Plasticity of photoreceptor-generating retinal progenitors revealed by prolonged retinoic acid exposure

Background

Epimorphic regeneration results in the restoration of lost tissues and structures from an aggregation of proliferating cells known as a blastema. Among amniotes the most striking example of epimorphic regeneration comes from tail regenerating lizards. Although tail regeneration is often studied in the context of ecological costs and benefits, details of the sequence of tissue-level events are lacking. Here we investigate the anatomical and histological events that characterize tail regeneration in the leopard gecko, Eublepharis macularius.

Results

Tail structure and tissue composition were examined at multiple days following tail loss, revealing a conserved pattern of regeneration. Removal of the tail results in a consistent series of morphological and histological events. Tail loss is followed by a latent period of wound healing with no visible signs of regenerative outgrowth. During this latent period basal cells of the epidermis proliferate and gradually cover the wound. An additional aggregation of proliferating cells accumulates adjacent to the distal tip of the severed spinal cord marking the first appearance of the blastema. Continued growth of the blastema is matched by the initiation of angiogenesis, followed by the re-development of peripheral axons and the ependymal tube of the spinal cord. Skeletal tissue differentiation, corresponding with the expression of Sox9, and muscle re-development are delayed until tail outgrowth is well underway.

Conclusions

We demonstrate that tail regeneration in lizards involves a highly conserved sequence of events permitting the establishment of a staging table. We show that tail loss is followed by a latent period of scar-free healing of the wound site, and that regeneration is blastema-mediated. We conclude that the major events of epimorphic regeneration are highly conserved across vertebrates and that a comparative approach is an invaluable biomedical tool for ongoing regenerative research.     

In vivo imaging indicates muscle fiber dedifferentiation is a major contributor to the regenerating tail blastema.

During tail regeneration in urodele amphibians such as axolotls, all of the tissue types, including muscle, dermis, spinal cord, and cartilage, are regenerated. It is not known how this diversity of cell types is reformed with such precision. In particular, the number and variety of mature cell types in the remaining stump that contribute to the blastema is unclear. Using Nomarski imaging, we followed the process of regeneration in the larval axolotl tail. Combining this with in vivo fluorescent labeling of single muscle fibers, we show that mature muscle dedifferentiates. Muscle dedifferentiation occurs by the synchronous fragmentation of the multinucleate muscle fiber into mononucleate cells followed by rapid cell proliferation and the extension of cell processes. We further show that direct clipping of the muscle fiber and severe tissue damage around the fiber are both required to initiate dedifferentiation. Our observations also make it possible to estimate for the first time how many of the blastema cells arise specifically from muscle dedifferentiation. Calculations based on our data suggest that up to 29% of nondermal-derived cells in the blastema come from dedifferentiation of mature muscle fibers. Overall, these results show that endogenous multinucleate muscle fibers can dedifferentiate into mononucleate cells and contribute significantly to the blastema.     

Cell differentiation and cell fate during urodele tail and limb regeneration

One of the most striking natural examples of adult tissue plasticity in vertebrates is limb and tail regeneration in urodele amphibians. In this setting, amputation triggers the destabilization of cell differentiation and the production of progenitor cells that extensively proliferate and pattern themselves to recreate a perfect replica of the missing part. A precise understanding of which cells dedifferentiate and how plastic they become has recently begun to emerge. Furthermore, information on which developmental gene programs are activated upon injury is becoming better understood. These studies indicate that, upon injury, an unusual cohort of genes are co-expressed. The future challenge will be to link the systems for studying dedifferentiation with activation of gene expression to understand on a molecular level how cells are 'pushed backward' to regenerate a complex structure such as a limb or tail.     

THE EFFECT OF COLCHICINE ON MYOGENESIS IN VIVO IN RANA PIPIENS AND RHODNIUS PROLIXUS (HEMIPTERA)

The effect of colchicine on myogenesis in vivo has been studied in the regenerating tadpole tail of the frog, Rana pipiens, and in the abdominal molting muscles of a blood-sucking bug, Rhodnius prolixus Stål. Colchicine is shown to disrupt microtubules in the differentiating muscle cells of both these organisms. The disruption of microtubules is correlated with a loss of longitudinal anisometry in the myoblasts and myotubes of the regeneration blastema in the tadpole tail. Before colchicine treatment, the myotubes contain longitudinally oriented myofibrils. After colchicine treatment, rounded, multinucleate myosacs containing randomly oriented myofibrils are present. It is suggested that the primary function of microtubules in myogenesis in the Rana pipiens tadpole is the maintenance of cell shape. The abdominal molting muscles of Rhodnius undergo repeated phases of differentiation and dedifferentiation of the sarcoplasm. However, the longitudinal anisometry of the muscle fibers is maintained in all phases by the attachments of the ends of the fibers to the exoskeleton, and microtubule disruption does not alter cell shape. The orientation of the developing myofibrils is also unaltered, indicating that the microtubules do not directly align or support the myofibrils in this system.     

Small molecules that recapitulate the early steps of urodele amphibian limb regeneration and confer multipotency

In urodele amphibians, an early step in limb regeneration is skeletal muscle fiber dedifferentiation into a cellulate that proliferates to contribute new limb tissue. However, mammalian muscle cannot dedifferentiate after injury. We have developed a novel, small-molecule-based method to induce dedifferentiation in mammalian skeletal muscle. Muscle cellularization was induced by the small molecule myoseverin. Candidate small molecules were tested for the induction of proliferation in the cellulate. We observed that treatment with the small molecules BIO (glycogen synthase-3 kinase inhibitor), lysophosphatidic acid (pleiotropic activator of G-protein-coupled receptors), SB203580 (p38 MAP kinase inhibitor), or SQ22536 (adenylyl cyclase inhibitor) induced proliferation. Moreover, these proliferating cells were multipotent, as confirmed by the chemical induction of mesodermal-derived cell lineages. Microarray analysis showed that the multipotent, BIO-treated cellulate possessed a markedly different gene expression pattern than lineage-restricted C2C12 myoblasts, especially for genes related to signal transduction and differentiation. Sequential small molecule treatment of the muscle cellulate with BIO, SB203580, or SQ22536 and the aurora B kinase inhibitor, reversine, induced the formation of cells with neurogenic potential (ectodermal lineage), indicating the acquirement of pluripotency. This is the first demonstration of a small molecule method that induces mammalian muscle to undergo dedifferentiation and rededifferentiation into alternate cell lineages. This method induces dedifferentiation in a simple, stepwise approach and has therapeutic potential to enhance tissue regeneration in mammals.     

PhOTO zebrafish: a transgenic resource for in vivo lineage tracing during development and regeneration

Background

Elucidating the complex cell dynamics (divisions, movement, morphological changes, etc.) underlying embryonic development and adult tissue regeneration requires an efficient means to track cells with high fidelity in space and time. To satisfy this criterion, we developed a transgenic zebrafish line, called PhOTO, that allows photoconvertible optical tracking of nuclear and membrane dynamics in vivo.

Methodology

PhOTO zebrafish ubiquitously express targeted blue fluorescent protein (FP) Cerulean and photoconvertible FP Dendra2 fusions, allowing for instantaneous, precise targeting and tracking of any number of cells using Dendra2 photoconversion while simultaneously monitoring global cell behavior and morphology. Expression persists through adulthood, making the PhOTO zebrafish an excellent tool for studying tissue regeneration: after tail fin amputation and photoconversion of a ∼100µm stripe along the cut area, marked differences seen in how cells contribute to the new tissue give detailed insight into the dynamic process of regeneration. Photoconverted cells that contributed to the regenerate were separated into three distinct populations corresponding to the extent of cell division 7 days after amputation, and a subset of cells that divided the least were organized into an evenly spaced, linear orientation along the length of the newly regenerating fin.

Conclusions/Significance

PhOTO zebrafish have wide applicability for lineage tracing at the systems-level in the early embryo as well as in the adult, making them ideal candidate tools for future research in development, traumatic injury and regeneration, cancer progression, and stem cell behavior.     

Requirement for Wnt and FGF signaling in Xenopus tadpole tail regeneration

We have investigated the requirement for the FGF and Wnt/beta-catenin pathways for Xenopus tadpole tail regeneration. Pathways were modified either by treatment with small molecules or by induction of transgene expression with heat shocks. Regeneration is inhibited by treatment with the FGF inhibitor SU5402, or by activation of a dominant negative FGF receptor, or by activation of expression of the Wnt inhibitor Dkk1. Agents promoting Wnt activity: the small molecule BIO, or a constitutively active form of beta-catenin, led to an increased growth rate. Combination of a Wnt activator with FGF inhibitor suppressed regeneration, while combination of a Wnt inhibitor with a FGF activator allowed regeneration. This suggests that the Wnt activity lies upstream of the FGF activity.Expression of both Wnt and FGF components was inhibited by activation of noggin, suggesting that BMP signalling lies upstream of both Wnt and FGF.The results show that the molecular mechanism of Xenopus tadpole tail regeneration is surprisingly similar to that of the Xenopus limb bud and the zebrafish caudal fin, despite the difference of anatomy.     

Regeneration of neural crest derivatives in the <Emphasis Type="Italic">Xenopus</Emphasis>tadpole tail

Background  

After amputation of the Xenopus tadpole tail, a functionally competent new tail is regenerated. It contains spinal cord, notochord and muscle, each of which has previously been shown to derive from the corresponding tissue in the stump. The regeneration of the neural crest derivatives has not previously been examined and is described in this paper.     

Differentiated skeletal cells contribute to blastema formation during zebrafish fin regeneration

The origin of cells that generate the blastema following appendage amputation has been a long-standing question in epimorphic regeneration studies. The blastema is thought to originate from either stem (or progenitor) cells or differentiated cells of various tissues that undergo dedifferentiation. Here, we investigate the origin of cells that contribute to the regeneration of zebrafish caudal fin skeletal elements. We provide evidence that the process of lepidotrichia (bony rays) regeneration is initiated as early as 24 hours post-amputation and that differentiated scleroblasts acquire a proliferative state, detach from the lepidotrichia surface, migrate distally, integrate into the blastema and dedifferentiate. These findings provide novel insights into the origin of cells in epimorphic appendage regeneration in zebrafish and suggest conservation of regeneration mechanisms between fish and amphibians.     

Plasticity and reprogramming of differentiated cells in amphibian regeneration: partial purification of a serum factor that triggers cell cycle re-entry in differentiated muscle cells

The reversal of cellular differentiation to form proliferating progenitor cells is a critical aspect of regenerative ability in the urodele amphibians. This process has been studied using skeletal muscle during limb or tail regeneration, or dorsal iris epithelium during lens regeneration. An unknown activity in serum triggers cell cycle re-entry from the differentiated state. Here we describe the biochemical properties and fractionation of this serum factor. The factor is a glycoprotein that associates with large molecular weight complexes. The purification and molecular identification of the serum factor represents an important avenue in understanding regenerative ability and dedifferentiation capacity on a molecular basis.     

Maternal topoisomerase II alpha, not topoisomerase II beta, enables embryonic development of zebrafish top2a -/- mutants

Background

Determining the type and source of cells involved in regenerative processes has been one of the most important goals of researchers in the field of regeneration biology. We have previously used several cellular markers to characterize the cells involved in the regeneration of the intestine in the sea cucumber Holothuria glaberrima.

Results

We have now obtained a monoclonal antibody that labels the mesothelium; the outer layer of the gut wall composed of peritoneocytes and myocytes. Using this antibody we studied the role of this tissue layer in the early stages of intestinal regeneration. We have now shown that the mesothelial cells of the mesentery, specifically the muscle component, undergo dedifferentiation from very early on in the regeneration process. Cell proliferation, on the other hand, increases much later, and mainly takes place in the mesothelium or coelomic epithelium of the regenerating intestinal rudiment. Moreover, we have found that the formation of the intestinal rudiment involves a novel regenerative mechanism where epithelial cells ingress into the connective tissue and acquire mesenchymal phenotypes.

Conclusions

Our results strongly suggest that the dedifferentiating mesothelium provides the initial source of cells for the formation of the intestinal rudiment. At later stages, cell proliferation supplies additional cells necessary for the increase in size of the regenerate. Our data also shows that the mechanism of epithelial to mesenchymal transition provides many of the connective tissue cells found in the regenerating intestine. These results present some new and important information as to the cellular basis of organ regeneration and in particular to the process of regeneration of visceral organs.     

In Vivo Dedifferentiation of Adult Adipose Cells

Introduction

Adipocytes can dedifferentiate into fibroblast-like cells in vitro and thereby acquire proliferation and multipotent capacities to participate in the repair of various organs and tissues. Whether dedifferentiation occurs under physiological or pathological conditions in vivo is unknown.

Methods

A tissue expander was placed under the inguinal fat pads of rats and gradually expanded by injection of water. Samples were collected at various time points, and morphological, histological, cytological, ultrastructural, and gene expression analyses were conducted. In a separate experiment, purified green fluorescent protein+ adipocytes were transplanted into C57 mice and collected at various time points. The transplanted adipocytes were assessed by bioluminescence imaging and whole-mount staining.

Results

The expanded fat pad was obviously thinner than the untreated fat pad on the opposite side. It was also tougher in texture and with more blood vessels attached. Hematoxylin and eosin staining and transmission electron microscopy indicated there were fewer monolocular adipocytes in the expanded fat pad and the morphology of these cells was altered, most notably their lipid content was discarded. Immunohistochemistry showed that the expanded fat pad contained an increased number of proliferative cells, which may have been derived from adipocytes. Following removal of the tissue expander, many small adipocytes were observed. Bioluminescence imaging suggested that some adipocytes survived when transplanted into an ischemic-hypoxic environment. Whole-mount staining revealed that surviving adipocytes underwent a process similar to adipocyte dedifferentiation in vitro. Monolocular adipocytes became multilocular adipocytes and then fibroblast-like cells.

Conclusions

Mature adipocytes may be able to dedifferentiate in vivo, and this may be an adipose tissue self-repair mechanism. The capacity of adipocytes to dedifferentiate into stem cell-like cells may also have a more general role in the regeneration of many tissues, notably in fat grafting.     

Contribution of human muscle-derived cells to skeletal muscle regeneration in dystrophic host mice

Background

Stem cell transplantation is a promising potential therapy for muscular dystrophies, but for this purpose, the cells need to be systemically-deliverable, give rise to many muscle fibres and functionally reconstitute the satellite cell niche in the majority of the patient''s skeletal muscles. Human skeletal muscle-derived pericytes have been shown to form muscle fibres after intra-arterial transplantation in dystrophin-deficient host mice. Our aim was to replicate and extend these promising findings.

Methodology/Principal Findings

Isolation and maintenance of human muscle derived cells (mdcs) was performed as published for human pericytes. Mdscs were characterized by immunostaining, flow cytometry and RT-PCR; also, their ability to differentiate into myotubes in vitro and into muscle fibres in vivo was assayed. Despite minor differences between human mdcs and pericytes, mdscs contributed to muscle regeneration after intra-muscular injection in mdx nu/nu mice, the CD56+ sub-population being especially myogenic. However, in contrast to human pericytes delivered intra-arterially in mdx SCID hosts, mdscs did not contribute to muscle regeneration after systemic delivery in mdx nu/nu hosts.

Conclusions/Significance

Our data complement and extend previous findings on human skeletal muscle-derived stem cells, and clearly indicate that further work is necessary to prepare pure cell populations from skeletal muscle that maintain their phenotype in culture and make a robust contribution to skeletal muscle regeneration after systemic delivery in dystrophic mouse models. Small differences in protocols, animal models or outcome measurements may be the reason for differences between our findings and previous data, but nonetheless underline the need for more detailed studies on muscle-derived stem cells and independent replication of results before use of such cells in clinical trials.     

Long-distance signals are required for morphogenesis of the regenerating Xenopus tadpole tail, as shown by femtosecond-laser ablation

Background

With the goal of learning to induce regeneration in human beings as a treatment for tissue loss, research is being conducted into the molecular and physiological details of the regeneration process. The tail of Xenopus laevis tadpoles has recently emerged as an important model for these studies; we explored the role of the spinal cord during tadpole tail regeneration.

Methods and Results

Using ultrafast lasers to ablate cells, and Geometric Morphometrics to quantitatively analyze regenerate morphology, we explored the influence of different cell populations. For at least twenty-four hours after amputation (hpa), laser-induced damage to the dorsal midline affected the morphology of the regenerated tail; damage induced 48 hpa or later did not. Targeting different positions along the anterior-posterior (AP) axis caused different shape changes in the regenerate. Interestingly, damaging two positions affected regenerate morphology in a qualitatively different way than did damaging either position alone. Quantitative comparison of regenerate shapes provided strong evidence against a gradient and for the existence of position-specific morphogenetic information along the entire AP axis.

Conclusions

We infer that there is a conduit of morphology-influencing information that requires a continuous dorsal midline, particularly an undamaged spinal cord. Contrary to expectation, this information is not in a gradient and it is not localized to the regeneration bud. We present a model of morphogenetic information flow from tissue undamaged by amputation and conclude that studies of information coming from far outside the amputation plane and regeneration bud will be critical for understanding regeneration and for translating fundamental understanding into biomedical approaches.     

Insulin-like growth factor 1 regulation of proliferation and differentiation of <Emphasis Type="Italic">Xenopus laevis</Emphasis> myogenic cells in vitro

To understand the mechanism of muscle remodeling during Xenopus laevis metamorphosis, we examined the in vitro effect of insulin-like growth factor 1 (IGF-1) on growth and differentiation of three different-fate myogenic cell populations: tadpole tail, tadpole dorsal, and young adult leg muscle. IGF-1 promoted growth and differentiation of both tail and leg myogenic cells only under conditions where these cells could proliferate. Inhibition of cell proliferation by DNA synthesis inhibitor cytosine arabinoside completely canceled the IGF-1’s cell differentiation promotion, suggesting the possibility that IGF-1’s differentiation-promotion effect is an indirect effect via IGF-1’s cell proliferation promotion. IGF-1 promoted differentiation dose dependently with maximum effect at 100–500 ng/ml. RT-PCR analysis revealed the upregulation (11-fold) of ifg1 mRNA expression in developing limbs, suggesting that IGF-1 plays a role in promoting muscle differentiation during limb development. The combined effect of triiodo-l-thyronine (T₃) and IGF-1 was also examined. In adult leg cells, IGF-1 promoted growth and differentiation irrespective of the presence of T₃. In larval tail cells, cell count was 76% lower in the presence of T₃, and IGF-1 did not promote proliferation and differentiation in T₃-containing medium. In larval dorsal cells, cell count was also lower in the presence of T₃, but IGF-1 enhanced proliferation and differentiation in T₃-containing medium. This result is likely due to the presence among dorsal cells of both adult and larval types (1:1). Thus, IGF-1 affects only adult-type myogenic cells in the presence of T₃ and helps accelerate dorsal muscle remodeling during metamorphosis.