early chick neuroretinal responses following direct exposure to methotrexate

Early Chick Neuroretinal Responses Following Direct Exposure to Methotrexate

EUGENE DANIELS AND KEITH L. MOORE Department of Anatomy, University of Manitoba, Winnipeg, Canada R3E OW3

ABSTRACT Explants of 4.5-day-old chick embryonic neuroretinas with mesenchyme were exposed to Methotrexate (MTX) in medium 199 with embryo extract. Proliferative responses of the cultured neuroretinas were followed radioauto- graphically by administration of "-thymidine to the cultures. The DNA synthetic, mitotic and pyknotic responses of the ventricular cells of the neuroretina were followed over a 16-hour period. The responses observed suggested that MTX caused a synchronization of the ventricular cells in the pre-mitotic phases with no direct inhibition of mitosis. Furthermore, prolonged exposure to MTX resulted in the accumulation of labeled pyknotic cells, indicating a decline in the regenerative capacity of the proliferative ventricular cells.

Studies on the mechanism of teratogenic action under induced folic acid-deficient conditions have largely been investigated in vivo (Johnson et al., '63; Johnson, '64; Jordan et al., '70; Wilson, '70; Berry, '71). In vivo studies are often complicated by systemic drug transformations making it difficult to evaluate particular target tissue responses. Malformations produced under induced folic acid-deficient conditions fre- quently involve the central nervous system. Other studies on the direct effects of folic acid antagonists mainly on mitosis have not been conclusive (Gelfant, '58; Jacobson, '64; Deysson, '68) and do not clarify the observations of disturbances in proliferation seen in the developing neural tube (Johnson et al., '63). Variations in the number of mitoses seen in histological sections of the developing neural tube could be in- directly caused by the experimental synchronization of DNA synthesis. Further- more, observations of increased numbers of mitoses need not necessarily indicate hyperplasia but either a wave of synchronized mitosis following synchronized DNA synthesis or direct mitotic arrest (stathmokinesis) . Arrest of cellular proliferation and also hyperplasia have been considered as possible initial neuroepithe-

J. MORPH., 150: 307-320.

lial responses in teratogenesis (Patten, '53; Hicks, '60; Fujita et al., '64).

The establishment of a ventricular zone in the pseudostratified neural tube contain- ing subzones of DNA synthesis, interkinesis and mitosis (Sauer, '35; Fujita et al., '64; Boulder Committee, '70) and also in the early neuroretina (Sidman, '61) facilitate studies on the direct effects of teratogenic agents on these phases of proliferation. Our studies have shown that the proliferative ventricular zone is maintained in cultures of early neworetinal explants. The explants cultured consist of a pseudostratified neuroretinal epithelium in association with the pigmented epithelium and mesenchyme. Structural events in the disorganization and reorganization of similar neuroretinal explants have been described in earlier in vitro studies (Daniels and Moore, '72).

In the present study the morphogenetic, DNA synthetic, mitotic and pyknotic responses of the neuroretinal epithelia were followed during the direct exposure to the folic acid antagonist methotrexate (MTX) in the presence of chick embryo extract. Cultured cells which accumulate at the G, - S phase of the cell cycle due to MTX inhibition can be released from this block by

307

308 EUGENE DANIELS AND KEITH L. MOORE

pulse labeling with tritiated thymidine (Jasinska et al., '70). The use of a thymidine rescue procedure has been informative in studying the responses of cells and tissues following inhibition of thymidine synthesis (Rueckert and Mueller, '60; Jasinska et al., '70; Williams, '70; Wilson, '70). Our objec- tive is to utilize the neuroretinal explants at the ventricular cell stage to study the direct effects of MTX on the proliferative responses (DNA synthesis and mitosis), and also to estimate the potential for proliferative recovery in the embryonic tissue. Detailed analyses were made of the pseudostratified neuroretinal epithelium, which showed improved structural maintenance with normal proliferative responses when cultured in association with the pigmented epithelium and associated mesenchyme. Observations were therefore also made on the morphological integrity of these associated tissues in culture.

MATERIALS AND METHODS

Neuroretinal tissues with associated mesenchyme were dissected from 4.5-day White Leghorn chick embryos in Hanks' balanced salt solution (HBSS). Estimates of embryonic age were based on Hamburger and Hamilton ('51) stage 25. Explants (1-2 mm2) were obtained from the equatorial region of the neuroretina and gently placed on Millipore filters (Gelman metri- cal; pore size, 0.45) with the aid of Pastew pipettes.

The explants on Millipore filters were cultured on Trowell-type metal platforms (described earlier by Daniels and Moore, '72). The culture medium consisted of medium 199 with glutamine (Gibco, New York) supplemented with 10% chick embryo extract (CEE101 prepared from live embryos of the same incubation age as the explants (Paul, '60). The control medium consisted of nine parts medium 199 and one part CEE10 (199+CEElO). No sera or antibiotics were used. Petri dishes contain- ing the buffered medium (pH 7.4-7.6) were kept in a humid incubator at 37°C. Isotonic sodium bicarbonate (1.4%) and CO, (5% in

air) were used to maintain the required pH range.

Explants on Millipore filters were pre- cultured in the medium for up to 0.5 hours before addition of MTX. This period of in vitro adaptation has been used to allow mitotic cells present in the explant to pass beyond the metaphase stage (Bullough, '54; Gelfant, '58). The MTX (Lederle, 4- amino-N 10-methyl pteroyl-glutamic acid) was dissolved in HBSS and millipore-fil- tered. The selection of a suitable concentration of MTX to use was partially based on the in vitro studies of Deysson ('681, who reported that a 3-day direct exposure of HeLa cells to MTX indicated that there was a mitodepressive effect at 7.5 x M solution and a lethal effect at M solution. In our preliminary studies explants were exposed to various concentrations of MTX (5.5 x M, 5.5 x M and 5.5 x lo-" M solutions for 1-12 hours in culture. The concentration selected for the detailed study was 5.5 x 1 0 - 4 M MTX, since the 5.5 x M solution was pro- gressively lethal (increased pyknosis, tissue disorganization) and the 10- M solution ap- peared not to affect the normal morpholo- gy of the cultured explants even after 12- hour exposure (Daniels, unpublished).

The selected concentration of MTX was added to experimental cultures as indicated in figure 1. The experiment was designed so that the neuroretinal responses could be followed over a 16-hour period of exposure to MTX. Thymidine rescue could be ascertained by following the readiness of the cells to incorporate the exogenous labeled thymidine (methylJH-thymidine, Amersham/Searle, 1 pC/ml., sp. act. 5 CilmM) following prolonged exposure to MTX. These techniques have been suc- cessfully employed in cell culture studies (Rueckert and Mueller, '60; Jasinska et al., '70).

Analytic studies with folic acid antagonists such as MTX are usually complicated by the unknown concentration of folic acid coenzymes present in experimental tissues (Grant, '60). Furthermore, the

EARLY NEURORETINAL RESPONSES 309

0 E D I I M 199

MEDILIM 199 + CEE 10

METHOTREXATE (0.25 m 1 / 5 m l WZDIUPI)

a TRITIATED THMIDINE (1 i.c/rnl)

6-10 EXPLANTS FIXED I B CARNOY 11 AT 4oc.

I

I I

1.0 2.0 3.0 4.0 12.0 16.0 h

Fig. 1 Schedule of exposure conditions for neuroepithelial explants. Control explants cultured si- multaneously in the absence of methotrexate were obtained for each of the above exposure conditions.

use of an embryo extract to promote proliferation may introduce unknown factors or influence the activation of the MTX in culture systems. Embryo extract was, however, used in this study to maintain proliferation and also since many in vitro studies with antifolates have been performed with embryo extracts (Kieler and Kieler, ’54; Jacobson, ’64). Explants cultured concurrently under identical conditions but exposed to medium 199 plus MTX without embryo extract are also recorded. These responses are not compared directly to those with embryo extract but provide an estimate of the effect or modification the embryo extract had on the explant responses to MTX.

Throughout the culture period, a number of responses were evaluated in explants fixed in Carnoy’s fluid at 4°C and prepared for histological study. The paraf- fin-embedded explants were cut at 3 p and prepared for histological study and radioautography. Sections for radioautography were prepared by dipping the slides in

Kodak NTB-2 emulsion (Kopriwa and Leblond, ’62). After an exposure period of five days at 4°C the sections were de- veloped, fixed and stained. Sections were routinely stained with toluidine blue 0 or Harris’ hematoxylin for radioautography. The percentage of cells in DNA synthesis (thymidine labeling index, LI), in mitosis (mitotic index, MI) and in stages of degen- eration (pyknotic index, PI) were micro- scopically counted with the aid of an ocu- lar grid and hand tally counter. Nuclei con- taining five grains or more were classified as being labeled. A minimum of 1,000 cells (1,000-2,000 cells) were counted under oil magnification ( x 1,000) in every fourth serial section. A total of 160 explants were cultured. Ninety-six explants were studied histologically to select the suitable dura- tion and concentration of MTX for the detailed study. Four explants were used to establish each mean and standard devia- tion shown in figure 2 except for the controls (199 plus CEE10) at 5 hours where eight explants were used. In an isolated control explant 1 x 10-5 gm/ml of the mitotic inhibitor colchicine was added to a single culture for one hour in order to confirm that a distinct mitotic zone forms in cultured neuroretinas (fig. 31.

RESULTS

In the neuroretinal epithelia analyzed the mitotic spindles, with few exceptions, were oriented parallel to the limiting membrane. This orientation ensured the normal cytokinesis and “elevator-like” movements of the ventricular cell nuclei as they pass through phases of their cycle. The occasional disoriented mitotic spindle observed was of the same incidence as in control explants and in vivo. The structural integrity of the cultured epithelia was not directly affected by the MTX since normal cytokinetic movements were well maintained, indicating intact pseudostratifica- tion of the neuroretina. During the later stages of exposure to MTX, the pseudo- stratification of the cultured neuroretina persisted even though there was distortion

3 10 EUGENE DANIELS AND KEITH L. MOORE

of the epithelium due to uneven cell death and exfoliation.

The neuroretinal epithelial responses to 5.5 x M MTX plus embryo extract during a 16-hour exposure period are sum- marized in figure 2. The labeling indices of all explants cultured showed a slight decline and rise during the 16-hour culture period. After the initial 5-hour exposure to MTX plus embryo extract, the mean labeling indices of the neuroretina were similar to control explants (53.42 4 1.4; 53.13 f 4.9). No significant differences (p < 0.05) were observed after six and seven hours of exposure to MTX plus embryo extract. However, following eight hours in the same media the experimental explants showed a significant (p < 0.05) rise in labeling index (50.18 2 2.7) compared to the control value of 40.0'7 * 2.6. Following 16 hours of exposure the labeling indices were ele- vated ('73.45 ? 13.5; 56.35 k 14.3) but not significantly different (p < 0.05). T h e mitotic indices in explants exposed to MTX plus embryo extract were lower but not significantly affected (p < 0.051. At no point during the 16-hour exposure period was the MI increased nor was there any accumulation of cells in any one particular mitotic stage suggestive of mitotic block. In control neuroretina cultured for eight and five hours (table 11, 92-98% of the mitotic figures observed were labeled following the 4-hour period of tritiated thymidine exposure. In contrast, the number of labeled mitosis in experimental neuroretina was considerably reduced (40.8-67.6% reduction).

Cell death in the control explants was minimal at five hours (PI = 0.2 k 0.0) and reached a maximum of 2.03 k 1.4 after 16 hours of culture. During the later exposure periods (8-16 hours) increasing numbers of labeled pyknotic cells were evident in the experimental neuroretina. Many of these cells were detached and in stages of exfoliation from the neuroretina (figs. 9, 11, 12). The distribution of the pyknotic cells amidst viable ventricular cells is clearly distinguished from the indiscriminate cell death of whole segments of neuroretina in

TABLE 1

Percentage labeled mitotic figures' seen after tritiated thymidine labeling in neuroretina

following exposure to methotrexate

199 + 199 + CEE .wrx only

Exposure 199 + CEE + Mrx

5 hours 98.0% 31,7% (67.6%) 23.7%

8 hours 92.0% 54.4% (40.8%) 52.0% Onc hundred mitotic figures classified.

2 Figures in parrntheses represent percentage reduction, with control value taken as 100%

highly folded explants (fig. 13) due to diffusional limitations. The few folded explants inadvertently cultured were not used in the final analysis.

Figure 2 also shows the neuroretinal epithelial responses observed when cultured in medium 199 plus MTX without embryo extract. The fluctuation and magnitude of the LI observed throughout the 16-hour culture period were similar to those observed in the presence of embryo extract. The mitotic indices were comparable in magnitude to those observed in the presence of embryo extract. Furthermore, the number of labeled mitoses observed after five and eight hours (table 1) was low (23.7-52.0%) and as such were comparable to responses observed in the presence of embryo extract. A marked difference in response was, however, seen in the viability of the experimental explants cultured in the absence of embryo extract. After 6 hours of culture a high pyknotic index was observed (16.0 f 3.7). This value was ap- proximately four times the maximum seen in explants cultured in the presence of embryo extract. After 16 hours, the pyknotic index was considerably lowered (4.3 ? 2.6).

DISCUSSION

The exposure to MTX followed by an in- terval in which thymidine is made availa- ble to tissues, allows the effects of transient or complete inhibition of DNA synthesis to be studied. This technique has been adapted in vivo and has proved useful in studying the repair processes during fetal

EARLY NEURORETINAL RESPONSES

Mean , 5

10 PI

5 -

31 1

- -

0--

T Control (199. CEE 10 1 199 + CEE 10 + Methotrexate 8or

70 I3 199+ Methotrexate

Mean 60-

//

L I 50-

40 - 30 -

20 r T

life (Berry, '71, '72). However, there has been no direct demonstration of the effects of the drug on the rapidly growing embryonic tissues. The sensitivity of target embryonic cells and their regenerative capacity have not been adequately described in the in vivo studies due to the difficulty in distinguishing primary from secondary in vivo responses.

Tabulation of the labeling, mitotic and pyknotic indices of the neuroretinal epithelial explants exposed to MTX in the presence of CEE allowed classification of the direct effects on DNA synthesis and on mitosis. The fluctuation of DNA labeling

characteristics seen in control explants throughout the 16-hour culture period could be attributed to the adaptation phase reported in explants in vitro (Biggers, '631. A similar fluctuation of DNA synthetic responses was observed in a standard- ization study of neuroepithelial responses in vitro (Daniels, '711, and in the "metabo- lic lab" phase which has been previously described for cultures of kidney rudiments (Saxen et al., '68). The observations of similar labeling indices in control and experimental neuroretinal epithelia together with the increase in LI at eight hours in experimental neuroretina can be interpreted


when considered together with the observations on the mitotic indices. The mitotic index in experimental explants did not ex- ceed that of controls and furthermore the percentage of labeled mitosis was considerably reduced. These observations indicate that although similar numbers of cells were synthesizing DNA, fewer numbers of these cells were progressing to mitosis. This suggests a synchronization of ceIls in the pre-mitotic phases seen in the embryonic tissues, and is therefore similar to the effect of MTX reported in other tissues of adult animals (Rueckert and Mueller, '60). This synchronizing effect on the ventricular cells of the neuroretina was present as early as five hours and was more pronounced at eight hours when an increase in LI was recorded.

The onset and distribution of cell death also allowed an estimation of the sensitivity of the embryonic cells throughout the exposure period. Although pyknotic indices were recorded for the neuroretina only, relatively few pyknotic cells were observed in the associated pigmented epithelium and mesenchyme. The associated mesenchyme was structurally well maintained throughout the culture period (figs. 5, 9, 12). It is noteworthy that the mesen- chymal component has been implicated in a number of in vivo studies as the primary site of alteration in various CNS malformations (Thiersch and Phillips, '50; Marin- Padilla, '66). The present in vitro study with MTX, using isolated embryonic tissues, indicates that considerable proliferative and degenerative changes were observed in the neuroretinal epithelium before minimal degenerative changes ap- peared in the associated mesenchyme. The neuroretinal epithelium with pyknotic cells showed conspicuous gaps (fig. 9) suggestive of rapid detachment and extrusion of de- generated cells. The surviving pseudostratified neuroretinal epithelium, though distorted by the uneven cell loss, showed cohorts of labeled ventricular cells sug- gesting that these cells could continue to synthesize DNA. The appearance of la-

beled pyknotic cells later (8-16 hours) in experimental cultures exposed to MTX indicates that the DNA synthesis was aborted and that there was rapid degen- eration during or immediately after incor- poration of the exogenous thymidine. Since labeled pyknotic cells were not seen during earlier culture periods (5, 6 and 7 hours), the capacity for rescued cells to survive and possibly repopulate the ventricular cell layer seems to decline with extended exposure to MTX. This observation in embryonic tissue is in accordance with the reported irreversible loss of proliferative capacity of HeLa cells cultured under thymidine-deficient conditions induced by MTX (Rueckert and Muellex-, '601. In vivo studies with other cytotoxic DNA synthesis inhibitors such as ARA-C, ARA-CP and hy- droxyurea (Ritter et al., '71, '73) have also suggested that prolonged inhibition of DNA synthesis results in more severe malformations, and that these were much more closely associated with the time of massive cell death rather than the time of drug administration. In our in vitro studies the direct responses to MTX by the ventricular cells were monitored without the complications of drug transference in- herent in the in vivo studies. The suscep- tibility of the DNA synthetic and post-synthetic zones of the neuroretinal epithelium (figs. 5, 9, 11, 12) was similar to that reported in neuroepithelia following X-irradiation of mice (Fujita et al., '64) and following radioactive colloidal gold injection into chick mesencephalon (Menkes et al., '70). Apart from interfering with the for- mation and regeneration of adequate cell numbers, synchronization of ventricular cells due to antifolates (or folic acid deficiency) could result in unbalanced growth of cohorts of ventricular cells thereby dis- turbing the normal timetable of ventricular cell proliferation and migration. Further- more, synchronization of ventricular cells in the developing pseudostratified neuroepithelium could conceivably distort critical periods in early morphogenesis in a manner analogous to the distortion and

EARLY NEURORETINAL RESPONSES 313

overgrowth of neuroepithelium due to accumulation of mitotic figures caused by vincristine block (Langman et al., ’66).

The magnitude and fluctuations of the LI and MI in explants cultured concurrently in medium 199 plus MTX only were comparable to those cultured under similar conditions but in the presence of embryo extract. The low number of labeled mitoses observed also suggested that a similar synchronization of proliferation occurred as in the presence of embryo extract. These observations show that though the inclusion of embryo extract in medium 199 is not es- sential for the initiation of DNA synthesis, it was associated with improved viability of explants exposed to MTX. Its presence therefore did not mask the primary effect of MTX on the synchronization of DNA synthesis in the embryonic cells.

An additional observation relevant to in vitro studies with early neuroepithelial explants is emphasis of the importance of limited epithelial thickness especially in studies using antifolates. A distinctive gra- dient of early moribund cells was seen only in folded explants exposed to MTX and was characteristically located distal from the medium and gas phases. These explants were not used in the above-mentioned analysis but suggest that under experimental conditions of nutritional and gaseous deprivation, as simulated in folded explants, the ventricular cells show a heightened sensitivity to MTX irrespective of the cell cycle status of the cells. Optimal conditions for the study of proliferative responses of ventricular cells were seen in the single layered early neuroretina plus associated mesenchyme.

In summary our studies have demonstrat- ed the use of a unique model for analysis of the direct effects of the antifolate MTX on embryonic ventricular cells of the central nervous system as seen in the early neuroretina. Our data indicate that the detectable direct effects of the drug are primarily on the pre-mitotic stages (S and G, phases). Extended exposure to MTX results in irreversible loss of proliferative ac-

tivity of the ventricular cells. Analysis of the neuroepithelial ventricular cells in vitro is ideally performed in early single layered neuroretina and is ideally maintained in the presence of embryo extract.

ACKNOWLEDGMENTS

The authors are thankful to Miss I. Krzywdzinski, Miss S . Smith, Mrs. B. Bell and Mr. G . Reid for technical assistance and to Dr. T. V. N. Persaud for critically reading the manuscript. Supported by the Medical Research Council of Canada (MA-3641) and by the Anatomical Re- search Fund of the University of Manitoba.

LITERATURE CITED Berry, C. L. 1971 Transient inhibition of DNA syn-

thesis by methotrexate, in the rat embryo and foetus. J. Embryol. Exp. Morph., 26: 469-474.

The effect of methotrexate on DNA synthesis and its reversal by folinic acid. J. Embryol. Exp. Morph., 28: 601-605.

Biggers, J. D. 1963 Studies on the development of embryonic cartilage long bone rudiments in vitro. Nat. Cancer Inst. Monogr. 11, pp. 1-21.

Boulder Committee, The 1970 Embryonic vertebrate central nervous system: revised terminology. Anat. Rec., 166: 257-262.

Bullough, W. S. 1954 A study of hormonal relations of epidermal mitotic activity in vitro. I. Technique. Exp. Cell Res., 7: 176-185.

Daniels, E. 1971 An In Vitro Teratological Analysis of Neuroretinal Explants with Methotrexate. Ph.D. Thesis, Faculty of Graduate Studies, University of Manitoba, Winnipeg, Canada.

Daniels, E., and K. L. Moore 1972 A direct analysis of e d y chick embryonic neuroepithelial responses following exposure to EDTA. Teratology, 6:

Devsson, G. 1968 Antimitotic substances. Int. Rev.

1972

2 15-226.

Cytol., 24: 99-143. Fuiita. S.. M. Horii. I. lanimura and H. Nishimura 1964 HJ-thymidine”&toradiographic studies on cytokinetic responses to X-ray irradiation and to Thio-TEPA in the neural tube of mouse embryos. Anat. Rec., 149: 37-48.

Gelfant, S. 1958 Aminopterin and nitrogen mustard as mitotic inhibitors in mouse ear epidermis in vitro. Exp. Cell. Res., 15: 451-453.

Grant, P. 1960 The influence of folic acid analogs on development and nucleic acid metabolism in Rana pipiens embryos. Deve. Biol., 2: 197-251.

Hamburger, W., and H. L. Hamilton 1951 A series of normal stages in the development of the chick embryo. J. Morph.. 88: 49-92.

Hicks, S. P. 1960 Acute necrosis and malformation


of developing mammalian brain caused by X-ray. Proc. SOC. Exp. Biol. Med., 75: 485-489.

Jacobson, W. 1964 Cellular injuries caused by folic acid antagonists and some corticosteroids. In: Ciba Foundation Symposium on Cellular Injury. A. V. S. De Reuck and J. Knight, eds. Churchill, London, pp.

Jasinska, J., J. S. Steffen and A. Michalowski 1970 Studies on in vitro lymphocyte proliferation in cultures synchronized by the inhibition of DNA synthesis. 11. Kinetics of the initiation of the proliferative response. Exp. Cell. Res., 61: 333-341.

Johnson, E. M. 1964 Effects of maternal folic acid deficiency on cytologic phenomena in the rat embryo. h a t . Rec., 149: 49-56.

Johnson, E. M., M. M. Nelson and I. W. Monie 1963 Effects of taansitory pteroylglutamic acid (PGA) deficiency on embryonic and placental development in the rat. h a t . Rec., 146: 215-225.

Jordan, R. L., J. F. Terapane and H. J. Schumacher 1970 Studies on the teratogenicity of methotrexate in rabbits. Teratology, 3: 203 (abstract).

Kieler, J., and E. Kieler 1954 The effect of amethopterin on sensitive and resistant leukemic cells in vitro. Cancer Res., 14: 428-452.

Kopriwa, B., and C. P. Leblond 1962 Improvements in the coating technique of radioautography. J. Histochem. Cytochem., 210: 269-284.

Langman, J., R. L. Guerrant and B. G. Freeman 1966 Behaviour of neuro-epithelial cells during closure of the neural tube. J. Comp. Neur., 127: 399-412.

Marin-Padilla, M. 1966 Mesodermal alterations induced by hypervitaminosis A. J. Embryol. Exp.

Menkes, B., S. Sander and A. Illies 1970 Cell death in

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teratogenesis. Adv. Terat., 4: 169-215.

Patten, B. M. 1953 Embryological stages in estab- lishing of myeloschisis with spina bifida. Am. J. Anat., 93: 365-395.

Paul, J. 1960 Cell and Tissue Culture. Second ed. Livingstone, Edinburgh.

Ritter, E. J., W. J. Scott and J. G. Wilson 1971 Terato- genesis and inhibition of DNA synthesis induced in rat embryos by cytosine arabinoside. Teratology, 4:

Relationship of temporal patterns of cell death and development to malformations in the rat limb. Possible mechanisms of teratogenesis with inhibitors of DNA synthesis. Teratology, 7:

Rueckert, R. R., and G . C. Mueller 1960 Studies on unbalanced growth in tissue culture. I. Induction and consequences of thymidine deficiency. Cancer Res., 20: 1584-1591.

Sauer, F. C. 1935 Mitosis in the neural tube. J. Comp. Neur., 62: 377-405.

Saxen, L., 0. Koskimies, A. Lahti, H. Miettinen, J . Rapola and J. Wartiovaara 1968 Differentiation of kidney mesenchyme in an experimental model system. Adv. Morph., 7: 251-288.

Sidman, R. L. 1961 Histogenesis of mouse retina studied with thymidine-H? In: The Structure of the Eye. G. K. Smelser, ed. Academic Press, New York,

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pp. 487-506. Thiersch, J. B., and F. S. Phillips 1950 Effects of 4-

aminopteroyl glutamic acid (aminopterin) on early pregnancy. Proc. SOC. Exp. Biol. Med., 74: 204-208.

Williams, J. P. G. 1970 DNA synthesis in embryos: effects of fluorouracil. Teratology, 3: 210 (abstract).

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PLATES

PLATE 1

EXPLANATION OF FIGURES

3 Hadioautograph of cultured explant showing description of zones. NR, neuroretina; LM, limiting membrane; V, ventricular zone showing regions of DNA synthesis (Vs); interkinetic movement (VJ and mitosis (V,,,); M, marginal zone; PE, pigmented epithelium; AM, associated mesenchyme. x 680.

4 Control neuroretinal explant cultured for five hours. Mitotic spindles (arrows) were usually oriented parallel to the limiting membrane. x 160.

5 Neuroretinal explant exposed to methetrexate for seven hours. Degenerating nuclei were abundant in the DNA synthetic and interkinetic zones of the neuroepithelium. The rare mitotic figure observed showed normal orientation of its spindle. The pigmented epithelium and associated mesenchyme are well preserved. x 160.

6 Radioautograph of pigmented epithelium and its associated mesenchyme exposed to methotrexate for five hours. Both tissues appear structurally normal and show heavy labeling. x 520.

Radioautograph of a neuroretinal explant after 5-hour exposure to methotrexate in the presence of embryo extract. Several mitotic figures are seen. x 400.

7

316

EARLY NEUHORETINAL RESPONSES Eugene Daniels and Keith L. Moore

PLATE 1

317

PLATE 2

EXPLANATION OF FIGURES

8 Radioautograph of a neuroretinal explant after 8-hour exposure to methotrexate in the presence of embryo extract. Many labeled pyknotic cells are seen (arrows). Mitotic figures are rare. x 400.

Neuroretinal explant exposed to methotrexate for seven hours. The DNA synthetic and interkinetic zone show conspicuous gaps where pyknotic cells have been extruded. The rare mitotic cell (arrow) show normal spindle orientation and the associated mesenchyme is well preserved. x 160.

10 Radioautograph of a control neuroretinal explant after eight hours of culture. Many labeled mitotic cells are seen. x 520.

11 Neuroretinal explant exposed to methotrexate for 16 hours. The cell- depleted neuroretina shows groups of surviving cells with rare mitoses. Pyk- notic cells are largely extruded. x 400.

12 Radioautograph of a neuroretinal explant after 16 hours of exposure to methotrexate. The cell-depleted neuroretina is distorted and shows labeled cells; pyknotic cells and mitotic figures are rarely observed. The pigmented epithelium shows many labeled cells and the mesenchyme shows a few pyknotic cells. x 400.

Radioautograph of a folded neuroretinal explant exposed to methotrexate. A uniform zone of cell death is seen in the mid-region of the explant. x 160.

9

13

318

EARLY NEURORETINAL RESPONSES Eugene Daniels and Keith L. Moore

PLATE 2

early chick neuroretinal responses following direct exposure to methotrexate

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