scat- tered in all retinal cell layers (Fig. lc). The retina of the P74 rat was not labelled.

Development of BrdU-labelled cells in transplants

BrdU-labelled donor cells in the transplants could be dis- tinctly identified at 9 weeks (Fig. 2a,b) and 4 months (Fig. 2c,d) after transplantation of BrdU-labelled rat E21 retina to Long-Evans rat host retina. Similar results were seen in the other transplants up to 1 year after surgery. The labelling of cell nuclei ranged from complete filling to faint and punctate. Labelled donor cells could be seen embedded in the host retina without intervening glial barriers (Fig. 2b,d).

Transplants of donor cells labelled at El4 and El5 (data not shown) showed on average fewer labelled nu- clei than transplants of donor cells labelled at El6 and E17. Transplants of donor cells labelled on three consec- utive days (El3, 14 and 15) were more intensely labelled than donor cells labelled on two consecutive days. How- ever, there were also variations in the proportion of la- belled to unlabelled ceils in transplants derived from do- nors that had been treated in the same way.

In most cases, nuclei of inner nuclear layer cells and of some photoreceptor cells (presumably cones) were la- belled. No effort was made to further characterize the identity of labelled cells.

X-gal histochemistry

In retinas of normal adult NSE-lacZ mice, the reaction product for E. coli ]3-galactosidase was seen as a tur- quoise blue punctate cytoplasmic label in the ganglion cell and inner nuclear layers, but not in photoreceptors (Fig. 3a). NSE-lacZ mouse El8 and El3 transplants at 6 weeks after transplantation to athymic, nude rat hosts (Fig. 3b-d) seemed to contain more reaction product than the normal NSE-lacZ mouse retina. In the trans- plants, label was also seen in the photoreceptor layers of rosettes (a rosette consists of photoreceptors and cells of other retinal layers around a central lumen defined by an outer limiting membrane) (Fig. 3b,e). No diffusion of la- bel into the host retina was seen. The label could also be detected in transplants 14 weeks after transplantation (not shown). The mouse transplants were well integrated in the nude rat host retina; punctate label could be seen in the host inner plexiform layer close to the transplants (Fig. 3d,e).

63

Fig. 3a-e X-gal histochemistry, a Section of normal retina of an adult NSE-lacZ mouse, reacted with X-gal. The blue punctate reac- tion product can be seen in the ganglion cell layer (GC) and the in- ner nuclear layer (IN). Cryostat section. IP Inner plexiform layer, OP outer plexiform layer, b Epiretinal transplant (7) of El8 NSE- lacZ mouse retina that has developed for 6 weeks in an athymic "nude" rat host retina (H) without immunosuppression. Specific punctate labelling can be seen throughout the transplant. However, some areas show little labelling. Cryostat section, c El8 NSE-lacZ mouse transplant in the subretinal space of a nude rat host retina. Cryostat section, d This epiretinal NSE-lacZ mouse transplant (El 3 donor) has developed for 6 weeks in an athymic "nude" rat host retina. Punctate X-gal label. Note the complete integration between transplant and host retina. Cryostat section, e Different transplant (El3 donor, 6 weeks survival after surgery). Heavy X-gal label in transplant rosette on the left. Note specific label (arrows) in the in- ner plexiform layer of the host retina, indicating fiber outgrowth from the transplant. Cryostat section. Bars 50 Bm

Immunoh i s tochemis t ry for K coli 13-galactosidase

Immunoh i s tochemis t ry was more sensit ive than the other h is tochemical procedure. In retinas of normal adult NSE- lacZ mice, E. coIi [~-galactosidase- immunoreact ivi ty was seen in the inner retinal layers (ganglion cell to outer p lex i fo rm layer) (Fig. 4a). Cells in the inner part o f the inner nuclear layer were most intensely stained. NSE- lacZ mouse retinal transplants were readily distinguished f rom unstained surrounding host tissue (Fig. 4b). When gli- al barriers were not apparent between host and graft, E. coli ~3-galactosidase-immunoreactive transplant cells and a few stained fibers could be seen inside the host retina, close to the graft (Fig. 4b). In contrast to the normal adult NSE lacZ retina, retinal transplants also contained immunoreact ivi ty for E. coli 13-galactosidase in the cytoplasm and inner seg- ments of some photoreceptors (Fig. 4c).

Discussion

The two methods presented (BrdU labell ing and trans- plants of NSE- IacZ t ransgenic donor tissue) can rel iably label neuronal donor tissue for t ransplantat ion without diffusion problems. However , neither labell ing me thod

64

Fig. 4a-e Immunohistochemistry for E. coli ~-galactosidase. a Normal retina of an adult NSE-lacZ mouse. E. coli ~-galactosi- dase-immunoreactive cells are seen in the inner retinal layers. The most intense label is seen close to the ganglion cell layer and in some cells in the inner nuclear layer (IN). Note unstained cells in the inner nuclear layer which are probably Mtiller glial cells. Cryostat section. IP Inner plexiform layer, OP outer plexiform layer, ON outer nuclear layer, b Interface between unstained nude rat host retina (it/) and E. coli ~-galactosidase-immunoreactive El8 NSE-lacZ mouse transplant (7). Arrows point to specifically labelled fibers in the host retina. Cryostat section, c Photoreceptor cells in the outer nuclear layer (ON) of a rosette in an NSE-lacZ mouse transplant are immunoreactive for E. coli ~-galactosidase. Note the labelled inner segments at the outer limiting membrane (OLM). Cryostat section, d This El3 NSE-lacZ mouse transplant has developed mostly in the subretinal space of a nude rat host ret- ina. Note the complete integration between host and transplant. Cryostat section, e Enlargement of d. Host cells immediately adja- cent to transplant cells, without glial barrier. Bars 50 gm

marks all donor cells. Therefore, unlabelled donor cells cannot be distinguished with certainty f rom host cells, but a labelled cell is unequivocally identified as a donor cell.

BrdU, like 3H-thymidine, is incorporated into the DNA, and its visualization by immunohistochemistry is much less t ime-consuming than that of 3H-thymidine by autoradiography. Since BrdU is a nuclear label, cell pro- cesses cannot be identified. However, only a limited number of donor cells can be labelled with BrdU be- cause (1) it is toxic for the embryos at higher concentra- tions than 40 mg/kg, (2) only cells that are synthesizing DNA during the S-phase take up the injected BrdU, and (3) since most donor cells continue to divide after trans- plantation, the labelling is diluted out with subsequent cell divisions. To obtain more complete labelling, BrdU could have been injected into the timed-pregnant rats several times a day. However, this disturbance to the rats by additional handling and the higher total dosage of BrdU would have increased the risk that the fetuses might be absorbed by the mother.

There may be differences in the amount of BrdU that is taken up by the embryos, especially in a large litter, depending on the distance from the injection site. One of the BrdU control rats that was killed at P74 showed no staining at all. This could not have been due to the time passed after labelling, because staining was found in

transplants up to 1 year after surgery. There were varia- tions in the proportion of labelled to unlabelled cells in transplants derived from donor embryos that had been treated the same way.

Therefore, BrdU is useful only to define the borders of a transplant, and not to identify every transplant cell. The different intensities of cell labelling indicate differ- ences either in the initial uptake of BrdU in the S-phase and/or in the dilution of the label by subsequent cell divi- sions. In any case, the best-labelled cells are the ones that stop dividing shortly after injection of BrdU. BrdU, like 3H-thymidine, has been used for determining cell birth dates (Miller and Nowakowski 1988; Soriano et al. 1993) and it can also be applied to study retinal develop- ment.

Despite its limitations, immunohistochemistry for BrdU can be combined with other immunohistochemical and tracing methods and is now used routinely by our laboratory.

Recombinant retroviruses with the E. coli ~3-galactosi- dase marker have been widely used for cell lineage stud- ies (Sanes et al. 1986; Turner and Cepko 1987; review in Cepko 1993) and recently also for neuronal tracing stud- ies at the light and electron microscopic level (Loewy et al. 1991). In addition, recombinant viruses have been used to create genetically transformed cells for grafting in the brain (for review, see Fisher and Gage 1993). This technique has been first characterized with E. coli ~-ga- lactosidase-marked viruses (Shimohama et al. 1989).

In contrast to BrdU, the NSE-lacZ transgene in our transgenic donor tissue is a cytoplasmic label. The ex- pression of the transgene in the transplants appeared to be upregulated, and could clearly be identified 14 weeks after transplantation. The transplant photoreceptors that express the transgene are probably cones, because cones are also NSE-immunoreactive (Seiler and Aramant 1994). When glial barriers were not apparent between host and graft, E. coli [3-galactosidase-immunoreactive transplant cells, and, in addition, a few stained fibers could be seen inside the host retina close to the graft. The cells could have been mixed with host tissue during transplantation. However, the presence of immunoreac- tive fibers in the host retina indicates outgrowth of donor cell processes. The transgene is not expressed by all do- nor cells. Therefore, many donor cells can not be differ- entiated from host cells on the base of staining.

Other retinal transplantation groups have used a dif- ferent transgenic mouse strain that expresses the trans- gene only in the photoreceptors (Gouras et al. 1991, 1992; Mosinger Ogilvie et al. 1994). However, the ex- pression of this transgene in the transplants decreases with time (Gouras et al. 1991, 1992).

In summary, we have applied two cell label methods for retinal transplantation that can unequivoca l ly identify donor cells. Both labelling methods have confirmed re- sults of our previous studies that embryonic transplants integrate with the host retina without glial barriers. In fu- ture studies, we will use these markers at the ultrastruc- tural level.

65

Acknowledgements The authors thank Ms. Ann Potter and Ms. Carla Porter for their excellent technical assistance and Drs. G. Sutcliffe and S. Forss-Petter, La Jolla, Calif., for providing us with the transgenic NSE-lacZ mouse strain. The editorial help by Dr. James B. Longley has been very valuable. The research was sup- ported by the Schepens Eye Research Institute, Boston; the Ken- tucky Lions Eye Foundation; the Vitreoretinal Research Founda- tion, Louisville; an unrestricted grant from Research To Prevent Blindness, Inc.; and NEI grant EY08519.

References

Aramant RB, Seiler MJ (1992) Retina-to-retina transplantation of embryonic donor cells labelled with BrdU or carrying a genet- ic marker. Extended abstract for IVth International symposium on neural transplantation. J Neural Transplant Plast 3:283- 284

Aramant RB, Seiler MJ (1993) Embryonic retinal cell transplanta- tion to adult retina. In: Christen Y, Doly M, Droy-Lefaix MT (eds) Retina, aging and transplantation. Proceedings of '~ Ophthalmologiques IPSEN". Elsevier, Paris, pp 31-42

Aramant RB, Seiler MJ (1994) Human embryonic retinal cell transplants to athymic immunodeficient rat hosts. Cell Trans- plant 3(6):461-474

Aramant RB, Seller MJ, Ehinger B, BergstriSm A, Adolph AR, Turner JE (1990a) Neuronal markers in rat retinal grafts. Dev Brain Res 53:47-61

Aramant RB, Seiler MJ, Ehinger B, Bergstr/Sm A, Gustavii B, Brundin P, Adolph AR (1990b) Transplantation of human em- bryonic retina to adult rat retina. Restor Neurol Neurosci 2:9-22

Aramant RB, Seiler MJ, Ehinger B, Bergstr6m A, Adolph AR, Gustavii B, Brundin P (1991) Transplanting embryonic retina to the retina of adult animals. In: Anderson RE, Hollyfield JG,. LaVail MM (eds) Retinal degenerations. CRC Press, Boca Ra- ton, pp 275-288

Cepko CL (1993) Retinal cell fate determination. Prog Retinal Res 12:1-12

Fisher LJ, Gage FH (1993) Grafting in the mammalian central ner- vous system. Physiol Rev 73:583-616

Forss-Petter S, Danielson PE, Catsicas S, Battenberg E, Price J, Nerenberg M, Sutcliffe JG (1990) Transgenic mice expressing [3-galactosidase in mature neurons under neuron-specific eno- lase promoter control. Neuron 5:187- 197

Gouras R Du J, Kjeldbye H, Kwun R, Lopez R, Zack DJ (1991) Transplanted photoreceptors identified in dystrophic mouse retina by a transgenic reporter gene. Invest Opthalmol Vis Sci 32:3167-3174

Gouras R Du J, Kjeldbye H, Yamamoto S, Zack DJ (1992) Recon- strnction of degenerate rd mouse retina by transplantation of transgenic mouse photoreceptors. Invest Ophthalmol Vis Sci 33:2579-2586

Kawamoto JC, Barrett JN (1986) Cryopreservation of primary neurons for tissue culture. Brain Res 384:84-93

Loewy AD, Bridgman PC, Mettenleiter TC (1991) ~-Galactosi- dase expressing recombinant pseudorabies virus for light and electron microscopic study of transneuronally labeled CNS neurons. Brain Res 555:346-352

Miller MW, Nowakowski RS (1988) Use of bromodesoxyuridine- immunohistochemistry to examine the proliferation, migration and time of origin of cells in the central nervous system. Brain Res 457:44-52

Mosinger Ogilvie, J, Landgraf M, Lett JM, Tenkova T, Wang X, Wu M, Silverman MS (1994) Synaptic connectivity of trans- planted photoreceptors in rd mice expressing the transgenic marker gene lacZ. Invest Ophthalmol Vis Sci [Suppl 35]:1523

Sanes JR, Rubenstein JLR, Nicolas J-F (1986) Use of a recombi- nant retrovirus to study post-implantation cell lineage in mouse embryos. EMBO J 5:3133-3142

Seiler MJ, Turner JE (1988) The activities of host and graft glial cells following retinal transplantation into the lesioned adult

66

rat eye: developmental expression of glial markers. Dev Brain Res 43:111-122

Seiler M, Aramant R (1992) Transplantation of embryonic donor retina, labelled with BrdU or carrying genetic non-diffusible markers, to adult retina. Abstract, 15th Annual Meeting of Eu- ropean Neuroscience Association, September 13-17 1992, Munich, Germany

Seiler MJ, Aramant RB (1994) Photoreceptor and glial markers in human embryonic retina and in human embryonic retinal transplants to rat retina. Dev Brain Res 80:81-95

Seiler MJ, Aramant RB, Ehinger B, Bergstr6m A, Adolph AR, Gustavii B, Brundin P (1991) Characteristics of embryonic retina transplanted to rat and rabbit retina. Neuroophthalmolo- gy 11:263-279

Shimohama S, Rosenberg MB, Fagan AM, Wolff JA, Short MR Braekefield XO, Friedmann T, Gage FH (1989) Grafting ge- netically modified cells into the rat brain: characteristics of E. coIi ~-galactosidase as a reporter gene. Mol Brain Res 5:271-278

Sieradzan K, Vrbovfi G (1989) Replacement of missing motoneu- rons by embryonic grafts in the rat spinal cord. Neuroscience 31:115-130

Soriano E, del Rio JA, Auladell C (1993) Characterization of the phenotype and birthdates of pyknotic dead cells in the nervous system by a combination of DNA staining and immunohisto- chemistry for 5'-bromodeoxyuridine and neural antigens. J Histochem Cytochem 41:819-827

Turner DL, Cepko CL (1987) A common progenitor for neurons and glia persists in rat retina late in development. Nature 328:131-136