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Transient axonal glycoprotein-1 (TAG-1) and laminin-alpha1 regulate dynamic growth cone behaviors and initial axon direction in vivo.

Wolman MA, Sittaramane VK, Essner JJ, Yost HJ, Chandrasekhar A, Halloran MC - Neural Dev (2008)

Bottom Line: Loss of either TAG-1 or laminin-alpha1 causes nucMLF axons to extend into surrounding tissue in incorrect directions and reduces axonal growth rate, resulting in stunted nucMLF axons that fail to extend beyond the hindbrain.However, defects in axon-axon interactions were found only after TAG-1 knockdown, while defects in initial nucMLF axon polarity and excessive branching of nucMLF axons occurred only in laminin-alpha1 mutants.Laminin-alpha1 does not regulate axon-axon interactions, but does influence neuronal polarity and directional guidance.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Zoology, and Neuroscience Training Program, University of Wisconsin, Madison, Wisconsin 53706, USA. mawolman@mail.med.upenn.edu

ABSTRACT

Background: How axon guidance signals regulate growth cone behavior and guidance decisions in the complex in vivo environment of the central nervous system is not well understood. We have taken advantage of the unique features of the zebrafish embryo to visualize dynamic growth cone behaviors and analyze guidance mechanisms of axons emerging from a central brain nucleus in vivo.

Results: We investigated axons of the nucleus of the medial longitudinal fascicle (nucMLF), which are the first axons to extend in the zebrafish midbrain. Using in vivo time-lapse imaging, we show that both positive axon-axon interactions and guidance by surrounding tissue control initial nucMLF axon guidance. We further show that two guidance molecules, transient axonal glycoprotein-1 (TAG-1) and laminin-alpha1, are essential for the initial directional extension of nucMLF axons and their subsequent convergence into a tight fascicle. Fixed tissue analysis shows that TAG-1 knockdown causes errors in nucMLF axon pathfinding similar to those seen in a laminin-alpha1 mutant. However, in vivo time-lapse imaging reveals that while some defects in dynamic growth cone behavior are similar, there are also defects unique to the loss of each gene. Loss of either TAG-1 or laminin-alpha1 causes nucMLF axons to extend into surrounding tissue in incorrect directions and reduces axonal growth rate, resulting in stunted nucMLF axons that fail to extend beyond the hindbrain. However, defects in axon-axon interactions were found only after TAG-1 knockdown, while defects in initial nucMLF axon polarity and excessive branching of nucMLF axons occurred only in laminin-alpha1 mutants.

Conclusion: These results demonstrate how two guidance cues, TAG-1 and laminin-alpha1, influence the behavior of growth cones during axon pathfinding in vivo. Our data suggest that TAG-1 functions to allow growth cones to sense environmental cues and mediates positive axon-axon interactions. Laminin-alpha1 does not regulate axon-axon interactions, but does influence neuronal polarity and directional guidance.

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Initial extension and convergence of MLF axons.(a-e) Ventral views, anterior to the left. (a) Schematic representation of MLF axons and hindbrain axons that grow along the MLF. The dashed line denotes the ventral midline and the dashed box surrounds the 'nucMLF zone'. r = rhombomere. (b) Confocal projection of 20 hpf Tg(pitx2c:gfp) embryo stained with anti-GFP (green) and ZN-12 antibodies (red). (c-e) Whole mount preparations of Tg(pitx2c:gfp) embryos stained with anti-GFP at 16 (c), 20 (d), and 24 (e) hpf. Midline is up. Asterisks denote the caudal-most nucMLF cell and arrows indicate the 'convergence point'. Scale bar = 25 μm.
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Figure 1: Initial extension and convergence of MLF axons.(a-e) Ventral views, anterior to the left. (a) Schematic representation of MLF axons and hindbrain axons that grow along the MLF. The dashed line denotes the ventral midline and the dashed box surrounds the 'nucMLF zone'. r = rhombomere. (b) Confocal projection of 20 hpf Tg(pitx2c:gfp) embryo stained with anti-GFP (green) and ZN-12 antibodies (red). (c-e) Whole mount preparations of Tg(pitx2c:gfp) embryos stained with anti-GFP at 16 (c), 20 (d), and 24 (e) hpf. Midline is up. Asterisks denote the caudal-most nucMLF cell and arrows indicate the 'convergence point'. Scale bar = 25 μm.

Mentions: To investigate the guidance cues and growth cone behaviors involved in the initial outgrowth of axons from an early developing brain nucleus, we studied the formation of the zebrafish MLF, one of the first axon tracts to develop in the zebrafish central nervous system (CNS; Figure 1a) [32]. The MLF arises from bilateral clusters of neuronal cell bodies, called the nucMLF, that lie in the ventral midbrain. At the onset of MLF formation (16 hours post fertilization (hpf)), the nucMLF consists of 6–8 neurons and by 24 hpf the cluster has grown to approximately 30–35 neurons [33,34]. During the initial wave of axonogenesis (16–30 hpf), unipolar nucMLF cells each extend an axon caudally along the ipsilateral ventral neural tube. The caudal-most nucMLF cell extends the leading MLF axon into the hindbrain and then the more rostrally positioned cells extend axons that fasciculate with the leading axon [35]. We have previously reported that Semaphorin3D, which is expressed both rostral and medial to the nucMLFs, plays a role in repelling nucMLF axons in the caudal direction [36,37]; however, nothing is known about other extracellular cues involved in guiding the initial pathfinding decisions of nucMLF axons. Here, we further investigate guidance mechanisms that control how nucMLF axons initially extend in the correct direction and fasciculate with the leading nucMLF axon.


Transient axonal glycoprotein-1 (TAG-1) and laminin-alpha1 regulate dynamic growth cone behaviors and initial axon direction in vivo.

Wolman MA, Sittaramane VK, Essner JJ, Yost HJ, Chandrasekhar A, Halloran MC - Neural Dev (2008)

Initial extension and convergence of MLF axons.(a-e) Ventral views, anterior to the left. (a) Schematic representation of MLF axons and hindbrain axons that grow along the MLF. The dashed line denotes the ventral midline and the dashed box surrounds the 'nucMLF zone'. r = rhombomere. (b) Confocal projection of 20 hpf Tg(pitx2c:gfp) embryo stained with anti-GFP (green) and ZN-12 antibodies (red). (c-e) Whole mount preparations of Tg(pitx2c:gfp) embryos stained with anti-GFP at 16 (c), 20 (d), and 24 (e) hpf. Midline is up. Asterisks denote the caudal-most nucMLF cell and arrows indicate the 'convergence point'. Scale bar = 25 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2278142&req=5

Figure 1: Initial extension and convergence of MLF axons.(a-e) Ventral views, anterior to the left. (a) Schematic representation of MLF axons and hindbrain axons that grow along the MLF. The dashed line denotes the ventral midline and the dashed box surrounds the 'nucMLF zone'. r = rhombomere. (b) Confocal projection of 20 hpf Tg(pitx2c:gfp) embryo stained with anti-GFP (green) and ZN-12 antibodies (red). (c-e) Whole mount preparations of Tg(pitx2c:gfp) embryos stained with anti-GFP at 16 (c), 20 (d), and 24 (e) hpf. Midline is up. Asterisks denote the caudal-most nucMLF cell and arrows indicate the 'convergence point'. Scale bar = 25 μm.
Mentions: To investigate the guidance cues and growth cone behaviors involved in the initial outgrowth of axons from an early developing brain nucleus, we studied the formation of the zebrafish MLF, one of the first axon tracts to develop in the zebrafish central nervous system (CNS; Figure 1a) [32]. The MLF arises from bilateral clusters of neuronal cell bodies, called the nucMLF, that lie in the ventral midbrain. At the onset of MLF formation (16 hours post fertilization (hpf)), the nucMLF consists of 6–8 neurons and by 24 hpf the cluster has grown to approximately 30–35 neurons [33,34]. During the initial wave of axonogenesis (16–30 hpf), unipolar nucMLF cells each extend an axon caudally along the ipsilateral ventral neural tube. The caudal-most nucMLF cell extends the leading MLF axon into the hindbrain and then the more rostrally positioned cells extend axons that fasciculate with the leading axon [35]. We have previously reported that Semaphorin3D, which is expressed both rostral and medial to the nucMLFs, plays a role in repelling nucMLF axons in the caudal direction [36,37]; however, nothing is known about other extracellular cues involved in guiding the initial pathfinding decisions of nucMLF axons. Here, we further investigate guidance mechanisms that control how nucMLF axons initially extend in the correct direction and fasciculate with the leading nucMLF axon.

Bottom Line: Loss of either TAG-1 or laminin-alpha1 causes nucMLF axons to extend into surrounding tissue in incorrect directions and reduces axonal growth rate, resulting in stunted nucMLF axons that fail to extend beyond the hindbrain.However, defects in axon-axon interactions were found only after TAG-1 knockdown, while defects in initial nucMLF axon polarity and excessive branching of nucMLF axons occurred only in laminin-alpha1 mutants.Laminin-alpha1 does not regulate axon-axon interactions, but does influence neuronal polarity and directional guidance.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Zoology, and Neuroscience Training Program, University of Wisconsin, Madison, Wisconsin 53706, USA. mawolman@mail.med.upenn.edu

ABSTRACT

Background: How axon guidance signals regulate growth cone behavior and guidance decisions in the complex in vivo environment of the central nervous system is not well understood. We have taken advantage of the unique features of the zebrafish embryo to visualize dynamic growth cone behaviors and analyze guidance mechanisms of axons emerging from a central brain nucleus in vivo.

Results: We investigated axons of the nucleus of the medial longitudinal fascicle (nucMLF), which are the first axons to extend in the zebrafish midbrain. Using in vivo time-lapse imaging, we show that both positive axon-axon interactions and guidance by surrounding tissue control initial nucMLF axon guidance. We further show that two guidance molecules, transient axonal glycoprotein-1 (TAG-1) and laminin-alpha1, are essential for the initial directional extension of nucMLF axons and their subsequent convergence into a tight fascicle. Fixed tissue analysis shows that TAG-1 knockdown causes errors in nucMLF axon pathfinding similar to those seen in a laminin-alpha1 mutant. However, in vivo time-lapse imaging reveals that while some defects in dynamic growth cone behavior are similar, there are also defects unique to the loss of each gene. Loss of either TAG-1 or laminin-alpha1 causes nucMLF axons to extend into surrounding tissue in incorrect directions and reduces axonal growth rate, resulting in stunted nucMLF axons that fail to extend beyond the hindbrain. However, defects in axon-axon interactions were found only after TAG-1 knockdown, while defects in initial nucMLF axon polarity and excessive branching of nucMLF axons occurred only in laminin-alpha1 mutants.

Conclusion: These results demonstrate how two guidance cues, TAG-1 and laminin-alpha1, influence the behavior of growth cones during axon pathfinding in vivo. Our data suggest that TAG-1 functions to allow growth cones to sense environmental cues and mediates positive axon-axon interactions. Laminin-alpha1 does not regulate axon-axon interactions, but does influence neuronal polarity and directional guidance.

Show MeSH