Fig. 4

Wld^S potently delays degeneration and requires Nmnat enzymatic activity. (A) Schematic of the transgene used to drive expression of Wld^S and GFP simultaneously in sensory neurons with the CREST3 enhancer. (B-E) Confocal time-lapse series of a trigeminal neuron expressing Wld^S. Image in B was collected before axotomy at 30 hpf. (C) 0.5 hours post-axotomy, arrow indicates site of axotomy. (D,E) The Wld^S-expressing fragment 22 hours and 48 hours post-axotomy, respectively. Arrowheads indicate the persisting severed fragment (see Movie 4 in the supplementary material). Scale bar: 50 μm. (F) Quantification of the lag phase in axons expressing Wld^S, Wld^S with a mutation compromising Nmnat enzymatic activity (Wld^S*) and wild type at 54 hpf. Hash marks on Wld^S bar indicate that the lag phase lasted at least 12 hours, as all fragments persisted throughout the 12-hour time-lapse imaging sessions. The lag phase in Wld^S* was significantly shorter than in Wld^S, but still significantly longer than wild type (P<0.0001, one-way ANOVA) (ne12 for all groups). (G) Quantification of the lag and clearance phases in wild type and Wld^S*. Despite the lengthened lag phase in Wld^S* compared with wild type, the clearance phase occurs at a wild-type rate (P=0.1129). Error bars indicate s.e.m. (H) Axon expressing KikGR and Wld^S severed at ~30 hpf. UV illumination of the cell body (white arrow) converted KikGR protein from green to red, which spread throughout the arbor but not into the severed fragment (white arrowhead). The regenerating red parent arbor did not overlap with the green fragment.