Evidence for dedifferentiation and metaplasia in amphibian limb regeneration from inheritance of DNA methylation

Amphibian limb regeneration is a process in which it has been suggested that cells of one differentiated type may dedifferentiate and give rise to cells of another type in the regenerate. We have used two tissue-specific hypomethylations in the newt cardioskeletal myosin heavy chain gene as lineage markers to follow the fate of cells during limb regeneration. Analysis of genomic DNA from different muscle cell populations allowed the assignment of one marker to the muscle (Hypo A) lineage and the other, more tentatively, to the 'connective tissue' (Hypo B) component of muscle. The contribution to regenerated limb cartilage and limb blastemal tissue by cells carrying these markers was estimated by quantitative analysis of Southern blot hybridizations using DNA from regenerate tissues. The results are consistent with a contribution of cells from both muscle and connective tissue lineages to cartilage in regenerated limbs. In addition, removal of the humerus at the time of amputation (eliminating any contribution from pre-existing cartilage), has provided evidence for an increased representation of cells carrying the connective tissue marker in regenerate cartilage but did not affect the representation of cells carrying the muscle cell marker.     

Cytoskeletal dynamics of the teleostean fin ray during fin epimorphic regeneration

Teleost fishes can regenerate their fins by epimorphic regeneration, a process that involves the transition of the formerly quiescent tissues of the stump to an active, growing state. This involves dynamic modifications of cell phenotype and behavior that must rely on alterations of the cytoskeleton. We have studied the spatial and temporal distribution of three main components of the cytoskeleton (actin, keratin and vimentin) in the regenerating fin, in order to establish putative relationships between cell cytoskeleton and cell behavior. According to our results, the massive rearrangement undergone by the epidermis right after injury, which takes place by cell migration, correlates with a transient down-regulation of keratin and a strong up-regulation of actin in the epidermal cells. During the subsequent epidermal growth, based on cell proliferation, keratin normal pattern is recovered while actin is down-regulated, although not to normal (quiescent) levels. The epidermal basal layer in contact with the blastema displays a particular cytoskeletal profile, different to that of the rest of the epidermal cells, which reflects its special features. In the connective tissue compartment, somatic cells do not contain vimentin, but keratin, as intermediate filament. Proliferative and migrative activation of these cells after injury correlates with actin up-regulation. Although this initial activation does not involve keratin down-regulation, blastemal cells were later observed to lack keratin, suggesting that such cytoskeletal modification might be needed for connective tissue cells to dedifferentiate and form the blastema. Cell differentiation in the newly formed, regenerated ray is accompanied by actin down-regulation and keratin up-regulation.     

Developmental aspects of spinal cord and limb regeneration

The ability of birds and mammals to regenerate tissues is limited. By contrast, urodele amphibians can regenerate a variety of injured tissues such as intestine, cardiac muscle, lens and neural retina, as well as entire structures such as limbs, tail and lower jaw. This regenerative capacity is associated with the ability to form masses of mesenchyme cells (blastemas) that differentiate into the missing tissues or parts. Understanding the mechanisms that underlie blastema formation in urodeles will provide valuable tools with which to achieve the goal of stimulating regeneration in mammalian tissues that do not naturally regenerate. Here we discuss an example of tissue regeneration (spinal cord) and an example of epimorphic appendage regeneration (limb) in the axolotl Ambystoma mexicanum , emphasizing analysis of the processes that produce the regeneration blastema and of the tissue interactions and blastemal products that contribute to the regeneration-promoting environment.     

Dermal fibroblasts contribute to multiple tissues in the accessory limb model

The accessory limb model has become an alternative model for performing investigations of limb regeneration in an amputated limb. In the accessory limb model, a complete patterned limb can be induced as a result of an interaction between the wound epithelium, a nerve and dermal fibroblasts in the skin. Studies should therefore focus on examining these tissues. To date, however, a study of cellular contributions in the accessory limb model has not been reported. By using green fluorescent protein (GFP) transgenic axolotl tissues, we can trace cell fate at the tissue level. Therefore, in the present study, we transgrafted GFP skin onto the limb of a non‐GFP host and induced an accessory limb to investigate cellular contributions. Previous studies of cell contribution to amputation‐induced blastemas have demonstrated that dermal cells are the progenitors of many of the early blastema cells, and that these cells contribute to regeneration of the connective tissues, including cartilage. In the present study, we have determined that this same population of progenitor cells responds to signaling from the nerve and wound epithelium in the absence of limb amputation to form an ectopic blastema and regenerate the connective tissues of an ectopic limb. Blastema cells from dermal fibroblasts, however, did not differentiate into either muscle or neural cells, and we conclude that dermal fibroblasts are dedifferentiated along its developmental lineage.     

Evidence that the nerve controls molecular identity of progenitor cells for limb regeneration

Adult urodele amphibians can regenerate their limbs after amputation by a process that requires the presence of axons at the amputation plane. Paradoxically, if the limb develops in the near absence of nerves (the 'aneurogenic' limb) it can subsequently regenerate in a nerve-independent fashion. The growth zone (blastema) of regenerating limbs normally contains progenitor cells whose division is nerve-dependent. A monoclonal antibody that marks these nerve-dependent cells in the normal blastema does not stain the mesenchymal cells of developing limb buds and only stains the amputated limb bud when axons have reached the plane of amputation. This report shows that the blastemal cells of the regenerating aneurogenic limb also fail to react with the antibody in situ. These data suggest that the blastemal cells arising during normal regeneration have been altered by the nerve. This regulation may occur either at the time of amputation (when the antigen is expressed) or during development (when the limb is first innervated).     

Glial growth factor and nerve-dependent proliferation in the regeneration blastema of Urodele amphibians

After amputation of a limb from Urodele amphibians, division of the blastemal cells (the progenitor cells of the regenerate) depends on one or more unidentified growth factors provided by the nerve supply. Here we show that glial growth factor (GGF), a mitogenic protein previously purified from the bovine pituitary, is present in newt nervous system extracts. It is also detectable in extracts of the forelimb regeneration blastema, and its level there decreases after denervation. We have previously shown that blastemal cells dependent on the nerve for division are marked by a monoclonal antibody called 22/18. When denervated blastemas are cultured in the presence of partially purified GGF from newt brain, or pure GGF from the bovine pituitary, the thymidine labeling index of blastemal cells that are 22/18-positive is increased as much as sevenfold. These data indicate that GGF plays a role in nerve-dependent proliferation in the blastema.     

Changes in the Distribution of Fibronectin During Limb Regeneration in Newts Using Immunocytochemistry

The distribution of fibronectin in regenerating newt limbs was studied using immunocytochemistry. At appropriate intervals after the initial amputation at the elbow (10–30 days), animals were reamputated at the shoulder and processed for light microscopy. The peroxidase-antiperoxidase technique was used to localize affinity-purified antibodies to fibronectin in limb tissues. At the amputation site, fibronectin was associated with basal laminae and connective tissues adjacent to dedifferentiating limb tissues destined to form the regeneration blastema. Accumulation and growth of the blastema was accompanied by the apparent de novo synthesis of fibronectin, where it appeared randomly in the interstitium between blastemal cells. The onset of chondrogenesis was characterized by a central condensation of prechondroblasts that formed the cartilage anlagen. Fibronectin formed an amorphous network between presumptive chondroblasts. As the mature cartilage phenotype was expressed and chondrocytes became isolated in lacunae, fibronectin was greatly reduced and then disappeared. The extracellular matrix surrounding undifferentiated blastemal cells still contained fibronectin. Fibronectin was also found in high concentrations between differentiating myoblasts. A condensation of fibronectin was also observed beneath the epidermis at the distal limb tip at the onset of digit formation. These observations are consistent with the hypothesis that fibronectin may play a key role in the morphogenetic events that result in the spatial organization and subsequent differentiation of cells during pattern formation in the regenerating limb.     

Organization of the Morphogenetic Field in Regenerating Amphibian Limbs

The amphibian limb is an example of a secondary embryonic fieldthat can be reactivated during larval or adult life so thatamputated parts are regenerated. Two major questions are: (1)what is the origin of the morphogenetic field of the regenerationblastema, and (2) what is the nature of this field and how doesit specify the spatial pattern of blastemal redifferentiation?Evidence is analyzed here which leads to the following propositions:(1) the field is represented in latent form by properties ofthe mature limb cells, and these properties are activated andinherited by the blastemal cells after amputation and dedifferentiation.At the same time, the inherited field is sensitive to the maturestump tissues and its spatial organization can be altered bya stump pattern alien to the one from which it was derived.(2) The properties of the mesodermal limb tissues representpositional values that are arranged in gradients along the proximal-distal,anterior-posterior and dorsal-ventral axes. These propertiesallow dedifferentiated mesodermal cells to change their positionalvalue to any value between their original one in the limb andthe value of any neighboring cell after creation of a discontinuity.The direction of change is always from proximal to distal inthe PD axis; it is uncertain as to whether change can take placeonly centripetally or both centripetally and centrifugally alongthe AP and DV axes. (3) Epidermal cells have the same positionalvalue everywhere in the limb and act as the distal and circumferentialboundaries up to which the mesodermal cells may change theirpositional values. The proximal boundary is represented by thelevel-specific properties of the mesodermal cells at the maximumextent of distal to proximal dedifferentiation. Normal regenerationcan then be visualized as occurring in the following way. Whendeletions are made in the limb pattern, cells with widely differentpositional values are confronted. During regeneration, blastemacells increase in number and continually interact with theirneighbors to adjust their positional values within the boundariesuntil discontinuities are eliminated. The multiple limbs resultingfrom rearrangement of stump tissue patterns can also be accountedfor by using these propositions. It is suggested that positionalinformation is encoded on the cell surface and/or in the extracellularmatrix.     

Culture of newt cells from different tissues and their expression of a regeneration-associated antigen

We have established culture conditions for cells from normal limb, early limb regenerate (blastema), heart, and liver of the newt Notophthalmus viridescens. Whereas heart and liver cells had a relatively short life in culture, limb cells have shown no sign of senescence over more than 1 year in culture. Cultured cells from all these tissues express to differing extents the regeneration-associated antigen 22/18. The antigen is intracellular and filamentous, and its expression appears to be regulated by culture density. Furthermore, 22/18 antigen is turned off in limb and blastemal cultures following differentiation into muscle, as also occurs in vivo.     

Elastic fibres in health and disease

Elastic fibres are a major class of extracellular matrix fibres that are abundant in dynamic connective tissues such as arteries, lungs, skin and ligaments. Their structural role is to endow tissues with elastic recoil and resilience. They also act as an important adhesion template for cells, and they regulate growth factor availability. Mutations in major structural components of elastic fibres, especially elastin, fibrillins and fibulin-5, cause severe, often life-threatening, heritable connective tissue diseases such as Marfan syndrome, supravalvular aortic stenosis and cutis laxa. Elastic-fibre function is also frequently compromised in damaged or aged elastic tissues. The ability to regenerate or engineer elastic fibres and tissues remains a significant challenge, requiring improved understanding of the molecular and cellular basis of elastic-fibre biology and pathology, and ability to regulate the spatiotemporal expression and assembly of its molecular components.     

Patterning of avian craniofacial muscles

Vertebrate voluntary muscles are composed of myotubes and connective tissue cells. These two cell types have different embryonic origins: myogenic cells arise from paraxial mesoderm, while in the head many of the connective tissues are formed by neural crest cells. The objective of this research was to study interactions between heterotopically transplanted trunk myotomal cells and presumptive connective tissue-forming cephalic neural crest mesenchyme. Presumptive or newly formed cervical somites from quail embryos were implanted lateral to the midbrain of chick hosts prior to the onset of neural crest emigration. Hosts were sacrificed between 7 and 12 days of incubation, and sections examined for the presence of quail cells. Some grafted tissues differentiated in situ, forming ectopic skeletal, connective, and muscle tissues. However, many myotomal cells broke away from the implant, became integrated into adjacent neural crest mesenchyme, and subsequently formed normal extrinsic ocular or jaw muscles. In these muscles it was evident that only the myogenic populations were derived from grafted trunk cells. Ancillary findings were that grafted trunk paraxial mesoderm frequently interfered with the movement of neural crest cells which form the corneal posterior epithelial and stromal tissues, and that some grafted cells formed ectopic intramembranous bones adjacent to the eye. These results verify that presumptive connective tissue-forming mesenchyme derived from the neural crest imparts spatial patterning information upon myogenic cells that invade it. Moreover, interactions between myotomal cells and both lateral plate somatic mesoderm in the trunk and neural crest mesenchyme in the head appear to operate according to similar mechanisms.     

Neural retinal regeneration in the anuran amphibian Xenopus laevis post-metamorphosis: transdifferentiation of retinal pigmented epithelium regenerates the neural retina

In urodele amphibians like the newt, complete retina and lens regeneration occurs throughout their lives. In contrast, anuran amphibians retain this capacity only in the larval stage and quickly lose it during metamorphosis. It is believed that they are unable to regenerate these tissues after metamorphosis. However, contrary to this generally accepted notion, here we report that both the neural retina (NR) and lens regenerate following the surgical removal of these tissues in the anuran amphibian, Xenopus laevis, even in the mature animal. The NR regenerated both from the retinal pigment epithelial (RPE) cells by transdifferentiation and from the stem cells in the ciliary marginal zone (CMZ) by differentiation. In the early stage of NR regeneration (5-10 days post operation), RPE cells appeared to delaminate from the RPE layer and adhere to the remaining retinal vascular membrane. Thereafter, they underwent transdifferentiation to regenerate the NR layer. An in vitro culture study also revealed that RPE cells differentiated into neurons and that this was accelerated by the presence of FGF-2 and IGF-1. The source of the regenerating lens appeared to be remaining lens epithelium, suggesting that this is a kind of repair process rather than regeneration. Thus, we show for the first time that anuran amphibians retain the capacity for retinal regeneration after metamorphosis, similarly to urodeles, but that the mode of regeneration differs between the two orders. Our study provides a new tool for the molecular analysis of regulatory mechanisms involved in retinal and lens regeneration by providing an alternative animal model to the newt, the only other experimental model.     

The role of cartilage and fibronectin during respecification of pattern induced in the regenerating amphibian limb by retinoic acid

When retinoic acid (RA) is applied to the regenerating limb the positional information of blastemal cells is respecified and extra limb segments develop. We are trying to elucidate the molecular basis of the action of RA and report here experiments focused on the role that fibronectin (FN) might play in the process. The FN distribution in stump tissues, regeneration blastemas and RA-treated blastemas was investigated by immunocytochemistry. Two effects of RA were observed. Firstly, excessive dedifferentiation of the severed cartilage at the amputation plane, resulting in lumps of FN-positive matrix being released into the blastema; secondly, blastemal cells tend to aggregate together into FN-positive accumulations. Excessive dedifferentiation of the cartilage plays no role in the RA-induced respecification of pattern, because we show that extra segments are still produced in RA-treated limbs from which all the cartilage has been removed. The effect on blastemal cell FN distribution was investigated in several ways. Axolotl plasma FN and cellular FN were characterised on immunoblots, and no obvious change was observed after RA treatment; neither were there changes in amounts of FN detected by ELISA. Levels of FN synthesis were measured by [35S]-methionine labelling and again no change observed after RA treatment. We conclude that the change in FN distribution observed by immunocytochemistry after RA treatment may be due to the retention of FN on the surface of the blastemal cells rather than to any effect on the levels of synthesis of this molecule.     

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.     

Self-Renewal of the Adult Skeletal Muscle Satellite Cell

The concept of the stem cell has evolved in dynamic systems such as those involved inembryonic development and, in the adult, in tissues such as blood and skin which arecontinuously renewed. It has proved difficult to establish whether stem cell mechanismsunderlie the maintenance of the more stable tissues that form the majority of the adultbody. We have investigated skeletal muscle, a low-turnover and largely postmitotictissue which nevertheless maintains a remarkable capacity to regenerate itself followinginjury. The contractile units of muscle are myofibers, elongated syncytial cells eachcontaining many hundreds of postmitotic myonuclei. Satellite cells are resident beneaththe basal lamina of myofibers and function as myogenic precursors during muscleregeneration. We have recently demonstrated that as few as seven Pax7+ satellite cellsassociated with one myofiber can regenerate a hundred or more new myofiberscontaining thousands of myonuclei. Satellite cells also undergo self-renewal, givingthem the ability to participate in multiple rounds of injury-induced regeneration. Thesatellite cell may thus serve as a prototype for stem cell function in stable adult tissues: atissue-specific progenitor which is normally quiescent but which has self-renewalproperties similar to those of better known stem cells.     

组蛋白乙酰化对成体干细胞生物学性状的影响

成体干细胞（adult stem cells,ASCs）是指存在于一种已经分化组织中的未分化细胞,它们可以再生修复损伤的组织和器官,是组织工程和细胞治疗的理想细胞。但是ASCs在体外扩增过程中容易发生自主分化和衰老,影响其在临床的广泛应用。组蛋白乙酰化作为表观遗传调节的重要机制,参与细胞分化、衰老及凋亡等众多细胞活动的调控。该文就组蛋白乙酰化对成体干细胞生物学性状的影响进行综述。     

Sources of adult mesenchymal stem cells and their applicability for musculoskeletal applications

There is significant potential for the use of adult mesenchymal stem cells in regenerating musckuloskeletal tissues. The sources of these stem cells discussed in this review are bone marrow, blood, adipose tissue, synovium, periosteum & cartilage. Adult mesenchymal stem cells of bone marrow origin are the cells which are heavily investigated in many studies and have been shown capable of producing a variety of connective tissues especially cartilage and bone. It has recently been suggested that bone marrow derived mesenchymal stem cells originate from microvascular pericytes, and, indeed, many of the tissues from which stem cells have been isolated have good vascularisation and they may give a varied source of cells for future treatments. Clinical trials have shown that these cells are able to be successfully used to regenerate tissues with good clinical outcome. Other sources are showing promise, however, is yet to be brought to the clinical level in humans.