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.     

A transitional extracellular matrix instructs cell behavior during muscle regeneration

Urodele amphibians regenerate appendages through the recruitment of progenitor cells into a blastema that rebuilds the lost tissue. Blastemal formation is accompanied by extensive remodeling of the extracellular matrix. Although this remodeling process is important for appendage regeneration, it is not known whether the remodeled matrix directly influences the generation and behavior of blastemal progenitor cells. By integrating in vivo 3-dimensional spatiotemporal matrix maps with in vitro functional time-lapse imaging, we show that key components of this dynamic matrix, hyaluronic acid, tenascin-C and fibronectin, differentially direct cellular behaviors including DNA synthesis, migration, myotube fragmentation and myoblast fusion. These data indicate that both satellite cells and fragmenting myofibers contribute to the regeneration blastema and that the local extracellular environment provides instructive cues for the regenerative process. The fact that amphibian and mammalian myoblasts exhibit similar responses to various matrices suggests that the ability to sense and respond to regenerative signals is evolutionarily conserved.     

ERK signaling controls blastema cell differentiation during planarian regeneration

The robust regenerative ability of planarians depends on a population of somatic stem cells called neoblasts, which are the only mitotic cells in adults and are responsible for blastema formation after amputation. The molecular mechanism underlying neoblast differentiation associated with blastema formation remains unknown. Here, using the planarian Dugesia japonica we found that DjmkpA, a planarian mitogen-activated protein kinase (MAPK) phosphatase-related gene, was specifically expressed in blastema cells in response to increased extracellular signal-related kinase (ERK) activity. Pharmacological and genetic [RNA interference (RNAi)] approaches provided evidence that ERK activity was required for blastema cells to exit the proliferative state and undergo differentiation. By contrast, DjmkpA RNAi induced an increased level of ERK activity and rescued the differentiation defect of blastema cells caused by pharmacological reduction of ERK activity. These observations suggest that ERK signaling plays an instructive role in the cell fate decisions of blastema cells regarding whether to differentiate or not, by inducing DjmkpA as a negative regulator of ERK signaling during planarian regeneration.     

A fine structural study of regeneration after fission in the planarian Dugesia japonica

We examined morphologically the process of regeneration before and after fission in a sexual strain of the freshwater planarian Dugesia japonica. Usually fission takes place in the post-pharyngeal region. Decapitation significantly accelerates the rate of fissioning. When decapitated worms were treated with substance P and neuropeptide K separately, the rate of fission markedly decreased in both cases. Before the onset of fission, a presumptive region of fission was recognized in the post-pharyngeal portion where undifferentiated cells, regenerative cells and newly differentiated cells were localized. Moreover a functional network of fixed parenchyma cells was noted in this region. After fission, cell distribution in the blastema became quite different from that of artificially amputated worms. This difference seems to be due to the process that occurs in the presumptive region of fission. This revised version was published online in July 2006 with corrections to the Cover Date.     

Role of c-Jun N-terminal kinase activation in blastema formation during planarian regeneration

The robust regenerative abilities of planarians absolutely depend on a unique population of pluripotent stem cells called neoblasts, which are the only mitotic somatic cells in adult planarians and are responsible for blastema formation after amputation. Little is known about the molecular mechanisms that drive blastema formation during planarian regeneration. Here we found that treatment with the c-Jun N-terminal kinase (JNK) inhibitor SP600125 blocked the entry of neoblasts into the M-phase of the cell cycle, while allowing neoblasts to successfully enter S-phase in the planarian Dugesia japonica. The rapid and efficient blockage of neoblast mitosis by treatment with the JNK inhibitor provided a method to assess whether temporally regulated cell cycle activation drives blastema formation during planarian regeneration. In the early phase of blastema formation, activated JNK was detected prominently in a mitotic region (the "postblastema") proximal to the blastema region. Furthermore, we demonstrated that undifferentiated mitotic neoblasts in the postblastema showed highly activated JNK at the single cell level. JNK inhibition by treatment with SP600125 during this period caused a severe defect of blastema formation, which accorded with a drastic decrease of mitotic neoblasts in regenerating animals. By contrast, these animals still retained many undifferentiated neoblasts near the amputation stump. These findings suggest that JNK signaling plays a crucial role in feeding into the blastema neoblasts for differentiation by regulating the G2/M transition in the cell cycle during planarian regeneration.     

Planarian fibroblast growth factor receptor homologs expressed in stem cells and cephalic ganglions

The strong regenerative capacity of planarians is considered to reside in the totipotent somatic stem cell called the 'neoblast'. However, the signal systems regulating the differentiation/growth/migration of stem cells remain unclear. The fibroblast growth factor (FGF)/FGF receptor (FGFR) system is thought to mediate various developmental events in both vertebrates and invertebrates. We examined the molecular structures and expression of DjFGFR1 and DjFGFR2, two planarian genes closely related to other animal FGFR genes. DjFGFR1 and DjFGFR2 proteins contain three and two immunoglobulin-like domains, respectively, in the extracellular region and a split tyrosine kinase domain in the intracellular region. Expression of DjFGFR1 and DjFGFR2 was observed in the cephalic ganglion and mesenchymal space in intact planarians. In regenerating planarians, accumulation of DjFGFR1-expressing cells was observed in the blastema and in fragments regenerating either a pharynx or a brain. In X-ray-irradiated planarians, which had lost regenerative capacity, the number of DjFGFR1-expressing cells in the mesenchymal space decreased markedly. These results suggest that the DjFGFR1 protein may be involved in the signal systems controlling such aspects of planarian regeneration as differentiation/growth/migration of stem cells.     

Dose-dependent disruptive effects of melatonin,light and temperature on regeneration in the planarian Phagocata gracilis

Many critical cellular processes, including cell proliferation and tissue repair, exhibit melatonin sensitivity. We examined the effects of exogenous melatonin, photoperiod and temperature on regeneration in Phagocata gracilis, a common North American turbellarian. Planarian regeneration involves the proliferation and migration of pluripotent stem cells capable of replacing missing body components. A characteristic mass of cells (blastema) forms during planarian regeneration, and its formation, growth and differentiation provide reliable endpoints for the study of regenerative processes. We tested the effects of exogenous melatonin at two concentrations and three photoperiods on both head and tail regenerates under seasonal diurnal temperature regimes. High temperatures and melatonin treatment increased mortality in P. gracilis, and surviving melatonin-treated subjects exhibited delayed regeneration. Asymmetrical and deficient regeneration was more common in melatonin-treated, high temperature and dark treated subjects. Additionally, high temperature and melatonin induced a novel locomotor dysfunction.     

The phorbol ester TPA dramatically inhibits planarian regeneration

The effect of phorbol ester TPA (12-O-tetradecanoylphorbol-13-acetate) on head regeneration in decapitated planarians (Dugesia lugubris s.1.) has been studied. TPA-treatment soon after head amputation dramatically inhibited the regenerative process. Ultrastructural analysis revealed that the migration of fixed parenchymal cells (FPCs) to the wound area was strongly activated by TPA. FPCs interacted with various types of cells inducing lysis and phagocyting cell debris. The resulting fluid was removed through diaphanous protrusions appearing at the level of the wound zone. Moreover the close association of FPCs with neoblastlike cell clusters in the parenchyma indicated their possible role in the modulation of neoblast migration.     

Effects of a Carcinogen, N-methyl-N'-nitro-N-nitrosoguanidine on Blastema Cells and Blastema Formation in Newt Limb Regeneration

The effects of a potent, carcinogen, N -methyl- N' -nitro- N -nitrosoguanidine (MNNG) on blastema formation and blastema cells were studied in the adult newt by means of scanning electron microscopy. By administration of MNNG to the blastema of the limbs the formation of the basement lamella, which lies between the epithelium and the mesenchyme, was effectively inhibited at least for a month. Basement lamella in treated limbs is formed 30 or 40 days after carcinogen administration. MNNG altered significantly cell surface morphology and cell motility of blastema cells isolated in vitro. Membrane's specializations such as the formation of filopodia were inhibited. Motility of the treated cells was much reduced compared to the control. The role of these alterations in the carcinogen-induced abnormal regeneration is discussed.     

Exploring the role of microRNAs in axolotl regeneration

The axolotl, Ambystoma mexicanum, is used extensively for research in developmental biology, particularly for its ability to regenerate and restore lost organs, including in the nervous system, to full functionality. Regeneration in mammals typically depends on the healing process and scar formation with limited replacement of lost tissue. Other organisms, such as spiny mice (Acomys cahirinus), salamanders, and zebrafish, are able to regenerate some damaged body components. Blastema is a tissue that is formed after tissue injury in such organisms and is composed of progenitor cells or dedifferentiated cells that differentiate into various cell types during regeneration. Thus, identifying the molecules responsible for initiation of blastema formation is an important aspect for understanding regeneration. Introns, a major source of noncoding RNAs (ncRNAs), have characteristic sizes in the axolotl, particularly in genes associated with development. These ncRNAs, particularly microRNAs (miRNAs), exhibit dynamic regulation during regeneration. These miRNAs play an essential role in timing and control of gene expression to order and organize processes necessary for blastema creation. Master keys or molecules that underlie the remarkable regenerative abilities of the axolotl remain to be fully explored and exploited. Further and ongoing research on regeneration promises new knowledge that may allow improved repair and renewal of human tissues.     

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.     

Migration of mesenchymal cell fated to blastema is necessary for fish fin regeneration

Urodeles and fish have higher regeneration ability in a variety of tissues and organs than do other vertebrate species including mammals. Though many studies have aimed at identifying the cellular and molecular basis for regeneration, relatively little is known about the detailed cellular behaviors and involved molecular basis. In the present study, a small molecule inhibitor was used to analyzed the role of phosphoinositide 3-kinase (PI3K) signaling during regeneration. We showed that the inhibitor disrupted the formation of blastema including the expression of characteristic genes. The failure of blastema formation was due to the impaired migration of mesenchymal cells to the distal prospective blastema region, although it had a little affect on cell cycle activation in mesenchymal cells. Moreover, we found that the epidermal remodeling including cell proliferation, distal cell migration and Akt phosphorylation was also affected by the inhibitor, implying a possible involvement of epidermis for proper formation of blastema. From these data, we propose a model in which distinct signals that direct the cell cycle activation, mesenchymal cell migration and epidermal remodeling coordinate together to accomplish the correct blastema formation and regeneration.     

Paxillin mediates sensing of physical cues and regulates directional cell motility by controlling lamellipodia positioning

Physical interactions between cells and the extracellular matrix (ECM) guide directional migration by spatially controlling where cells form focal adhesions (FAs), which in turn regulate the extension of motile processes. Here we show that physical control of directional migration requires the FA scaffold protein paxillin. Using single-cell sized ECM islands to constrain cell shape, we found that fibroblasts cultured on square islands preferentially activated Rac and extended lamellipodia from corner, rather than side regions after 30 min stimulation with PDGF, but that cells lacking paxillin failed to restrict Rac activity to corners and formed small lamellipodia along their entire peripheries. This spatial preference was preceded by non-spatially constrained formation of both dorsal and lateral membrane ruffles from 5-10 min. Expression of paxillin N-terminal (paxN) or C-terminal (paxC) truncation mutants produced opposite, but complementary, effects on lamellipodia formation. Surprisingly, pax-/- and paxN cells also formed more circular dorsal ruffles (CDRs) than pax+ cells, while paxC cells formed fewer CDRs and extended larger lamellipodia even in the absence of PDGF. In a two-dimensional (2D) wound assay, pax-/- cells migrated at similar speeds to controls but lost directional persistence. Directional motility was rescued by expressing full-length paxillin or the N-terminus alone, but paxN cells migrated more slowly. In contrast, pax-/- and paxN cells exhibited increased migration in a three-dimensional (3D) invasion assay, with paxN cells invading Matrigel even in the absence of PDGF. These studies indicate that paxillin integrates physical and chemical motility signals by spatially constraining where cells will form motile processes, and thereby regulates directional migration both in 2D and 3D. These findings also suggest that CDRs may correspond to invasive protrusions that drive cell migration through 3D extracellular matrices.     

Quantitative analysis of cell types during growth,degrowth and regeneration in the planarians Dugesia mediterranea and Dugesia tigrina

A method of tissue maceration (dissociation) of planarian tissues into single cells was used to characterize the basic cell types in the planarians Dugesia mediterranea and Dugesia tigrina, and to determine the total cell number and distribution of cell types during growth, degrowth and regeneration.Using this method, 13 basic cell types have been determined for both species. The total number of cells increases with body length and volume whereas the distribution of cell types is only slightly affected. Growth and degrowth occur mainly through changes in total cell number leaving cell distribution only moderately affected. During regeneration, an increase in neoblast density in the blastema followed later on by increases in nerve cells are the more significant changes detected.These results are discussed in relation to mechanisms of cell renewal, blastema formation and maintenance of tissue polarity.Abbreviations nb neoblasts - nv nerve cells - ep epidermal cells - fp fixed parenchyma cells - g gastrodermal cells     

General characterization of regeneration in Aeolosoma viride (Annelida,Aeolosomatidae)

Regeneration has long been the focus of scientific interest for its potential to restore lost, damaged, or aged tissues and organs. A wide range of regenerative studies have been conducted on different vertebrate and invertebrate model organisms. Annelids are known for their regenerative capacities, and because of their relatively complex organ systems, they are an ideal organism for regeneration study. Our present work focused on the freshwater annelid Aeolosoma viride, an asexually reproducing annelid capable of regenerating both anteriorly and posteriorly. Even though regenerative ability has been documented in this animal in previous studies, detailed characterization of the process is still unavailable. The objective of this study was to evaluate the regenerative ability of A. viride. We described the sequential morphological events during the process of regeneration, such as wound healing and the formation of blastema, mouth, and pygidium. In order to clarify the capacity and type of regeneration, we conducted a series of observations and experiments using a cell proliferation assay. Massive proliferation and the absence of cell migration indicated that the animal regenerates primarily through epimorphosis. Our study of the epimorphic regenerative process of A. viride provides a clearer picture of the evolutionary origin of regeneration in annelids.     

The Fine Structure of Blastema Cells and Differentiating Cartilage Cells in Regenerating Limbs of Amblystoma Larvae

Regenerating forelimbs of larval salamanders, Amblystoma punctatum, were fixed in OsO₄ at various intervals after amputation and were sectioned for study with the electron microscope. The dedifferentiated cells comprising the early blastema were found to have a fine structure similar to that of other undifferentiated cells and to have lost all of the identifying morphological features of their tissues of origin. The cytoplasm of such cells is characterized by numerous free ribonucleoprotein granules and a discontinuous vesicular endoplasmic reticulum. The cells have more abundant cytoplasm and are in closer contact with each other than was previously realized. The layer of condensed ground substance investing most differentiated cell types is lacking. After a period of rapid cell division, the morphology of the blastema cell changes. Cytoplasm is now sparse and contains a high concentration of free ribonucleoprotein granules, but little endoplasmic reticulum. The differentiating cartilage cell, however, develops an extensive, highly organized endoplasmic reticulum and the Golgi apparatus also appears to become more highly differentiated and more extensive at this time. Small vesicles appear throughout the cytoplasm at the time the new cisternae originate and may contribute to their formation. These and other changes in the cytoplasmic organelles are discussed.     

Integrin bi‐directional signaling across the plasma membrane

Integrins are heterodimeric cell adhesion molecules that are important in many biological functions, such as cell migration, proliferation, differentiation, and survival. They can transmit bi‐directional signals across the plasma membrane. Inside‐out activating signal from some cell surface receptors bound with soluble agonists triggers integrins conformational change leading to high affinity for extracellular ligands. Then binding of ligands to integrins results in outside‐in signaling, leading to formation of focal adhesion complex at the integrin cytoplasmic tail and activation of downstream signal pathways. This bi‐directional signaling is essential for rapid response of cell to surrounding environmental changes. During this process, the conformational change of integrin extracellular and transmembrane/cytoplasmic domains is particularly important. In this review, we will summarize recent progress in both inside‐out and outside‐in signaling with specific focus on the mechanism how integrins transmit bi‐directional signals through transmembrane/cytoplasmic domains. J. Cell. Physiol. 228: 306–312, 2013. © 2012 Wiley Periodicals, Inc.     

Immunolocalization of c‐myc‐positive cells in lizard tail after amputation suggests cell activation and proliferation for tail regeneration

After tail amputation in lizard, a regenerative response is elicited leading to the formation of a new tail. The stimulation of the proliferation process may involve the proto‐oncogene c‐myc. The immunocytochemical analysis detects the c‐myc protein few days after wound in free cells accumulating over the injured tissues of the tail stump. Western blot detects a protein band at 68–70 kDa that is more intense in the regenerating blastema than in normal tail tissues. Nuclei positive for the c‐myc protein are seen in mesenchymal‐like cells located among muscles, connectives and fat tissues of the tail stump 4 days postamputation. Proliferating cells labelled for 5BrdU are seen at 4 days postamputation and are sparse in the mesenchyme of the regenerating blastema formed at 12 days postamputation. Fine immunolocalization of the c‐myc protein shows it is mainly located over euchromatin or poorly condensed chromatin to indicate gene activation. The study correlates the detection of the c‐myc protein with activation of cell division in the injured tissues leading to the formation of the regenerative blastema. The lizard c‐myc protein probably activates a controlled proliferation process through a mechanism that can give information on the uncontrolled process occurring in cancer.