Abstract
BackgroundHow 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.ResultsWe 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-α1, 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-α1 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-α1 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-α1 mutants.ConclusionThese results demonstrate how two guidance cues, TAG-1 and laminin-α1, 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-α1 does not regulate axon-axon interactions, but does influence neuronal polarity and directional guidance.
Highlights
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 identify two guidance signals critical for initial nucleus of the medial longitudinal fascicle (nucMLF) axon guidance: laminin, a component of the extracellular matrix (ECM) through which axons grow, and transient axonal glycoprotein-1 (TAG-1), a cell adhesion molecule (CAM) present on axons
Characterization of normal MLF axon convergence 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]
Summary
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. We have focused on the initial pathfinding steps that nucMLF axons must make: emergence from the cell body with correct polarity, extension in the correct direction, and convergence into an axon tract or fascicle. These crucial first steps are required by axons of many developing brain nuclei, little is known about how they are regulated. We identify two guidance signals critical for initial nucMLF axon guidance: laminin, a component of the extracellular matrix (ECM) through which axons grow, and transient axonal glycoprotein-1 (TAG-1), a cell adhesion molecule (CAM) present on axons
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