Brain Research, 98 (1975) 291-302 © ElsevierScientificPublishingCompany,Amsterdam- Printed in The Netherlands
291
THE RETROGRADE TRANSPORT OF HORSERADISH PEROXIDASE FROM THE DEVELOPING LIMB OF THE CHICK EMBRYO
R O N A L D W. O P P E N H E I M AND M A R I E T A B. H E A T O N
Neuroembryology Laboratory, Research Division, North Carolina Department of Mental Health, Box 7532, Raleigh, N. C. 27611 (U.S.A.) (Accepted May 3rd, 1975)
SUMMARY
Chick embryos ranging in age from 4.0 to 18 days of incubation and 1-2-dayold hatchlings received injections of HRP solutions directly into the leg musculature. After survival periods of from 5 to 25 h motoneurons and cells in the spinal sensory ganglia were found to be stained with the HRP reaction product. It was found that the first appearance of a positive HRP reaction coincided with the time when nerve processes are first detected in the limb-bud by silver techniques (i.e. at 4.5 days of incubation). Only neurons with processes in the region of injection showed a positive reaction. The demonstration of a retrograde transport mechanism in neurons and axons which are still undergoing growth and differentiation provides a possible mechanism for the peripheral regulation of certain features of CNS neurogenesis. The application of this technique during development may also allow one to map neuroanatomical pathways during their formation.
INTRODUCTION
Many years ago Hamburger 15-n demonstrated that the numerical development of motoneurons in the spinal cord of the chick embryo can be radically altered by removing one limb-bud or by transplanting an additional limb-bud adjacent to the normal one. He found that in the former case, as long as the limb removal was complete, almost all of the motoneurons on one side in the lumbar lateral motor column degenerated and disappeared by the 9th day of incubation, a loss of about 20,000 cells. Conversely, the transplantation of a supernumary limb resulted in a motor hyperplasia of about 40 ~ in those spinal segments which successfully innervated the transplant. Because these two effects were found to be strictly limited to those segments and cell groups which directly innervate the leg, and since the opera-
292 tions were done at a stage prior to normal innervation - - thereby ruling out direct injury to the growing axons - - Hamburger 1:, suggested that he was dealing with a normal developmental process whereby the number of motoneurons that survive during development is rather directly related to the size of the peripheral field to be innervated. At the time, the exact mechanism responsible for this central-peripheral interaction was unknown, although it was later shown to be unlikely that proliferation, migration or initial differentiation were the critical events being regulated16,17. A remaining possibility suggested by Hamburger was that the phenomenon was mediated by a direct 'trophic' interaction between the growing tip of the motor axons and the non-neural periphery outside of the spinal cord 15. The possibility that in the normal course of development cell death might also occur in this system was suggested by the data of Hamburger 17 which indicated an apparent loss of cells on the non-operated side of the spinal cord in one set of embryos with limb-bud removal. Subsequently, work with amphibians by Hughes 26 and Prestige 37 demonstrated a dramatic reduction of spinal motoneurons during normal development, resulting in the loss of 4 out of every 5 cells produced. Recently this phenomenon has also been seen in the chick 19. Hamburger has reported a loss of approximately 8000 motoneurons (about 40 ~ ) on one side in the lumbar lateral motor column between days 6 and 9 during normal development. These observations are consistent with several other recent reports of normal cell death in many regions of the embryonic nervous system1,2,8, 9,~2,40, all of which underscore the prospect that cell death is of fundamental importance in the development and organization of the nervous system 7,~6,27,a7. At the moment, the most popular explanation of cell death during neurogenesis is still the original proposal of Hamburger 17 that there is a competition of some sort between the axonal processes of growing neurons for innervation sites; those that succeed survive, while the remainder degenerate. The acceptance of this explanation as a working hypothesis, however, requires the assumption of several propositions, the most crucial of which, in our opinion, are the following: (a) there is a temporal coincidence between the period of cell death and the time of innervation in a given system, (b) all or at least most cells develop axons which initially grow into the zone of innervation, and (c) there is a cellular mechanism for the centripetal or retrograde transfer of information from the axonal terminals back to the cell body. This last factor need not necessarily involve axoplasmic flow of'trophic' substances but rather could be related to some aspect of functional innervation in the periphery, or indeed to another, entirely different, retrograde mechanism aT. With regard to the limb-spinal cord model of the chick the first assumption apparently is correct 17, although there is a need for more details on this point on the ultrastructural level. Cell death in the ciliary ganglion of the chick embryo also occurs almost simultaneously with the formation of peripheral connections a3. The second assumption, while currently under study in this laboratory with the chick, has received some support from a recent study with amphibians. Prestige and Wilson a8 have reported an almost one-to-one relation between the number of motoneurons and ventral root fibers at different stages of development before, during and after the
293 period of cell death. It has also recently been reported 4 that, in the postnatal rat and the chick embryo, synaptic sites in muscle are initially functionally innervated by as many as 3-4 terminals, whereas later in development only one functional terminal is found on each such site; correlated ultrastructural examination of the same material by these investigators was consistent with this interpretation. There is also additional physiological evidence from neonatal mammals 3,39 that muscle fibers, which in the adult are normally innervated by only one axon, are multiply innervated at these younger stages again suggesting that many excess motoneurons may make functional connections before they degenerate. The final assumption (c) concerning an embryonic mechanism for the retrograde transfer of information from the growing tips of axons has been greatly strengthened by the recent reports that the injection of horseradish peroxidase into muscle 28-~0, or the CNS 6,al,a4,36,44, results in the uptake by axonal tips or terminals and the retrograde transport of the protein back to the cell body where it can be demonstrated histochemically. Retrograde axonal transport has also been reported to occur in the axons of spinal motoneurons in tadpoles of the toad Xenopus laevis 32. We report here evidence for the retrograde transport of horseradish peroxidase from axons in the growing limb-bud to the motoneurons and cells of the spinal sensory ganglia at the very inception of leg innervation in the chick embryo. MATERIALS AND METHODS
Fifty-nine chick embryos and hatchlings were injected with a 50 ~o solution of HRP (Sigma, types II or VI) in avian Ringers. In preliminary studies with 15 day embryos it was determined that the type II HRP resulted in fewer cells being stained and a less intense reaction in those cells that did stain. For this reason, all results discussed below were obtained on animals injected with type VI HRP. The ages chosen for injection were 4, 4.5, 5, 6.5, 8, 10, 15 and 18 days of incubation (embryonic stages 23-44) and 24-48 h posthatch. Injections were made through a window in the egg into the right leg, typically into the region of the semitendinosus or quadriceps femoris muscles, although a few injections were also made into the gastrocnemius. In the younger embryos prior to day 8 the injections were made into the middle of the limb-bud adjacent or distal to the growing tips of the axons (Fig. 1). The animals received between 0.05 #1 and 4.0 #1 of the HRP solution, with the amounts injected increasing with increasing age (Table I). The injections were made using a 10 #1 Hamilton syringe with a 33-gauge needle. The syringe was held in a micromanipulator and was slowly advanced under visual control until the tip of the needle penetrated approximately 1-3 mm into the muscle. The HRP was injected over a 30-45-sec period after which the needle remained in place for another 60 sec. Some slight hemorrhaging and leakage of the HRP frequently occurred after withdrawal of the needle, particularly at the younger ages before days 6-8. During the injection the embryo was carefully held and stabilized by the use of fine hair loops 18. Following injection the window in the egg was sealed with Parafilm and the eggs returned to the incubator. After survival times ranging from 5 to 48 h (Table I), the spinal cords from the lumbosacral region were carefully dissected out within 1-3 min and refrigerated in a cold
294 TABLE
I
S U M M A R Y OF E X P E R I M E N T S W I T H
HRP
I N J E C T I O N S IN C H I C K E M B R Y O S A N D H A T C H L I N ( i S
Age (days)
Histochemical reaction
Samph" size
Survival (h)
Amount it~/ected (/tl)
4 4.5 5
-t -i
6 10 8
5-24 5 6 6-7
0.1 0.2 0.05-1.0 O. 1-0.5
0.1--0.2 0.5 0.5-1.0 1.0 2.0 2.0 1.0-4.0 4.0
6.5 8 10
i ~ ~
5 8 6
57 5--6 5-6
15
~
4
5, 10,24
18 I p.h.* 2 p.h.
i
4 5 3
10 8, 24 10, 24, 48
=
* p.h., p o s t h a t c h e d .
phosphate buffered solution of 2 ~ glutaraldehyde in 5 ~ sucrose for a period of 4 h. The tissues were then washed in running tap water overnight or longer. They were then refrigerated in a 0.08~ solution of 3,3-diaminobenzidine tetrahydrochloride (DAB) for 4 h. After this time, hydrogen peroxide was added to make a 0.02 ~ solution and the tissues were left at room temperature for an additional 4 h. They were then transferred to 70 ~ ethyl alcohol overnight and the following day were dehydrated, cleared, infiltrated and embedded in paraplast according to standard procedures (Lamb, personal communication). The blocks were serially sectioned at 12 #m, mounted on slides, de-paraffinized with xylene and cover-slipped. No counter-staining was used. Several control embryos were processed in a manner identical to the experimentals, except that no HRP was injected. The purpose of this control was to guard against spurious conclusions resulting from non-specific staining, especially of red blood cells which have endogenous peroxidase activity and endothelial cells which phagocytose the protein 6,36. RESULTS
The youngest age at which we were able to demonstrate the retrograde transport of H R P from the leg was at stage 24 or 4.5 days of incubation (Fig. 2). Despite repeated attempts with different survival times up to 24 h (Table I), we have not been successful in obtaining evidence for H R P transport prior to this time; even embryos only one stage earlier (stage 23, 4 days of incubation) failed to show a positive HRP reaction (Fig. 3). By 4.5 days and thereafter the H R P reaction product was always localized ipsilateral to the side of injection and was only found in the motoneurons of the lateral motor column or in the sensory ganglion cells (Fig. 4). The intensity and clarity of the H R P reaction product in the motor and sensory neurons during these early stages appeared similar regardless of whether the embryo was allowed to
i:f
Fig. 1. Cross-section of 4.5 day embryo stained with the Cajal reduced silver technique. Circle in limb-bud indicates the approximate site of H R P injection, p, peripheral nerve; so, spinal cord; he, notocord; lb, right limb-bud, x 70. Fig. 2. Cross-section through lumbar spinal cord of 4.5 day embryo (stage 24-25) showing the selective staining of motoneurons in the lateral motor column (line) on the injected side only. Also note positively stained dorsal root fibers (asterisk) ending in the dorsal funiculus (df). Outlined regions are enlarged in Figs. 11 and 12. rbe, red blood cells, x 165. Fig. 3. Cross-section through lumbar spinal cord of a 4 day embryo (stage 23) which failed to show any positive staining of motoneurons after HRP. lmc, lateral motor column on injected side; co, central canal; no, notoeord, x 220. Fig. 4. Ganglion cells in the spinal sensory ganglion on the side of H R P injection of the limb-bud of a 5 day embryo. Several cells (arrows) exhibit a granular H R P reaction product, x 2400.
297 survive for 5, 10 or 24 h after the injection. Both types o f cells typically exhibited a granular-like distribution o f the H R P , which occurred most intensely over the cytoplasm (Fig. 5), although motoneurons, sensory cells, and their processes occasionally displayed a rather diffuse, non-granular distribution o f H R P reaction product (Figs. 6 and 7). After hatching it was necessary to use longer survival times (10-24 h) and larger injections o f H R P (2-4 #!) to demonstrate the staining o f motoneurons. In m a n y cases m o t o n e u r o n axons containing the reaction p r o d u c t could be traced from the site o f injection all the way into the spinal cord. Within the spinal cord the dendritic extensions o f motoneurons were frequently clearly stained (Fig. 8), and in some instances, particularly in those cells with a diffuse reaction product, they could be followed into the lateral and ventral marginal zones (prospective white matter) (Figs. 9 and 10). With regard to the sensory ganglion cells there did not appear to be any tendency for cells in only a particular part o f the ganglion to react positively at the early stages 2°. It was o f interest, however, that both the peripheral and central processes of these cells contained the H R P reaction p r o d u c t (Fig. 7), and, indeed in some cases it was possible to follow the central processes o f sensory ganglion cells into the dorsal funiculus (Figs. 2, 11 and 12). Because after day 8 the spinal cords were dissected out o f the vertebral canal it was not possible to observe the reaction o f sensory ganglion cells at later stages. Since we made no attempt to study the extent o f diffusion at the site o f injection it was not possible to systematically determine whether the injection into different muscles resulted in differential patterns o f uptake by specific groups o f motoneurons. Nevertheless, it was our impression that this was the case. F o r example, when the injection was limited primarily to the semitendinosus muscle only ceils immediately adjacent to the lateral funiculus were stained positively, whereas injections into the gastrocnemius muscle resulted in the staining o f motonenrons in the central or medial part o f the lateral m o t o r column. At no time did we observe the H R P reaction product in neurons other than those whose processes are k n o w n to innervate the region o f injection, lpsilateral
Fig. 5. Two motoneurons in the ventral horn of a 15 day embryo after HRP injection into the leg. Note the granular distribution of HRP primarily over the cytoplasm and in the axons (ax) and dendrites (d). x 2400. Fig. 6. Motoneurons in the ventral horn of a 5 day embryo on the side of HRP injection of the limbbud. Two cells with a diffuse distribution of reaction product in the soma and axons (asterisks) as well as several cells with a granular distribution of HRP (arrows) are visible. Because the motoneurons only contain a thin rim of cytoplasm the outline of the cells with the granular reaction product are not easily detectable at this stage, x 2400. Fig. 7. Spinal sensory ganglion (ssg) from a 5 day embryo (st. 26) on the side of HRP injection. The sensory ganglion cells (stars) and their distal (short arrows) and proximal (long arrow) processes exhibit a positive but diffuse HRP reaction. Most positive cells in this same ganglion, however, contain a granular distribution of HRP (e.g. see Fig. 4). dr, dorsal root. x 460. Fig. 8. Several motoneurons in the ventral horn of a 15 day embryo. Note especially the HRP staining of dendrites, x 500.
Fig. 9. Ventral horn of a 5 day embryo. One diffusely stained motoneuron dendrite (arrows) extends out of the lateral motor column (Imc) and into the lateral funiculus (If). vf, ventral funiculus. ~'. 370 Fig. 10. A higher magnification of diffusely stained motoneuron dendrites (arrows) extending into the lateral funiculus (If)..: 1000. Fig. 11. Enlargement of the region outlined on the left in Fig. 2 from the dorsal funiculus of the t,ninjected side. Note the absence of HRP positive dorsal root fibers, dr, dorsal funiculus; p, pial surface. 2400. Fig. 12. Enlargement of the region outlined on the right ill Fig. 2 from the dorsal funiculus of the HRP injected side. Numerous small black dots (arrows) are positively stained dorsal root fibers Compare with Fig. 11. p, pial surface. 2400.
299 medial motoneurons, contralateral motoneurons, other CNS neurons, cells in the sympathetic ganglia and cells in Hofmann's nucleus major and minor consistently failed to show any uptake of HRP.
DISCUSSION
The innervation of the leg in the chick embryo is a gradual process occurring over a period of several days11,TM. At stage 23 (4 days) motor axons have reached the base of the limb-bud but have not yet penetrated it. By stage 24 (4.5 days) numerous nerve fibers have entered the leg and by stage 25 they have penetrated to the level of the knee. It is stage 24-25 that we have first been able to demonstrate a retrograde transport of HRP in the present study. At stage 26 (5 days) fine nerve fibers can be found extending close to the distal toe plate. During day 6 (stages 27-28) myotube formation is underway and individual nerve fibers can be seen to branch off from the main nerve bundles and end in close proximity to the myotubes. Throughout days 6 and 7 there is a continuation ofneurotization ofmyotubes, and myofibrils first appear. Although spontaneous neuromuscular activity begins in the leg by about stage 29 (6.5 days)21, histochemica124 and ultrastructuralz5 studies indicate that mature-like neuromuscular end-plates are not formed until about 12 days of incubation. Yet even at the earliest stages of functional innervation nerve branches are already somewhat selective in the muscles they will innervateTM. According to Visintini and Levi-Montalcini4z and Hamburger and LeviMontalcini~° the outgrowth of the proximal and distal branches of the spinal sensory cells in the lumbar region is well underway by 4 days, and by 5 days some of the distal branches have entered the leg and may already contact the dermis. At 4.5 to 5 days we have observed the positive HRP staining of sensory ganglion ceils after limb-bud injections. In the present report we have shown that at the earliest stage that nerve fibers are detectable in the limb-bud by silver techniques (stage 24-25, 4.5 days) these fibers are capable of transporting material from their still growing tips back to the cell bodies in the lateral motor column and spinal sensory ganglia. Along with the recent report of Lamb a2 with Xenopus tadpoles these observations now extend the initial reports of a retrograde transport mechanism in postnatal 6,2s-3x,a4 and adult 6,14,31,33,36 animals to embryos. Additionally, these observations provide evidence for a mechanism whereby the tips of growing nerve fibers may sample and transport information from their environment back to the cell body, a necessary requirement for most conceptions of the peripheral regulation of CNS neurogenesis7,19,2a,4a. Experiments are now underway in this laboratory to determine whether b2¢ blocking the retrograde transport mechanism5 in the leg of the chick embryo one can mimic the effects of limb-bud removal on the CNS. This may allow one to more specifically determine the contribution of retrograde axonal transport to the maintenance and differentiation of neurons during developmentl~,28.
300 It has been previously suggested that neurons and processes which display it diffuse, non-granular distribution of the H R P reaction product have been injured at the time of injection or that they are in some way pathological, perhaps due to fixation or other technical artifactsS,35, 36. Although there have been no systematic studies devoted exclusively to this question, it does indeed, appear that such a diffuse reaction is indicative of injury or artifact. A more critical question, however, is whether the occurrence of a granular H RP reaction product can always be taken as evidence against injury and in favor of a physiological uptake and transport mechanism. For example, it has been shown in two recent reports10, r' that peripheral nerves which have been purposely cut and the proximal stump dipped into a H R P solution not only take up the H R P and transport it back to the cell body but that the reaction product in the cell body is granular in distribution, and appears not to differ from that seen in apparently non-injured preparations. Therefore, while we assume that in the present experiment with the chick embryo the presence of cells and axons with a diffuse reaction product indicates injury, whereas a granular reaction product suggests the uptake and transport of H R P by a normal physiological mechanism, at the moment we can not rule out the other alternative discussed above. This question may be especially critical in the present case where we are dealing with an embryonic system which may have an entirely different, or perhaps an immature mechanism for H R P uptake and transport compared to that of a more mature or adult preparation. We are presently conducting experiments which are aimed at resolving this question. The present demonstration of embryonic retrograde transport of H R P may allow one to use this technique to trace neuroanatomical pathways in the CNS during their development. It will also prove useful in mapping the position of motoneurons within the CNS 14,4~ which innervate specific muscles, a problem still to be solved in the avian spinal cord. Although we have not yet attempted to use the H R P technique with older, more mature chickens the numerous recent reports of its success with adult mammalsS,6,3J,36,44 suggests that it will also prove applicable to adult avian forms. The report of LaVail and LaVai134 that 21- and 34-day-old chicks failed to show a retrograde transport of H R P from the retina to the isthmo-optic nucleus may not reflect any fundamental difference in the retrograde transport mechanism between young and old chickens since in the same report these authors were in fact able to demonstrate the retrograde transport of H R P from the optic tectum to the retina in 21-day-old chicks. Indeed, in a more recent report, LaVail and LaVai135 have found retrograde transport of H R P from the retina to the isthmo-optic nucleus of 63-day-old chickens. In conclusion, we believe that the neuroanatomical technique of retrograde H R P transport will prove to be a valuable new tool not only for adult studies of pathways in the avian CNS, but also for developmental studies at early embryonic stages. ACKNOWLEDGEMENTS
This research was supported in part by NSF Grant GB-31874 and by funds from the North Carolina Department of Mental Health.
301 W e w o u l d like to t h a n k R a n d y Pittman, Car o l Willard, an d Dr. I - W u C h u W a n g for their assistance, and Prof. V i k t o r H a m b u r g e r for the loan o f a series o f silver p r e p a r a t i o n s depicting limb i n n e r v at i o n in the chick embryo.
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291
THE RETROGRADE TRANSPORT OF HORSERADISH PEROXIDASE FROM THE DEVELOPING LIMB OF THE CHICK EMBRYO
R O N A L D W. O P P E N H E I M AND M A R I E T A B. H E A T O N
Neuroembryology Laboratory, Research Division, North Carolina Department of Mental Health, Box 7532, Raleigh, N. C. 27611 (U.S.A.) (Accepted May 3rd, 1975)
SUMMARY
Chick embryos ranging in age from 4.0 to 18 days of incubation and 1-2-dayold hatchlings received injections of HRP solutions directly into the leg musculature. After survival periods of from 5 to 25 h motoneurons and cells in the spinal sensory ganglia were found to be stained with the HRP reaction product. It was found that the first appearance of a positive HRP reaction coincided with the time when nerve processes are first detected in the limb-bud by silver techniques (i.e. at 4.5 days of incubation). Only neurons with processes in the region of injection showed a positive reaction. The demonstration of a retrograde transport mechanism in neurons and axons which are still undergoing growth and differentiation provides a possible mechanism for the peripheral regulation of certain features of CNS neurogenesis. The application of this technique during development may also allow one to map neuroanatomical pathways during their formation.
INTRODUCTION
Many years ago Hamburger 15-n demonstrated that the numerical development of motoneurons in the spinal cord of the chick embryo can be radically altered by removing one limb-bud or by transplanting an additional limb-bud adjacent to the normal one. He found that in the former case, as long as the limb removal was complete, almost all of the motoneurons on one side in the lumbar lateral motor column degenerated and disappeared by the 9th day of incubation, a loss of about 20,000 cells. Conversely, the transplantation of a supernumary limb resulted in a motor hyperplasia of about 40 ~ in those spinal segments which successfully innervated the transplant. Because these two effects were found to be strictly limited to those segments and cell groups which directly innervate the leg, and since the opera-
292 tions were done at a stage prior to normal innervation - - thereby ruling out direct injury to the growing axons - - Hamburger 1:, suggested that he was dealing with a normal developmental process whereby the number of motoneurons that survive during development is rather directly related to the size of the peripheral field to be innervated. At the time, the exact mechanism responsible for this central-peripheral interaction was unknown, although it was later shown to be unlikely that proliferation, migration or initial differentiation were the critical events being regulated16,17. A remaining possibility suggested by Hamburger was that the phenomenon was mediated by a direct 'trophic' interaction between the growing tip of the motor axons and the non-neural periphery outside of the spinal cord 15. The possibility that in the normal course of development cell death might also occur in this system was suggested by the data of Hamburger 17 which indicated an apparent loss of cells on the non-operated side of the spinal cord in one set of embryos with limb-bud removal. Subsequently, work with amphibians by Hughes 26 and Prestige 37 demonstrated a dramatic reduction of spinal motoneurons during normal development, resulting in the loss of 4 out of every 5 cells produced. Recently this phenomenon has also been seen in the chick 19. Hamburger has reported a loss of approximately 8000 motoneurons (about 40 ~ ) on one side in the lumbar lateral motor column between days 6 and 9 during normal development. These observations are consistent with several other recent reports of normal cell death in many regions of the embryonic nervous system1,2,8, 9,~2,40, all of which underscore the prospect that cell death is of fundamental importance in the development and organization of the nervous system 7,~6,27,a7. At the moment, the most popular explanation of cell death during neurogenesis is still the original proposal of Hamburger 17 that there is a competition of some sort between the axonal processes of growing neurons for innervation sites; those that succeed survive, while the remainder degenerate. The acceptance of this explanation as a working hypothesis, however, requires the assumption of several propositions, the most crucial of which, in our opinion, are the following: (a) there is a temporal coincidence between the period of cell death and the time of innervation in a given system, (b) all or at least most cells develop axons which initially grow into the zone of innervation, and (c) there is a cellular mechanism for the centripetal or retrograde transfer of information from the axonal terminals back to the cell body. This last factor need not necessarily involve axoplasmic flow of'trophic' substances but rather could be related to some aspect of functional innervation in the periphery, or indeed to another, entirely different, retrograde mechanism aT. With regard to the limb-spinal cord model of the chick the first assumption apparently is correct 17, although there is a need for more details on this point on the ultrastructural level. Cell death in the ciliary ganglion of the chick embryo also occurs almost simultaneously with the formation of peripheral connections a3. The second assumption, while currently under study in this laboratory with the chick, has received some support from a recent study with amphibians. Prestige and Wilson a8 have reported an almost one-to-one relation between the number of motoneurons and ventral root fibers at different stages of development before, during and after the
293 period of cell death. It has also recently been reported 4 that, in the postnatal rat and the chick embryo, synaptic sites in muscle are initially functionally innervated by as many as 3-4 terminals, whereas later in development only one functional terminal is found on each such site; correlated ultrastructural examination of the same material by these investigators was consistent with this interpretation. There is also additional physiological evidence from neonatal mammals 3,39 that muscle fibers, which in the adult are normally innervated by only one axon, are multiply innervated at these younger stages again suggesting that many excess motoneurons may make functional connections before they degenerate. The final assumption (c) concerning an embryonic mechanism for the retrograde transfer of information from the growing tips of axons has been greatly strengthened by the recent reports that the injection of horseradish peroxidase into muscle 28-~0, or the CNS 6,al,a4,36,44, results in the uptake by axonal tips or terminals and the retrograde transport of the protein back to the cell body where it can be demonstrated histochemically. Retrograde axonal transport has also been reported to occur in the axons of spinal motoneurons in tadpoles of the toad Xenopus laevis 32. We report here evidence for the retrograde transport of horseradish peroxidase from axons in the growing limb-bud to the motoneurons and cells of the spinal sensory ganglia at the very inception of leg innervation in the chick embryo. MATERIALS AND METHODS
Fifty-nine chick embryos and hatchlings were injected with a 50 ~o solution of HRP (Sigma, types II or VI) in avian Ringers. In preliminary studies with 15 day embryos it was determined that the type II HRP resulted in fewer cells being stained and a less intense reaction in those cells that did stain. For this reason, all results discussed below were obtained on animals injected with type VI HRP. The ages chosen for injection were 4, 4.5, 5, 6.5, 8, 10, 15 and 18 days of incubation (embryonic stages 23-44) and 24-48 h posthatch. Injections were made through a window in the egg into the right leg, typically into the region of the semitendinosus or quadriceps femoris muscles, although a few injections were also made into the gastrocnemius. In the younger embryos prior to day 8 the injections were made into the middle of the limb-bud adjacent or distal to the growing tips of the axons (Fig. 1). The animals received between 0.05 #1 and 4.0 #1 of the HRP solution, with the amounts injected increasing with increasing age (Table I). The injections were made using a 10 #1 Hamilton syringe with a 33-gauge needle. The syringe was held in a micromanipulator and was slowly advanced under visual control until the tip of the needle penetrated approximately 1-3 mm into the muscle. The HRP was injected over a 30-45-sec period after which the needle remained in place for another 60 sec. Some slight hemorrhaging and leakage of the HRP frequently occurred after withdrawal of the needle, particularly at the younger ages before days 6-8. During the injection the embryo was carefully held and stabilized by the use of fine hair loops 18. Following injection the window in the egg was sealed with Parafilm and the eggs returned to the incubator. After survival times ranging from 5 to 48 h (Table I), the spinal cords from the lumbosacral region were carefully dissected out within 1-3 min and refrigerated in a cold
294 TABLE
I
S U M M A R Y OF E X P E R I M E N T S W I T H
HRP
I N J E C T I O N S IN C H I C K E M B R Y O S A N D H A T C H L I N ( i S
Age (days)
Histochemical reaction
Samph" size
Survival (h)
Amount it~/ected (/tl)
4 4.5 5
-t -i
6 10 8
5-24 5 6 6-7
0.1 0.2 0.05-1.0 O. 1-0.5
0.1--0.2 0.5 0.5-1.0 1.0 2.0 2.0 1.0-4.0 4.0
6.5 8 10
i ~ ~
5 8 6
57 5--6 5-6
15
~
4
5, 10,24
18 I p.h.* 2 p.h.
i
4 5 3
10 8, 24 10, 24, 48
=
* p.h., p o s t h a t c h e d .
phosphate buffered solution of 2 ~ glutaraldehyde in 5 ~ sucrose for a period of 4 h. The tissues were then washed in running tap water overnight or longer. They were then refrigerated in a 0.08~ solution of 3,3-diaminobenzidine tetrahydrochloride (DAB) for 4 h. After this time, hydrogen peroxide was added to make a 0.02 ~ solution and the tissues were left at room temperature for an additional 4 h. They were then transferred to 70 ~ ethyl alcohol overnight and the following day were dehydrated, cleared, infiltrated and embedded in paraplast according to standard procedures (Lamb, personal communication). The blocks were serially sectioned at 12 #m, mounted on slides, de-paraffinized with xylene and cover-slipped. No counter-staining was used. Several control embryos were processed in a manner identical to the experimentals, except that no HRP was injected. The purpose of this control was to guard against spurious conclusions resulting from non-specific staining, especially of red blood cells which have endogenous peroxidase activity and endothelial cells which phagocytose the protein 6,36. RESULTS
The youngest age at which we were able to demonstrate the retrograde transport of H R P from the leg was at stage 24 or 4.5 days of incubation (Fig. 2). Despite repeated attempts with different survival times up to 24 h (Table I), we have not been successful in obtaining evidence for H R P transport prior to this time; even embryos only one stage earlier (stage 23, 4 days of incubation) failed to show a positive HRP reaction (Fig. 3). By 4.5 days and thereafter the H R P reaction product was always localized ipsilateral to the side of injection and was only found in the motoneurons of the lateral motor column or in the sensory ganglion cells (Fig. 4). The intensity and clarity of the H R P reaction product in the motor and sensory neurons during these early stages appeared similar regardless of whether the embryo was allowed to
i:f
Fig. 1. Cross-section of 4.5 day embryo stained with the Cajal reduced silver technique. Circle in limb-bud indicates the approximate site of H R P injection, p, peripheral nerve; so, spinal cord; he, notocord; lb, right limb-bud, x 70. Fig. 2. Cross-section through lumbar spinal cord of 4.5 day embryo (stage 24-25) showing the selective staining of motoneurons in the lateral motor column (line) on the injected side only. Also note positively stained dorsal root fibers (asterisk) ending in the dorsal funiculus (df). Outlined regions are enlarged in Figs. 11 and 12. rbe, red blood cells, x 165. Fig. 3. Cross-section through lumbar spinal cord of a 4 day embryo (stage 23) which failed to show any positive staining of motoneurons after HRP. lmc, lateral motor column on injected side; co, central canal; no, notoeord, x 220. Fig. 4. Ganglion cells in the spinal sensory ganglion on the side of H R P injection of the limb-bud of a 5 day embryo. Several cells (arrows) exhibit a granular H R P reaction product, x 2400.
297 survive for 5, 10 or 24 h after the injection. Both types o f cells typically exhibited a granular-like distribution o f the H R P , which occurred most intensely over the cytoplasm (Fig. 5), although motoneurons, sensory cells, and their processes occasionally displayed a rather diffuse, non-granular distribution o f H R P reaction product (Figs. 6 and 7). After hatching it was necessary to use longer survival times (10-24 h) and larger injections o f H R P (2-4 #!) to demonstrate the staining o f motoneurons. In m a n y cases m o t o n e u r o n axons containing the reaction p r o d u c t could be traced from the site o f injection all the way into the spinal cord. Within the spinal cord the dendritic extensions o f motoneurons were frequently clearly stained (Fig. 8), and in some instances, particularly in those cells with a diffuse reaction product, they could be followed into the lateral and ventral marginal zones (prospective white matter) (Figs. 9 and 10). With regard to the sensory ganglion cells there did not appear to be any tendency for cells in only a particular part o f the ganglion to react positively at the early stages 2°. It was o f interest, however, that both the peripheral and central processes of these cells contained the H R P reaction p r o d u c t (Fig. 7), and, indeed in some cases it was possible to follow the central processes o f sensory ganglion cells into the dorsal funiculus (Figs. 2, 11 and 12). Because after day 8 the spinal cords were dissected out o f the vertebral canal it was not possible to observe the reaction o f sensory ganglion cells at later stages. Since we made no attempt to study the extent o f diffusion at the site o f injection it was not possible to systematically determine whether the injection into different muscles resulted in differential patterns o f uptake by specific groups o f motoneurons. Nevertheless, it was our impression that this was the case. F o r example, when the injection was limited primarily to the semitendinosus muscle only ceils immediately adjacent to the lateral funiculus were stained positively, whereas injections into the gastrocnemius muscle resulted in the staining o f motonenrons in the central or medial part o f the lateral m o t o r column. At no time did we observe the H R P reaction product in neurons other than those whose processes are k n o w n to innervate the region o f injection, lpsilateral
Fig. 5. Two motoneurons in the ventral horn of a 15 day embryo after HRP injection into the leg. Note the granular distribution of HRP primarily over the cytoplasm and in the axons (ax) and dendrites (d). x 2400. Fig. 6. Motoneurons in the ventral horn of a 5 day embryo on the side of HRP injection of the limbbud. Two cells with a diffuse distribution of reaction product in the soma and axons (asterisks) as well as several cells with a granular distribution of HRP (arrows) are visible. Because the motoneurons only contain a thin rim of cytoplasm the outline of the cells with the granular reaction product are not easily detectable at this stage, x 2400. Fig. 7. Spinal sensory ganglion (ssg) from a 5 day embryo (st. 26) on the side of HRP injection. The sensory ganglion cells (stars) and their distal (short arrows) and proximal (long arrow) processes exhibit a positive but diffuse HRP reaction. Most positive cells in this same ganglion, however, contain a granular distribution of HRP (e.g. see Fig. 4). dr, dorsal root. x 460. Fig. 8. Several motoneurons in the ventral horn of a 15 day embryo. Note especially the HRP staining of dendrites, x 500.
Fig. 9. Ventral horn of a 5 day embryo. One diffusely stained motoneuron dendrite (arrows) extends out of the lateral motor column (Imc) and into the lateral funiculus (If). vf, ventral funiculus. ~'. 370 Fig. 10. A higher magnification of diffusely stained motoneuron dendrites (arrows) extending into the lateral funiculus (If)..: 1000. Fig. 11. Enlargement of the region outlined on the left in Fig. 2 from the dorsal funiculus of the t,ninjected side. Note the absence of HRP positive dorsal root fibers, dr, dorsal funiculus; p, pial surface. 2400. Fig. 12. Enlargement of the region outlined on the right ill Fig. 2 from the dorsal funiculus of the HRP injected side. Numerous small black dots (arrows) are positively stained dorsal root fibers Compare with Fig. 11. p, pial surface. 2400.
299 medial motoneurons, contralateral motoneurons, other CNS neurons, cells in the sympathetic ganglia and cells in Hofmann's nucleus major and minor consistently failed to show any uptake of HRP.
DISCUSSION
The innervation of the leg in the chick embryo is a gradual process occurring over a period of several days11,TM. At stage 23 (4 days) motor axons have reached the base of the limb-bud but have not yet penetrated it. By stage 24 (4.5 days) numerous nerve fibers have entered the leg and by stage 25 they have penetrated to the level of the knee. It is stage 24-25 that we have first been able to demonstrate a retrograde transport of HRP in the present study. At stage 26 (5 days) fine nerve fibers can be found extending close to the distal toe plate. During day 6 (stages 27-28) myotube formation is underway and individual nerve fibers can be seen to branch off from the main nerve bundles and end in close proximity to the myotubes. Throughout days 6 and 7 there is a continuation ofneurotization ofmyotubes, and myofibrils first appear. Although spontaneous neuromuscular activity begins in the leg by about stage 29 (6.5 days)21, histochemica124 and ultrastructuralz5 studies indicate that mature-like neuromuscular end-plates are not formed until about 12 days of incubation. Yet even at the earliest stages of functional innervation nerve branches are already somewhat selective in the muscles they will innervateTM. According to Visintini and Levi-Montalcini4z and Hamburger and LeviMontalcini~° the outgrowth of the proximal and distal branches of the spinal sensory cells in the lumbar region is well underway by 4 days, and by 5 days some of the distal branches have entered the leg and may already contact the dermis. At 4.5 to 5 days we have observed the positive HRP staining of sensory ganglion ceils after limb-bud injections. In the present report we have shown that at the earliest stage that nerve fibers are detectable in the limb-bud by silver techniques (stage 24-25, 4.5 days) these fibers are capable of transporting material from their still growing tips back to the cell bodies in the lateral motor column and spinal sensory ganglia. Along with the recent report of Lamb a2 with Xenopus tadpoles these observations now extend the initial reports of a retrograde transport mechanism in postnatal 6,2s-3x,a4 and adult 6,14,31,33,36 animals to embryos. Additionally, these observations provide evidence for a mechanism whereby the tips of growing nerve fibers may sample and transport information from their environment back to the cell body, a necessary requirement for most conceptions of the peripheral regulation of CNS neurogenesis7,19,2a,4a. Experiments are now underway in this laboratory to determine whether b2¢ blocking the retrograde transport mechanism5 in the leg of the chick embryo one can mimic the effects of limb-bud removal on the CNS. This may allow one to more specifically determine the contribution of retrograde axonal transport to the maintenance and differentiation of neurons during developmentl~,28.
300 It has been previously suggested that neurons and processes which display it diffuse, non-granular distribution of the H R P reaction product have been injured at the time of injection or that they are in some way pathological, perhaps due to fixation or other technical artifactsS,35, 36. Although there have been no systematic studies devoted exclusively to this question, it does indeed, appear that such a diffuse reaction is indicative of injury or artifact. A more critical question, however, is whether the occurrence of a granular H RP reaction product can always be taken as evidence against injury and in favor of a physiological uptake and transport mechanism. For example, it has been shown in two recent reports10, r' that peripheral nerves which have been purposely cut and the proximal stump dipped into a H R P solution not only take up the H R P and transport it back to the cell body but that the reaction product in the cell body is granular in distribution, and appears not to differ from that seen in apparently non-injured preparations. Therefore, while we assume that in the present experiment with the chick embryo the presence of cells and axons with a diffuse reaction product indicates injury, whereas a granular reaction product suggests the uptake and transport of H R P by a normal physiological mechanism, at the moment we can not rule out the other alternative discussed above. This question may be especially critical in the present case where we are dealing with an embryonic system which may have an entirely different, or perhaps an immature mechanism for H R P uptake and transport compared to that of a more mature or adult preparation. We are presently conducting experiments which are aimed at resolving this question. The present demonstration of embryonic retrograde transport of H R P may allow one to use this technique to trace neuroanatomical pathways in the CNS during their development. It will also prove useful in mapping the position of motoneurons within the CNS 14,4~ which innervate specific muscles, a problem still to be solved in the avian spinal cord. Although we have not yet attempted to use the H R P technique with older, more mature chickens the numerous recent reports of its success with adult mammalsS,6,3J,36,44 suggests that it will also prove applicable to adult avian forms. The report of LaVail and LaVai134 that 21- and 34-day-old chicks failed to show a retrograde transport of H R P from the retina to the isthmo-optic nucleus may not reflect any fundamental difference in the retrograde transport mechanism between young and old chickens since in the same report these authors were in fact able to demonstrate the retrograde transport of H R P from the optic tectum to the retina in 21-day-old chicks. Indeed, in a more recent report, LaVail and LaVai135 have found retrograde transport of H R P from the retina to the isthmo-optic nucleus of 63-day-old chickens. In conclusion, we believe that the neuroanatomical technique of retrograde H R P transport will prove to be a valuable new tool not only for adult studies of pathways in the avian CNS, but also for developmental studies at early embryonic stages. ACKNOWLEDGEMENTS
This research was supported in part by NSF Grant GB-31874 and by funds from the North Carolina Department of Mental Health.
301 W e w o u l d like to t h a n k R a n d y Pittman, Car o l Willard, an d Dr. I - W u C h u W a n g for their assistance, and Prof. V i k t o r H a m b u r g e r for the loan o f a series o f silver p r e p a r a t i o n s depicting limb i n n e r v at i o n in the chick embryo.
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