Early ganglion cell differentiation in the mouse retina: An electron microscopic analysis utilizing serial sections

DEVELOPMENTAL

BIOLOGY

37, 381-416 (1974)

Early Ganglion Electron

Cell Differentiation

Microscopic

in the Mouse

Analysis

Utilizing

Retina:

An

Serial Sections’

JAMES W. HINDS AND PATRICIA L. HINDS Department

of Anatomy,

Boston University Accepted

School of Medicine. December

Boston, Massachusetts

02118

10, 1973

The retina of a mouse embryo on day 13 of gestation, the first day when ganglion cells with axons are detectable, has been studied both qualitatively and quantitatively by reconstructing a large number of cells (more than 100) from an electron microscopic serial section series. Direct evidence has been obtained for migration of prophase nuclei of ventricular cells to the ventricle within an intact process which spans the thickness of the retinal wall. At metaphase most of the vitreal process appears to be pinched off, and the cell completely rounds up. After cytokinesis, cells take one of two courses: (1) regrowth of their vitreal process to the vitreal surface while keeping their ventricular process attached at the ventricular surface by a junctional complex; these cells will undergo another round of DNA synthesis and division; (2) regrowth of their vitreal process only so far as the marginal layer with detachment of their ventricular process from the junctional complex and beginning migration of their centrioles and cilium away from the ventricle. These changes represent the earliest detectable quantitative or qualitative changes undergone by cells that will subsequently differentiate into ganglion cells. The sequence of events for the formation of unipolar ganglion cells from these early bipolar cells involves transformation of the simple vitreal process ending in the marginal layer into an axonal growth cone insinuating itself between the tangential axons of the marginal layer and growing toward the optic stalk: at the same time the Golgi complex and centrioles migrate to the perikaryon. and the ventricular process completely withdraws. Usually, but not always, both daughter cells of a mitotic division appear to have the same fate, either both remain ventricular cells or both become ganglion cells. This result is used to construct a simple hypothesis explaining some of the apparently contradictory results of neuronal development, both in the retina and in the rest of the central nervous system. INTRODUCTION

Dramatic morphological and cellular changes must occur in the developing central nervous system (CNS) to bring about the transformation of proliferative ventricular (matrix) cells into postmitotic young neurons with axons. The initial stages of differentiation are poorly understood in most regions, yet they are clearly important in the development of the complex and highly ordered mature CNS. One of the principal reasons that early morphological changes have remained largely unexplored is the difficulty in most regions of examining the entire cell with electron microscopy. The length and tortuosity of ‘This work was supported by Research Grant NS-08655 from the National Institute of Neurological Diseases and Stroke.

most developing neuron processes is such that single thin sections cannot possibly display the whole cell. In general, only when the cell has highly regular morphology, as for example the granule cell of the cerebellum (Mugnaini and Forstr@nen, 1967; Rakic, 1971; Altman, 1972), or when serial sections and reconstruction are employed, as in a recent study on the olfactory bulb (Hinds, 1972b), can significant progress be made in unraveling the early morphological events of neurogenesis at an ultrastructural level. The present study was undertaken to obtain detailed information on early neuron differentiation in a region favorable for such an analysis, the retina. A long series of serial sections was cut through the embryonic mouse retina on the first day in which differentiated neurons could be detected

382

DEVELOPMENTALBIOLOGY

light microscopically. Based on previous silver (Rambn y Cajal, 1911) and autoradiographic studies (Sidman, 1961), the expectation would be that at this early stage a high proportion of the differentiating neurons would be ganglion cells. By reconstructing a large number of cells at this time, therefore, it should be possible to discover cells transitional between proliferative cells and ganglion cells. Arrangement of these transitional cells into a transitional series could then lead to a plausible interpretation of the sequence of morphological events of early neuron differentiation in the retina. To avoid biasing the selection of transitional cells, it was felt important in the present study initially to reconstruct all the cells whose nuclei appeared in a section approximately midway through the serial section series, since it could not be ascertained a priori which ones would be important for a transitional series. Once the general sequence of development of retinal ganglion cells had become clear from this initial study, it would then be possible to search the rest of the sections for additional transitional cells to arrive at a numerically larger and more closely spaced series. MATERIALS

AND

METHODS

Embryos at day 13 of gestation were removed from the uterus of pregnant Charles River mice and fixed by immersion of the entire embryo in 1% glutaraldehyde and 4% formaldehyde in 0.1 M cacodylate buffer to which 1 ml of 5% CaCl,/lOO ml of fixative had been added. After fixation overnight or longer, small pieces containing the entire developing eye were cut, and then they were dehydrated in a graded series of alcohols and propylene oxide and embedded in Araldite 502. Blocks were thick sectioned at 1 pm and stained with methylene blue and azure II for general orientation. The region chosen for serial sectioning was trimmed to a precise rectangular face, approximately 0.1

VOLUME 37,1974

mm x 0.2 mm x 0.04 mm high, using the corner edge of a glass knife (Hinds and Hinds, 1972). A set of 441 serial sections was cut at a thickness of approximately 600 A each and mounted on 20 grids in ribbons of 17-26 sections. The size of the block of tissue cut was approximately 100 pm x 200 pm x 26 Nrn. The ribbons were picked up from the boat using g-barred (100 “mesh”), parallel-bar grids (Ted Pella Co., Tustin, California) previously coated with 0.25% Formvar (Ladd). The ribbons were carefully oriented perpendicular to the bars. In this way only part of every third section was obscured by the grid bars. Since the only practical way to pick up ribbons of sections so that they were consistently perpendicular to the bars was to catch the first section of the ribbon on the edge of the grid, it was necessary initially to trim off most of this wide edge on one side with a razor blade. This left only a thin barlike edge, so that only one section at the beginning of the ribbon was obscured. Sections were stained for 5 min in saturated aqueous uranyl acetate followed by 2 min staining in 0.2% lead citrate. At every third section (or occasionally at intervals of 2 or 4 sections) the full thickness of the retina was photographed in a montage consisting of two pictures and a primary magnification approximately 1400 x, using a low magnification polepiece for an RCA EMU 3F electron microscope. In the case of ribbons that were curved slightly, consistent orientation of each successive section was achieved by means of a rotating stage (Ernest F. Fullam, Schenectady, New York). Prints (8 x 10 inches) at a magnification of approximately 3400 x were covered by transparent acetate sheets. Cells to be followed were assigned a number, and that number was placed directly over the profile on the acetate sheets. An extremely fine No. 0000 (O/4) LeRoy standard pen point was used for inking in numbers. The advantage of using acetate sheets rather than inking directly on the electron micrographs

HINDS AND HINDS

Retinal Ganglion Cell Deuelopment

is that the numbers do not obscure the subcellular structures of the profiles. With experience and careful attention to detail, all the cells and even their smallest processes could be followed and numbered accurately. After numbering the cells, some of them were graphically reconstructed by superimposing outline drawings of profiles of every fifth electron micrograph (or sometimes at closer intervals for fine details) in the “best-fit” position relative to other profiles (Fig. 3,C). The profiles drawn for this plane of section reconstruction (Fig. 3,A) could also be used for a reconstruction at right angles to the plane of section (Fig. 3,B). Distances from the vitreous body or optic ventricle were measured to the top and bottom of the drawing of the profile, and these distances were replotted on a separate drawing after correcting measurements for slight variations in primary magnification (assessed by measuring the distance on the print from vitreous body to ventricle). Measurements for each profile were plotted in correct spacing on the paper to correspond with the actual distance that the profiles were separated in the block of tissue. Following the initial analysis of cells in the low magnification series, selected regions were rephotographed at a higher primary magnification in order to elucidate certain subcellular details. Axonal growth cones were reconstructed by stacking profiles cut out of wax plates. Drawings of the profiles were made on acetate sheets for every section available (every third section) through the entire growth cone and then enlarged by placing the acetate sheet in a photographic enlarger until in the reconstruction the thickness of one wax plate was equivalent to three thin sections. The models were then used as an aid for the final drawing. A quantitative study was carried out, correlating the size of certain organelles with the stage of differentiation of the cells in which they were found. A relative measure of the amount of granular endoplas-

383

mic reticulum in a given cell was determined by drawing, with the aid of a stereobinocular microscope, a line which was equal to the length of every cistern of granular endoplasmic reticulum that was seen in all the electron micrographs containing the profiles of a given cell (usually every third section). The lengths of all the lines were then estimated using a 7x magnifier with a reticule, and the total length and average length per measured profile were determined. A relative measure of the size of the Golgi complex was obtained in a similar way by drawing the irregular outline of the extent of the structure on all profiles where it occurred in a given cell. Owing to their small size, these outlines were enlarged three times using a photographic enlarger before measuring them. Total area of the enlarged outlines for each cell was then determined by counting the number of l-mm squares contained within each outline. Finally, an estimate of the nuclear volume was obtained by treating the nucleus as a prolate spheroid and calculating the volume from measurements of the major and minor axes. For certain irregular nuclei, volume was also estimated directly from the nuclear areas determined on every electron micrograph through the entire nucleus. For tests of statistical significance we used a nonparametric test (Mann-Whitney U test) since we did not feel justified in assuming a normal distribution for the size of organelles. This was both on the basis of our own data and the known fact that ganglion cells in the adult exist in small and giant varieties (Ramon y Cajal, 1972). For the same reasons, we used for the graphs the median and range rather than the mean and standard deviation. RESULTS

The Mouse Retina at Day 13 of Gestation (El3)

The general appearance of the retina on E13, the first day when ganglion cells are

384

DEVELOPMENTALBIOLOGY

seen, is shown in Figs. 1 and 2. A relatively thick layer, consisting largely of the nuclei of radially oriented ventricular cells in various phases of their generation cycle, extends inward from the ventricular surface. At the vitreal surface is found a light staining layer (marginal layer) without cell bodies and consisting of optic nerve axons and expanding terminals of vitreal processesof ventricular cells. Axons are clearly grouped into irregular fascicles (Fig. 2). Between the marginal and ventricular layers is the ganglion cell layer which, besides ganglion cell bodies, also contains vitreal processes of ventricular cells. The ganglion cell layer is somewhat more loosely organized than the ventricular layer, and the ganglion cell nuclei are lighter, more spherical, and more irregular in shape than the ovoid nuclei of ventricular cells. The mouse retina on day 13 of gestation corresponds to the retina of a stage 3+ mouse eye (Pei and Rhodin, 1970), the rat retina on day 15 of gestation (Braekevelt and Hollenberg, 1970), the rabbit retina on day 14 of gestation (Uza and Smelser, 1973), and the chick retina on day 4 of incubation (Coulombre, 1955). It appears, however, to differ significantly from any of the early stages of retinal development in the human embryo (Mann, 1964; O’Rahilly, 1966; Spira and Hollenberg, 1973). Cell reconstructions were made of all nuclei which appear in the electron microscopic montage of section 249 (Fig. 2). Of the 69 cells that were completely contained within the set of serial sections, 51 were ventricular cells, 17 were postmitotic young neurons, and 1 was a macrophage (cell 74, Figs. 2 and 12). Seventeen of the ventricular cells had a vitreal process which did not reach the vitreal surface (e.g., cells 9, 10, 5, 18, and 45, Fig. 4); they were presumed to be early interphase cells still regrowing their vitreal process after having rounded up at the ventricle during metaphase and anaphase stages of mitosis (Hinds and Ruffett, 1971). The majority

VOLUME 37,1974

(26) of ventricular cells were interphase cells whose processes spanned the retinal wall thickness (e.g., cells 16, 17, 44, Fig. 4), while seven of the ventricular cells were in prophase (e.g., cells 50, 15, 6, 11, Fig. 4). Only one cell was found on section 249 that was in metaphase (cell 3, Fig. 4), and none in anaphase or telophase. Of the 17 postmitotic young neurons, the great majority (14) were clearly unipolar ganglion cells with an axon projecting into the marginal layer and coursing toward the optic nerve (e.g., all cells drawn in Fig. 10,A). Two cells, however, appeared to be in a bipolar stage transitional between ventricular cells and unipolar ganglion cells (cells 26 and 37, Fig. 17,A). Finally, one cell (cell 58, Fig. 10,B) could not be easily classified, since it appeared to be a postmitotic young neuron but possessed a thick process extending into the layer of ganglion cells rather than a thin one projecting to the marginal layer. Thus it is clear that transitional cells make up a very small sample of the total number of cells. However, by searching the entire set of serial sections (a usable population of 250-300 cells) for transitional cells, we were able to obtain a total of 13 complete and 5 partially complete reconstructions of cells transitional between ventricular and unipolar ganglion cells (Figs. 17,A; 18). Ventricular

Cells

The data on ventricular cells in the present study directly confirm in the retina the inferences on the ventricular cell generation cycle made previously by combined Golgi and electron microscopic analysis of the cerebral vesicle (Hinds and Ruffett, 1971). It is likely, therefore, that the ventricular cell cycle as described here is quite general throughout the early development of the CNS. In the present study we have been able to confirm by complete cell reconstruction that prophase nuclei, which in early prophase tend to be located in the vitreal part of the ventricular layer (Fig. 4,

HINDS

AND HINDS

Retinal

Ganglion Cell Development

385

FIG. 1. Light micrograph of optic cup of mouse embryo of 13 days gestation. In the posterior retina, the inner two layers of nuclei in the neural retina are somewhat lighter than the rest; they belong to ganglion cells whose axons form a layer of optic nerves in the adjacent marginal layer. The rest of the nuclei, which are crowded together and radially oriented, belong to the proliferative ventricular cells. The ventricular lumen (vi of the optic vesicle is nearly obliterated and is located between the pigment epithelium (PE) and the neural retina (DIR). Rectangle outlines approximate size and position of the block face which was serially sectioned. L. lens; Vi, vitreous body. x 210.

cell 50; Fig. 26, *), migrate to the ventricular surface within an intact process reaching to the vitreal surface (cells 50, 15, 6, and 11, Fig. 4). Furthermore, we also have given support to the suggestion (Hinds and Ruffett, 1971) that much of the peripheral process (vitreal process in the case of the retina) is lost at metaphase when the cell completely rounds up to divide. A prometaphase cell was found (cell 116, Fig. 4) which shows an extreme attenuation of the vitreal process just distal to the cell body; it seems extremely unlikely that much more cytoplasm would be able to migrate

into the cell body through such a small constriction in the short time left before rounding up at metaphase. Furthermore, just vitreal to a metaphase cell (cell 3, Fig. 6) was found a bundle of tiny processes. smaller than any intact processes that were found and in the same position as a vitreal process, but not connecting with the cell body. The most likely explanation for this bundle is that it represents the breakdown of the vitreal process when it becomes separated from the cell body as the latter completely rounds up. During early interphase the two daughter cells remain at-

DEVELOPMENTALBIOLOGY

VOLUME %‘,I974

halfway thro wh FIG. 2. Montage of two electron micrographs of the neural retina taken from approximately the seri al section series (section 249). The border (dashed line) between the ganglion cell layer (GCL) and the

HINDS

[CELL

AND HINDS

Retinal

Ganglion Cell Development

#441

FIG. 3. A semidiagrammatic illustration of the method used for cell reconstructions (see Materials and Methods). Profiles drawn at correct distance from vitreous body and ventricle (AI were superimposed for a plane of section view (C), and the same profiles were used to plot a view at right angles to the plane of section. taking into account the thickness of the thin sections (B). Except where noted, all reconstructions in subsequent figures are the right-angle view. This interphase ventricular cell (44) has a vitreal process reaching to the vitreous body (bottom line) and a ventricular process reaching the ventricular surface (top line) and containing a pair of centrioles and a cilium projecting into the obliterated optic ventricle between the neural retina and pigment epithelium. Note that the.vitreal process contains a thin. sheetlike portion. whose shape can be best appreciated by looking at both views. Diagonal lines indicate the sheetlike areas of processes in this figure and in Fig. 4. Black irregular shape inside cell indicates the position of the Golgi complex in this and subsequent drawings. x 800.

tached to one another by a midbody, and their perikarya and processes tend to lie very close to one another (Fig. 4, cells 1 and 2, 9 and 10). As demonstrated also in Hinds and Ruffett (1971) the growth cone of the vi.treal process of early interphase ventricular cells is a relatively simple structure (Fig. 4, cells 5, 18, and 45; Fig. 9) compared with axonal growth cones (Figs. 11 and 15). The ultrastructure of ventricular cells resembles that described in Hinds and Ruffett (197 1); some additional features

are shown in Figs. 5-9. Other structures found in ventricular cells but not depicted in these figures are multivesicular bodies, lipid droplets (particularly in the vitreal processes passing through the marginal layer), and occasional attachment junctions between ventricular processes and pigment epithelial cells. A great deal of’ difference in cytoplasmic density is often apparent between adjacent cell bodies or processes of ventricular cells. For example. in Fig. 7 the cell bodies of cells 1 and 2 are

ventricular layer (VL) is not sharp because the cell bodies of each cell type (as determined from analysis of serial sections) can sometimes penetrate a short way into the other layer. Fascicles of optic nerve axons in crops section can be seen in the marginal layer (MgL). All cells whose nuclei appear in this picture were reconstructed in their entirety. Two cells (26 and 37) are labeled which on the basis of the reconstructions were found to be transitional between ventricular cells and ganglion cells with axons. However. at this stage their nuclei and perikarya are indistinguishable from those of ventricular cells. Cell 74 is unusual in containing numerous large dense bodies and lipid droplets; it is interpreted as a macrophage. PE, pigment epithelium; Vi, vitreous body. 2000.

388

DEVELOPMENTALBIOLOGY

VOLUME 37.1974

FIG. 4. Reconstructions of a representative sample of the variety of ventricular cells, arranged with interphase cells on the left (16, 17, 44) followed by a succession of prophase cells (50, 15, 6, ll), a prometaphase cell (116), a metaphase cell (3), two early interphase cell pairs still attached by a midbody (cells 1 and 2, 9 and lo), and three early interphase cells regrowing their processes toward tthe internal limiting membrane at the vitreal surface. Note that all interphase ventricular cells have a pair of centrioles and a cilium near the ventricular surface. Cells 9 and 10 have been shown in the plane of section view; all others at right angles to the plane of section. In this and subsequent drawings, horizontal dashed lines separate the three principal layers at this stage: marginal layer (MgL), ganglion cell layer (GCL), and ventricular layer (VL). Vertical dashed lines show where the process of cell 1 leaves the serial section series. x 800.

much lighter than adjacent ventricular processes. We have been unable to correlate this difference in cytoplasmic density with the stage in the generation cycle of ventricular cells or with any other characteristic. In fact, the cytoplasmic density can vary considerably from one part of a cell to another. Sometimes the decrease in cytoplasmic density in ventricular processes appears to be accompanied by an increase in the number of microtubules; other instances can be found, however, where few microtubules can be found even in quite light cytoplasm. Thus the significance of these cytoplasmic density variations remains completely unknown. Unipolar

Ganglion

Cells

The variety of unipolar ganglion cells seen at El3 in the mouse retina is depicted in Fig. 10,A. All are without any ventricu-

lar process or with only a very small one. The cell body is located at various levels in the ganglion cell layer, or sometimes entirely (cell 91) or partially (cells 56 and 62) in the ventricular layer. The axon arises at or near the vitreal pole of the cell, either close to (cell 56, Figs. 10 and 14) or at some distance from the perinuclear region (cell 64, Figs. 10 and 13). Some cell bodies are already in definitive position near the level of the optic nerve layer (cells 72,70, and 73, Fig. 10); in these cells the axon emerges from the perikaryon and courses in a tangential direction from the start. A few ganglion cells (e.g., cells 67, 68, Figs. 10 and 14) have a short process projecting from near the base of the axon. It is likely that this represents vestiges of an axonal growth cone, but it is also possible that it is the first dendritic process sprouting, since such sprouts can occur on the base of the

HINDS

AND HINDS

Retinal Ganglion Cell Development

axonal processes in young ganglion cells (Ramon y Caja, 1972). As is apparent in Fig. 10,A the Golgi complex and pair of centrioles of unipolar ganglion cells are located either in lateral or ventricular juxtanuclear position in the cell body. Many unipolar ganglion cells also contain a ciliurn, but others do not, particularly ones near the marginal layer. No simple generalization was found that consistently predicted which cells would have cilia and which would not. We also failed to find any pattern for correlating the position of the optic nerve axon in the marginal layer with the shape or stage of development of the ganglion cell. Perhaps a pattern could be found if it were possible to follow the axons for longer distances. Figure 10,B depicts a cell (58) which, although presumably a differentiated neuron, does not resemble the unipolar ganglion cell. It has a dendritelike process coursing near the border of the ganglion cell layer and the ventricular layer. Furthermore, unlike all the unipolar cells encountered in this study, its Golgi complex and centrioles are located in the vitreal part of the perikaryon. By its resemblance to the mature amacrine cell (Ramon y Cajal, 1972; Raviola and Raviola, 1967), it is thus possibly an immature form of this retinal cell type, which is thought from autoradiographic studies to be a relatively early forming neuron (Sidman, 1961). It is also possible, however, that it is an aberrant form of a ganglion cell or even an early stage of a macrophage. Only with further work at later stages when amacrine cell formation is more prevalent (Sidman, 1961) can we hope to settle this question. The general ultrastructure of unipolar ganglion cells is depicted in Figs. 13-15. In addition to the structures depicted there, the following have also been found in unipolar ganglion cells: coated vesicles, both in the perikaryon and in the axon; lipid droplets (rare); and puncta adherentia joining the ganglion cell with adja-

389

cent ganglion cells or with ventricular cell processes. Puncta adherentia, however, are extremely scarce, most cells not showing any clear examples even when the cell membrane is examined on every third section all the way through the cell. Cells Transitional between Ventricular Cells and Unipolar Ganglion Cells A total of 18 cells has been found that are intermediate in morphology between ventricular cells and unipolar ganglion cells. All have been reconstructed and are shown in Figs. 17,A and 18. In Fig. 17,A the cells have been arranged approximately in increasing degree of development from left to right. In the El3 mouse retina the first morphological changes that clearly distinguish future ganglion cells from early interphase ventricular cells are the beginning detachment of the ventricular process from the junctional complex near the ventricular surface and the beginning migration of the centrioles and cilium from the ventricular region (cells 26, 132, 131, 92, 37. and 111, Fig. 17; cell 92, Fig. 5). These changes start when the vitreal process is still growing into the ganglion cell and marginal layers (cells 26, 132, 131, 92, Fig. 17); at later stages of ventricular process withdrawal and centriole migration (cells 37, 111, 85, 86 and 99, Fig. 17) the tip of the vitreal process remains in the marginal layer and starts to arborize slightly, The perikaryon of these early bipolar ganglion cells usually has migrated further from the ventricle following division than that of most early interphase cells at a similar stage after mitosis (Figs. 4, 17, 26). The next important development is the transformation of the region near the end of the vitreal process into an axonal growth cone oriented tangentially (cells 81, 75, 102, 110, Fig. 17). Sometimes this tangentially directed axon branches and includes processes coursing in an opposite direction from the optic stalk (cells 102, 110, Fig. 17); presumably these incorrectly directed

390

DEVELOPMENTALBIOLOGY

VOLUME 37,1974

HINDS

AND HINDS

Retinal

processes will be withdrawn during further development. The time at which the centrioles and Golgi complex finish migrating from the ventricular process to the perikaryon appears to be quite variable, as does the time of complete withdrawal of the ventricular process. In one bipolar cell (cell 99) the centrioles and Golgi complex have migrated to the perikaryon before any clear axonal growth cone has appeared at all; more usually these structures are still in the ventricular process at this bipolar stage (cells 26, 132, 131, 92, 37, 111, 85, 86, Fig. 17). Sometimes the centrioles and cilium appear to migrate passively as the ventricular process is withdrawn, since they are found at or near the very end (cells 111,

Ganglion Cell Development

391

85, Fig. 17). In other cases, however, the centrioles and cilium migrate partly or entirely into the perikaryon while the ventricular process remains in an extended condition (cells 37,86,99, Fig. 17). A similar variation occurs in later stages after the axon has started growing out. For example, cells 102 and 110 (which have both lost their cilium) have their centrioles and Golgi complex in the perikaryon while still trailing a relatively long ventricular process. Cell 81 is similar to cell 102 in the extent of its axon outgrowth and centriole migration, but no trailing process is found. In two other cells with beginning axon growth (75 and 101) the centrioles and cilium still remain at some distance from the nucleus _--.-

-

FIGS. 5-9. Electron micrographs of selected aspects of ventricular cells from El3 mouse retina. FIG. 5. A prometaphase (or possibly an early metaphase) cell (116) just before completion of rounding up. As seen in the reconstruction of this cell (Fig. 4), a markedly attenuated portion of the vitreal process occurs near the cell body; the initial part of this attenuated process can be seen (arrow). In other sections this cell reaches the ventricular surface and forms a junctional complex with adjacent ventricular end feet. Cross section of a ventricular cell cilium in the ventricle is circled. Even at this low magnification it can be seen to have a light center, and higher magnification pictures confirms that it has 9 pairs of peripheral microtubules and no central ones. Double arrow points to a centriole at a short distance from the ventricular surface; reconstruction of this cell (cell 92. Fig. 17) discloses that it is at a very early stage of transformation into a neuron. x 4100. FIG. 6. The large round mitotic cell (3) near the ventricle is a metaphase cell; in its center is a centriole with spindle microtubules inserting themselves near it. In adjacent sections the cell forms typical junctional complexes with adjacent ventricular processes. In the vitreal direction the cell gives off irregular blebs that in serial sections do not continue for an appreciable distance. A bundle of very small profiles (between large arrows) could be followed for a considerable distance toward the layer of ganglion cells before breaking up. A typical ventricular cell Golgi complex (GC) can be seen in one of the ventricular processes, while in another one considerable amounts of granular endoplasmic reticulum (GER) are visible. At the ventricular surface numerous end feet are joined by junctional complexes; isolated puncta adherentia (small arrows) may also be seen joining ventricular cells. Note how the ventricular process of the starred cell (*) appears to have been pushed into a curve by the rounding up of the metaphase cell (3). x 9700. FIG. 7. A pair of early interphase cells (1 and 2) still attached by a midbody (not visible in this section). Note the similarity of their nuclei and their widened nuclear envelopes as well as their position one above the other. A drawing of the reconstructed cells appears in Fig. 4. Base of cilium of cell 2 indicated at arrow; the short cilium of this stage projects into a closed vacuole, not yet communicating with the ventricle. At left a section of a metaphase cell (3) near its equatorial region. x 4100. FIG. 8. Vitreal processes of a daughter pair of early interphase cells, one at its growing tip (9) and the other (10) taken proximal to the growing tip. In the simple growing tip of cell 9, microfilaments are abundant as well as a few ribosomes and vesicles; farther away from the growing tip (cell 10) ribosomes are more numerous and mitochondria occur. x 24,000. FIG. 9. Three end feet of interphase ventricular cells forming the internal limiting layer adjacent to the vitreous body. Separating the end feet from scattered collagen fibers and mesenchymal cells of the vitreous body is a continuous basal lamina. Dark staining end feet contain mitochondria, dense bodies (double arrows), ribosomes. smooth endoplasmic reticulum, and coated vesicles in various stages of formation (arrows). Just subjacent to the dense-staining cell membrane facing the basal lamina is a feltwork of microfilaments. Light profiles cut in cross section are optic nerve axons (Ax) of the marginal layer. They contain longitudinally oriented microtubules, abundant smooth endoplasmic reticulum, and a few ribosomes. x 14,000.

392

DEVELOPMENTALBIOLOGY

VOLUME 37.1974

FIG. 10. (A) Reconstructions of representative ganglion cells with axons. All have an axon running tangential to the surface in the marginal layer (MgL). All are essentially unipolar in configuration with Golgi complexes and centrioles (sometimes with a cilium) located in the lateral or ventricular parts of the perikaryon. Vertical dashed line on end of axons shows where axons leave the serial section series. (B) Reconstructed cell which did not fit the configuration of the unipolar ganglion cells. See text for explanation. VL, ventricular layer; GCL, ganglion cell layer. x 800.

in the bluntly ending ventricular process. The only branched axons found in the present study occur on late transitional cells 102 and 110 that appear to have just started growing their axons. These two cells closely resemble a cell drawn by Ramon y Cajal (1909: Fig. 242) in a reduced silver study of the early chick retina. In agreement with these findings, Goldberg and Coulombre (1972) have recently reported, in a whole-mount silver study of the developing chick retina, that the only branched axons that occur bifurcate close to the cell body, with each branch extending only 5-10 pm. In Fig. 17,B is pictured a cell (82) which could not easily be placed in the sequence of ganglion cell formation. It appears to be a relatively young neuron since the centrioles and cilium are still at a distance from the nucleus, in the bluntly ending ventricular process. Although the cell has two processes extending radially, one of which just reaches the marginal layer, it also possesses another branching process cours-

ing obliquely into the ganglion cell layer; and this latter process appears to be the one with more growth activity. This cell may be an aberrant ganglion cell precursor in which either the oblique or radial process is destined to grow eventually into the marginal layer and become an optic nerve axon, or the cell may be destined to degenerate. Another possibility, however, is that it is a very immature amacrine cell. If cell 58 (Fig. 10,B) is assumed to be an immature amacrine cell, this cell could then be a cell transitional between it and the ventricular cells. Under this interpretation the radial process might be a remnant of an earlier, more epithelial-like stage of development that is destined to degenerate. The ultrastructure of cells transitional between ventricular and unipolar ganglion cells (Figs. 20-32) is unremarkable. In general, early stages resemble ventricular cells and later forms resemble unipolar ganglion cells. The only consistent ultrastructural difference that we could detect between bipolar ganglion cells and early

HINDS AND HINDS

Retinal

393

Ganglion Cell Development

011

FIG. 11. Drawings of wax reconstructions of optic nerve growth cones in the marginal layer. Actual outline of the profiles as seen in electron micrographs is shown for selected sites. One growth cone is relatively simple, the other quite complex, but both are marked by prominent sheetlike foliopodia and occasional threadlike filopodia. x 1800

012

FIG. 12. Reconstructions of the two macrophages found in the serial sections. Note the extremely complicated shape of one of them (107). An electron micrograph through the other one (74) appears in Fig. 2. Cell 107 is shown in the plane of section view; cell 74 in the right-angle view. x 800.

interphase ventricular cells is the position of the centrioles and cilium. Some of the bipolar ganglion cells have very lightly stained cytoplasm, especially in the ven-

tricular process, which is almost entirely filled with microtubules. These cells can perhaps be distinguished from ventricular cells by this characteristic alone. However, other bipolar ganglion cells (e.g., cell 37, Fig. 2 or cell 85, Figs. 20-25) have a cytoplasmic density and organelle content indistinguishable from that of ventricular cells. Thus, microtubule-rich and lightstaining processes are a sufficient but not a necessary condition for identifying a postmitotic ganglion cell precursor at, this time. The criterion is somewhat difficult to apply, since some ventricular cells have quite light-staining processes with a fairly high number of microtubules. All the transitional cells encountered except two advanced ones (cells 102 and 110, Fig. 17) still have a cilium. A much larger percentage oi unipolar ganglion cells do not have a ciliurn. Thus the cilium is apparently always formed after mitosis, but in some cells it is lost later in development. Daughter

Cell Pair Analysis

It is possible by examination of the serial electron micrographs to identify daughter

394

DEVELOPMENTALBIOLOGY

VOLUME 37,1974

HINDS ANDHINDS

Retinal Ganglion Cell DeL~elopment

pairs of’ recently divided cells with a high degree of certainty, even in cases where their midbody connection has been lost. The ventricular processes always touch each other along nearly their whole extent, as do the cell bodies and parts of the vitreal processes (Fig. 4, cells 1 and 2, 9 and 10; Figs. 20-26). Furthermore, the density of the nucleoplasm and the width of the nuclear envelope is the same, and the density of the cytoplasm is usually the same, in both members of the daughter cell pair (Fig. 7). For a given early interphase or early transitional cell there is only one other cell that has all the above characteristics, and this cell is presumed to be its daughter cell mate. In the present study the daughter cell mates of nearly all the early transitional cells (bipolar ganglion cells before axon outgrowth) have been identified as well as some of the late transitional cells, and these pairs have been reconstructed together to show their

:19.5

interrelationships (Fig. 18). It is also possible to plot a projection onto the vitreal surface of the nuclei of all the 18 cells transitional between ventricular cells and unipolar ganglion cells. along wit,h their daughter cell mates where known (Fig. 19). The results show that of the 18 transitional cells, 10 are definitely paired with another transitional cell in five pairs (R/78. 83184, 261132, 85192, 75/86), and another transitional-transitional cell pair is likely but cannot be definitely established since the ventricular processes have been largely withdrawn (81/110). In the case of cells 77178 and 83184 it might appear that there is not enough of the cells contained in the sections to be sure that they are pairs. However, in both these cases both members of the pairs have ventricular processes strikingly lighter than the surrounding processes of ventricular cells and virtually identical to each other. Two transitional cells have ventricular cell mates ( I:< 1: X 1;

FIG. 13. Electron micrograph of a unipolar ganglion cell (64) showing an irregular nucleus, blindly ending ventricular cytoplasm, and a vitreal cytoplasm which gradually tapers into an axon (Ax) that turns in subsequent sections into tangential orientation. With increasing distances from the perikaryon. the relative number of ribosomes gradually decreases and the relative number of microtubules increases. A reconstruction of this cell appears in Fig. 10. Perikarya of two more unipolar ganglion cells (63. 71) are also shown. Dashed line separates the layer of ganglion cells (GCL) from the marginal layer (MgL) containing mostly optic nerve axons cut in cross section. Also visible in the marginal layer are darkly stained vitreal processes of Interphase ventricular cells expanding at the surface to form the limiting layer. ,< 4900. FIG. 14. Electron micrograph of portions of several unipolar ganglion cells. Reconstructions of four of these (56, 67. 68, 73) are shown in Fig. 10. The typical irregularly scattered granular endoplasmic reticulum of unipolar ganglion cells is shown in cells 73 and 67. and cell 67 also displays a portion of the Golgi complex (GC). Both cells have dense bodies, and cell 67 shows in addition one member of a pair of centrioles (double arrow). Cell 68 shows a blindly ending projection (arrows) from its vitreally directed axonal process: this is unusual for unipolar ganglion cells at this stage. A sharp transition is visible between the ribosome-rich perikaryon of cell 5ti (just visible in the upper right-hand corner) and the axon proper, containing largely microtubules and smooth endoplasmic reticulum. Note the isolated profile of the axon of cell 67; its connection occurs in a nearby section. x 10,000. FIG. 15. Electron micrograph of marginal layer showing a bundle of axons cut in cross section (Axi and two axonal growth cone profiles (*) wrapping around fascicles of axons in a manner reminiscent of an astroglial process in an adult. Axons contain abundant, longitudinally oriented microtubules, smooth endoplasmic reticulum, mitochondria, and occasional ribosomes. The core of the growth cone (*I contains smooth endoplasmic reticulum, microtubules, and mitochondria, while the foliopodia arising from the cores appear to contain only microfilaments and vesicles. These axonal growth cones could be followed back in serial sections to completely typical optic nerve axons. Reconstruction of similar axonal growth cones are shown in Fig. 1 I, 13,000. FIG. 16. Electron micrograph of two portions of a macrophage cell (107). Lipid droplets are prominent in the cytoplasm, which sometimes attenuates into exceedingly thin sheets (arrows). A reconstruction of this cell appears in Fig. 12. x 3700.

396

DEVELOPMENTALBIOLOGY

VOLUME 37, I974

FIG. 17. (A) Reconstructions of nearly all the cells found which were transitional between ventricular celh and unipolar ganglion cells. Dotted lines on outline of cell 101 indicate that this part was not in the seria sections; it was added in order to complete the outline of the cell. This cell shows a trough-shaped foliopodiun in the marginal layer just before it leaves the serial section series. Vertical dashed lines show where axons leave th4 serial section series, (B) A cell which could not easily be fitted into the main sequence of ganglion cell formation from ventricular cells. MgL, marginal layer; GCL, ganglion cell layer; VL, ventricular layer. x 800.

FIG. 18. Reconstructions of transitional cells for which probable daughter cell mates could be found. Both right angle view (A) and plane of section view (B) have been drawn to show the close apposition of the members of each pair; one member of each pair has been shaded. All the pairs shown appear to consist of two transitional cells except 31/37 and 131/133 in which one member is a transitional cell and the other is a ventricular cell. Dotted line completing profiles of cells 77/78 and 38/84 indicates that portion was outside the serial section series and was drawn in probable position to make drawing clearer. Vertical dashed line in pair 83/84 in (A) indicates the end of the serial section series; for clarity, such a line has been omitted from cells 77/78. In cell 110 in (A) the full extent of the reconstructed axon (see Fig. 17) was not drawn because of lack of space; this is indicated by two perpendicular lines. x 800.

HINDS AND HINDS

Retinal

Ganglion Cell Development

397

FIG. 19. A diagram of the tissue block that was serially sectioned, as viewed looking from the ventricular surface toward the vitreal surface. The transitional cell nuclei. stylized as circles, are plotted in accurate position within the block and shown projected onto the vitreal surface. Short side of rectangle represents the thickness of the block that was serially sectioned, while the long side is the width of the electron microscopic montage. Since daughter cell pairs tend to be in approximately the same position but one above the other (e.g.. Fig. 8), one would expect to see their nuclei overlapping or touching on this diagram; this expectation is in fact borne out. Based on examinations of the serial sections and reconstructions (Fig. 18) nine pairs have been identified. In the case of 84 and 77 only the ventricular process was available in the serial sections: these cells are represented by a smaller circle. The missing corner arises because at section 145 we switched from a montage overlapped on the shorter dimension of a 3’ 4 x 4 inch plate to one overlapped along the longer dimension. Transitional cell nuclei have been indicated by shaded nuclei, ventricular cells by clear circles. The identity of cell 134 (the mate of cell 111) could not be determined because the end of its ventricular process was beyond the last section of the series and no centriole could be found: its unknown status is indicated by parallel diagonal lines. It was not possible to find the mate of certain more advanced transitional cells (99. 101, 10“). b 1300.

the one at an earlier stage of development (131) is combined with an early interphase cell, and the one at a later stage (37) is combined with an interphase cell spanning the retinal wall. One more transitional cell (99) almost certainly has a ventricular cell mate since no other unpaired transitional cell is found anywhere near it (Fig. 19); its daughter cell mate could not be definitely determined, however. Finally, in the case of three transitonal cells no statement can be made about their daughter cell mates, in one case (111) because the pair of centrioles of its mate is off the edge of the serial sections and in two other cases (cells 101 and 102) because the cells themselves are advanced transitional cells that have had more time since division to lose their close relationship with daughter cell mates. Thus there is definite evidence: (1) that some cells divide at the ventricle and give rise to two transitional cells and (2) that

others divide and give rise to one ventricular cell and one transitional cell. The first possibility to consider in attempting to explain this result is that the transitional cells have as mates either transitional cells or ventricular cells at random, according to the proportion of these two cell types in the total population; perhaps because of our small sample size, by chance we obtained a few more transitional-transitional cell pairings than would be predicted on the average. The proportion of transitional cells to ventricular cells at similar times after mitosis can be roughly estimated for the mouse, if we assume that as in the early chick retina, it takes approximately 10 hr from the end of the DNA synthetic phase (S phase) for the perikaryon to migrate into the ganglion cell layer (Sechrist, 1969). Allowing 1 hr for the postsynthetic period (G,) and 1 hr for mitosis, we are left with 8 hr for the time to differentiate through

398

DEVELOPMENTALBIOLOGY

early and late transitional cell stages. The generation time of retinal ventricular cells at El3 in the mouse can be estimated from Sinitsina’s (1971) data to be approximately 12.5 hr; thus the population of ventricular cells within 8 hr of mitosis would be 8112.5 of the total number of ventricular cells. This latter number is approximately 204 (four times the number, 51, found on section 249), since the average thickness of the nucleus is about 100 sections and there are somewhat more than four times as many sections in the whole series (441). Thus there are approximately 8/12.5 (204) = 131 ventricular cells within 8 hr from the last mitosis, compared with only 18 transitional cells. Thus, in round figures, transitional cells make up 18/150 or one-twelfth of the total number of cells postmitotic within 8 hr of mitosis, and ventricular cells make up the other eleven-twelfths. It can be calculated2 that on the assumption of random pairings the chances of getting the five transitional-transitional cell pairs out of the nine possible pairs is only about 0.0005. Thus we can safely conclude that there is a definite tendency for both daughter cells of certain mitotic cells to give rise to postmitotic ganglion cell precursors. On the other hand, it is also clear that sometimes one daughter cell gives rise to a 2Given the presence of transitional cells (T) and upon random pairing the ‘M’ pairs can be expressed

150 cells, of which 18 are 132 are ventricular cells, probability of obtaining k by the following formula:

(114 + 2k)! 2’s’+k’ .(57 + k)! (150)! 2’5’(75)!

1

When computed, the following probabilities are obtained: Pr (0 TT) = 0.3141; Pr (1 TT) = 0.4142; Pr (2 TT) = 0.2108; Pr (3 lT) = 0.0532; Pr (4 TT) = 0.00720; Pr (5 TT) = 0.000523. The sum of the above six probabilities (rounded to four decimal places) is 1 .oOOo.

VOLUME 37,1974

bipolar ganglion cell and one remains a ventricular cell presumably destined for another round of DNA synthesis and division. The possible implications of these findings are taken up below in the Discussion. Quantitative Study of the Development of the Nucleus, Go&i Complex, and Granular Endoplasmic Reticulum during Early Ganglion Cell Differentiation

The quantitative development of the nucleus, Golgi complex and granular endoplasmic reticulum (granular ER) during the differentiation of unipolar ganglion cells from ventricular cells is given in Table 1 and Fig. 33. The size of the nucleus of unipolar ganglion cells shows considerable overlap with that of early interphase cells (Table 1) and is significantly smaller (I’ < 0.01) than that of prophase cells. There is a significant increase in the size of the nucleus between early and late transitional cell stages (P < 0.05) and between early transitional and unipolar ganglion cell stages (P < 0.01). The amount of granular ER shows a definite trend upward (Fig. 33) from early transitional cell to unipolar ganglion cell (P < 0.01); nevertheless, the amount of granular ER in unipolar ganglion cells has extensive overlap (Table 1) with that of prophase cells, and even overlaps somewhat with that of early interphase cells. In ventricular cells, however, the amount of granular ER is not as obvious as in unipolar ganglion cells, since it is mostly in the ventricular process at a distance from the nucleus and thus not readily recognized in single sections. It is interesting to note that a few late transitional and unipolar ganglion cells (Table 1) have much larger amounts of granular ER than the others. The average length of ER profiles shows a striking dichotomy between ventricular cells and young neurons (Table 1; Fig. 33). The three types of young neurons (early transitional, late transitional, and unipolar ganglion cells) show no

HINDS AND HINDS

Retinal

significant differences between them in average length of ER profiles, but all three have significantly shorter average lengths than either of the two types of ventricular cells measured (early interphase and prophase cells). The significant difference between early interphase and early transitional cells (P < 0.05) is particularly noteworthy since this difference represents the only significant difference between these two cell types of any of the structures studied quantitatively. Another significant trend during early development of ganglion cells is the increase in the size of the Golgi complex. Although considerable variation exists from cell to cell within groups, nevertheless from early transitional through late transitional to unipolar ganglion cells the average size of the Golgi complex increases (Fig. 33), and the increase is particularly striking between early and late transitional cell stages (P < 0.01). Note, however, that there is still considerable overlap in the size of the Golgi complex between unipolar ganglion cells and prophase cells. DISCUSSION

Proposed Sequence of Differentiation A summary of the proposed sequence for the differentiation of ganglion cells is outlined in Fig. 34. Reconstructed cells from El3 mouse retina have been selected and arranged into a plausible sequence. According to this scheme the first morphological sign of ganglion cell differentiation is the detachment of the ventricular process from the junctional complex at the ventricular surface and the beginning of the migration of the centrioles and cilium toward the cell body. This occurs in bipolar daughter cells of a mitotic division at a time when their vitreal process has grown toward, but not yet reached, the vitreal surface of the retina. These cells clearly resemble in overall morphology the early bipolar stage of ganglion cell development described in reduced silver stain

Ganglion

CellDevelopment

399

studies of chick embryo retinas by Ramon y Cajal (1911) and Sechrist (1969), and in Golgi impregnations of early postnatal rat retina by Morest (1970b). Transformation of the vitreal process of these bipolar cells into a tangentially oriented axonal growth cone, which insinuates its sheetlike foliopodia among the tangential axons of the marginal layer, generally occurs at about the same time that the centrioles and Golgi complex are completing their migration to a juxtanuclear position. At still later stages of differentiation, as the axon lengthens, the axon shaft near the cell body loses its foliopodia and filopodia and becomes a smooth structure of even caliber. At this time there is usually no sign of a ventricular process, and the cell conforms to the shape of the unipolar neuroblast of His (1889), as described in the early chick retina by Ramon y Cajal (1911) and by Sechrist (1969). Axons are grouped in irregular fascicles, and in serial sections it can be appreciated that individual axons are unbranched, although they can meander from one fascicle to another, just as recently described in a silver-stain, wholemount study of the embryonic chick retina (Goldberg and Coulombre, 1972). In the present study there are no signs of dendrites arising from the cell body, but these will presumably sprout at later stages of differentiation (Ramon y Cajal, 1972, 1911). The perikarya of many of the unipolar ganglion cells are located at some distance from the marginal layer; these must subsequently migrate vitreally (in the direction of the initial portion of their axon) as the ganglion cell layer becomes a single layer of cell bodies during later development (Ramon y Cajal, 1911; Caley et al.. 1972). Some variation occurs in the exact timing of certain of the morphological events, particularly in the timing of perikaryal migration and ventricular process withdrawal. For example, sometimes the perikaryon migrates almost to its definitive position while the ventricular process still

HINDS AND HINDS

Retinal

extends toward the ventricular surface (Fig. 17, cells 102 and 110). Similarly, Ram6n y Cajal (1960) noted that, although almost all bipolar cells with axons in the retina of chicks at the third or the fourth day of incubation show only very small ventricular appendages, exceptions could be found in which the ventricular prolongation still terminated near the ventricular border. In the early postnatal rat retina, Morest (1970b) has shown that the withdrawal of the ventricular process can be even more delayed; in Golgi impregnations he has found such a process on ganglion cells with well developed axons and even on those which have started to sprout dendritic processes. Perhaps, as suggested by results in the olfactory bulb (Hinds, 1972a,b), in older animals the trailing or

Ganglion

Cell Development

301

ventricular process generally takes longer to withdraw. The above summary of the morphological differentiation of ganglion cells needs to be examined critically in order to delimit areas of uncertainty and to make sure that there have been no errors of interpretation. The first important point to establish is whether most of the transitional forms we have described are really ganglion cell precursors, rather than a mixture of precursors of ganglion cells and of one or more other cell types. Although we cannot be sure that all the transitional ceils are ganglion cell precursors, it is extreme11 likely that the great majority are, for the following reason: the reconstruction of all cells whose nuclei were found on section 249 reveals that the great majority of

FIGS. 20-32. Electron micrographs of transitional cells. El3 mouse retina. FIGS. 20-25. Selected sections through the end of the ventricular process of an early transitional cell (85). Shows the appearance of the pairs of centrioles and the cilium (circled in Fig. 22) and also the termination of the ventricular process at a distance from the ventricular surface. Reconstruction of this cell shown in Fig. 17. The ventricular process of the daughter cell mate of cell 85 (cell 92) can be seen to be in close proximity. This daughter cell pair (Fig. 18) is unusual in that the cytoplasmic density of the ventricular processes of the two cells differs. Section numbers of Figs. 20-25 are 284, 290, 296, 302, 305, and 310. x 3700. FIG. 26. Portions of cell bodies of cells 85 and 92 and the proximal part of the vitreal process of 92. From section 152. Note that at the level of the cell bodies the densities of the two cells is approximately the same. Dashed line is at the approximate boundary of ventricular layer (VL) and ganglion cell layer (GCL); these cells thus have migrated nearly to the ganglion cell layer. *, an early prophase nucleus. I 3500. FIG. 27. A portion of the ventricular layer (VL) and ganglion cell layer (GCL) showing several transitional cells. From section 162. Cell 75 is a late transitional cell (reconstruction on Fig. 17) showing in this electron micrograph a broad ventricular process with a pair of centrioles and a cilium near its end (encircled), a small perikaryon, and a vitreal process extending into the ganglion cell layer. A small portion of the nucleus and perikaryon of its daughter cell mate (86) can be seen cut tangentially just ventricular to its cell body; the vitreal process of 86 is next to the vitreal process of cell 75 in the ganglion cell layer. Another daughter cell pair of two transitional cells (132, 26) can be seen to the left of cells 75 and 86. The cell body of 132, the proximal part of its vitreal process and three isolated profiles of its vitreal process further distally are visible in this electron micrograph; next to the proximal part of the vitreal process of cell 132 is that of cell 26; and another profile of 26 is visible in the ganglion cell layer in the same relative position with respect to the process of cell 132. Finally. the cell body, proximal ventricular process, and distal vitreal process of cell 83 is visible still farther to the left x 3300. FIG. 28. Vitreal process of cell 75 as it courses through the ganglion cell layer (GCL) and extends Into the marginal layer (MgL). From section 187. Y 3300. FIG. 29-32. Four views of the axonal growth cones of cell 75 at progressively more distal sections from the cell body: Fig. 29. section 234; Fig. 30, section 261; Fig. 31, section 302; Fig. 32, section 331. Figure 29 shows the axon near where it turns into tangential orientation in the marginal layer: ribosomes, smooth endoplasmic reticulum and mitochondria are visible. In Fig. 30 a foliopodium (arrow) extends away from the main shaft. Figure :{I shows a bulbous enlargement shortly before the termination of the growth cone; it contains many small vesicles, ribosomes, and one very large vacuole. In Fig. 32 is shown a cross section of a foliopodium extending toward the optic nerve. II_ 8000.

402

DEVELOPMENTALBIOLOGY

VOLUME 37,1974

TABLE 1 SIZE OF NUCLEUS, GOLGI COMPLEX, GRANULAR ER, AND AVERAGE LENGTH OF GRANULAR ER PROFILES IN DIFFERENT CELLS OF THE El3 MOUSE RETINA Cell type”

Pro

EI

ET

Cell No.

6 11 15 33 43 48 50 53 1 2 9 10 18 22 36 108 26 37 85 86 92 99 111 131 132

Nuclear volume bmY

2102 1988 1944 2054 1626 1908 2006 2293 1137 1326 1082 1132 1434 1243 1236 1297 1149 1132 962 1211 1142 1616 1343 1075 1096

Golgi complex size (rm’)’ 17.1 35.7 42.9 33.2 11.7 43.6 17.3 22.7 9.9 10.6 18.9 15.8 15.9 24.4 25.8 14.8 8.8 13.5 12.6 17.3 16.9 13.3 29.3 10.2 17.8

Amount Average of gran- length ular ER of granular ER (P)” profiles 150 153 115 114 105 65 101 103 65 85 74 61 24 82 112 92 46 58 45 32 48 43 69 38 87

0.744 0.696 0.720 0.717 0.780 0.795 0.842 0.783 0.726 0.616 0.777 0.732 0.634 0.943 1.077 0.542 0.562 0.589 0.595 0.542 0.699 0.324 0.494 0.568 0.717

Cell type”

Cell No.

Nuclear volume (~m3”

Golgi complex size

w

(prn*Y -

LT

GC

M

75 81 101 102 110 56 57 62 63 64 65 66 67 68 69 70 71 72 73 98 100 74 107

1834 1484 1272 1597 1890 1863 1212 1240 1274 1354 1413 1240 1837 1141 1179 1528 1379 1645 1575 1700 1646 1237 2099

29.0 29.2 22.2 70.6 50.8 62.8 25.1 35.7 36.8 37.9 35.6 54.0 45.7 33.9 60.2 86.2 30.6 36.3 41.3 62.9 52.4 100.3 104.1

Amount Average of gran- length ular ER of granular ER profiles

-

65 68 71 164 282 124 123 145 116 118 109 153 126 110 124 295 62 108 166 193 62 273 202

0.467 0.402 0.524 0.506 0.536 0.574 0.634 0.571 0.458 0.548 0.536 0.655 0.497 0.473 0.545 0.557 0.448 0.750 0.497 0.705 0.378 0.616 0.854

“Pro, prophase ventricular cells; EI, early interphase ventricular cells; ET, early transitional cells (preaxonic, bipolar ganglion cells); LT, late transitional cells (axon containing, bipolar ganglion cells); GC, unipolar ganglion cells; M, macrophage cells. b Estimated by finding major and minor axes and using the formula for the volume of a prolate spheroid. CRelative measure obtained by summing the estimated area of the Golgi complex as measured on every electron micrograph (approximately every third section) through the entire Golgi complex. d Relative measure obtained by measuring the total length of all the ER profiles on every electron micrograph through the entire cell.

nonventricular cells (14 out of 18) are clearly ganglion cells with axons in the marginal layer. Thus, unless one assumes a sudden shift from ganglion cell production to production of some other cell type in a matter of a few hours-a shift which seems precluded by the autoradiographic evidence of Sidman (1961)-the transitional cells should also be largely ganglion cell precursors. Of the four nonventricular cells that are not clearly ganglion cells, one is a

macrophage (74), and two are early transitional cells (26 and 37). Only one cell (58) is a possible candidate for a differentiated young neuron of a type other than a ganglion cell, in this case perhaps an amacrine cell. Another important question is how we know that we have not missed some important transitional forms. For example, Morest (1970b) has described in Golgi impregnations of the early postnatal rat retina an

HINDS

Retinal

AND HINDS

033 2OO@l3-

T

NUCLEUS 1 I

lOOOjI3-

PRO

El

LT

GC

ET

LT

CC

ET

11

CC

ET

GO&l COMPLEX

PRO 3oop-

El

GRANULARER

PRO

El

AVERAGELENGTHOF GRANULAR ER PROfIlES lo4

r

PRO

I

El

ET

11

I

CC

FIG. 33. Data of Table 1 in graphical form. Graphs show relative amounts of certain cell organelles at five different stages: prophase (PRO) and early interphase (EI) ventricular cells, early transitional (ET) and late transitional (L7’j cells, and unipolar ganglion cells (GC). Height of bars equals the median, with the range shown by a line extending above and below it.

apparently typical interphase ventricular cell except that instead of having a vitreal process extending to the vitreal surface, it turns tangential to the surface and courses

Ganglion

Cell Development

40:1

in the marginal layer as an axon. If this type of cell had a pair of centrioles and a cilium at the ventricular surface, there would he no way of detecting it by looking only at forms whose centrioles and cilium had started to migrate vitreally. However, we initially reconstructed all the cells whose nuclei appeared on section 249 of the serial section series. If this form had been common we would have expected to have discovered it. We conclude that the numerous transitional forms that we did find represent the most important, perhaps the only, major varieties of transitional cells at this time. A third question is, even assuming that the sample of transitional cells that we have found represents stages in the differentiation of ganglion cells from ventricular cells, how do we know that the sequence we have portrayed is the correct one. In the present study the extent of axon development has been the primary way of staging the various transitional cells. It is clear that, on the average, forms with no axon should temporally precede ones with a short axon tipped by a growth cone in the marginal layer. This form, in turn, must precede ones with longer axons which leave the set of serial sections. In addition, the extent of migration of the centrioles and cilium and the extent of withdrawal of the ventricular process were used. In agreement with a previous light microscopic study on the retina (Leboucq, 19091, all ventricular cells that we have reconstructed have a pair of centrioles and a cilium near the ventricle and a ventricular process (or the cell body itself in the case of mitotic cells) forming part of the ventricular surface. Furthermore, all unipolar ganglion cells have centrioles and cilium lo cated in juxtanuclear position at a considerable distance away from the ventricle and do not have a ventricular process reaching the ventricular surface. Thus, in the development of ganglion cells the pair of centrioles and the cilium must migrate

404

DEVELOPMENTALBIOLOGY VOLUME37,1974

FIG. 34. Summary of development of unipolar ganglion cells in the El3 mouse retina. All drawings of cells in this summary represent reconstructions from serial sections of actual cells; they have been selected and arranged to indicate a possible sequence of development. 1, an interphase ventricular cell with ventricular and vitreal processes spanning the thickness of the retinal wall; 2, a prophase cell, whose nucleus and perikaryon have migrated to near the ventricular surface, but which still has a vitreal process reaching to the surface; 3, a late prometaphase cell, probably about to lose its vitreal process; 4, a rounded metaphase cell with no intact vitreal process; 5, a daughter cell pair which has just been formed by a mitotic division, with the vitreal processes starting to grow toward the vitreal surface; 6, an early transitional cell (early bipolar cell stage), apparently committed to ganglion cell differentiation, that has lost or nearly lost its attachment to the ventricular surface and whose vitreal process is still growing toward the surface; 7, a later stage of the bipolar ganglion cell in which the centrioles and cilium have started to migrate toward the perikaryon, the ventricular process has fully detached from the junctional complex at the ventricular surface, and the vitreal process is starting to enlarge and branch in the marginal layer; 8, a late transitional cell that has nearly finished withdrawing its ventricular process and its centriole and cilium and has started forming an axonal growth cone in the marginal layer; 9, a nearly mature unipolar ganglion cell whose axon leaves the serial section series, but whose immaturity is revealed by an incompletely migrated pair of centrioles and cilium and by a trough-shaped foliopodium coming off the axon in the marginal layer; 10, a mature unipolar ganglion cell with centrioles and cilium near the nucleus, no ventricular process and a smooth axon coursing into tangential orientation in the marginal layer and running toward the optic stalk. Dotted line on end of process indicates that the process could not be followed farther because it left the serial section series. Centrioles and cilium are shown schematically and the outline of the position of the Golgi complex is shown in black. MgL, marginal layer; GCL, ganglion cell layer; VL, ventricular layer. x 900.

from near the ventricular surface to the layer of ganglion cells, and at some point the ventricular process must be withdrawn. A sequence obtained on the basis of centriole migration or ventricular process withdrawal is similar to but not exactly the same as that obtained on the basis of the extent of axon development. This implies

that the time of axon outgrowth is somewhat variable with respect to the migration of the centrioles and withdrawal of the ventricular process. In the above discussion it has been assumed that all ganglion cells are derived from ventricular cells rather than from, for example, a population of subventricular

HINDS AND HINDS

Retinal

cells, detached from the ventricular surface and dividing near the border of the ventricular layer and the layer of young ganglion cells. This subventricular layer probably exists in many parts of the CNS even quite early in development (Hinds and Ruffett, 1971; Hinds, 1972b), but on the basis of the present study, it appears to be completely absent in the mouse retina at E13. A final point to consider is whether all the cells spanning the distance from ventricular to vitreal surface are undifferentiated and pluripotent ventricular cells or whether some of them might be differentiated Miiller cells. Certainly interphase ventricular cells, particularly ones with sheetlike processes and projections, bear a resemblance to the mature Miiller cell and are presumably performing some of the same supportive functions. In fact it has been suggested on the basis of both Golgi impregnations (Ram6n y Cajal, 1972) and electron microscopic studies (Uza and Smelser, 1973) that Miiller cells are the first cells to differentiate in the retina. Although we cannot exclude the possibility that some of the ventricular cells at El3 are determined already to be future Miiller cells, we do think it unlikely that there are any presumptive Miller cells which have become permanently postmitotic, for the following reasons: (1) Sechrist (1969) has shown in the chick embryo retina, at a stage comparable (3.5 days in incubation) with that of the present study on the mouse, that after 10 hr of cumulative labeling with tritiated thymidine all ventricular cells are labeled and the only unlabeled cells are the ganglion cells; (2) Waechter and Jaensch (1972) have shown in the rat by means of cumulative labeling with tritiated thymidine that all cells of the ventricular layer of the E12-El8 cerebral hemisphere eventually take up the label and therefore, presumably, eventually divide (Thrasher, 1966; Cameron, 1968); (3) specific ultrastructural characteristics of adult Miiller cells such as high concentration of glycogen and

Ganglion Cell Deuelopment

405

filaments (Magalhges and Coimbra, 1972; Uza and Smelser, 1973) were not found in the present study in any cell. Comparison of the Sequence of Differentiation of Retinal Ganglion Cells with Neuronal Differentiation Elsewhere in the CNS On the basis of his silver studies on the retina, spinal cord, and cerebral vesicle of early chick embryos, Rambn y Cajal (1960) summarized his concept of the early differentiation of CNS neurons by stating that they usually go through five st,ages: (1) germinative cell of His (1889), (2) apolar or polygonal cell, (3) bipolar cell, (4) unipolar cell (neuroblast of His), and (5) multipolar cell. It is now known (Sauer, 1935; Sidman et al., 1959; Hinds and Ruffett, 1971) that the germinative cell of His, which was unstained in Cajal’s reduced silver preparations. is the mitotic phase of the ventricular cell population. whose interphase members form a pseudostratified epithelium that makes up the rest of the ventricular layer (the spongioblasts of His and Ram6n y Cajal). Cajal’s apolar cells are similar to germinative cells but since they have an affinity for silver, he thought they were specific neuronal precursors. Sechrist (1968, 1969, and personal communication) has confirmed the presence of apolar cells positive for silver in the chick retina and elsewhere in the embryonic chick CNS and has shown by autoradiography and electron microscopy that: (1) the apolar neuroblasts form a small percentage of the mitotic cells next to the ventricle. and (2) they contain considerable quantities of 60 A microfilaments in a loose coil about the nucleus. Possible correlations of Cajal’s apolar cells with cells found in the present study will be taken up below, in the next two sections of the Discussion. The bipolar and unipolar cells of Cajal have clearly been confirmed and documented in the present study so that, at least for the early retina, there should no longer be any ques-

406

DEVELOPMENTALBIOLOGY

tion as to their existence. The present study appears to have been carried out at too early a stage for any multipolar ganglion cells to occur, but presumably they arise simply by sprouting of dendrites from the perikaryon. Thus it can be said that the results of the present study in the mouse retina, as well as those of Sechrist (1969) on the chick retina, have confirmed the classical ideas on the general sequence of early differentiation of neurons (Ramon y Cajal, 1960; Cowdry, 1914; Windle and Austin, 1936; Kershman, 1938; Barron, 1946; Sechrist, 1968). However, this sequence is now known not to be the only one for early neuron differentiation in the CNS, not even in the early CNS, since recent studies have disclosed several other varieties. In the cerebral cortex some of the future cortical cells appear to go through a bipolar, unipolar, and multipolar sequence (Stensaas and Stensaas, 1968), while others clearly migrate to the cortical plate before elaboration of any axon and then either grow out an axon as a separate process or elaborate it from the trailing process (Morest, 1970a; Rakic, 1972). In the olfactory bulb, mitral cells initially form a tangentially oriented cell without any axon; later a precisely oriented axon develops and the cell body migrates toward the surface, forming an extension of the axon in its trail (Hinds, 1972a,b; Hinds and Ruffett, 1973). Still other varieties of early neuron differentiation have been described in Golgi impregnations by Morest in the medulla (1969a), optic tectum (1968a), caudate nucleus (1970a), and hippocampal formation (1970a). It is clear, therefore, that it is not possible to fit the morphological differentiation of all early-forming CNS neurons into a single sequence. It appears that the wide variety of forms of adult neurons (Ramon y Cajal, 1909, 1911) is also reflected in perhaps a narrower but still wide variety of early morphogenesis. Nevertheless, it is possible that the devel-

VOLUME 37,1974

opmental sequence of retinal ganglion cells demonstrated in the present study may closely resemble that of other early forming projecting neurons studied with silver studies: for example, motoneurons of the spinal cord (Ramon y Cajal, 1960; Kershman, 1938; Barron, 1946, Sechrist, personal communication) and brain stem (Cowdry, 1914; Windle and Baxter, 1936; Windle and Austin, 1936; Sechrist, 1968), and neurons in the wall of the early diencephalon (Sechrist, 1968) and cerebral vesicle (Ramon y Cajal, 1960). Further detailed studies of other CNS regions, preferably at the electron microscopical level, will be necessary to determine the truth of the above statement. Ultrastructural Differentiation

Features

of Ganglion

Cell

It is of interest to compare the ultrastructural features of the retinal ganglion cells at defined developmental stages in the present study with previous electron microscopic studies of the early differentiation of CNS neurons. Particular attention will be paid to results on the differentiation of motoneurons in the spinal cord, a neuronal type resembling the retinal ganglion cell in its general development (Ramon y Cajal, 1911) and the subject of several electron microscopic studies (e.g., Lyser, 1964, 1968; Tennyson, 1970a). Nucleus and nucleolus. The present study has shown a significant increase in the size of the nucleus of axon-containing ganglion cells (late transitional or unipolar ganglion cells) compared with preaxonic ones (early transitional cells). Accompanying this increase in size is a change to a more irregular shape with a lighter and ’ more homogeneous nucleoplasm. A dispersal of the nuclear chromatin, resulting in a less dense staining, has been noted by LaVelle and LaVelle (1970) in the spinal cord and by Caley and Maxwell (1968) in the cerebral cortex as characteristic of early stages of neuron development. We

HINDS

AND HINDS

Retinal

would suggest, however, that these changes in the nuclear appearance are not reliable for the very earliest stages in neuron differentiation, at least in the retina, since some of our early transitional cells (bipolar, preaxonic ganglion cells) have nuclei closely resembling those of ventricular cells. We found no consistent difference in the appearance of the nucleolus in ventricular cells and in ganglion cells with axons, even when the whole nucleus was serially sectioned. In both types the nucleolus usually was dispersed into two to four small structures which were either located next to the nuclear envelope or isolated nearer the center of the nucleus. It appears then that in the retina, as in the spinal cord (Tennyson, 1970a; LaVelle and LaVelle, 1970), the development of a single, large, and centrally located nucleolus is a characteristic of later stages of neuron development. Cerztrioles and cilium. Lyser (1968) appears to have been the first author to make a detailed ultrastructural study on the location of the centriole during early neuronal development. She suggested that in the development of spinal cord motoneurons the centriole migrates from the juxtaventricular area to the apical (ventricular) perikaryon during the early bipolar stage and finally into a position adjacent to the nucleus or at the base of the axon during the unipolar stage. In the present study we have been able to confirm this suggestion for retinal ganglion cells. In 11 of the 12 early transitional cells, a pair of centrioles and a cilium were found in the apical (ventricular) process, and in the exceptional cell (cell 99) they were adjacent to the nucleus. In later stages of development the pair of centrioles was located in a juxtanuclear position except for a few late transitional cells, where it was still in the blindly ending ventricular process. As Lyser (1968) has already pointed out. the apical location of the centrioles at all stages of early develop-

Ganglion

Cell Development

407

ment is incompatible with the idea of Martin and Langman (1965) that young neurons in the spinal cord and elsewhere in the CNS might stem from the peripherally located daughter cell of a mitosis whose spindle was oriented perpendicular to the ventricular surface (see also Langman et al., 1966; Martin, 1967). The presence of a pair of centrioles in early neurons had been noted before (e.g., Lyser, 1964, 1968; Gonatas and Robbins, 1965; Hinds, 1972b), but only in the present study has it been shown that this organelle is a constant feature of all early ganglion cells, and that furthermore, it is a useful indicator of the stage of differentiation of the cell in which it lies. In fact, in the present study the beginning migration of the pair of centrioles and cilium, along with the concurrent detachment of the ventricular process from the ventricular surface, was the first morphological change that definitely could be attributed to specific differentiation of a ganglion cell from a ventricular cell. Golgi complex. In the El3 mouse retina the extent of the Golgi complex is highly variable from cell to cell; nevertheless, a clear progression toward larger Golgi complexes is evident during the course of differentiation into a unipolar ganglion cell with axons. It is also clear, despite assertions to the contrary, that a well formed Golgi complex is found in all ventricular cells except cells in metaphase and anaphase; however, the Golgi complex is not conspicuous because of its location in the ventricular process, often at a considerable distance from the perikaryon. In fact, particularly in some interphase and prophase ventricular cells, the Golgi complex can be quite large and well developed, more so than in many bipolar or even in some of the unipolar ganglion cells. In the developing ganglion cells the greatest increase in the size of the Golgi complex occurs between early and late transitional cell stages, correlating with the initiation of axon forma-

408

DEVELOPMENTALBIOLOGY

tion in the late transitional cell stage. In addition, the Golgi complex shifts from the ventricular process, where it is located in all ventricular cells except in ones just before or just after division, into a juxtanuclear position, where it is located in most of the late transitional cells and all of the unipolar ganglion cells. Tennyson (1970a) has noted that the increase in the Golgi complex is the most striking change in the bipolar stage motoneurons of the spinal cord when they are compared with ventricular cells. On the basis of the present study it could be conjectured that the increase in the Golgi complex noted by Tennyson is not so much an absolute increase in the size of the Golgi complex as a shift in its position to the perikaryon, with the result that it becomes visible in single sections through the nucleus. Endoplasmic reticulum. In the present study the granular endoplasmic reticulum (ER) was relatively common in ventricular cells, particularly in late interphase and prophase cells, but was inconspicuous because of its location in the ventricular process, often far from the nucleus. At least in the early mouse retina, therefore, these “undifferentiated” ventricular cells have considerable amounts of granular ER. In fact, some of them contain more than many unipolar ganglion cells. The granular ER becomes more obvious in its perikaryal position in unipolar cells, however, and is, on the average, slightly greater in amount and with a shorter average length of the measured membrane profiles than in ventricular cells. The average length of ER profiles was significantly smaller for all stages of young neuron formation compared with ventricular cells, including early transitional cells compared with early interphase cells. A few of the unipolar ganglion cells of the present study (and one of the later transitional cells) contain relatively large amounts of granular ER compared with the rest; perhaps these cells are destined to become the giant ganglion cells

VOLUME 37,1974

seen in the mature mouse brain (Ramon y Cajal, 1972). In the present study smooth ER has been encountered in large amounts only in differentiated axons, where it is abundant and resembles that previously described in growing axons (Tennyson, 1970b; Hinds, 1972b). Some smooth ER also occurs in the vitreal end feet of ventricular cells. Mitochondria. We have been unable to detect any consistent differences between the mitochondria of ventricular cells and those of unipolar ganglion cells. We would agree with LaVelle and LaVelle (1970) that “practically anything that has been said about the changes in form or character of neuronal mitochondria during the developmental sequence has not yet . . . been satisfactorily separated from the possible influences of extrinsic factors” (p. 143). Dense bodies, multivesicular bodies, and coated vesicles. According to Tennyson (1970a), single membrane-bound dense bodies presumed to be lysosomes are infrequently encountered in ventricular cells but are much more common in later stages of motoneuron development (unipolar and multipolar stages). In the early mouse retina we would agree with this view to the extent that the perikaryon of ventricular cells has relatively few lysosomes and that of the unipolar ganglion cell considerably more. However, lysosomes are quite frequent in vitreal processes of ventricular cells as they pass through the marginal layer and insert themselves as end feet in the external limiting membrane. Thus it is not clear that there is any striking change in total number of lysosomes in the entire cell as development proceeds from ventricular cell to unipolar ganglion cell. Multivesicular bodies can also be found in smaller numbers than dense bodies in both ventricular cells and unipolar ganglion cells. Pale bodies not apparently membrane-bound are encountered fairly commonly in vitreal processes of ventricular cells in the marginal layer, but are scarce

HINDS

AND HINDS

Retinal

elsewhere; they are only very rarely encountered in unipolar ganglion cells. They are extremely abundant, however, in the two cells found in the present study (cells 74 and 107, Figs. 2, 12, and 16) which have been interpreted as macrophages. These cells also have large numbers of dense bodies of various sizes and large vesicles with irregular contents presumed to be phagosomes. Macrophages in the developing CNS have been previously described (Stensaas and Reichert, 1971), and they presumably serve to remove excess debris; for example, sloughed vitreal processes of prometaphase cells (Hinds and Ruffett, 1971) and degenerating whole cells (Gliicksmann, 1951). The ubiquity and abundance of coated vesicles in various stages of formation came as a surprise, since, aside from brief mention (Tennyson, 1965; Rosenbluth, 1966), these structures have not been commented on in early CNS development. Their function in developing systems is unknown. Altman (1971) has suggested that in the early postnatal rat cerebellum coated vesicles may be formed in the Golgi complex and migrate to the plasma membrane, where they fuse and give rise to dense membranes for attachment sites or for synapses. In contrast, Birks et al. (1972) have shown with ferritin labeling that, in the axonal growth cone in vitro, coated vesicles form by invagination of the surface membrane and eventually their contents empty into multivesicular bodies and dense bodies, by a process somewhat similar to that described in mature nonnervous tissue (Friend and Farquhar, 1967). In the mature nervous system, coated vesicles have been suggested to be involved with membrane recycling in synaptic transmission (Gray and Willis, 1970; Heuser and Reese, 1973; Turner and Harris, 1973). Cell-to-cell junctions. Aside from the junctional complex between ventricular cells at the ventricular surface, which resembles that described previously (e.g.,

Gangli-m Cell Development

409

Hinds and Ruffett, 1971), the only other specialized cell-to-cell junction detected in the early mouse retina is a small, symmetrical junction resembling the punctum adherens of the adult CNS (Palay, 1967) and the macula adherens diminuta of other developing tissues (Hay, 1968). Puncta adherentia of ganglion cells in the present study were extremely few, but the fact that examples were found both with other ganglion cells and with processes of ventricular cells suggests that these junctions lack specificity at this stage. At later stages of development, at least in the chick (Sheffield and Fischman, 1970), puncta adherentia appear to be much more common. Dixon and Cronly-Dillon (1972) have shown that in the amphibian retina before the beginning of ganglion cell formation, tight or gap junctions exist outside of the junctional complex region. We have not looked carefully at very early stages. and whether such junctions exist in the mouse retina at an earlier, preganglionic cell stage remains to be determined. Microfilaments and microtubules. In the present study we did not encounter aggregations of microfilaments in the perikaryon of mitotic cells, nor in bipolar and unipolar ganglion cells, not even the small aggregations that had occasionally been seen in mitotic cells in a previous study on the cerebral vesicle (Hinds and Ruffett. 197 1) In this regard, the mouse retina differs from the chick retina, where conspicuous aggregates of microfilaments have been found in a small proportion of mitotic cells (about 10%) as well as in bipolar and unipolar young neurons (Sechrist, 1969 and personal communication). Similar cells have been found to be positive for silver staining in the early chick retina (Ramon y Cajal, 1960; Sechrist, 1969). In the chick. therefore, the apolar neuroblast of Cajal appears to be represented by mitotic cells containing large aggregations of microfilaments that displace other organelles from their location. Such an apolar neuroblast.

410

DEVELOPMENTALBIOLOGY

however, appears not to occur in the early stages of development of the mammalian CNS (Windle and Baxter, 1936; Sechrist, 1968). Likewise, in the amphibian retina, Sechrist (1968) found no silver staining prior to the unipolar stage, and this is correlated with a paucity or lack of microfilaments (and microtubules) in electron micrographs of cells of the ventricular layer of the early amphibian retina (Fisher and Jacobson, 1970; Grill0 and Rosenbluth, 1972; Dixon and Cronly-Dillon, 1972). Thus there does seem to be real species difference in the appearance of the earliest stages of differentiation, and recognition of this fact should help to explain some of the discrepancies in the literature. Another important variable is differences among different CNS regions. For example, in the early embryonic mouse spinal cord (unpublished observations) some axons are filled with 60 A microfilaments, while in the present study the only noticeable aggregation of microfilaments are in axonal growth cones and in the vitreal end feet of ventricular cells. It is interesting that Sechrist (1968) has noted that in the chick the end feet of ventricular cells often are positive for silver, a finding which had previously been depicted by Ramon y Cajal (1960). Microtubules are present in all ventricular cells, but particularly common in the ventricular processes of certain of them. Some of the bipolar, early transitional cells have conspicuous numbers of microtubules in the ventricular processes while others in the same stage did not. In the unipolar ganglion cell stage an accumulation of microtubules always occurs in the axon shaft, which starts either at some distance away from the perikaryon, and is separated from it by a ribosome-rich process, or arises directly from the perikaryon. No specializations of the axon initial segment (Palay et al., 1968) could be detected at this time. Axonal growth cone. The present study appears to be the first in which the accu-

VOLUME 37,1974

rate three-dimensional architecture of an axonal growth cone in vivo has been reconstructed from electron micrographs. All axonal growth cones encountered have thin but extensive flanges of cytoplasm (foliopodia) wrapping around axon shafts in the marginal layer. Some of them have, in addition, threadlike filopodia. The axonal growth cones in the retinal marginal layer thus resemble the axonal growth cones seen in Golgi impregnations of the chick spinal cord and medulla in vivo (Ramon y Cajal, 1909; Morest, 196813)and in electron micrographs of the mammalian spinal cord and olfactory bulb (Tennyson, 1970b; Skoff, 1973; Hinds, 1972b). Their chief characteristic seems to be sheetlike extensions of membrane. This is in marked contrast with dendritic growth cones that appear to be characterized only by filopodia (Morest, 1968b; Hinds and Hinds, 1972). In the foliopodia themselves microfilaments and vesicles are the chief or only organelles, while in the preterminal enlargement other organelles, such as smooth endoplasmic reticulum, mitochondria, microtubules, and large vacuoles occur, thus conforming to previous descriptions of axonal growth cones in situ (Tennyson, 1970b; Hinds, 1972b) and in tissue culture (Yamada et al., 1971; Bunge, 1973). Since the axonal growth cones of the present study have such intimate association with fascicles of optic axons, they might be difficult to discern in silver stains. It is perhaps not surprising, therefore, that Goldberg and Coulombre (1972) were able to visualize growth cones only near the vitreal surface in their whole-mount study. In addition to axonal and dendritic varieties of growth cones, a third type of growing tip also can be distinguished in the present study. In this type little or no enlargement or specialization appears at the tip. This is characteristic of the growing vitreal processes of early interphase ventricular cells seen in the present study and in the cerebral vesicle (Hinds and

HINDS

AND HINDS

Retinal

Ruffett, 1971). Unspecialized growth tips have also been previously described in the leading processes of migrating young neurons of the monkey cerebellar and cerebral cortices (Rakic, 1971, 1972). In all these cases the unspecialized growing tip appears to be growing along radial glial or ventricular processes. A similar relationship has been well documented in the visual system of the crustacean Daphnia by Lopresti et al. (1973). Of the eight optic axons growing toward the optic lamina only the lead axon has a growth cone; the follower fibers grow nearly as fast but have no specialized tip. Lopresti et al. (1973) suggest that the growth cone per se is “functionally associated with the process of ‘recognizing’ cell surfaces or otherwise detecting position in space” (p. 437). This idea fits well with the present results which demonstrate a simple growing tip in regrowing ventricular cell processes that appears to grow along other ventricular cell processes. In this instance sophisticated position detection would perhaps not be necessary. On the other hand, axonal growth cones of the optic nerve axons encountered in the present study are extremely elaborate, as would correlate with the sophisticated position detection that these axons presumably are capable of performing (Gaze, 1970; Jacobson, 1970). The relatively simple dendritic growth cones (Hinds and Hinds, 1972) would perhaps be intermediate in their position detection capabilities and requirements. Daughter Cell Pair Analysis and Time of Commitment for Neuron Formation The present results have shown that there is a marked tendency for both daughter cells to have the same fate, either both become ventricular cells or both become postmitotic, presumptive ganglion cells, The simplest, although certainly not the only, way to explain the tendency of transitional cells to differentiate as daughter cell pairs is to assume that some cells become

Ganglion Cell Development

311

committed for ganglion cell formation and thus both of their premitotically, daughter cells are also committed and both differentiate into ganglion cells following division. This hypothesis would agree with Sechrist’s findings in the chick retina (1969 and personal communication) using silver stains, autoradiography with tritiated thymidine, and electron microscopy. He observed aggregations of microfilaments. similar to those in bipolar and unipolar ganglion cells, in a small percentage of the prophase cells near the ventricle as well as in telophase daughter cell pairs. Thus in the chick an apparently specific neuronal organelle (large aggregations of microfilaments) appears prior to mitosis, strongly implying that commitment to ganglion cell differentiation has already occurred at this time. Furthermore, Sechrist (1968 and personal communication) has observed. in agreement with results of the present study, that examples are common in which both daughter cells appear to be differentiating into neurons. They are both positively stained with silver or both contain conspicuous aggregations of microfilaments. even though such silver positive cells or cells with aggregations of microfilaments generally make up only a relatively small percentage of the total population of cells. In addition, the present results have suggested that sometimes each member of a daughter cell pair have differing fates. in which one becomes a transitional cell apparently destined to differentiate into a postmitotic young neuron, while its mate remains a ventricular cell, presumably destined for another round of DNA synthesis and division. One explanation for this difference is to assume that in these cases commitment for immediate neuron differentiation takes place postmitotically. after the two daughter cells become independent, and that this commitment only occurs in one of the two daughter cells. The two separate hypotheses for the two

412

DEVELOPMENTALBIOLOGY

cases discussed above can be combined into one simple hypothesis, namely that the decision for immediate neuron differentiation in the retina can be reached at any time throughout the cell cycle, or at least during the postmitotic, prereplicative phase (G,) as well as the DNA-synthetic phase (S). Cells that became committed during S phase (or G, phase) for neuron differentiation would go into the obligatory mitosis which follows these stages (Thrasher, 1966; Gelfant, 1966; Cameron, 1968) in a committed state, and both of the daughter cells would be expected to show early signs of neuron formation following division. In contrast, cells that reached a decision for neuron differentiation after daughter cell separation in G, phase would be expected to be capable of differentiating immediately and showing a separate fate for the two daughter cells. The above hypothesis, which suggests that important decisions regarding a cell fate can occur without any particular regard to their exact point in the cell cycle, conflicts with some previous evidence in certain systems that a mitotic division must follow an inductive event before differentiation can occur (reviewed by Holtzer et al., 1972). On the other hand, it is in agreement with some recent evidence showing the unimportance of mitosis in induction, determination, and differentiation. For example, it has been shown (Tomkins et al., 1969) that increased synthesis of tryosine aminotransferase in cultured hepatoma cells can be induced by corticosteroids during the later part of G 1and during S, and no mitosis is required. Cooke (1973) has found that in Xenopus, cell division in beginning gastrulae may be entirely inhibited, either with Colcemid or mitomycin C, and that this is followed by essentially normal development up to early tailbud stages. Furthermore, if an additional stage 10 dorsal blastoporal lip is implanted into a host early gastrula that has been completely inhibited in its cell

VOLUME 37,1974

cycle, a secondary “anterior pattern of differentiation tendency” is induced. In the latter case there was no possibility of progression of the cell cycle since the host embryo was fully inhibited at the time of blastoporal lip transplantation. Another system where differentiation in relation to the generation cycle has been studied is differentiating skeletal muscle in uitro, a system characterized, as in the nervous system, by a loss of proliferative ability of the differentiating cells. Recently, O’Neil and Stockdale (1972) have found that, upon subculturing, myoblasts will enter an additional cell cycle prior to fusion into myotubes when plated at a low density and will fuse without cycling when plated at high density. They conclude that the initiating events in the differentiation of muscle cells cannot necessarily be bound to a particular mitosis or the cell cycle preceding that mitosis. Finally, Schubert and Jacob (1970) have evidence that the differentiation of neuroblastoma cells that is induced by 5-bromodeoxyuridine can occur in the absence of DNA synthesis or mitosis. Thus, it is not unreasonable to suggest that in the development of the nervous system, cells can become committed for neuron differentiation either in G, or S phase, a somewhat different sequence of development occurring in each of the two cases. These ideas can also perhaps account for some of the apparently contradictory results in the literature on the development of other CNS regions. In the olfactory bulb an external or pial process extending toward or even reaching the outer brain surface has been detected in some early mitral cell precursors, both in Golgi impregnations and in electron micrographs (Hinds, 1972a,b). Since these radial external processes are never found in later tangential stages of mitral cell differentiation, it is reasonable to deduce that precursor cells grew out an external process only to lose it again in the course of further differentiation. One way to account for this

HINDS

AND HINDS

Retinal Ganglion Cell Development

seemingly paradoxical result would be to assume that at the time of initial outgrowth of an external process the cell was not yet committed to neuron formation in that generation; but later, because of unknown influences in G1, it became so; and at that time it had already grown out an external process, sometimes even to the outer surface (Hinds, 1972a,b). Results of the present study would predict that in the olfactory bulb, besides this G1-committed sequence of development, there should be another sequence in which cells become committed for neuron formation in the previous interphase (during S or Gz) and so take a more direct sequence to tangential mitral cells. In retrospect, there do appear to be transitional cells depicted in the olfactory bulb study (Hinds, 1972a.b) that would fit a more direct sequence, although they were not so interpreted in that study. Cells with a perikaryon in the layer of tangential mitral cells, an internal process extending toward the ventricular surface, and little or no external process were interpreted (Hinds, 1972a,b) as cells that had withdrawn their external process in preparation for further mitral cell differentiation. It is perhaps more likely, however, that these cells never had an extensive external process but developed directly from mitotic daughter cells with only a short external process associated with migration. The ideas being developed here can also account for the existence later in retinal development (Morest, 1970b) of ganglion cells with axons that still have a ventricular process attached to the ventricular surface (G l-committed cells) existing sideby-side with ganglion cell precursors detached from the ventricle but not yet possessing an axon (S-committed). It need only be assumed that the G1-committed cells attain an epithelial or interphase ventricular cell configuration before becoming committed for neuron differentiation and acquiring an axon. The result

413

would be a mixture of epithelial cell characteristics with neuronlike characteristics. S-committed cells, on the other hand, would start differentiating into ganglion cells immediately after mitosis and thus not show any attachment to the ventricular surface even in very early stages of neuron formation. If the G, phase lengthens during later stages of development of the retina relative to the length of S phase (Sinitsina. 1971), one would expect, as Morest ( 1970b) in fact has apparently observed, a larger number of G1-committed cells than were observed in the present study on younger stages. In other parts of the CNS two types of neuron formation have also been depicted. one in which epithelial or ventricular characteristics coexist with neuronal ones. and another in which cells appear to be neuronal, starting immediately after final cell division. Thus. Ramhn y Cajal (1960: Fig. 111) has depicted in a chick embryo of 8 days’ incubation differentiating neurons of the dorsal horn of the spinal cord which possess an axon but also still clearly have a ventricular process attached at the ventricular surface. Such forms were not common (although they did occur apparently-see Fig. 43, Ramon y Cajal, 1960) in the early stages of spinal cord development (Ramon y Cajal, 1960), perhaps because of the relatively short G, phase in the early spinal cord (Kauffman, 1968) and the faster withdrawal of ventricular processes earlier in development (Hinds, 1972b). In his study on the basal ganglia of pouch young opossum, Morest (1970a) has pictured Golgi-impregnated neurons possessing axons that still have a ventricular process reaching the ventricle. Likewise, in the cerebral cortex he has described neurons with perikarya migrating toward or in the cortical plate which still have their ventricular process reaching the ventricle. In contrast, Stensaas and Stensaas (1968) and Rakic (1972) have described in the developing cerebral cortex of rabbit and

414

DEVELOPMENTAL

BIOLOGY

monkey unattached neurons, with no obvious ventricular cell characteristics, migrating to the cortical plate. In these cases, as well as in others discussed above, the hypothesis that G,-committed cells have one type of early development and S-committed cells another can help explain the coexistence of two types of neuron formation. It is hoped that the validity of these ideas will be tested by further studies of early neuron development in these and other CNS regions. The authors would like to thank Mrs. Kathleen Rockland for her helpful comments on the manuscript and Dr. Charles Rockland for his help with the probability problem. REFERENCES J. (1971). Coated vesicles and synaptogenesis. A ( evelopmental study in the cerebellar cortex of the iat. Brain Res. 30, 311-322. ALTMAN, J. (1972). Postnatal development of the cerebellar cortex in the rat. I. The external germinal layer and the transitional molecular layer. J. Comp. Neurol. 145, 353-398. BARRON, D. H. (1946). Observations on the early differentiation of the motor neuroblasts in the spinal cord of the chick. J. Comp. Neural. 85, 149-170. BIRKS, R. I., MACKEY, M. C., and WELDON, P. R. (1972). Organelle formation from pinocytotic elements in neurites of cultured sympathetic ganglia. J. Neurocytol. 1, 311-340. BRAEKEVELT, C. R., and HOLLENBERG, M. J. (1970). Development of the retinal pigment epithelium, choriocapillaris and Bruch’s membrane in the albino rat. Exp. Eye Res. 9, 124-131. BUNGE, M. B. (1973). Fine structure of nerve fibers and growth cones of isolated sympathetic neurons in culture. J. Cell Biol. 56, 713-735. CALEY, D. W., and MAXWELL, D. S. (1968). An electron microscopic study of neurons during postnatal development of the rat cerebral cortex. J. Comp. Neurol. 133, 17-44. CALEY, D. W., JOHNSON, C., and LIEBELT, R. A. (1972). The postnatal development of the retina in the normal and rodless CBA mouse: a light and electron microscopic study. Amer. J. Anat. 133, 179-212. CAMERON, I. L. (1968). A method for the study of cell proliferation and renewal in the tissues of mammals. In “Methods in Cell Physiology” (D. M. Prescott, ed.) Vol. III, pp. 261-276. Academic Press, New York. COOKE, J. (1973). Morphogenesis and regulation in ALTMAN,

VOLUME 37, I974

spite of continued mitotic inhibition in Xenopus embryos. Nature (London) 242, 55-57. COULOMBRE, A. J. (1955). Correlations of structural and biochemical changes in the developing retina of the chick. Amer. J. Anat. 96, 153-190. COWDRY, E. V. (1914). The development of the cytoplasmic constituents of the nerve cells of the chick. I. Mitochondria and neurofibrils. Amer. J. Anat. 15, 389-429. DIXON, J. S., and CRONLY-DILLON, J. R. (1972). The fine structure of the developing retina in Xenopus laevis. FISHER,

J. Embryol.

Exp.

Morphol.

28, 659-666.

S., and JACOBSON,M. (1970). Ultrastructural changes during early development of retinal ganglion cells in Xenopus. 2. Zellforsch. Mikrosk. Anat. 104, 165-177. FRIEND, D. S., and FARQUHAR,M. G. (1967). Functions of coated vesicles during protein absorption in the rat vas deferens. J. Cell Biol. 35, 357-376. GAZE, R. M. (1970). “The Formation of Nerve Connections.” Academic Press, New York. GELFANT, S. (1966). Patterns of cell division; the demonstration of discrete cell populations. In “Methods in Cell Physiology” (D. M. Prescott, ed.), Vol. II, pp. 359-395. Academic Press, New York. GLUCKSMANN, A. (1951). Cell deaths in normal vertebrate ontogeny. Biol. Rev. CambridgePhil. Sot. 26, 59-86. GOLDBERG,

S., and COULOMBRE, A. J. (1972). Topographical development of the ganglion cell fiber layer in the chick retina. A whole mount study. J. Comp. Neurol. 146, 507-518. GONATAS, N. K., and ROBBINS, E. (1965). The homology of spindle tubules and neuro-tubules in the chick embryo retina. Protoplasma 59, 377-391. GRAY, E. G., and WILLIS, R. A. (1970). On synaptic vesicles, complex vesicles and dense projections. Brain Res. 24, 149-168. GRILLO, M. A., and ROSENBLUTH, J. (1972). Ultrastructure of developing Xenopus retina before and after ganglion cell specifications. J. Comp. Neural. 145, 131-140. GURDON, J. B., and WOODLAND, H. R. (1970). On the long-term control of nuclear activity during cell differentiation. Curr. Top. Develop. Biol. 5,39-70. HAY, E. D. (1968). Organization and fine structure of epithelium and mesenchyme in the developing ’ chick embryo. In “Epithelial-Mesenchymal Interactions” (R. W. Fleischmajer, ed.), pp. 31-55. Williams & Wilkins, Baltimore, Maryland. HEUSER, J. E., and REESE, T. S. (1973). Evidence for recycling of synaptic vesicle membrane during transmitter release at the frog neuromuscular junction. J. Cell Biol. 57, 315-344. HINDS, J. W. (1972a). Early neuron differentiation in the mouse olfactory bulb. I. Light microscopy. J. Comp. Neurol. 146, 233-252.

HINDS AND HINDS

Retinal

HINDS, J. W. (1972b). Early neuron differentiation in the mouse olfactory bulb. II. Electron microscopy. J. Comp. Neurol. 146, 25?-276. HINDS, J. W., and HINDS, P. L. (1972). Reconstruction of dendritic growth cones in neonatal mouse olfactory bulb. J. Neurocytol. 1, 169-187. HINDS, J. W., and RUFFETT, T. L. (1971). Cell proliferation in the neural tube: an electron microscopic and Golgi analysis in the mouse cerebral vesicle. Z. Zellforsch. Mikrosk. Anat. 115, 226264. HINDS, J. W. and RUFFETT, T. L. (1973). Mitral cell development in the mouse olfactory bulb: reorientation of the perikaryon and maturation of the axon initial segment. J. Comp. Neural. 151, 281-306. HIS, W. (1889). Die Neuroblasten und deren Entestehung im embryonalen Mark. Arch. Anat. Physiol., Anut. Abt. 1889, 249-300. HOLTZER, H., WEINTRAUB, H., MAYNE R., and MOCHAN, B. (1972). The cell cycle, cell lineages and cell differentiation. Curr. Top. Deuelop. Biol. 7, 229-256. JACOBSON,M. (1970). “Developmental Neurobiology.” Holt, Rinehart & Winston, New York. KAUFFMAN, S. L. (1968). Lengthening of the generation cycle during embryonic differentiation of the mouse neural tube. Exp. Cell Res. 49, 420-424. KERSHMAN, J. (1938). The medulloblast and the medulloblastoma. A study of the human embryo. Arch. Neural. Psychiat. 40, 937-967. LANGMAN, J., GUERRANT, R. L., and FREEMAN, B. G. (1966). Behavior of neuroepithelial cells during closure of the neural tube. J. Comp. Neural. 127, 399-411. LAVELLE, A. and LAVELLF, F. W. (1970). Cytodifferentiation in the neuron. In “Developmental Neurobiology” (W. A. Himwich, ed.), pp. 117-164. Thomas, Springfield, Illinois. LEBOUCQ, G. (1909). Contribution a l’etude de l’histogenese de la retine chez les mammiferes Arch. Anat. Microsc. 10, 555-605. LOPRESTI, V., MACAGNO, E. R., and LEVINTHAL, C. (1973). Structure and development of neuronal connections in isogenic organisms: cellular interactions in the development of the optic lamina of Daphnia. Proc. Nat. Acad. Sci. U.S. 70, 433-437. LYSE~ K. M. (1964). Early differentiation of motor neuroblasts in the chick embryo as studied by electron microscopy. I. General aspects. Deuelop. Biol. 10, 433-466. LYSER, K. M. (1968). An electron-microscope study of centrioles in differentiating motor neuroblasts. J. Embryo/. Exp. Morphol. 20, 343-354. MAOALHAES, M. M. and COIMBRA, A. (1972). The rabbit retina Miiller cell. A fine structural and cytochemical study. J. Ultrastruct. Res. 39, 310-326.

Ganglion Cell Development

415

MANN, I. (1964). “The Development of the Human Eye.” Grune & Stratton, New York, New York. MARTIN, A. H. (1967). Significance of mitotic spindle fibre orientation in the neural tube. Nature (Lendon) 216, 1133-1134. MARTIN, A., and LANGMAN, J. (1965). The development of the spinal cord examined by autoradiography. J. Embtyol. Erp. Morphol. 14, 25-35. MOREST, D. K. (1968a). Growth of cerebral dendrites and synapses. Anat. Rec. 160, 516. MOREST, D. K. (196813). The growth of synaptic endings in the mammalian brain: a study of the calyces of the trapezoid body. Z. Anat. Entwicklungsgesch. 127, 201-220. MOREST, D. K. (1969a). The differentiation of cerebral dendrites: a study of the post-migratory neuroblast in the medial nucleus of the trapezoid body. Z. Anat. Entwicklungsgesch. 128, 271-289. MOREST, D. K. (1969b). The growth of dendrites in the mammalian brain. Z. Anat. Entwicklungsgesch. 128, 290-317. MOREST, D. K. (1970a). A study of neurogenesis in the forebrain of opossum pouch young. 2. Anat. Entwicklungsgesch. 130, 265-305. MOREST, D. K. (1970b). The pattern of neurogenesis in the retina of the rat. Z. Anat. Entwicklungsgesch. 131, 45-67. MUGNAINI, and FORSTR~NEN, P. F. (1967). Ultrastructural studies on the cerebellar histogenesis. I. Differentiation of granule cells and development of glomeruli in the chick embryo. Z. Zellforsch. Mikrosk. Anat. 77, 115-143. O’NEIL, M. C., and STOCKDALE,F. E. (1972). Differentiation without cell division in cultured skeletal muscle. Develop. Biol. 29, 410-418. O’RAHILLY, R. (1966). The early development of the eye in staged human embryos. Contrib. Embryol. 38, l-42. PALAY, S. L. (1967). Principles of cellular organization in the nervous system. In “The Neurosciences. A Study Program” (G. C. Quarton, T. Melnechuk. and F. 0. Schmitt, eds.), pp. 24-31. PALAY, S. L., SOTELO, C., PETERS, A., and ORKAND, P. M. (1968). The axon hillock and the initial segment. J. Cell Biol. 38, 193-201. PEI, Y. F., and RHODIN, J. A. G. (1970). The prenatal development of the mouse eye. Anat. Rec. 168, 105-126. RAKIC, P. (1971). Neuron-glia relationship during granule cell migration in developing cerebellar cortex. A Golgi and electronmicroscopic study in Macacus rhesus. J. Comp. Neural. 141, 283-312. RAKIC, P. (1972). Mode of cell migration to the superficial layers of fetal monkey neocortex. J. Comp. Neural. 145, 61-84. RAM~N Y CAJAL, S. (1909). “Histologie du Systeme Nerveux de 1’Homme et des Vertebres,” Vol. I(1952

416

DEVELOPMENTALBIOLOGY

reprint). Instituto Rambn y Cajal, Madrid. RAM~N Y CAJAL, S. (1911). “Histologie du Systeme Nerveux de I’Homme et des Vertebres,” Vol. II (1955 reprint). Instituto Ramon y Cajal, Madrid. RAM~N, v CAIN., S. (1960). “Studies on Vertebrate Neurogenesis” (revised and translated by L. Guth). Thomas, Springfield, Illinois. (Revision of a 1929 edition.) RAM~N, Y CAJAL, S. (1972). “The Structure of the Retina” (compiled and translated by S. A. Thorpe and M. Glickstein). Thomas, Springfield, Illinois (English edition of work originally published in 1892.) RAVIOLA, G., and RAVIOLA, E. (1967). Light and electron microscopic observations on the inner plexiform layer of the rabbit retina. Amer. J. Anat. 120, 403-426. ROSENBLUTH,J. (1966). Cell contacts in the developing central nervous system of the toad. Anat. Rec. 154, 412-413. SAUER, F. C. (1935). Mitosis in the neural tube. J. Comp. Neurol. 62, 377-405. SCHUBERT, D., and JACOB, F. (1970). &Bromodeoxyuridine-induced differentiation of a neuroblastoma. F’roc. Nat. Acad. Sci. U.S. 67, 247-254. SECHRIST, J. W. (1968). “Initial Cytodifferentiation of Neuroblasts.” Ph.D. Dissertation, University of Illinois. SECHRIST, J. W. (1969). Neurocytogenesis. I. Neurofibrils, neurofilaments and the terminal mitotic cycle. Amer. J. Anat. 124, 117-134. SHEFFIELD, J. B. and FISCHMAN, D. A. (1970). Intercellular junctions in the developing neural retina of the chick embryo. Z. Zellforsch. Mikrosk. Anat. 104, 405-418. SIDMAN, R. L. (1961). Histogenesis of mouse retina studied with thymidine-Hz. In “The Structure of the Eye” (G. K. Smelser, ed.), pp. 487-506. Academic Press, New York. SIDMAN, R. L., MIALE, I. L., and FEDER, N. (1959). Cell proliferation and migration in the primitive ependymal zone; an autoradiographic study of histogenesis in the nervous system. Exp. Neural. 1, 322-333. SINITSINA, V. F. (1971). DNA synthesis and kinetics of cellular populations in embryohistogenesis of mice. Arch. Anat. Gistol. Embryiol. 61, 58-67. (In Russian.) SKOFF, R. P. (1973). Ultrastructural identification of axonal and dendritic profiles in the white matter of chick embryo spinal cord. Anat. Rec. 175, 444. SPIRA, A. W., and HOLLENBERG, M. J. (1973). Human

VOLUME 37,1974 retinal retinal

development: ultrastructure of the inner layers. Develop. Biol. 31, l-21. STENSAAS, L. J., and REICHERT, W. H. (1971). Round and amoeboid microglial cells in the neonatal rabbit brain. 2. Zellforsch. Mikrosk. Anat. 119, 147-163. STENSAAS, L. J., and STENSAAS, S. S. (1968). An electron microscope study of cells in the matrix and intermediate laminae of the cerebral hemisphere of the 45 mm rabbit embryo. Z. Zellforsch. Mikrosk. Annt. 91, 341-365. TENNYSON, V. M. (1965). Electron microscopic study of the developing neuroblast of the dorsal root ganglion of the rabbit embryo. J. Comp. Neurol. 124, 267, 318. TENNYSON, V. M. (1970a). The fine structure of the developing nervous system. In “Developmental Neurobiology” (W. A. Himwich, ed.), pp. 47-116. Thomas, Springfield, Illinois. TENNYSON, V. M. (1970b). The fine structure of the axon and growth cone of the dorsal root neuroblast of the rabbit embryo. J. Cell Biol. 44, 62-79. THRASHER, J. D. (1966). Analysis of renewing epitheha1 cell populations. In “Methods in Cell Physiology” (D. M. Prescott, ed.), Vol. II, pp. 324-357. Academic Press, New York. TOMKINS, G. M., GELEHRTER, T. D., GRANNER, D., MARTIN, D., JR., SAMUELS, H. H., ~~~THOMPSON, E. B. (1969). Control of specific gene expression in higher organisms. Science 166, 1474-1480. TURNER, P. T., and HARRIS, A. B. (1973). Ultrastructure of synaptic vesicle formation in the cerebral cortex. Nature (London) 242, 57-59. UZA, S., and SMELSER, G. K. (1973). Electron microscopic study of the development of retinal Milllerian cells. Znuest. Ophthulmol. 12, 295-307. WAECHTER, R., and JAENSCH, B. (1972). Generation times of the matrix cells during embryonic brain development: an autoradiographic study in rats. Brain Res. 46, 235-250. WINDLE, W. F., and AUSTIN, M. F. (1936). Neurofibrillar development in the central nervous system of chick embryos up to 5 days incubation. J. Comp. Neural. 63, 431-463. WINDLE, W. F., and BAXTER, R. E. (1936). The first neurofibrillar development in albino rat embryos. J. Comp. Neurol. 63, 173-187. YAMADA, K. M., SPOONER, B. S., and WESSELLS, N. K. (1971). Ultrastructure and function of growth cones and axons of cultured nerve cells. J. Cell Biol. 49, 614-635.