FIGURE SUMMARY
Title

Neuronal regeneration in a zebrafish model of adult brain injury

Authors
Kishimoto, N., Shimizu, K., and Sawamoto, K.
Source
Full text @ Dis. Model. Mech.

The healing process in the adult zebrafish telencephalon. (A) A zebrafish model of adult telencephalon lesions. A lesion was formed by inserting a needle into the dorsolateral domain of one hemisphere of the telencephalon. (A′) The inset is an illustration of the coronal hemisphere section obtained by a cut at the red line. Tel, telencephalon; mPa, medial pallium; dPa, dorsal pallium; Pa, pallium; lPa, lateral pallium; Sub, subpallium. (B,C) Dorsal views of an injured adult zebrafish brain at 0 days post-lesion (dpl) (B) and 35 dpl (C). Note the reduction of the lesion at 35 dpl. (D–F) Hu protein detected in coronal brain sections at the level indicated in the illustration in panel D (dorsal up) of zebrafish at 1 (D), 14 (E) and 35 (F) dpl. Higher magnifications of the boxed regions in panels D–F are shown in D2–F2, respectively. Arrows indicate accumulated Hu-positive cells at the injury site, which is circled in white. Scale bars: 100 μm.

Cell proliferation induced by telencephalon injury. (A,B) Immunodetection of PCNA in coronal brain sections at the level indicated in the illustration in panel A (dorsal up): (A) control; (B) 3 dpl. Higher magnifications of panels A and B are shown in A′ and B′, respectively. Notice the injury-induced cell proliferation in the vicinity of the injury site (B) and in the telencephalic ventricular zone (B′). Arrows show the zones where cell proliferation was induced by injury. White dotted circles indicate the injury site. V, telencephalic ventricle. Scale bars: 100 μm. (C,D) Histograms showing the PCNA-positive cell counts in the telencephalic ventricular zone (C), and in the medial-dorsal-lateral domain of the telencephalic pallium (mdlPa) (D) over time. Student’s t-test was used to determine significant differences in expression. Error bars represent s.e.m. *P<0.05, **P<0.01. VZ, ventricular zone; Sub, subpallium; Pa, pallium; mPa, medial pallium; dPa, dorsal pallium; lPa, lateral pallium.

Notch intracellular domain immunoreactivity after telencephalic injury. (A,B) Immunodetection of the Notch intracellular domain (NICD) in coronal brain sections (dorsal up) at 1 dpl. Higher magnifications of panels A and B are shown in panels A′ and B′, respectively. (C) Immunodetection of a proliferation marker (PCNA) and the NICD in a coronal brain section (dorsal up) at 1 dpl. Arrowheads indicate NICD and PCNA double-positive cells in the telencephalic VZ. V, telencephalic ventricle. (D,E) Histograms showing the counts of NICD-positive cells (D) and PCNA-positive cells (E) in the telencephalic VZ. Notice the accumulation of NICD in proliferating cells in the telencephalic VZ induced in the injured hemisphere, compared with the uninjured one (A,C,D). This accumulation was prevented by treatment with the Notch inhibitor DAPT (B,D,E). Student’s t-test was used to determine significant differences in expression. Error bars represent s.e.m. **P<0.01, ***P<0.001. Scale bars: 50 μm (A,B,C); 10 μm (A′,B′).

Distribution of BrdU-labeled cells in the injured adult zebrafish telencephalon. (A) Adult zebrafish were placed in water containing 10 mM BrdU for 4 hours, and then in BrdU-free water. After creating the lesion, they were then sacrificed at the indicated time points. (B–E′) Immunodetection of BrdU in the coronal brain sections (dorsal up) at 0 (B,B′), 3 (C,C′), 7 (D,D′) and 10 (E,E′) dpl. Higher magnifications of the boxed areas in panels BE are shown in B′–E′, respectively. Arrows show injury-induced accumulations of BrdU-positive cells. White dotted circles indicate the injury site. VZ, ventricular zone; Sub, subpallium; Pa, pallium; mPa, medial pallium; dPa, dorsal pallium; lPa, lateral pallium. Scale bars: 100 ¼m. (F–H) Histograms showing the counts of BrdU-positive cells in the telencephalic VZ (F), in the subpallium (Sub) and pallium (Pa) (G), and in the medial-dorsal-lateral domain of the telencephalic pallium (mdlPa) (H). Student’s t-test was used to determine significant differences in expression. Error bars represent s.e.m. *P<0.05, **P<0.01, ***P<0.001.

Distribution of neuronal precursor cells in the injured adult zebrafish telencephalon. (A–C,E,F) Distribution of GFP-positive cells in the lesioned brain of adult Tg(ngn1:gfp) fish (coronal views, dorsal up). (A) Control; (B) 3 dpl; (C) 7 dpl; (E) 3 dpl treated with DMSO; (F) 3 dpl treated with DAPT. White dotted circles indicate the injury site. VZ, ventricular zone; Sub, subpallium; Pa, pallium; mPa, medial pallium; dPa, dorsal pallium; lPa, lateral pallium. Scale bars: 100 μm. (D,G) Histograms showing the GFP-positive cell counts in the VZ, in the subpallium (Sub) and pallium (Pa), and in the medial-dorsal-lateral domain of the telencephalic pallium (mdlPa) in the injured brains of adult Tg(ngn1:gfp) fish, with (D) no treatment, and (G) DMSO or DAPT treatment. Student’s t-test was used to determine significant differences in expression. Error bars represent s.e.m. *P<0.05, **P<0.01.

Neuronal precursor cells differentiate into neurons adjacent to the injury site. (A–C) Immunodetection of Hu (blue), GFAP (red) and GFP (green) in coronal brain sections of the lesioned adult Tg(ngn1:gfp) fish brain (dorsal up): (A) control; (B) 3 dpl; (C) 7 dpl. Higher magnifications of the boxed areas in panels A–C are shown in A′–C′, respectively. Insets in B, B′ and C′ show high-magnification views of the boxed areas in B, B2, and C′, respectively. (D) The total count of GFP-positive cells, and the percentage that was also positive for Hu, in the telencephalic VZ, subpallium (Sub) and pallium (Pa), and the medial-dorsal-lateral domain of the telencephalic pallium (mdlPa). Student’s t-test was used to determine significant differences in expression. Error bars represent s.e.m. *P<0.05. (E) Immunodetection of Tbr1 (red) and GFP (green) in coronal brain sections (dorsal up) of injured adult Tg(ngn1:gfp) zebrafish at 7 dpl. Higher magnifications of the boxed areas in panel E are shown in panels E′–E′′ ′. The ngn1:gfp-positive cells expressed Tbr1 protein. (F) Immunodetection of BrdU (red) and Tbr1 (green) in coronal brain sections (dorsal up) of the injured adult wild-type fish at 14 dpl. White arrows indicate BrdU and Tbr1 double-positive cells. BrdU-labeling protocol: see Fig. 4A. White dotted circles indicate the injury site. Scale bars: 100 μm (A–C,E); 50 μm (F).

Distribution of PSA-NCAM-positive newborn cells in the adult zebrafish telencephalon. (A) Immunodetection of BrdU (red) and PSA-NCAM (green) in coronal brain sections (dorsal up) of injured adult wild-type zebrafish at 2 d.p.l. Higher magnifications of the boxed areas in panel A are shown in panels B-D (B, vicinity of the VZ; C, medial pallium; D, injury site), respectively. White arrows indicate BrdU and PSA-NCAM double-positive cells. BrdU-labeling protocol: see Fig. 4A. White dotted circles indicate the injury site. Scale bars: 100 μm (A), 20 μm (B), 50 μm (C, D).

Distribution of Tbr1-positive cells in the adult zebrafish telencephalon. (A-D) Immunodetection of Tbr1 protein in coronal brain sections (dorsal up) of the lesioned adult zebrafish brain: (A, A′) control; (B) 0 d.p.l.; (C) 21 d.p.l.; (D) 31 d.p.l.. Yellow dotted circles indicate the injury site. (E) Histogram showing the Tbr1-positive cell counts in the medial-dorsal-lateral domain of the telencephalic pallium (mdlPa) in the injured brains of adult wild-type zebrafish. Scale bars: 100 μm. Abbreviations: see Fig. 2.

Newborn cells differentiate into Hu-positive neurons in the injury site. Immunodetection of BrdU (red) and Hu protein (green) in coronal brain sections (dorsal up) of injured adult wild-type zebrafish at 21 d.p.l.. White arrowheads indicate BrdU and Hu double-positive cells. BrdU-labeling protocol: see Fig. 4A. Scale bars: 50 μm.

Acknowledgments
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