The expression pattern of tomoregulin-1 in urodele limb regeneration and mouse limb development

Tomoregulin-1 (TMEFF1) was first identified as a gene implicated in pituitary secretion in Xenopus laevis. The predicted structure of TMEFF1 is that of a transmembrane protein with a highly conserved cytoplasmic tail, two follistatin domains and one modified EGF domain in its extracellular region. We report the cloning of the newt orthologue, and show that the expression of TMEFF1 is upregulated in the blastema during limb regeneration, and is also expressed in mouse embryonic limb development.     

In vitro effects of insulin on macromolecular events in newt limb regeneration blastemata

This work provides data demonstrating a stimulatory effect of insulin on macromolecular events occurring in cultured regeneration blastemata and demonstrates a synergistic interdependence between nerves and insulin in newt limb regeneration. The current experiments provide evidence for the following: (1) Insulin is paramount for expression of the mitogenic effect of nerves on cultures blastemata. (2) Insulin stimulates the incorporation of (3H)uridine into the acid-insoluble fraction of blastemal homogenates, but it does not alter the turnover rate of incorporated labeled uridine. (3) Insulin also stimulates the incorporation of 35SO4 and (3H)leucine into both chondroitinase-sensitive and chondroitinase-resistant blastemal proteoglycans. (4) Insulin increases the uptake of radiolabeled precursors by the blastemata, namely, (3H)leucine, (3H)uridine, 35SO4, (3H)alpha-aminoisobutyrate, and (3H)2-deoxy-D-glucose. The importance of insulin in the regulation of newt limb regeneration is discussed.     

Characterization of a cloned histone gene cluster of the newt Notophthalamus viridescens.

We report the cloning and characterization of a histone gene cluster of the newt Notophthalamus viridescens. Fragments containing newt histone genes were identified in whole genome Southern blots; these fragments were cloned into a bacteriophage lambda cloning vector constructed for this purpose. The positions of most of the histone genes were determined by hybridizing subcloned sea urchin histone genes to digests of the cloned newt gene cluster. The position of each gene was verified, and its polarity determined by sequencing a portion of each. The order of the genes in the cloned segment is H1-H3-H2B-H2A-H4, with each of the genes but H2B being transcribed in the same direction. Subcloned segments of the histone gene repeat were used to determine the size of each newt oocyte histone mRNA.     

Normal newt limb regeneration requires matrix metalloproteinase function

Newts regenerate lost limbs through a complex process involving dedifferentiation, migration, proliferation, and redifferentiation of cells proximal to the amputation plane. To identify the genes controlling these cellular events, we performed a differential display analysis between regenerating and nonregenerating limbs from the newt Notophthalmus viridescens. This analysis, coupled with a direct cloning approach, identified a previously unknown Notophthalmus collagenase gene (nCol) and three known matrix metalloproteinase (MMP) genes, MMP3/10a, MMP3/10b, and MMP9, all of which are upregulated within hours of limb amputation. MMP3/10b exhibits the highest and most ubiquitous expression and appears to account for the majority of the proteolytic activity in the limb as measured by gel zymography. By testing purified recombinant MMP proteins against potential substrates, we show that nCol is a true collagenase, MMP9 is a gelatinase, MMP3/10a is a stromelysin, and MMP3/10b has an unusually broad substrate profile, acting both as a stromelysin and noncanonical collagenase. Exposure of regenerating limbs to the synthetic MMP inhibitor GM6001 produces either dwarfed, malformed limb regenerates or limb stumps with distal scars. These data suggest that MMPs are required for normal newt limb regeneration and that MMPs function, in part, to prevent scar formation during the regenerative process.     

The newt ortholog of CD59 is implicated in proximodistal identity during amphibian limb regeneration

The proximodistal identity of a newt limb regeneration blastema is respecified by exposure to retinoic acid, but its molecular basis is unclear. We identified from a differential screen the cDNA for Prod 1, a gene whose expression in normal and regenerating limbs is regulated by proximodistal location and retinoic acid: Prod 1 is the newt ortholog of CD59. Prod 1/CD59 was found to be located at the cell surface with a GPI anchor which is cleaved by PIPLC. A proximal newt limb blastema engulfs a distal blastema after juxtaposition in culture, and engulfment is specifically blocked by PIPLC, and by affinity-purified antibodies to two distinct Prod 1/CD59 peptides. Prod 1 is therefore a cell surface protein implicated in the local cell-cell interactions mediating positional identity.     

Structure and expression of a newt cardio-skeletal myosin gene. Implications for the C value paradox

As part of our studies on the fate of the muscle lineage during amphibian limb regeneration, we have isolated genomic and cDNA sequences from a myosin heavy chain in the newt (Notophthalmus viridescens). Notwithstanding the technical problems inherent in analysing the large newt genome, genomic and cDNA sequences have been isolated and subjected to analysis by restriction mapping. Northern hybridization, Southern hybridization and DNA sequencing. We believe these to be the first single copy newt gene sequences to have been subjected to this type of analysis. The newt gene sequences showed a striking difference from mammalian myosins in both the estimated sizes of the gene and its intervening sequences; these being much larger than in the mammalian models, it is speculated that this could contribute to the exceptional size of the newt genome. By contrast, the coding sequences displayed very high levels of sequence homology to mammalian myosins. In particular, the amino acid sequence of the newt myosin was found to have greatest homology with rat and human myosin isotypes having a similar cardio-skeletal muscle expression pattern. Despite a long evolutionary separation, newt and mammalian cardio-skeletal myosins have remained more similar to each other than have the human or rat cardiac forms to skeletal myosins within their own respective species.     

In vitro and in vivo characterization of blastemal cells from regenerating newt limbs.

To better characterize the cells involved in newt limb regeneration, blastemal cells from accumulation and differentiation phase blastemas were grown in dissociated cell culture, and their morphology and antigenic phenotype determined using a variety of antibodies directed against intermediate filaments, cell adhesion molecules, and extracellular matrix molecules. In addition to previously described blastemal cell morphologies, many of the cells in these cultures had a round cell body, with an eccentrically placed nucleus and a cytoplasm filled with autofluorescent granules. The majority of accumulation phase blastemal cells labeled with antibodies against GFAP, vimentin, 22/18 as well as with antibodies against NCAM, L-1, laminin, and fibronectin. The majority of differentiation phase blastemal cells had a similar phenotype but lacked expression of vimentin and fibronectin. Comparison of the blastemal phenotype in vitro and in vivo showed similar expression characteristics. However, in differentiation phase blastemas, laminin immunoreactivity was concentrated in specific locations. In addition, the proliferation of cultured blastemal cells is stimulated by the addition of a crude brain extract, consistent with previous studies in vivo and in vitro. Taken together, these observations suggest that dissociated cultures of newt limb blastemal cells provide a suitable model for the analysis of the cell and molecular mechanisms involved in limb regeneration.     

Expression of complement 3 and complement 5 in newt limb and lens regeneration

Some urodele amphibians possess the capacity to regenerate their body parts, including the limbs and the lens of the eye. The molecular pathway(s) involved in urodele regeneration are largely unknown. We have previously suggested that complement may participate in limb regeneration in axolotls. To further define its role in the regenerative process, we have examined the pattern of distribution and spatiotemporal expression of two key components, C3 and C5, during limb and lens regeneration in the newt Notophthalmus viridescens. First, we have cloned newt cDNAs encoding C3 and C5 and have generated Abs specifically recognizing these molecules. Using these newt-specific probes, we have found by in situ hybridization and immunohistochemical analysis that these molecules are expressed during both limb and lens regeneration, but not in the normal limb and lens. The C3 and C5 proteins were expressed in a complementary fashion during limb regeneration, with C3 being expressed mainly in the blastema and C5 exclusively in the wound epithelium. Similarly, during the process of lens regeneration, C3 was detected in the iris and cornea, while C5 was present in the regenerating lens vesicle as well as the cornea. The distinct expression profile of complement proteins in regenerative tissues of the urodele lens and limb supports a nonimmunologic function of complement in tissue regeneration and constitutes the first systematic effort to dissect its involvement in regenerative processes of lower vertebrate species.     

Appendage-restricted gene induction using a heated agarose gel for studying regeneration in metamorphosed Xenopus laevis and Pleurodeles waltl

Amphibians and fish often regenerate lost parts of their appendages (tail, limb, and fin) after amputation. Limb regeneration in adult amphibians provides an excellent model for appendage (limb) regeneration through 3D morphogenesis along the proximodistal, dorsoventral, and anteroposterior axes in mammals, because the limb is a homologous organ among amphibians and mammals. However, manipulating gene expression in specific appendages of adult amphibians remains difficult; this in turn hinders elucidation of the molecular mechanisms underlying appendage regeneration. To address this problem, we devised a system for appendage-specific gene induction using a simplified protocol named the “agarose-embedded heat shock (AeHS) method” involving the combination of a heat-shock-inducible system and insertion of an appendage in a temperature-controlled agarose gel. Gene expression was then induced specifically and ubiquitously in the regenerating limbs of metamorphosed amphibians, including a frog (Xenopus laevis) and newt (Pleurodeles waltl). We also induced gene expression in the regenerating tail of a metamorphosed P. waltl newt using the same method. This method can be applied to adult amphibians with large body sizes. Furthermore, this method enables simultaneous induction of gene expression in multiple individuals; further, the data are obtained in a reproducible manner, enabling the analysis of gene functions in limb and tail regeneration. Therefore, this method will facilitate elucidation of the molecular mechanisms underlying appendage regeneration in amphibians, which can support the development of regenerative therapies for organs, such as the limbs and spinal cord.     

Hormone action in newt limb regeneration: insulin and endorphins

Although several hormones have been linked to newt limb regeneration, a cohesive hypothesis as to how these hormones control the process is yet to emerge. A critical review of the traditional approaches and a reevaluation of currently operative assumptions and interpretations of results precede the data on insulin and beta-endorphin. Results from in vivo and in vitro experiments on insulin are summarized, showing that insulin not only promotes various cellular events but also is essential for the expression of the mitogenic effect of nerves on cultured newt limb blastemata. Furthermore, the strong likelihood that insulin may be the common link in promoting limb regeneration in hypophysectomized newts that received pituitary hormone replacement therapy or a nutritional supplement is discussed. The status of beta-endorphin in regeneration is also explored. Data are presented to show that vertebrates with regenerating capacity (newts, tadpoles) have higher levels of plasma beta-endorphin than that found in species where the capacity to regenerate is either restricted (frogs) or totally lost (mammals). beta-Endorphin-like immunoreactivity has been localized in the epidermis of a regenerating newt blastema, as well as in the intermediate lobe of the pituitary gland of axolotl, newt, and Xenopus. A possible opiate connection in vertebrate limb regeneration, in particular, wound healing, is discussed.     

Highly efficient transfection system for functional gene analysis in adult amphibian lens regeneration

The analysis of newt lens regeneration has been an important subject in developmental biology. Recently, it has been reported that the genes involved in the normal eye development are also expressed in the regenerative process of lens regeneration in the adult newt. However, functional analysis of these genes has not been possible, because there is no system to introduce genes efficiently into the cells involved in the regeneration. In the present study, lipofection was used as the method for gene transfer in cultured pigmented iris cells that can transdifferentiate into lens cells in newt lens regeneration. Positive expression of a reporter gene was obtained in more than 70% of cells. In addition, the aggregate derived from gene-transfected cells maintained its expression at a high level for a long time within the host tissue. To verify the effectiveness of this model system with a reporter gene in lens regeneration, Pax6, which is suggested to be involved in normal eye development and lens regeneration, was transfected. Ectopic expression of lens-specific crystallins was obtained in cells that show no such activity in normal lens regeneration. These results made it possible for the first time to analyze the molecular mechanism of lens regeneration in the adult newt.     

Generation of transgenic newt Cynops pyrrhogaster for regeneration study

To take advantage of the ample potential for tissue regeneration by the newt, a technique to create transgenic newt was developed. The technique was based on a procedure for producing transgenic Xenopus, but modified to adapt to the different sperm morphology and to overcome the refractoriness of newt eggs to activation by normal cleavage. Sperm was collected from mature testes early in winter, permeabilized with digitonin, but without treatment of egg extract. Efficient egg activation was achieved by coinjection of inositol 1,4,5-trisphosphate (IP3) with DNA-sperm nucleus complex. Transgenic Cynops for EGFP/DsRed2 genes under the control of cytomegalovirus (CMV) enhancer/promoter showed nonmosaic widespread expression of reporter genes in embryos, swimming larvae, and adults after metamorphosis. Transgenic newt carrying EGFP gene under regulation of betaB1-crystallin promoter expressed the transgene uniquely in the lens. During lens regeneration after lens removal, EGFP expression occurred, reflecting the lens regeneration process. The newt transgenesis technique described here is likely to be of wide use in monitoring and manipulating gene expression in the study of molecular mechanisms underlying tissue regeneration.     

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.