Axonal injury signaling is restrained by a spared synaptic branch
- Department of Molecular, Cellular and Developmental Biology, University of Michigan, United States
- Department of Neurosciences, Case Western Reserve University, United States
- Department of Cell and Developmental Biology, University of Michigan, United States
Figures
Figure 1 with 1 supplement
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 24 hr 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 < 0.001; **p < 0.01; ns = not significant.
Figure 1—figure supplement 1
Further characterization of branched injury assays to SNc and aCC motoneurons.
(A) Cartoon denoting nomenclature of MNSNc-26/29 (red) and MNSNc-27 (blue) branches. (B) Example images of nerve terminals and cell bodies of m12-Gal4; UAS-Bitbow2 labeled MNSNc neurons, used to confirm the anatomy. (C) Similarly to laser axotomy in Figure 1C, nerve crush injury (24 hr) induces puc-lacZ expression in both MNSNc neurons, but not in MNSNc neurons that co-express wnd-RNAi. (D) Synaptic terminals (top row) and cell bodies (bottom row) of aCC motoneurons on muscle 1, labeled in Dpr4-Gal4, UAS-mCD8-GFP larvae. puc-lacZ expression (red) is induced following injuries that result in loss of all synaptic boutons but not following injuries to one branch that leave the other branch intact. (E) Quantification of puc-lacZ intensities in aCC neurons. A one-way ANOVA with Tukey test for multiple comparisons was performed. ****p < 0.0001; ns = not significant. Full (F) but not partial (G) removal of synaptic branches induces stability and trafficking ectopically expressed kinase-dead GFP-Wnd-KD (in UAS-GFP-Wnd-KD; m12-Gal4, UAS-mCD8-RFP animals). Synaptic branches from muscle 27 (F), or muscle 29 (G) were axotomized by laser surgery and imaged following 24 hr. The white asterisk (*) marks the injury location. (F) GFP-Wnd-KD protein accumulates at the proximal tip of axons that have lost all synaptic boutons. (G) GFP-Wnd-KD is barely detectable in axons following injuries that leave spared synaptic branches. Not shown, GFP-Wnd-KD levels in G are similar in uninjured MNSNc axons.
Figure 2 with 1 supplement
Restraint of Wnd-mediated injury signaling by spared branch in bifurcated neurons.
(A) Cartoon of ventral unpaired median (VUM) neurons, 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 significant. Scale bars = 20 µm.
Figure 2—figure supplement 1
Anatomy and laser surgery of Tdc2 bifurcated neurons.
(A–E) Views of Tdc2-Gal4, UAS-mCD8-GFP expressing VUM neurons to illustrate their anatomy. (A) Individual confocal plane that shows three cell bodies in individual segments, which lie in the middle of the nerve cord. (B) Side view and (C) top view that show the locations of the bifurcations. (D) Cartoon of the three neurons from one segment, showing their bifurcations to symmetrical sides of the animal. (E) Composite and camera lucida views of the NMJ terminals for the three neurons on one abdominal hemisegment. The three Tdc2 neurons each form stereotyped branches to innervate a unique group of muscles. (F) Example results from laser axotomies to individual Tdc2/VUM neurons (24 hr after injury) on either one side or both sides of the animal. Surgeries were carried out at the indicated locations which lie upstream of the final synaptic branches (at the transition zone between the segmental nerve and abdominal muscles). The stereotyped anatomy allows for identification of each VUM neuron (labeled 1, 2, and 3). Injured branches are marked with an asterisk while spared branches are marked with squares. For each neuron, only injuries to both bifurcations allowed for induction of puc-lacZ. Scale bars = 20 um.
Figure 3 with 1 supplement
Presence of spared synaptic branch restrains Wnd signaling independently of Hiw.
(A) Laser axotomy is carried out to MNSNc neurons at a location (indicated by asterisk (*)) 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) 24 and 48 hr 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.
Figure 3—figure supplement 1
Confirmation that elevated puc-lacZ expression inhiw mutants requires Wnd.
(A) Top row shows puc-lacZ expression (red) together with a marker for nuclei (Draq5, gray). Control (lexA)-RNAi or wnd-RNAi UAS lines are driven by the BG380Gal4 driver in the background of control versus hiwΔN. Bottom row shows example NMJs in these genotypes. Scale bars = 20 µm. (B) Quantification of puc-lacZ expression in A. A one-way ANOVA with Tukey test for multiple comparisons was performed. ****p < 0.0001; ns = not significant.
Figure 4
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 (e.g., 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 favor 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.
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Gene (Drosophila melanogaster) | wnd (wallenda) | Flybase | FBgn0036896 | |
| Gene (Drosophila melanogaster) | puc (puckered) | Flybase | FBgn0243512 | |
| Gene (Drosophila melanogaster) | hiw (highwire) | Flybase | FBgn0030600 | |
| Genetic reagent (Drosophila melanogaster) | UAS-mCD8-GFP | Bloomington Drosophila Stock Center (BDSC) | RRID:BDSC_5137 | Lee and Luo, 1999 |
| Genetic reagent (Drosophila melanogaster) | m12-gal4 (P(Gal4)5053A) | Bloomington Drosophila Stock Center (BDSC) | RRID:BDSC_2702 | Ritzenthaler et al., 2000 |
| Genetic reagent (Drosophila melanogaster) | BG380-Gal4 | Bloomington Drosophila Stock Center (BDSC) | RRID:BDSC_42736 | Budnik et al., 1996; Sanyal, 2009 |
| Genetic reagent (Drosophila melanogaster) | puc-lacZ[E69] | Bloomington Drosophila Stock Center (BDSC) | RRID:BDSC_98329 | Martin-Blanco et al., 1998 |
| Genetic reagent (Drosophila melanogaster) | puc-GFP | Melissa Rolls | Rao and Rolls, 2017 | |
| Genetic reagent (Drosophila melanogaster) | hiwΔN | Bloomington Drosophila Stock Center (BDSC) | RRID:BDSC_51637 | Wu et al., 2005 |
| Genetic reagent (Drosophila melanogaster) | UAS-Bitbow2 | Dawen Cai | Li et al., 2021 | |
| Genetic reagent (Drosophila melanogaster) | Tdc2-Gal4 | Bloomington Drosophila Stock Center (BDSC) | RRID:BDSC_9313 | |
| Genetic reagent (Drosophila melanogaster) | UAS-wnd-RNAi | Bloomington Drosophila Stock Center (BDSC) | RRID:BDSC_35369 | |
| Genetic reagent (Drosophila melanogaster) | UAS-lexA -RNAi | Bloomington Drosophila Stock Center (BDSC) | RRID:BDSC_67947 | |
| Genetic reagent (Drosophila melanogaster) | Additional Drosophila lines are summarized in Supplementary file 1 | |||
| Antibody | Mouse monoclonal anti-lacZ | DSHB Cat# 40-1a | RRID:AB_528100 | 1:100 dilution |
| Antibody | Rabbit polyclonal anti-DsRed | Takara Bio Cat# 632496 | RRID:AB_10013483 | 1:1000 dilution |
| Antibody | A488 rabbit polyclonal anti-GFP | Molecular Probes Cat# A-21311 | RRID:AB_221477 | 1:1000 dilution |
| Antibody | AlexFluor 568 goat polyclonal anti-mouse | Thermo Fisher, A11004 | RRID:AB_2534072 | 1:1000 dilution |
| Antibody | AlexFluor 488 goat polyclonal anti-mouse | Thermo Fisher A32723 | RRID:AB_2633275 | 1:1000 dilution |
| Antibody | AlexFluor 568 goat polyclonal anti-rabbit | Thermo Fisher A-11011 | RRID:AB_143157 | 1:1000 dilution |
| Chemical compound, drug | Paraformaldehyde Aqueous Solution EM Grade | Electron Microscopy Sciences | Cat #15710 | 16% aqueous solution diluted to 4% in PBS. Used within 1 week of dilution. |
| Biological sample (goat) | Normal goat serum (NGS) | Fisher Scientific | Cat #16210064 | Diluted to 5% in PBS |
| Chemical compound, drug | Prolong Diamond Antifade media | Thermo Fisher Scientific, P36970 | Cat #P36970 | |
| Other | PDMS microfluidic chip for immobilizing larvae | MicroKosmos | ‘Mechanical Immobilization Chip' for Drosophila larvae | (specialized equipment) Larva chip is available for purchase from https://www.ukosmos.com |
| Other | Dumont #5 fine forceps | Roboz Surgical | Cat #RS4978 | (specialized equipment) Forceps for carrying out the peripheral nerve crush assay |
| Software, algorithm | Volocity software 6.2 | Improvision, PerkinElmer | ||
| Software, algorithm | GraphPad Prism | |||
Additional files
-
MDAR checklist
- https://cdn.elifesciences.org/articles/104896/elife-104896-mdarchecklist1-v1.pdf
-
Supplementary file 1
Summary of negative results from genetic manipulations that impair synaptic transmission and/or signaling at synapses.
- https://cdn.elifesciences.org/articles/104896/elife-104896-supp1-v1.docx
Download links
A two-part list of links to download the article, or parts of the article, in various formats.
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
Axonal injury signaling is restrained by a spared synaptic branch
eLife 13:RP104896.
https://doi.org/10.7554/eLife.104896.3
