- Email: [email protected]
Motor axon pathfinding
Motor
axon pathfinding
Monte Westerfield University Recent studies motor
of Oregon,
Eugene, Oregon,
have provided a significant
axon pathfinding.
of the mechanisms
In particular,
used by motor
USA
advance in our understanding
sources
growth
of
of guidance cues and some
cones during
pathfinding
have
been identified.
Current
Opinion
in Neurobiology
1992, 2:28-30
Introduction
When faced with both a normal and an ectopic fin bud, some motor nerves branched and extended to both.
An outstanding problem in developmental neurobiology is how specific synaptic connections form between motor neurons and muscles. Studies of several vertebrate species during the past decade have demonstrated that specific connections arise, at least in part, by the cellspecific pathfinding of motor growth cones [ 11. In both chicks [ 2] and zebrafish [3] individual growth cones, including the first (pioneer) growth cones, exhibit cellspecific behaviors and extend stereotypically towards the regions where appropriate muscle targets arise. Their behavior suggests that they recognize cues in the environment which guide them along their pathways. These observations are contrary to an older hypothesis that initial outgrowth is unpatterned and that specific connections arise because errors are eliminated by retraction of incorrect projections or by selective survival of appropriately connected motor neurons. The major challenge now is to understand how cell-specific pathfinding is regulated. What are the guidance cues, where do they come from, and how do growth cones respond to them? The past year’s literature provides some new clues.
O’Brian et al. [6,7] have obtained complementary results by moving motor neurons relative to the limb bud in chicks. When they replaced the lumbosacral spinal cord, which normally innervates the hindlimb, with thoracic spinal cord, which normally never innervates the hindlimb, thoracic motor neurons innervated specific hindlimb muscles and fom7ed motor pools in appropri ate positions, The pattern of muscle activation, however, was appropriate for thoracic rather than hindlimb segments suggesting that although the foreign motor neurons innervated the limb muscles, their central connections were unchanged. Taken together, these results from fish and chick suggest that the limb provides cues which can attract and guide motor growth cones.
limbs
are a source of guidance
Pathfinding
One possible source of these guidance cues is muscle. Recent experiments by Phelan and Hollyday [8] , however, demonstrate that pathfinding in muscle-less chick wings is remarkably normal. They eliminated the muscles by removing the somites that give rise to the myogenic cells of the wings, Although the nerves that formed in these abnormal wings were somewhat stubby and smooth because they formed fewer intramuscular branches, the basic pattern, including nerves that would normally innervate individual muscles, was present. The axial organization of motor pools within the spinal cord was also normal, suggesting that motor axons selected appropriate pathways distal to the plexus, despite the absence of muscle cells in the developing wing. It is not known, however, whether sensory or motor growth cones initially formed the nerves.
cues
There are a variety of sources of potential guidance cues that might be used by motor growth cones for pathfinding. Recent experiments in the medaka fish suggest that the limb is a source of cues that direct axonal outgrowth. Okamoto and Kuwada have examined the nerves that form in embryos which are missing fins due to a muta tion [4], surgical ablation [5-l, or which have ectopitally placed fins IS**]. They found that the segmental nerves that normally innervate the fin failed to converge when the fin was absent. Instead, the axons followed a trajectory similar to the nerves in segments without fins. When the lin developed in an ectopic position, the nerves followed abnormal pathways to reach it. Moreover, motor nerves from nearby segments, which do not normally innervate the Iin, also extended to the ectopic Iin bud.
In contrast to these results in the chick, Eisen and Pike [9] have found that pathfinding by motor growth cones is seriously disturbed in zebra&h embryos homozygous for a recessive mutation, @t-Z, which produces a deficiency of trunk muscles. The mutation alters the mor-
Abbreviation PSA-polysialic
28
@
Current
Biology
in the absence of muscle
acid.
Ltd ISSN 09594388
Motor axon pathfinding Westerfield phology, number, and arrangement of motor neurons. transplanting motor neurons between wild-type and mutant embryos, Eisen and Pike found that the effect of the mutation was non-autonomous; the motor neurons developed normally in wild-type hosts and abnormally in mutants, regardless of their own cellular genotype. Surgical removal of muscle cells from wild-type embryos produced similar results. One explanation for the differences between the observations in muscle-less chicks and zebrafish is that elimination of muscle in the fish may have affected aspects of the myotomes, other than the muscle, that are required for normal pathlinding by the motor growth cones. By
Connective
tissue may provide
guidance
The connective tissue, through which motor growth cones navigate to reach their targets, is a non-muscle component of the periphery that might provide a source of guidance cues. In the limb, connective tissues arise from the somatopleure, a two-layered structure composed of the upper layer of the lateral plate mesoderm and its overlying ectoderm. The mesodermal component gives rise to all the limb connective tissues including the dermis, muscle sheaths, tendons, and cartilage [lO,ll], while the ectoderm produces the limb epidermis. Lance-Jones and Dias [12-l have recently examined the role of the limb connective tissues in pathway guidance by shifting the somatopleure anteriorly from the lumbosacral to the thoracic level in chick embryos. The limb formed ectopically in the thoracic region adjacent to the transplanted somatopleure and contained muscles derived from both lumbosacral and thoracic precursors, whereas the connective tissue came exclusively from the transplanted lumbosacral somatopleure. The normal lumbosacral motor neurons correctly innervated muscles in the ectopic limb even though their growth cones had to follow abnormal routes. Moreover, the growth cones formed a remarkably normal plexus and pattern of nerve branches, including branches to individual muscles in the ectopic limb. There was little or no motor innervation from thoracic segments. Because both the muscles and the neural crest that populated the limb were derived from thoracic segments, these results argue that only the somatopleure component of the limb was of normal origin and that this component provides the primary source of guidance cues for the motor growth cones. The cues probably come from the mesodermal rather than the ectodermal contribution of the somatopleure, because the cues are thought to be local [2,13], and it is the extracellular mattix derived from the mesoderm that immediately surrounds the outgrowing axons.
Mechanisms
of guidance
How do motor growth cones recognize and respond to guidance cues? One possibility is that they use activitydependent mechanisms to interact with environmental cues. Several recent observations argue against this no-
tion. My colleagues and I have found that both pathfinding [14] and the formation of terminal arbors [15] by motor growth cones are normal in mutant zebrafish embryos ( nit- 2) that lack functional acetylcholine receptors. Similarly, muscles are appropriately innervated [ 151, and synaptogenesis proceeds [ 161, in the presence of pharmacological agents that block neuronal activity or neuromuscular transmission, and in the absence of competitive interactions with other motor neurons [ 151. Another possibility is that contacts between growth cones and environmental cues are important in regulating ax onal extension. Although no cell surface molecules have yet been shown to guide axonal pathlinding, recent experiments by Iandmesser et al. [ 17.1 implicate polysialic acid (PSA) in the final steps of intramuscylar branch formation PSA is a component of cell surface molecules, like Ll and N-CAM, which mediate -tiell-cell adhesion. Iandmesser and colleagues found that developmental changes in the pattern of muscle innervation were correlated with the amount of PSA at the axon surface, rather than with levels of the adhesion molecules. Removal of PSA by endoneuraminidase treatment increased axonal fasciculation and reduced nerve branching. In contrast, nerve trunk defasciculation and increased branching produced by neuromuscular activity blockade were associated with an increase in axonal PSA levels. Thus, PSA may be important for regulating the amount of axonal branching on the surface of the target muscle.
Conclusion Studies reported during the past year have significantly advanced our thinking about motor axon pathfinding. Several potential sources of guidance cues have been identified and others have been eliminated. For exam ple, recent evidence suggests that connective tissues, perhaps extracellular matrices, rather than muscles, provide guidance in the limbs. Similarly, we have learned that the molecular form of cell surface molecules, rather than activity-dependent interactions, regulate pathfinding choices. Further analyses of these guidance cues will provide a better understanding of the mechanisms underlying axon pathfinding.
References
and recommended
Papers of particular interest, published view, have been highlighted as: of special interest . .. of outstanding interest
reading
within the annual period of re-
1.
WESTERFIEID M,
EISEN JS: Neuromuscular Specilicity: Pathfinding by Identified Motor Growth Cones in a Vertebrate Embryo. Trends Neurosci 1988, 11:1%22.
2.
TOSNEY KW, LNDMESSER LT: Specificity of Early Motor Neu-
ron Growth Cone Outgrowth in the Chick Embryo. J Neurosci 1985, 5~23362344. 3.
EISEN JS, MYERS PZ, WESTERFIELD M: Pathway Selection by Growth Cones of Identified Motor Neurons in Live Zebra Fish Embryos. Nature 1986, 320:26%271.
29
30
Development 4.
OKAMOTOH, KUWADA JY: Outgrowth by Fm Motor Axons in WiIdtypc and a FinIess Mutant in the Japanese Medaka Fish. Dev Biol 1391, 146:4-l.
5. ..
OKAMOTOH, KUWADAJY: Alteration of Pectoral Fii Nerves Following Ablation of Fin Buds and by Ectopic Fin Buds in the Japanese Medaka Fish. Dev Biol 1991, 146:62-71. This manuscript provides an extremely clear demonstration that the limb is required for correct outgrowth of nerves. It also suggests that limbs provide a source of guidance cues for motor growth cones.
6.
7.
O’BRL%NMK, OPPENHEIM RW: Development and Survival of Thoracic Motoneurons and HindIimb Musculature FoIIowing Transplantation of the Thoracic Neural Tube to the Lumbar Region of the Chick Embryo: Anatomical Aspects. J Neurobiol 1990, 21:31%340. O’BRIANMK, LWDMESSERLT, OPPENHEIMRW: Development and Survival of ‘Ihoracic Motoneurons and HindIimb Musculature Following Transplantation of the Thoracic Neural Tube to the Lumbar Region of the Chick Embryo: Functional Aspects. J Neurobiol 1990, 21:341-355.
8.
PHEIAN KA, HOLLVDAYM: Axon Guidance in Muscleless Chick Wings - the Role of Muscle CeIIs in Motoneuronal Pathway Selection and Muscle Nerve Formation. J Neurosci 1990, lO:2633-2716.
9.
EISENJS, PIKE SH: The sptl Mutation Ahers Segmental Arrangement and_AxonaI Development of Identified Neurons in the Spinal Cord of the Embryonic Zebrafish. Neuron 1991, 6:767-776.
10.
CHEVALWER A, KIENYM, MAUCERA: Limb-Somite Relationships: Origin of the Limb Musculature. Roux’s Arch Dev Bioll977, 191:227-280.
11.
CHRISTB, JACOBHJ, JACOBM: Experimental Analysis of the Origin of the Wing Musculature in Avian Embryos. Anal Embryo1 1977, 150:171-186.
12. ..
LANCE-JONES C, DIASM: The InJluence of Presumptive Limb Connective Tissue on Motoneuron Axon Guidance. Dev Biol 1991, 143:9~110. The authors shifted chick limb bud precursors anteriorly and found that individual muscles were innervated specifically according to the axial level of origin of their connective tissues, rather than their muscles or neural crest cells. 13.
14.
TOSNEYKW, LANDMESSER LT: Pattern and Specificity of Axonal
Outgrowth Following Varying Degrees of Chick Limb Bud Ablation. J Neuraxi 1984, 4:251%2527. WESTERFIELD M, LIU DW, KIMMELCB, WAIKER C: Normal
PathfindIng by Pioneer Motor Growth Cones in Mutant Zebralish Lacking Functional AcetylchoIine Receptors. Neuron 1990, 4:867+374. 15.
LIU DW, WES’IXRFIEU) M: The Formation of Terminal Fields in the Absence of Competitive Interactions Among Primary Motor Neurons in the Zebrafish. J Neurosci 1990, 10:3947-3959.
16.
DAHMM, L%NDMESSER LT: The Regulation of Sy-naptogenesis During Normal Development and Following Activity Blockade. J Neurasci 1991, 11:23%255.
L+NDMESSER LT, DAHML, TANGJC, RUTISHAUSER U: PolysiaIic Acid as a Regulator of Intramuscular Nerve Branching During Embryonic Development. Neuron 1990, 4:655_667. This study shows that the amount of polysialic acid, rather than the levels of adhesion molecules expressed on axonal surfaces, regulates the degree of motor nerve branching. 17. .
M Westedield, Institute of Neuroscience, Universityof Oregon, Eugene, Oregon 97403, USA
axon pathfinding
Monte Westerfield University Recent studies motor
of Oregon,
Eugene, Oregon,
have provided a significant
axon pathfinding.
of the mechanisms
In particular,
used by motor
USA
advance in our understanding
sources
growth
of
of guidance cues and some
cones during
pathfinding
have
been identified.
Current
Opinion
in Neurobiology
1992, 2:28-30
Introduction
When faced with both a normal and an ectopic fin bud, some motor nerves branched and extended to both.
An outstanding problem in developmental neurobiology is how specific synaptic connections form between motor neurons and muscles. Studies of several vertebrate species during the past decade have demonstrated that specific connections arise, at least in part, by the cellspecific pathfinding of motor growth cones [ 11. In both chicks [ 2] and zebrafish [3] individual growth cones, including the first (pioneer) growth cones, exhibit cellspecific behaviors and extend stereotypically towards the regions where appropriate muscle targets arise. Their behavior suggests that they recognize cues in the environment which guide them along their pathways. These observations are contrary to an older hypothesis that initial outgrowth is unpatterned and that specific connections arise because errors are eliminated by retraction of incorrect projections or by selective survival of appropriately connected motor neurons. The major challenge now is to understand how cell-specific pathfinding is regulated. What are the guidance cues, where do they come from, and how do growth cones respond to them? The past year’s literature provides some new clues.
O’Brian et al. [6,7] have obtained complementary results by moving motor neurons relative to the limb bud in chicks. When they replaced the lumbosacral spinal cord, which normally innervates the hindlimb, with thoracic spinal cord, which normally never innervates the hindlimb, thoracic motor neurons innervated specific hindlimb muscles and fom7ed motor pools in appropri ate positions, The pattern of muscle activation, however, was appropriate for thoracic rather than hindlimb segments suggesting that although the foreign motor neurons innervated the limb muscles, their central connections were unchanged. Taken together, these results from fish and chick suggest that the limb provides cues which can attract and guide motor growth cones.
limbs
are a source of guidance
Pathfinding
One possible source of these guidance cues is muscle. Recent experiments by Phelan and Hollyday [8] , however, demonstrate that pathfinding in muscle-less chick wings is remarkably normal. They eliminated the muscles by removing the somites that give rise to the myogenic cells of the wings, Although the nerves that formed in these abnormal wings were somewhat stubby and smooth because they formed fewer intramuscular branches, the basic pattern, including nerves that would normally innervate individual muscles, was present. The axial organization of motor pools within the spinal cord was also normal, suggesting that motor axons selected appropriate pathways distal to the plexus, despite the absence of muscle cells in the developing wing. It is not known, however, whether sensory or motor growth cones initially formed the nerves.
cues
There are a variety of sources of potential guidance cues that might be used by motor growth cones for pathfinding. Recent experiments in the medaka fish suggest that the limb is a source of cues that direct axonal outgrowth. Okamoto and Kuwada have examined the nerves that form in embryos which are missing fins due to a muta tion [4], surgical ablation [5-l, or which have ectopitally placed fins IS**]. They found that the segmental nerves that normally innervate the fin failed to converge when the fin was absent. Instead, the axons followed a trajectory similar to the nerves in segments without fins. When the lin developed in an ectopic position, the nerves followed abnormal pathways to reach it. Moreover, motor nerves from nearby segments, which do not normally innervate the Iin, also extended to the ectopic Iin bud.
In contrast to these results in the chick, Eisen and Pike [9] have found that pathfinding by motor growth cones is seriously disturbed in zebra&h embryos homozygous for a recessive mutation, @t-Z, which produces a deficiency of trunk muscles. The mutation alters the mor-
Abbreviation PSA-polysialic
28
@
Current
Biology
in the absence of muscle
acid.
Ltd ISSN 09594388
Motor axon pathfinding Westerfield phology, number, and arrangement of motor neurons. transplanting motor neurons between wild-type and mutant embryos, Eisen and Pike found that the effect of the mutation was non-autonomous; the motor neurons developed normally in wild-type hosts and abnormally in mutants, regardless of their own cellular genotype. Surgical removal of muscle cells from wild-type embryos produced similar results. One explanation for the differences between the observations in muscle-less chicks and zebrafish is that elimination of muscle in the fish may have affected aspects of the myotomes, other than the muscle, that are required for normal pathlinding by the motor growth cones. By
Connective
tissue may provide
guidance
The connective tissue, through which motor growth cones navigate to reach their targets, is a non-muscle component of the periphery that might provide a source of guidance cues. In the limb, connective tissues arise from the somatopleure, a two-layered structure composed of the upper layer of the lateral plate mesoderm and its overlying ectoderm. The mesodermal component gives rise to all the limb connective tissues including the dermis, muscle sheaths, tendons, and cartilage [lO,ll], while the ectoderm produces the limb epidermis. Lance-Jones and Dias [12-l have recently examined the role of the limb connective tissues in pathway guidance by shifting the somatopleure anteriorly from the lumbosacral to the thoracic level in chick embryos. The limb formed ectopically in the thoracic region adjacent to the transplanted somatopleure and contained muscles derived from both lumbosacral and thoracic precursors, whereas the connective tissue came exclusively from the transplanted lumbosacral somatopleure. The normal lumbosacral motor neurons correctly innervated muscles in the ectopic limb even though their growth cones had to follow abnormal routes. Moreover, the growth cones formed a remarkably normal plexus and pattern of nerve branches, including branches to individual muscles in the ectopic limb. There was little or no motor innervation from thoracic segments. Because both the muscles and the neural crest that populated the limb were derived from thoracic segments, these results argue that only the somatopleure component of the limb was of normal origin and that this component provides the primary source of guidance cues for the motor growth cones. The cues probably come from the mesodermal rather than the ectodermal contribution of the somatopleure, because the cues are thought to be local [2,13], and it is the extracellular mattix derived from the mesoderm that immediately surrounds the outgrowing axons.
Mechanisms
of guidance
How do motor growth cones recognize and respond to guidance cues? One possibility is that they use activitydependent mechanisms to interact with environmental cues. Several recent observations argue against this no-
tion. My colleagues and I have found that both pathfinding [14] and the formation of terminal arbors [15] by motor growth cones are normal in mutant zebrafish embryos ( nit- 2) that lack functional acetylcholine receptors. Similarly, muscles are appropriately innervated [ 151, and synaptogenesis proceeds [ 161, in the presence of pharmacological agents that block neuronal activity or neuromuscular transmission, and in the absence of competitive interactions with other motor neurons [ 151. Another possibility is that contacts between growth cones and environmental cues are important in regulating ax onal extension. Although no cell surface molecules have yet been shown to guide axonal pathlinding, recent experiments by Iandmesser et al. [ 17.1 implicate polysialic acid (PSA) in the final steps of intramuscylar branch formation PSA is a component of cell surface molecules, like Ll and N-CAM, which mediate -tiell-cell adhesion. Iandmesser and colleagues found that developmental changes in the pattern of muscle innervation were correlated with the amount of PSA at the axon surface, rather than with levels of the adhesion molecules. Removal of PSA by endoneuraminidase treatment increased axonal fasciculation and reduced nerve branching. In contrast, nerve trunk defasciculation and increased branching produced by neuromuscular activity blockade were associated with an increase in axonal PSA levels. Thus, PSA may be important for regulating the amount of axonal branching on the surface of the target muscle.
Conclusion Studies reported during the past year have significantly advanced our thinking about motor axon pathfinding. Several potential sources of guidance cues have been identified and others have been eliminated. For exam ple, recent evidence suggests that connective tissues, perhaps extracellular matrices, rather than muscles, provide guidance in the limbs. Similarly, we have learned that the molecular form of cell surface molecules, rather than activity-dependent interactions, regulate pathfinding choices. Further analyses of these guidance cues will provide a better understanding of the mechanisms underlying axon pathfinding.
References
and recommended
Papers of particular interest, published view, have been highlighted as: of special interest . .. of outstanding interest
reading
within the annual period of re-
1.
WESTERFIEID M,
EISEN JS: Neuromuscular Specilicity: Pathfinding by Identified Motor Growth Cones in a Vertebrate Embryo. Trends Neurosci 1988, 11:1%22.
2.
TOSNEY KW, LNDMESSER LT: Specificity of Early Motor Neu-
ron Growth Cone Outgrowth in the Chick Embryo. J Neurosci 1985, 5~23362344. 3.
EISEN JS, MYERS PZ, WESTERFIELD M: Pathway Selection by Growth Cones of Identified Motor Neurons in Live Zebra Fish Embryos. Nature 1986, 320:26%271.
29
30
Development 4.
OKAMOTOH, KUWADA JY: Outgrowth by Fm Motor Axons in WiIdtypc and a FinIess Mutant in the Japanese Medaka Fish. Dev Biol 1391, 146:4-l.
5. ..
OKAMOTOH, KUWADAJY: Alteration of Pectoral Fii Nerves Following Ablation of Fin Buds and by Ectopic Fin Buds in the Japanese Medaka Fish. Dev Biol 1991, 146:62-71. This manuscript provides an extremely clear demonstration that the limb is required for correct outgrowth of nerves. It also suggests that limbs provide a source of guidance cues for motor growth cones.
6.
7.
O’BRL%NMK, OPPENHEIM RW: Development and Survival of Thoracic Motoneurons and HindIimb Musculature FoIIowing Transplantation of the Thoracic Neural Tube to the Lumbar Region of the Chick Embryo: Anatomical Aspects. J Neurobiol 1990, 21:31%340. O’BRIANMK, LWDMESSERLT, OPPENHEIMRW: Development and Survival of ‘Ihoracic Motoneurons and HindIimb Musculature Following Transplantation of the Thoracic Neural Tube to the Lumbar Region of the Chick Embryo: Functional Aspects. J Neurobiol 1990, 21:341-355.
8.
PHEIAN KA, HOLLVDAYM: Axon Guidance in Muscleless Chick Wings - the Role of Muscle CeIIs in Motoneuronal Pathway Selection and Muscle Nerve Formation. J Neurosci 1990, lO:2633-2716.
9.
EISENJS, PIKE SH: The sptl Mutation Ahers Segmental Arrangement and_AxonaI Development of Identified Neurons in the Spinal Cord of the Embryonic Zebrafish. Neuron 1991, 6:767-776.
10.
CHEVALWER A, KIENYM, MAUCERA: Limb-Somite Relationships: Origin of the Limb Musculature. Roux’s Arch Dev Bioll977, 191:227-280.
11.
CHRISTB, JACOBHJ, JACOBM: Experimental Analysis of the Origin of the Wing Musculature in Avian Embryos. Anal Embryo1 1977, 150:171-186.
12. ..
LANCE-JONES C, DIASM: The InJluence of Presumptive Limb Connective Tissue on Motoneuron Axon Guidance. Dev Biol 1991, 143:9~110. The authors shifted chick limb bud precursors anteriorly and found that individual muscles were innervated specifically according to the axial level of origin of their connective tissues, rather than their muscles or neural crest cells. 13.
14.
TOSNEYKW, LANDMESSER LT: Pattern and Specificity of Axonal
Outgrowth Following Varying Degrees of Chick Limb Bud Ablation. J Neuraxi 1984, 4:251%2527. WESTERFIELD M, LIU DW, KIMMELCB, WAIKER C: Normal
PathfindIng by Pioneer Motor Growth Cones in Mutant Zebralish Lacking Functional AcetylchoIine Receptors. Neuron 1990, 4:867+374. 15.
LIU DW, WES’IXRFIEU) M: The Formation of Terminal Fields in the Absence of Competitive Interactions Among Primary Motor Neurons in the Zebrafish. J Neurosci 1990, 10:3947-3959.
16.
DAHMM, L%NDMESSER LT: The Regulation of Sy-naptogenesis During Normal Development and Following Activity Blockade. J Neurasci 1991, 11:23%255.
L+NDMESSER LT, DAHML, TANGJC, RUTISHAUSER U: PolysiaIic Acid as a Regulator of Intramuscular Nerve Branching During Embryonic Development. Neuron 1990, 4:655_667. This study shows that the amount of polysialic acid, rather than the levels of adhesion molecules expressed on axonal surfaces, regulates the degree of motor nerve branching. 17. .
M Westedield, Institute of Neuroscience, Universityof Oregon, Eugene, Oregon 97403, USA