Labeled cells in the cerebellar cortex by IUE of transposon plasmids.

<p>(A) Low magnification view of a sagittal section of a Tol2-GFP electroporated cerebellum. (B) A basket cell extending its axon branches towards the Purkinje cell layer (lower left). (C) A stellate cell in the upper ML extending ramified processes. Black dots in the insets in B and C show the location of the respective cell bodies. (D) A granule cell with radiating dendrites (arrow) and an axon extending towards the ML (arrowheads). (E,F) Numerous parallel fibers occupy the ML. F is a high-magnification view of the boxed region in E. (G) A UBC in the IGL. (H) A Bergmann glia. (I) An astrocyte in the IGL. (J) An oligodendrocyte in the WM. (K) A large-diameter DN neuron (arrowhead). (L) A small-diameter DN neuron. (M1, N1, O1, P1) Maximum projection images for each series of confocal images of each cell. (M2–M4, N2–N4, O2–O4, P2–P4) Confocal plane images. (M1–M4) A Tol2-GFP labeled cell in the upper part of the ML shows the morphology of stellate cells. Double-labeling immunohistochemistry for parvalbumin (magenta) and GFP (green) confirmed the stellate cell identity. (N1–N4) A Tol2-GFP labeled cell in the lower part of the ML shows the morphology of basket cells. Double-labeling immunohistochemistry for parvalbumin (magenta) and GFP (green) confirmed the basket cell identity. (O1–O4) A Tol2-GFP labeled cell in the IGL shows the morphology of astrocytes. Double-labeling immunohistochemistry for GFAP (magenta) and GFP (green) confirmed the astrocyte identity. (P1–P4) A Tol2-GFP labeled cell in the white matter shows the morphology of oligodendrocytes. Double-labeling immunohistochemistry for MBP (magenta) and GFP (green) confirmed the oligodendrocyte identity. Scale bars: 10 µm ML, molecular layer; PCL, Purkinje cell layer; IGL, internal granular layer. Scale bars: A, 1 mm; B, C, E, F, K, L, M1, N1, O1, and P1, 10 µm; in G, 10 µm for D and G; in J, 10 µm for H, I, and J.</p

Schwann cells instruct axonal regrowth.

(A) Pre-transection maximum projection of axons labeled with Tubb:dsRed and Schwann cells labeled with 37a> EGFP. Boxed inset outlines the region shown in B. DP labels the dorsal plexus. (B) Timelapse imaging captures pioneer axons (arrows) navigating a choice point. Axons remain tightly associated with Schwann cell membranes. (C) Maximum projections of abductor-specific innervation labeled with zCrest:GFP in animals that also express 37a> NTR-tagRFP before injury and at 2 dpt. The addition of ronidazole ablated Schwann cells. (D) Maximum projection and traces of mistargeted zCrest:GFP-labeled axons on the adductor muscle in 37a> NTR-tagRFP animals treated with ronidazole or control. (E) Quantification of total amount of mistargeted zCrest:GFP+ axon growth on adductor muscle. Data points labeled with hollow circles denote the examples shown in D. Data are represented as mean ± SD. *p S1 Data. DP, dorsal plexus; dpt, day post transection; GFP, green fluorescent protein.</p

Summary of cells in the DN labeled at each stage of electroporation.

<p>All sagittally sectioned samples in which both VZ- and uRL-derived cells were labeled were analyzed. Coronally sectioned samples were excluded from this table because it was difficult to systematically identify the labeled cells from their morphology in the coronal planes. mCherry, pCAGGS-mCherry; Tol2-GFP, a mixture of pCAGGS-T2TP and pT2K-CAGGS-EGFP; Cre*, pCAGGS-Cre was introduced into Ai9 mouse embryos. Number of samples from Tol2-GFP-introduced mice is three for each stage of electroporation. Number of samples from Cre introduced Ai9 mice is as follows: n = 1 (electroporated at E10.5), n = 2 (electroporated at E11.5), n = 3 (electroporated at E12.5). +, ++: more than 4 cells were labeled in all samples; −: no cells were labeled in all samples; +/−: more than 4 cells were labeled in subsets of all samples. NA: not available.</p

Functional recovery of pectoral fin movements.

High-speed movies of pectoral fin movements were captured at 250 frames per second, but are slowed down 25× for this movie. The same fish was recorded for multiple time points. Prior to axon transection, both pectoral fins move spontaneously. Nerves that innervate the right fin were transected while the left fin remained intact. At 5 h and at 1 day post transection, the right fin does not move. However, at 2 days post transection, the right fin has regained movement. (MP4)</p

Schwann cells organize axonal regrowth through the plexus.

Timelapse imaging during regeneration of zCrest:GFP to label abductor-specific axons. Schwann cells express 37a> NTR-tagRFP. The timelapse begins approximately 15 hpt with 30-min intervals between frames, showing a maximum projection of zCrest:GFP+ axons growing through and near the dorsal plexus during regeneration. In the control, axons are fasciculated to form the “X” shape of the dorsal plexus and extend orderly projections into the fin musculature. The orange arrow points to abductor axons while the pink arrow points to adductor axons. When Schwann cells are ablated after adding ronidazole, axon growth is disorganized and axons are defasciculated. (MP4)</p

Mistargeted axons are selectively retracted.

zCrest:GFP labels axons that project only to the abductor muscle prior to axon injury, whereas Tubb:dsRed labels all axons. The timelapse begins at 10 hpt with 15-min intervals. In the first part, axon growth onto the adductor muscle is shown. Here, zCrest:GFP+ axons are incorrectly targeted to the adductor muscle and some of these axons are retracted while correctly targeted Tubb:dsRed+ axons remain. In the second part, axon growth onto the abductor muscle of the same fin is shown. Correctly targeted zCrest:GFP+ axons grow robustly. (MP4)</p

Differentiated Schwann cells organize axons at the plexus.

(A) Maximum projections of abductor muscle innervation labeled with Tubb:dsRed in sibling and sox10cls mutant larvae. The same fin is shown pre-transection, at 10 hpt (axon degeneration), and at 2 dpt (axon regeneration). (B) sox10 mutants display a subtle increase in axon defasciculation, as measured by a higher number of individual axon fascicles quantified dorsal to the dorsal plexus, prior to axon transection that increases after axon regeneration. (C) Maximum projection through the plexus region before transection and at 2 dpt. sox10 mutants display a disordered plexus region that worsens after injury. (D) sox10 mutants more frequently display severely disorganized axon patterning at the plexus at 2 dpt; ** p (TIF)</p

Target-selective regeneration of motor axons.

(A) Schematic of a lateral view of pectoral fin abductor muscle motor innervation. An example trajectory of a single axon is shown in magenta. DP labels dorsal plexus, motor nerves in the body wall are labeled 1–4. (B, C) A timecourse of sparsely labeled axon(s) showing the innervation pattern pre-transection, after axon degeneration, and at 2 dpt. Sparsely labeled axons are labeled in white (B) and magenta (C) and all adductor motor axons are labeled in green (C). The white arrow points to a fascicle that did not degenerate and the orange arrow points to ectopic growth during regeneration. (D) Quantification of the muscle localization of sparsely labeled axons pre-transection compared to where these labeled axons innervated after regeneration. Small numbers on pre-transection data represent n. Diagonal lines indicate the axon mistargeted during regeneration. (E) Schematic defining domains for axon domain scoring. For category 3, which are the minority, axons entered the fin at the DP but innervated the ventral region of the fin, overlapping with domain 4 (like the sparsely labeled axon in A). The domains used to quantify sparsely labeled axon targeting are a broader categorization than the topographic territories schematized in Fig 1A. (F) Quantification of fin domain localization of sparsely labeled axons pre-transection compared to where these labeled axons innervated after regeneration. Small numbers on pre-transection data represent n. (G) Example of sparsely labeled axons (magenta) that form new trajectories and reestablish previous trajectories during regeneration with both motor axons (mnx1:GFP) and muscle fibers (α-actin:GFP) labeled. (H) The original (green) and regenerated (orange) trajectories of the sparsely labeled axons in G. Here, part of the pre-injury and regenerated trajectory can be overlayed precisely (arrows). Additionally, an axon does not follow its original trajectory (double arrows) but instead is mistargeted along the base of the fin (triangle). Original data for panels D and F are in S1 Data. DP, dorsal plexus; dpt, day post transection; GFP, green fluorescent protein.</p

Schwann cells organize axon regeneration through the plexus.

(A) Maximum projection of the DP of zCrest:GFP to label abductor-specific axons at 2 dpt from 2 control animals. Schwann cells also express 37a> NTR-tagRFP. Nerves 1/2 and 3, the DP, and the abductor (Ab) and adductor (Ad) innervation are labeled. Both were categorized as “none/mild” plexus disorder. (B) Two examples of plexus disorganization category “severe” in animals with Schwann cells ablated after adding ronidazole. (C) Categorization of plexus organization in regenerated fins. Numbers indicate n. NTR = nitroreductase, Ron = ronidazole. (D, E) Timelapse imaging during regeneration of zCrest:GFP. (D) In the control, the plexus is organized with fascicles correctly targeting the abductor muscle (green) and incorrectly targeting the adductor muscle (magenta). Axons extend orderly projections into the fin musculature; n = 4/5 (E). When Schwann cells are ablated after adding ronidazole, axon growth through the plexus is disorganized and axons are defasciculated; n = 4/4. Scale bars are 25 microns (D, E) and 10 microns (A). Original data for panel C is in S1 Data. DP, dorsal plexus; dpt, day post transection; GFP, green fluorescent protein.</p

Motor axons regenerate to their original muscle fibers.

(A) Maximum projection prior to axon transection of abductor muscle in Tg(α-actin:GFP);Tg(mnx1:GFP) larvae showing muscles and motor axons (green motor axons are faint in this example) with a single axon trajectory in magenta. (B) After 2 days, this labeled regenerated axon has reoccupied its original muscle fibers but has formed unique branches. Insets are expanded in A’ and B’ with individual muscle fibers labeled with the dotted lines to compare the axon location. The single labeled axon is shown in A” and B”. (TIF)</p