Nonmuscle stem cells fail to significantly contribute to regeneration of normal muscle.

Whole, normal extensor digitorum longus muscles (EDL) were orthtopically transplanted into transgenic mice, expressing nuclear localing beta-galactosidase (nlsbeta-gal) under control of a muscle-specific promoter, in order to determine the extent to which nonmuscle derived, multipotent stem cells (which under experimental conditions exhibit myogenic potential) are spontaneously recruited from distal, nonmuscle organs to participate in the graft's regeneration. The host's contribution to the graft's regeneration was determined by evaluating the number and distribution of beta-gal positive myonuclei in regenerated grafts. Fibers with beta-gal positive nuclei accounted for approximately 1% of the long-term (28- and 56-day) graft's myofibers. All were confined to the graft's periphery, adjacent to host's muscles. Failure to find myofibers with beta-gal positive nuclei across the revascularized graft's girth demonstrated that there was no meaningful recruitment of nonmuscle stem cells from distal host organs, which must arrive at the graft via the circulation. Rather, stem cells residing in the graft at the time of transplantation accounted for approximately 99.9% of the regenerated graft's myonuclei, with a minor contribution from the surrounding host muscles' myosatellite cells (that occurred when the epimysia of graft or host muscles were damaged during transplantation). The significance of these findings to gene therapy for Duchenne muscular dystrophy is discussed.

Effect of chronic denervation and denervation-reinnervation on cytoplasmic creatine kinase transcript accumulation.

The extensor digitorum longus (EDL) and soleus muscles of adult mice were chronically denervated or denervated and allowed to reinnervate. Muscles were evaluated 1, 5, 14, 21, and 52 days after sciaticectomy. In terms of weight loss, myofiber atrophy, degeneration, and fibrosis, the soleus muscle was more affected than the EDL by chronic denervation. Fifty-two days after chronic denervation, the number of molecules of MCK/ng total RNA in both muscles (determined with competitive PCR) decreased, with the soleus muscle being more affected. At that stage, BCK mRNA levels in the denervated soleus were unchanged, but they were increased (>50%) in the EDL. Reinnervation restored MCK transcript accumulation in the EDL, whereas, in the soleus MCK, transcripts exceeded control values by 57%, approaching levels in the reinnervated EDL. Despite restoration of MCK mRNA levels, the number of molecules of BCK mRNA/ng total RNA was four- to fivefold higher in reinnervated versus control muscles, suggesting that the genes encoding the CK mRNAs are not coordinately regulated in adult muscle. The role of denervation induced, fiber type changes in regulating CK mRNA accumulation has been evaluated. Electron microscopic analyses have established that fibrosis is not a factor that determines BCK mRNA levels in the chronically denervated or denervated-reinnervated muscles. CK isozyme analyses support the hypothesis that a greater proportion of BCK mRNA found in 52 day chronically denervated and denervated-reinnervated muscles is produced in myofibers vs. nonmuscle cells than in control muscles.

Enhancement of adult muscle regeneration by primary myoblast transplantation.

Extensor digitorum longus muscles (EDL) of SCID mice were induced to undergo degeneration-regeneration subsequent to orthotopic, whole-muscle transplantation. Two days after transplantation some of these muscles received injections of primary myoblasts derived from EDL muscles of transgenic mice, which express nuclear localizing beta-galactosidase under the control of the myosin light-chain 3F promoter and enhancer. Nine weeks after transplantation, regenerated muscles that received exogenous myoblasts were compared to similarly transplanted muscles that received no further treatment and to unoperated EDL muscles in order to determine the effect of myoblast transfer on muscle regeneration. Many myofibers containing donor derived myonuclei could be identified in the regenerated muscles that had received exogenous myoblasts. The mass of the muscles subjected to transplantation only was significantly less (31% less) than that of unoperated muscles. The addition of exogenous myoblasts to the regenerating EDL resulted in a muscle mass similar to that of unoperated muscles. The absolute twitch and tetanic tensions and specific twitch and tetanic tensions of transplant-only muscles were 28%, 36%, 32%, and 41%, respectively, of those of unoperated muscles. Myoblast transfer increased the absolute twitch and tetanic tensions of the regenerated muscles by 65% and 74%, respectively, and their specific twitch and tetanic tensions were increased by 41% and 48%, respectively. These data suggest a possible role for the addition of exogenous, primary myoblasts in the treatment of traumatized and/or diseased muscles that are characterized by myofiber loss.

Creatine kinase transcript accumulation: effect of nerve during muscle development.

To determine the role of the nerve in regulating the accumulation of cytoplasmic creatine kinase (CK) mRNAs in hindleg muscles of the developing mouse, the lumbosacral spinal cords of 14-day gestation mice (E14) were laser ablated, and the accumulation of muscle CK (MCK) and brain CK (BCK) mRNAs was evaluated just prior to birth with in situ hybridization. Numbers of molecules of each of these transcripts/ng total RNA in the soleus and extensor digitorum longus (EDL) muscles were determined with competitive PCR and compared to transcripts found in innervated crural muscles. Data suggest that: 1) the level of BCK mRNA accumulation in innervated hindlimb muscles peaks at E16.5 and remains at fetal levels until the second month postnatal, when it falls to the level found in the adult. Given that MCK transcripts meet or exceed adult levels by day 28 postnatal, the "down-regulation" of the BCK gene and the "up-regulation" of the MCK gene are not tightly coupled; 2) the developmental switch from BCK to MCK, as the dominant cytoplasmic CK mRNA, occurs in innervated and aneural leg muscles between E14 and E16.5, indicating this switch is not nerve dependent; 3) the absence of innervation has no effect on BCK mRNA accumulation. MCK transcripts/ng total RNA continue to increase in aneural muscle throughout the late fetal period, but from E16.5-E19.5 the MCK transcript levels in aneural muscles become progressively lower than in age-matched innervated muscles. Thus, the accumulation of the muscle specific cytoplasmic CK, but not BCK, transcripts is affected by the absence of innervation during the fetal period. Dev Dyn 1999;215:285-296.

Role of the nerve in determining fetal skeletal muscle phenotype.

To determine the role of the nerve on the establishment of myofiber diversity in skeletal muscles, the lumbosacral spinal cord of 14-day gestation mice (E14) was laser ablated, and the accumulation of the myosin alkali light chains (MLC) mRNAs in crural (hindleg) muscles was evaluated just prior to birth with in situ hybridization. Numbers of molecules of each alkali MLC/ng total RNA in the extensor digitorum longus (EDL) and soleus muscles were determined with competitive polymerase chain reaction. Transcripts for all four alkali MLCs accumulate in aneural muscles. Data suggest that: (1) the absence of the nerve to either future fast or slow muscles results in less accumulation of MLC1V transcript. Moreover, the presence of the nerve is required for the enhanced accumulation of this transcript in future slow muscles; (2) the absence of innervation of future slow, but not fast, muscles decreases the accumulation of MLC1A transcript. Since increased accumulation of MLC1A and MLC1V transcripts are found in future slow muscles at birth, the nerve is necessary for the development of the slow phenotype during myogenesis; (3) MLC1F and MLC3F transcripts do not display any preferential accumulation in future fast muscles during the fetal period. Therefore, the establishment of the differential distribution of these mRNAs, based on fiber type, is a postnatal phenomenon. The nerve is required during the fetal period to allow accumulation of MLC3F messages above a basal level in future fast as well as slow muscles; whereas, the absence of the innervation to future fast, but not slow, muscles reduces the accumulation of MLC1F. Thus, the accumulation of the various alkali MLC mRNAs shows a differential, rather than coordinate, response to the absence of the nerve, and this response may vary depending on the future fiber type of the muscles.

Mass and functional capacity of regenerating muscle is enhanced by myoblast transfer.

Morphology and functional capacity of homotopically transplanted extensor digitorum longus muscles (EDL) of adult SCID mice that received 1 x 10(6) myoblasts [stably transfected to express nuclear localizing beta-galactosidase under the control of the myosin light-chain 3F promoter/enhancer] 2 days posttransplantation were evaluated 9 weeks after transplantation, to determine whether the injection of exogenous myoblasts had an effect on muscle regeneration. Regenerated muscles that received exogenous myoblasts were compared to similarly transplanted muscles that received (a) no further treatment, or (b) sham injection of the vehicle (without myoblasts) and to unoperated EDL. Nine weeks after myoblast transfer, myofibers containing donor-derived nuclei could be identified after staining with X-gal solution. Judging from its size and poor functional performance compared to muscles subjected to transplantation only, sham injection provided a secondary trauma to the regenerating muscle from which it failed to fully recover. In comparison to the sham-injected muscle, the myoblast-injected muscles weighed 61% more and had 50% more myofibers and 82% more cross-sectional area occupied by myofibers at the muscles' widest girths. Their absolute twitch and tetanic tensions were threefold and twofold greater, respectively, and their specific twitch and tetanic tensions were 71% and 50% greater, respectively, than those of sham-injected muscles. In many parameters, the regenerating muscle subjected to myoblast transfer equaled or exceeded those of muscles that were transplanted only (received only one trauma). Absolute twitch and tetanic tensions were 73% and 65% greater, respectively, and specific twitch tensions of the muscles receiving myoblasts were 50% greater than forces generated by muscles subjected to whole-muscle transplantation only.

Can insights into urodele limb regeneration be achieved with cell cultures and retroviruses?

Recent advances in the field of amphibian limb regeneration have provided insights into its cellular and molecular events. This review summarizes the development of cell lines from limb tissues and their application to the study of transdifferentiation and limb regeneration. In addition, the availability of suitable retroviral vectors for salamanders is discussed for it has opened new avenues for experimentation at the molecular level.

Cloning of homoeobox sequences expressed in the intact and the regenerating newt eye.

In this report we present the sequences of four different homeoboxes cloned from the intact and regenerating eye of the newt Notophthalmus viridescens. All these homeoboxes are novel for this species.

Transdifferentiation as a basis for amphibian limb regeneration.

Limb regeneration is a phenomenon occurring only in some urodeles. The process seems to be initiated by the dedifferentiation of the terminally differentiated cells. These cells differentiate, subsequently, to the tissues that comprise the limb, thus reconstructing the pattern of the missing limb part. In this paper we review and present evidence that certain cell types of the limb have the capacity to differentiate to different cell types than their original one by cellular metaplasia. This switch is called transdifferentiation. The focus of this review is the process of dedifferentiation which is the necessary prerequisite for differentiation, and the possible mechanisms involved.

Expression of pax-6 during urodele eye development and lens regeneration.

Regeneration of eye tissues, such as lens, seen in some urodeles involves dedifferentiation of the dorsal pigmented epithelium and subsequent differentiation to lens cells. Such spatial regulation implies possible action of genes known to be specific for particular cell lineages and/or axis. Hox genes have been the best examples of genes for such actions. We have, therefore, investigated the possibility that such genes are expressed during lens regeneration in the newt. The pax-6 gene (a gene that contains a homeobox and a paired box) has been implicated in the development of the eye and lens determination in various species ranging from Drosophila to human and, because of these properties, could be instrumental in the regeneration of the urodele eye tissues as well. We present data showing that pax-6 transcripts are present in the developing and the regenerating eye tissues. Furthermore, expression in eye tissues, such as in retina, declines when a urodele not capable of lens regeneration (axolotl) surpasses the embryonic stages. Such a decline is not seen in adult newts capable of lens regeneration. This might indicate a vital role of pax-6 in newt lens regeneration.

Mononuclear leukocytes in the newt limb blastema: in vitro behavior.

Since the precise interactions of the various newt limb blastema cell types (pleiomorphic, bipolar, signet and multinucleated cells) with one another are difficult to ascertain in vivo, in this study we describe the in vitro behavior and interactions of these cell types with one another. The data show that the mononuclear signet cells interact (fuse) with other signet cells or fuse directly with the multinucleated cells. Further, they indicate that the signet cells are in fact mononuclear leukocytes and the giant cells are osteoclasts. Therefore, we conclude that these two cell types are not formed by dedifferentiation but are of hematopoietic origin and may play minor roles in blastema production.

Pantropic retroviral vector-mediated gene transfer, integration, and expression in cultured newt limb cells.

Limb regeneration is a unique developmental phenomenon restricted to certain urodeles in which limb cells dedifferentiate and produce the blastema and then redifferentiate into the tissues that compose the missing part. Genetic modification of the blastema cells would greatly facilitate understanding the programmed gene expression that results in the reconstitution of the limb. To test whether pantropic retroviral vectors pseudotyped with the vesicular stomatitis virus G glycoprotein could mediate gene transfer into blastema cells, we infected a stable newt limb cell line and demonstrated integration and expression of the provirus. Thus, pantropic retroviral vectors offer a new tool for the study of limb regeneration and other developmental phenomena in amphibia.

Protein synthesis in the brain of newts undergoing limb regeneration.

The nervous system plays an important role during the process of amphibian limb regeneration. However, the molecules that are involved in such a control of regeneration are largely unknown. We have attempted to map protein synthesis in the brains of intact newts and from newts undergoing limb or tail regeneration. Our results show unique protein synthesis in the brain of newts undergoing limb regeneration. Such an analysis can lead to the identification and characterization of these proteins.

The mutant axolotl Short toes exhibits impaired limb regeneration and abnormal basement membrane formation.

The mutant axolotl Short toes develops with abnormal kidneys, Mullerian ducts, and limbs and provides one of the few experimental systems for developmental studies in amphibia. The present paper describes another deviation from this animal's normal physiology, which is very characteristic of the wild type: amputated limbs of Short toes fail to regenerate. A blastema is formed but differentiation does not occur. Detailed histological analysis provides evidence of abnormal formation of the basement membrane and accumulation of extracellular matrix within the blastema, which could be attributed to an imbalance of extracellular matrix and basement membrane proteins. The basement membrane develops much thicker and is convoluted in the arrested blastema of mutant animals. In contrast to the limbs, the tails of Short toes regenerated normally with no apparent abnormalities. No gross genomic aberrations have been detected between normal and mutant DNA, indicating that a large deletion or insertion is not likely to be the cause of this mutation.

Histological analysis of forelimb regeneration in the California newt Taricha granulosa.

The regenerative ability of the forelimbs of the California rough-skinned newt, Taricha granulosa was determined and compared to the same ability of the adult Mexican axolotl, Ambystoma mexicanum. Forelimbs were amputated distally at the wrist and limbs removed at 1, 2, 3, 4, 5 and 6 weeks post-amputation were examined by histological analysis. Since vitamin A and its derivatives cause extreme changes in pattern formation in the regenerating amphibian limb, we decided to study the ability of retinoic acid to proximalize the distal amputation. Animals injected with RA displayed apparent proximalization of the distal amputation.