The state of determination of the anterior-posterior, dorsal-ventral and proximal-distal axes of the undifferentiated limb regeneration blastema was evaluated by heterografting and autografting experiments in which these axes were... more
The state of determination of the anterior-posterior, dorsal-ventral and proximal-distal axes of the undifferentiated limb regeneration blastema was evaluated by heterografting and autografting experiments in which these axes were reversed with respect to the limb stump. Species-specific size differences in skeletal elements were used as markers to trace the origin of regenerate tissues in the heterografting experiments, and differences in the skeletal patterns of hindlimbs and forelimbs were used as markers in the autografting experiments. The resulting primary regenerates fell into two categories, those composed wholly or partly of donor tissues, and those composed entirely of host tissues. Regenerate structures formed from donor tissues always maintained the handedness of origin, while regenerates formed from host tissues always displayed host-side handedness. These results demonstrate that the axes of the blastema are determined from the start of regeneration, and that previous ...
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Science is at the core of many of the political, social, and economic issues that dominate modern society. These issues demand that citizens have a level of scientific literacy sufficient to understand the science on which policy... more
Science is at the core of many of the political, social, and economic issues that dominate modern society. These issues demand that citizens have a level of scientific literacy sufficient to understand the science on which policy arguments and decisions need to be based. Scientific literacy is also vital to maintaining a workforce increasingly reliant on science, and to link with the humanities in a search for truth to better understand our world as a whole.
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Investigate the developmental physiology of the temporomandibular joint (TMJ), a unique articulation between the cranium and the mandible. Principal regulatory factors for TMJ and disc development are Indian hedgehog (IHH) and bone... more
Investigate the developmental physiology of the temporomandibular joint (TMJ), a unique articulation between the cranium and the mandible. Principal regulatory factors for TMJ and disc development are Indian hedgehog (IHH) and bone morphogenetic protein (BMP-2). The mechanism is closely associated with ear morphogenesis. Secondary condylar cartilage emerges as a subperiosteal blastema on the medial surface of the posterior mandible. The condylar articular surface is immunoreactive for tenascin-C, so it is a modified fibrous periosteum with an underlying proliferative zone (cambrium layer) that differentiates into fibrocartilage. The latter cushions high loads and subsequently produces endochondral bone. The TMJ is a heavily loaded joint with three cushioning layers of fibrocartilage in the disc, as well as in subarticular zones in the fossa and mandibular condyle. The periosteal articular surface produces fibrocartilage to resist heavy loads, and has unique healing and adaptive prop...
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This review explores the historical and current state of our knowledge about urodele limb regeneration. Topics discussed are (1) blastema formation by the proteolytic histolysis of limb tissues to release resident stem cells and... more
This review explores the historical and current state of our knowledge about urodele limb regeneration. Topics discussed are (1) blastema formation by the proteolytic histolysis of limb tissues to release resident stem cells and mononucleate cells that undergo dedifferentiation, cell cycle entry and accumulation under the apical epidermal cap. (2) The origin, phenotypic memory, and positional memory of blastema cells. (3) The role played by macrophages in the early events of regeneration. (4) The role of neural and AEC factors and interaction between blastema cells in mitosis and distalization. (5) Models of pattern formation based on the results of axial reversal experiments, experiments on the regeneration of half and double half limbs, and experiments using retinoic acid to alter positional identity of blastema cells. (6) Possible mechanisms of distalization during normal and intercalary regeneration. (7) Is pattern formation is a self-organizing property of the blastema or dicta...
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This chapter discusses the mechanisms by which epithelial cells of the intestine, airways, and kidney tubule epithelium, are regenerated. The primary regenerative mechanism for most of these tissues is adult stem cell proliferation and... more
This chapter discusses the mechanisms by which epithelial cells of the intestine, airways, and kidney tubule epithelium, are regenerated. The primary regenerative mechanism for most of these tissues is adult stem cell proliferation and differentiation. The location, molecular markers and niche regulation of these stem cells are discussed. The liver and pancreas, however, use compensatory hyperplasia as their primary mechanism of regeneration, with stem activation as a back-up system. Interestingly, stem cells for nephron regeneration are found in some fish, and a similar cell has recently been isolated from mammalian Bowman’s capsule.
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Urodele salamander limbs regenerate by the accumulation of undifferentiated cells under an apically thickened wound epidermis to form a blastema that grows and redifferentiates into the missing limb parts. Successful regeneration requires... more
Urodele salamander limbs regenerate by the accumulation of undifferentiated cells under an apically thickened wound epidermis to form a blastema that grows and redifferentiates into the missing limb parts. Successful regeneration requires several early signals generated within seconds to hours after amputation. The initial accumulation of blastema cells forms largely in the absence of mitosis, after which the cells proliferate rapidly. The accumulation and proliferation of blastema cells is dependent on both nerves and apical epidermis. The nerves and epidermis are linked in a circuit in which nerve axons induce the apical epidermis to make and secrete a mitogen, the anterior gradient protein (AGP), which binds to its receptor Prod1 on the blastema cell surface. Dependence of the epidermis on the nerve for AGP production during regeneration arises during late stages of limb bud development as axons innervate the epidermis, but aneurogenic limbs never acquire axon dependence for the production of AGP after amputation.
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All vertebrates regenerate epidermis, periodontal tissues, and corneal epithelium, but only a few species can regenerate teeth and lens. Epidermis, hair, nails, periodontium, and cornea turn over continually and maintain their structure... more
All vertebrates regenerate epidermis, periodontal tissues, and corneal epithelium, but only a few species can regenerate teeth and lens. Epidermis, hair, nails, periodontium, and cornea turn over continually and maintain their structure by maintenance regeneration as well as regenerating in response to injury. The epidermis of the mammalian skin is a continuous, multilayered epithelium interspersed with several appendages such as hair follicles, sebaceous glands, and sweat glands. The regions of epidermis between hair follicles and their associated sebaceous as well as sweat glands are termed as interfollicular epidermis (IFE). In excisional wounds of adult skin, epidermis recovers the wound via the centripetal migration of basal cells. Basal cells of interfollicular epidermis detach from the basement membrane and begin migrating into the fibrin matrix of the wound within a day or two after injury. Nails are hardened epidermal variants of hair. The nail regenerates directly from a matrix of transient amplifying cells located at the proximal end of the nail bed, but the actual stem cells may be located further distally in a region under the nail bed that is equivalent to the bulge of the hair follicle. Teeth, such as nails, are variants of hair follicles that develop by a combination of epithelial–mesenchymal interactions. Although adult mammals cannot regenerate lost teeth, adult urodele amphibians and crocodilians can do so. Lens regeneration has been studied extensively in the adult newt. orneal epithelium is regenerated continuously or after injury by adult stem cells (ASCs) located in the limbus, the region where the cornea undergoes a transition into the sclera of the eye.
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This chapter introduces the subject of regenerative biology. The chapter begins by defining regeneration as opposed to fibrosis, followed by a brief history of the origins of the study of regenerative biology. Next, the mechanisms by... more
This chapter introduces the subject of regenerative biology. The chapter begins by defining regeneration as opposed to fibrosis, followed by a brief history of the origins of the study of regenerative biology. Next, the mechanisms by which vertebrate tissues regenerate and the regulation of these mechanisms by niche signaling molecules via various signal transduction pathways are introduced. The final segment of the chapter deals with approaches to the study of regeneration, including genetic marking methods to track the origin of stem cells, approaches to analyzing niche regulation of cell activities, cell imaging and identification, and comparative analysis of regeneration and fibrosis.
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Bone marrow stem cells, in particular, have been reported capable of differentiating into a wide range of cells other than their normal prospective fate. In many cases, autogeneic or allogeneic adult stem cells (ASCs) expanded in culture... more
Bone marrow stem cells, in particular, have been reported capable of differentiating into a wide range of cells other than their normal prospective fate. In many cases, autogeneic or allogeneic adult stem cells (ASCs) expanded in culture have been used as a source of transplantable cells. Autogeneic ASCs are preferable, because both pre- and posttransplant immune suppressions are not required for cell survival. Merkel cells are neural sensory receptors found in the dermis of the skin. In rats, the number of these receptors is diminished by denervation but is restored by innervation, suggesting that the skin might contain a precursor cell capable of differentiating into neural cell types. Analysis of the hearts at a late stage of fetal development showed that the human cells engrafted in the heart and differentiated into Purkinje fibers. On average, over 43% of the total Purkinje fibers in random areas of both ventricles were of human origin, as compared to only 0.01% of cardiomyocytes. Dental pulp cells are a heterogeneous population of cells that includes progenitor odontoblasts and two types of stem cells that have similarities to MSCs of the bone marrow.
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The blood cells and the endothelial cells of the vascular system arise during embryogenesis from common precursor cells called hemangioblasts. In the mouse embryo, hemangioblasts are already present in the posterior region of the mid... more
The blood cells and the endothelial cells of the vascular system arise during embryogenesis from common precursor cells called hemangioblasts. In the mouse embryo, hemangioblasts are already present in the posterior region of the mid primitive streak. Blood consists of plasma and a myeloid component consisting of erythrocytes, platelets, granulocytes, monocytes, and myeloid dendritic cells. Mature erythroid, myeloid, and lymphoid cells, with the exception of memory B and T cells, have half-lives of only days to weeks, and their numbers must be maintained by continual regeneration from multipotent hematopoietic stem cells (HSCs) residing in the bone marrow stroma of the ilium, vertebrae, and endochondral bones. Blood cells are regenerated by HSCs in the bone marrow, and blood vessels are regenerated both by endothelial cells in the walls of venules and circulating EnSCs from the bone marrow. Mammalian myocardium harbors cardiac stem cells that initiate a regenerative response after injury, but this response is quickly overtaken by fibroblast proliferation and scar formation. By contrast, amphibian and fish myocardium are able to regenerate new heart muscles.
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Urodele limbs regenerate via a blastema of mesenchymal cells derived from muscle, connective tissue and nerve sheath cells in the vicinity of the amputation surface by a process of histolysis, dedifferentiation and release of stem cells.... more
Urodele limbs regenerate via a blastema of mesenchymal cells derived from muscle, connective tissue and nerve sheath cells in the vicinity of the amputation surface by a process of histolysis, dedifferentiation and release of stem cells. Blastema cells replicate their DNA, arrest in G2 and accumulate under an apical epidermal cap (AEC). G2 arrest is broken by factor(s) expressed by the AEC that bind to receptors on the blastema cell surface. Expression of these factors is dependent on factor(s) provided by axons reinnervating the AEC. The blastema cells proliferate and are patterned into new limb structures by suites of signalling factors such as retinoic acid, sonic hedgehog and Wnt, in addition to homeobox transcription factors that endow the cells with positional identities expressed as axial gradients on the cell surface. The degree to which the early blastema is determined is controversial, with some evidence arguing for developmental plasticity and other evidence arguing that the blastema is self-organising. Key Concepts Urodeles are unique in their ability to form a regeneration blastema in response to amputation. The cells of the regeneration blastema are derived from resident stem cells and/or by the reprogramming (dedifferentiation) of differentiated cells. Blastema cells derived from the different limb tissues redifferentiate in a lineage-specific manner, but blastema cells derived from fibroblasts can also transdifferentiate into cartilage and tendon. The apical epidermal cap (AEC) of the wound epidermis is induced by regenerating nerve axons to produce a mitogen(s) that drives the proliferation of blastema cells. Patterning of the blastema involves region-specific contributions of limb cells, lineage-specific redifferentiation of blastema cells, interactions between blastema cells of different positions to eliminate discontinuities and sorting out of blastema cells with different cell surface adhesion. Cell interactions are mediated during axial patterning of the blastema by several signalling molecules and homeobox transcription factors. How the axial pattern and limb type of the early blastema is determined, whether by induction, self-organisation or both, remains controversial. Keywords: blastema origin and formation; cell cycling; determination; patterning; retinoic acid; Hox genes
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Epimorphic limb regeneration in amphibians, by definition, must include replacement of those parts that are amputated from the whole. But to make a functional regenerate, a stringent constraint is that the regeneration blastema not... more
Epimorphic limb regeneration in amphibians, by definition, must include replacement of those parts that are amputated from the whole. But to make a functional regenerate, a stringent constraint is that the regeneration blastema not duplicate any structure proximal to its level of origin. Redifferentiation of only those structures distal to the amputation plane by the blastema is known as the “rule of distal transformation” (Rose, 1970). The mechanism underlying the rule of distal transformation has been investigated since the end of the 19th century. At a first level, we can ask whether the mechanism is intrinsic or extrinsic to the blastema. If the former, the blastema is an independently differentiating tissue; if the latter, it is a nullipotent or pluripotent tissue requiring an inductive signal from the adjacent differentiated limb tissues to specify its pattern of redifferentiation.
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Background Following amputation, urodele salamander limbs reprogram somatic cells to form a blastema that self-organizes into the missing limb parts to restore the structure and function of the limb. To help understand the molecular basis... more
Background Following amputation, urodele salamander limbs reprogram somatic cells to form a blastema that self-organizes into the missing limb parts to restore the structure and function of the limb. To help understand the molecular basis of blastema formation, we used quantitative label-free liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS)-based methods to analyze changes in the proteome that occurred 1, 4 and 7 days post amputation (dpa) through the mid-tibia/fibula of axolotl hind limbs. Results We identified 309 unique proteins with significant fold change relative to controls (0 dpa), representing 10 biological process categories: (1) signaling, (2) Ca2+ binding and translocation, (3) transcription, (4) translation, (5) cytoskeleton, (6) extracellular matrix (ECM), (7) metabolism, (8) cell protection, (9) degradation, and (10) cell cycle. In all, 43 proteins exhibited exceptionally high fold changes. Of these, the ecotropic viral integrative factor 5 (EVI5),...
Research Interests: Chromatography, Mass Spectrometry, Biology, Cell Cycle, Proteomics, and 15 moreMedicine, Extracellular Matrix, Biological Sciences, Protein Structure and Function, Animals, Calcium Signaling, Amputation, Inositol, High Pressure Liquid Chromatography, Biological Process, Proteome analysis, Complex Structure, Ambystoma, Extremities, and BMC
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... 2. Martin P. (1997) Wound healingaiming for perfect skin regeneration ... 6. Masinde GL, Li X, Gu W, Davidson H, Mohan S, Baylink D. (2001) Identification of wound healing/regeneration quantitative trait loci (QTL) at multiple time... more
... 2. Martin P. (1997) Wound healingaiming for perfect skin regeneration ... 6. Masinde GL, Li X, Gu W, Davidson H, Mohan S, Baylink D. (2001) Identification of wound healing/regeneration quantitative trait loci (QTL) at multiple time points that explain seventy percent of variance in ...
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... From the present data, the un-coupling has already occurred for the ra-dial-ulnar and carpal regions by the cone stage, but does not occur for the meta-carpals until the late palette stage and for the phalangeal ... Stocum, D. L. 1968... more
... From the present data, the un-coupling has already occurred for the ra-dial-ulnar and carpal regions by the cone stage, but does not occur for the meta-carpals until the late palette stage and for the phalangeal ... Stocum, D. L. 1968 The urodele limb regen-eration blastema. ...
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Research Interests: Regeneration and Els
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Tests of the morphogenetic potency of the urodele limb regeneration blastema by a variety of transplantation experiments indicate that blastema is a self-organizing system. The cells of blastema inherit a memory of their position on the... more
Tests of the morphogenetic potency of the urodele limb regeneration blastema by a variety of transplantation experiments indicate that blastema is a self-organizing system. The cells of blastema inherit a memory of their position on the three cardinal axes of the limb. This memory is called positional memory, and it is fundamental to recognizing loss of structure, triggering cellular interactions to replace missing structures, and specifying the level from which new structures can begin. Retinoic acid (RA) modifies the positional memory in all three cardinal axes of the limb. The vitamin proximalizes positional memory in the proximodistal axis, posteriorizes it in the anteroposterior axis, and ventralizes it in the dorsoventral axis. In vivo and in vitro assays of interactions between cell derived from different proximodistal limb levels, before and after treatment with RA, suggest that the cellular basis of positional memory resides in recognition/affinity property of the cell surface. RA probably modifies positional memory by modulating transcription, via a nuclear retinoid receptor. It is suggested that the genes involved in specifying positional memory, and whose transcription is therefore affected in regenerating limbs, are homeo box genes and genes that encode for molecules involved in determining the composition and architecture of the cell surface and extracellular matrix.
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A series of normal stages describing the regeneration of larval A. maculatum limbs after amputation through the upper arm or wrist is described. Nine discrete stages were recognized, based on external morphological and associated... more
A series of normal stages describing the regeneration of larval A. maculatum limbs after amputation through the upper arm or wrist is described. Nine discrete stages were recognized, based on external morphological and associated histological features. These stages are Initial Dedifferentiation (ID), Early Bud (EB), Medium Bud (MB), Late Bud (LB), Early Redifferentiation (ER), Notch (N), 2-Fingerbud (2-FB), 3-Fingerbud (3-FB) and 4-Fingerbud (4-FB). Similarities and differences between this and other staging systems for urodele limb regeneration are discussed. The absence of osteoclasts was a striking feature during dedifferentiation of the wrist, in contrast to their presence in large numbers during dedifferentiation of the upper arm.
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The central nervous system (CNS) and the heart muscle regenerate poorly after injury, yet evidence is mounting that both harbor cells capable of rebuilding neural and cardiac tissue. The reason for the poor regenerative response of CNS... more
The central nervous system (CNS) and the heart muscle regenerate poorly after injury, yet evidence is mounting that both harbor cells capable of rebuilding neural and cardiac tissue. The reason for the poor regenerative response of CNS tissue and myocardium must therefore lie in the nature of the injury environment, which promotes fibrosis over regeneration. Strategies for regenerating these tissues thus rely on overcoming the fibrotic response by filling lesions with tissue-specific regeneration-competent cells that replace or rescue dying cells, or by activating endogenous regeneration-competent cells that do likewise. There has also been considerable excitement about the possibility of transplanting bone marrow cells into CNS or cardiac lesions to repair them, because bone marrow cells have been reported to be pluripotent. In this chapter, contemporary evidence for the existence of regeneration-competent cells in the CNS and heart is discussed, as well as attempts to use these ce...
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The cost of tissue damage due to degenerative disease and injury is enormous in terms of health care costs, lost economic productivity, diminished quality of life and premature death. Advances in cell, developmental and molecular biology,... more
The cost of tissue damage due to degenerative disease and injury is enormous in terms of health care costs, lost economic productivity, diminished quality of life and premature death. Advances in cell, developmental and molecular biology, and the discovery of regeneration-competent cells in many non-regenerating mammalian tissues, have given impetus to systematic investigations that will enable us to regenerate these tissues by cell transplantation or the pharmaceutical induction of regeneration from the body's own tissues. A significant avenue of research is the identification of the soluble and insoluble signals and their transduction pathways that govern the proliferation and differentiation of regeneration-competent cells, and the signals that inhibit their activity after injury. The most direct experimental strategy to identify the chemical and physical signals that promote regeneration and the factors that inhibit it is to make genomic and proteomic comparisons, using bioi...
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Larval and adult urodeles and anuran tadpoles readily regenerate their limbs via a process of histolysis and dedifferentiation of mature cells local to the amputation surface that accumulate under the wound epithelium as a blastema of... more
Larval and adult urodeles and anuran tadpoles readily regenerate their limbs via a process of histolysis and dedifferentiation of mature cells local to the amputation surface that accumulate under the wound epithelium as a blastema of stem cells. These stem cells require growth and trophic factors from the apical epidermal cap (AEC) and the nerves that re-innervate the blastema for their survival and proliferation. Members of the fibroblast growth factor (FGF) family synthesized by both AEC and nerves, and glial growth factor, substance P, and transferrin of nerves are suspected survival and proliferation factors. Stem cells derived from fibroblasts and muscle cells can transdifferentiate into other cell types during regeneration. The regeneration blastema is a self-organizing system based on positional information inherited from parent limb cells. Retinoids, which act through nuclear receptors, have been used in conjunction with assays for cell adhesivity to show that positional id...
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Over the past 50 years, we have made remarkable advances in the use of bionic devices and solid organ transplants as replacement parts for failing tissues and organs. These approaches to tissue restoration, however, have a number of... more
Over the past 50 years, we have made remarkable advances in the use of bionic devices and solid organ transplants as replacement parts for failing tissues and organs. These approaches to tissue restoration, however, have a number of drawbacks. Thus, a new approach, regenerative biology and engineering, has been developed, consisting of the strategies of cell transplantation, bioartificial tissue constructs, and stimulation of regeneration in vivo. Cell transplants have been successfully used to restore articular cartilage and to treat Parkinson's disease in humans. In rats, transplanted fetal and embryonic stem cell line-derived cardiomyocytes have been shown to differentiate and integrate well with the ventricular myocardium, suggesting the feasibility of using such transplants to restore damaged cardiac muscle. Diabetic symptoms in humans have been alleviated by implanting a bioartificial pancreas consisting of islet cells microencapsulated in alginate. Hydroxyapatite matrixes can stimulate the regeneration of bone across large gaps. Collagenous artificial matrixes can stimulate the regeneration of dermis, and peripheral nerve grafts embedded in a fibrin clot containing fibroblast growth factor-1 stimulate some regeneration of spinal cord axons in adult rats. Future research in regenerative biology will focus on several issues: (1) providing adequate sources of cells for transplantation and bioartificial tissue construction and determining ways to prevent these cells from coming under attack by the immune system, (2) developing new and better materials to build better bionic devices and bioartificial constructs and to stimulate regeneration in vivo, (3) determining how many tissues of the body might contain reserve cells for regeneration in vivo, (4) analyzing the molecular differences between cells and environments of regenerating versus nonregenerating tissues, and (5) understanding the factors and mechanisms involved in the proliferation and patterning of regenerating tissues.
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This article summarizes the results of three basic research approaches directed toward achieving the restoration of injured or diseased human tissues. The three approaches are (1) to understand the differences in the molecular... more
This article summarizes the results of three basic research approaches directed toward achieving the restoration of injured or diseased human tissues. The three approaches are (1) to understand the differences in the molecular characteristics of cells and their environments in tissues which exhibit regenerative capacity at one stage of the life cycle but not at another; (2) to understand the molecular mechanisms whereby tissues regenerate by means of reserve progenitor cells or progenitor cells formed by dedifferentiation; and (3) to design artificial tissues for implantation into the body. These strategies should allow us to locate the key switchpoints which determine regeneration versus repair and how to reconfigure those switchpoints into a regenerative circuit where necessary or to build tissues in vitro that can serve as in vivo substitutes. For these strategies to be successful, we must understand the role of the immune system in repair and regeneration and the developmental roles of regulatory molecules, such as growth factors, trophic factors, extracellular matrix components, and the protein products of patterning genes, as well as the intracellular signaling systems activated by these molecules. The examples used to illustrate these themes are repair versus regeneration in wounded mammalian skin, the regeneration of injured mammalian bone and muscle by reserve progenitor cells, and the regeneration of the urodele limb, neural retina, and lens by progenitor cells produced by dedifferentiation. In addition, several approaches used in the construction of bioartificial tissues are discussed.