Mps1 defines a proximal blastemal proliferative compartment essential for zebrafish fin regeneration

One possible reason why regeneration remains enigmatic is that the dominant organisms used for studying regeneration are not amenable to genetic approaches. We mutagenized zebrafish and screened for temperature-sensitive defects in adult fin regeneration. The nightcap mutant showed a defect in fin regeneration that was first apparent at the onset of regenerative outgrowth. Positional cloning revealed that nightcap encodes the zebrafish orthologue of mps1, a kinase required for the mitotic checkpoint. mps1 expression was specifically induced in the proximal regeneration blastema, a group of cells that normally proliferate intensely during outgrowth. The nightcap mutation caused severe defects in these cells. However, msxb-expressing blastemal cells immediately distal to this proliferative region did not induce mps1 and were retained in mutants. These results indicate that the proximal blastema comprises an essential subpopulation of the fin regenerate defined by the induction and function of Mps1. Furthermore, we show that molecular mechanisms of complex tissue regeneration can now be dissected using zebrafish genetics.     

Temperature-Sensitive Mutations That Cause Stage-Specific Defects in Zebrafish Fin Regeneration

When amputated, the fins of adult zebrafish rapidly regenerate the missing tissue. Fin regeneration proceeds through several stages, including wound healing, establishment of the wound epithelium, recruitment of the blastema from mesenchymal cells underlying the wound epithelium, and differentiation and outgrowth of the regenerate. We screened for temperature-sensitive mutations that affect the regeneration of the fin. Seven mutations were identified, including five that fail to regenerate their fins, one that causes slow growth during regeneration, and one that causes dysmorphic bumps or tumors to develop in the regenerating fin. reg5 mutants fail to regenerate their caudal fins, whereas reg6 mutants develop dysmorphic bumps in their regenerates at the restrictive temperature. Temperature-shift experiments indicate that reg5 and reg6 affect different stages of regeneration. The critical period for reg5 occurs during the early stages of regeneration before or during establishment of the blastema, resulting in defects in subsequent growth of the blastema and failure to differentiate bone-forming cells. The critical period for reg6 occurs after the onset of bone differentiation and during early stages of regenerative outgrowth. Both reg5 and reg6 also show temperature-sensitive defects in embryonic development or in ontogenetic outgrowth of the juvenile fin.     

Both Hoxc13 orthologs are functionally important for zebrafish tail fin regeneration

Hox genes are re-expressed during regeneration in many species. Given their important role in body plan development, it has been assumed, but not directly shown, that they play a functional role in regeneration. In this paper we show that morpholino-mediated knockdown of either Hoxc13a or Hoxc13b during the process of zebrafish tail fin regeneration results in a significant reduction of regenerative outgrowth. Furthermore, cellular proliferation within the blastema is directly affected in both knockdowns. Hence, similar to the demonstration of unique functions of multiple Hox genes during limb formation, both Hoxc13 orthologs have distinct functions in regeneration.     

Nerve-dependent and -independent events in blastema formation during Xenopus froglet limb regeneration

Blastema formation, the initial stage of epimorphic limb regeneration in amphibians, is an essential process to produce regenerates. In our study on nerve dependency of blastema formation, we used forelimb of Xenopus laevis froglets as a system and applied some histological and molecular approaches in order to determine early events during blastema formation. We also investigated the lateral wound healing in comparison to blastema formation in limb regeneration. Our study confirmed at the molecular level that there are nerve-dependent and -independent events during blastema formation after limb amputation, Tbx5 and Prx1, reliable markers of initiation of limb regeneration, that start to be expressed independently of nerve supply, although their expressions cannot be maintained without nerve supply. We also found that cell proliferation activity, cell survival and expression of Fgf8, Fgf10 and Msx1 in the blastema were affected by denervation, suggesting that these events specific for blastema outgrowth are controlled by the nerve supply. Wound healing, which is thought to be categorized into tissue regeneration, shares some nerve-independent events with epimorphic limb regeneration, although the healing process results in simple restoration of wounded tissue. Overall, our results demonstrate that dedifferentiated blastemal cells formed at the initial phase of limb regeneration must enter the nerve-dependent epimorphic phase for further processes, including blastema outgrowth, and that failure of entry results in a simple redifferentiation as tissue regeneration.     

The regenerative capacity of the zebrafish caudal fin is not affected by repeated amputations

Background

The zebrafish has the capacity to regenerate many tissues and organs. The caudal fin is one of the most convenient tissues to approach experimentally due to its accessibility, simple structure and fast regeneration. In this work we investigate how the regenerative capacity is affected by recurrent fin amputations and by experimental manipulations that block regeneration.

Methodology/Principal Findings

We show that consecutive repeated amputations of zebrafish caudal fin do not reduce its regeneration capacity and do not compromise any of the successive regeneration steps: wound healing, blastema formation and regenerative outgrowth. Interfering with Wnt/ß-catenin signalling using heat-shock-mediated overexpression of Dickkopf1 completely blocks fin regeneration. Notably, if these fins were re-amputated at the non-inhibitory temperature, the regenerated caudal fin reached the original length, even after several rounds of consecutive Wnt/ß-catenin signalling inhibition and re-amputation.

Conclusions/Significance

We show that the caudal fin has an almost unlimited capacity to regenerate. Even after inhibition of regeneration caused by the loss of Wnt/ß-catenin signalling, a new amputation resets the regeneration capacity within the caudal fin, suggesting that blastema formation does not depend on a pool of stem/progenitor cells that require Wnt/ß-catenin signalling for their survival.     

In vivo Electroporation of Morpholinos into the Regenerating Adult Zebrafish Tail Fin

Certain species of urodeles and teleost fish can regenerate their tissues. Zebrafish have become a widely used model to study the spontaneous regeneration of adult tissues, such as the heart¹, retina², spinal cord³, optic nerve⁴, sensory hair cells⁵, and fins⁶.The zebrafish fin is a relatively simple appendage that is easily manipulated to study multiple stages in epimorphic regeneration. Classically, fin regeneration was characterized by three distinct stages: wound healing, blastema formation, and fin outgrowth. After amputating part of the fin, the surrounding epithelium proliferates and migrates over the wound. At 33 °C, this process occurs within six hours post-amputation (hpa, Figure 1B)^6,7. Next, underlying cells from different lineages (ex. bone, blood, glia, fibroblast) re-enter the cell cycle to form a proliferative blastema, while the overlying epidermis continues to proliferate (Figure 1D)⁸. Outgrowth occurs as cells proximal to the blastema re-differentiate into their respective lineages to form new tissue (Figure 1E)⁸. Depending on the level of the amputation, full regeneration is completed in a week to a month.The expression of a large number of gene families, including wnt, hox, fgf, msx, retinoic acid, shh, notch, bmp, and activin-betaA genes, is up-regulated during specific stages of fin regeneration^9-16. However, the roles of these genes and their encoded proteins during regeneration have been difficult to assess, unless a specific inhibitor for the protein exists¹³, a temperature-sensitive mutant exists or a transgenic animal (either overexpressing the wild-type protein or a dominant-negative protein) was generated^7,12. We developed a reverse genetic technique to quickly and easily test the function of any gene during fin regeneration.Morpholino oligonucleotides are widely used to study loss of specific proteins during zebrafish, Xenopus, chick, and mouse development^17-19. Morpholinos basepair with a complementary RNA sequence to either block pre-mRNA splicing or mRNA translation. We describe a method to efficiently introduce fluorescein-tagged antisense morpholinos into regenerating zebrafish fins to knockdown expression of the target protein. The morpholino is micro-injected into each blastema of the regenerating zebrafish tail fin and electroporated into the surrounding cells. Fluorescein provides the charge to electroporate the morpholino and to visualize the morpholino in the fin tissue.This protocol permits conditional protein knockdown to examine the role of specific proteins during regenerative fin outgrowth. In the Discussion, we describe how this approach can be adapted to study the role of specific proteins during wound healing or blastema formation, as well as a potential marker of cell migration during blastema formation.     

Comparative Expression Profiling Reveals an Essential Role for Raldh2 in Epimorphic Regeneration

Zebrafish have the remarkable ability to regenerate body parts including the heart and fins by a process referred to as epimorphic regeneration. Recent studies have illustrated that similar to adult zebrafish, early life stage larvae also possess the ability to regenerate the caudal fin. A comparative microarray analysis was used to determine the degree of conservation in gene expression among the regenerating adult caudal fin, adult heart, and larval fin. Results indicate that these tissues respond to amputation/injury with strikingly similar genomic responses. Comparative analysis revealed raldh2, a rate-limiting enzyme for the synthesis of retinoic acid, as one of the most highly induced genes across the three regeneration platforms. In situ localization and functional studies indicate that raldh2 expression is critical for the formation of wound epithelium and blastema. Patterning during regenerative outgrowth was considered to be the primary function of retinoic acid signaling; however, our results suggest that it is also required for early stages of tissue regeneration. Expression of raldh2 is regulated by Wnt and fibroblast growth factor/ERK signaling.     

Dynamics of actinotrichia regeneration in the adult zebrafish fin

The skeleton of adult zebrafish fins comprises lepidotrichia, which are dermal bones of the rays, and actinotrichia, which are non-mineralized spicules at the distal margin of the appendage. Little is known about the regenerative dynamics of the actinotrichia-specific structural proteins called Actinodins. Here, we used immunofluorescence analysis to determine the contribution of two paralogous Actinodin proteins, And1/2, in regenerating fins. Both proteins were detected in the secretory organelles in the mesenchymal cells of the blastema, but only And1 was detected in the epithelial cells of the wound epithelium. The analysis of whole mount fins throughout the entire regenerative process and longitudinal sections revealed that And1-positive fibers are complementary to the lepidotrichia. The analysis of another longfin fish, a gain-of-function mutation in the potassium channel kcnk5b, revealed that the long-fin phenotype is associated with an extended size of actinotrichia during homeostasis and regeneration. Finally, we investigated the role of several signaling pathways in actinotrichia formation and maintenance. This revealed that the pulse-inhibition of either TGFβ/Activin-βA or FGF are sufficient to impair deposition of Actinodin during regeneration. Thus, the dynamic turnover of Actinodin during fin regeneration is regulated by multiple factors, including the osteoblasts, growth rate in a potassium channel mutant, and instructive signaling networks between the epithelium and the blastema of the regenerating fin.     

Large-scale analysis of the genes involved in fin regeneration and blastema formation in the medaka, Oryzias latipes

Medaka is an attractive model to study epimorphic regeneration. The fins have remarkable regenerative capacity and are replaced about 14 days after amputation. The formation of blastema, a mass of undifferentiated cells, is essential for regeneration; however, the molecular mechanisms are incompletely defined. To identify the genes required for fin regeneration, especially for blastema formation, we constructed cDNA libraries from fin regenerates at 3 days postamputation and 10 days postamputation. A total of 16,866 expression sequence tags (ESTs) were sequenced and subjected to BLASTX analysis. The result revealed that about 60% of them showed strong matches to previously identified proteins, and major signaling molecules related to development, including FGF, BMP, Wnt, Notch/Delta, and Ephrin/Eph signaling pathways were isolated. To identify novel genes that showed specific expression during fin regeneration, cDNA microarray was generated based on 2900 independent ESTs from each library which had no sequence similarity to known proteins. We obtained 6 candidate genes associated with blastema formation by gene expression pattern screening in competitive hybridization analyses and in situ hybridization. Olrfe16d23 and olrfe14k04 were expressed only in early regenerating stages when blastema formation was induced. The expression of olrf5n23, which encodes a novel signal peptide, was detected in wound epidermis throughout regeneration. Olrfe23l22, olrfe20n22, and olrfe24i02 were expressed notably in the blastema region. Our study has thus identified the gene expression profiles and some novel candidate genes to facilitate elucidation of the molecular mechanisms of fin regeneration.     

The regenerating tail of lizard transits through a tumour-like stage represented by the regenerative blastema

Review. The regenerating tail of lizard transits through a tumour-like stage represented by the regenerative blastema. Acta Zoologica (Stockolm). Molecular studies on lizard tail regeneration indicate that the blastema stage is a tumour-like outgrowth capable of self-regulate to produce a new tail. Various oncogenes and tumour suppressors are expressed, and their proteins are localized in specific regions of the growing blastema. SnoRNAs are exclusively overexpressed in the tail blastema suggesting changes in ribosome translation efficiency in blastema cells, like in cancer. Blastema cells secrete high levels of hyaluronate and adopt an anaerobic metabolism (Warburg effect). These studies indicate that the lizard blastema represents a unique case among terrestrial vertebrates of physiological tumour remission. Mesenchymal cells and fibroblasts forming the blastema are turned within 1–2 months into a functional organ, the tail. In vitro studies on isolated mesenchymal cells from the regenerative blastema shows that these cells do not undergo contact inhibition but continue proliferation after confluence, and contain nestin, vimentin and K17. After 2–3 weeks they stratify into 5–7 layers forming a pellicle of loose connective tissue. Future molecular studies on genes and proteins that allow the control of growth in the lizard blastema may help to determine how lizards turn a tumour into a new organ with numerous differentiated and functional tissues, providing clues on cancer growth regulation.     

The chemokine SDF-1 regulates blastema formation during zebrafish fin regeneration

The work presented in this study focuses on blastema formation in epimorphic regeneration. We describe the expression pattern of Sdf1a and Sdf1b (the chemokines stromal-cell-derived factor-1a and 1b) and their two receptors Cxcr4a and Cxcr4b during zebrafish fin regeneration. We demonstrate that Sdf1a/Cxcr4a plays a critical role in fin regeneration and more precisely in epidermal cell proliferation, an important process for blastema formation. In mammals, a single cxcr4 gene is involved both in chemotaxis and cell proliferation and survival; we discuss in this study a possible functional division of the two cxcr4 zebrafish genes.     

Three genes control the timing, the site and the size of blastema formation in Drosophila

Regeneration is a vital process to maintain and repair tissues. Despite the importance of regeneration, the genes responsible for regenerative growth remain largely unknown. In Drosophila, imaginal disc regeneration can be induced either by fragmentation and in vivo culture or in situ by ubiquitous expression of wingless (wg/wnt1). Imaginal discs, like appendages in lower vertebrates, initiate regeneration by wound healing and proliferation at the wound site, forming a regeneration blastema. Most blastema cells maintain their disc-specific identity during regeneration; a few cells however, exhibit stem-cell like properties and switch to a different fate, in a phenomenon known as transdetermination. We identified three genes, regeneration (rgn), augmenter of liver regeneration (alr) and Matrix metalloproteinase-1 (Mmp1) expressed specifically in blastema cells during disc regeneration. Mutations in these genes affect both fragmentation- and wg-induced regeneration by either delaying, reducing or positioning the regeneration blastema. In addition to the modifications of blastema homeostasis, mutations in the three genes alter the rate of regeneration-induced transdetermination. We propose that these genes function in regenerative proliferation, growth and regulate cellular plasticity.