Immunohistological staining for P-Smad3 as an indicator of TGFβ pathway activation.

<p>The red channel with P-Smad3 staining is shown in the figures above, whereas overlay with the green (autofluorescence of photoreceptor outer segments) and blue channel (DAPI) is shown below. No relevant staining for P-Smad3 (red) was observed in the uninjured retina and one day after induction of retina degeneration with MNU. Starting at day 3 and until day 8, immunohistochemical staining for P-Smad3 revealed the activation of the TGFβ pathway (exemplarily, day 5 is shown). At day 15 and thereafter, no relevant activation was observed anymore (exemplarily, day 30 is shown). When the TGFβ pathway was inhibited (small molecule inhibitor SB431542), reduced staining for P-Smad3 was observed, when compared to the non-inhibited group in 0.1% dimethyl sulfoxide (DMSO). Lower magnification of retina 3 days after MNU treatment, including the peripheral retina is shown on the right. Cell nuclei are stained with DAPI (blue). The scale bar indicates 50 μm. GC: ganglion cells; INL: inner nuclear layer; ONL: outer nuclear layer.</p

Schematic representation of distinct effects of TGF-β inhibition on Col XII and Col Iα deposition in the epicardium and the fibrotic tissue during zebrafish heart regeneration.

<p>Illustrations of longitudinal heart sections at 14 dpci in normal conditions (left side) and after inhibition of the TGF-β signaling pathway (right side). The uninjured part of the heart displays the presence of Col XII along the heart surface, while Col Iα is expressed in the bulbus arteriosus and in the atrio-ventricular valves. The injured myocardial wall heals by enhanced Col XII deposition along the outer margin of the wound, forming a Col XII-rich epicardial cap. The inner part of the damaged myocardium is replaced with fibrotic tissue that contains <i>tgf-β</i>-expressing cells. The activity of this pathway stimulates deposition of fibrillar Col Iα and fibril-associated Col XII in the fibrotic tissue, but it is not required for the formation of the epicardial cap. The provisional matrix maintains the organ function during the regenerative process, until its completion at 30 dpci.</p

P-Smad3 is activated in proliferating cells.

<p>Top: The co-localization of P-Smad3 and proliferating cell nuclear antigen (PCNA) indicates that Smad3 is activated in proliferating cells. Bottom: P-Smad3-positive cells in the inner nuclear layer (INL) co-localized with glutamine synthetase (GS), suggesting that these cells are Müller glia. Representative immunohistochemical staining at day 3 is depicted. Cell nuclei are stained with DAPI (blue). The scale bar indicates 25 μm. GC: ganglion cells, ONL: outer nuclear layer.</p

Col XII is expressed in the epicardium of the adult zebrafish heart.

<p>(A) Aniline blue staining of a ventricle transversal section visualizes collagen (blue). Framed areas encompass parts with the atrio-ventricular valve (A/V-Valve) and ventricular wall (V-Wall). The thickness of the compact myocardial layer is depicted as a bar in this and subsequent panels. Ep, epicardium; CoM, compact myocardium; TrM, trabecular myocardium. N = 5. (B-D) <i>In -situ</i> hybridization of ventricle sections detected by a color reaction (purple). Probe names are to the left. Framed areas encompass the parts that are enlarged in the panels to the right. N ≥ 4. (E) Superposition of a bright-field image with <i>in -situ</i> hybridization using <i>col12a1b</i> probe (purple) and fluorescent immunodetection of muscle protein Tropomyosin, TPM (red). <i>col12a1b</i> is expressed in the epicardium that is located externally from the myocardial border (dashed line). A few <i>col12a1b</i>-expressing cells are Tropomyosin-negative (arrows) and are interspersed within the compact myocardium (the thickness of the compact myocardium is indicated with a white bar). N = 4. (F) Immunofluorescence with anti-Tropomyosin (blue) to label cardiomyocytes and anti-Col XII (green) of transgenic fish <i>wt1a(-6</i>.<i>8kb)</i>:<i>GFP</i> (red), which labels cardiac subepicardial fibroblasts (white arrows) located mainly along the junction between the outer compact myocardium (white bar) and inner trabecular myocardium. N = 4. (G, H) Aniline blue, acid Fuchsin, Orange G (AFOG) staining detects collagen (blue) in bulbus arteriosus (G, longitudinal heart section) and the leaflets of the atrioventricular valve (H, transversal heart section). N = 6. (I, J) Triple immunofluorescence staining against Col XII (green), Col Iα (red) and Tropomyosin (blue) of the structures shown in above panels. (I’) Col XII is detected on the myocardial surface. (I”) In the bulbus arteriosus, Col Iα fibers are in the interstitium, while Col XII is restricted to its surface. (J’) The atrio-ventricular connection displays Col Iα, but little Col XII in the valve leaflets. N = 6. (A’, B’, C’, D’, E’, F’) Higher magnifications of the framed areas shown in images that are labeled with the same letter without prime symbol. The same rule applies to all subsequent figures.</p

Collagen XII distribution correlates with the activated epicardium and fibroblast-like cells.

<p>(A-F) Analysis of transversal heart sections at 14 dpci. (A and D) AFOG staining of the sections used for immunostaining. (B and C) Raldh2 expression (red) demarcates the activated epicardium and endocardium. (C’) Col XII (green) and Raldh2 are colocalized in the intact epicardium (epicard). (C”) Raldh2-positive cells invade the post-cryoinjured area that is labeled by Col XII expression. Cardiac muscle is detected by Tropomyosin antibody staining (blue). N = 5. (E and F) Triple antibody staining against Col XII (green), intermediate filament Vimentin (blue) and alpha-Smooth Muscle Actin (αSMA; red). (F’ and F”) αSMA- and Vimentin-positive cells are non-overlapping cell populations in the epicardium and post-cryoinjured area. Both of them are associated with Col XII-labeled fibrils. N = 6.</p

TGF-β signaling is activated in Collagen XII-positive fibrotic tissue.

<p>(A-C) Analyses of ventricle sections at 14 dpci. (A) <i>In -situ</i> hybridization detects <i>tgf-β2</i> expression in the cryoinjury area. N = 5. (B, C) Immunofluorescence staining reveals co-distribution of Col XII fibrils (green) and p-Smad3 (red) in the post-cryolesion zone that is recognized by the absence of Tropomyosin (blue in B). p-Smad3 reactivity is detected in the nuclei visualized by DAPI (blue in C). N = 5.</p

The upregulation of fibrillar Collagen Iα in the post-cryoinjured area is induced by TGF-β signaling.

<p>(A-D) Immunofluorescence staining of heart at 14 dpci using Col Iα (green) and Tropomyosin (red). The post-infarcted tissue is tropomyosin-negative (encircled with a dashed line). (A-B’) Control heart displays the presence of fibrillar collagen in the fibrotic tissue. N = 4.(C-D’) The treatment with the inhibitor of the TGF-β receptors, SB431542, suppresses Col Iα (green) in the inner wound site. In the epicardium, Col Iα can be still detected (arrowhead). N = 6. (E-H’) <i>In-situ</i> hybridization against <i>col1a2</i> (purple) and immunostaining with Tropomyosin (red). (E-F’) Control hearts display expression of <i>col1a2</i> in the post-infarcted tissue. (G-H’) The inhibition of TGF-β signaling with SB431542-treated suppresses <i>col1a2</i> expression in the inner part of the wound, without affecting the epicardial expression. N = 6.</p

Cell proliferation in the zebrafish retina exposed to 150 mg/l MNU.

<p>Proliferating cell nuclear antigen (PCNA) positive cells (red) indicate proliferation. Cell proliferation in the inner nuclear layer (INL) was highest at day 3 and 5, with no relevant difference between the non-inhibited (0.1% dimethyl sulfide, DMSO) and inhibited group (small molecule inhibitor SB431542). In contrast, proliferation in the outer nuclear layer (ONL) was higher in the inhibited group between 3 and 8. Cell nuclei are stained with DAPI (blue). Scale bar indicates 50 μm. GC: ganglion cells.</p

Developmental dynamics of Col XII expression in the zebrafish heart.

<p>(A-D) Longtudinal sections of the zebrafish heart after double immunostaining against Col XII (green) and Tropomyosin (red), with DAPI contrastain (blue). dpf, days post-fertilization. Three chambers of the zebrafish heart: v, ventricle; a atrium; b.a., bulbus arteriosus (non-muscular structure). N ≥ 5. (A) At 3 dpf, embryos express Col XII in the pericardium, but not in the heart. The pericardial fibers seem to invade the surface of the heart. (B) At 14 dpf, the three chambers of the larval heart are surrounded by Col XII-positive fibrils within 10 μm of outer myocardial layer (white bar). (C) At 30 dpf, the juvenile fish heart contains a thickened myocardium (red), but the size of Col XII-positive layer remains unaltered. (D) At 120 days post fertilization, young adult fish maintain Col XII-labeled fibers along the heart circumference in a pattern similar to the one seen at the larval stage.</p

TUNEL positive cells in the zebrafish retina after exposure to MNU.

<p>In uninjured zebrafish retina there are merely no TUNEL positive cells. Three days after exposure to 150 mg/l MNU, both the non-inhibited (0.1% dimethyl sulfide, DMSO) and the inhibited group (small molecule inhibitor SB431542) show a considerable amount of TUNEL positive cells (green) in the outer nuclear layer (ONL) and to a lesser degree in the inner nuclear layer (INL). Cell nuclei are stained with DAPI (blue). Scale bar indicates 50 μm. GC: ganglion cells.</p