In this study, we use in vivo time-lapse imaging to show how TAG-1 and laminin-α1 affect dynamic growth cone behaviors, initial axon direction, and interactions among nucMLF axons in the zebrafish brain. Normally, nucMLF axons converge and fasciculate soon after extending from their cell bodies. Our results suggest this growth pattern is driven by a combination of guidance by surrounding tissue and axon-axon fasciculation. TAG-1 or laminin-α1 loss of function disrupted this stereotypical outgrowth pattern. First, TAG-1 or laminin-α1 loss of function caused MLF axons to aberrantly extend into the surrounding tissue. TAG-1 knockdown also caused MLF growth cones to retract from neighboring MLF axons, which led to aberrant directional extension of MLF axons and possibly influenced changes in neuronal polarity. Axon-axon interactions appeared normal in bal embryos; however, neuronal polarity was affected, since MLF axons often emerged from their cell bodies in the incorrect direction. MLF axons were also excessively branched in bal embryos. Finally, MLF axons in TAG1MO injected or bal embryos exhibited reduced growth rates and were stalled in the hindbrain. Taken together, these results reveal critical roles for TAG-1 and laminin-α1 in controlling specific growth cone behaviors, neuronal polarity, and directional outgrowth to guide proper formation of a major CNS pathway.
Interestingly, TAG-1 may serve dual functions in mediating nucMLF axon pathfinding as it appears to be required for sensing a signal in the surrounding tissue and for mediating axon-axon interactions. Several Ig superfamily CAMs, including TAG-1, have been shown to mediate axon fasciculation via homophilic and heterophilic binding partners [17
]. Therefore, the defective nucMLF axon-axon interactions caused by TAG-1 knockdown were not surprising. It is highly possible that TAG-1 promotes adhesion among nucMLF axons either homophilically or heterophilically, although a heterophilic partner has not been determined in this case. The apparent consequences of these failed interactions were more intriguing. Often, growth cone retraction upon contacting a neighboring axon was followed by a change in direction and extension into the surrounding tissue, suggesting that these defective interactions may have contributed to further pathfinding errors. Furthermore, in a few cases, defective axon-axon interactions led to the complete retraction of the original axon and the emergence of a new axon on the opposite side of the neuron, thus altering the polarity and directional extension of nucMLF axons. Collectively, these results exemplify the significance of TAG-1 mediated axon-axon interactions not only to fasciculation, but also to directional guidance and neuronal polarity. Finally, it is possible that TAG-1 mediates interactions between nucMLF and nucTPOC axons, which also extend through the midbrain and join the MLF. However, nucTPOC axons grow into this region 2–3 hours after the initial outgrowth of nucMLF axons and appeared unaffected by TAG-1 knockdown.
In addition to this expected role for TAG-1, our results also suggest TAG-1 is required for nucMLF axons to sense a guidance signal from the environment because TAG-1 knockdown also caused nucMLF axons to grow in aberrant directions without first contacting other axons. This signal potentially could be an inhibitory cue located in tissue surrounding the nucMLF and/or an attractive cue emanating from the convergence point. This finding is interesting because TAG-1 is not known to function as a receptor for repulsive or attractive ligands. However, related Ig superfamily CAMs, such as L1 and NrCAM, have recently been shown to be required receptor components for repulsive semaphorin signaling [42
]. Thus, these observations raise the intriguing possibility that TAG-1 may also serve as a receptor component for repulsive and/or attractive ligands in vivo
Laminin also appears to be involved in preventing MLF axon growth into inappropriate surrounding tissues and orienting outgrowth direction. Laminin is expressed throughout the basal lamina upon which MLF axons extend, and thus it could function directly to guide nucMLF axons by signaling via integrins or other receptor molecules on nucMLF growth cones. Alternatively, laminin-α1 may affect axons indirectly through more global effects on brain organization or by binding and presenting other guidance signals. An interesting possibility is that laminin-α1 may regulate growth cone responsiveness to other cues expressed by the surrounding tissue, such as netrin or ephrin, which have been shown to elicit repulsion in the presence of laminin [7
]. Without functional laminin-α1, nucMLF axons may be attracted or non-responsive to such cues. Interestingly, in bal
, misguided axons appeared biased to extend towards the ventral midline rather than wandering in all directions equally. This observation suggests that laminin-α1 indirectly regulates axon repulsion by another cue in the midline region because laminin-α1 is expressed broadly, not only at the midline [46
Loss of laminin-α1 also caused defects in polarity and branching. Establishing appropriate neuronal polarity is arguably the first step towards properly guiding an axon because it determines the initial direction of axon outgrowth. Normally, almost all nucMLF neurons immediately extended their axon in the direction of the convergence point. However, in bal
, we observed a significant number of neurons that initially extended axons in aberrant directions, suggesting that laminin-α1 is critical for establishing the proper polarity of these neurons. Again, whether nucMLF neuronal polarity is directly or indirectly regulated by laminin-α1 remains to be determined. Recently, netrin was shown to regulate neuronal asymmetry and define the site of axon formation [10
]. It is possible that these roles may also require the presence of laminin, similar to netrin-mediated repulsion. Functionally, the nucMLF polarity defect may also have contributed to the increased percentage of time that axons extended off-track. In addition to polarity defects, axons were excessively branched in bal
. It might be surprising that loss of laminin-α1, which usually functions as a permissive growth substratum, would directly stimulate increased branching. However, it is possible that the loss of laminin-α1 created an inhibitory substratum causing growth cone stalling or retraction, which has been correlated with back branching [47
]. These observations reveal novel roles for laminin-α1 in establishing neuronal polarity and regulating axon branching.
TAG-1 or laminin-α1 loss of function ultimately caused nucMLF axons to prematurely stall in the hindbrain. Interestingly, the stalled axons were almost always observed in the anterior hindbrain, instead of being positioned along a continuum from the anterior hindbrain to the anterior spinal cord, where they should have reached by 24 hpf. This observation suggests that an additional cue may be required to promote axon extension through this region and that late arriving axons may miss the temporal expression window of this factor. Alternatively, nucMLF axons may become non-responsive to the potential factor in the absence of TAG-1 and/or laminin-α1 function. In any case, it is interesting that the position of stalled nucMLF axons is the same in embryos deficient for TAG-1 or laminin-α1.