Wallenda/DLK protein levels are temporally downregulated by Tramtrack69 to allow R7 growth cones to become stationary boutons

Abstract

Dual leucine zipper kinase (DLK) promotes growth cone motility and must be restrained to ensure normal development. PHR (Pam/ Highwire/RPM-1) ubiquitin ligases therefore target DLK for 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 become 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, as R7 growth cones become boutons, Wnd levels are further repressed by a temporally expressed transcription factor, Tramtrack69 (Ttk69). Previously unobserved negative feedback from JNK also contributes to Wnd repression at both time points. We conclude that neurons deploy additional mechanisms to downregulate DLK as they form stable, synaptic connections. We use live imaging to probe the effects of Wnd and Ttk69 on R7 bouton development and conclude that Ttk69 coordinates multiple regulators of this process.

An axonal stress response pathway: degenerative and regenerative signaling by DLK

“…More dramatic defects in developmental wiring of the nervous system have been linked to lost regulation of DLK: DLK protein is held in check by a highly conserved ubiquitin ligase, Pam/Highwire/Rpm-1 (PHR) [17–20]. This restraint appears to be important for some axon guidance decisions [17,21], axon termination at correct locations [22,23], assembly of presynaptic machinery [19,20,24••], and elaboration of dendrite branches [25]. Hence restraint verses activity of DLK appears to be important at specific time points in nervous system development.…”

“…How does DLK become activated? A growing number of conditions, kinases and some phosphatases have been implicated in its regulation [23,41,84,86•,87–95], and activated DLK is heavily phosphorylated across multiple sites [41,86•]. However, the molecular mechanisms that link various stressors in axons (in Figures 1 and 2) to DLK activation are still poorly understood.…”

RPM-1 regulates axon termination by affecting growth cone collapse and microtubule stability

“…Time-lapse imaging studies in cultured and intact fly retina (Akin and Zipursky, 2016;Feoktistov and Herman, 2016;Ozel et al, 2015) and mammalian brain slice (Halloran and Kalil, 1994;Hand et al, 2015) have begun to explore how growth cones transition at axonal branch points and during terminal bouton synapse formation in vivo and ex vivo. Although careful temporal and spatial analysis can aid in distinguishing axon termination from synapse formation in fly retinal axons (Ting et al, 2005), axon termination has yet to be explored under normal and perturbed conditions using time-lapse imaging in this system.…”

“…C. elegans regulator of presynaptic morphology 1 (RPM-1) is an intracellular signaling hub that regulates axon termination, and is orthologous to Drosophila Highwire, mouse Phr1 and human Pam/ MYCBP2 (Borgen et al, 2017;Feoktistov and Herman, 2016;Grill et al, 2016;Opperman and Grill, 2014;Schaefer et al, 2000). RPM-1 acts as a ubiquitin ligase to inhibit MAP3K proteins, such as DLK-1 and MLK-1 (Baker et al, 2015;Collins et al, 2006;Nakata et al, 2005).…”

Untangling the wiring of theDrosophilavisual system: developmental principles and molecular strategies

“…This is achieved through specific filopodial types and their dynamic patterns at different developmental steps of column restriction, layer separation and synaptogenesis (Özel et al, 2015), and the regulated intrinsic axonal growth of the R7 cell ( Fig. 4) (Feoktistov & Herman, 2016;Kniss, Holbrook & Herman, 2013).…”