An autoradiographic analysis of the time of appearance of neurons in the developing chick neural retina

DEVELOPMENTAL

BIOLOGY

38, 30-40 (1974)

An Autoradiographic

Analysis

of the Time

in the Developing

Chick

of Appearance

Neural

of Neurons

Retina

A. J. KAHN Department

of Anatomy,

Washington

University

Accepted

School of Medicine,

December

St. Louis, Missouri

63110

17, 197,?

The pattern of incorporation of [3H]thymidine into the chick neural retina has been used to establish the time and order in which different classes of neuroepithelial cells withdraw from the cell cycle and initiate migration and differentiation. The posterior pole of the retina is the first to form during development. In this region most neuroepithelial cells complete mitotic activity between the third and sixth day of incubation. Presumptive ganglion cells initiate the withdrawal process, and they are soon followed by the neuroepithelial precursors of amacrine, horizontal, and receptor cells. Bipolar cell precursors are the last to begin and the last to complete cell cycle activity. It is worthy of note, however, that, in any given region of the retina, neuroepithelial cells of all types cease mitosis in close, overlapping succession. These results are in reasonable agreement with those previously published on the chick retina by Fujita and Horii (1963), and other investigators on the mouse (MM), killifish (Fundulus), and toad (Xenopus). The present data are also consistent with those proposals of Angevine (1970). Jacobson (1968a, b, 1970), and others that relate the cessation of mitotic activity of neuroepithelial cells to the determination of neuronal size, axon length, and the specification of neuronal connections.

Jacobson (1970) has designated the large neurons of the first type as Class I cells, and has postulated that they are specified with regard to the neuronal connections that they will make in an invariant fashion early in development. Small, late-appearing Class II neurons, on the other hand, are postulated to form modifiable synaptic connections that depend at least to some extent upon the pattern of activity to which they are subjected. The specification of neuronal connections can also be correlated with the cessation of mitotic activity. In a series of experiments on Xenopus laeuis, Jacobson (1968a, b) reported that retinal ganglion cells are specified with regard to their sites of termination in the optic tectum coincidental with their withdrawal from th,e cell cycle. Crossland et al. (1973) and Kahn (1973) have obtained essentially the same result in a series of similar experiments on the developing chick neural retina. In the present experiments, an effort was

INTRODUCTION

In recent years a number of observations have been made on the developing nervous system which suggest that there is a close relationship between the time that neuroblasts withdraw from the cell cycle and the determination of their subsequent developmental history. For example, neuroblasts that cease division early in embryogenesis tend to differentiate into neurons that are larger than their counterparts that continue to divide for some time (literature summarized in Altman, 1970; Angevine, 19’70; Hinds, 1968). Such macroneurons usually possess long axons, contribute to primary afferent or efferent pathways and are usually characterized by a high degree of topographic organization in their connectivity. By contrast, the smaller microneurons cease division later in development, generally have short axons, and make up a large portion of the interneuronal pool (Altman, 1970; Jacobson, 1970). 30

KAHN

Time

of

Appearance

made to establish when, and in what order, retinal neurons cease to divide during the development of the chick neural retina. In a similar study, Fujita and Horii (1963) reported that ganglion cell precursors are the first to withdraw from the cell cycle and that this event occurs on day 5 of development. They further reported that the neuroepithelial cells that give rise to rods and cones cease division shortly after the presumptive ganglion cells but before the cells of the inner nuclear layer. The results of the present experiments are consistent with those of Fujita and Horii (1963) in describing the order in which events occur, but clearly deviate from their estimates of the time at which they occur. As in Fujita and Horii’s experiments, the cessation of mitotic activity has been assessed utilizing the “cumulative labeling” technique of Fujita (1963). In this method, sufficient [3H]thymidine is made available to the developing embryo to ensure that every neuroepithelial cell still passing through the cell cycle will incorporate the isotope into DNA and, as a consequence, appear labeled in autoradiographs prepared at some later stage of development. Any cell that has completed mitotic activity at the time the isotope is introduced, will not incorporate [3H]thymidine and will appear unlabeled in autoradiographs. Therefore, by preparing autoradiographs of retinal tissue taken from embryos that have been exposed to isotope at different stages of development, it is possible to determine from the appearance of cells with unlabeled nuclei at what stage of development these cells ceased mitotic activity. In the present study advantage was also taken of the fact that cells exposed to high concentrations of isotope early in the S phase of their terminal mitotic cycle appear to be heavily labeled in autoradiographs. Since such heavily labeled neurons are derived from neuroepithelial cells that were about to complete mitotic activity at the time the isotope was introduced, their

31

of Neurons

appearance and distribution can also be used to assess the time at which these cells withdraw from cell cycle activity (Sidman, 1961, 1970). MATERIALS

AND

METHODS

The present experiments were conducted on White Leghorn chick embryos raised to the desired stage of development in a forced-draft incubator at 38°C. The eggs were candled on the morning of the second day, and oval windows were ground into the shell overlying the developing blastoderms. These windows allowed for the staging of the embryos according to the Hamburger and Hamilton series (1951) and for the introduction of [3H]thymidine. The isotope (TudR methyl-‘H, sp. act. 17 to 20 Ci/mmole) was usually obtained from Schwarz BioResearch and was diluted with sterile water or saline to a concentration of 100 &i/ml before use. Twenty to 25 &i (0.2 or 0.25 ml) was introduced into each egg. Except for the brief periods required for staging and the introduction of the isotope, the windows were kept sealed with transparent cellophane tape. Older embryos, with their investment of extraembryonic membranes and deeper position within the egg, were difficult to see through a shell window and were staged according to the length of the incubation period. In these older eggs, the isotope was generally introduced through a small hole bored into the shell in a region where there was a well established extraembryonic circulation. In most of these experiments, the animals were sacrificed on day 12 or day 16 of incubation, and the heads or eyes were fixed in Bouin’s solution or a modification of it referred to as Alien’s B-15 (Coulombre, 1955; Lillie, 1965). Following a rinse in water, the tissues were freed of residual picric acid with ammoniated 70% alcohol, dehydrated through an ethanol-water series and cleared in a 1% (w/v) solution of celloidin in methyl benzoate. The cleared

32

DEVELOPMENTALBIOLOGY

tissues were then passed through several changes of benzene and embedded in paraffin. The tissues were sectioned at 10 pm and the sections mounted on slides with egg albumin either serially or as a one-in-ten series. After deparaffinization with xylene and hydration in an alcohol-water series, the slides were placed in dust-free glass dishes and dried at 37” in an incubator. Once a sufficiently large group of slides was collected, the sections were coated with Kodak NTB-2 or NTB-3 emulsion that had been diluted 1: 1 with distilled water. The slides were then dried slowly in humid air and stored at 4°C for 10 days to several weeks in light-tight boxes containing a desiccant. After the storage period, the autoradiographs were developed in Kodak D-19 developer and the tissues were stained through the emulsion with 1% thionin. RESULTS

Two different experimental protocols were utilized in preparing these autoradiographs. In the first, the [3H]thymidine was introduced into chick embryos from late on day 2 to day 6 of incubation, and the animals were sacrificed on day 12 or day 16. In this series, particular attention was paid to the distribution of heavily labeled and unlabeled cells in the posterior pole of the retina in the region near the optic nerve. It is this region of the neural retina that forms first during development and is the most readily comparable in autoradiographs from different animals. In the second series, the [3H]thymidine was introduced on day 6, 7, 8, or 9 of embryonic development and, after fixing and processing the tissue on day 12 or 16 of incubation, the pattern of labeling was compared in different regions of the same retina. In the initial phases of development mitotic activity occurs uniformly throughout the neural retina. Beginning late on

VOLUME 38, 1974

day 5 or early on day 6, there is a marked reduction in cell division in a small area of the posterior pole. The zone of reduced mitotic activity increases in size until by day 8 most of the cell division is confined to the ciliary margin (ora serrata) (Coulombre, 1955; Weysse and Burgess, 1906). The appearance of postmitotic, differentiating cells follows the same pattern. Such cells first appear in the posterior pole and later in the equatorial and marginal regions of the retina. It is this sequential aspect of retinal development that makes it possible to visualize the condition of the retina as it appears at different stages of development by systematically examining a single specimen from the ciliary margin to the posterior pole. Because it is impractical to silver impregnate autoradiographs to show details of neuronal morphology, different cell types were identified utilizing the orderly arrangement of cell nuclei and the various plexiform layers. This order is apparent in the iron hematoxylin stained section of chick neural retina shown in Fig. 1. It can be seen that the nuclei of ganglion (gc) and receptor cells (rc) are isolated from other nuclei by the inner and outer plexiform layers (IPL, OPL), respectively. The horizontal cells (hc), on the other hand, are marked by a single row of nuclei at the border of the inner nuclear layer (INL) and OPL. The amacrine cell (ac) nuclei constitute a zone within the INL 3 or 4 rows deep, but along the border of the IPL. The nuclei of both bipolar and Miiller cells (bc, mc) are found in the “inner” portion of the INL and are difficult to distinguish in autoradiographs prepared from paraffin sections. Because of this, the data and observations made relating to these two cell types are grouped and treated together as a single category (Bi-Ml. In practice, the Bi-M nuclei are considered to extend from one row beneath the border of the OPL to a point approximately midway through the

KAHN

Time of Appearance

of Neurons

Fro. 1. Chick neural retina on day 16 of incubation. Note that within the inner nuclear layer (INL) it is possible to distinguish different areas corresponding to where horizontal (hc), bipolar (bc), Miiller (mc), and amacrine cell (ac) nuclei are located, rc refers to receptor cells; gc, ganglion cells; OPL and ZPL, outer and inner plexiform layers, respectively. Iron hematoxylin stain; original magnification approximately x 400.

34

DEVELOPMENTALBIOLOGY

INL. The greater part of this zone is usually discernible as a region of relatively greater staining density.

Qualitative Observations Virtually all the evidence pertaining to the stage of development at which retinal neuroepithelial cells withdraw from the cell cycle came from examining autoradiographs of the posterior pole of retinas of 12 and 16-day-old embryos that had been injected with [3H]thymidine prior to day 6 of development. In these experiments, the first animals to possess heavily labeled nuclei were those in which the isotope had been administered at stage 14 of the Hamburger and Hamilton (1951) series, that is, early on day 3 of incubation. As can be seen in the tracings in Fig. 2, the positions of these first heavily labeled nuclei correspond to those regions in which one would expect to find horizontal, receptor, and ganglion cells. This observation suggests that the neuroepithelial cells that give rise to these retinal neurons are the earliest to withdraw from the cell cycle and initiate differentiation.

VOLUME 38, 1974

The introduction of isotope later on day 3 and through day 4 of incubation results in a steady increase in the number of heavily labeled receptor, ganglion, horizontal, and amacrine cells (Fig. 2). The number of such heavily labeled cells appears to reach a maximum when the isotope is introduced on day 4 of incubation, and to decline thereafter. These observations suggest that the majority of the ganglion, amacrine, horizontal, and receptor cell precursors begin to withdraw from the cell cycle on day 3 of development and complete the process by day 5. Heavily labeled nuclei are first apparent in the Bi-M portion of the INL in animals injected with isotope late on day 3 of incubation. However, in contrast to the cell types just described, significant numbers of neuroepithelial cells that contribute to the Bi-M area continue to incorporate isotope into day 6 of incubation (Fig. 2). This pattern of isotope incorporation indicates that presumptive Bi-M cells begin to withdraw from the cell cycle slightly later in development than other ret&al neuroepithelial cells and do not

*

m

GCl~+----

-

*QQ

@ s I Day

6

Day

8

FIG. 2. Camera lucida drawings showing the distribution of heavily labeled nuclei in the posterior pole of 12-day-old embryonic retinas exposed to [3H]thymidine at different times early in development. Differences in retinal thickness in these samples are largely due to variations in paraffin technique and plane of section. ONL, outer nuclear layer; ZNL, inner nuclear layer; GCL, ganglion cell layer.

KAHN

Time

of Appearance of Neurons

35

of mitotic activity is supported by observacomplete the withdrawal process until tions made on those further developed some later time. In fact, some presumptive portions of the retina that lie still closer to Bi-M cells continue to incorporate [3H]thymidine until at least day 12 of the optic nerve. No labeled receptor or incubation. These late dividing cells are ganglion cells are apparent in this region, probably Miiller cells which, as a form of and most of the labeled nuclei that can be glia, are likely to maintain their ability to observed are confined to the Bi-M portion divide throughout the life of the animal. of the INL. The latter is indicative of an Information on the order of withdrawal increased percentage of unlabeled (postmiof retinal neuroepithelial cells from the cell totic) amacrine and horizontal cells and cycle was also obtained by systematically suggests that amacrine and horizontal preexamining the same retinal autoradiograph cursors complete mitotic activity after the from the ciliary margin to the posterior receptor and ganglion cells. pole. A sample of the type of information The most developed region of the retina, that can be obtained by using this ap- near and superior to the optic nerve, has a proach is shown in Fig. 3, in which portions labeling pattern similar to the one just of a single autoradiograph from a retina described. No labeled ganglion cells or that had been exposed to isotope at stage receptors are in evidence and compara30 (day 6) and was fixed at stage 38 (day tively few labeled horizontal and amacrine 12) are reproduced. It will be noticed that cells can be seen. The fact that most of the in the last-formed region of the neural labeled nuclei are located in the Bi-M area retina-the area near the ciliary marginof the INL would again indicate that Bi-M -all the cells are labeled, but the intensity cell precursors are the last to complete of the labeling is not very great and only an mitotic activity. occasional cell in the ganglion cell layer is Quantitative Observations heavily labeled. In this respect, the labeling pattern near the margin very much It is interesting to compare the observaresembles that seen in the posterior poles of tions made on the appearance and distriretinas that had been injected at about bution of heavily labeled nuclei with the stage 13 or 14 of development. information obtained from calculating the By contrast, portions of the retina that percentage of unlabeled cells in different lie in the equatorial region of the eye regions of the retina. It is assumed that the possess a good many heavily labeled nu- higher the percentage of unlabeled cells, clei-indicating that on day 6 of developthe more complete the withdrawal from ment the cells in this region were nearer the cell cycle activity at the time the isotope completion of mitotic activity than those was introduced. In Figs. 4 and 5, the at the ciliary margin. What is particularly percentage of unlabeled cells in adjacent striking about this region of the retina is sectors of a 12-day embryonic retina that the virtual absence of labeled ganglion cells had received isotope on day 8 of incubation and the reduction in the number of labeled is plotted against the relative position of receptor cells. These observations support each sector. It will be noted that in the the view that ganglion cells begin to withtransit from the least developed sector, at draw from the cell cycle before neuroepithe ciliary margin, toward the most develthelial cells of any other type and indicate oped sector, in the posterior pole, the that receptor cell progenitors are the next ganglion cell population is the first to type of neuroepithelial cell to complete demonstrate a conspicuous percentage of mitotic activity. unlabeled cells and is the first to achieve This proposed sequence for the cessation completely unlabeled status. These obser-

36

DEVELOPMENTALBIOLOGY

VOLUME 38, 1974

FIG. 3. Sequence of photographs showing the distribution of heavily labeled nuclei in different regions of a stage 38 (12 day) retina that had been exposed to [9H]thymidine at stage 30 (day 6). Note that the labeling patterns seen in different regions of the retina are similar to those encountered in animals injected at different stages of development (Fig. 2).

vations are consistent with the results obtained from the analysis of the distribution of heavily labeled cells, uiz., that presumptive ganglion cells are the first to cease mitotic activity.

It is more difficult to interpret the quantitative data obtained for other classes of retinal cells. For example, it is clear that in this particular analysis, receptor cells are the only other class of retinal neuron to

KAHN

M 1

3

Time of Appearance

5

7 SCORING

FIG. 4. Percentage of unlabeled cells retina exposed to [3H]thymidine on day toward the posterior pole for a distance zontal cells; n , amacrine cells; V, Bi-M

9

37

of Neurons

11

TRANSIT

13

15

17

-

of different types in contiguous 180 PM sectors of a 12-day embryonic 8 of incubation. Scoring was initiated at the margin (AI) and continued of approximately 3.2 mm. 0, ganglion cells; 0, receptor cells; 0, horicells.

RETINAL TRANSIT FIG. 5. Percentage of unlabeled ganglion and receptor cells in contiguous 180 FM sectors of a 12-day embryonic retina exposed to [SH]thymidine on day 8 of incubation. These data were collected from the same animal used in Fig. 4 and are intended to demonstrate the reproducibility of the scoring technique. 0, ganglion cells; 0, receptor cells. Filled and open symbols indicate that the counts were made on different sections of the retina.

achieve completely unlabeled status. Yet because some unlabeled amacrine and horizontal cells are present in the same sectors of the retina that contain unlabeled rods and cones, it is impossible to establish which of these three cell types was the first to withdraw from the cell cycle. In fact, these data suggest that neuroepithelial cells of all three types withdraw from the cell cycle at about the same time. However, since neither the amacrine nor the horizontal cell populations in any sector become

totally unlabeled in a manner comparable to receptor cells, it seems evident that it takes longer for the amacrine and horizontal cell populations to complete mitotic activity. It is worthy to note that the Bi-M population shows virtually no unlabeled members in the region of the retina surveyed in Fig. 4. The absence of such unlabeled cells is consistent with the observations made on cells with heavily labeled nuclei and supports the proposal that bipo-

38

DEVELOPMENTALBIOLOGY

lar and Miiller cell precursors are the last to withdraw from the cell cycle and to initiate differentiation.

VOLUME 38, 1974

[3H]thymidine no longer labels all receptor cells, then it would appear that presumptive receptor cells begin to withdraw from the cell cycle at about the same time as DISCUSSION some amacrine and horizontal cell precurThe data from the present study made it sors but before those of bipolar cells. If, on clear that the major conclusions drawn by the other hand, the time of withdrawal is Fujita and Horii (1963) on the withdrawal determined by noting that stage of develof neuroepithelial cells from the cell cycle opment at which the isotope no longer are generally correct. However, their esti- labels any receptors, then it would appear mates on the times of withdrawal from the that presumptive receptors precede all the cell cycle are not in accord with the present neuroepithelial cells contributing to the study or with some of the anatomical INL in completing mitotic activity. (Some observations of Cajal (1911), Coulombre appreciation for the differences between (1955), (Goldberg and Coulombre (1972), these alternatives can be obtained by comRogers (1957), and Kahn (1973) on the paring in Fig. 4 the unlabeled cell percentdeveloping retina. For example, Fujita and ages near the margin of the retina with Horii (1963) indicate that the presumptive those nearer the posterior pole.) ganglion cells begin to withdraw from the In some ways, the least surprising and cell cycle on day 5 of incubation. By most important result to emerge from the contrast, Cajal, Rogers, Goldberg, and autoradiographs is the observation that in Kahn describe the first appearance of juveany given region of the developing neural nile postmitotic ganglion cells as occurring retina most neuroepithelial cells leave the on day 3 of development, while Coulombre cell cycle at about the same time or in succession. Even ganstates that they appear on day 4. Since not close, overlapping all of these authors specify the temperature glion cells, which are the first to cease at which the embryos were incubated, it is mitosis, are soon joined by postmitotic, amacrine, horizontal and possible that the discrepancies in time are differentiating due to differences in the conditions of receptor cells. Other investigators working with different species have obtained simiincubation. lar results but, in general, have not elaboIf allowance is made for the time discrepancies, the present study and that of Fujita rated upon the question of overlap in and Horii also agree that the neuroepidiscussing their data. Sidman (1961), for stated that cells in all thelial cells that give rise to rods and cones example, explicitly begin to withdraw from the cell cycle three cellular layers of the mouse retina are shortly after the ganglion cell precursors. formed simultaneously, throughout develHollyfield (1972) has The early withdrawal of receptor cells is opment. Similarly, somewhat surprising, since rods and cones shown that when retinal neuroepithelial do not complete differentiation in the chick cells begin to withdraw from the cell cycle embryo until just before hatching (Cou- at stages 28 and 29 of Fundulus developlombre, 1955). Whether receptor cell proge- ment, some retinal neurons of every type nitors precede neuroepithelial cells of the are represented. Jacobson (1968a) had earINL in withdrawing from the cell cycle, as lier published less complete, but similar, Fujita and Horii suggest, depends upon the findings for Xenopus at stages 29 and 30. Despite the fact that there is an overlap criterion adopted in interpreting the autoradiographs. If the time of withdrawal is in the appearance of different types of established by noting that stage in devel- retinal neurons, results of the present study opment at which the introduction of are in reasonable accord with some of the

KAHN

Time of Appearance

proposals made by Altman (1970), Angevine (1970), Jacobson (1970) and others, that relate the cessation of mitotic activity to other events in the development of the nervous system. For example, retinal ganglion cells which cease division early in development match the characteristics of a class I or macroneuron very well. They are large cells with long axons that contribute to a primary efferent pathway that is topographically organized. Furthermore, they are specified with regard to their neuronal connections shortly after they cease mitotic activity (Crossland et al., 1973; Kahn, 1973). Bipolar cells, on the other hand, which are the last retinal neurons to withdraw from the cell cycle, fit rather closely the characteristics of the class II neuron or microneuron. They are small with short axons, and they make up a large portion of the interneuronal connectives of the neural retina. Beyond this general level, it is difficult to relate the withdrawal of cells from cell cycle activity to subsequent developmental events. For one thing, there are significant gaps between the times when neuroepithelial cells in the retina complete mitotic activity, and when plexiform layers and synapses are formed. As the data show, most cells in the posterior pole of the retina cease mitotic activity sometime between day 3 and day 6 of incubation. In the same region of the retina, the IPL does not make its appearance until about day 8 of development, and the OPL is not recognizable until about a day or two later (Coulombre, 1955). Furthermore, synapse formation does not begin until day 14 of incubation (Hughes, 1972; Kahn, unpublished observations; Sheffield and Fischman, 1970). Thus, there is an interval of 8-11 days between the times most cells have completed mitotic activity and the times that they have formed synaptic connections. To make meaningful correlations over such a long time span would require a technique capable of precisely labeling single or small

39

of Neurons

groups of retinal neurons early in development in a manner that would allow them to be identified at the electron microscope level at some later time. No such technique is currently available. The author wishes to thank Miss Ginger Tamplin for her assistance in preparing the slides and autoradiographs, and Drs. W. M. Cowan and M. T. Price for their kindness in reviewing the manuscript. This work was supported by U. S. Public Health Service Research Grant EY-00720. REFERENCES ALTMAN, J. (1970). Postnatal neurogenesis and the problem of neural plasticity. “Developmental Neurobiology” (W. A. Himwich, ed.), pp. 197-237, Thomas, Springfield, Illinois. ANGEVINE, J. B., JR. (1970). Critical cellular events in the shaping of neural centers. “The Neurosciences, Second Study Program” (F. 0. Schmitt, ed.), pp. 62-72, Rockefeller Univ. Press, New York. CAJAL, S. RAM6N y (1911). Histologie du Syst&me Nerveux de 1’Homme et des VertBbr&, Vol. 2. Maloine, Paris. COULOMBRE, A. J. (1955). Correlations of structural and biochemical changes in the developing retina of the chick. Amer. J. Anat. 96, 153-189. CROSSLAND, W. J., COWAN, W. M., ROGERS, L., and KELLY, J. (1973). The specification of the retinotectal projection in the chick. Submitted to J. Comp. Neural. FUJITA, S. (1963). The matrix cell and cytogenesis in the developing central nervous system. J. Comp. Neural. 120, 37-42. FUJITA, S., and HORII, M. (1963). Analysis of cytogenesis in chick retina by tritiated thymidine autoradiography. Arch. Histol. Jap. 23, 359-366. GOLDBERG,S., and COULOMBRE,A. (1972). Topographical development of the ganglion cell fiber layer in the chick retina. A whole mount study. J. Comp. Neural. 146, 507-518. HINDS, J. W. (1968). Autoradiographic study of histogenesis in the mouse olfactory bulb. I. Time of origin of neurons and neuroglia. J. Comp. Neural. 134, 287-304. HOLLYFIELD, J. G. (1972). Histogenesis of the retina in the killifish, F~ndulus heteroclitus. J. Comp. Neurol. 144, 373-380. HUGHES, F. (1972). Personal communication. JACOBSON,M. (1968a). Development of neuronal specificity in retinal ganglion cells of Xenopus. Deuelop. Biol. 17, 202-218. JACOBSON,M. (1968b). Cessation of DNA synthesis in retinal ganglion cells correlated with the time of specification of their central connections. Deuelop. Biol. 17,219-232.

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M. (1969). Development of specific neuronal connections. Science 163. 5433547. JACOBSON, M. (1970). “Developmental Neurobiology.” Holt, New York. KAHN, A. J. (1973). Ganglion cell formation in the chick neural retina. Brain Res. 63, 285-290. LILLIE, R. D. (1965). “Histopathologic Technique and Practical Histochemistry.” McGraw-Hill, New York. ROGERS, K. T. (1957). Early development of the optic nerve in the chick. Amt. Rec. 127, 97-107. SIDMAN, R. (1961). Histogenesis of mouse retina studied with thymidine-H3. “The Structure of the Eye” JACOBSON,

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(G. K. Smelser, ed.), pp. 487-506. Academic Press, New York. SIDMAN, R. (1970). Autoradiographic methods and principles for study of the nervous system with thymidine-H3. “Contemporary Research Methods in Neuroanatomy” (W. J. H. Nauta and S. 0. E. Ebbesson, eds.), pp. 252-274. Springer-Verlag, New York. SHEFFIELD, J. B., and FISCHMAN, D. A. (1970). Intercellular junctions in the developing neural retina of the chick. Z. Zellforsch. 104, 405-418. WEYSSE, A. W., and BURGESS, W. S. (1906). Histogenesis of the retina. Amer. Nutur. 40, 611-637.