Spinal motor neurons are regenerated after mechanical lesion and genetic ablation in larval zebrafish

Abstract

In adult zebrafish, relatively quiescent progenitor cells show lesion-induced generation of motor neurons. Developmental motor neuron generation from the spinal motor neuron progenitor domain (pMN) sharply declines at 48 hours post-fertilisation (hpf). After that, mostly oligodendrocytes are generated from the same domain. We demonstrate here that within 48 h of a spinal lesion or specific genetic ablation of motor neurons at 72 hpf, the pMN domain reverts to motor neuron generation at the expense of oligodendrogenesis. By contrast, generation of dorsal Pax2-positive interneurons was not altered. Larval motor neuron regeneration can be boosted by dopaminergic drugs, similar to adult regeneration. We use larval lesions to show that pharmacological suppression of the cellular response of the innate immune system inhibits motor neuron regeneration. Hence, we have established a rapid larval regeneration paradigm. Either mechanical lesions or motor neuron ablation is sufficient to reveal a high degree of developmental flexibility of pMN progenitor cells. In addition, we show an important influence of the immune system on motor neuron regeneration from these progenitor cells.

Keywords: Dopamine; Hb9; Macrophage; Microglia; Nitroreductase; Olig2; Sox10.

Fig. 1.

A mechanical lesion to the spinal cord heals within 48 h and leads to motor neuron regeneration close to the lesion site. (A) Time line for experiments. (B) A zebrafish larva with a lesion in the dorsal trunk area, leaving the notochord intact at 3 dpf. (C,D) The same larva imaged at 0 and 48 h post lesion shows closure of the wound. (E,F) The lesion area is outlined with double-labelled Hb9:GFP⁺/EdU⁺ neurons indicated by arrowheads. A lesion leads to increased numbers of Hb9:GFP⁺/EdU⁺ double-labelled motor neurons (compare E and F). E′ to F‴ show higher magnifications of areas boxed in E and F, respectively, in single optical sections indicating double labelling. (G) Motor neurons are regenerated close to (<50 µm rostral and caudal), but not far from (100 µm>×>50 µm rostral and caudal) the lesion site (one-way ANOVA with Bonferroni's multiple comparisons test, ***P<0.001, ****P<0.0001; unlesioned, n=10; far, n=11; close, n=11). (H) Time line of the increase in the number of EdU-labelled motor neurons (t-test, ****P<0.0001). Values are means±s.e.m. Lateral views of larvae are shown (rostral is left; dorsal is up). Scale bars: 100 µm in B; 500 µm in D for C,D; 50 µm in F for E,F; 15 µm in F‴ for E′-F‴.

Fig. 2.

After a lesion, the pMN domain shows increased proliferation and gives rise to motor neurons. (A) Time line of the experiment. (B,C) olig2:DsRed⁺ cells (arrowheads) in the pMN domain that incorporated EdU within the last 4 h. (B′-C‴) Higher magnifications of single optical sections of the cells indicated by asterisks in B and C, respectively, showing double labelling. (D) The number of proliferating cells in the pMN domain is significantly increased in the vicinity of the lesion site (Mann–Whitney test; **P=0.0049). (E) In Hb9:GFP and olig2:DsRed double-transgenic larvae (lesion: 3 dpf; analysis: 5 dpf), newly generated motor neurons (Hb9:GFP⁺/EdU⁺) that retain DsRed protein are indicated by arrowheads. Lateral views are shown; rostral is left, dorsal is up. Values are means±s.e.m. Scale bars: 50 µm in C for B,C; 20 µm in C‴ for B″-C‴ and 10 µm for B′,C′; 15 µm in E.

Fig. 3.

Oligodendrocyte generation is reduced after a spinal lesion. (A) Time line of the experiment. (B,C) Newly generated oligodendrocytes and their precursors, triple labelled by olig2:GFP, sox10:mRFP and EdU (arrowheads), are reduced in number after lesion. (B′-B″″) A triple-labelled cell (indicated with asterisk in B) in a single optical section at higher magnification. (D) The number of triple-labelled cells is reduced (Student's t-test, ****P<0.0001). (E) mbp:GFP⁺ oligodendrocytes incorporate EdU (indicated by arrowheads) in unlesioned larvae. (E′-E‴) Two double-labelled neurons indicated in E at higher magnification in a single optical section. (F) Fewer double-labelled cells are observed after a lesion. (G) The number of new oligodendrocytes is significantly reduced after a lesion (Mann–Whitney U-test; ***P=0.0005). Lateral views are shown; rostral is left, dorsal is up. The lesion site is indicated by a dashed line. Values are means±s.e.m. Scale bars: 100 µm in B for B,C; 20 µm in B″″ for B′-B″″; 50 µm in F for E,F; 20 µm in E‴.

Fig. 4.

Motor neuron regeneration is enhanced by application of a dopamine agonist. (A) Experimental time line. (B,C) Double-labelled Hb9:GFP⁺/EdU⁺ neurons are indicated by arrowheads. (B′-C‴) Double-labelled cells from B,C (asterisks) are indicated by arrowheads in single optical sections at higher magnification. (D) Pergolide treatment during the regeneration phase significantly increases the number of Hb9:GFP⁺/EdU⁺ double-labelled motor neurons (t-test, **P=0.0092; DMSO, n=12; Pergolide, n=9). Lateral views are shown; rostral is left, dorsal is up. The lesion site is indicated by a dashed line. Values are means±s.e.m. Scale bars: 50 µm in B for B,C; 10 µm in B‴ for B′-C‴.

Fig. 5.

Suppression of the immune response inhibits motor neuron regeneration. (A) Time line for the experiments. (B-F) Incubation with dexamethasone does not lead to visible changes in unlesioned larvae (B,C), but strongly reduces the immune reaction at the lesion site (D,E) as indicated by reduced 4C4 (arrowheads indicate 4C4⁺ cells) and L-plastin immunoreactivity. Quantification of immunoreactivity is shown in F (Student's t-test, ***P<0.001). (G-I) Dexamethasone treatment reduces the number EdU-labelled Hb9:GFP⁺ motor neurons (arrowheads). Higher magnifications of double-labelled neurons indicated by asterisks in G,H are shown in single optical sections in G′-H‴. (I) Quantification of the reduction in newly generated motor neurons (t-test; **P=0.0085; unlesioned, n=16; dexamethasone, n=13). Lateral views are shown; rostral is left, dorsal is up. The lesion site is indicated by a dashed line. Values are means±s.e.m. Scale bars: 100 µm in E for B-E; 100 µm in H for G,H; 50 µm in H‴ for G′-H‴.

Fig. 6.

The Tg(mnx1:Gal4, UAS:nfsB-mCherry) transgene is expressed in motor neurons. (A) Lateral view of a whole larva (rostral left, dorsal up, 3 dpf) indicates labelling in spinal motor neurons, the pancreas and heart. (B,C) Spinal cross sections (3 dpf) indicate that most mCherry⁺ cells are also Hb9⁺ or ChAT⁺, or both. Arrowheads indicate triple-labelled cells. (C) Venn diagram showing the overlap of mCherry expression with motor neuron markers. Scale bars: 500 µm in A; 25 µm in B‴ for B-B‴.

Fig. 7.

MTZ treatment leads to ablation of all transgene-expressing cells and to microglia/macrophage activation. (A) Treatment time line. (B-D) mCherry⁺ motor neurons and their axons are visible in untreated control larvae at 3 and 4 dpf (B,B′), but mCherry labelling is completely lost after 24 h treatment with MTZ (C,C′), quantified in D (Mann–Whitney U-test, ****P<0.0001). (E-G) Cross sections show that mCherry-labelled cells fragment during MTZ treatment and that microglia/macrophages appear and phagocytose the cell debris (arrow in F,F′). Microglia/macrophages are quantified in G (Mann–Whitney U-test, ***P<0.0001). Values are means±s.e.m. Scale bars: 100 µm in C′ for B-C′; 25 µm in F′ for E-F′.

Fig. 8.

Motor neuron ablation leads to regeneration of motor neurons. (A) Experimental timeline. (B-H) In spinal cross sections, Hb9⁺/EdU⁺ motor neurons are only observed in MTZ-treated larvae (B,C, shown in a whole cross section of the spinal cord including orthogonal views in F), whereas Pax2⁺ interneurons are labelled by EdU in untreated and MTZ-treated larvae, quantified in G (Mann–Whitney U-test, **P=0.0063) and H (Mann–Whitney U-test, P>0.99), respectively. (I) Overall numbers of Hb9⁺ and ChAT⁺ profiles are reduced after a lesion, but return to control values at 7 dpf (Hb9 only) or 10 dpf (ChAT) (t-test, *P=0.0188; **P=0.0012; ***P=0.0004). Values are means±s.e.m. Scale bars: 25 µm in B for B-E; 5 µm in inset in C for all insets; 25 µm in F.