Cell proliferation and movement during early fin regeneration in zebrafish.

Cell proliferation and cell movement during early regeneration of zebrafish caudal fins were examined by injecting BrdU and Di-I, respectively. In normal fins of adult fish, a small number of proliferating cells are observed in the epidermis only. Shortly following amputation, epithelial cells covered the wound to form the epidermal cap but did not proliferate. However, by 24 hr, epithelial cells proximal to the level of amputation were strongly labeled with BrdU. Label incorporation was also detected in a few mesenchymal cells. Proliferating cells in the basal epithelial layer were first observed at 48 hr at the level of the newly formed lepidotrichia. At 72 hr, proliferating mesenchymal cells were found distal to the plane of amputation whereas more proximal labeled cells included mainly those located between the lepidotrichia and the basal membrane. When BrdU-injected fins were allowed to regenerate for longer periods, labeled cells were observed in the apical epidermal cap, a location where cells are not thought to proliferate. This result is suggestive of cell migration. Epithelial cells, peripheral to the rays or in the tissue between adjacent rays, were labeled with Di-I and were shown to quickly migrate towards the site of amputation, the cells closer to the wound migrating faster. Amputation also triggered migration of cells of the connective tissue located between the hemirays. Although cell movement was induced up to seven segments proximal from the level of amputation, cells located within two segments from the wound provided the main contribution to the blastema. Thus, cell proliferation and migration contribute to the early regeneration of zebrafish fins.     

Notch signaling regulates venous arterialization during zebrafish fin regeneration

To protect against blood pressure, a mature artery is supported by mural cells which include vascular smooth muscle cells and pericytes. To regenerate a functional vascular system, arteries should be properly reconstructed with mural cells although the mechanisms underlying artery reconstruction remain unclear. In this study, we examined the process of artery reconstruction during regeneration of the zebrafish caudal fin as a model to study arterial formation in an adult setting. During fin regeneration, the arteries and veins form a net‐like vasculature called the vascular plexus, and this plexus undergoes remodeling to form a new artery and two flanking veins. We found that the new vascular plexus originates mainly from venous cells in the stump but very rarely from the arterial cells. Interestingly, these vein‐derived cells contributed to the reconstructed arteries. This arterialization was dependent on Notch signaling, and further analysis showed that Notch signaling was required for the initiation of arterial gene expression. In contrast, venous remodeling did not require Notch signaling. These results provide new insights toward understanding mechanisms of vascular regeneration and illustrate the utility of the adult zebrafish fin to study this process.     

Osteoblast maturation occurs in overlapping proximal‐distal compartments during fin regeneration in zebrafish

During fin regeneration, osteoblasts must continually differentiate for outgrowth of the bony fin rays. Bone maturity increases in a distal‐proximal manner, and osteoblast maturation can be detected similarly when following gene expression. We find that early markers for osteoblast differentiation are expressed in a discrete domain at the distal end of the fin, just proximal to the adjacent germinal compartment of dividing cells. Matrix genes, required at later stages developmentally, are expressed in a population of cells proximally to the early genes. A marker for mature osteoblasts is expressed in cells further proximal. These domains of gene expression are partially overlapping, perhaps revealing additional levels of osteoblast maturity. We suggest a model for growth where new cells are continually added to the distal‐most osteoblast compartment, while osteoblasts in more proximal locations differentiate, thus translating developmental time to location on the proximal‐distal axis. Developmental Dynamics 238:2922–2928, 2009. © 2009 Wiley‐Liss, Inc.     

Elastoidin turn-over during tail fin regeneration in teleosts

Summary During teleostean fin regeneration the actinotrichia, rods of a collagen-like protein, the elastoidin, are immersed in the blastema, maintaining their apical position. In this epimorphic event the latter fact might be achieved by either a cellular carriage or a continuous turn-over of these hyperpolimerized fibrils. By means of a ³H-proline pulse and radioautographic chase experiment of the isolated actinotrichia we have found a turn-over of collagen within the structure. From these and additional morphometric results, we present in this work an operational hypothesis of how gradually differentiating blastemal cells and an appropriately shaped basal lamina, can control the number and distribution of actinotrichia which might be under the balanced control of their synthesis and degradation.     

Early fin primordia of zebrafish larvae regenerate by a similar growth control mechanism with adult regeneration.

Some vertebrate species, including urodele amphibians and teleost fish, have the remarkable ability of regenerating lost body parts. Regeneration studies have been focused on adult tissues, because it is unclear whether or not the repairs of injured tissues during early developmental stages have the same molecular base as that of adult regeneration. Here, we present evidence that a similar cellular and molecular mechanism to adult regeneration operates in the repair process of early zebrafish fin primordia, which are composed of epithelial and mesenchymal cells. We show that larval fin repair occurs through the formation of wound epithelium and blastema-like proliferating cells. Cell proliferation is first induced in the distal-most region and propagates to more proximal regions, as in adult regeneration. We also show that fibroblast growth factor signaling helps induce cell division. Our results suggest that the regeneration machinery directing cell proliferation in response to injury may exist from the early developmental stages.     

Expression of the fras1/frem gene family during zebrafish development and fin morphogenesis

Mouse studies have highlighted the requirement of the extracellular matrix Fras and Frem proteins for embryonic epidermal adhesion. Mutations of the genes encoding some of these proteins underlie the blebs mouse mutants, whereas mutations in human FRAS1 and FREM2 cause Fraser syndrome, a congenital disorder characterized by embryonic blistering and renal defects. We have cloned the zebrafish homologues of these genes and characterized their evolutionary diversification and expression during development. The fish gene complement includes fras1, frem1a, frem1b, frem2a, frem2b, and frem3, which display complex overlapping and complementary expression patterns in developing tissues including the pharyngeal arches, hypochord, musculature, and otic vesicle. Expression during fin development delineates distinct populations of epidermal cells which have previously only been described at a morphological level. We detect relatively little gene expression in epidermis or pronephros, suggesting that the essential role of these proteins in mediating their development in humans and mice is recently evolved.     

Tales of regeneration in zebrafish.

Complex tissue regeneration involves exquisitely coordinated proliferation and patterning of adult cells after severe injury or amputation. Certain lower vertebrates such as urodele amphibians and teleost fish have a greater capacity for regeneration than mammals. However, little is known about molecular mechanisms of regeneration, and cellular mechanisms are incompletely defined. To address this deficiency, we and others have focused on the zebrafish model system. Several helpful tools and reagents are available for use with zebrafish, including the potential for genetic approaches to regeneration. Recent studies have shed light on the remarkable ability of zebrafish to regenerate fins.     

Growth in the larval zebrafish pectoral fin and trunk musculature.

After initial patterning, muscle in the trunk and fins of teleosts grows extensively. Here, we describe muscle growth in zebrafish, with emphasis on the pectoral fin musculature. In the trunk, slow muscle fibers differentiate first. In contrast, slow muscle does not appear in the pectoral fin until the beginning of the juvenile period. Mosaic hyperplasia contributes to trunk muscle growth, and new fibers are apparent within the muscle as early as 6 mm standard length. In the pectoral fin muscle, mosaic hyperplasia is not evident at any examined stage. Instead, the predominant mode of hyperplasia is stratified. In larval pectoral fin muscle new fibers appear subjacent to the skin, and this correlates with the expression of myogenic genes such as muscle regulatory factors and Pax7. Our results suggest that regulation of fiber type development and muscle growth may differ in the pectoral fin and trunk.     

Heparan sulfate proteoglycan expression in the regenerating zebrafish fin

Background

Heparan sulfate proteoglycan (HSPG) expression is found in many animal tissues and regulates growth factor signaling such as of Fibroblast growth factors (Fgf), Wingless/Int (Wnt) and Hedgehog (HH). Glypicans, which are GPI (glycosylphosphatidylinositol)-anchored proteins, and transmembrane-anchored syndecans represent two major HSPG protein families whose involvement in development and disease has been demonstrated. Their participation in regenerative processes both of the central nervous system and of regenerating limbs is well documented. However, whether HSPG are expressed in regenerating zebrafish fins, is currently unknown.

Results

Here, we carried out a systematic screen of glypican and syndecan mRNA expression in regenerating zebrafish fins during the outgrowth phase. We find that 8 of the 10 zebrafish glypicans and the three known zebrafish syndecans show specific expression at 3 days post amputation. Expression is found in different domains of the regenerate, including the distal and lateral basal layers of the wound epidermis, the distal most blastema and more proximal blastema regions.

Conclusions

HSPG expression is prevalent in regenerating zebrafish fins. Further research is needed to delineate the function of glypican and syndecan action during zebrafish fin regeneration.

     

Effect of naproxen on tail fin regeneration in teleost

In the present investigation we studied the effect of naproxen, a non-steroidal anti-inflammatory drug used for the treatment of rheumatic disease, on the synthesis of the lepidotrichial matrix of the tail fin of the teleost fish Cyprinus carpio (carp) during the regeneration process. The lepidotrichial synthesis was observed by standard and polarized light microscopy and by transmission electron microscopy. In general, naproxen at the dose used in the present study did not affect the organization of the extracellular matrix components and the mineralization of the fundamental substance of the lepidotrichia during the process of tail fin regeneration. Since the effect of naproxen, as well as of other non-steroidal anti-inflammatory drugs, depends on the dose used, the route of administration and the metabolism of the animal in which the drug is being tested, higher doses of the drug may perhaps delay or even fully inhibit this process, possibly also provoke disorganization of the lepidotrichial matrix.