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Growth pattern of pioneering chick spinal cord axons
DEVELOPMENTAL
BIOLOGY
123,375-388
(1987)
Growth Pattern of Pioneering Chick Spinal Cord Axons’ JOHN Department
of Developmental
A.
ANDJERRYSILVER
HOLLEY’
Genetics and Anatomy,
Received April
Case Western Reserve University,
9, 1985; accepted in revised
Cleveland, Ohio 44106
form May 26, 1987
The early growth pattern of axons in the embryonic chick spinal cord was studied by electron microscopy. Serial perisagittal thin sections were obtained from the lateral margins of spinal cords of stage 17 (S17) and S19 embryos. A simple stereotypic pattern of axonal growth was found. Axons originated from a dispersed population of presumptive interneurons located along the lateral spinal cord margin. They first grew ventrally in a nonfasciculative pattern and later turned at right angles and grew in a fasciculative manner longitudinally in the ventrolateral fasciculus. Growth along the circumferential pathway was analyzed in detail by reconstructing individual axons and growth cones from the S17 specimen. Most circumferential axons, regardless of their site of origin, grew in a parallel orientation, and each of their growth cones projected ventrally. This pattern suggested that circumferential growth cones were guided at many, if not all, points along their path. Study of the region in front of these seven growth cones, however, revealed no apparent structural basis for their guidance. Alternative guidance mechanisms are discussed, In conjunction with previous studies (e.g., Windle and Baxter, 1936; Lyser, 1966), these findings suggest that the circumferential-nonfasciculative and the longitudinal-fasciculative patterns of axonal growth are the two fundamental patterns followed by most early forming axons in the brain stem and spinal cord of all higher vertebrates. o 1987 Academic press, IIIC.
INTRODUCTION
The early growth pattern of axons of spinal cord interneurons is similar in all higher vertebrates ranging from chick to human (Rambn y Cajal, 1960; Windle and Orr, 1934; Windle and Austin, 1936; Windle and Fitzgerald, 1937; Holley, 1982). Axons of early forming interneurons first grow circumferentially along the lateral margin of the embryonic spinal cord and in the transverse plane. After coursing ventrally, they turn and grow longitudinally in either the ipsi- or contralateral ventrolateral fasciculus (Ramon y Cajal, 1960; Windle and Austin, 1936; Windle and Baxter, 1936b). The apparent simplicity of this pattern lends itself to the study of the mechanisms of axonal guidance. Various mechanisms of axonal guidance have been proposed based on a large number of studies (reviews, Jacobson, 1978; Horder and Martin, 1978; ConstantinePaton, 1979). Several guidance mechanisms have been considered more recently; one involves the formation of oriented extracellular spaces (channels) prior to axonal growth (Singer et al, 1979, Silver and Robb, 1979; Silver and Sidman, 1980). A second mechanism, demonstrated in grasshopper appendages, involves guidance by closely spaced guidepost or landmark cells (Bentley and Kesh-
i Portions of this study have been reported elsewhere (Holley and Lasek, 1982; Katz and Lasek, 1985). ‘To whom correspondence and reprint requests should be addressed at Department of Neurology, Yale University School of Medicine, 333 Cedar St., New Haven, CT 06510. 375
ishan, 1982; Ho and Goodman, 1982). Growth cones extend long filopodia and form gap junctions with guidepost cells (Bentley and Caudy, 1983; Taghert et aL, 1982). A third mechanism involves the guidance of axons according to local variations in the relative adhesiveness of the tissue substrate for growth cones (Nardi, 1983; Berlot and Goodman, 1984). Whether these or other guidance mechanisms direct the early axonal growth of vertebrate interneurons was investigated in this and the following paper (Holley, 1987). In the present study, serial sections were obtained from the lateral margin of chick spinal cords at two early stages of development. The pattern of early developing axons, their shape and morphological relationship with surrounding cells, was determined. Several pioneering growth cones were found within the series and were studied in detail. MATERIALS
AND
METHODS
White Leghorn chicken embryos were obtained after various times of incubation at 38°C and staged according to Hamburger and Hamilton (1951). Embryos were immersed in fixative containing 2% glutaraldehyde, 2% formaldehyde, 0.5% acrolein, 0.5% dimethyl sulfoxide, and 3 mM CaCl, in 0.08 M cacodylate buffer at pH 7.4. After 30 min, the brachial spinal cord region adjacent to somites 17-19 was dissected and stored overnight at 4°C in fresh fixative. Specimens were postfixed in 1% OsOl for 1 hr in the same buffer, rinsed in 0.08 M phosphate buffer, and stained en bloc with 2% uranyl acetate for 30 min. Specimens were dehydrated in ethanol and embedded in Spurr’s plastic. 0012-1606/87 $3.00 Copyright All rights
0 1987 by Academic Press, Inc. of reproduction in any form reserved.
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A stage 1’7 (S17) chick spinal cord of apparently normal size and form was chosen for serial sectioning. After careful trimming, the specimen was serially thin sectioned at -80 nm thickness in an obliquely sagittal plane (see Fig. 1). A total of 100 serial sections were collected, beginning at the lateral edge of the neural tube, and were transferred to single slot, formvar-coated grids.
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After drying, the sections were stained with uranyl acetate and lead citrate and examined with a JEOL 100 TEM. Every fifth section in the series was completely micrographed at a primary magnification of 2000x, and montages were then constructed on cardboard sheets at a total enlargement of 5500X. Outlines of the spinal cord margin and all long cellular processes and perikarya were drawn on plastic overlays. Structures of special interest and those with unclear boundries were reexamined and micrographed at higher magnifications. The entire length of all the long cellular processes in the series was reconstructed by overlapping consecutive plastic overlays and aligning contiguous structures. In addition to manual reconstruction, the patterns of axons and selected growth cones were reconstructed by computer-aided graphics as described by Carney and Silver (1983). A S19 chick brachial spinal cord was also prepared and sectioned in the same manner as described above. Approximately 150 serial sections were obtained. Five sections within the series were selected and completely micrographed at 3300x. They were each located between 1 to 2 pm apart as judged by changes in the sizes and shapes of identified cell nuclei. An additional 5 sections were micrographed at 1000X, and these together with the higher magnification montages made it possible to identify the pattern of axons within the series. After serial thin sectioning, the S17 and S19 spinal cord specimens were reoriented and sectioned in a slightly oblique transverse plane. The resulting semithin sections showed the final depth and plane of the serialthin sections. In addition, the pattern of longitudinal axons was examined in transverse thin sections of the S19 spinal cord. RESULTS
FIG. 1. (A) Light micrograph of an obliquely transverse section of S17 spinal cord after serial thin sectioning. The perisagittal plane and final depth of thin sectioning are seen along the right margin (sect. pln.). The total depth of tissue reconstructed is illustrated as the area between the dashed lines along the left margin. Extracellular space (es) is more abundant in the dorsal than the ventral region of the brachial neural tube. Bar = 50 Wm. Similar semithin section of S19 spinal cord shown after thin sectioning and reorientation. Circumferential axons (ca) are seen medial to the primordial motor column and are included in the zone of serial thin sections. f, ventrolateral fasciculus. Bar = 50 pm.
Figure 1 shows slightly oblique transverse sections of the S17 and S19 spinal cord specimens obtained after thin sectioning. The final depth and plane of serial thin sections is illustrated. In both specimens, as well as others of similar age, there is relatively more extracellular space in the dorsal as compared to ventral regions of the spinal cord. The extracellular spaces are more prevalent along the lateral margin, but are not restricted to the marginal zone. The spaces have various shapes and no discernible preferential orientation. Sl7 Axonal Pattern The serial thin sections of the S17 specimen numbered 1 to 100, beginning at the lateral spinal margin. Figure 2 shows section 80. The principle ponents of the marginal zone include the endfeet distal processes of neuroepithelial cells, as well as
were cord comand peri-
.
-
.
FIG. 2. Low magnification montage of section 80 from S17 spinal cord series. Numerous circumferential of growth cones l-5 and 7. Motor axons (arrows) are along the ventral region. Bar = 10 Km.
---
axons are seen, as well as portions
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karya located more centrally. Most of these cellular profiles are seen in cross section and are oval in shape. In addition, there are a number of processes that course for longer distances in the marginal zone, i.e., parallel the plane of section. Each of these longer cellular processeshave a more electron-lucent cytoplasm and therefore could be readily traced in the low magnification EM montages. The outlines of all long cellular processes and most perikarya in every fifth section of the series were drawn on plastic overlays. Figure 3 shows several of these drawings, including that of section 80. Individual cells and processes were identified and then reconstructed by following each one in the sequential sections. A composite illustration showing all of the long cellular processes and most perikarya in the S17 marginal zone is presented in Fig. 4. Most of the processes continue to course beyond the limits of the serial sections and thus appear as blunt or cut ends in the drawing. In the dorsal and middle regions all long cellular processes are oriented dorsoventrally. They have been numbered l26. Each one is identifiable ultrastructurally as an em-
showing lateral view of S17 spinal cord with FIG. 4. Reconstruction . _. all axons and most perikarya in the serial sections. Several of the total 26 circumferential axons are numbered. Seven axonal growth cones are present, and each projects ventrally. Growth cone 2 contacts circumferential process 11 at the location of the arrow. Longitudinal axons and apparent growth cones (a,b) are at the level of ventral rootlets (vr). Bar = 50 pm.
FIG. 3. Drawings of all long cellular processes and selected perikarya present in several sections of 517 spinal cord. Section number corresponding shown in parentheses. Continuity of many axons can be followed between sections. All axons in dorsal and middle regions course dorsoventrally. Several shorter, longitudinally oriented axons appear in the last section (F) and are located at the level of ventral rootlets. Bar = 100 pm. From Holley (1983).
bryonic axon based on its electron-lucent cytoplasm that contains numerous longitudinally oriented microtubules and intermediate filaments, dense mitochondria, and few nonaggregated ribosomes (Figs. 2 and 5-7). Thus based on shape, orientation, and ultrastructure these processes are identified as circumferential axons. Most circumferential axons occur singly within the tissue and are surrounded by perikarya and neuroepithelial cell processes (Figs. 2 and 5-7). In addition, several pairs of circumferential axons are present which course side-by-side for a variable distance. These adjacent axons may then diverge and continue separately. In one instance three axons are located in juxtaposition (Fig. 4). Noteably, all long axons are oriented in a stereotypic dorsoventral direction. Their course is markedly
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FIG. 5. Ventral margin of S17 spinal cord from section 49. Endfeet (ef) are irregularly shaped, have no preferential have surface junctions (j). Circumferential axons occasionally lie subjacent to the basal lamina (bl). Bar = 4 ym.
straight with no more than a few bends at low angles of curvature. These axons do not branch. The direction of growth can be determined with certainty for 14 of the 26 circumferential axons. Seven axons terminate with growth cones and another 7 axons have their cell bodies of origin at least partially within the serial sections (Fig. 4). In four additional cases, circumferential axons (9,11, 12, and 13) are found which turn at a right angle and have a medially directed segment that continues several micrometers toward the ventricular zone before the series end. Presumably the cell bodies of these axons are located more medially within the ventricular zone. Axons 11 and 12 are two such axons that appear to project out from the ventricular zone, turn, and grow ventrally. Unlike the other two axons, they have an enlarged region of cytoplasm at the corner where they turn circumferentially (Figs. 2 and 4). Each of these 18 circumferential axons, except one, is directed ventrally. The single dorsally directed axon (25) is noteably among the single set of three fasciculating axons, and its cell body is located in the ventrolateral region.
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orientation,
and often
As shown in Fig. 4, circumferential axons appear to originate from cell bodies located at nearly all levels along the lateral spinal cord wall. They are not aligned in dorsoventrally arranged sets, which may indicate an interdependent mechanism of growth. The distribution of circumferential axons was also analyzed statistically to test for certain types of growth patterns. If contact inhibition directs growth, for example, then a nonrandom, segregated distribution would be expected along the rostrocaudal axis. Hence, the composite illustration of circumferential axons was blindly divided into 10 dorsoventrally oriented areas. The number of axons within each area was counted along seven equally spaced lines, representing an even sample of all dorsoventral regions of the marginal zone. The observed frequency of axons within each test division was compared with the expected frequency based on a Poisson distribution (x = 48163). The results showed that the distribution was not significantly different from random (X = 4.37, df = 2, P = 0.2). Thus, there is no statistical evidence of significant repulsion or attraction
FIG. 6. Growth cones 1 and 2 have several filopodia (f) and project ventrally (direction arrow). Growth cone 2 is atypical with mark1 edly electron lucent cytoplasm; it contacts circumferential axon 11 (arrow). Extracellular space (es) is common in dorsal spinal cord, but is not arranged in aligned channels. r, radial processes. Bar = 4 pm. 380
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within this small sample of early growing circumferential axons. In addition to circumferential axons, a number of ventral motor axons appear to exit the ventrolateral region of the spinal cord (Fig. 2). At the same level, a few relatively short, longitudinally oriented axons are found (Fig. 4). The origin of these longitudinal axons could not be traced because they are located at the edge of the serial sections. Sl7 Growth Cones
Seven of the 26 circumferential axons terminate with a definitive growth cone. Each has a bulbous body 3-4 pm in diameter and several filopodia that extend in various directions for distances of up to 6 /*rn (Figs. 6 and 7). The growth cones are located between 4 and 6 pm from the basal lamina. Although none of the growth cone filopodia appear to reach the basal lamina, contacts with small processes could have escaped detection. Substantial ultrastructural variation is seen among the growth cones. Most are relatively electron-lucent such as growth cones 4 and 5 (Fig. 7). Others are more electron-dense and have fewer membranous organelles such as growth cone 1 (Fig. 6). Growth cone 2 is distinct. As illustrated in Fig. 4, it contacts the radially oriented segment of circumferential axon 11, whose cell body presumably resides deeper within the ventricular zone. In contrast to the other growth cones, the surface of circumferential axon and growth cone 2 is not fully covered by surrounding cells (Fig. 6). The distended cytoplasm that is largely devoid of ground substance indicates that growth cone 2 may be regressing. Noteably, all seven growth cones are directed ventrally from a dorsally located parent axon (Fig. 4). Growth cones 1,3,4, and 7 are not contacting other growth cones or nearby axons. By definition, these are pioneering circumferential growth cones. The region surrounding and immediately in front of the pioneering growth cones was analyzed in detail for the following features that might guide circumferential axons: Shape and alignment
of marginal
zone cellular
ele-
ments. The circumferential axonal growth cones are surrounded by distal processes of neuroepithelial cells and perikarya. As shown in figures 6 and 7, the cellular elements in front of the growth cones have various shapes, but tend to be rounded with no distinct dorsoventral polarity. There is also no indication that cells or processes are aligned in a dorsoventral pattern that would physically guide circumferential growth. Aligned extracellular spaces. Although extracellular spaces are present, they are rarely found immediately in front of growth cones (Figs. 6 and 7). Furthermore,
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there is no indication in the serial sections that dorsoventrally aligned sets of extracellular spaces (i.e., channels) are present at any level in the lateral spinal cord. Guidepost and landmark cells. Most circumferential axons grow in a parallel, nonfasciculative pattern and show no apparent tendency to congregate locally at specific (guidepost or landmark) sites (Fig. 4). Since circumferential growth cone filopodia are apparently less than 7 pm long, several potential guidepost and landmark cells must be positioned in dorsoventrally oriented sets in order to guide circumferential axons. Cells and processes in front of these pioneering circumferential growth cones, however, appear structurally indistinguishable from other cells in the marginal zone (Figs. 6 and 7). In no case, are sets of nonaxonal filopodia found aligned in advance of circumferential growth cones. Intercellular junctions. Despite extensive search for specialized contacts on the surfaces of each axonal growth cone and filopodia, no instance was found of a localized specialization, such as close membrane apposition or membrane-associated electron-dense material, that would indicate the presence of an intercellular junction. As a morphological control, numerous desmosomal-like junctions are observed between adjacent neuroepithelial endfeet (Fig. 5). S19 Axonal Patterns and Growth Cones
As shown in Fig. lB, the serial thin sections of the S19 brachial spinal cord included most of the circumferential axonal pathway. At this stage of development, immature neurons have migrated into the primordial motor column, and the early forming circumferential axons are displaced medially from the ventrolateral margin (Holley, 1982). Hence the serial sagittal sections included the marginal zone in the dorsal region, as well as the region between the motor column and the ventricular zone. All axons and many cell nuclei present in four sections of the series are shown in Fig. 8. Except for two short portions of axons in Fig. 8B, all axons in the dorsal region are oriented dorsoventrally. Dorsal root fibers evidently have not entered at least this region of the spinal cord. Circumferential axons tend to be arranged in a layer along the lateral surface of the ventricular zone. The axons are often positioned side-by-side in the layer and seldom fasciculate in large bundles. The density of axons found in this S19 specimen is about twice as large as that in the S17 specimen. Also shown in Fig. 8 are the shapes and locations of all four growth cones found in the serial sections. Growth cones 1 and 2 are located within the middle of the spinal cord, Figs. 8C and 8D, respectively. Both are directed ventrally and are about 8 pm from the neural tube sur-
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FIG. 8. Drawings of four sagittal sections from S19 spinal cord series. Basal lamina (bl) along dorsal and ventral borders is drawn, as well as all cell nuclei. Symbols (m, 0, A) denote nuclei seen in adjacent sections. All axons are drawn, and except for two axons in section B, all in the dorsal and middle regions course dorsoventrally (black processes). Growth cones are labeled G4 and G5. Along the ventral margins of(D) are several short profiles of longitudinal axons (la) and growth cone 4. Bar = 50 pm.
face. Growth cone 3 is located in the ventral area of Fig. longitudinal axons is present at this stage and is located 8D and is about 10 pm from the surface. Each of these near the ventral edge of the primordial motor column circumferential growth cones are located near several (Fig. 1B). This ventrolateral fascicle contains 12 axons circumferential axons; however, there is no direct sur- (Fig. 10). As compared to the circumferential axons, face contact (Figs. 8 and 9A). These observations indicate these longitudinal axons grow in a single bundle with that later forming circumferential axons tend to grow extensive axon-to-axon surface contact. in regions of earlier forming axons, and yet there is little DISCUSSION tendency to fasciculate. Along the ventral margin of Fig. 9D several profiles Fasciculative vs Nonfasciculative Growth of longitudinal axons are present as well as two cellular processes, labeled G4 and G5. G4 appears to be a cirThe overall pattern of early axonal growth shown in cumferential growth cone that is contacting an area of the present and companion study (Holley, 1987) is in longitudinal axons (Fig. 9B). G5 is an L-shaped process accord with earlier light microscopic investigations with axonal-like cytoplasm (Fig. SC). Adjacent serial (Ramon y Cajal, 1960; Windle and Austin, 1936). The sections showed that both G4 and G5 were in contact first axons that grow within the spinal cord are from with a fascicle of longitudinal axons (Fig. 9D). G4 and early forming interneurons; they first grow circumferG5 thus appear to be axons at different stages of turning entially and later turn and grow longitudinally in the from circumferential to longitudinal orientations. primitive ventrolateral fasciculus. Axons from motor To further analyze the axonal patterns in this S19 neurons also develop at this time and exit the cord. spinal cord, the specimen was reoriented and thin-secOne remarkable aspect of the pattern is the distinct tioned in the transverse plane. One major fascicle of difference between circumferential and longitudinal
FIG. 7. (A) Growth cone 5 located adjacent to axons 4, from section 75 of Sli’ series. Several dark filopodia (f’) project from surrounding cells and contact growth cone 5 filopodia. Filopodium of growth cone 4 can be seen further ventrally (4f). The nucleus (n) is labeled for reference in (A-C). Bar = 2 Frn. (B) Computer-aided reconstruction of growth cones 4 and 5 with their axons. Filopodia extend from each growth cone up to 5 pm in various directions. Bar = 4 pm. (C) Growth cone 4 seen in section 90. Note lack of specialized contacts and processes extending from surrounding cells. Bar = 2 pm.
FIG. 9. (A) Growth cone 3 (gc) located medial to primordial motor column of S19 neural tube (labeled G3 in Fig. 8D). Filopodia (f) extend ventrally. Bar = 2 pm. (B) Growth cone 4 (gc) appears to approach longitudinal axons (a) at the ventral margin (labeled G4 in Fig. 8D). Large diameter process from growth cone has a microfilamentous cytoplasm, but no microtubules or intermediate filaments. Bar = 2 pm. (C) Process with axonal-like cytoplasm (a’) located along the ventral margin (labeled G5 in Fig. 8D). The process forms a right angle turn from circumferential to longitudinal. Bar = 2 pm. (D) Longitudinal axons located medial to the presumptive turning axon. Bar = 2 pm. 384
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velop along the entire lateral wall of the neural tube from the spinal cord rostrally through the mesencephalon (Windle and Baxter, 1936b,Windle and Austin, 1936, Lyser, 1966). These axons are derived from a dispersed population of interneurons that grow circumferentially in a nonfasciculative pattern until they turn and grow longitudinally in a variety of fascicle tracts. The concept of these two fundamental growth patterns was first suggested by Holley (1983; J. A. Holley, J. Silver, and R. J. Lasek, unpublished data) and has also been discussed by Katz and Lasek (1985). The distinct difference in axonal growth patterns suggests that at least two basic sets of factors guide axons in the vertebrate nervous system. One of the factors that may coordinate the growth of the axons along the longitudinal pathway is the preferential accumulation of the cell surface adhesion molecule NILE along fasciculating spinal cord axons (Stallcup et aZ.,1985). Growth Pattern
FIG. 10. Transverse section S19 specimen. Its location is axons (la) are grouped into cones (gc) are located near axon (ca) is located medially ef, endfeet. Bar = 2 pm.
of ventrolateral fasciculus from the same illustrated in Fig. 1B. Twelve longitudinal the bundle, and two presumptive growth the basal lamina (bl). A circumferential and is in direct contact with the fascicle.
growth. Circumferential axons grow in a nonfasciculative pattern, whereas longitudinal axons grow in fascicles. Even after numerous circumferential axons had developed by S19, circumferential axons were distributed in a layer along the ventricular zone margin and showed little tendency to fasciculate. Furthermore, several of the growth cones in both the S1’7 and S19 specimens were located within filopodial reach of preformed axons but did not fasciculate. Longitudinal axons, on the other hand, grow in close axon-to-axon contact in the developing funiculi. The first fascicles of longitudinal interneuronal axons develop along the ventrolateral spinal cord margin and subsequent axons accumulate in an orderly fasciculative manner (Windle and Orr, 1934; Windle and Austin, 1936; Nornes et al., 1980). The distinction between circumferential-nonfasciculative growth and longitudinal-fasciculative growth appears to be a general organizing feature of the vertebrate brain stem and spinal cord. Circumferential axons de-
of Circumferential
Axons
Most early forming axons that grow along the lateral margin of the spinal cord are oriented in a markedly straight dorsoventral orientation (Holley, 1987). They originate from presumptive interneurons that are distributed throughout the lateral wall of the neural tube, including the far dorsal and ventral aspects. The cellular milieu through which circumferential axons grow consists of a maze of radially oriented neuroepithelial cell processes and other cells that might physically deflect linear growth, and indeed, many of the axons had shallow bends. Nevertheless, axons continued to grow circumferentially without appreciable wandering, indicating that the factor(s) which guide circumferential growth are functioning at many, if not all, points along the embryonic spinal cord’s lateral margin. The direction of many circumferential axons was determined based on the relative positions of their cell bodies or growth cones. Each of these circumferential axons were directed ventrally, except one. The lone dorsally projecting axon originated from a cell body in the far ventrolateral region of the S1’7 spinal cord. This atypical axon may be from an “aberrant” motor neuron described by Ramon y Cajal(l960) which grows dorsally from the motor column and exits the cord in the dorsal root. This axon was among the single set of three fasciculating axons, and coincidentally, motor axons are known to grow in fascicles (Ramon y Cajal, 1960). A detailed study of early interneuron development by Wentworth (1984) supplements and supports the present ultrastructural findings. Based on Golgi preparations of embryonic mouse spinal cord at comparable stages of development, all early forming axons from association and commissural neurons appear to grow dorsoventrally
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along the lateral margin. The first axons were found to grow in an ipsilateral ventrolateral fasciculus and therefore may be primitive associational axons. Commissural axons begin to cross the ventral commissure shortly thereafter (Windle and Baxter, 1936a,b; Wentworth, 1984). Guidance of Circumferential
Axons
In the following sections, the detailed growth pattern of nonfasciculating circumferential axons is analyzed in reference to several proposed mechanisms of axonal guidance. Basal lamina. The guidance of pioneering circumferential axons by the neural tube basal lamina was suggested earlier (Holley, 1982), based on their close proximity at the initial stage of development and the known guidance of regenerating axons (Sanes et al, 1978). None of the growth cones or filopodia reconstructed in the present and succeeding studies (Holley, 1987), however, were found to contact the basal lamina. Furthermore, several nonfasciculating circumferential growth cones were found in the S19 specimen which were located more than 10 pm away from the basal lamina. Since the filopodia of circumferential growth cones are about 5 km long, with the longest one reaching only up to 6 pm (Holley, 198’7),possible contact with the basal lamina is unlikely, especially at later stages of development. Pioneering axMzs. The preferential growth and fasciculation of axons upon early formed, pioneering axons has been shown in a number of developing axonal systems (e.g., Lopresti et al., 1973; Edwards et al, 1981; Murray et ak, 1984). However, fasciculation is clearly not a major force in directing axons within the circumferential pathway, at least, through S19. The factor(s) which guide the very first circumferential axons may also direct the somewhat later growing axons which follow a similar course. Thus, there is no evidence suggesting the presence of unique pioneering (pathfinding) axons. It has been shown experimentally in developing grasshopper leg that late forming axons can also navigate along their normal pathways in the absence of pioneering fibers (Keshishian and Bentley, 1983). Physical guidance and aligned extracellular space. The physical substratum through which axons grow is a major factor influencing their direction (Ram& y Cajal, 1960; Weiss, 1941). Within the lateral margin of the spinal cord, however, there was no evidence of preformed structures which would serve to guide circumferential axons. By comparison, it should be noted that in each of the axonal systems where prealigned structures have been found, axons grow in a fasciculative pattern (Singer et al., 1979; Silver and Robb, 1979; Silver and Sidman, 1980; Silver et al, 1982; Carney and Silver, 1983). Clearly,
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the factors which guide fasciculating and nonfasciculating axons would be expected to differ at least quantitatively. Guidepost/landmark cells. In the developing appendages of grasshoppers, a set of closely spaced guidepost/ landmark cells with numerous filopodia appear to orient axonal growth (Ho and Goodman, 1982; Bentley and Caudy, 1983). A similar set of guidepost cells may exist in the embryonic fish spinal cord which guide RohonBeard axons (Kuwada, 1986). By comparison, the present findings suggest that circumferential axons are not guided by an analogous mechanism. Circumferential axons at S17 and S19 do not group together at certain (guidepost) locations, but instead tend to course in a parallel, dorsoventral direction with no apparent centers of congregation. Furthermore, there was no indication of specialized sets of dorsoventrally arranged cells or groups of filopodia in front of circumferential growth cones. One explanation for these differences is simply that different guidance cues are used in the two systems. In the grasshopper peripheral nerve, axons grow in fascicles along crooked pathways; whereas, circumferential axons grow in a nonfasciculative manner along relatively straight courses. Population-dependent guidance. Directionality may be determined by a combination of factors acting concurrently on growth cones. Katz and Lasek (1985) have tested a set of hypothetical factors by computer-aided modeling. Although this model may help explain certain aspects of the later developing patterns of circumferential pathway, it is lacking in its ability to explain how axons are initially directed ventrally. The model proposes that directionality depends on a minimal density of axons necessary for sufficient population interaction; however, the first circumferential axons can grow relatively long distances in the correct direction when their density is far below the proposed minimum (Holley, 1987). Gradients. The growth pattern of circumferential axons can be explained, in part, by a gradient mechanism of guidance. Three types of such guidance mechanisms are chemotatic (Sperry, 1963; Gundersen and Barrett, 1980), electromagnetic (Jaffe and Poo, 1979) and adhesive (Steinberg, 1970; Gunderson and Park, 1984) gradients. If a continuous guiding gradient were distributed along the lateral spinal cord margin and with a dorsal-to-ventral polarity, then circumferential growth would be expected. Early forming axons, regardless of their relative site or time of origin, would grow ventrally along the margin. Moreover, early forming axons would grow relatively independent of one another when there is a large substrate-to-axon ratio. At later stages, additional circumferential axons would tend to grow parallel in the rostrocaudal axis to maximize contacts with the pro-
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29,526posed chemotatic or adhesive substances rather than CONSTANTINE-PATON, M. (1979). Axonal navigation. BioW 532. each other. Hence the gradient model appears to account EDWARDS, J. S., CHEN, S.-W., and BERNS,M. W. (1981). Cereal sensory for the pattern of circumferential pathway. development following laser microlesions of embryonic apical cells If a gradient mechanism guides circumferential axons in Acheta domesticus. J. Neurosci 1.250-258. along the lateral spinal cord wall, other factors, in ad- GUNDERSEN, R. W., and BARRETT, J. N. (1980). Characterization of the turning response of dorsal root neurites toward nerve growth factor. dition, may be operating in the developing ventral comJ. Cell BioL 87, 546-554. missure where axons from the two spinal cord halves GUNDERSEN,R. W., and PARK,K. H. C. (1984). The effects of conditioned grow in opposite directions. The ventral floor plate differs media on spinal neurites: Substrate-associated changes in neurite from lateral neural tube margin in two major respects. direction and adherence. Dev. BioL 104,18-27. First, all cells in the floor plate are presumptive glio- HAMBURGER,V., and HAMILTON,H. L. (1951). A series of normal stages in the development of the chick embryo. J. MorphoL 88,49-92. blasts, which in many species have distinctive pigment Ho, R. K., and GOODMAN,C. S. (1982). Peripheral pathways are pigranules. No axons originate from this region (Ramon oneered by an array of central and peripheral neurones in grassy Cajal, 1960; Baker, 1927; Kingsbury, 1930). Second, axhopper embryos. Nature @.mcZtm) 297,404-406. ons never appear to grow longitudinally in any part of HOLLEY, J. A. (1982). Early development of the circumferential axonal the brain or spinal cord floor plate (Kingsburg, 1930; pathway in mouse and chick spinal cord. J. Comp. NeuroL 205,371382. Windle, 1931). Once circumferential axons cross the floor HOLLEY, J. A. (1983). “Development of the Circumferential Axonal plate, however, they apparently never continue dorsally Pathway,” Ph.D. Thesis. Case Western Reserve Univ., Cleveland, up the contralateral side, but always turn and grow lonOH. gitudinally (Windle, 1931; Windle and Austin, 1936; HOLLEY, J. A. (1987). Differential adhesivity of neuroepithelial cells and pioneering circumferential axons. Dev. BioL 123,389-400. Nornes et al, 1980). The mechanism that induces the change from non- HOLLEY, J. A., and LASEK, R. J. (1982). Development of circumferential axons in chick embryos: The first spinal cord axons. Sot. Neurosti. fasciculative to fasciculative growth is presently unreAbstr. 8, 927. solved. It is conceivable that as circumferential axons HOLLEY, J. A., SILVER, J., and LASEK, R. J. Growth pattern of pioneering approach the ventrolateral fasciculus, or cross the midchick spinal cord axons. Unpublished, earlier version of the present line as commissural axons, local cues induce change in manuscript. the surface properties of their growth cones which favor HORDER, T. J., and MARTIN, K. A. C. (1978). Morphogenetics as an alternative in the formation of nerve connections. Svmp. Sot. Exp. fasciculation. One potentially inducible factor that may BioL 32,275-358. coordinate the growth of axons along the longitudinal JACOBSON, M. (1978). “Developmental Neurobiology.” Plenum, New pathway is the cell surface adhesion molecule NILE York. (Stallcup et al., 1985), which has been shown to accu- JAFFE, L. F., and Poo, M. M. (1979). Neurites grow faster toward the cathode than the anode in a steady field. J. Exp. ZooL 209,115-128. mulate preferentially along fasciculating spinal cord KATZ, M. J., and LASEK, R. J. (1985). Early axon patterns of the spinal axons (Stallcup et al., 1985; Holley and Schachner, uncord: Experiments with a computer. Dev. BioL 109,140-149. published data.) KESHISHIAN, H., and BENTLEY, D. (1983). Embryogenesis of peripheral The authors thank Dr. Susanne Kuhlmann-Krieg for reviewing the manuscript, and Alicia Lasek and Maggie Willett for fine technical assistance. We are grateful to Dr. Raymond L. Lasek for his contributions and acknowledge Dr. Michael J. Katz for his interest. This work was supported by Alexander von Humbolt and NIH fellowships to J.A.H., and NIH grants awarded to J.S. (NS1573-03) and Dr. Lasek (NS07118-03).
REFERENCES BARKER, R. C. (1927). The early development of the ventral part of the neural plate of amblystoma. J. Camp, NeuroL 44, l-27. BENTLEY, D., and CAUDY, M. (1983). Pioneer axons lose directed growth after selective killing of guidepost cells. Nature (Londcm) 304, 6% 65. BENTLEY, D., and KESHISHIAN, H. (1982). Pioneer neurons and pathways in insect appendages. Trends Neurosci 5,354-358. BERLOT, J., and GOODMAN, C. S. (1984). Guidance of peripheral pioneer neurons in the grasshopper: Adhesive hierarchy of epithelial and neuronal surfaces. Nature (London) 223,493-496. CARNEY, P. R., and SILVER, J. (1983). Studies on cell migration and axon guidance in the developing distal auditor system of the mouse. J. Camp. NeuroL 215,359-369.
nerve pathways in grasshopper legs. III. Development without pioneering neurons. Dev. BioL 96,116-124. KINGSBURY, B. F. (1930). The developmental significance of the floorplate of the brain and spinal cord. J. Camp. NeuroL 50,177-207. KUWADA, J. L. (1986). Cell recognition by neuronal growth cones in a simple vertebrate embryo. Science 233, 740-746. 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. NatL Acad Sci. USA 70,433-437. LETOURNEAU, P. C. (1975). Cell-to-substratum adhesion and guidance of axonal elongation. Dev. BioL 44, 92-101. LYSER, K. M. (1966). The development of the chick embryo diencephalon and mesencephalon during the initial phases of neuroblast differentiation. J. EmbqyoL Exp. MorphoL 16, 497-517. MURRAY, M. A., SCHUBIGER, M., and FALKA, J. (1984). Neuron differentiation and axon growth in the developing wing of Drosophila melanogaster. Dev. BioL 104, 259-273. NARDI, J. B. (1983). Neuronal pathfinding in developing wings of the moth Mauduca sexta Deu. BioL 95,163-174. NORNES, H. O., HART, H., and CARRY, M. (1980). Pattern of development of ascending and descending fibers in embryonic spinal cord of chick. I. Role of position information. J. Comp. NeuroL 192.119-132. RAM~N Y CAJAL, S. (1960). “Studies in Vertebrate Neurogenesis” (translated by L. Guth). Thomas, Springfield, IL.
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SANES,J. R., MARSHALL,L. M., and MCMAHAN, U. J. (1978). Reinnervation of muscle fiber basal lamina after removal of myofibers. Differentiation of regenerating axons at original synaptic sites. J. CeU BioL 78.1’76-198. SILVER,J., LORENZ,S. E., WAHLSTEN,D., and COUGHLIN,J. (1982). Axon guidance during development of the great cerebral commissures: Descriptive and experimental studies, in vivo, on the role of preformed glial pathways. J. Camp. Neural 210,10-29. SILVER,J., and ROBB,R. M. (1979). Studies on the development of the eye cup and optic nerve in normal mice and in mutants with cogenital optic nerve aplasia. Dm. Biol 68,175-190. SILVER,J., and SIDMAN, R. L. (1980). A mechanism for the guidance and topographic patterning of retinal ganglion cell axons. J. Camp. Neural 189,101-111. SINGER,M., NORDLANDER,R. H., and EGAR,M. (1979). Axonal guidance during embryogenesis and regeneration in spinal cord of the newt: The blueprint hypothesis of neuronal pathway patterning. J. Comp. NeuroL 185,1-22. SPERRY,R. W. (1963). Chemoaffinity in the orderly growth of nerve fiber patterns and connections. Proc. Nat1 Ad. Sci USA 50,703710. STALLCUP,W. B., BEASLEY,L. L., and LEVINE, J. M. (1985). Antibody against nerve growth factor-inducible lare external (NILE) glycoprotein labels nerve fiber tracts in the developing rat nervous system. J. Neurosti 5,1090-1101. STEINBERG, M. S. (1970). Does differential adhesion govern self-assembly processes in histogenesis? Equilibrium configurations and the
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P. H., BASTIANI, M. J., Ho, R. K., and GOODMAN,C. S. (1982). Guidance of pioneering growth cones: Filopodial contacts and coupling revealed with an antibody to lucifer yellow. DGV. BioL 94,391399. WEISS,P. A. (1941). Nerve patterns. The mechanics of nerve growth. Growth 5, (Suppl.), 163-203. WENTWORTH, L. E. (1984). The development of the cervical spinal cord of the mouse embryo. II. A Golgi analysis of sensory, commissural and association cell differentiation. J. Cmp. NeuroL 222, 96-115. WINDLE, W. F. (1931). The neurofibrillar structure of the spinal cord of cat embryos correlated with the appearance of early somatic movements. J. Cmp. NeuroL 53,71-113. WINDLE, W. F., and AUSTIN, M. F. (1936). Neurofibrillar development in the central nervous system of chick embryos up to 5 days incubation. J. Cmp. NeuroL 63,431-463. WINDLE, W. F., and BAXTER,R. E. (1936a). The first neurofibrillar development in albino rat embryos. J. Camp. NeuroL 63.173-187. WINDLE, W. F., and BAXTER, R. E. (1936b). Development of reflex mechanisms in the spinal cord of albino rat embryos. Correlations between structure and function, and comparisons with the cat and the chick. J. Cmp. NeuroL 63,189-209. WINDLE, W. F., and FITZGERALD, J. E. (1937).Development of the spinal reflex mechanism in human embryos. J. Cmp. NeuroL 67,493-509. WINDLE, W. F., and ORR,D. W. (1934). The development of behavior in chick embryos: Spinal cord structures correlated with early somatic motility. J. Camp. NeuroL 60, 287-308. TAGHERT,
BIOLOGY
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(1987)
Growth Pattern of Pioneering Chick Spinal Cord Axons’ JOHN Department
of Developmental
A.
ANDJERRYSILVER
HOLLEY’
Genetics and Anatomy,
Received April
Case Western Reserve University,
9, 1985; accepted in revised
Cleveland, Ohio 44106
form May 26, 1987
The early growth pattern of axons in the embryonic chick spinal cord was studied by electron microscopy. Serial perisagittal thin sections were obtained from the lateral margins of spinal cords of stage 17 (S17) and S19 embryos. A simple stereotypic pattern of axonal growth was found. Axons originated from a dispersed population of presumptive interneurons located along the lateral spinal cord margin. They first grew ventrally in a nonfasciculative pattern and later turned at right angles and grew in a fasciculative manner longitudinally in the ventrolateral fasciculus. Growth along the circumferential pathway was analyzed in detail by reconstructing individual axons and growth cones from the S17 specimen. Most circumferential axons, regardless of their site of origin, grew in a parallel orientation, and each of their growth cones projected ventrally. This pattern suggested that circumferential growth cones were guided at many, if not all, points along their path. Study of the region in front of these seven growth cones, however, revealed no apparent structural basis for their guidance. Alternative guidance mechanisms are discussed, In conjunction with previous studies (e.g., Windle and Baxter, 1936; Lyser, 1966), these findings suggest that the circumferential-nonfasciculative and the longitudinal-fasciculative patterns of axonal growth are the two fundamental patterns followed by most early forming axons in the brain stem and spinal cord of all higher vertebrates. o 1987 Academic press, IIIC.
INTRODUCTION
The early growth pattern of axons of spinal cord interneurons is similar in all higher vertebrates ranging from chick to human (Rambn y Cajal, 1960; Windle and Orr, 1934; Windle and Austin, 1936; Windle and Fitzgerald, 1937; Holley, 1982). Axons of early forming interneurons first grow circumferentially along the lateral margin of the embryonic spinal cord and in the transverse plane. After coursing ventrally, they turn and grow longitudinally in either the ipsi- or contralateral ventrolateral fasciculus (Ramon y Cajal, 1960; Windle and Austin, 1936; Windle and Baxter, 1936b). The apparent simplicity of this pattern lends itself to the study of the mechanisms of axonal guidance. Various mechanisms of axonal guidance have been proposed based on a large number of studies (reviews, Jacobson, 1978; Horder and Martin, 1978; ConstantinePaton, 1979). Several guidance mechanisms have been considered more recently; one involves the formation of oriented extracellular spaces (channels) prior to axonal growth (Singer et al, 1979, Silver and Robb, 1979; Silver and Sidman, 1980). A second mechanism, demonstrated in grasshopper appendages, involves guidance by closely spaced guidepost or landmark cells (Bentley and Kesh-
i Portions of this study have been reported elsewhere (Holley and Lasek, 1982; Katz and Lasek, 1985). ‘To whom correspondence and reprint requests should be addressed at Department of Neurology, Yale University School of Medicine, 333 Cedar St., New Haven, CT 06510. 375
ishan, 1982; Ho and Goodman, 1982). Growth cones extend long filopodia and form gap junctions with guidepost cells (Bentley and Caudy, 1983; Taghert et aL, 1982). A third mechanism involves the guidance of axons according to local variations in the relative adhesiveness of the tissue substrate for growth cones (Nardi, 1983; Berlot and Goodman, 1984). Whether these or other guidance mechanisms direct the early axonal growth of vertebrate interneurons was investigated in this and the following paper (Holley, 1987). In the present study, serial sections were obtained from the lateral margin of chick spinal cords at two early stages of development. The pattern of early developing axons, their shape and morphological relationship with surrounding cells, was determined. Several pioneering growth cones were found within the series and were studied in detail. MATERIALS
AND
METHODS
White Leghorn chicken embryos were obtained after various times of incubation at 38°C and staged according to Hamburger and Hamilton (1951). Embryos were immersed in fixative containing 2% glutaraldehyde, 2% formaldehyde, 0.5% acrolein, 0.5% dimethyl sulfoxide, and 3 mM CaCl, in 0.08 M cacodylate buffer at pH 7.4. After 30 min, the brachial spinal cord region adjacent to somites 17-19 was dissected and stored overnight at 4°C in fresh fixative. Specimens were postfixed in 1% OsOl for 1 hr in the same buffer, rinsed in 0.08 M phosphate buffer, and stained en bloc with 2% uranyl acetate for 30 min. Specimens were dehydrated in ethanol and embedded in Spurr’s plastic. 0012-1606/87 $3.00 Copyright All rights
0 1987 by Academic Press, Inc. of reproduction in any form reserved.
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A stage 1’7 (S17) chick spinal cord of apparently normal size and form was chosen for serial sectioning. After careful trimming, the specimen was serially thin sectioned at -80 nm thickness in an obliquely sagittal plane (see Fig. 1). A total of 100 serial sections were collected, beginning at the lateral edge of the neural tube, and were transferred to single slot, formvar-coated grids.
VOLUME 123, 1987
After drying, the sections were stained with uranyl acetate and lead citrate and examined with a JEOL 100 TEM. Every fifth section in the series was completely micrographed at a primary magnification of 2000x, and montages were then constructed on cardboard sheets at a total enlargement of 5500X. Outlines of the spinal cord margin and all long cellular processes and perikarya were drawn on plastic overlays. Structures of special interest and those with unclear boundries were reexamined and micrographed at higher magnifications. The entire length of all the long cellular processes in the series was reconstructed by overlapping consecutive plastic overlays and aligning contiguous structures. In addition to manual reconstruction, the patterns of axons and selected growth cones were reconstructed by computer-aided graphics as described by Carney and Silver (1983). A S19 chick brachial spinal cord was also prepared and sectioned in the same manner as described above. Approximately 150 serial sections were obtained. Five sections within the series were selected and completely micrographed at 3300x. They were each located between 1 to 2 pm apart as judged by changes in the sizes and shapes of identified cell nuclei. An additional 5 sections were micrographed at 1000X, and these together with the higher magnification montages made it possible to identify the pattern of axons within the series. After serial thin sectioning, the S17 and S19 spinal cord specimens were reoriented and sectioned in a slightly oblique transverse plane. The resulting semithin sections showed the final depth and plane of the serialthin sections. In addition, the pattern of longitudinal axons was examined in transverse thin sections of the S19 spinal cord. RESULTS
FIG. 1. (A) Light micrograph of an obliquely transverse section of S17 spinal cord after serial thin sectioning. The perisagittal plane and final depth of thin sectioning are seen along the right margin (sect. pln.). The total depth of tissue reconstructed is illustrated as the area between the dashed lines along the left margin. Extracellular space (es) is more abundant in the dorsal than the ventral region of the brachial neural tube. Bar = 50 Wm. Similar semithin section of S19 spinal cord shown after thin sectioning and reorientation. Circumferential axons (ca) are seen medial to the primordial motor column and are included in the zone of serial thin sections. f, ventrolateral fasciculus. Bar = 50 pm.
Figure 1 shows slightly oblique transverse sections of the S17 and S19 spinal cord specimens obtained after thin sectioning. The final depth and plane of serial thin sections is illustrated. In both specimens, as well as others of similar age, there is relatively more extracellular space in the dorsal as compared to ventral regions of the spinal cord. The extracellular spaces are more prevalent along the lateral margin, but are not restricted to the marginal zone. The spaces have various shapes and no discernible preferential orientation. Sl7 Axonal Pattern The serial thin sections of the S17 specimen numbered 1 to 100, beginning at the lateral spinal margin. Figure 2 shows section 80. The principle ponents of the marginal zone include the endfeet distal processes of neuroepithelial cells, as well as
were cord comand peri-
.
-
.
FIG. 2. Low magnification montage of section 80 from S17 spinal cord series. Numerous circumferential of growth cones l-5 and 7. Motor axons (arrows) are along the ventral region. Bar = 10 Km.
---
axons are seen, as well as portions
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karya located more centrally. Most of these cellular profiles are seen in cross section and are oval in shape. In addition, there are a number of processes that course for longer distances in the marginal zone, i.e., parallel the plane of section. Each of these longer cellular processeshave a more electron-lucent cytoplasm and therefore could be readily traced in the low magnification EM montages. The outlines of all long cellular processes and most perikarya in every fifth section of the series were drawn on plastic overlays. Figure 3 shows several of these drawings, including that of section 80. Individual cells and processes were identified and then reconstructed by following each one in the sequential sections. A composite illustration showing all of the long cellular processes and most perikarya in the S17 marginal zone is presented in Fig. 4. Most of the processes continue to course beyond the limits of the serial sections and thus appear as blunt or cut ends in the drawing. In the dorsal and middle regions all long cellular processes are oriented dorsoventrally. They have been numbered l26. Each one is identifiable ultrastructurally as an em-
showing lateral view of S17 spinal cord with FIG. 4. Reconstruction . _. all axons and most perikarya in the serial sections. Several of the total 26 circumferential axons are numbered. Seven axonal growth cones are present, and each projects ventrally. Growth cone 2 contacts circumferential process 11 at the location of the arrow. Longitudinal axons and apparent growth cones (a,b) are at the level of ventral rootlets (vr). Bar = 50 pm.
FIG. 3. Drawings of all long cellular processes and selected perikarya present in several sections of 517 spinal cord. Section number corresponding shown in parentheses. Continuity of many axons can be followed between sections. All axons in dorsal and middle regions course dorsoventrally. Several shorter, longitudinally oriented axons appear in the last section (F) and are located at the level of ventral rootlets. Bar = 100 pm. From Holley (1983).
bryonic axon based on its electron-lucent cytoplasm that contains numerous longitudinally oriented microtubules and intermediate filaments, dense mitochondria, and few nonaggregated ribosomes (Figs. 2 and 5-7). Thus based on shape, orientation, and ultrastructure these processes are identified as circumferential axons. Most circumferential axons occur singly within the tissue and are surrounded by perikarya and neuroepithelial cell processes (Figs. 2 and 5-7). In addition, several pairs of circumferential axons are present which course side-by-side for a variable distance. These adjacent axons may then diverge and continue separately. In one instance three axons are located in juxtaposition (Fig. 4). Noteably, all long axons are oriented in a stereotypic dorsoventral direction. Their course is markedly
HOLLEY
AND
SILVER
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of Pimeering
FIG. 5. Ventral margin of S17 spinal cord from section 49. Endfeet (ef) are irregularly shaped, have no preferential have surface junctions (j). Circumferential axons occasionally lie subjacent to the basal lamina (bl). Bar = 4 ym.
straight with no more than a few bends at low angles of curvature. These axons do not branch. The direction of growth can be determined with certainty for 14 of the 26 circumferential axons. Seven axons terminate with growth cones and another 7 axons have their cell bodies of origin at least partially within the serial sections (Fig. 4). In four additional cases, circumferential axons (9,11, 12, and 13) are found which turn at a right angle and have a medially directed segment that continues several micrometers toward the ventricular zone before the series end. Presumably the cell bodies of these axons are located more medially within the ventricular zone. Axons 11 and 12 are two such axons that appear to project out from the ventricular zone, turn, and grow ventrally. Unlike the other two axons, they have an enlarged region of cytoplasm at the corner where they turn circumferentially (Figs. 2 and 4). Each of these 18 circumferential axons, except one, is directed ventrally. The single dorsally directed axon (25) is noteably among the single set of three fasciculating axons, and its cell body is located in the ventrolateral region.
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Axons
orientation,
and often
As shown in Fig. 4, circumferential axons appear to originate from cell bodies located at nearly all levels along the lateral spinal cord wall. They are not aligned in dorsoventrally arranged sets, which may indicate an interdependent mechanism of growth. The distribution of circumferential axons was also analyzed statistically to test for certain types of growth patterns. If contact inhibition directs growth, for example, then a nonrandom, segregated distribution would be expected along the rostrocaudal axis. Hence, the composite illustration of circumferential axons was blindly divided into 10 dorsoventrally oriented areas. The number of axons within each area was counted along seven equally spaced lines, representing an even sample of all dorsoventral regions of the marginal zone. The observed frequency of axons within each test division was compared with the expected frequency based on a Poisson distribution (x = 48163). The results showed that the distribution was not significantly different from random (X = 4.37, df = 2, P = 0.2). Thus, there is no statistical evidence of significant repulsion or attraction
FIG. 6. Growth cones 1 and 2 have several filopodia (f) and project ventrally (direction arrow). Growth cone 2 is atypical with mark1 edly electron lucent cytoplasm; it contacts circumferential axon 11 (arrow). Extracellular space (es) is common in dorsal spinal cord, but is not arranged in aligned channels. r, radial processes. Bar = 4 pm. 380
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within this small sample of early growing circumferential axons. In addition to circumferential axons, a number of ventral motor axons appear to exit the ventrolateral region of the spinal cord (Fig. 2). At the same level, a few relatively short, longitudinally oriented axons are found (Fig. 4). The origin of these longitudinal axons could not be traced because they are located at the edge of the serial sections. Sl7 Growth Cones
Seven of the 26 circumferential axons terminate with a definitive growth cone. Each has a bulbous body 3-4 pm in diameter and several filopodia that extend in various directions for distances of up to 6 /*rn (Figs. 6 and 7). The growth cones are located between 4 and 6 pm from the basal lamina. Although none of the growth cone filopodia appear to reach the basal lamina, contacts with small processes could have escaped detection. Substantial ultrastructural variation is seen among the growth cones. Most are relatively electron-lucent such as growth cones 4 and 5 (Fig. 7). Others are more electron-dense and have fewer membranous organelles such as growth cone 1 (Fig. 6). Growth cone 2 is distinct. As illustrated in Fig. 4, it contacts the radially oriented segment of circumferential axon 11, whose cell body presumably resides deeper within the ventricular zone. In contrast to the other growth cones, the surface of circumferential axon and growth cone 2 is not fully covered by surrounding cells (Fig. 6). The distended cytoplasm that is largely devoid of ground substance indicates that growth cone 2 may be regressing. Noteably, all seven growth cones are directed ventrally from a dorsally located parent axon (Fig. 4). Growth cones 1,3,4, and 7 are not contacting other growth cones or nearby axons. By definition, these are pioneering circumferential growth cones. The region surrounding and immediately in front of the pioneering growth cones was analyzed in detail for the following features that might guide circumferential axons: Shape and alignment
of marginal
zone cellular
ele-
ments. The circumferential axonal growth cones are surrounded by distal processes of neuroepithelial cells and perikarya. As shown in figures 6 and 7, the cellular elements in front of the growth cones have various shapes, but tend to be rounded with no distinct dorsoventral polarity. There is also no indication that cells or processes are aligned in a dorsoventral pattern that would physically guide circumferential growth. Aligned extracellular spaces. Although extracellular spaces are present, they are rarely found immediately in front of growth cones (Figs. 6 and 7). Furthermore,
381
there is no indication in the serial sections that dorsoventrally aligned sets of extracellular spaces (i.e., channels) are present at any level in the lateral spinal cord. Guidepost and landmark cells. Most circumferential axons grow in a parallel, nonfasciculative pattern and show no apparent tendency to congregate locally at specific (guidepost or landmark) sites (Fig. 4). Since circumferential growth cone filopodia are apparently less than 7 pm long, several potential guidepost and landmark cells must be positioned in dorsoventrally oriented sets in order to guide circumferential axons. Cells and processes in front of these pioneering circumferential growth cones, however, appear structurally indistinguishable from other cells in the marginal zone (Figs. 6 and 7). In no case, are sets of nonaxonal filopodia found aligned in advance of circumferential growth cones. Intercellular junctions. Despite extensive search for specialized contacts on the surfaces of each axonal growth cone and filopodia, no instance was found of a localized specialization, such as close membrane apposition or membrane-associated electron-dense material, that would indicate the presence of an intercellular junction. As a morphological control, numerous desmosomal-like junctions are observed between adjacent neuroepithelial endfeet (Fig. 5). S19 Axonal Patterns and Growth Cones
As shown in Fig. lB, the serial thin sections of the S19 brachial spinal cord included most of the circumferential axonal pathway. At this stage of development, immature neurons have migrated into the primordial motor column, and the early forming circumferential axons are displaced medially from the ventrolateral margin (Holley, 1982). Hence the serial sagittal sections included the marginal zone in the dorsal region, as well as the region between the motor column and the ventricular zone. All axons and many cell nuclei present in four sections of the series are shown in Fig. 8. Except for two short portions of axons in Fig. 8B, all axons in the dorsal region are oriented dorsoventrally. Dorsal root fibers evidently have not entered at least this region of the spinal cord. Circumferential axons tend to be arranged in a layer along the lateral surface of the ventricular zone. The axons are often positioned side-by-side in the layer and seldom fasciculate in large bundles. The density of axons found in this S19 specimen is about twice as large as that in the S17 specimen. Also shown in Fig. 8 are the shapes and locations of all four growth cones found in the serial sections. Growth cones 1 and 2 are located within the middle of the spinal cord, Figs. 8C and 8D, respectively. Both are directed ventrally and are about 8 pm from the neural tube sur-
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FIG. 8. Drawings of four sagittal sections from S19 spinal cord series. Basal lamina (bl) along dorsal and ventral borders is drawn, as well as all cell nuclei. Symbols (m, 0, A) denote nuclei seen in adjacent sections. All axons are drawn, and except for two axons in section B, all in the dorsal and middle regions course dorsoventrally (black processes). Growth cones are labeled G4 and G5. Along the ventral margins of(D) are several short profiles of longitudinal axons (la) and growth cone 4. Bar = 50 pm.
face. Growth cone 3 is located in the ventral area of Fig. longitudinal axons is present at this stage and is located 8D and is about 10 pm from the surface. Each of these near the ventral edge of the primordial motor column circumferential growth cones are located near several (Fig. 1B). This ventrolateral fascicle contains 12 axons circumferential axons; however, there is no direct sur- (Fig. 10). As compared to the circumferential axons, face contact (Figs. 8 and 9A). These observations indicate these longitudinal axons grow in a single bundle with that later forming circumferential axons tend to grow extensive axon-to-axon surface contact. in regions of earlier forming axons, and yet there is little DISCUSSION tendency to fasciculate. Along the ventral margin of Fig. 9D several profiles Fasciculative vs Nonfasciculative Growth of longitudinal axons are present as well as two cellular processes, labeled G4 and G5. G4 appears to be a cirThe overall pattern of early axonal growth shown in cumferential growth cone that is contacting an area of the present and companion study (Holley, 1987) is in longitudinal axons (Fig. 9B). G5 is an L-shaped process accord with earlier light microscopic investigations with axonal-like cytoplasm (Fig. SC). Adjacent serial (Ramon y Cajal, 1960; Windle and Austin, 1936). The sections showed that both G4 and G5 were in contact first axons that grow within the spinal cord are from with a fascicle of longitudinal axons (Fig. 9D). G4 and early forming interneurons; they first grow circumferG5 thus appear to be axons at different stages of turning entially and later turn and grow longitudinally in the from circumferential to longitudinal orientations. primitive ventrolateral fasciculus. Axons from motor To further analyze the axonal patterns in this S19 neurons also develop at this time and exit the cord. spinal cord, the specimen was reoriented and thin-secOne remarkable aspect of the pattern is the distinct tioned in the transverse plane. One major fascicle of difference between circumferential and longitudinal
FIG. 7. (A) Growth cone 5 located adjacent to axons 4, from section 75 of Sli’ series. Several dark filopodia (f’) project from surrounding cells and contact growth cone 5 filopodia. Filopodium of growth cone 4 can be seen further ventrally (4f). The nucleus (n) is labeled for reference in (A-C). Bar = 2 Frn. (B) Computer-aided reconstruction of growth cones 4 and 5 with their axons. Filopodia extend from each growth cone up to 5 pm in various directions. Bar = 4 pm. (C) Growth cone 4 seen in section 90. Note lack of specialized contacts and processes extending from surrounding cells. Bar = 2 pm.
FIG. 9. (A) Growth cone 3 (gc) located medial to primordial motor column of S19 neural tube (labeled G3 in Fig. 8D). Filopodia (f) extend ventrally. Bar = 2 pm. (B) Growth cone 4 (gc) appears to approach longitudinal axons (a) at the ventral margin (labeled G4 in Fig. 8D). Large diameter process from growth cone has a microfilamentous cytoplasm, but no microtubules or intermediate filaments. Bar = 2 pm. (C) Process with axonal-like cytoplasm (a’) located along the ventral margin (labeled G5 in Fig. 8D). The process forms a right angle turn from circumferential to longitudinal. Bar = 2 pm. (D) Longitudinal axons located medial to the presumptive turning axon. Bar = 2 pm. 384
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velop along the entire lateral wall of the neural tube from the spinal cord rostrally through the mesencephalon (Windle and Baxter, 1936b,Windle and Austin, 1936, Lyser, 1966). These axons are derived from a dispersed population of interneurons that grow circumferentially in a nonfasciculative pattern until they turn and grow longitudinally in a variety of fascicle tracts. The concept of these two fundamental growth patterns was first suggested by Holley (1983; J. A. Holley, J. Silver, and R. J. Lasek, unpublished data) and has also been discussed by Katz and Lasek (1985). The distinct difference in axonal growth patterns suggests that at least two basic sets of factors guide axons in the vertebrate nervous system. One of the factors that may coordinate the growth of the axons along the longitudinal pathway is the preferential accumulation of the cell surface adhesion molecule NILE along fasciculating spinal cord axons (Stallcup et aZ.,1985). Growth Pattern
FIG. 10. Transverse section S19 specimen. Its location is axons (la) are grouped into cones (gc) are located near axon (ca) is located medially ef, endfeet. Bar = 2 pm.
of ventrolateral fasciculus from the same illustrated in Fig. 1B. Twelve longitudinal the bundle, and two presumptive growth the basal lamina (bl). A circumferential and is in direct contact with the fascicle.
growth. Circumferential axons grow in a nonfasciculative pattern, whereas longitudinal axons grow in fascicles. Even after numerous circumferential axons had developed by S19, circumferential axons were distributed in a layer along the ventricular zone margin and showed little tendency to fasciculate. Furthermore, several of the growth cones in both the S1’7 and S19 specimens were located within filopodial reach of preformed axons but did not fasciculate. Longitudinal axons, on the other hand, grow in close axon-to-axon contact in the developing funiculi. The first fascicles of longitudinal interneuronal axons develop along the ventrolateral spinal cord margin and subsequent axons accumulate in an orderly fasciculative manner (Windle and Orr, 1934; Windle and Austin, 1936; Nornes et al., 1980). The distinction between circumferential-nonfasciculative growth and longitudinal-fasciculative growth appears to be a general organizing feature of the vertebrate brain stem and spinal cord. Circumferential axons de-
of Circumferential
Axons
Most early forming axons that grow along the lateral margin of the spinal cord are oriented in a markedly straight dorsoventral orientation (Holley, 1987). They originate from presumptive interneurons that are distributed throughout the lateral wall of the neural tube, including the far dorsal and ventral aspects. The cellular milieu through which circumferential axons grow consists of a maze of radially oriented neuroepithelial cell processes and other cells that might physically deflect linear growth, and indeed, many of the axons had shallow bends. Nevertheless, axons continued to grow circumferentially without appreciable wandering, indicating that the factor(s) which guide circumferential growth are functioning at many, if not all, points along the embryonic spinal cord’s lateral margin. The direction of many circumferential axons was determined based on the relative positions of their cell bodies or growth cones. Each of these circumferential axons were directed ventrally, except one. The lone dorsally projecting axon originated from a cell body in the far ventrolateral region of the S1’7 spinal cord. This atypical axon may be from an “aberrant” motor neuron described by Ramon y Cajal(l960) which grows dorsally from the motor column and exits the cord in the dorsal root. This axon was among the single set of three fasciculating axons, and coincidentally, motor axons are known to grow in fascicles (Ramon y Cajal, 1960). A detailed study of early interneuron development by Wentworth (1984) supplements and supports the present ultrastructural findings. Based on Golgi preparations of embryonic mouse spinal cord at comparable stages of development, all early forming axons from association and commissural neurons appear to grow dorsoventrally
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along the lateral margin. The first axons were found to grow in an ipsilateral ventrolateral fasciculus and therefore may be primitive associational axons. Commissural axons begin to cross the ventral commissure shortly thereafter (Windle and Baxter, 1936a,b; Wentworth, 1984). Guidance of Circumferential
Axons
In the following sections, the detailed growth pattern of nonfasciculating circumferential axons is analyzed in reference to several proposed mechanisms of axonal guidance. Basal lamina. The guidance of pioneering circumferential axons by the neural tube basal lamina was suggested earlier (Holley, 1982), based on their close proximity at the initial stage of development and the known guidance of regenerating axons (Sanes et al, 1978). None of the growth cones or filopodia reconstructed in the present and succeeding studies (Holley, 1987), however, were found to contact the basal lamina. Furthermore, several nonfasciculating circumferential growth cones were found in the S19 specimen which were located more than 10 pm away from the basal lamina. Since the filopodia of circumferential growth cones are about 5 km long, with the longest one reaching only up to 6 pm (Holley, 198’7),possible contact with the basal lamina is unlikely, especially at later stages of development. Pioneering axMzs. The preferential growth and fasciculation of axons upon early formed, pioneering axons has been shown in a number of developing axonal systems (e.g., Lopresti et al., 1973; Edwards et al, 1981; Murray et ak, 1984). However, fasciculation is clearly not a major force in directing axons within the circumferential pathway, at least, through S19. The factor(s) which guide the very first circumferential axons may also direct the somewhat later growing axons which follow a similar course. Thus, there is no evidence suggesting the presence of unique pioneering (pathfinding) axons. It has been shown experimentally in developing grasshopper leg that late forming axons can also navigate along their normal pathways in the absence of pioneering fibers (Keshishian and Bentley, 1983). Physical guidance and aligned extracellular space. The physical substratum through which axons grow is a major factor influencing their direction (Ram& y Cajal, 1960; Weiss, 1941). Within the lateral margin of the spinal cord, however, there was no evidence of preformed structures which would serve to guide circumferential axons. By comparison, it should be noted that in each of the axonal systems where prealigned structures have been found, axons grow in a fasciculative pattern (Singer et al., 1979; Silver and Robb, 1979; Silver and Sidman, 1980; Silver et al, 1982; Carney and Silver, 1983). Clearly,
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the factors which guide fasciculating and nonfasciculating axons would be expected to differ at least quantitatively. Guidepost/landmark cells. In the developing appendages of grasshoppers, a set of closely spaced guidepost/ landmark cells with numerous filopodia appear to orient axonal growth (Ho and Goodman, 1982; Bentley and Caudy, 1983). A similar set of guidepost cells may exist in the embryonic fish spinal cord which guide RohonBeard axons (Kuwada, 1986). By comparison, the present findings suggest that circumferential axons are not guided by an analogous mechanism. Circumferential axons at S17 and S19 do not group together at certain (guidepost) locations, but instead tend to course in a parallel, dorsoventral direction with no apparent centers of congregation. Furthermore, there was no indication of specialized sets of dorsoventrally arranged cells or groups of filopodia in front of circumferential growth cones. One explanation for these differences is simply that different guidance cues are used in the two systems. In the grasshopper peripheral nerve, axons grow in fascicles along crooked pathways; whereas, circumferential axons grow in a nonfasciculative manner along relatively straight courses. Population-dependent guidance. Directionality may be determined by a combination of factors acting concurrently on growth cones. Katz and Lasek (1985) have tested a set of hypothetical factors by computer-aided modeling. Although this model may help explain certain aspects of the later developing patterns of circumferential pathway, it is lacking in its ability to explain how axons are initially directed ventrally. The model proposes that directionality depends on a minimal density of axons necessary for sufficient population interaction; however, the first circumferential axons can grow relatively long distances in the correct direction when their density is far below the proposed minimum (Holley, 1987). Gradients. The growth pattern of circumferential axons can be explained, in part, by a gradient mechanism of guidance. Three types of such guidance mechanisms are chemotatic (Sperry, 1963; Gundersen and Barrett, 1980), electromagnetic (Jaffe and Poo, 1979) and adhesive (Steinberg, 1970; Gunderson and Park, 1984) gradients. If a continuous guiding gradient were distributed along the lateral spinal cord margin and with a dorsal-to-ventral polarity, then circumferential growth would be expected. Early forming axons, regardless of their relative site or time of origin, would grow ventrally along the margin. Moreover, early forming axons would grow relatively independent of one another when there is a large substrate-to-axon ratio. At later stages, additional circumferential axons would tend to grow parallel in the rostrocaudal axis to maximize contacts with the pro-
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of Pimzeering
Axons
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29,526posed chemotatic or adhesive substances rather than CONSTANTINE-PATON, M. (1979). Axonal navigation. BioW 532. each other. Hence the gradient model appears to account EDWARDS, J. S., CHEN, S.-W., and BERNS,M. W. (1981). Cereal sensory for the pattern of circumferential pathway. development following laser microlesions of embryonic apical cells If a gradient mechanism guides circumferential axons in Acheta domesticus. J. Neurosci 1.250-258. along the lateral spinal cord wall, other factors, in ad- GUNDERSEN, R. W., and BARRETT, J. N. (1980). Characterization of the turning response of dorsal root neurites toward nerve growth factor. dition, may be operating in the developing ventral comJ. Cell BioL 87, 546-554. missure where axons from the two spinal cord halves GUNDERSEN,R. W., and PARK,K. H. C. (1984). The effects of conditioned grow in opposite directions. The ventral floor plate differs media on spinal neurites: Substrate-associated changes in neurite from lateral neural tube margin in two major respects. direction and adherence. Dev. BioL 104,18-27. First, all cells in the floor plate are presumptive glio- HAMBURGER,V., and HAMILTON,H. L. (1951). A series of normal stages in the development of the chick embryo. J. MorphoL 88,49-92. blasts, which in many species have distinctive pigment Ho, R. K., and GOODMAN,C. S. (1982). Peripheral pathways are pigranules. No axons originate from this region (Ramon oneered by an array of central and peripheral neurones in grassy Cajal, 1960; Baker, 1927; Kingsbury, 1930). Second, axhopper embryos. Nature @.mcZtm) 297,404-406. ons never appear to grow longitudinally in any part of HOLLEY, J. A. (1982). Early development of the circumferential axonal the brain or spinal cord floor plate (Kingsburg, 1930; pathway in mouse and chick spinal cord. J. Comp. NeuroL 205,371382. Windle, 1931). Once circumferential axons cross the floor HOLLEY, J. A. (1983). “Development of the Circumferential Axonal plate, however, they apparently never continue dorsally Pathway,” Ph.D. Thesis. Case Western Reserve Univ., Cleveland, up the contralateral side, but always turn and grow lonOH. gitudinally (Windle, 1931; Windle and Austin, 1936; HOLLEY, J. A. (1987). Differential adhesivity of neuroepithelial cells and pioneering circumferential axons. Dev. BioL 123,389-400. Nornes et al, 1980). The mechanism that induces the change from non- HOLLEY, J. A., and LASEK, R. J. (1982). Development of circumferential axons in chick embryos: The first spinal cord axons. Sot. Neurosti. fasciculative to fasciculative growth is presently unreAbstr. 8, 927. solved. It is conceivable that as circumferential axons HOLLEY, J. A., SILVER, J., and LASEK, R. J. Growth pattern of pioneering approach the ventrolateral fasciculus, or cross the midchick spinal cord axons. Unpublished, earlier version of the present line as commissural axons, local cues induce change in manuscript. the surface properties of their growth cones which favor HORDER, T. J., and MARTIN, K. A. C. (1978). Morphogenetics as an alternative in the formation of nerve connections. Svmp. Sot. Exp. fasciculation. One potentially inducible factor that may BioL 32,275-358. coordinate the growth of axons along the longitudinal JACOBSON, M. (1978). “Developmental Neurobiology.” Plenum, New pathway is the cell surface adhesion molecule NILE York. (Stallcup et al., 1985), which has been shown to accu- JAFFE, L. F., and Poo, M. M. (1979). Neurites grow faster toward the cathode than the anode in a steady field. J. Exp. ZooL 209,115-128. mulate preferentially along fasciculating spinal cord KATZ, M. J., and LASEK, R. J. (1985). Early axon patterns of the spinal axons (Stallcup et al., 1985; Holley and Schachner, uncord: Experiments with a computer. Dev. BioL 109,140-149. published data.) KESHISHIAN, H., and BENTLEY, D. (1983). Embryogenesis of peripheral The authors thank Dr. Susanne Kuhlmann-Krieg for reviewing the manuscript, and Alicia Lasek and Maggie Willett for fine technical assistance. We are grateful to Dr. Raymond L. Lasek for his contributions and acknowledge Dr. Michael J. Katz for his interest. This work was supported by Alexander von Humbolt and NIH fellowships to J.A.H., and NIH grants awarded to J.S. (NS1573-03) and Dr. Lasek (NS07118-03).
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