Setting the Limit on Axon Growth: Multiple Overlapping Mechanisms Repress the MAP3K Wnd/DLK So That Growth Cones Can Remodel into Stationary Synaptic Boutons
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The development of a stereotyped pattern of neural connectivity depends upon the behavior of growth cones, motile structures at the tips of axons that propel axon growth and steer the axon to its targets. When growth cones reach their appropriate target cells, they halt and ultimately remodel into stationary presynaptic boutons. The influence of extracellular cues in directing growth cones to their targets is well studied, but cell-intrinsic factors are also increasingly appreciated for their role in driving much of growth cone behavior. Dual leucine zipper kinases (DLKs) promote growth cone motility and must be kept in check to ensure normal development. PHR (Pam/Highwire/RPM-1) ubiquitin ligases therefore target DLK for proteosomal degradation unless axon injury occurs. Overall DLK levels decrease during development, but how DLK levels are regulated within a developing growth cone has not been examined. We analyzed the expression of the fly DLK Wallenda (Wnd) in R7 photoreceptor growth cones as they halt at their targets and as they remodel into presynaptic boutons. We found that Wnd protein levels are repressed by the PHR protein Highwire (Hiw) during R7 growth cone halting, as has been observed in other systems. However, during remodeling, Wnd levels are further repressed by a temporally-expressed transcription factor, Tramtrack69 (Ttk69). Previously unobserved negative feedback from JNK also contributes to Wnd repression. We conclude that maturing neurons progressively deploy additional mechanisms to keep DLK off and thereby protect their connectivity. We use live imaging to directly probe the effects of Wnd and Ttk69 on remodeling R7 growth cones and conclude that Ttk69 coordinates multiple regulators of this process. Preliminary results indicate that excess Wnd signaling requires the transcription factor Fos to disrupt growth cone remodeling in R7s. This opens up new strategies to identify how Wnd exerts its motility-promoting effects on growth cone cytoskeletons. Additional findings point to a later requirement for Wnd in normal R7 synapse development, suggesting that Wnd expression is not fully silenced in R7s. Further investigation into these findings would greatly advance our understanding of how the neuronal cytoskeleton is regulated as neurons undergo profound morphological and functional changes while developing. This dissertation includes both unpublished and published co-authored material. This dissertation also includes supplemental movie files, which can be found online and are described in Appendix B.