Bone morphogenetic protein signaling promotes morphogenesis of blood vessels, wound epidermis, and actinotrichia during fin regeneration in zebrafish

Zebrafish fin regeneration involves initial formation of the wound epidermis and the blastema, followed by tissue morphogenesis. The mechanisms coordinating differentiation of distinct tissues of the regenerate are poorly understood. Here, we applied pharmacologic and transgenic approaches to address the role of bone morphogenetic protein (BMP) signaling during fin restoration. To map the BMP transcriptional activity, we analyzed the expression of the evolutionarily conserved direct phospho-Smad1 target gene, id1, and its homologs id2a and id3. This analysis revealed the BMP activity in the distal blastema, wound epidermis, osteoblasts, and blood vessels of the regenerate. Blocking the BMP function with a selective chemical inhibitor of BMP type I receptors, DMH1, suppressed id1 and id3 expression and arrested regeneration after blastema formation. We identified several previously uncharacterized functions of BMP during fin regeneration. Specifically, BMP signaling is required for remodeling of plexus into structured blood vessels in the rapidly growing regenerate. It organizes the wound epithelium by triggering wnt5b expression and promoting Collagen XIV-A deposition into the basement membrane. BMP represents the first known signaling that induces actinotrichia formation in the regenerate. Our data reveal a multifaceted role of BMP for coordinated morphogenesis of distinct tissues during regeneration of a complex vertebrate appendage.—Thorimbert, V., König, D., Marro, J., Ruggiero, F., Jaźwińska, A. Bone morphogenetic protein signaling promotes morphogenesis of blood vessels, wound epidermis, and actinotrichia during fin regeneration in zebrafish

Distinct effects of inflammation on preconditioning and regeneration of the adult zebrafish heart

The adult heart is able to activate cardioprotective programmes and modifies its architecture in response to physiological or pathological changes. While mammalian cardiac remodelling often involves hypertrophic expansion, the adult zebrafish heart exploits hyperplastic growth. This capacity depends on the responsiveness of zebrafish cardiomyocytes to mitogenic signals throughout their entire life. Here, we have examined the role of inflammation on the stimulation of cell cycle activity in the context of heart preconditioning and regeneration. We used thoracotomy as a cardiac preconditioning model and cryoinjury as a model of cardiac infarction in the adult zebrafish. First, we performed a spatio-temporal characterization of leucocytes and cycling cardiac cells after thoracotomy. This analysis revealed a concomitance between the infiltration of inflammatory cells and the stimulation of the mitotic activity. However, decreasing the immune response using clodronate liposome injection, PLX3397 treatment or anti-inflammatory drugs surprisingly had no effect on the re- entry of cardiac cells into the cell cycle. In contrast, reducing inflammation using the same strategies after cryoinjury strongly impaired cardiac cell mitotic activity and the regenerative process. Taken together, our results show that, while the immune response is not necessary to induce cell-cycle activity in intact preconditioned hearts, inflammation is required for the regeneration of injured hearts in zebrafish

Collagen XII contributes to epicardial and connective tissues in the Zebrafish heart during ontogenesis and regeneration

Zebrafish heart regeneration depends on cardiac cell proliferation, epicardium activation and transient reparative tissue deposition. The contribution and the regulation of specific collagen types during the regenerative process, however, remain poorly characterized. Here, we identified that the non-fibrillar type XII collagen, which serves as a matrix-bridging component, is expressed in the epicardium of the zebrafish heart, and is boosted after cryoinjury-induced ventricular damage. During heart regeneration, an intense deposition of Collagen XII covers the outer epicardial cap and the interstitial reparative tissue. Analysis of the activated epicardium and fibroblast markers revealed a heterogeneous cellular origin of Collagen XII. Interestingly, this matrix-bridging collagen co-localized with fibrillar type I collagen and several glycoproteins in the post-injury zone, suggesting its role in tissue cohesion. Using SB431542, a selective inhibitor of the TGF-β receptor, we showed that while the inhibitor treatment did not affect the expression of collagen 12 and collagen 1a2 in the epicardium, it completely suppressed the induction of both genes in the fibrotic tissue. This suggests that distinct mechanisms might regulate collagen expression in the outer heart layer and the inner injury zone. On the basis of this study, we postulate that the TGF-β signaling pathway induces and coordinates formation of a transient collagenous network that comprises fibril-forming Collagen I and fiber-associated Collagen XII, both of which contribute to the reparative matrix of the regenerating zebrafish heart

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

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

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

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

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