MLK-1/MEK-1/KGB-1 MAPK pathway is required for axon regeneration. (A) Age-dependent decline of axon regeneration in the wild type. Regeneration to the dorsal nerve cord (DNC) fails first, followed by failure of axotomized axons to initiate growth cones (GC). %GC is the percentage of severed axons that form a growth cone (GC). % to DNC is the percentage of growth cone-forming axons that regrow to the dorsal nerve cord. (B) Regeneration is blocked in mutants of the MLK-1/MEK-1/KGB-1 MAPK module, whereas MLK-1 overexpression improves regeneration. (C–E) Improved regeneration in animals overexpressing MLK-1 as scored by: (C) the percentage of regenerating axons that successfully migrate back to targets in the dorsal nerve cord (DNC), (D) growth cone (GC) formation/extension in older animals, and (E) timing of first growth cone (GC) appearance. All axotomy performed at L4 stage unless indicated. Ex[mlk-1], mlk-1 transgene expression from an extrachromosomal array. P determined by Fisher's exact test. **P < 0.01, ***P < 0.001, NS not significant. Error bars indicate 95% confidence interval.

Time-lapse recording illustrates characteristic features of axon regeneration by wild-type L4 GABA motor neurons. Dorsal nerve cord is at the top and ventral nerve cord is at the bottom of each frame. 0 min shows site of laser axotomy and beginning formation of retraction bulbs. 138 and 324 min show microspike activity and remodeling of the axon stump. At 360 min, the right stump initiates a growth cone. By 414 min, both growth cones are beginning to migrate. 519 and 597 min show dystrophic growth cones that make poor progress toward the dorsal nerve cord. At 960 min the dystrophic processes are stalled and it is apparent that regeneration has failed. See Movie S1. Arrowheads point to axon stump and growth cones. Arrows point to microspikes. (Scale bar, 20 μm.)

Axon regeneration by GABA neurons expressing higher levels of MLK-1. Dorsal nerve cord is at the top and ventral nerve cord is at the bottom of every frame. 0 min shows retraction bulb formation shortly after laser axotomy. The first sign of stump remodeling is seen at 6 min. At 45 min growth cones are initiated from both stumps. 114 min shows the growth cones extending around their respective sites of laser damage (asterisks). Notice as the growth cone migrates it successfully remodels to reconstitute an embryonic like growth cone (156, 183, and 227 min). By 417 min, both axons have successfully reached the dorsal nerve cord. See Movie S2. Arrowheads point to proximal axon stump and growth cones. (Scale bar, 20 μm.)

Crosstalk between p38 and JNK MAPK pathways. (A) Diagram of two MAPK pathways essential for axon regeneration with arrows indicating crosstalk between kinases. (B and C) Crosstalk by MAPKKK. DLK-1 can activate both MKK-4 and MEK-1, whereas MLK-1 activates only MEK-1. (B) DLK-1 overexpression suppresses mlk-1 loss of function and MLK-1 overexpression suppresses dlk-1 loss of function. (C) mkk-4 mutants are rescued by overexpressing either DLK-1 or MLK-1. mkk-4 rescue by MAPKKK overexpression requires functional MEK-1. (D) Crosstalk by MAPKK. MKK-4 and MEK-1 each activate PMK-3 and KGB-1. Ex[dlk-1] and Ex[mlk-1], dlk-1 and mlk-1 transgene expression from an extrachromosomal array, respectively. P determined by Fisher's exact test. **P < 0.01, ***P < 0.001, NS not significant. Error bars indicate 95% confidence interval. GC, growth cone.

VHP-1 acts on both PMK-3 and KGB-1 MAPK. (A) vhp-1 mutants show improved regeneration of growth cones (GC) and suppress the loss of either pmk-3 or kgb-1. (B) Successful migration back to dorsal nerve cord (DNC) is improved in vhp-1 mutants. (C) dlk-1 or pmk-3 mutants suppress vhp-1 larval arrest. Loss of mkk-4 does not suppress vhp-1 larval arrest. Scale bar is 100 μm. (D) Complexes immunoprecipitated (IP) with anti-T7 (targeting T7-VHP-1) and immunoblotted (IB) with anti-HA (targeting HA-PMK-3). Whole cell extracts (WCE) were also probed with anti-T7 and anti-HA antibodies. P determined by Fisher's exact test. *P < 0.05, **P < 0.01, ***P < 0.001. Error bars indicate 95% confidence interval.

MLK-1 is targeted for degradation by the E3 ubiquitin ligase RPM-1. The red (A1–D1) is cytoplasmic mCherry to show the GABA neuron morphology. The green (A–D) is MLK-1:GFP. There is no visible MLK-1:GFP in the wild-type background (dorsal nerve cord in A and ventral nerve cord in B). (C) The punctate localization of MLK-1 to synaptic zones in the dorsal nerve cord and diffuse labeling in axons. (D) Punctate localization in ventral nerve cord synaptic zones and diffuse labeling in the cell cytoplasm. Notice that MLK-1:GFP is excluded from the nucleus. (Scale bar, 5 μm.)

Upstream regulation and downstream targets of MAPK signaling in regeneration. (A) rpm-1 mutants show improved regeneration of growth cones (GC) which requires functional KGB-1. (B) RPM-1 does not influence the critical period for regeneration. (C) PMK-3 and KGB-1 promote regeneration via different downstream effectors. Improved regeneration in vhp-1 mutants was not affected by loss of mak-2. cebp-1 mutants fail to regenerate, but can be partially suppressed by vhp-1 loss-of-function. The cebp-1; vhp-1; pmk-3 triple mutant reveals that suppression occurs through KGB-1 activity. (D) Summary of signaling interactions highlighting the coordinated regulation of both PMK-3(p38) and KGB-1(JNK) MAPK pathways by the E3 ubiquitin ligase RPM-1(Phr1), the MAPKKKs DLK-1 and MLK-1, and the MKP VHP-1(MKP7). P determined by Fisher's exact test. *P < 0.05, **P < 0.01, ***P < 0.001, NS not significant. Error bars indicate 95% confidence interval. GC, growth cone. HS, heat shock. Ex[Phsp-16.2:dlk-1], dlk-1 transgene under the control of a heat shock promoter and expressed as an extrachromosomal array.