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The specificity of retinotectal connections studied by retinal grafts onto the optic tectum in chick embryos
DEVELOPMENTAL
BIOLOGY
16, 513-531 (1967)
The Specificity of Retinotectal Connections Studied by Retinal Grafts onto the Optic Tectum in Chick Embryos G. ROBERT DELONG~ AND ALFRED J. COULOMBRE Section on Experimental Embryology, Laboratory of Neuroanatomical National Institute of Neurological Diseases and Blindness, National Institutes of Health, Bethesda, Maryland 20014 Accepted
Sciences,
May 18, 1967
INTRODUCTION
The optic nerve fibers in nonmammalian vertebrates connect local areas of the retina with corresponding local areas of the optic tectum in a specific topographic manner. Possible mechanisms controlling the development of this orderly projection have been the subject of considerable interest (Wkely, 1966; Roberts and Flexner, 1966). Recent histological studies have provided evidence that in fact, during both embryogenesis and regeneration, optic fibers attain their proper local termination areas on the tectum. Attardi and Sperry (1963) sh owed in fish that regenerating optic fibers preferentially grew toward their proper local area of termination on the tectum. Arora and Sperry (1962) displaced one bundle of the optic tract from its normal pathway during regeneration and found that as the fibers continued to grow they regained their proper channel and continued to their appropriate terminations. The factors determining this specifically directed fiber growth are unknown. Sperry (1963) has suggested that the proper matching of optic fibers with their tectal terminations depends on a system of local specification of retinal and tectal areas by cell-bound markers, determined by cytochemical gradients operating over the extent of the retina and the tectum. Complementary or matching markers, arising independently in the retina and the tectum, then provide a mechanism for linking specific optic fibers with the corresponding tectal cells. 1 Present address: Department Boston, Massachusetts 02114.
of Neurology, 513
@ 1967 by Academic
Press Inc.
Massachusetts
General
Hospital,
514
DELONG
AND
COULOMBRE
We have recently reported that developing optic nerve fibers in the chick embryo likewise selectively innervate their proper local termination area on the tectum, as shown by the distribution of retinal ganglion cell axons on the tectum following quadrantal retinal ablations performed at a stage before the optic fibers reach the tectum ( DeLong and Coulombre, 1965). In that study the retinal projection onto the embryonic chick tectum was mapped and its pattern was found to develop uniformly and independently of the retinal fibers, regardless of which group of them was allowed to reach the tectum. The present investigation extends the analysis of the formation of retinotectal connections in the chick embryo. Small fragments of 4day retina from known quadrants of the eye were grafted directly onto the surface of the tectum of 6- or 7-day hosts (the contralateral eye of the host having been removed previously to prevent the normal complement of optic fibers from reaching the tectum) and the directional orientation and destination of outgrowing fibers from the grafts were subsequently examined. In this way, it was hoped to discover whether fibers from a specific area of the retina would preferentially grow toward a specific area of the tectum, regardless of the position of the graft on the tectum and despite the disruption of normal anatomic relationships between eye and brain. MATERIALS
AND METHODS
Fertile White Leghorn eggs were incubated at 37.5-38.O”C in a forced-draft incubator. At 4 days of incubation the right eye was excised to remove the normal retinal source of innervation of the contralateral optic lobe. All operations were performed with watchmaker’s forceps and fine tungsten needles through a window in the shell. After an operation the window was sealed with cellophane tape and the eggs were returned to the incubator. In a second operation at 6 or 7 days of incubation, donor retina was grafted to the left side of the tectum. The graft was a fragment of normal 4-day retina and adjacent pigment epithelium about OS-O.8 mm square, cut from one quadrant (nasal, dorsal, ventral, temporal) of the eye. The graft was first transferred to the chorioallantoic membrane of the host. This membrane and the amnion were then opened allowing access to the left side of the head. With watchmaker’s forceps a small slit was made in the skin covering the cranium to expose a vascular meningeal membrane closely applied to the optic tectum.
RETINOTECTAL
CONNECTIONS
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By careful manipulation with forceps this membrane usually could be opened and gently separated from the tectal surface to form a small subarachnoid pocket. The graft, with the internal (vitreal) surface of retina facing the tectum, was then slipped between the membrane and the tectal surface. During subsequent growth the graft often rounded up to form a hollow sphere, with the internal surface of the retina directed away from the tectum. When the forceps were withdrawn, the pressure of the membrane against the brain surface held the graft in place; in favorable cases the graft lay flat against the tectal surface. The dorsoventral and anterior-posterior orientations of the retina could not be maintained in transferring the graft and are considered to have been randomized. The window was sealed and the egg returned to the incubator. After 5 or 6 days, at 11 or 12 (rarely 13) days of incubation, the embryos were examined with a dissecting microscope and fixed for histological study. The grafts, now l-2 mm in diameter, were readily identified; however, outgrowing fiber bundles could not be identified regularly with the dissecting microscope, even after vital staining with methylene blue. In the course of the work, grafts from each retinal quadrant were placed in all quadrants of the 7-day tectum. Grafts were not distributed in equal numbers in all areas of the tectum, however, because it proved more difficult to approach the posterior-inferior tectum and fewer successful grafts were placed in that position. The initial position of the graft on the tectum was recorded on a diagram; this was then compared with the location in which the graft was found at I2 days. In three-fourths of the cases the relative position of the graft on the tectum did not change; in one-fourth the position appeared to have shifted randomly. The heads were fixed for 48 hours in Heidenhain’s Susa, dehydrated, embedded in paraffin, and serially sectioned at 30 p. Mercuric chloride crystals were removed with 0.5% iodine in 80% alcohol and the sections were silver impregnated by the Protargol method. Serial sections were examined microscopically to determine the degree of differentiation of the retinal graft as well as the extent, direction, and termination of outgrowing retinal fibers. The position of the graft and the length and course of the fibers arising from it were then charted on a two-dimensional equal-area representation of the surface of the in left tectum of the I2-day embryo similar to that used previously mapping the optic fiber distribution on the tectum (&Long and
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FIG. 1, 2
RETINOTECTAL
CONNECTIONS
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Coulombre, 1965). The left and right tabs of the diagram represent the medial surfaces which have been folded out (Figs. 7-11). This representation of the tectum was constructed from a series of area1 strips, one dimension of each strip representing the length of the curve of the tectal surface in a given transverse section and the other representing the distance between the measured sections in the series. As a check, similar maps were plotted from parasagittal sections. RESULTS
Histological
Description
The retinal graft generally rested directly on the pial membrane, which thus formed a distinct continuous barrier about 5 p thick between graft and underlying brain. The grafts matured at approximately the normal rate and closely resembled normal retina. Both pigment epithelium and neural retina were present. The innermost cells of the graft formed a distinct layer that appeared identical to the normal ganglion cell layer of retina, and internal to this was a layer of parallel fine fibers resembling the optic fiber layer of the normal retina (Fig. 1). These fibers gathered into one or two fascicles, then coursed through the wall of the graft and into the surrounding loose mesenchymal tissue ( Figs. l-6). The fibers passing out from the graft formed one bundle, or two or three parallel fascicles, which were situated either immediately on the pial membrane or in the loose, mesenchymal tissue above this membrane. They coursed in one well-defined direction over the surface of the tectum, for distances up to 2.5 mm (Fig. 3) (nineteen
FIG. 1. Transverse section through anterior left optic lobe at 12 days. To the right of the tectum is the retinal graft (from the temporal quadrant). The differentiation of retinal layers is clearly visible in the portion of the graft adjacent to the tectum. The innermo& layer of the graft consists of a band of fibers presumed to be retinal ganglion-cell fibers. These course through the wall of the retina (arrow), then continue as a straight fiber bundle through loose mesenchymal tissue to reach the tectal surface, where they continue dorsally (out of the plane of the section). Note the absence of the normal stratum opticum in this and the following specimens. x 38. FIG. 2. An example of fiber bundles penetrating the pial membrane from the graft in the upper part of the picture and ramifying within the superficial tectal layers. In this case the fibers enter the tectum directly beneath the graft. x 428.
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FIG. 8
RETINOTECTAL
CONNECTIONS
519
specimens had fibers traced for 1.0 mm or more). Very occasionally a fiber bundle, advancing from a graft toward the tectum, turned sharply upon nearing the pia and continued in a different direction now parallel to a second fascicle from the same graft (Fig. 5). The mesenchymal tissue overlying the tectum deserves mention. In some cases it appeared to originate from the graft, and in others to be part of the normal cranial tissue, probably contributing to the developing arachnoidal membrane. The fiber bundles invariably ran within its loose matrix, and in cases where it incompletely covered the surface they did not venture beyond its margins. Fiber growth was examined in 53 specimens. In 32 the fiber bundles pierced the pial membrane and were lost among the cells of the superficial tectal layers (Fig. 4). In another 8 specimens the fibers penetrated into the tectum immediately beneath the graft and were identifiable as a fiber mass in the subpial layer; they could not be traced further among the cells of the tectum (Fig. 2). In 3 cases, a group of fibers was seen to enter the tectum near the graft, while a larger bundle coursed for a long distance over the surface. In the remaining 10 specimens the fiber bundles coursed along the tectal surface, sometimes for long distances, then appeared to end without entering the tectum. Direction
of Fiber
Growth
For the fiber bundles of each graft, the source, direction, length, and site of penetration of the pia, determined by examination of serial sections, were plotted as arrows on a two-dimensional representation of the tectal surface. (Specimens with fibers penetrating into the tectum directly beneath the graft were represented by circles.) What is represented by the arrows, thus, is the course and approximate relative length of the identifiable fiber bundles emanating from the graft along their path on the surface of the tectum. The head of the arrow indicates the point where the fibers pierce the pia to enter the tectum, FIG. 3. (a) Low-power view of the left side of the optic tectum in a lz-day embryo. The graft (dorsal retina) is visible in the upper left. Two parallel bundles arising from the graft are seen coursing ventrolaterally over the surface of the tectum. x 38. (b and c) High-power views of the same section. The fibers rest directly on the pial membrane, which separates them from the underlying tectal tissue. (b) x 95. (cl x 428.
RETINOTECTAL
CONNECXIONS
52,l
in cases where this occurred. In cases where this was not observed, the arrow represents the furthest point to which the fibers could be clearly followed on the surface. The specimens have been grouped according to the retinal quadrant from which the grafts were taken; in this way the fiber growth was analyzed with regard to both the retinal quadrant from which the graft was taken and also the position of the graft on the tectum. Grafts from left and right donor eyes were initially analyzed separately, but since there was no difference attributable to the side of origin of the graft both groups have been combined. The results are presented in Figs. 7-10. These figures may be analyzed visually. First, the position of the grafts (the base of each arrow) tends to be concentrated in the central part, the anterior part, and to a lesser extent in the dorsal part of the tectum. The paucity of posterior grafts reflects the technical difficulties in their placement mentioned above, and possibly a disproportionate growth of the posterior tectum in the period after the grafts were placed. Second, the directions of growth of fiber bundles exhibited by the specimens as a whole warrants comment. In any quadrant of the tectum, arrows are seen oriented in several directions. Particularly in the central area several arrows point dorsally, ventrally, and posteriorly. However, only five grafts sent fibers anteriorly; and in the FIG. 4. Transverse section of left optic lobe. A retinal graft (nasal) partly visible in the lower right corner, lies in a broad depression in the tectal surface. Pigment epithelium from the graft has formed a branching figure containing no neural epithelium (upper right) and below this forms a partial sleeve around the fiber bundle coursing dorsoposteriorly from the graft. In the plane of this section, the fiber bundle from the graft is seen coursing over the tectal surface and finally penetrating the surface to be lost within the superficial tectal layer. Inset: Highpower view of fibers turning downward into the substance of the tectum. FIG. 5. (a) Another example of a retinal graft (dorsal). Part of the graft is seen at the upper left. The graft and the surface membrane are artifactually pulled away from the tectum. Two fiber bundles are visible passing from the right margin of the graft to the pial membrane and then continuing laterally (down-pointing arrows). Beneath the graft, in the left half of the photograph, there is another stout fiber bundle resting immediately on the membrane (uppointing arrow). This bundle arises from the left (medial) side of the graft, bends around it, and continues laterally under the graft to join the other fibers. The first part of its course is not included in this section. x 114. (b) Higher-power view of the same specimen, showing parallel fiber bundles coursing laterally. x 285.
522
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COULOMBRE
6
RPTT~NOTECTAL CoNmcTIoNs
523
four of these with fiber bundles of significant length, the grafts were located posteriorly (the exception is in Fig. 10, near the dorsal margin). No clear explanation offers itself for the rather striking observation that of the many grafts placed centrally or anteriorly none sent fibers anteriorly, though collectivelv they sent fibers in all other directions. Finally, the directions taken by fiber bundles may be considered in relation to the specific retinal origin of the grafts (as grouped in the diagrams). Examination of the group of 17 grafts of dorsal retina reveals that, with one exception, the fiber bundles originating from the grafts are directed ventrally and laterally, regardless of the position of the graft on the tectum (Fig. 7). In contrast, eleven grafts of ventral retina, situated generally on the same areas of tectum as the dorsal group, with one exception have their fiber bundles directed dorsally and medially (Fig. 8). Ten grafts of nasal retina are represented in Fig. 9. The general direction of the fiber bundles dorsally and posteriorly is clear; even the one possible exception (the most dorsal graft) has a fiber bundle which though directed laterally penetrated the pial membrane within the posterodorsal “target zone” defined by the direction of the other fibers. However, the very anteriorly placed grafts with short fiber bundles can be given little weight as indicating any preferential orientation of fibers. More significant are the long fiber bundle arising at the anterior pole and passing dorsoposteriorly, and the other long fascicles ending in the posterodorsal quadrant. The group of 15 grafts of temporal retina (Fig. 10) are particularly important. Several features may be stressed: (1) This group of grafts is remarkably consistent in the direction of fiber growth, more so than any of the other groups. A relatively small “target area” may be described. The grafts tend to be concentrated centrally and anteriorly, but comparison with the dorsal group shows that this in itself cannot account for the consistency of the findings. (2) The temporal group FIG. 6. (a) Lateral surface of the optic tectum. A retinal graft (temporal) is out of the field at the right, and a small fiber bundle arising from it enters the plane of the section about the middle of the photograph and traversesthe surface to the left margin of the picture, where it turns downward to enter the tectum. x 160. (b) High-power view of the same specimen showing the course of the fibers. x 450.
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I mm.
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COULOMBBE
V
FIGS. 7-10. These diagrams are two-dimensional representatives of the surface of the left tectum of the U-day chick embryo. In each, the origin, direction, and length of fiber fascicles arising from retinal grafts have been indicated by arrows (see text). The circles are diagrammatic representations of grafts whose fibers penetrated directly into the tectum beneath the graft; their diameter is roughly proportioned to that of the area innervated. The results found with retinal grafts from each quadrant of the eye are presented separately. FIG. 7. Seventeen grafts of dorsal retina. FIG. 8. Eleven grafts of ventral retina.
differs from the nasal group less clearly than does the ventral from the dorsal, as seen above. (3) The temporal group is not significantly different from the ventral group in the orientation of the fiber fascicles; however, both can clearly be separated from the dorsal group. In fact, the strongest conclusion that arises from this analysis is that retinal grafts of dorsal origin quite consistently send fiber bundles ventro-
RETINOTECTAL
525
CONNECTIONS
FIG. 9. Ten grafts of nasal retina. FIG. 10. Fifteen grafts of temporal retina. Specimens bundles are shown by two arrows from a common source.
with
divergent
fiber
laterally, while retinal grafts of temporal or ventral origin, placed on the same areas of the tectum, consistently send fiber bundles dorsally and medially. The group of nasal retina grafts send fibers toward a third distinct field, in general more like ventral than dorsal retina. There thus appears a consistent differential property of pieces of retinal tissue that correlates with the part of the eye from which the retina was originally taken. Three different distinct areas within the 4-day chick embryo retina can be identified by the available data; dorsal, ventrotemporal, and nasal. Whether the “map” of local specificities has finer definition at this early embryonic stage cannot be answered from the present data. It may be that as the retina and tectum develop
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COULOMBRE
further, a progressively finer mosaic of differentiated local areas appears. Are the differential properties demonstrated for different retinal areas simply a curiosity of an artificial experimental system, or may they be related to the normal mechanisms for constructing specific orderly connections between the retina and tectum during organogenesis? Data from the mapping of normal retinotectal interconnections tends to support the second possibility. The representation of the retina on the embryonic chick tectum derived independently from retinal ablation studies ( DeLong and Coulombre, 1965) is reproduced in Fig. 11. This shows ventral retina projecting on dorsal tectum,
FIG. 11. Retinotopic map of the 12-day optic tectum, derived from ablations of retinal quadrants in the 4-day chick embryo with subsequent determination of the patterns of fiber distribution on the optic tectum, for comparison with the preceding figures. The labels refer to areas of retina, showing their topographic representation on the surface of the tectum.
dorsal retina on ventral tectum, nasal retina on posterodorsal tectum, and temporal retina on anterodorsal tectum but overlapping widely with the ventral projection. This mapping is consistent with the fiber orientations observed in the present experiments, even to the difficulty in distinguishing ventral and temporal areas, and the sharp, clear-cut distinction between dorsal and ventral-temporal areas. Since both sets of experiments presumably reflect the state of the retina at 4-5 days-when the grafting and ablations were performed-the parallels between the two sets of data strengthen the likelihood that the fiber orientations in the present experiments reflect the normal mechanisms for establishment of retinotectal interconnections.
RETINOTECTAL
CONNECTIONS
527
DISCUSSION
The system utilized herein offered several advantages for confrontation of source (retina) and target (tectum) tissues. The chick tectum is readily accessible to manipulation during embryonic stages. The normal afferent innervation to the tectal surface, which in the early embryo consists only of the optic fibers, is easily prevented from reaching the tectum by excising the contralateral eye, thus leaving the tectum free of surface fibers and receptive to the fibers emanating from the graft. Furthermore, the graft fibers are then conspicuous on the otherwise bare tectal surface. Since the tectum has a relatively wide expanse, placement and mapping can be fairly accurate. Finally, the retinotopic map of the embryonic tectum had been outlined previously. A disadvantage is that the silver stains employed do not permit visualization of the terminations of fibers from the grafts. Where the fascicles penetrate the pia and form a subpial mass (as in Fig. 2) in the normal position for optic fibers, it is reasonable to suggest that they may terminate in the same way as normal optic fibers reaching the same location; however, from the present preparations this cannot be determined. In contrast to the uncertainty about precise terminations, it was possible to identify accurately fibers advancing out of the graft and across the tectal surface. The fibers generally issued from the graft at well-defined points and traversed an empty, cell- and fiber-free space where the smallest fascicles of fibers stood out clearly. In almost all cases fibers grew out from a retinal graft in only one direction, Where more than one fascicle formed, the fascicles were parallel to one another. Occasionally fiber bundles curved around the graft or changed direction and assumed a straight course on nearing the tectal surface. It was necessary to ask, however, whether the final course of the fibers reflected some nonspecific or extraneous force. Conceivably, the graft might have been displaced, towing the fibers behind it, by a shift of the overlying membranes in relation to the surface of the tectum; such a shearing action could conceivably result from the continued growth of the tectum while the graft was held in place by the meninges. While this factor may have operated to a minor degree, it cannot explain the results found, for the following reasons: 1. Under any kind of passive movement, the fibers of all grafts
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placed in any one area of the tectum would be expected to be oriented predominantly in the same direction. The results were otherwise; fibers of some grafts coursed in opposite direction or at right angles to those from other grafts on the same area. 2. The initial placement and the final location of the grafts at the time of sacrifice were identical in three-fourths of the cases. When a shift of position was recorded, there was no consistency in the direction of the shift and no demonstrable relation between the latter and the course of fibers from the graft. 3. Regardless of the site of the graft on the tectum, the direction taken by outgrowing fibers correlated with the retinal site of origin of the graft. By similar reasoning, one can dismiss the possibility that the fibers simply followed the channels that would be taken by normal optic fibers over the tectum. One further possibility must be considered: that the orientation of the retinal graft on the surface of the tectum determined the direction of fiber outgrowth, i.e., that the direction of outgrowth depended only on the retina and was independent of the tectum. Were this the case, the fiber orientations should have been random and independent of the quadrant of origin of the graft since, as stated above, the grafts were positioned in random orientation with regard to their dorsoventral and anteroposterior axes. The question was raised above (see Introduction) whether the normal orderly connection of retinal fibers to specific areas of the tectum depends on the integrity of the overall configuration of the entire optic system, as if the axons simply advance along preexisting tissue channels to their destination. The inadequacy of this interpretation has been shown by the experiments of Arora and Sperry (1962) in which they displaced bundles of optic fibers from their normal pathways and observed that they regained their proper positions, and by experiments with fused half-eyes in embryonic toads which showed that fibers from one-half of the retina would spread out over the entire tectum under certain circumstances (Gaze et al., 1963, 1965). It is further contradicted by the results reported herein, in which the normal relationships were grossly disrupted, with the fragments of grafted retina of 4-day embryos in direct apposition to the tectum of 6- or 7-day hosts. Under these conditions the fibers arose in abnormal locations and traversed the tectum in directions very different from
HETINOTECTAL
CONNECTIONS
529
those normally taken by optic fibers. Nevertheless, the direction of fiber outgrowth correlated with the local retinal origin of the graft, and was generally directed toward the appropriate local area of termination on the tectum. This result indicates a specific active matching of retinal fibers and tectal areas, and supports the hypothesis advanced by Sperry (1963). The embryologic and cellular mechanisms underlying the specific fiber orientations found in the present experiments are not known. Specific matching requires that each retinal graft contain some localityspecific marker which differentiates it from other parts of the retina; similarly each area of the tectum must be differentiated from other areas by some specific marker. Finally, the retinal and tectal markers must have some means for mutual recognition. The nature of the postulated markers is unknown. Some evidence as to the intermediate mechanisms controlling oriented fiber growth may be gained from the data presented herein. One possible mechanism is that pioneering fibers, searching randomly, may find matching terminal fields by trialand-error, with secondary enlargement of the successful tract by “selective fasciculation” (Weiss, 1950) and atrophy of the unsuccessful pioneering fibers. The available data give no evidence of such a process. No randomly straying fibers were seen, even in the near vicinity of the graft where they should be most numerous. While all such fibers may have grown out to all areas of the tectum and then atrophied without trace in all cases during the interval between grafting at 7 days and harvesting at 12 days, this seems unlikely. The normal rate of growth is such that the advancing “front” of optic fibers grows from the ventrolateral margin of the tectum on the sixth day of incubation to the dorsomedial margin on the twelfth day (DeLong and Coulombre, 1965), a rate approximating that of the longest graft fascicles; for random pioneer fibers to have grown to all areas and disappeared within this period, one must postulate a much faster rate of growth. Further, the fascicles clearly do not follow any random or meandering path; they course in a straight and direct line, so much so that they may be followed in a single histological section for as much as 2 mm. This strongly suggests that the fibers are accurately guided throughout their course. The fact that fibers advanced for considerable distances over the tectum above the pial membrane, apparently directed toward a specific goal, suggests that factors responsible for guiding the fibers were
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acting at or immediately above the surface of the membrane. The membrane was about 5 p thick and appeared to separate the retinal fibers from direct contact with the tectal cells, up to the point where the fibers penetrated the membrane. One may speculate either that the pial membrane (or the connective tissue outside it) contains the guiding cues and thus determines the retinotopic map, or alternatively that the relevant factors originate in the superficial tectal cells and act over a short distance. One final suggestion arises from the present work. Since the retinotopic “map” on the tectum appears to arise independently of that on the retina, one may speculate that the “tectotopic map” (of some center which receives tectal efferents) might arise independently of the tectum. One would expect that the factors determining the local topographic specificity of other areas within the central nervous system might have some common genetic and developmental basis with those of the retina and tectum. This possibility of a shared specificity would seem to be susceptible to test by confrontation experiments similar to those reported here. SUMMARY
Pieces of retina from known quadrants of 4-day chick embryo eyes were grafted onto the surface of the o’ptic tectum of 6- or 7-day embryos (the contralateral eye of the hosts having been excised in an earlier operation to prevent the normal complement of optic fibers from reaching the tectum). The embryos with grafts were incubated 5 days, then prepared for histological study. Examination of serial sections stained with Protargol revealed that the retinal grafts had differentiated normally and produced fibers which passed onto the surface of the tectum as a single bundle, or as parallel fascicles. These coursed for varying distances on the surface membrane above the tectum, ending in many cases by piercing the membrane and entering the superficial tectal layers. The location of the graft on the tectal surface and the course and length of the fiber bundles were recorded, and the results grouped according to the quadrant of origin of the retinal graft. Fibers from a particular quadrant of retina preferentially grew toward specific areas of the tectum (dorsal retina to lateroventral tectum; ventral retina to dorsal tectum; nasal retina to posterodorsal tectum; temporal retina to anterodorsal tectum). These findings correlated with the known retinotopic distribution of normal optic fiber projec-
RETINOTECTAL
531
CONNECTIONS
tions onto the tectum. Possible mechanisms guidance of fibers are discussed.
mediating
the specific
REFERENCES AHORA, H. L., and SPERRY, R. W. ( 1962). Optic nerve regeneration after surgical cross-union of medial and lateral optic tracts. Am. Zoologist 2, 389. ATTAHDI, D. G., and SPERRY, R. W. (1963). P re f erential selection of central pathways by regenerating optic fibers. Exptl. Neural. 7, 46-64. DELONG, G. R., and COULOMBRE, A. J. (1965). D evelopment of the retinotectal topographic projection in the chick embryo. Exptl. Neural. 13, 350463. GAZE, R. hf., JACOBSON, M., and SZBKELY, G. (1963). The retinotectal projection in Xenopcs with compound eyes. 1. Physiol. (London) 165, 484499. GAZE, R. hf., JACOBSON, M., and SZ~KELY, G. (1965). On the formation of connexions by compound eyes in Xenopus. J. Physiol. (London) 176, 409417. ROBERTS, R. B., and FLEXNEH, L. B. (1966). A model for the development of retina-cortex connections. Am. Scientist 54, 174-183. SPERRY, R. W. ( 1963). Chemoaffinity in the orderly growth of nerve fiber patterns and connections. Proc. Natl. Acad. Sci. U.S. 50, 703-710. SZ~KELY, G. ( 1966). Embryonic determination of neural connections. A&an. Morphogenesis 5, 181-219. WEISS, P. ( 1950). The deplantation of fragments of nervous system in amphibians. I. Central reorganization and the formation of nerves. J. Exptl. Zool. 113, 397461.
BIOLOGY
16, 513-531 (1967)
The Specificity of Retinotectal Connections Studied by Retinal Grafts onto the Optic Tectum in Chick Embryos G. ROBERT DELONG~ AND ALFRED J. COULOMBRE Section on Experimental Embryology, Laboratory of Neuroanatomical National Institute of Neurological Diseases and Blindness, National Institutes of Health, Bethesda, Maryland 20014 Accepted
Sciences,
May 18, 1967
INTRODUCTION
The optic nerve fibers in nonmammalian vertebrates connect local areas of the retina with corresponding local areas of the optic tectum in a specific topographic manner. Possible mechanisms controlling the development of this orderly projection have been the subject of considerable interest (Wkely, 1966; Roberts and Flexner, 1966). Recent histological studies have provided evidence that in fact, during both embryogenesis and regeneration, optic fibers attain their proper local termination areas on the tectum. Attardi and Sperry (1963) sh owed in fish that regenerating optic fibers preferentially grew toward their proper local area of termination on the tectum. Arora and Sperry (1962) displaced one bundle of the optic tract from its normal pathway during regeneration and found that as the fibers continued to grow they regained their proper channel and continued to their appropriate terminations. The factors determining this specifically directed fiber growth are unknown. Sperry (1963) has suggested that the proper matching of optic fibers with their tectal terminations depends on a system of local specification of retinal and tectal areas by cell-bound markers, determined by cytochemical gradients operating over the extent of the retina and the tectum. Complementary or matching markers, arising independently in the retina and the tectum, then provide a mechanism for linking specific optic fibers with the corresponding tectal cells. 1 Present address: Department Boston, Massachusetts 02114.
of Neurology, 513
@ 1967 by Academic
Press Inc.
Massachusetts
General
Hospital,
514
DELONG
AND
COULOMBRE
We have recently reported that developing optic nerve fibers in the chick embryo likewise selectively innervate their proper local termination area on the tectum, as shown by the distribution of retinal ganglion cell axons on the tectum following quadrantal retinal ablations performed at a stage before the optic fibers reach the tectum ( DeLong and Coulombre, 1965). In that study the retinal projection onto the embryonic chick tectum was mapped and its pattern was found to develop uniformly and independently of the retinal fibers, regardless of which group of them was allowed to reach the tectum. The present investigation extends the analysis of the formation of retinotectal connections in the chick embryo. Small fragments of 4day retina from known quadrants of the eye were grafted directly onto the surface of the tectum of 6- or 7-day hosts (the contralateral eye of the host having been removed previously to prevent the normal complement of optic fibers from reaching the tectum) and the directional orientation and destination of outgrowing fibers from the grafts were subsequently examined. In this way, it was hoped to discover whether fibers from a specific area of the retina would preferentially grow toward a specific area of the tectum, regardless of the position of the graft on the tectum and despite the disruption of normal anatomic relationships between eye and brain. MATERIALS
AND METHODS
Fertile White Leghorn eggs were incubated at 37.5-38.O”C in a forced-draft incubator. At 4 days of incubation the right eye was excised to remove the normal retinal source of innervation of the contralateral optic lobe. All operations were performed with watchmaker’s forceps and fine tungsten needles through a window in the shell. After an operation the window was sealed with cellophane tape and the eggs were returned to the incubator. In a second operation at 6 or 7 days of incubation, donor retina was grafted to the left side of the tectum. The graft was a fragment of normal 4-day retina and adjacent pigment epithelium about OS-O.8 mm square, cut from one quadrant (nasal, dorsal, ventral, temporal) of the eye. The graft was first transferred to the chorioallantoic membrane of the host. This membrane and the amnion were then opened allowing access to the left side of the head. With watchmaker’s forceps a small slit was made in the skin covering the cranium to expose a vascular meningeal membrane closely applied to the optic tectum.
RETINOTECTAL
CONNECTIONS
515
By careful manipulation with forceps this membrane usually could be opened and gently separated from the tectal surface to form a small subarachnoid pocket. The graft, with the internal (vitreal) surface of retina facing the tectum, was then slipped between the membrane and the tectal surface. During subsequent growth the graft often rounded up to form a hollow sphere, with the internal surface of the retina directed away from the tectum. When the forceps were withdrawn, the pressure of the membrane against the brain surface held the graft in place; in favorable cases the graft lay flat against the tectal surface. The dorsoventral and anterior-posterior orientations of the retina could not be maintained in transferring the graft and are considered to have been randomized. The window was sealed and the egg returned to the incubator. After 5 or 6 days, at 11 or 12 (rarely 13) days of incubation, the embryos were examined with a dissecting microscope and fixed for histological study. The grafts, now l-2 mm in diameter, were readily identified; however, outgrowing fiber bundles could not be identified regularly with the dissecting microscope, even after vital staining with methylene blue. In the course of the work, grafts from each retinal quadrant were placed in all quadrants of the 7-day tectum. Grafts were not distributed in equal numbers in all areas of the tectum, however, because it proved more difficult to approach the posterior-inferior tectum and fewer successful grafts were placed in that position. The initial position of the graft on the tectum was recorded on a diagram; this was then compared with the location in which the graft was found at I2 days. In three-fourths of the cases the relative position of the graft on the tectum did not change; in one-fourth the position appeared to have shifted randomly. The heads were fixed for 48 hours in Heidenhain’s Susa, dehydrated, embedded in paraffin, and serially sectioned at 30 p. Mercuric chloride crystals were removed with 0.5% iodine in 80% alcohol and the sections were silver impregnated by the Protargol method. Serial sections were examined microscopically to determine the degree of differentiation of the retinal graft as well as the extent, direction, and termination of outgrowing retinal fibers. The position of the graft and the length and course of the fibers arising from it were then charted on a two-dimensional equal-area representation of the surface of the in left tectum of the I2-day embryo similar to that used previously mapping the optic fiber distribution on the tectum (&Long and
516
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FIG. 1, 2
RETINOTECTAL
CONNECTIONS
517
Coulombre, 1965). The left and right tabs of the diagram represent the medial surfaces which have been folded out (Figs. 7-11). This representation of the tectum was constructed from a series of area1 strips, one dimension of each strip representing the length of the curve of the tectal surface in a given transverse section and the other representing the distance between the measured sections in the series. As a check, similar maps were plotted from parasagittal sections. RESULTS
Histological
Description
The retinal graft generally rested directly on the pial membrane, which thus formed a distinct continuous barrier about 5 p thick between graft and underlying brain. The grafts matured at approximately the normal rate and closely resembled normal retina. Both pigment epithelium and neural retina were present. The innermost cells of the graft formed a distinct layer that appeared identical to the normal ganglion cell layer of retina, and internal to this was a layer of parallel fine fibers resembling the optic fiber layer of the normal retina (Fig. 1). These fibers gathered into one or two fascicles, then coursed through the wall of the graft and into the surrounding loose mesenchymal tissue ( Figs. l-6). The fibers passing out from the graft formed one bundle, or two or three parallel fascicles, which were situated either immediately on the pial membrane or in the loose, mesenchymal tissue above this membrane. They coursed in one well-defined direction over the surface of the tectum, for distances up to 2.5 mm (Fig. 3) (nineteen
FIG. 1. Transverse section through anterior left optic lobe at 12 days. To the right of the tectum is the retinal graft (from the temporal quadrant). The differentiation of retinal layers is clearly visible in the portion of the graft adjacent to the tectum. The innermo& layer of the graft consists of a band of fibers presumed to be retinal ganglion-cell fibers. These course through the wall of the retina (arrow), then continue as a straight fiber bundle through loose mesenchymal tissue to reach the tectal surface, where they continue dorsally (out of the plane of the section). Note the absence of the normal stratum opticum in this and the following specimens. x 38. FIG. 2. An example of fiber bundles penetrating the pial membrane from the graft in the upper part of the picture and ramifying within the superficial tectal layers. In this case the fibers enter the tectum directly beneath the graft. x 428.
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FIG. 8
RETINOTECTAL
CONNECTIONS
519
specimens had fibers traced for 1.0 mm or more). Very occasionally a fiber bundle, advancing from a graft toward the tectum, turned sharply upon nearing the pia and continued in a different direction now parallel to a second fascicle from the same graft (Fig. 5). The mesenchymal tissue overlying the tectum deserves mention. In some cases it appeared to originate from the graft, and in others to be part of the normal cranial tissue, probably contributing to the developing arachnoidal membrane. The fiber bundles invariably ran within its loose matrix, and in cases where it incompletely covered the surface they did not venture beyond its margins. Fiber growth was examined in 53 specimens. In 32 the fiber bundles pierced the pial membrane and were lost among the cells of the superficial tectal layers (Fig. 4). In another 8 specimens the fibers penetrated into the tectum immediately beneath the graft and were identifiable as a fiber mass in the subpial layer; they could not be traced further among the cells of the tectum (Fig. 2). In 3 cases, a group of fibers was seen to enter the tectum near the graft, while a larger bundle coursed for a long distance over the surface. In the remaining 10 specimens the fiber bundles coursed along the tectal surface, sometimes for long distances, then appeared to end without entering the tectum. Direction
of Fiber
Growth
For the fiber bundles of each graft, the source, direction, length, and site of penetration of the pia, determined by examination of serial sections, were plotted as arrows on a two-dimensional representation of the tectal surface. (Specimens with fibers penetrating into the tectum directly beneath the graft were represented by circles.) What is represented by the arrows, thus, is the course and approximate relative length of the identifiable fiber bundles emanating from the graft along their path on the surface of the tectum. The head of the arrow indicates the point where the fibers pierce the pia to enter the tectum, FIG. 3. (a) Low-power view of the left side of the optic tectum in a lz-day embryo. The graft (dorsal retina) is visible in the upper left. Two parallel bundles arising from the graft are seen coursing ventrolaterally over the surface of the tectum. x 38. (b and c) High-power views of the same section. The fibers rest directly on the pial membrane, which separates them from the underlying tectal tissue. (b) x 95. (cl x 428.
RETINOTECTAL
CONNECXIONS
52,l
in cases where this occurred. In cases where this was not observed, the arrow represents the furthest point to which the fibers could be clearly followed on the surface. The specimens have been grouped according to the retinal quadrant from which the grafts were taken; in this way the fiber growth was analyzed with regard to both the retinal quadrant from which the graft was taken and also the position of the graft on the tectum. Grafts from left and right donor eyes were initially analyzed separately, but since there was no difference attributable to the side of origin of the graft both groups have been combined. The results are presented in Figs. 7-10. These figures may be analyzed visually. First, the position of the grafts (the base of each arrow) tends to be concentrated in the central part, the anterior part, and to a lesser extent in the dorsal part of the tectum. The paucity of posterior grafts reflects the technical difficulties in their placement mentioned above, and possibly a disproportionate growth of the posterior tectum in the period after the grafts were placed. Second, the directions of growth of fiber bundles exhibited by the specimens as a whole warrants comment. In any quadrant of the tectum, arrows are seen oriented in several directions. Particularly in the central area several arrows point dorsally, ventrally, and posteriorly. However, only five grafts sent fibers anteriorly; and in the FIG. 4. Transverse section of left optic lobe. A retinal graft (nasal) partly visible in the lower right corner, lies in a broad depression in the tectal surface. Pigment epithelium from the graft has formed a branching figure containing no neural epithelium (upper right) and below this forms a partial sleeve around the fiber bundle coursing dorsoposteriorly from the graft. In the plane of this section, the fiber bundle from the graft is seen coursing over the tectal surface and finally penetrating the surface to be lost within the superficial tectal layer. Inset: Highpower view of fibers turning downward into the substance of the tectum. FIG. 5. (a) Another example of a retinal graft (dorsal). Part of the graft is seen at the upper left. The graft and the surface membrane are artifactually pulled away from the tectum. Two fiber bundles are visible passing from the right margin of the graft to the pial membrane and then continuing laterally (down-pointing arrows). Beneath the graft, in the left half of the photograph, there is another stout fiber bundle resting immediately on the membrane (uppointing arrow). This bundle arises from the left (medial) side of the graft, bends around it, and continues laterally under the graft to join the other fibers. The first part of its course is not included in this section. x 114. (b) Higher-power view of the same specimen, showing parallel fiber bundles coursing laterally. x 285.
522
DELONC
AND
FIG.
COULOMBRE
6
RPTT~NOTECTAL CoNmcTIoNs
523
four of these with fiber bundles of significant length, the grafts were located posteriorly (the exception is in Fig. 10, near the dorsal margin). No clear explanation offers itself for the rather striking observation that of the many grafts placed centrally or anteriorly none sent fibers anteriorly, though collectivelv they sent fibers in all other directions. Finally, the directions taken by fiber bundles may be considered in relation to the specific retinal origin of the grafts (as grouped in the diagrams). Examination of the group of 17 grafts of dorsal retina reveals that, with one exception, the fiber bundles originating from the grafts are directed ventrally and laterally, regardless of the position of the graft on the tectum (Fig. 7). In contrast, eleven grafts of ventral retina, situated generally on the same areas of tectum as the dorsal group, with one exception have their fiber bundles directed dorsally and medially (Fig. 8). Ten grafts of nasal retina are represented in Fig. 9. The general direction of the fiber bundles dorsally and posteriorly is clear; even the one possible exception (the most dorsal graft) has a fiber bundle which though directed laterally penetrated the pial membrane within the posterodorsal “target zone” defined by the direction of the other fibers. However, the very anteriorly placed grafts with short fiber bundles can be given little weight as indicating any preferential orientation of fibers. More significant are the long fiber bundle arising at the anterior pole and passing dorsoposteriorly, and the other long fascicles ending in the posterodorsal quadrant. The group of 15 grafts of temporal retina (Fig. 10) are particularly important. Several features may be stressed: (1) This group of grafts is remarkably consistent in the direction of fiber growth, more so than any of the other groups. A relatively small “target area” may be described. The grafts tend to be concentrated centrally and anteriorly, but comparison with the dorsal group shows that this in itself cannot account for the consistency of the findings. (2) The temporal group FIG. 6. (a) Lateral surface of the optic tectum. A retinal graft (temporal) is out of the field at the right, and a small fiber bundle arising from it enters the plane of the section about the middle of the photograph and traversesthe surface to the left margin of the picture, where it turns downward to enter the tectum. x 160. (b) High-power view of the same specimen showing the course of the fibers. x 450.
524
DELONG
I mm.
AND
COULOMBBE
V
FIGS. 7-10. These diagrams are two-dimensional representatives of the surface of the left tectum of the U-day chick embryo. In each, the origin, direction, and length of fiber fascicles arising from retinal grafts have been indicated by arrows (see text). The circles are diagrammatic representations of grafts whose fibers penetrated directly into the tectum beneath the graft; their diameter is roughly proportioned to that of the area innervated. The results found with retinal grafts from each quadrant of the eye are presented separately. FIG. 7. Seventeen grafts of dorsal retina. FIG. 8. Eleven grafts of ventral retina.
differs from the nasal group less clearly than does the ventral from the dorsal, as seen above. (3) The temporal group is not significantly different from the ventral group in the orientation of the fiber fascicles; however, both can clearly be separated from the dorsal group. In fact, the strongest conclusion that arises from this analysis is that retinal grafts of dorsal origin quite consistently send fiber bundles ventro-
RETINOTECTAL
525
CONNECTIONS
FIG. 9. Ten grafts of nasal retina. FIG. 10. Fifteen grafts of temporal retina. Specimens bundles are shown by two arrows from a common source.
with
divergent
fiber
laterally, while retinal grafts of temporal or ventral origin, placed on the same areas of the tectum, consistently send fiber bundles dorsally and medially. The group of nasal retina grafts send fibers toward a third distinct field, in general more like ventral than dorsal retina. There thus appears a consistent differential property of pieces of retinal tissue that correlates with the part of the eye from which the retina was originally taken. Three different distinct areas within the 4-day chick embryo retina can be identified by the available data; dorsal, ventrotemporal, and nasal. Whether the “map” of local specificities has finer definition at this early embryonic stage cannot be answered from the present data. It may be that as the retina and tectum develop
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further, a progressively finer mosaic of differentiated local areas appears. Are the differential properties demonstrated for different retinal areas simply a curiosity of an artificial experimental system, or may they be related to the normal mechanisms for constructing specific orderly connections between the retina and tectum during organogenesis? Data from the mapping of normal retinotectal interconnections tends to support the second possibility. The representation of the retina on the embryonic chick tectum derived independently from retinal ablation studies ( DeLong and Coulombre, 1965) is reproduced in Fig. 11. This shows ventral retina projecting on dorsal tectum,
FIG. 11. Retinotopic map of the 12-day optic tectum, derived from ablations of retinal quadrants in the 4-day chick embryo with subsequent determination of the patterns of fiber distribution on the optic tectum, for comparison with the preceding figures. The labels refer to areas of retina, showing their topographic representation on the surface of the tectum.
dorsal retina on ventral tectum, nasal retina on posterodorsal tectum, and temporal retina on anterodorsal tectum but overlapping widely with the ventral projection. This mapping is consistent with the fiber orientations observed in the present experiments, even to the difficulty in distinguishing ventral and temporal areas, and the sharp, clear-cut distinction between dorsal and ventral-temporal areas. Since both sets of experiments presumably reflect the state of the retina at 4-5 days-when the grafting and ablations were performed-the parallels between the two sets of data strengthen the likelihood that the fiber orientations in the present experiments reflect the normal mechanisms for establishment of retinotectal interconnections.
RETINOTECTAL
CONNECTIONS
527
DISCUSSION
The system utilized herein offered several advantages for confrontation of source (retina) and target (tectum) tissues. The chick tectum is readily accessible to manipulation during embryonic stages. The normal afferent innervation to the tectal surface, which in the early embryo consists only of the optic fibers, is easily prevented from reaching the tectum by excising the contralateral eye, thus leaving the tectum free of surface fibers and receptive to the fibers emanating from the graft. Furthermore, the graft fibers are then conspicuous on the otherwise bare tectal surface. Since the tectum has a relatively wide expanse, placement and mapping can be fairly accurate. Finally, the retinotopic map of the embryonic tectum had been outlined previously. A disadvantage is that the silver stains employed do not permit visualization of the terminations of fibers from the grafts. Where the fascicles penetrate the pia and form a subpial mass (as in Fig. 2) in the normal position for optic fibers, it is reasonable to suggest that they may terminate in the same way as normal optic fibers reaching the same location; however, from the present preparations this cannot be determined. In contrast to the uncertainty about precise terminations, it was possible to identify accurately fibers advancing out of the graft and across the tectal surface. The fibers generally issued from the graft at well-defined points and traversed an empty, cell- and fiber-free space where the smallest fascicles of fibers stood out clearly. In almost all cases fibers grew out from a retinal graft in only one direction, Where more than one fascicle formed, the fascicles were parallel to one another. Occasionally fiber bundles curved around the graft or changed direction and assumed a straight course on nearing the tectal surface. It was necessary to ask, however, whether the final course of the fibers reflected some nonspecific or extraneous force. Conceivably, the graft might have been displaced, towing the fibers behind it, by a shift of the overlying membranes in relation to the surface of the tectum; such a shearing action could conceivably result from the continued growth of the tectum while the graft was held in place by the meninges. While this factor may have operated to a minor degree, it cannot explain the results found, for the following reasons: 1. Under any kind of passive movement, the fibers of all grafts
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placed in any one area of the tectum would be expected to be oriented predominantly in the same direction. The results were otherwise; fibers of some grafts coursed in opposite direction or at right angles to those from other grafts on the same area. 2. The initial placement and the final location of the grafts at the time of sacrifice were identical in three-fourths of the cases. When a shift of position was recorded, there was no consistency in the direction of the shift and no demonstrable relation between the latter and the course of fibers from the graft. 3. Regardless of the site of the graft on the tectum, the direction taken by outgrowing fibers correlated with the retinal site of origin of the graft. By similar reasoning, one can dismiss the possibility that the fibers simply followed the channels that would be taken by normal optic fibers over the tectum. One further possibility must be considered: that the orientation of the retinal graft on the surface of the tectum determined the direction of fiber outgrowth, i.e., that the direction of outgrowth depended only on the retina and was independent of the tectum. Were this the case, the fiber orientations should have been random and independent of the quadrant of origin of the graft since, as stated above, the grafts were positioned in random orientation with regard to their dorsoventral and anteroposterior axes. The question was raised above (see Introduction) whether the normal orderly connection of retinal fibers to specific areas of the tectum depends on the integrity of the overall configuration of the entire optic system, as if the axons simply advance along preexisting tissue channels to their destination. The inadequacy of this interpretation has been shown by the experiments of Arora and Sperry (1962) in which they displaced bundles of optic fibers from their normal pathways and observed that they regained their proper positions, and by experiments with fused half-eyes in embryonic toads which showed that fibers from one-half of the retina would spread out over the entire tectum under certain circumstances (Gaze et al., 1963, 1965). It is further contradicted by the results reported herein, in which the normal relationships were grossly disrupted, with the fragments of grafted retina of 4-day embryos in direct apposition to the tectum of 6- or 7-day hosts. Under these conditions the fibers arose in abnormal locations and traversed the tectum in directions very different from
HETINOTECTAL
CONNECTIONS
529
those normally taken by optic fibers. Nevertheless, the direction of fiber outgrowth correlated with the local retinal origin of the graft, and was generally directed toward the appropriate local area of termination on the tectum. This result indicates a specific active matching of retinal fibers and tectal areas, and supports the hypothesis advanced by Sperry (1963). The embryologic and cellular mechanisms underlying the specific fiber orientations found in the present experiments are not known. Specific matching requires that each retinal graft contain some localityspecific marker which differentiates it from other parts of the retina; similarly each area of the tectum must be differentiated from other areas by some specific marker. Finally, the retinal and tectal markers must have some means for mutual recognition. The nature of the postulated markers is unknown. Some evidence as to the intermediate mechanisms controlling oriented fiber growth may be gained from the data presented herein. One possible mechanism is that pioneering fibers, searching randomly, may find matching terminal fields by trialand-error, with secondary enlargement of the successful tract by “selective fasciculation” (Weiss, 1950) and atrophy of the unsuccessful pioneering fibers. The available data give no evidence of such a process. No randomly straying fibers were seen, even in the near vicinity of the graft where they should be most numerous. While all such fibers may have grown out to all areas of the tectum and then atrophied without trace in all cases during the interval between grafting at 7 days and harvesting at 12 days, this seems unlikely. The normal rate of growth is such that the advancing “front” of optic fibers grows from the ventrolateral margin of the tectum on the sixth day of incubation to the dorsomedial margin on the twelfth day (DeLong and Coulombre, 1965), a rate approximating that of the longest graft fascicles; for random pioneer fibers to have grown to all areas and disappeared within this period, one must postulate a much faster rate of growth. Further, the fascicles clearly do not follow any random or meandering path; they course in a straight and direct line, so much so that they may be followed in a single histological section for as much as 2 mm. This strongly suggests that the fibers are accurately guided throughout their course. The fact that fibers advanced for considerable distances over the tectum above the pial membrane, apparently directed toward a specific goal, suggests that factors responsible for guiding the fibers were
530
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COULOMBRE
acting at or immediately above the surface of the membrane. The membrane was about 5 p thick and appeared to separate the retinal fibers from direct contact with the tectal cells, up to the point where the fibers penetrated the membrane. One may speculate either that the pial membrane (or the connective tissue outside it) contains the guiding cues and thus determines the retinotopic map, or alternatively that the relevant factors originate in the superficial tectal cells and act over a short distance. One final suggestion arises from the present work. Since the retinotopic “map” on the tectum appears to arise independently of that on the retina, one may speculate that the “tectotopic map” (of some center which receives tectal efferents) might arise independently of the tectum. One would expect that the factors determining the local topographic specificity of other areas within the central nervous system might have some common genetic and developmental basis with those of the retina and tectum. This possibility of a shared specificity would seem to be susceptible to test by confrontation experiments similar to those reported here. SUMMARY
Pieces of retina from known quadrants of 4-day chick embryo eyes were grafted onto the surface of the o’ptic tectum of 6- or 7-day embryos (the contralateral eye of the hosts having been excised in an earlier operation to prevent the normal complement of optic fibers from reaching the tectum). The embryos with grafts were incubated 5 days, then prepared for histological study. Examination of serial sections stained with Protargol revealed that the retinal grafts had differentiated normally and produced fibers which passed onto the surface of the tectum as a single bundle, or as parallel fascicles. These coursed for varying distances on the surface membrane above the tectum, ending in many cases by piercing the membrane and entering the superficial tectal layers. The location of the graft on the tectal surface and the course and length of the fiber bundles were recorded, and the results grouped according to the quadrant of origin of the retinal graft. Fibers from a particular quadrant of retina preferentially grew toward specific areas of the tectum (dorsal retina to lateroventral tectum; ventral retina to dorsal tectum; nasal retina to posterodorsal tectum; temporal retina to anterodorsal tectum). These findings correlated with the known retinotopic distribution of normal optic fiber projec-
RETINOTECTAL
531
CONNECTIONS
tions onto the tectum. Possible mechanisms guidance of fibers are discussed.
mediating
the specific
REFERENCES AHORA, H. L., and SPERRY, R. W. ( 1962). Optic nerve regeneration after surgical cross-union of medial and lateral optic tracts. Am. Zoologist 2, 389. ATTAHDI, D. G., and SPERRY, R. W. (1963). P re f erential selection of central pathways by regenerating optic fibers. Exptl. Neural. 7, 46-64. DELONG, G. R., and COULOMBRE, A. J. (1965). D evelopment of the retinotectal topographic projection in the chick embryo. Exptl. Neural. 13, 350463. GAZE, R. hf., JACOBSON, M., and SZBKELY, G. (1963). The retinotectal projection in Xenopcs with compound eyes. 1. Physiol. (London) 165, 484499. GAZE, R. hf., JACOBSON, M., and SZ~KELY, G. (1965). On the formation of connexions by compound eyes in Xenopus. J. Physiol. (London) 176, 409417. ROBERTS, R. B., and FLEXNEH, L. B. (1966). A model for the development of retina-cortex connections. Am. Scientist 54, 174-183. SPERRY, R. W. ( 1963). Chemoaffinity in the orderly growth of nerve fiber patterns and connections. Proc. Natl. Acad. Sci. U.S. 50, 703-710. SZ~KELY, G. ( 1966). Embryonic determination of neural connections. A&an. Morphogenesis 5, 181-219. WEISS, P. ( 1950). The deplantation of fragments of nervous system in amphibians. I. Central reorganization and the formation of nerves. J. Exptl. Zool. 113, 397461.