A spared synaptic branch restrains Wnd-dependent injury signaling in SNc motoneurons.

A) Schematic representation of the two SNc motoneurons innervating muscles 26 and 29 (MNSNc-26/29, red) and muscle 27 (MNSNc-27, blue), which are labeled by expression of the m12-Gal4 driver.

B) Example images of NMJ terminals from m12-Gal4/+; UAS-BitBow2 (Li et al., 2021) /+ third instar larvae, used to define the connectivity shown in A. The neuron that innervates muscle 27 (MNSNc-27) expresses a distinct set of colors from the Bitbow2 (Li et al., 2021) reporter than the neuron that innervates muscles 26 and 29 (MNSNc-26/29).

C) Confirmation of puc-lacZ induction following laser axotomy. The cartoons on the top row show the location used to injure both axons; this location removes all of the synaptic branches from both MNSNc-26/29 and MNSNc-27. The middle row shows example injuries (versus uninjured, right) at the indicated location in m12-Gal4, UAS-mCD8GFP/puc-lacZ larvae. The bottom row shows examples of puc-lacZ expression (red channel) in the MNSNc cell bodies 24h following injury.

D) Example MNSNc-26/29 (blue) and MNSNc-27 (red) neurons injured at different locations.

E) Quantification of puc-lacZ intensity measurements in MNSNc-26/29 (blue) and MNSNc-27(red) following injuries that remove all synaptic branches versus injuries that leave a spared synaptic branch. Injury location (a) removes the small number of boutons on muscle 29 while sparing the boutons on muscle 26. Injury location (b) removes boutons from muscle 29 and the posterior sub-branch on muscle 26. Injury location (c) removes all branches except for the small number of boutons on muscle 29. Note that all injuries that leave spared boutons (hatched shading) show no puc-lacZ induction, regardless of the number of boutons lost or spared. A one-way ANOVA with Tukey test for multiple comparisons was performed for each neuron. **** p < 0.0001; ** p = 0.0011; ns = not significant.t.

Restraint of Wnd-mediated injury signaling by spared branch in bifurcated neurons.

A) Cartoon of Ventral Unpaired Motoneurons (VUM), which have bifurcated axons that symmetrically innervate body wall muscles on both the left and right sides of the animal. Nerve crush to either left or right side of the animal can axotomize a single bifurcation while leaving the other bifurcated axon intact.

B) Example images of VUM axons (visualized in Tdc2-Gal4, UAS-mCD8-GFP larvae) in segmental nerves on the uninjured and injured sides following nerve crush to a single side.

C) Example images of puc-lacZ expression in the VNC (ventral nerve cord) of larvae following nerve crush to a single side (half crush) versus crush to all the segmental nerves (full crush). puc-lacZ expression (red) is induced in VUM neurons (white) only after full crush. In contrast, other motoneurons, which innervate a single side, are induced by both half and full crush injuries. Co-expression of UAS-wnd-RNAi in VUM neurons cell autonomously inhibits puc-lacZ induction.

D) Quantification of puc-lacZ intensity measurements in VUM neurons. A one-way ANOVA with Tukey test for multiple comparisons was performed. **** p < 0.0001; ns = not signifcant. Scale bars = 20 um.

Presence of spared synaptic branch restrains Wnd signaling independently of Hiw.

A) Laser axotomy is carried out to MNSNc neurons at a location that completely removes the synaptic terminal of MNSNc-27 (red neuron). The injury also leads to loss of the MNSNc-26/29 (blue) terminal on muscle 29 but not 26 (hence leaves a spared synaptic branch. The final column shows an axotomy that fully removes the terminals for both MNSNc neurons. These injuries were repeated in control animals versus the background of a hiw null mutant, hiwΔN.

B) Quantification of puc-lacZ expression for individual MNSNc neurons after full versus spared axotomies, compared to uninjured neurons. Basal puc-lacZ expression is already elevated in uninjured hiwΔN neurons compared to control. This can be further elevated in axotomies that remove all synapses, but not in axotomies that leave spared branches.

C) Quantification of puc-lacZ in VUM neurons (labeled by Tdc-2-Gal4; UAS-mCD8-GFP) 24h and 48h following full nerve crush in control versus hiwΔN mutants. A two-way ANOVA with Tukey test for multiple comparisons was performed. **** p < 0.0001; *** p < 0.001; **p<0.01; ns = not significant.

Potential mechanisms for regulation of Wnd signaling from synaptic terminals.

In green, Wnd signaling may be regulated in the cell body downstream of a retrogradely transported signal. (For example, neurotrophin signaling). In blue, Wnd signaling activation is restrained locally at synaptic terminals, perhaps by regulating the levels or activation of Wnd itself. Activated Wnd or a downstream signaling factor is then retrogradely transported to the cell body. Previous observations that inhibition of retrograde transport blocks the induction of Wnd signaling following axonal injury favors the latter (blue) possibility. However the restraint conferred by a physically separate bifurcation suggests that an inhibitor of Wnd signaling activation can be retrogradely transported (green). We speculate that both mechanisms act as dual checkpoints to restrain Wnd signaling activation in the context of healthy circuits.