Zebrafish ProVEGF-C Expression, Proteolytic Processing and Inhibitory Effect of Unprocessed ProVEGF-C during Fin Regeneration

BACKGROUND: In zebrafish, vascular endothelial growth factor-C precursor (proVEGF-C) processing occurs within the dibasic motif HSIIRR(214) suggesting the involvement of one or more basic amino acid-specific proprotein convertases (PCs) in this process. In the present study, we examined zebrafish proVEGF-C expression and processing and the effect of unprocessed proVEGF-C on caudal fin regeneration. METHODOLOGY/PRINCIPAL FINDINGS: Cell transfection assays revealed that the cleavage of proVEGF-C, mainly mediated by the proprotein convertases Furin and PC5 and to a less degree by PACE4 and PC7, is abolished by PCs inhibitors or by mutation of its cleavage site (HSIIRR(214) into HSIISS(214)). In vitro, unprocessed proVEGF-C failed to activate its signaling proteins Akt and ERK and to induce cell proliferation. In vivo, following caudal fin amputation, the induction of VEGF-C, Furin and PC5 expression occurs as early as 2 days post-amputation (dpa) with a maximum levels at 4-7 dpa. Using immunofluorescence staining we localized high expression of VEGF-C and the convertases Furin and PC5 surrounding the apical growth zone of the regenerating fin. While expression of wild-type proVEGF-C in this area had no effect, unprocessed proVEGF-C inhibited fin regeneration. CONCLUSIONS/SIGNIFICANCES: Taken together, these data indicate that zebrafish fin regeneration is associated with up-regulation of VEGF-C and the convertases Furin and PC5 and highlight the inhibitory effect of unprocessed proVEGF-C on fin regeneration

Cellular and Animal Models of Striated Muscle Laminopathies

The lamin A/C (LMNA) gene codes for nuclear intermediate filaments constitutive of the nuclear lamina. LMNA has 12 exons and alternative splicing of exon 10 results in two major isoforms—lamins A and C. Mutations found throughout the LMNA gene cause a group of diseases collectively known as laminopathies, of which the type, diversity, penetrance and severity of phenotypes can vary from one individual to the other, even between individuals carrying the same mutation. The majority of the laminopathies affect cardiac and/or skeletal muscles. The underlying molecular mechanisms contributing to such tissue-specific phenotypes caused by mutations in a ubiquitously expressed gene are not yet well elucidated. This review will explore the different phenotypes observed in established models of striated muscle laminopathies and their respective contributions to advancing our understanding of cardiac and skeletal muscle-related laminopathies. Potential future directions for developing effective treatments for patients with lamin A/C mutation-associated cardiac and/or skeletal muscle conditions will be discussed

Effects of fin fold mesenchyme ablation on fin development in zebrafish

<div><p>The evolution of the tetrapod limb involved an expansion and elaboration of the endoskeletal elements, while the fish fin rays were lost. Loss of fin-specific genes, and regulatory changes in key appendicular patterning genes have been identified as mechanisms of limb evolution, however their contributions to cellular organization and tissue differences between fins and limbs remains poorly understood. During early larval fin development, <i>hoxa13a/hoxd13a</i>-expressing fin fold mesenchyme migrate through the median and pectoral fin along actinotrichia fibrils, non-calcified skeletal elements crucial for supporting the fin fold. Fin fold mesenchyme migration defects have previously been proposed as a mechanism of fin dermal bone loss during tetrapod evolution as it has been shown they contribute directly to the fin ray osteoblast population. Using the nitroreductase/metronidazole system, we genetically ablated a subset of <i>hoxa13a/hoxd13a</i>-expressing fin fold mesenchyme to assess its contributions to fin development. Following the ablation of fin fold mesenchyme in larvae, the actinotrichia are unable to remain rigid and the median and pectoral fin folds collapse, resulting in a reduced fin fold size. The remaining cells following ablation are unable to migrate and show decreased <i>actinodin1</i> mesenchymal reporter activity. Actinodin proteins are crucial structural component of the actinotrichia. Additionally, we show a decrease in <i>hoxa13a</i>, <i>hoxd13a</i>, <i>fgf10a</i> and altered <i>shha</i>, and <i>ptch2</i> expression during larval fin development. A continuous treatment of metronidazole leads to fin ray defects at 30dpf. Fewer rays are present compared to stage-matched control larvae, and these rays are shorter and less defined. These results suggest the targeted <i>hoxa13a/hoxd13a</i>-expressing mesenchyme contribute to their own successful migration through their contributions to actinotrichia. Furthermore, due to their fate as fin ray osteoblasts, we propose their initial ablation, and subsequent disorganization produces truncated fin dermal bone elements during late larval stages.</p></div

<i>A</i>ltered gene expression profiles in the median and pectoral fin of <i>Tg(Inta11:NTR)</i> larvae following metronidazole treatment.

<p><b>(A-N)</b><i>in situ hybridization</i> and <i>and1</i> reporter data showing gene expression profiles in the median and pectoral fin at 60, and 72hpf in Inta11: NTR—MTZ and Inta11: NTR—MTZ larvae. Inta11: NTR—MTZ are present in the left panels (A, C, E, G, I, K, M, O, Q) and Inta11: NTR + MTZ are present in the right panels (B, D, F, H, J, L, N, P, R). Inta11: NTR + MTZ show a decrease in distal <i>hoxa13a</i> expression (red arrow) in the median fin at 60hpf (B), and in the pectoral fin at 72hpf (D) compared to Inta11: NTR—MTZ (green arrow) (A, C). Note unaltered <i>hoxa13a</i> expression in the trunk region of Inta11: NTR + MTZ (red asterisks) (B). Inta11: NTR + MTZ show a decrease in distal <i>hoxd13a</i> expression (red arrow) in the pectoral fin at 72hpf (F) compared to Inta11: NTR—MTZ larvae (green arrow) (E). Note unaltered <i>hoxd13a</i> expression in the proximal disc region of Inta11: NTR + MTZ larvae (red asterisks) (F). Inta11: NTR + MTZ double transgenic larvae show decreased <i>and1</i> reporter activity (red arrow) (J) in the pectoral fin compared to Inta11: NTR—MTZ double transgenic larvae (Red arrow) (I) at 72hpf. Brightfield (G-H) and fluorescent (I-J) images are included. Inta11: NTR + MTZ larvae show an increased anterior-posterior, and decreased proximal–distal expression domain of both <i>shha</i> and its receptor <i>ptch2</i> in the pectoral fin at 72hpf (yellow arrows) (L, N) compared to Inta11: NTR—MTZ larvae (green arrow) (K, M). Inta11: NTR + MTZ show a decrease in distal/distal posterior <i>fgf10a</i> expression at 72hpf, in the median and pectoral fin respectively (red arrows) (P, R) compared to Inta11: NTR—MTZ larvae (green arrows) (O, Q). Note unaltered expression of <i>fgf10a</i> in the anterior pectoral fin mesenchyme of Inta11: NTR + MTZ larvae (red asterisks) (N). Dotted lines indicate fin fold and disk boundary (C-F, K-N, Q-R)). Probe or reporter line is indicated in the top right corner of each panel in the left column, age is indicated in the top right corner of each panel in the right column (A-R). Number of larvae displaying gene expression pattern, for <i>in situ</i> hybridization data, are indicated in the bottom right corner of each panel (A-F, K-R). WT-MTZ+DMSO images are contained in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0192500#pone.0192500.s001" target="_blank">S1 Fig</a>, and show similar expression profiles to Inta11: NTR—MTZ larvae (A, C, E, K, M, O, Q). ED, Endoskeletal disc; T, Trunk. Scale bars: 100μm in A, B, K, L; 50μm in I, J, M, N; 30μm in C-H.</p

Metronidazole-treated <i>Tg(Inta11:NTR)</i> larvae show defects in median fin fold mesenchyme migration, a reduction in median and pectoral fin fold size and a reduction in endoskeletal disc size.

<p><b>(A-B)</b> Schematic of median fin fold measurements. <b>(C-E, J)</b> Graphs displaying measurements of median fin mesenchyme displacement (%), median fin fold width (mm) and height (mm), and pectoral fin fold and endoskeletal disc area (mm²). <b>(F-G)</b> Inta11: NTR—MTZ and <b>(H-I)</b> Inta11: NTR + MTZ pectoral fin at 7dpf outcrossed with <i>Tg(kr19)</i> to highlight endoskeletal disc. Fin fold mesenchyme cell displacement is represented as a percentage displaced relative to the overall fin fold length (trunk to distal tip) (Measurement 1), length of median fin fold is measured from trunk to distal tip (measurement 2), and height of median fin fold is measured from dorsal to ventral tips at the trunk (measurement 3) (A-B). Inta11: NTR + MTZ larvae display a reduction in median fin fold mesenchyme cell displacement at 48, 60, and 72hpf compared to control larvae (C). Inta11: NTR + MTZ larvae show a reduction in median fin fold width and height at 60, 72hpf, and 7dpf compared to control larvae (D, E). No difference is observed for either measurement at 48hpf (D, E). Inta11: NTR + MTZ larvae show a decrease in pectoral fin fold area at 72hpf, and 7dpf, as well as a reduction in endoskeletal disc size at 7dpf (J). Example of Inta11: NTR—MTZ (F, G) and Inta11: NTR + MTZ (H, I) pectoral fin used for distal fin fold, endoskeletal disc measurements. Region used for measurement is indicated by dotted line (F, G). Note the decreased disc size in the Inta11: NTR + MTZ pectoral fin (white asterisks) (I). Scapulocoracoid not included in the disc area measurements (yellow asterisks) (G, I). All bar values are an average of 10 measurements (n = 10 fins) with standard deviation indicated, with the exception of endoskeletal disc size (J). Endoskeletal disc values are based on measurements of 5, 5, and 8 fins (n = 5 fins, n = 5 fins, n = 8 fins) for treatment controls and Inta11: NTR + MTZ larvae respectively. Standard one-way ANOVA was performed. Each mean was compared against both other means. Tukey’s correction was applied. No statistically relevant difference was ever detected between treatment controls (WT + MTZ, Inta11: NTR—MTZ). Inta11: NTR + MTZ P-values (asterisks) are representative of comparisons with both treatment controls, with the exception of median fin fold width at 7dpf, where unique P-values are indicated for comparisons with each control (D). Brightfield (A-B, F, H), fluorescence (G, I). P-values: ** P = 0.001>0.005, **** P = <0.0001. ED, Endoskeletal disc; MFF, Median fin fold. Scale bars: 100μm (F-I).</p

Actinotrichia defects in 72hpf, 7dpf pectoral and median fins of <i>Tg(Inta11:NTR)</i> following metronidazole treatment.

<p>Collagen II Immunostaining of <b>(A-F, M-R)</b> pectoral and <b>(G-L, S-X)</b> median fins of Inta11: NTR + MTZ and Inta11: NTR–MTZ control larvae at 72hpf and 7dpf. At 72hpf, and 7dpf untreated larvae show rigid, parallel actinotrichia throughout the pectoral and median fin fold (A, C, G, I, M, O, S, U), with DAPI staining revealing proper fin fold mesenchymal cell migration (Yellow arrow) (B, H, N, T). Note the fin fold mesenchyme elongate along the proximal distal axis, aligning with the actinotrichia (Yellow arrow) (B-C, H-I, N-O, T-U). At 72hpf, and 7dpf, actinotrichia of MTZ-treated larvae are unable to remain rigid and bend within the fin fold (Purple arrow) (D, F, J, L, O, R, V, X). This correlates with fin fold collapse. The actinotrichia are unable to remain parallel to one another, creating gaps within the fin fold (D, F, J, L, O, R, V, X). Note the apparent unbundling of Collagen II stained strands at 72hpf (Purple arrow) (D, J). At 72hpf, DAPI staining reveals fin fold mesenchyme cluster next to the pectoral fin endoskeletal disc and the trunk region proximal to the median fin fold (Teal arrow) (E, K), having failed to migrate correctly. At 7dpf, surviving fin fold mesenchyme fails to migrate correctly (Teal arrow) (Q, W). In the pectoral fin, migration is restricted to the central region of the fin fold (Teal arrow) (Q) and in both the pectoral and median fin, these cells display elongation along various different axes, correlating with actinotrichia defects (Teal arrow) (P-R, V-X). Collagen II staining (A, D, G, J, M, P, S, V), DAPI (B, E, H, K, N, Q, T, W) and merged (C, F, I, L, O, R, U, X) images are presented. ED, Endoskeletal disc, T, Trunk Scale bars: 50μm in A-X.</p

Morphological and migratory defects of the pectoral and median fin fold mesenchyme in <i>Tg(Inta11:NTR)</i> larvae following metronidazole treatment.

<p><b>(A-J)</b> Pectoral and <b>(M-W)</b> median fin of 60, and 72hpf Inta11: NTR + MTZ and Inta11: NTR—MTZ larvae outcrossed with <i>Tg(Inta11-β-globin:eGFP)</i> transgenic larvae. At 60hpf, Inta11: NTR—MTZ show the beginning of fin fold migration in the pectoral fin (white arrow) (A-C). Migration is absent/delayed in the pectoral fin of the Inta11: NTR + MTZ group (yellow arrow) (D-F). At 72hpf, Inta11: NTR + MTZ larvae display reduced fin fold mesenchyme migration in the pectoral fin (J-L) compared to the control (G-I). Fin fold mesenchyme are less elongated/branched and are clustered close to the endoskeletal disk (red arrow) (J-L), compared to control pectoral fins (white arrow) (G-I). At 60, and 72hpf median fin fold mesenchyme of Inta11: NTR + MTZ larvae cluster next to the trunk, and are more round and less elongated/branched (red arrow) (P-R, V-X), compared to control larvae (white arrow) (M-O, S-U). Brightfield (A, D, G, J, M, P, S, V), fluorescence (B, E, H, K, N, Q, T, W), and brightfield/fluorescence merged images (C, F, I, L, O, R, U, X). ED, Endoskeletal disc; T, Trunk. Scale bars: 50μm in A-X.</p

Fin fold collapse in 72hpf, 7dpf pectoral and median fins of <i>Tg(Inta11:NTR)</i> following metronidazole treatment.

<p><b>(A-H)</b> Pectoral and <b>(I-L)</b> median fins of Inta11: NTR + MTZ and Inta11: NTR–MTZ control larvae at 72hpf and 7dpf. Inta11: NTR + MTZ larvae display pectoral fin fold collapse at 72hpf (B, F) and 7dpf (D, H), compared to Inta11: NTR—MTZ (A, C, E, G). Note the collapse of the fin fold (red arrows) (B, D). Panels E-H are magnifications of dotted box in panels A-D. Note the appearance of bending actinotrichia fibrils (yellow arrows) in Inta11: NTR + MTZ larvae (F, H) compared to straight actinotrichia (black arrows) in the Inta11: NTR—MTZ larvae (E, G). Inta11: NTR + MTZ larvae display major median fin fold defects at 72hpf (J) compared to Inta11: NTR—MTZ larvae (I). Note the collapse of the fin fold (red arrows) (J). By 7dpf, Inta11: NTR + MTZ larvae continue to show a reduction in median fin fold size compared to Inta11: NTR—MTZ larvae (K), however defects are ameliorated compared to Inta11: NTR + MTZ larvae at 72hpf (J, L). Note the minor folding of distal tip of the median fin (red arrow) (L). ED, Endoskeletal disc; FF, Fin fold. Scale bars: 100μm in A-D, F, H, I-L; 50μm in E, G.</p

Subset of <i>hoxa13a/hoxd13a</i>-expressing cells specifically ablated in <i>Tg(Inta11:NTR)</i>fish following metronidazole treatment.

<p><b>(A-C, E-I)</b> Median fin fold of 72hpf larvae from 3 treatment groups (2 control, 1 experimental), YFP expression levels and TUNEL assay are shown. <b>(D)</b> Schematic of “Larval 1” treatment, larvae are exposed from 30-60hpf. Median fin morphology unaffected in treatment control groups (WT + MTZ, Inta11: NTR—MTZ) (A, B) compared to Inta11: NTR + MTZ (C). Inat11: NTR + MTZ larvae show median fin fold collapse (black arrow) (C). YFP expression drastically reduced in Inta11: NTR + MTZ larvae (red arrow) (F), when compared to Inta11: NTR—MTZ (green arrow) (E). A small percentage of treated control larvae (10% and 6.66%) display single TUNEL-positive cells in the median fin fold (white arrow) (G, H). All treated Inta11: NTR + MTZ larvae (n = 16) show TUNEL-positive cells in the median fin fold (white arrow) (I). Brightfield (A—C), fluorescence (E, F), and brightfield/fluorescence merged images (G-I). Scale bars: 100μm in A-C, E-I.</p

Laser ablation of the sonic hedgehog-a-expressing cells during fin regeneration affects ray branching morphogenesis

The zebrafish fin is an excellent system to study the mechanisms of dermal bone patterning. Fin rays are segmented structures that form successive bifurcations both during ontogenesis and regeneration. Previous studies showed that sonic hedgehog (shha) may regulate regenerative bone patterning based on its expression pattern and functional analysis. The present study investigates the role of the shha-expressing cells in the patterning of fin ray branches. The shha expression domain in the basal epidermis of each fin ray splits into two prior to ray bifurcation. In addition, the osteoblast proliferation profile follows the dynamic expression pattern of shha. A zebrafish transgenic line, 2.4shh:gfpABC#15, in which GFP expression recapitulates the endogenous expression of shha, was used to specifically ablate shha-expressing cells with a laser beam. Such ablations lead to a delay in the sequence of events leading to ray bifurcation without affecting the overall growth of the fin ray. These results suggest that shha-expressing cells direct localized osteoblast proliferation and thus regulate branching morphogenesis. This study reveals the fin ray as a new accessible system to investigate epithelial–mesenchymal interactions leading to organ branching