Denervation impairs regeneration of amputated zebrafish fins

Abstract

Background: Zebrafish are able to regenerate many of its tissues and organs after damage. In amphibians this process is regulated by nerve fibres present at the site of injury, which have been proposed to release factors into the amputated limbs/fins, promoting and sustaining the proliferation of blastemal cells. Although some candidate factors have been proposed to mediate the nerve dependency of regeneration, the molecular mechanisms involved in this process remain unclear.

Results: We have used zebrafish as a model system to address the role of nerve fibres in fin regeneration. We have developed a protocol for pectoral fin denervation followed by amputation and analysed the regenerative process under this experimental conditions. Upon denervation fins were able to close the wound and form a wound epidermis, but could not establish a functional apical epithelial cap, with a posterior failure of blastema formation and outgrowth, and the accumulation of several defects. The expression patterns of genes known to be key players during fin regeneration were altered upon denervation, suggesting that nerves can contribute to the regulation of the Fgf, Wnt and Shh pathways during zebrafish fin regeneration.

Conclusions: Our results demonstrate that proper innervation of the zebrafish pectoral fin is essential for a successful regenerative process, and establish this organism as a useful model to understand the molecular and cellular mechanisms of nerve dependence, during vertebrate regeneration.

Figure 1

Adult zebrafish pectoral fin denervation assay. a) Pectoral fin innervation. The zebrafish pectoral fin is innervated by both sensory and motor nerves that descend from the spinal cord (SC) and enter the pectoral fin region medially, as a combined brachial plexus (BP) (*). Sensory and motor axons then branch to serve the pectoral muscles and fin rays. Sensorial nerves run both along the intra and inter-ray regions (adapted from [38,39]). b) Denervation assay. The right pectoral fin was denervated (DEN) by transecting the nerve fibres in the brachial plexus region, while the left fin served as an innervated control (CTRL). In the next day, the right fin was re-denervated to assure total nerve degeneration. After 6–8 hours both fins were amputated and the discarded tissue (*) was collected for ac. α-tub staining. Fish were placed in 33°C water tanks and regeneration was allowed to proceed. Re-denervation took place every day after amputation, to avoid nerve recovery. Regenerates were collected for further analysis at specific time points post-amputation. c,d) Pectoral fin denervation efficiency. Staining for ac. α-tub in whole mount fins shows that nerve ablation at the level of the brachial plexus is efficient to deprive pectoral fins from its innervation. An innervated control fin (c,c’), with bundles of axons running in the inter and intra-ray region (c* - magnification of the boxed region in c), contrasts with a denervated fin (d,d’), with fewer or any presence of the axonal marker ac. α-tub, inside and outside bony rays (d*- magnification of the boxed region in d). The images are a projection of confocal optical slices. Scale bar - 100 μm.

Figure 2

Analysis of pectoral fin regeneration, upon denervation. a-l) Staining for ac. α-tub in whole mount fins confirms the absense of nerve fibres at the amputation site of denervated fins. Brightfield images demonstrate the difference in the extent of regeneration among fins (a’-l’). At 0.5 and 1 dpa denervated fins (b,d) have a WE that is thinner than controls (a,c). From 1.5 to 5 dpa, denervated fins are not able to form a normal blastema and regenerate (f,h,j,l). In some cases the blastema is completely absent (h’,l’), while in others a smaller and defective blastema is formed and fins regenerate partially (f’,j’). Red arrowed solid lines indicate regenerated tissue length. m-p) Defective denervated regenerating fins. Denervated fins regenerate defectively and form “merged blastemas” on adjacent rays. The “wavy” appearance of new control fins, where each ray has its own cone shaped blastema (m-bracket), contrasts with denervated fins, where apparently several rays share a single, merged blastema (obracket). At 9 dpa denervated fins with a similar extent of regeneration as the controls (n), present a defective patterning (p). q) Quantification of the area and length of regenerated tissue. Measurements taken from the amputation site to the most distal tip reveal a consistent significant reduction (***p < 0.0001, *p < 0.05) of denervated fins in relation to controls. r-t) Influence of nerve quantity in fin regeneration. Staining for ac. α-tub. in whole mount fins at 3 dpa show equal innervation and regeneration of control rays within the same fin (r), while denervated fins, the rays with less or no innervation present less or no regeneration (s,t), suggesting that the success of regeneration depends on the quanity of innervation for each ray. a-q and s-t) The images are a projection of confocal optical slices. Dashed lines mark amputation plane. Scale bar - 100 μm.

Figure 3

Width of regenerated fins, upon denervation. a-d) Brightfield images show that the inter-ray region of denervated fins is reduced in relation to controls. Additionally, “merged blastemas” (bracket) are observed on consecutive rays at 2 dpa (d), in contrast to single-ray blastemas found in control fins (c). Solid red arrowed lines indicate the inter-ray width. e) Quantification of inter-ray width. Measurements of rays and inter-ray units show that width of denervated fins is significantly smaller than that of the controls (***p < 0.0001, *p < 0.05). f-i) Apoptosis in whole mount amputated fins. Staining for activated caspase3 and ac. α-tub/p63 in whole mount fins activity reveals an increase in apoptotic cells in the WE and in the inter-ray region (arrowhead) of denervated fins, during the first day after amputation (g’). At 2 dpa, activated caspase-3 is expressed in the epidermis of both control (h’) and denervated and fins (i’). a-d, f-i) The images are a projection of confocal optical slices. Dashed lines mark amputation plane. Scale bar - 100 μm.

Figure 4

Analysis of cell cycle markers in mesenchymal cells, upon denervation. a-f) Staining for PCNA in whole mount fins shows equal expression in epidermal and mesenchymal cells of both control and denervated fins at 0.5 dpa (a,b). At 1 dpa, PCNA-positive cells start to accumulate at the level of amputation in the region that will give rise to the blastema in control fins (c - arrowhead), which is not observed in denervated fins (d). At 2 dpa control fins show an accumulation of PCNA-positive cells in the blastema region (e), while denervated fins resemble as 0.5 dpa fins (f). g-j) Live imaging with the Tg(Ef1α:mAG:zGem). At 1 dpa Geminin-positive cells are equally expressed in control (g) and denervated fins (h). GFP nuclei started to be evident in the mesenchyme above the amputation plane of control fins at 1.5 dpa (i), but not in the denervated ones (j). k-o) Staining for H3P in whole mount fins. H3P starts to be expressed at 1 dpa in some mesenchymal cells of control fins (k). At 2 dpa H3P-positive cells are present only in control fins (m) and in reduced number in partially regenerating denervated fins (o). Note: H3P has a non-specific label in distal epidermal cells, as previously reported [42]. a-o) The images are a projection of confocal optical slices. Dashed lines mark amputation plane. Scale bar - 50 μm. p) qRT-PCR formps1. Mps1 levels of expression increase in denervated fins at 1 dpa and decrease at 1.5 and 2 dpa, in relation to controls (***p < 0.0001, *p < 0.05).

Figure 5

Gene expression in the WE of control and denervated fins. a-x) mRNA ISH on whole mount amputated fins: lef1, wnt5b, pea3, krt8. a’-x’) Longitudinal sections of the rays using whole mount ISH. a-g) Lef1 is expressed from 0.5 to 1.5 dpa in the BEL of both control (a,c,e) and denervated fins (b,d,f,g). At 1.5 dpa lef1 is expressed in the inter-ray region of non-regenerating (f) and partially regenerating denervated fins, where it forms what seems a shared BEL on contiguous rays (g*). h-o) Wnt5b is expressed in the WE of both control (h,j,l,n) and denervated fins (i,k,m,o) from 0.5 to 4 dpa. [The arrowhead in h’ indicates expression, staining in the WE is an artefact]. In denervated fins, after 1.5 dpa, wnt5b presents a spread and de-regulated expression domain (m). p-s) Pea3 is expressed in both control and denervated fins at 0.5 and 1 dpa, with a reduced expression in denervated fins (q,s). In controls pea3 is expressed in the whole WE (p’,r’), while in denervates is restricted to the distal WE cells (q’,s’). t-x) Krt8 is expressed in both control (t,v) and denervated fins (u,x) at 0.5 and 1 dpa, with a reduced expression in denervates (u,x). While in controls krt8 is strongly expressed in the whole WE (t’,v’), in denervates it is restricted to some epidermal cells in the distal tip (u’,x’). a,a’-x,x’) Dashed lines mark amputation plane. Scale bar - 100 μm in whole mount; 25 μm in sections. y) qRT-PCR for genes expressed in the WE. qRT-PCR analysis shows a decrease in the expression levels of analysed genes on denervated fins, in relation to controls, from 0.5 to 2 dpa, except for wnt5b, which is increased at 0.5 dpa. Note: ***p < 0.0001, *p < 0.05. Data not evaluated for krt8 and fgf24, at 1.5 and 2 dpa.

Figure 6

Gene expression in the blastema of control and denervated fins. a-v) mRNA ISH on whole mount amputated fins: fgf20a, mkp3, msxb, msxc. a’-l’, p’-t’) Longitudinal sections of the rays using whole mount ISH. a-d) Fgf20a expression is detected at 0.5 and 1 dpa in control fins (a,c) and only a residual expression is detected in denervated fins (b,d). e-k) Mkp3 is detected at 1.5 dpa in control (e) and denervated fins (f). At 2 dpa the expression in denervated fins is reduced but stronger (h) than in controls (g). i-o) Msxb expression starts at 1 dpa in both control and denervated fins. At 1.5 dpa denervated rays that have partially regenerated (m) present msxb expression spread in the inter-ray tissue (*). msxb expression is still detected at 2 dpa, however, in denervated fins is reduced to a thin domain (o). p-v) Msxc expression is present in both fins at 1 dpa, but with a stronger expression in denervated fins (q,q’). Expression is maintained after 1.5 dpa in non-regenerating (s,s’) and partially regenerating fins (t,t’). At 2 dpa, msxc is expressed as a continuum in the tip of partially regenerating denervated fins, in what seems a “merged blastema” (Adult zebrafish pectoral fin denervation assay.,v’). a,a’-v,v’) Dashed lines mark amputation plane. Scale bar - 100 μm in whole mount fins; 25 μm in sections. x) qRT-PCR for genes expressed in the blastema. qRT-PCR analysis shows an increase in the expression of msxc and fgfr1 on denervated fins at 0.5 and 1 dpa, as well as in mkp3 at 1 and 2 dpa, when compared to controls. Fgf20a levels of expression are always decreased in relation to the controls, as it is msxb at 1.5 and 2 dpa. Note: ***p < 0.0001, *p < 0.05.

Figure 7

Scleroblasts alignment in amputated fins, upon denervation. a-d) Staining for Zns5 and DAPI in longitudinal sections shows that between 1 and 1.5 dpa, Zns5-positive cells start to accumulate just distal to the amputation plane in control fins (a-arrowhead). However, in denervated fins scleroblasts are not aligned with the stump rays, but instead are deposited between the 2 hemi-rays (b-arrowhead). At later time points the scleroblast deposition covers the tip of denervated rays (d). e,f) Live imaging with the Tg (oc:GFP) co-stained with Alizarin red-S (ARS). In vivo imaging of Tg (oc:GFP) stained with ARS shows that mature bone cells (oc-positive) of control fins are localized at the amputation level and in the blastema (e-arrowhead). However, in denervated fins oc-positive cells do not migrate further than the amputation plane (f-arrowhead). g,h) Live imaging with the transgenics Tg (oc:GFP), Tg (osx:mCherry) and ARS. Zebrafish transgenic line resulting from an outcross between Tg(oc:GFP) and Tg(osx:mCherry) shows that at 2 dpa only control fins present oc-positive and osx-positive cells in the blastema (g-arrowhead). a-h) The images are a projection of confocal optical slices. Dashed lines mark amputation plane. a-d) Scale bar - 25 μm. e-h) Scale bar - 100 μm.

Figure 8

Shh and ptc1 expression in control and denervated fins. a-h) mRNA ISH on whole mount amputated fins. Shh expression is first detected at 1.5 dpa in the blastema of control fins (a), but not in the denervated ones (b). Ptc1 mRNA starts to be expressed in the stump at 1 dpa both in control (e) and denervated fins (f). While in control fins ptc1 is expressed in every ray at 2 dpa, in denervated fins it is expressed only in the rays with a small blastema (h). a-h) Dashed lines mark amputation plane. Scale bar - 100 μm. i) qRT-PCR for shh and ptc1. qRT-PCR reveals lower levels of shh expression on denervated fins in relation to controls, at 0.5 dpa. These levels abruptly increase at 1 dpa, decreasing again at 1.5 and 2 dpa. Ptc1 expression is also higher at 1 dpa and lower at 1.5 and 2 dpa on denervated fins, in relation to controls (**p < 0.001, *p < 0.05).

Figure 9

Contribution of innervation to fin regeneration. a) Illustration of the putative role of nerves in zebrafish fin regeneration. The WE is formed after fin amputation, in a process that is independent of nerves. Innervation may be important to the subsequent thickening of the WE and the establishment of the AEC, which contributes to the formation and outgrowth of the blastema and to the progression of the regenerative process. The role of nerves may be to release factor(s) (“factor x”) that regulate the expression of target genes in the WE, such as pea3, fgf24 and lef1, which are important to the thickening and maintenance of the WE and to the communication established with the underlying cells. At the same time nerves may be involved in the inhibition of wnt5b in the WE, as well as in the pathways that lead to apoptosis. “Factor x” may also be directly released by nerves into the stump to induce cell proliferation and dedifferentiation. b) Illustration of the cellular and molecular changes occurred upon fin denervation. In the absence of innervation fins do not establish a functional AEC and several signalling pathways are affected. Wnt5b, an inhibitor of Wnt signalling and regeneration, is upregulated at 0.5 dpa, but is downregulated at 1 dpa. During this period krt8 and pea3, which are essential to WE maintenance, as well as fgf24 and lef1, important for the communication established with the underlying cells, are downregulated. At the same time, apoptosis activity is increased in the WE. The Fgf signalling molecules fgfr1, msxc, and mkp3 are upregulated, while fgf20a is downregulated. Shh is early downregulated at 0.5 dpa, but is then upregulated at 1 dpa. These signalling defects result in a breakdown of communication with mesenchymal cells, and impairment of the formation of the blastema and fin regeneration.