Wnt signaling controls pro-regenerative Collagen XII in functional spinal cord regeneration in zebrafish

The inhibitory extracellular matrix in a spinal lesion site is a major impediment to axonal regeneration in mammals. In contrast, the extracellular matrix in zebrafish allows substantial axon re-growth, leading to recovery of movement. However, little is known about regulation and composition of the growth-promoting extracellular matrix. Here we demonstrate that activity of the Wnt/β-catenin pathway in fibroblast-like cells in the lesion site is pivotal for axon re-growth and functional recovery. Wnt/β-catenin signaling induces expression of col12a1a/b and deposition of Collagen XII, which is necessary for axons to actively navigate the non-neural lesion site environment. Overexpression of col12a1a rescues the effects of Wnt/β-catenin pathway inhibition and is sufficient to accelerate regeneration. We demonstrate that in a vertebrate of high regenerative capacity, Wnt/β-catenin signaling controls the composition of the lesion site extracellular matrix and we identify Collagen XII as a promoter of axonal regeneration. These findings imply that the Wnt/β-catenin pathway and Collagen XII may be targets for extracellular matrix manipulations in non-regenerating species.

(c) 6xTCF:dGFP Wnt reporter activity in the spinal lesion site is detectable 12 hours (0.5 dpl) after a stab lesion or dorsal incision lesion (arrows). Note that larvae were treated with PTU to suppress pigmentation. (d) Detection of gfp mRNA in the 6xTCF:dGFP transgenic reporter line shows transient activity of the Wnt/β-catenin pathway in the lesion site during regeneration, analysed in stab-lesioned animals. (e) Representative images of a dorsal incision lesion or less invasive stab lesion taken immediately after lesion. Note that stab lesions do not compromise the dorsal edge of the animal, resulting in reduced injury size as compared to dorsal incision lesions. Arrowhead points to the lesion site. (f) 6xTCF:dGFP Wnt reporter activity in the lesion site is strongly reduced upon IWR-1 treatment for 12 hours, indicating specificity of the transgenic Wnt reporter. (g) 6xTCF:dGFP Wnt reporter activity in the lesion site is strongly reduced upon heat shock-induced axin1 overexpression for 6 hours in 6xTCF:dGFP;hs:Axin1 double transgenic animals, indicating specificity of the transgenic Wnt reporter. (h) GFP protein is largely confined to the lesion site in 6xTCF:dGFP transgenic animals at 1 dpl and 2 dpl. A few additional cells are labelled within the presumptive spinal cord region in the periphery of the lesion site (empty arrows). Maximum intensity projections and a single optical section through the center of a whole mount larvae are shown. (i) Anti-GFP immunohistochemistry on sections of 6xTCF:dGFP transgenic animals confirms labelling in whole mount preparations. 6xTCF:dGFP Wnt reporter activity at 2 dpl is largely confined to the lesion site. In the peripheral lesion area limited Wnt reporter activity is also detected in the spinal cord (empty arrows). (j) Detection of gfp mRNA by in situ hybridization in 5 day-old her4.3:EGFP transgenic zebrafish, labelling ependymoradial glia cells, reveals efficient probe penetration in whole mount preparations. (a-j) Views are lateral (a-h, j; dorsal is up, rostral is left) or transversal (i; dorsal is up). BF: brightfield. Scale bars: whole mounts, 200 µm (j,e) and 100 µm (a-d, f-h); sections, 100 µm.
Supplementary Figure 3 | Wnt/β-catenin pathway is upregulated in basal keratinocytes and fibroblast-like cells but not muscle or immune cells after a lesion.
(a-b) Subepidermal 6xTCF:dGFP + /p63 − cells (arrows) do not co-label with MyHC antibodies, a marker for slow twitch muscle cells. Arrowheads indicate 6xTCF:dGFP + /p63 + basal keratinocytes. Note that close to the disorganized lesion center some subepidermal 6xTCF:dGFP + /p63 − cells are found sandwiched between p63 + basal keratinocytes and MyHC + slow twitch muscle fibers (insets), which represents a typical dermal fibroblast location in zebrafish larvae. (c) 6xTCF:dGFP + cells do not co-label with L-Plastin, a marker for innate immune cells.     (a) Anti-Col XII immunoreactivity is increased in the lesion site (arrows) at 1 dpl and 2 dpl compared to adjacent unlesioned tissue and to unlesioned animals at the same trunk position. (b) Inhibition of Wnt/β-catenin signaling via heat shock-induced systemic overexpression of the pathway antagonist dkk1 interferes with Col XII deposition in the lesion site, as determined by quantification of lesion site immunoreactivity (t-test: **P<0.01). (c) Double immunolabelling of axons (anti-acetylated Tubulin + ) and Col XII shows little to no Col XII deposition in the lesion site at 0.5 dpl (12 hpl) and axon are yet to enter the lesion site (asterisk). At 1 dpl, anti-Col XII immunoreactivity is markedly increased in the lesion site and axon have entered the Col XII-rich lesion site (arrow). Confocal depth was limited to spinal cord. Note, that the 1 dpl dataset is the same as presented in Fig. 4f.  A minimum intensity threshold is applied to the raw image (a-b), limiting pixels to only those of equal or higher intensity (c). After thresholding, a ROI is defined manually in which the thresholded pixel area is measured (d). The analysis is done without knowledge of the experimental condition.

Supplementary Notes
Supplementary Note 1 | Supplemental information on transgenic zebrafish lines used.

Detection of Wnt/β-catenin pathway activity 6xTCF/Lef-miniP:2dGFP (abbreviated as 6xTCF:dGFP)
Reporter of β-catenin-dependent transcription. Destabilized EGFP under the control of a pGL4 minimal promoter plus 6 consensus Tcf/Lef binding sites. Previously shown to reliably report Wnt/β-catenin signaling during zebrafish development and adult zebrafish tail fin regeneration 1,2 . In this study lesion-induced 6xTCF:dGFP reporter activity is specific for Wnt/β-catenin signaling because reporter activity is suppressed after overexpression of the pathway antagonist axin1 for 6 hours or after 12 hours of treatment with the pharmacological pathway inhibitor IWR-1 (see Supplementary Fig. 2f-g).

Top:dGFP
Reporter of β-catenin-dependent transcription. Destabilized GFP under the control of the TOPFLASH promoter containing 4 consensus Tcf/Lef binding sites. Previously shown to reliably report Wnt/β-catenin signaling during zebrafish development and caudal fin regeneration 2,4,5 . Astroglia-like cell-specific TetActivator line. Tetracycline-inducible transcriptional activator tagged with p2a and AmCyan under the control of regulatory sequences of the her4.3 gene known to be selectively expressed in astroglia-like cells 9 .

TetRE:Mmu.Axin1-YFP (abbreviated as TetRE:Axin1-YFP)
Mouse axin1 (cytoplasmic antagonist of Wnt/β-catenin signaling) lacking the N-terminal RGS domain fused at the C-terminus to YFP under the control of a tetracycline response element. Potently inhibits Wnt/β-catenin signaling during zebrafish development or adult zebrafish tail fin regeneration when induced via a Tet Activator expressing transgene 2,14 .