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The growth of motor axons in the spinal cord of Xenopus embryos
DEVELOPMENTAL
BIOLOGY
The Growth
(1985)
109,96-101
of Motor Axons MONTE The Institute Received
WESTERFIELDAND
of Neuroscience, June
in the Spinal Cord of Xenopus
18, 19X&;
University accepted
JUDITH of Oregon,
in revised
form
Embryos
S.EISEN Eugene, November
C?regon 97403 29, 1984
The innervation of the myotomal muscles in the trunk region of Xen0pu.s embryos has been examined to see how the path taken by motoneurons within the spinal cord is formed. The growth of motor axons has been studied by retrograde labeling with horseradish peroxidase and the growth of the spinal cord and myotomes has been studied by labeling with fluorescent beads. Results show that motoneurons initially innervate the nearest muscles. Then through a process of differential growth whereby the muscles elongate more than the spinal cord, the axonal terminals in the muscles become displaced caudally relative to their cell bodies. In this manner the central pathway taken by the motor axons develops after initial innervation of their peripheral targets. Q 1985 Academic press, he.
INTRODUCTION
By labeling the extracellular space in the myotomes and the spinal cord in young animals with fluorescent latex beads, we have compared the longitudinal growth of the spinal cord and the myotomes after the initial neuromuscular connections are formed, and have observed that the beads in the muscles move caudally relative to those within the spinal cord. We propose that the central pathway of the motor axons develops by a process of differential growth, whereby the muscles elongate more than the spinal cord, thus, moving the location of the terminals of motor axons in the muscles caudally relative to their cell bodies. A preliminary report of these results has appeared (Westerfield and Eisen, 1983).
A distinctive feature of the amphibian spinal cord is the displacement of the cell bodies of motoneurons relative to their axon terminals in the myotomal muscles (Hughes, 1959; Roberts and Clarke, 1982). The motor axons course within the cord for a distance of several segments before emerging in a ventral root. This phenomenon was first observed in Ambystoma by Coghill (1913). Hughes (1959) described the development of this motor system based upon silver-stained material in Xenopus and suggested that the displacement is present from the earliest developmental stages. Thus the longitudinal motor tracts within the spinal cord are thought to develop before the ventral roots. This would require that the axons of motoneurons grow for some distance within the spinal cord before entering the ventral roots. We have reexamined the innervation of the myotomal muscles in the trunk region of Xenopus embryos to determine the origin of the central path taken by motor axons within the spinal cord. We have studied the early projections of motor axons by retrogradely labeling them with horseradish peroxidase (HRP) injected into the muscles of individual myotomes. Contrary to the earlier studies cited above, our results show that motoneurons initially innervate muscles of the nearest myotome and that their long central projections appear during subsequent development. By labeling motoneurons early in development and letting them complete growth before processing, we have shown that the same population of cells, projecting to the same muscles, is present throughout embryonic and larval development. This eliminates the possibility that the motoneurons with the long axonal projections represent a separate class of cells that develops later. 0012-1606/85 $3.00 Copyright All rights
0 1985 by Academic Press, Inc. of reproduction in any form reserved.
MATERIALS
AND METHODS
Embryos and larvae of Xenopus laevis were maintained at 23°C and were staged according to the criteria of Nieuwkoop and Faber (1956). Animals were immobilized by embedding them in 1.2% agar in Ringer of the following composition: NaCl, 115 mM; KCl, 3 mM; CaClz, 10 mM; Hepes buffer, 5 mM, pH 7.2. To label motoneurons, the peripheral nerves that innervate the myotomal muscles were lesioned in the clefts between adjacent segments with a fine needle and a 40% solution of horseradish peroxidase (Sigma) in Ringer was injected into the wound. To label the local extracellular space, a suspension of 0.57-pm diameter polycarboxylated latex beads labeled with coumarin (Fluoresbrite, Polysciences, Inc.) was injected into the spinal cord and the adjacent myotomal muscle using pipets of 5-25 pm inner diameter. A pipet was advanced through the myotomal muscles and through the spinal cord in a horizontal plane perpendicular to the long axis of the embryo. As the pipet was withdrawn, 96
WESTERFIELD
AND
EISEN
Growth
of Motor
Axons
pressure (0.5 kg/cm2) was applied to the back of the pipet to eject beads into the lesion. In this manner, the beads labeled a particular myotome and the adjacent region of spinal cord as observed in two animals examined immediately. The beads were too large to be taken up by neurons and were only found in the extracellular spaces around the cells. The animals were left in the agar for 20 min and were then allowed to recover in an oxygenated, 50% Ringer solution for 2 hr. They were then either transferred to their normal holding tanks for additional development or were fixed by immersion in 1.5% gluteraldehyde and 0.5% paraformaldehyde for HRP labeling or 8% paraformaldehyde for fluorescence labeling in 0.1 M phosphate buffer at pH 7.4. After fixatilon the specimens were either sectioned at 50 pm on a. freezing microtome (Frank et al., 1980) or were processed as whole mounts after removal of the skin and muscle contralateral to the injection site. To visualize the HRP, sections were reacted with 0.2% 3,3’-diamino benzidine (DAB, Frank et al, 1980) or with Hanker et al. (1977) reagent. The whole mounts were re,acted with DAB by standard procedures (Lamb, 1974). The HRP-labeled material was examined using Nomarski optics. The fluorescent beads were visualized in wet mount (95% glycerol) sections with epifluorescence illumination. The results presented here are based upon HRP injections in 95 animals and fluorescent bead injections in 59 animals.
MN
8 MM
FlESULTS
The Central Projection
of Motor Axons
The myotomal muscles are located caudal to the cell bodies of the motoneurons that innervate them. This finding is illustrated in Fig. 1 which shows a horizontal section through a stage 48 tadpole. The cell bodies and axons of motoneurons were labeled retrogradely with HRP that was injected into the muscles of two adjacent segments (segments 11 and 12) in the trunk. Motoneurons are contained within a discreet region of the spinal cord aligned with body segments 6 and 7. Their axons course within the spinal cord along a relatively medial path. They then pass through the ventral white matter, enter the ventral roots, and finally innervate the injected muscles four body segments (900 pm) caudal of their cell bodies. As described by Hughes (1959) the axons of these motoneurons projected for a considerable distance within the spinal cord. In tadpoles, the length of the motor axons depended upon which body segment they innervated. Motor axons innervating more cauda-I segments projected very long distances within the cord. Shorter projections were observed after labeling more rostrally located muscles. The axons of motoneurlons innervating segments 4-5
10 AA
b
*
FIG. 1. The central path of motor axons. Left, photomicrograph of a horizontal section (50-pm thick, rostra1 to the top) through the trunk of a stage 48 Xe?zopz~ tadpole. The motoneurons innervating body segments 11 and 12 were labeled by retrograde transport of HRP injected into the myotomal muscles of these segments. Right, camera lucida drawing of the section shown on left. The body segment numbers of the myotomes are labeled. Spinal cord (SC), myotomal muscles (MM), motoneurons (MN), motor axons (MA), injection site (star). Scale bar, 100 pm.
projected nearly straight out of the cord while the motor axons innervating segments 2 and 3 projected rostrally within the cord before entering the ventral roots, in agreement with previous observations (Hughes, 1959; Roberts and Clarke, 1982).
98 The Growth
DEVELOPMENTAL
of Motor Axons
within
the Spinal
BIOLOGY
109, 1985
Cord
To see how these central projections of motor axons form, we injected HRP into the myotomal muscles of young embryos shortly after the time that motoneurons first innervate muscles (Kullberg et al, 1977). We found that motoneurons in the trunk region of Xenopus embryos initially innervate nearby muscles. This is illustrated in Fig. 2A which shows a camera lucida drawing of a motoneuron retrogradely labeled after 31 hr of development (stage 27) by injection of HRP into muscles of the 10th segment. As shown, the axon of this motoneuron projected straight out of the cord to innervate muscles in the nearest segment. The soma of this early neuron lacked any other conspicuous processes and was located on the peripheral edge of the cord. In 13 animals injected at stages 26 to 29, each labeled motoneuronal soma within the trunk region (segments 5-15) was in longitudinal register with the muscle segment it innervated. Approximately l-2 days later (stages 30-40), injections in 10 animals showed that the cell bodies of the motoneurons had become displaced relative to their terminals in the muscles. As illustrated in the example of Fig. 2B, the soma of this motoneuron was rostra1 of the axon terminals, such that the motor axon ran for 180 pm (1.5 segments) within the spinal cord before entering the ventral root. By this stage the motoneuron had begun to develop dendrites and was separated from the peripheral edge of the spinal cord by fibers in the lateral tracts. After another 11 days of development (stages 40-50, 11 animals) injection of muscles in the trunk labeled motoneurons whose terminals were significantly displaced from their somata. As shown in Fig. 2C, injection of the 10th segment labeled a motoneuron whose soma was located three segments rostrally. This motoneuron had an extensive dendritic arbor. The motor axon projected caudally from the cell body in a relatively medial position within the cord. After approximately 300 pm (two segments) it coursed laterally and left the cord in the 10th ventral root. Displacement of the Myotomes to the Spinal Cord
VOLUME
Relative
The observation of direct initial projections suggests that the path taken by the axons of motoneurons in the trunk region of the spinal cord develops after these neurons have innervated their peripheral targets. However, it could be that there are different types of motoneurons present at different developmental times. In this case we would label a type that projects straight out of the cord at early stages (Fig. 2A). Later these neurons would be replaced by another class of
Stage
27
-
I-I$zJz!! Stage
40
-
Stage
49
H
cFIG. 2. Growth of motor axons. Camera lucida drawings of motoneurons that were retrogradely labeled with HRP applied via a lesion to their axons in the myotomes. (A) Stage 27 embryo. A motoneuron that innervated the 10th segment was labeled. (B) Stage 40 embryo. A motoneuron that innervated the 10th and 11th segments was labeled. (C) Stage 49 embryo. A motoneuron was labeled by HRP applied to a lesion of the 10th segment. Horizontal section, 50pm thick, rostra1 to the left, scale bar, 50 Frn. Note that this section was slightly oblique. Reconstruction of the spinal cord (not shown) demonstrated that the axon ran in a relatively medial position within the spinal cord.
motoneurons with long projecting axons (as in Fig. 2C). This hypothesis can be ruled out by several observations. First, the progressive development of the displacement (Figs. 2A-C) suggests that it is a continuous process. Second, we have been able to demonstrate that the same motoneurons are present in younger and older animals. In 12 animals, motoneurons were labeled at stage 27. Half of these animals were examined 2 hr after labeling and the other half were examined after they had developed for 5 more days. All the motoneurons examined at stage 27 projected straight out of the cord (as in Fig. 2A), whereas the motoneurons that were allowed to grow had the long axonal projections. An example of our results is shown in Fig. 3 which
WESTERFIELD
AND
EISEN
presents a camera lucida drawing of a stage 47 motoneuron that was labeled at the earlier time when its axon projected straight out of the cord at stage 27. The terminals of this motoneuron had been displaced caudally and its axon projected within the longitudinal tract before entering the 10th ventral root. Thus it is clear that the early motoneurons persist, and that their central axons elongate. A second possibility to account for our observations is that the peripheral connections of the motoneurons change during developlment. Under this hypothesis, motoneurons initially innervate nearby muscles. Then at a later time they withdraw these initial connections, send their axons caudally, and innervate different muscles. This idea also seems unlikely because it predicts that, in the experiment illustrated in Fig. 3, we should see labeled motor axons projecting to unlabeled muscles caudal to the lesion. In 16 out of 16 animals we observed labeled motoneurons projecting only to labeled muscles. A third explanation for the relative displacement of somata and axon terminals, is that the cell bodies of motoneurons migrate rostrally within the spinal cord after innervating their targets. We tested this hypothesis in 22 animals by marking a location within a myotome and the adjacent region of spinal cord and seeing if labeled motoneurons innervating that myotome moved relative to the marked site within the cord. After several days of further development, the labeling pattern illustrated in Fig. 4 was observed. As shown, the labeled myotome was caudal to the site of the beads within the spinal cord. In some cases, the injection grazed the surface of the spinal cord so that some beads were deposited in the connective tissue surrounding the cord a:s well as within the cord. In these instances, two regions of label were found centrally, one within the connective tissue which was
r
8
______-------__-&L+LL
.-__e------
Stage FIG.
changes labeled to the continue cessing. bar, 50
47
---
_
-
3. The projection of motor axons within the spinal cord during development. Camera lucida drawing of a motoneuron with HRP. This motoneuron was labeled by a lesion applied 11th somite at stage 2 7. The animal was then allowed to development until stage 4’7 before fixation and HRP proHorizontal section, 50-pm thick, rostra1 to the left, scale pm.
Growth
of Motor
Axons
FIG. 4. Differential growth of the muscles and the spinal cord. Left, a photomicrograph of a horizontal section (50-pm diameter, rostra1 at the top) through the trunk of a tadpole. The 11th and 12th myotomes and the corresponding region of the spinal cord of this animal were labeled at stage 27 by injection of fluorescent beads. After subsequent development to stage 50, the tadpole was fixed and sectioned. The location of the original injection site is marked by the bright fluorescence of the beads. Right, drawing of the section shown on the left. The body segment numbers of the myotomes are labeled. The locations of fluorescent beads in the muscle (large arrow) and in the spinal cord (small arrow) are shown. Scale bar, 100 pm.
lined up with the label in the myotome, and one which was rostra1 within the cord. The amount of this difference depended upon which segment had been labeled, being greater for more caudally located segments (triangles in Fig. 5). Thus we can account for the displacement of motoneurons as being passive, produced by the muscles elongating more than cord. To obtain evidence for a superimposed active migration of motoneurons we compared the location of HRP-labeled motoneurons innervating these same myotomes (in different animals, circles in Fig. 5) with the location of the beads within the cord. As shown in Fig. 5 there is no significant difference between the displacement of the beads and the displacement of the motoneurons relative to the labeled myotomes suggesting that both displacements are due to the same process. But is this developmental scheme also followed by neurons which differentiate later? Several pieces of information suggest that the axons of later developing motoneurons may, indeed, grow for some distance within the spinal cord before entering a ventral root.
100
DEVELOPMENTAL
BIOLOGY
VOLUME
109, 1985
and their displacement relative to the spinal cord at this stage may require considerable central navigation by the growing axons of motoneurons within the tail. DISCUSSION
A
l l e
l
l
5 Segment
10 No.
of
Injected
15 Myotome
FIG. 5. Relative displacement of the spinal cord and myotomes. The location of motoneuron cell bodies (circles) and fluorescent beads (triangles) is plotted as a function of the segment injected. Each point represents a separate animal. Motoneurons were labeled by HRP injected into myotomes in stage 49-51 tadpoles. The rostralcaudal midpoint of the pool of labeled cells was determined and a line perpendicular to the long axis of the animal was drawn. The segment number of the myotome intersected by this line was plotted as the ordinate and the abscissa was the segment number of the injected myotome. The extracellular space was labeled by injection of fluorescent beads into the myotomes and spinal cord of stage 2% 30 embryos. The animals were allowed to grow until stage 50-51. The midpoint of the distribution of the beads within the spinal cord was determined as for the HRP-labeled motoneurons and plotted as a function of the injected segment. The slopes of lines fit by linear regression analysis to the two sets of points (+ the standard error of the slope) were 0.70 + 0.07 (T = 0.94) for the motoneurons and 0.74 + 0.05 (r = 0.96) for the beads. The broken line on the plot indicates a slope of 1.0 which would be predicted if there were no displacement of the central label relative to the injection sites.
First, examination of the relative size of motoneurons in the spinal cords of young animals (Fig. 2A) suggests that there is only space for a maximum of approximately four motoneurons per hemisegment. This idea is consistent with our inability to label more than two motoneurons per hemisegment (12 animals) before stage 29, although, of course, young axons may not transport adequate amounts of HRP to become visible. By stage 49 we consistently label about 12 motoneurons per hemisegment (9 animals). This suggests that the axons of these additional motoneurons are growing at a time when there has already been some relative displacement of their peripheral targets. And second, it is not clear if this scheme is followed during development of the tail. Somites continue to be added to the tail through stage 40 (Nieuwkoop and Faber, 1956)
We have shown that motoneurons in Xenopus tadpoles initially project to nearby muscles and only later become displaced from their muscles. Thus the central path taken by their axons forms after their initial innervation of muscles. We conclude that the growth cones of the motoneurons do not navigate for any appreciable distance within the CNS before contacting their peripheral targets. Although several different mechanisms could account for the relative displacement of the cell bodies and axonal terminals of the motoneurons, the most likely explanation is differential growth whereby the body elongates more than the spinal cord. Since both the body and the spinal cord of the embryo are fixed at the rostra1 end by attachment to the head, continued growth results in a relative caudal displacement of the somites, the amount of displacement being greater in more caudal segments. This idea has been previously proposed (Patten, 1946) as an explanation for the cauda equina in human development, a major difference in Xenopus being that much of the intervening growth of the motor axons occurs within the spinal cord. The growth of axons due directly to mechanical tension has recently been demonstrated in tissue culture (Bray, 1984). This type of differential growth may well be a common process in vertebrate development since it has also been seen in the developing spinal cord of zebrafish (Westerfield and Myers, unpublished observations). The relative movement of the motor axons into a medial location within the spinal cord may be explained by considering the development of the longitudinal tracts. At the time that the motoneurons first innervate muscles, there are very few longitudinally running axons present. Nordlander and Singer (1982) have shown that as these tracts develop, new axons are preferentially added to the peripheral margin. The growth cones of newly forming axons are always found at the peripheral edge of the spinal cord, pushing the older axons toward a more medial position within the developing tract. Thus the axons of the motoneurons in the trunk, which may be among the first axons to develop within the cord, end up lying within the longitudinal tracts due to the greater growth of the myotomal muscles relative to the spinal cord. Within these tracts they are pushed into a relatively medial location by later developing fibers that preferentially grow along the peripheral edge of the tract.
WESTERFIELD
AND
EISEN
We are grateful for thoughtful conversations with W. Metcalfe and C. Kimmel during the course of this work, technical assistance from D. Brumbley, S. Powell, Y. Tseng, M. Shelley, and H. Howard, critical reading of the manuscript by R. Nordlander, and support from the NSF BNS 81-03573, the NIH T32-GM07257, the Muscular Dystrophy Association, and the Medical Research Foundation of Oregon. M.W. was an Alfred P. Sloan Fellow. REFERENCES BRAY, D. (1984). Axonal growth in response to experimentally applied mechanical tension. Dev. BloL 102, 3’79-389. COGHILL, G. E. (1913). The primary ventral roots and somatic motor column of Amblystcnna. J. Camp. Neural. 13, 121-143. FRANK, E., HARRIS, W. A., and KENNEDY, M. B. (1980). Lysophosphatidy1 choline facilitates labeling of CNS projections with horseradish peroxidase. J. Neurosci Methods 2, 183-189. HANKER, J. S., YATES, P. E., METZ, C. B., and RUSTIONI, A. (1977). A new specific, sensitive and non-carcinogenic reagent for the demonstration of horseradish peroxidase. Histochem. J. 9, 789-792. HUGHES, A. (1959). Studies in embryonic and larval development in
Growth
of Motor
Azons
101
amphibia. II. The spinal motor-root. J. Embryol. Exp. MorphoL 7, 128-145. KULLBERG, R. W., LENTZ, T. L., and COHEN, M. W. (1977). Development of myotomal neuromuscular junction in Xenopus Iaevis: An elec trophysiological and fine-structural study. Dev. BioL 60, 101-129. LAMB, A. H. (1981). Target dependency of developing motoneurons in Xenopus laevis. J. Camp. NeuroL 203, 157-171. LAMB, A. H. (1974). The timing of the earliest motor innervation to the hind limb in the Xenow tadpole. Brain Res. 67, 527-530. NIEUWKOOP, P. D., and FABER, J. (eds.) (1956). “Normal Table of Xenopus laevis (Daudin).” North-Holland, Amsterdam. NORDLANDER, R. H., and SINGER, M. (1982). Morphology and position of growth cones in the developing Xenopus spinal cord. Dev. Brain Res. 4, 181-193. PATTEN, B. M. (1946). “Human Embryology.” pp. 336-337. Blakiston, Philadelphia. ROBERTS, A., and CLARKE, J. D. W. (1982). The neuroanatomy of an amphibian embryo spinal cord. Phil. Trans. R. Sot. London Ser. B 296, 195-212. WESTERFIELD, M., and EISEN, J. S. (1983). The growth of motor axons in Xenopus spinal cord. Sot. Neurosci. Abstr. 9, 210.
BIOLOGY
The Growth
(1985)
109,96-101
of Motor Axons MONTE The Institute Received
WESTERFIELDAND
of Neuroscience, June
in the Spinal Cord of Xenopus
18, 19X&;
University accepted
JUDITH of Oregon,
in revised
form
Embryos
S.EISEN Eugene, November
C?regon 97403 29, 1984
The innervation of the myotomal muscles in the trunk region of Xen0pu.s embryos has been examined to see how the path taken by motoneurons within the spinal cord is formed. The growth of motor axons has been studied by retrograde labeling with horseradish peroxidase and the growth of the spinal cord and myotomes has been studied by labeling with fluorescent beads. Results show that motoneurons initially innervate the nearest muscles. Then through a process of differential growth whereby the muscles elongate more than the spinal cord, the axonal terminals in the muscles become displaced caudally relative to their cell bodies. In this manner the central pathway taken by the motor axons develops after initial innervation of their peripheral targets. Q 1985 Academic press, he.
INTRODUCTION
By labeling the extracellular space in the myotomes and the spinal cord in young animals with fluorescent latex beads, we have compared the longitudinal growth of the spinal cord and the myotomes after the initial neuromuscular connections are formed, and have observed that the beads in the muscles move caudally relative to those within the spinal cord. We propose that the central pathway of the motor axons develops by a process of differential growth, whereby the muscles elongate more than the spinal cord, thus, moving the location of the terminals of motor axons in the muscles caudally relative to their cell bodies. A preliminary report of these results has appeared (Westerfield and Eisen, 1983).
A distinctive feature of the amphibian spinal cord is the displacement of the cell bodies of motoneurons relative to their axon terminals in the myotomal muscles (Hughes, 1959; Roberts and Clarke, 1982). The motor axons course within the cord for a distance of several segments before emerging in a ventral root. This phenomenon was first observed in Ambystoma by Coghill (1913). Hughes (1959) described the development of this motor system based upon silver-stained material in Xenopus and suggested that the displacement is present from the earliest developmental stages. Thus the longitudinal motor tracts within the spinal cord are thought to develop before the ventral roots. This would require that the axons of motoneurons grow for some distance within the spinal cord before entering the ventral roots. We have reexamined the innervation of the myotomal muscles in the trunk region of Xenopus embryos to determine the origin of the central path taken by motor axons within the spinal cord. We have studied the early projections of motor axons by retrogradely labeling them with horseradish peroxidase (HRP) injected into the muscles of individual myotomes. Contrary to the earlier studies cited above, our results show that motoneurons initially innervate muscles of the nearest myotome and that their long central projections appear during subsequent development. By labeling motoneurons early in development and letting them complete growth before processing, we have shown that the same population of cells, projecting to the same muscles, is present throughout embryonic and larval development. This eliminates the possibility that the motoneurons with the long axonal projections represent a separate class of cells that develops later. 0012-1606/85 $3.00 Copyright All rights
0 1985 by Academic Press, Inc. of reproduction in any form reserved.
MATERIALS
AND METHODS
Embryos and larvae of Xenopus laevis were maintained at 23°C and were staged according to the criteria of Nieuwkoop and Faber (1956). Animals were immobilized by embedding them in 1.2% agar in Ringer of the following composition: NaCl, 115 mM; KCl, 3 mM; CaClz, 10 mM; Hepes buffer, 5 mM, pH 7.2. To label motoneurons, the peripheral nerves that innervate the myotomal muscles were lesioned in the clefts between adjacent segments with a fine needle and a 40% solution of horseradish peroxidase (Sigma) in Ringer was injected into the wound. To label the local extracellular space, a suspension of 0.57-pm diameter polycarboxylated latex beads labeled with coumarin (Fluoresbrite, Polysciences, Inc.) was injected into the spinal cord and the adjacent myotomal muscle using pipets of 5-25 pm inner diameter. A pipet was advanced through the myotomal muscles and through the spinal cord in a horizontal plane perpendicular to the long axis of the embryo. As the pipet was withdrawn, 96
WESTERFIELD
AND
EISEN
Growth
of Motor
Axons
pressure (0.5 kg/cm2) was applied to the back of the pipet to eject beads into the lesion. In this manner, the beads labeled a particular myotome and the adjacent region of spinal cord as observed in two animals examined immediately. The beads were too large to be taken up by neurons and were only found in the extracellular spaces around the cells. The animals were left in the agar for 20 min and were then allowed to recover in an oxygenated, 50% Ringer solution for 2 hr. They were then either transferred to their normal holding tanks for additional development or were fixed by immersion in 1.5% gluteraldehyde and 0.5% paraformaldehyde for HRP labeling or 8% paraformaldehyde for fluorescence labeling in 0.1 M phosphate buffer at pH 7.4. After fixatilon the specimens were either sectioned at 50 pm on a. freezing microtome (Frank et al., 1980) or were processed as whole mounts after removal of the skin and muscle contralateral to the injection site. To visualize the HRP, sections were reacted with 0.2% 3,3’-diamino benzidine (DAB, Frank et al, 1980) or with Hanker et al. (1977) reagent. The whole mounts were re,acted with DAB by standard procedures (Lamb, 1974). The HRP-labeled material was examined using Nomarski optics. The fluorescent beads were visualized in wet mount (95% glycerol) sections with epifluorescence illumination. The results presented here are based upon HRP injections in 95 animals and fluorescent bead injections in 59 animals.
MN
8 MM
FlESULTS
The Central Projection
of Motor Axons
The myotomal muscles are located caudal to the cell bodies of the motoneurons that innervate them. This finding is illustrated in Fig. 1 which shows a horizontal section through a stage 48 tadpole. The cell bodies and axons of motoneurons were labeled retrogradely with HRP that was injected into the muscles of two adjacent segments (segments 11 and 12) in the trunk. Motoneurons are contained within a discreet region of the spinal cord aligned with body segments 6 and 7. Their axons course within the spinal cord along a relatively medial path. They then pass through the ventral white matter, enter the ventral roots, and finally innervate the injected muscles four body segments (900 pm) caudal of their cell bodies. As described by Hughes (1959) the axons of these motoneurons projected for a considerable distance within the spinal cord. In tadpoles, the length of the motor axons depended upon which body segment they innervated. Motor axons innervating more cauda-I segments projected very long distances within the cord. Shorter projections were observed after labeling more rostrally located muscles. The axons of motoneurlons innervating segments 4-5
10 AA
b
*
FIG. 1. The central path of motor axons. Left, photomicrograph of a horizontal section (50-pm thick, rostra1 to the top) through the trunk of a stage 48 Xe?zopz~ tadpole. The motoneurons innervating body segments 11 and 12 were labeled by retrograde transport of HRP injected into the myotomal muscles of these segments. Right, camera lucida drawing of the section shown on left. The body segment numbers of the myotomes are labeled. Spinal cord (SC), myotomal muscles (MM), motoneurons (MN), motor axons (MA), injection site (star). Scale bar, 100 pm.
projected nearly straight out of the cord while the motor axons innervating segments 2 and 3 projected rostrally within the cord before entering the ventral roots, in agreement with previous observations (Hughes, 1959; Roberts and Clarke, 1982).
98 The Growth
DEVELOPMENTAL
of Motor Axons
within
the Spinal
BIOLOGY
109, 1985
Cord
To see how these central projections of motor axons form, we injected HRP into the myotomal muscles of young embryos shortly after the time that motoneurons first innervate muscles (Kullberg et al, 1977). We found that motoneurons in the trunk region of Xenopus embryos initially innervate nearby muscles. This is illustrated in Fig. 2A which shows a camera lucida drawing of a motoneuron retrogradely labeled after 31 hr of development (stage 27) by injection of HRP into muscles of the 10th segment. As shown, the axon of this motoneuron projected straight out of the cord to innervate muscles in the nearest segment. The soma of this early neuron lacked any other conspicuous processes and was located on the peripheral edge of the cord. In 13 animals injected at stages 26 to 29, each labeled motoneuronal soma within the trunk region (segments 5-15) was in longitudinal register with the muscle segment it innervated. Approximately l-2 days later (stages 30-40), injections in 10 animals showed that the cell bodies of the motoneurons had become displaced relative to their terminals in the muscles. As illustrated in the example of Fig. 2B, the soma of this motoneuron was rostra1 of the axon terminals, such that the motor axon ran for 180 pm (1.5 segments) within the spinal cord before entering the ventral root. By this stage the motoneuron had begun to develop dendrites and was separated from the peripheral edge of the spinal cord by fibers in the lateral tracts. After another 11 days of development (stages 40-50, 11 animals) injection of muscles in the trunk labeled motoneurons whose terminals were significantly displaced from their somata. As shown in Fig. 2C, injection of the 10th segment labeled a motoneuron whose soma was located three segments rostrally. This motoneuron had an extensive dendritic arbor. The motor axon projected caudally from the cell body in a relatively medial position within the cord. After approximately 300 pm (two segments) it coursed laterally and left the cord in the 10th ventral root. Displacement of the Myotomes to the Spinal Cord
VOLUME
Relative
The observation of direct initial projections suggests that the path taken by the axons of motoneurons in the trunk region of the spinal cord develops after these neurons have innervated their peripheral targets. However, it could be that there are different types of motoneurons present at different developmental times. In this case we would label a type that projects straight out of the cord at early stages (Fig. 2A). Later these neurons would be replaced by another class of
Stage
27
-
I-I$zJz!! Stage
40
-
Stage
49
H
cFIG. 2. Growth of motor axons. Camera lucida drawings of motoneurons that were retrogradely labeled with HRP applied via a lesion to their axons in the myotomes. (A) Stage 27 embryo. A motoneuron that innervated the 10th segment was labeled. (B) Stage 40 embryo. A motoneuron that innervated the 10th and 11th segments was labeled. (C) Stage 49 embryo. A motoneuron was labeled by HRP applied to a lesion of the 10th segment. Horizontal section, 50pm thick, rostra1 to the left, scale bar, 50 Frn. Note that this section was slightly oblique. Reconstruction of the spinal cord (not shown) demonstrated that the axon ran in a relatively medial position within the spinal cord.
motoneurons with long projecting axons (as in Fig. 2C). This hypothesis can be ruled out by several observations. First, the progressive development of the displacement (Figs. 2A-C) suggests that it is a continuous process. Second, we have been able to demonstrate that the same motoneurons are present in younger and older animals. In 12 animals, motoneurons were labeled at stage 27. Half of these animals were examined 2 hr after labeling and the other half were examined after they had developed for 5 more days. All the motoneurons examined at stage 27 projected straight out of the cord (as in Fig. 2A), whereas the motoneurons that were allowed to grow had the long axonal projections. An example of our results is shown in Fig. 3 which
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presents a camera lucida drawing of a stage 47 motoneuron that was labeled at the earlier time when its axon projected straight out of the cord at stage 27. The terminals of this motoneuron had been displaced caudally and its axon projected within the longitudinal tract before entering the 10th ventral root. Thus it is clear that the early motoneurons persist, and that their central axons elongate. A second possibility to account for our observations is that the peripheral connections of the motoneurons change during developlment. Under this hypothesis, motoneurons initially innervate nearby muscles. Then at a later time they withdraw these initial connections, send their axons caudally, and innervate different muscles. This idea also seems unlikely because it predicts that, in the experiment illustrated in Fig. 3, we should see labeled motor axons projecting to unlabeled muscles caudal to the lesion. In 16 out of 16 animals we observed labeled motoneurons projecting only to labeled muscles. A third explanation for the relative displacement of somata and axon terminals, is that the cell bodies of motoneurons migrate rostrally within the spinal cord after innervating their targets. We tested this hypothesis in 22 animals by marking a location within a myotome and the adjacent region of spinal cord and seeing if labeled motoneurons innervating that myotome moved relative to the marked site within the cord. After several days of further development, the labeling pattern illustrated in Fig. 4 was observed. As shown, the labeled myotome was caudal to the site of the beads within the spinal cord. In some cases, the injection grazed the surface of the spinal cord so that some beads were deposited in the connective tissue surrounding the cord a:s well as within the cord. In these instances, two regions of label were found centrally, one within the connective tissue which was
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3. The projection of motor axons within the spinal cord during development. Camera lucida drawing of a motoneuron with HRP. This motoneuron was labeled by a lesion applied 11th somite at stage 2 7. The animal was then allowed to development until stage 4’7 before fixation and HRP proHorizontal section, 50-pm thick, rostra1 to the left, scale pm.
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FIG. 4. Differential growth of the muscles and the spinal cord. Left, a photomicrograph of a horizontal section (50-pm diameter, rostra1 at the top) through the trunk of a tadpole. The 11th and 12th myotomes and the corresponding region of the spinal cord of this animal were labeled at stage 27 by injection of fluorescent beads. After subsequent development to stage 50, the tadpole was fixed and sectioned. The location of the original injection site is marked by the bright fluorescence of the beads. Right, drawing of the section shown on the left. The body segment numbers of the myotomes are labeled. The locations of fluorescent beads in the muscle (large arrow) and in the spinal cord (small arrow) are shown. Scale bar, 100 pm.
lined up with the label in the myotome, and one which was rostra1 within the cord. The amount of this difference depended upon which segment had been labeled, being greater for more caudally located segments (triangles in Fig. 5). Thus we can account for the displacement of motoneurons as being passive, produced by the muscles elongating more than cord. To obtain evidence for a superimposed active migration of motoneurons we compared the location of HRP-labeled motoneurons innervating these same myotomes (in different animals, circles in Fig. 5) with the location of the beads within the cord. As shown in Fig. 5 there is no significant difference between the displacement of the beads and the displacement of the motoneurons relative to the labeled myotomes suggesting that both displacements are due to the same process. But is this developmental scheme also followed by neurons which differentiate later? Several pieces of information suggest that the axons of later developing motoneurons may, indeed, grow for some distance within the spinal cord before entering a ventral root.
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and their displacement relative to the spinal cord at this stage may require considerable central navigation by the growing axons of motoneurons within the tail. DISCUSSION
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FIG. 5. Relative displacement of the spinal cord and myotomes. The location of motoneuron cell bodies (circles) and fluorescent beads (triangles) is plotted as a function of the segment injected. Each point represents a separate animal. Motoneurons were labeled by HRP injected into myotomes in stage 49-51 tadpoles. The rostralcaudal midpoint of the pool of labeled cells was determined and a line perpendicular to the long axis of the animal was drawn. The segment number of the myotome intersected by this line was plotted as the ordinate and the abscissa was the segment number of the injected myotome. The extracellular space was labeled by injection of fluorescent beads into the myotomes and spinal cord of stage 2% 30 embryos. The animals were allowed to grow until stage 50-51. The midpoint of the distribution of the beads within the spinal cord was determined as for the HRP-labeled motoneurons and plotted as a function of the injected segment. The slopes of lines fit by linear regression analysis to the two sets of points (+ the standard error of the slope) were 0.70 + 0.07 (T = 0.94) for the motoneurons and 0.74 + 0.05 (r = 0.96) for the beads. The broken line on the plot indicates a slope of 1.0 which would be predicted if there were no displacement of the central label relative to the injection sites.
First, examination of the relative size of motoneurons in the spinal cords of young animals (Fig. 2A) suggests that there is only space for a maximum of approximately four motoneurons per hemisegment. This idea is consistent with our inability to label more than two motoneurons per hemisegment (12 animals) before stage 29, although, of course, young axons may not transport adequate amounts of HRP to become visible. By stage 49 we consistently label about 12 motoneurons per hemisegment (9 animals). This suggests that the axons of these additional motoneurons are growing at a time when there has already been some relative displacement of their peripheral targets. And second, it is not clear if this scheme is followed during development of the tail. Somites continue to be added to the tail through stage 40 (Nieuwkoop and Faber, 1956)
We have shown that motoneurons in Xenopus tadpoles initially project to nearby muscles and only later become displaced from their muscles. Thus the central path taken by their axons forms after their initial innervation of muscles. We conclude that the growth cones of the motoneurons do not navigate for any appreciable distance within the CNS before contacting their peripheral targets. Although several different mechanisms could account for the relative displacement of the cell bodies and axonal terminals of the motoneurons, the most likely explanation is differential growth whereby the body elongates more than the spinal cord. Since both the body and the spinal cord of the embryo are fixed at the rostra1 end by attachment to the head, continued growth results in a relative caudal displacement of the somites, the amount of displacement being greater in more caudal segments. This idea has been previously proposed (Patten, 1946) as an explanation for the cauda equina in human development, a major difference in Xenopus being that much of the intervening growth of the motor axons occurs within the spinal cord. The growth of axons due directly to mechanical tension has recently been demonstrated in tissue culture (Bray, 1984). This type of differential growth may well be a common process in vertebrate development since it has also been seen in the developing spinal cord of zebrafish (Westerfield and Myers, unpublished observations). The relative movement of the motor axons into a medial location within the spinal cord may be explained by considering the development of the longitudinal tracts. At the time that the motoneurons first innervate muscles, there are very few longitudinally running axons present. Nordlander and Singer (1982) have shown that as these tracts develop, new axons are preferentially added to the peripheral margin. The growth cones of newly forming axons are always found at the peripheral edge of the spinal cord, pushing the older axons toward a more medial position within the developing tract. Thus the axons of the motoneurons in the trunk, which may be among the first axons to develop within the cord, end up lying within the longitudinal tracts due to the greater growth of the myotomal muscles relative to the spinal cord. Within these tracts they are pushed into a relatively medial location by later developing fibers that preferentially grow along the peripheral edge of the tract.
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We are grateful for thoughtful conversations with W. Metcalfe and C. Kimmel during the course of this work, technical assistance from D. Brumbley, S. Powell, Y. Tseng, M. Shelley, and H. Howard, critical reading of the manuscript by R. Nordlander, and support from the NSF BNS 81-03573, the NIH T32-GM07257, the Muscular Dystrophy Association, and the Medical Research Foundation of Oregon. M.W. was an Alfred P. Sloan Fellow. REFERENCES BRAY, D. (1984). Axonal growth in response to experimentally applied mechanical tension. Dev. BloL 102, 3’79-389. COGHILL, G. E. (1913). The primary ventral roots and somatic motor column of Amblystcnna. J. Camp. Neural. 13, 121-143. FRANK, E., HARRIS, W. A., and KENNEDY, M. B. (1980). Lysophosphatidy1 choline facilitates labeling of CNS projections with horseradish peroxidase. J. Neurosci Methods 2, 183-189. HANKER, J. S., YATES, P. E., METZ, C. B., and RUSTIONI, A. (1977). A new specific, sensitive and non-carcinogenic reagent for the demonstration of horseradish peroxidase. Histochem. J. 9, 789-792. HUGHES, A. (1959). Studies in embryonic and larval development in
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amphibia. II. The spinal motor-root. J. Embryol. Exp. MorphoL 7, 128-145. KULLBERG, R. W., LENTZ, T. L., and COHEN, M. W. (1977). Development of myotomal neuromuscular junction in Xenopus Iaevis: An elec trophysiological and fine-structural study. Dev. BioL 60, 101-129. LAMB, A. H. (1981). Target dependency of developing motoneurons in Xenopus laevis. J. Camp. NeuroL 203, 157-171. LAMB, A. H. (1974). The timing of the earliest motor innervation to the hind limb in the Xenow tadpole. Brain Res. 67, 527-530. NIEUWKOOP, P. D., and FABER, J. (eds.) (1956). “Normal Table of Xenopus laevis (Daudin).” North-Holland, Amsterdam. NORDLANDER, R. H., and SINGER, M. (1982). Morphology and position of growth cones in the developing Xenopus spinal cord. Dev. Brain Res. 4, 181-193. PATTEN, B. M. (1946). “Human Embryology.” pp. 336-337. Blakiston, Philadelphia. ROBERTS, A., and CLARKE, J. D. W. (1982). The neuroanatomy of an amphibian embryo spinal cord. Phil. Trans. R. Sot. London Ser. B 296, 195-212. WESTERFIELD, M., and EISEN, J. S. (1983). The growth of motor axons in Xenopus spinal cord. Sot. Neurosci. Abstr. 9, 210.