Fig 1.
Dynein and Kinesin-1 are required for peripheral nerve regeneration in vivo.
(A) Wild type motor nerve pre-lesion (red box, transection site; scale bar = 20 μm). (B) By 48 hpt, several fascicles have regrown fully across the ventral myotome (green arrowheads, regrown axons, strong regeneration). (C) kif5aa-/- motor nerve pre-lesion. (D) At 48 hpt, some axons have extended across the myotome (blue arrowheads, regrown axons, moderate regeneration). (E) Quantification of kif5aa mutant regeneration at 48 hpt (wild type siblings, n = 66 nerves; kif5aa-/-, n = 20 nerves, p = 0.0487, Fisher’s exact test). (F) dync1h1-/- motor nerve pre-lesion. (G) By 48 hpt, regrowing axons have extended slightly but failed to reach the ventral extend of the myotome (red arrowheads, stalled axons, no/weak regeneration). (H) Quantification of dync1h1 mutant regeneration at 48 hpt (dync1h1+/+, n = 59 nerves; dync1h1+/-, n = 21 nerves; dync1h1-/-, n = 25 nerves; p = 0.007; p = 0.0006; p<0.0001, respectively, Fisher’s exact test).
Fig 2.
Dynein is required for injury-induced Schwann cell morphology changes.
(A-F) Schwann cells in 5 dpf larvae labeled by Tg(sox10:mRFP). (A) Prior to injury, wild type Schwann cells have smooth, straight membranes that are tightly associated with the axonal track (scale bar = 5 μm). (B) After axonal fragmentation, Schwann cell membranes change morphology and widen to accommodate axonal debris. (C) Prior to injury, gpr126-/- Schwann cells are loosely associated with axons as they do not myelinate. (D) After axonal fragmentation, gpr126-/- Schwann cells are able to change morphology and widen. (E) Prior to injury, dync1h1-/- Schwann cells are loosely associated with axons as they also do not myelinate. (F) After axonal fragmentation, dync1h1-/- Schwann cell membranes maintain an elongated conformation and do not dramatically change morphology, indicating a disrupted injury response. (G) Quantification of Schwann cell width pre- and post-fragmentation in gpr126 and dync1h1 mutants.
Fig 3.
Neuronal dynein is sufficient to promote axonal regrowth.
(A) ~10 rhodamine-labeled cells were transplanted from wild type blastulas to dync1h1-/- blastulas. (B) At 5 dpf, nerves contained wild type neurons (transplanted cells labeled by rhodamine-dextran, magenta) in a dync1h1-/- larva (host motor neurons labeled by Tg(mnx1:GFP), green; scale bar = 10 μm). (C-E) After transection, wild type axons (magenta arrowheads) are able to regrow robustly in the dync1h1-/- embryo, while dync1h1-/- host axons regrow significantly less (green arrowheads; scale bar = 10 μm). (F) Quantification of growth cone displacement in dync1h1-/- host axons and transplanted wild type axons. Open circles indicate dync1h1-/- mutant axons that grew along transplanted wild type axons. (G-I) Some dync1h1-/- axons demonstrated improved regeneration in the presence of wild type axons in the same nerve. Here, a dync1h1-/- axon (green arrowheads) follows along a previously regrown wild type axon (magenta arrowheads; scale bar = 5 μm).
Fig 4.
Dynein stabilizes axonal extensions during regeneration.
(A-B) In wild type animals, regenerating axons begin probing the environment by extending and retracting (green and red arrowheads, respectively; scale bar = 5 μm). (C-D) dync1h1-/- axons also extend and retract after injury. (E) Quantification of extension and retraction events in wild type siblings (n = 13 axons) and dync1h1-/- axons (n = 13 axons). (F-G) Measurement of overall growth cone displacement from transection site ~16 hpt in wild type siblings (F; blue arrowheads, growth cones; scale bar = 10 μm) and dync1h1-/- (G; red arrowheads, growth cones). (H) Quantification of growth cone displacement ~16 hpt (wild type siblings, n = 15; dync1h1-/-, n = 10; p = 0.0005, unpaired t-test).
Fig 5.
Dynein stabilizes microtubules to promote persistent regrowth.
(A-D) Regenerating wild type axons first extend actin protrusions then extended microtubules, leading to stable growth (scale bar = 5 μm; green arrowheads, actin; magenta arrowheads, microtubules). (E-H) dync1h1-/- axons extend actin protrusions followed by microtubule growth that arrests during growth cone engorgement and leads to axon retraction (G,H). (I-L) dync1h1-/- axons extend actin protrusions but microtubules form aberrant loop structures (magenta arrowheads), preventing further regrowth. (M) Quantification of microtubule organization in regrowing axons of dync1h1 mutants (siblings, n = 19 axons; dync1h1-/-, n = 37 axons; p<0.0001, Fisher’s exact test). (N) Quantification of growth cone displacement ~12 hpt with and without taxol treatment (dync1h1-/- with DMSO, n = 8, dync1h1-/- with 5 μM taxol, n = 12, p = 0.0159; wild type siblings with DMSO, n = 4, wild type siblings with 5 μM taxol, n = 4, p = 0.0571).