New regulators of vertebrate appendage regeneration

Appendage regeneration is a complex and fascinating biological process exhibited in vertebrates by urodele amphibians and teleost fish. A current focus in the field is to identify new molecules that control formation and function of the regeneration blastema, a mass of proliferative mesenchyme that emerges after limb or fin amputation and serves as progenitor tissue for lost structures. Two studies published recently have illuminated new molecular regulators of blastemal proliferation. After amputation of a newt limb, the nerve sheath releases nAG, a blastemal mitogen that facilitates regeneration. In amputated zebrafish fins, regeneration is optimized through depletion of the microRNA miR-133, a mechanism that requires Fgf signaling. These discoveries establish research avenues that may impact the regenerative capacity of mammalian tissues.     

Retinoic acid signaling controls the formation, proliferation and survival of the blastema during adult zebrafish fin regeneration

Adult teleosts rebuild amputated fins through a proliferation-dependent process called epimorphic regeneration, in which a blastema of cycling progenitor cells replaces the lost fin tissue. The genetic networks that control formation of blastema cells from formerly quiescent stump tissue and subsequent blastema function are still poorly understood. Here, we investigated the cellular and molecular consequences of genetically interfering with retinoic acid (RA) signaling for the formation of the zebrafish blastema. We show that RA signaling is upregulated within the first few hours after fin amputation in the stump mesenchyme, where it controls Fgf, Wnt/β-catenin and Igf signaling. Genetic inhibition of the RA pathway at this stage blocks blastema formation by inhibiting cell cycle entry of stump cells and impairs the formation of the basal epidermal layer, a signaling center in the wound epidermis. In the established blastema, RA signaling remains active to ensure the survival of the highly proliferative blastemal population by controlling expression of the anti-apoptotic factor bcl2. In addition, RA signaling maintains blastema proliferation through the activation of growth-stimulatory signals mediated by Fgf and Wnt/β-catenin signaling, as well as by reducing signaling through the growth-inhibitory non-canonical Wnt pathway. The endogenous roles of RA in adult vertebrate appendage regeneration are uncovered here for the first time. They provide a mechanistic framework to understand previous observations in salamanders that link endogenous sources of RA to the regeneration process itself and support the hypothesis that the RA signaling pathway is an essential component of vertebrate tissue regeneration.     

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.     

Expression of FGF2 in the limb blastema of two Salamandridae correlates with their regenerative capability

Limb regenerative potential in urodeles seems to vary among different species. We observed that Triturus vulgaris meridionalis regenerate their limbs significantly faster than T. carnifex, where a long gap between the time of amputation and blastema formation occurs, and tried to identify cellular and molecular events that may underlie these differences in regenerative capability. Whereas wound healing is comparable in the two species, formation of an apical epidermal cap (AEC), which is required for blastema outgrowth, is delayed for approximately three weeks in T. carnifex. Furthermore, fewer nerve fibres are present distally early after amputation, consistent with the late onset of blastemal cell proliferation observed in T. carnifex. We investigated whether different expression of putative blastema mitogens, such as FGF1 and FGF2, in these species may underlie differences in the progression of regeneration. We found that whereas FGF1 is detected in the epidermis throughout the regenerative process, FGF2 onset of expression in the wound epidermis of both species coincides with AEC formation and initiation of blastemal cell proliferation, which is delayed in T. carnifex, and declines thereafter. In vitro studies showed that FGF2 activates MCM3, a factor essential for DNA replication licensing activity, and can be produced by blastemal cells themselves, indicating an autocrine action. These results suggest that FGF2 plays a key role in the initiation of blastema growth.     

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.     

Positional cloning of a temperature-sensitive mutant emmental reveals a role for sly1 during cell proliferation in zebrafish fin regeneration

Here, we used classical genetics in zebrafish to identify temperature-sensitive mutants in caudal fin regeneration. Gross morphological, histological, and molecular analyses revealed that one of these strains, emmental (emm), failed to form a functional regeneration blastema. Inhibition of emm function by heat treatment during regenerative outgrowth rapidly blocked regeneration. This block was associated with reduced proliferation in the proximal blastema and expansion of the nonproliferative distal blastemal zone. Positional cloning revealed that the emm phenotype is caused by a mutation in the orthologue of yeast sly1, a gene product involved in protein trafficking. sly1 is upregulated in the newly formed blastema as well as during regenerative outgrowth. Thus, sly1 is essential for blastemal organization and proliferation during two stages of fin regeneration.     

Fibroblast growth factor pathway component expression in the regenerating zebrafish fin

Adult zebrafish regenerate their appendages (fins) after amputation including the regeneration of bone structures (fin rays). Fibroblast growth factor (Fgf) signaling, which is involved in morphogenetic processes during development, has been shown to be essential for the process of fin regeneration. Moreover, mutations in Fgf pathway component genes lead to abnormal skeletal growth in teleosts and mammals, including humans, illustrating the importance of Fgf signaling in the growth control of tissues. Here, we revisited Fgf signaling pathway component expression by RNA in situ hybridization to test for the expression of about half of the ligands and all receptors of the pathway in the regenerating zebrafish fin. Expression patterns of fgf7, fgf10b, fgf12b, fgf17b and fgfr1b have not been reported in the literature before. We summarize and discuss known and novel localization of expression and find that all five Fgf receptors (fgfr1a, fgfr1b, fgfr2, fgfr3 and fgfr4) and most of the tested ligands are expressed in specific regions of the regenerate. Our work provides a basis to study domain specific functions of Fgf signaling in the regenerating teleost appendage.     

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.     

Fgf signaling instructs position-dependent growth rate during zebrafish fin regeneration

During appendage regeneration in urodeles and teleosts, tissue replacement is precisely regulated such that only the appropriate structures are recovered, a phenomenon referred to as positional memory. It is believed that there exists, or is quickly established after amputation, a dynamic gradient of positional information along the proximodistal (PD) axis of the appendage that assigns region-specific instructions to injured tissue. These instructions specify the amount of tissue to regenerate, as well as the rate at which regenerative growth is to occur. A striking theme among many species is that the rate of regeneration is more rapid in proximally amputated appendages compared with distal amputations. However, the underlying molecular regulation is unclear. Here, we identify position-dependent differences in the rate of growth during zebrafish caudal fin regeneration. These growth rates correlate with position-dependent differences in blastemal length, mitotic index and expression of the Fgf target genes mkp3, sef and spry4. To address whether PD differences in amounts of Fgf signaling are responsible for position-dependent blastemal function, we have generated transgenic fish in which Fgf receptor activity can be experimentally manipulated. We find that the level of Fgf signaling exhibits strict control over target gene expression, blastemal proliferation and regenerative growth rate. Our results demonstrate that Fgf signaling defines position-dependent blastemal properties and growth rates for the regenerating zebrafish appendage.     

Divergent requirements for fibroblast growth factor signaling in zebrafish maxillary barbel and caudal fin regeneration

The zebrafish maxillary barbel is an integumentary organ containing skin, glands, pigment cells, taste buds, nerves, and endothelial vessels. The maxillary barbel can regenerate (LeClair & Topczewski 2010); however, little is known about its molecular regulation. We have studied fibroblast growth factor (FGF) pathway molecules during barbel regeneration, comparing this system to a well‐known regenerating appendage, the zebrafish caudal fin. Multiple FGF ligands (fgf20a, fgf24), receptors (fgfr1‐4) and downstream targets (pea3, il17d) are expressed in normal and regenerating barbel tissue, confirming FGF activation. To test if specific FGF pathways were required for barbel regeneration, we performed simultaneous barbel and caudal fin amputations in two temperature‐dependent zebrafish lines. Zebrafish homozygous for a point mutation in fgf20a, a factor essential for caudal fin blastema formation, regrew maxillary barbels normally, indicating that the requirement for this ligand is appendage‐specific. Global overexpression of a dominant negative FGF receptor, Tg(hsp70l:dn‐fgfr1:EGFP)^pd1 completely blocked fin outgrowth but only partially inhibited barbel outgrowth, suggesting reduced requirements for FGFs in barbel tissue. Maxillary barbels expressing dn‐fgfr1 regenerated peripheral nerves, dermal connective tissue, endothelial tubes, and a glandular epithelium; in contrast to a recent report in which dn‐fgfr1 overexpression blocks pharyngeal taste bud formation in zebrafish larvae (Kapsimali et al. 2011), we observed robust formation of calretinin‐positive tastebuds. These are the first experiments to explore the molecular mechanisms of maxillary barbel regeneration. Our results suggest heterogeneous requirements for FGF signaling in the regeneration of different zebrafish appendages (caudal fin versus maxillary barbel) and taste buds of different embryonic origin (pharyngeal endoderm versus barbel ectoderm).     

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.     

SDF-1α/CXCR4 signaling mediates digit tip regeneration promoted by BMP-2

Previously we demonstrated that BMP signaling is required for endogenous digit tip regeneration, and that treatment with BMP-2 or -7 induces a regenerative response following amputation at regeneration-incompetent levels (Yu et al., 2010 and Yu et al., 2012). Both endogenous regeneration and BMP-induced regeneration are associated with the transient formation of a blastema, however the formation of a regeneration blastema in mammals is poorly understood. In this study, we focus on how blastema cells respond to BMP signaling during neonatal digit regeneration in mice. First, we show that blastema cells retain regenerative properties after expansion in vitro, and when re-introduced into the amputated digit, these cells display directed migration in response to BMP-2. However, in vitro studies demonstrate that BMP-2 alone does not influence blastema cell migration, suggesting a requirement of another pivotal downstream factor for cell recruitment. We show that blastema cell migration is stimulated by the cytokine, SDF-1α, and that SDF-1α is expressed by the wound epidermis as well as endothelial cells of the blastema. Blastema cells express both SDF-1α receptors, CXCR4 and CXCR7, although the migration response is inhibited by the CXCR4-specific antagonist, AMD3100. Mice treated with AMD3100 display a partial inhibition of skeletal regrowth associated with the regeneration response. We provide evidence that BMP-2 regulates Sdf-1α expression in endothelial cells but not cells of the wound epidermis. Finally, we show that SDF-1α-expressing COS1 cells engrafted into a regeneration-incompetent digit amputation wound resulted in a locally enhanced population of CXCR4 positive cells, and induced a partial regenerative response. Taken together, this study provides evidence that one downstream mechanism of BMP signaling during mammalian digit regeneration involves activation of SDF-1α/CXCR4 signaling by endothelial cells to recruit blastema cells.     

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.     

Analysis of gene expressions during Xenopus forelimb regeneration

Xenopus laevis can regenerate an amputated limb completely at early limb bud stages, but the metamorphosed froglet gradually loses this capacity and can regenerate only a spike-like structure. We show that the spike formation in a Xenopus froglet is nerve dependent as is limb regeneration in urodeles, since denervation concomitant with amputation is sufficient to inhibit the initiation of blastema formation and fgf8 expression in the epidermis. Furthermore, in order to determine the cause of the reduction in regenerative capacity, we examined the expression patterns of several key genes for limb patterning during the spike-like structure formation, and we compared them with those in developing and regenerating limb buds that produce a complete limb structure. We cloned Xenopus HoxA13, a marker of the prospective autopodium region, and the expression pattern suggested that the spike-like structure in froglets is accompanied by elongation and patterning along the proximodistal (PD) axis. On the other hand, shh expression was not detected in the froglet blastema, which expresses fgf8 and msx1. Thus, although the wound epidermis probably induces outgrowth of the froglet blastema, the polarizing activity that organizes the anteroposterior (AP) axis formation is likely to be absent there. Our results demonstrate that the lost region in froglet limbs is regenerated along the PD axis and that the failure of organization of the AP pattern gives rise to a spike-like incomplete structure in the froglet, suggesting a relationship between regenerative capacity and AP patterning. These findings lead us to conclude that the spike formation in postometamorphic Xenopus limbs is epimorphic regeneration.     

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.     

Unraveling tissue regeneration pathways using chemical genetics

Identifying the molecular pathways that are required for regeneration remains one of the great challenges of regenerative medicine. Although genetic mutations have been useful for identifying some molecular pathways, small molecule probes of regenerative pathways might offer some advantages, including the ability to disrupt pathway function with precise temporal control. However, a vertebrate regeneration model amenable to rapid throughput small molecule screening is not currently available. We report here the development of a zebrafish early life stage fin regeneration model and its use in screening for small molecules that modulate tissue regeneration. By screening 2000 biologically active small molecules, we identified 17 that specifically inhibited regeneration. These compounds include a cluster of glucocorticoids, and we demonstrate that transient activation of the glucocorticoid receptor is sufficient to block regeneration, but only if activation occurs during wound healing/blastema formation. In addition, knockdown of the glucocorticoid receptor restores regenerative capability to nonregenerative, glucocorticoid-exposed zebrafish. To test whether the classical anti-inflammatory action of glucocorticoids is responsible for blocking regeneration, we prevented acute inflammation following amputation by antisense repression of the Pu.1 gene. Although loss of Pu.1 prevents the inflammatory response, regeneration is not affected. Collectively, these results indicate that signaling from exogenous glucocorticoids impairs blastema formation and limits regenerative capacity through an acute inflammation-independent mechanism. These studies also demonstrate the feasibility of exploiting chemical genetics to define the pathways that govern vertebrate regeneration.     

A dynamic epicardial injury response supports progenitor cell activity during zebrafish heart regeneration

Zebrafish possess a unique yet poorly understood capacity for cardiac regeneration. Here, we show that regeneration proceeds through two coordinated stages following resection of the ventricular apex. First a blastema is formed, comprised of progenitor cells that express precardiac markers, undergo differentiation, and proliferate. Second, epicardial tissue surrounding both cardiac chambers induces developmental markers and rapidly expands, creating a new epithelial cover for the exposed myocardium. A subpopulation of these epicardial cells undergoes epithelial-to-mesenchymal transition (EMT), invades the wound, and provides new vasculature to regenerating muscle. During regeneration, the ligand fgf17b is induced in myocardium, while receptors fgfr2 and fgfr4 are induced in adjacent epicardial-derived cells. When fibroblast growth factors (Fgf) signaling is experimentally blocked by expression of a dominant-negative Fgf receptor, epicardial EMT and coronary neovascularization fail, prematurely arresting regeneration. Our findings reveal injury responses by myocardial and epicardial tissues that collaborate in an Fgf-dependent manner to achieve cardiac regeneration.