Increased protein kinase C activity in the central nervous system of the newt during limb regeneration.

Protein kinase C (PKC) activity was examined in the CNS of the newt Pleurodeles waltlii undergoing regeneration after limb amputation. In the spinal cord and brain of control newts, the level of PKC activity was virtually the same for the cytosolic and the particulate fractions. At days 7 and 14 after amputation of two limbs, a twofold increase in overall PKC activity occurred in the spinal cord and accounted for increased membrane-bound activity, while cytosolic activity was not significantly impaired. In contrast, overall PKC activity was not affected in brain. However, a twofold increase in the brain particulate fraction occurred at day 14 while cytosolic activity decreased proportionately. Similar alterations were observed in newts undergoing one or multiple limb amputations. Such changes in PKC activity neither occurred in the CNS of newt after limb denervation nor in the CNS of limb amputated frog Rana temporaria, an Amphibian which is unable to regenerate. Taken together, these results provide evidence that PKC of the CNS is involved in the regeneration process of newts. Changes in activation-associated PKC distribution proceeded through different mechanisms: long-lasting increase in membrane bound activity with a net increase of overall activity in the spinal cord, and long-term redistribution of enzyme activity to the particulate fraction in brain.     

Control of blastema cell proliferation by possible interplay of calcium and cyclic nucleotides during newt limb regeneration

The effects of the divalent cation ionophore A23187, papaverine, and chlorpromazine on the mitotic index and cyclic nucleotide levels in newt limb regeneration blastemata (Notophthalmus viridescens) were assessed. The results of the experiments suggest that an intracellular increase in divalent cation (Ca2+) concentration results in elevated cGMP levels, suppressed cAMP levels, and a corresponding increase in blastema cell proliferation. The results also suggest that the converse conditions, namely, calcium efflux or inhibition of calmodulin activation (i.e., inhibition of Ca2+ binding), yields elevated cAMP levels, suppressed cGMP levels, and a corresponding decrease in blastema cell divisions.     

The cell cycle during amphibian limb regeneration.

The duration of the cell cycle in the blastema of regenerating limbs of axolotls has been measured by means of [3H]thymidine pulse labelling and autoradiography. A chase was required to define the pulse period. An average cell cycle at 20 degrees C takes 53 h, S-phase takes 38 h; including parts of mitosis, G1 is 10 h and G2 is 5 h long. The protracted cycle and S-phase are consonant with the large genome in axolotis and other urodeles. The rapidly growing blastema probably contains a steady population of about 5000 proliferating cells, as there is a regular withdrawal of differentiating cells from the population. The kinds of determination which exist in this population of cells, or are exerted on it, are briefly considered.     

An investigation of the prolactin-thyroxine synergism in newt limb regeneration

The use of hormone replacement to support limb regeneration in hypophysectomized newts has been the subject of many investigations. Growth hormone, as well as prolactin (PL) in combination with exogenously supplied thyroxine, have all been shown to he effective. However, the bovine growth hormone used to support limb regeneration was contaminated by prolactin and thyroidstimulating hormone (TSH). The present investigation evaluates the significance of (1) prolactin contamination and (2) endogenous thyroxine synthesis resulting from TSH contamination on limb regeneration in hypophysectomized newts. The effect of supplying exogenous thyroxine was also evaluated. Our studies showed that when hypophysectomized newts were injected with contamination levels of PL and TSH, regeneration occurred, suggesting that the newt's thyroid synthesized sufficient thyroxine to support a prolactin-thyroxine synergism. The endogenous thyroxine was synthesized by thyroid glands that were indistinguishable from those of saline-injected, hypophysectomized controls.     

Plasticity of retrovirus-labelled myotubes in the newt limb regeneration blastema

Two important indices of myogenic differentiation are the formation of syncytial myotubes and the postmitotic arrest from the cell cycle, both of which occur after fusion of mononucleate cells. We show here that these indices are reversed in the environment of the urodele limb regeneration blastema. In order to introduce an integrated (genetic) marker into newt myotubes, we infected mononucleate cells in culture with a pseudotyped retrovirus expressing human placental alkaline phosphatase (AP). After fusion the myotubes expressed AP and could be purified by sieving and micromanipulation so as to remove all mononucleate cells. When such purified retrovirus-labelled myotubes were implanted into a limb blastema they gave rise to mononucleate progeny with high efficiency. Purified myotubes labelled with fluorescent lipophilic cell tracker dye also gave rise to mononucleate cells; myotubes which were double labelled with the tracker dye and a nuclear stain gave rise to double-labelled mononucleate progeny. Nuclei within retrovirus-labelled myotubes entered S phase as evidenced by widespread labelling after injection of implanted newts with BrdU. The relation between the two aspects of plasticity is a critical further question.     

Vesicle and granule content of sympathetic ganglion cells during limb regeneration of the newt Triturus

Summary Fine structural observations were made on the vesicle and granule content of ganglion cells in the posterior subclavian ganglion and peripheral nerve fibers of the upper forelimb of the newt Triturus. The populations of vesicles and granules in normal ganglion cells and nerve fibers were compared with those observed after limb transection. In normal neurons, clear vesicles range in size from 250 to 1000 Å in diameter, but are most frequently 400–500 Å. Vesicles with dense contents (granules) also vary greatly in size, but most are 450–550 Å in diameter and correspond to dense-core vesicles. Large granules that contain acid phosphatase activity are thought to be lysosomes. During limb regeneration, in both the ganglion cells and peripheral nerves, the ratio of dense vesicles to clear vesicles increases. There is a large increase in number of dense granules with a diameter over 800 Å, particularly in the peripheral regenerating fibers. This study shows that regenerating neurons differ from normal in their content of vesicular structures, especially large, membrane-bounded granules.This work was supported by grants from the National Science Foundation (GB 7912) and from the National Cancer Institute (TICA-5055), National Institutes of Health, United States Public Health Service.     

Association of mesenchyme with attenuated basement membranes during morphogenetic stages of newt limb regeneration

The interface between epithelium and mesenchyme may be involved in inductive interactions which occur during development. This interface within the growth bud, or blastema, of a regenerating limb has been examined to determine whether changes in basement-membrane structures are visible in regions of putative epithelial-mesenchymal inductive interaction. Regenerating forelimbs of adult newts were fixed by perfusion with osmotically balanced aldehydes. Late-bulb to early-digit stage regenerates were collected and processed either for light and transmission electron microscopy or for scanning electron microscopy. Light microscopy confirmed that regions characterized by increased numbers of subepithelial mesenchymal cells were covered by a diffusely stained basement membrane. Transmission electron microscopy of these regions revealed two structural components of the basement membrane. The thin basal lamina was continuous in all regions of all stages examined, but it was attenuated apically in areas of mesenchymal cell accumulation. The thicker underlying reticular lamina was markedly attenuated in these regions near the blastemal apex. Scanning electron microscopy of de-epithelialized blastemas revealed that, apically, the reticular lamina formed only a delicate lacelike network. On the base of the blastema, it formed a dense fibrillar meshwork which was further organized into a geometric pattern in the adjacent stump skin. Cumulatively, these observations suggest that physical contact between epithelial and mesenchymal cells is not essential at these stages, but that regions of putative epithelial-mesenchymal interaction are characterized by a distinctly diminished reticular lamina. Structural changes in basement-membrane components may be related to termination of local inductive events.     

Scanning electron microscopy of epidermal cell migration in wound healing during limb regeneration in the adult newt, Notophthalmus viridescens

The epidermal cells which migrate over the wound surface of the amputated limb of the adult newt were examined using the scanning electron microscope. Specimens were prepared routinely for scanning electron microscopy or were embedded in Epon 812 for light microscopic observations. A cuff of epidermal cells was seen at the edge of the wound, from which cells appeared to migrate over the wound surface. As early as five hours after transection of the limb, the basal layers of this cuff appeared to send out pseudopodial projections. These seemed to establish a physical contact with a fibrin-like substratum, which apparently served as a means of support for the migrating cells. Subsequently, the epidermal cells became elongate and had the appearance of streaming toward the center of the wound. Between 10 and 13 hours post-amputation, the cells in the central region of the stump were rounded up and some possessed microappendages resembling microplicae and microvilli. Throughout the entire period of wound coverage, the cells seemed to maintain contact with the fibrin network, which appeared to be the first structural element of wound architecture. As a result of these observations, the mechanism by which the epidermal cells migrate has been clarified.     

An immunofluorescent study of the distribution of fibronectin and laminin during limb regeneration in the adult newt

Fibronectin and laminin are two extracellular glycoproteins which are involved in various processes of cellular development and differentiation. The present investigation describes changes in their distribution during regeneration of the newt forelimb, as determined by indirect immunofluorescence. The distribution of fibronectin and laminin was similar in normal limb tissue components. These glycoproteins were localized in the pericellular region of the myofibers corresponding to its basement membrane; the perineurium and endoneurium of the nerves; and the basement membranes of blood vessels, skin epithelium, and dermal glands. The cytoplasm of myofibers, axons, skin epithelium, and bone matrix lacked fluorescence for both glycoproteins. After limb amputation in the regenerating blastema, extensive presence of fibronectin, but not laminin, was seen in and around the undifferentiated blastemal cells. Increased fluorescence for fibronectin was also seen during blastema growth, blastemal cell aggregation, and early stages of redifferentiation. As redifferentiation continued, staining for fibronectin slowly disappeared from the cartilage matrix and the myoblast fusion zone. Laminin was first observed around the regenerated myotubes; this was followed by the appearance of fibronectin suggesting a sequential formation of these two components of the new myotube basement membrane. In the regenerated limb, the distribution of laminin and fibronectin was similar to that seen in normal limb. Based on the distribution pattern of these glycoproteins, it is concluded that fibronectin may play an important role in blastemal cell aggregation, cell alignment, and initiation of redifferentiation. After redifferentiation, both laminin and fibronectin may be important in the determination of the architecture of the regenerated limb.     

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.     

A rapid and simple estimation of cell cycle parameters by continuous labeling with bromodeoxyuridine

The DNA synthesis time (Ts) and other related cell cycle parameters were roughly estimated in HeLa cells labeled with bromodeoxyuridine (BrdUrd) for various durations by using the flow cytometrical technique. The labeling indices increased in proportion to time after addition of BrdUrd. The Ts can be calculated from the slope of the regression line obtained by plotting the serial labeling indices against the labeling time and was equivalent to the value determined by fraction labeled cells in mid S-phase (FLSm) method. These parameters would be determined by only two samples labeled for different times. This simple method using BrdUrd provides rough but rapid estimation of Ts and other cell cycle parameters without complicated mathematical procedures, in addition to cell cycle partition of cell populations.     

Ultrastructural labeling of cell surface lectin receptors during the cell cycle.

The distribution of specific surface receptors in the course of the cell cycle has been studied on two transformed cell lines by means of ultrastructural labelling techniques employing Concanavalin A (ConA) and wheat germ agglutinin (WGA). Synchronized cultures of Cl₂TSV₅, an SV40-transformed hamster cell line and of CHO cells were labelled as monolayers or in suspension in the different phases of the cell cycle. In cells labelled in monolayers, a moderately discontinuous pattern of surface labelling was present during G 1, S, and G 2. On cells in mitosis, however, this pattern changes strikingly and becomes very discontinuous. These results indicate that the degree of receptor clustering is greater in mitosis than in interphase. In cells labelled in suspension, the differences in pattern between mitosis and interphase were absent. Colcemid treatment did not modify the distribution of the label, either in interphase or in mitosis. Moreover, cells in mitosis collected by Colcemid treatment and labelled at a moment in which parallel unblocked cultures had completed mitosis and passed into G 1 showed an interphase-type labelling pattern; this indicates that a certain dissociation exists between surface events and nuclear events during mitosis. These results are discussed in terms of several factors that may contribute to the production of receptor clustering, namely, direct lectin action, surface movement and membrane flow, participation of cytoplasmic structures and, finally, attachment of cells to a substratum.     

Inhibition and axial deviation of limb regeneration in the newt by means of a digit implanted into the amputated limb

This research was designed to follow up the observation of Thornton and Kraemer ('51) that regressed, denervated limbs of Ambystoma larvae will not regenerate upon reinnervation if all digits on the limbs were not completely resorbed. The object of this experiment was to determine whether the presence of an apical structure, protruding past the amputation surface, would affect the regenerative process. Both forearms of adult newts were amputated midway between the elbow and the wrist. One limb served as a normal regeneration control, and in the other limb the third digit from the removed hand was implanted in place of the removed radius, so that the three distal phalangeal segments protruded past the plane of amputation. Blastema formation in the experimental limbs was delayed by several weeks as compared with control limbs. Approximately one third of the experimental limbs did not regenerate. The regenerates that did form were strongly deviated (45–90°) radially from the longitudinal axis of the limb. Experimental analysis showed that the delay in regeneration is due largely to the projecting part of the digit. The radial deviation of the regenerates is not due to the digital implant, but rather to the removal of the radius. Trauma alone does not account for this phenomenon.     

Control of cell fate choices by lateral signaling in the adult peripheral nervous system of Drosophila melanogaster

The thoracic integument of the adult fruit fly is a relatively simple but highly patterned structure. It is composed of sensory organ cells distributed within a monolayer of epidermal cells. Both cell types are easily detected at the cuticular surface, as each external sense organ forms a sensory bristle and each epidermal cell secretes a small nonsensory hair. Inhibitory cell—cell interactions play a key role in regulating the distribution as well as the formation of the sense organs. This review focuses on the role of these cell—cell interactions in the adoption of alternative cell fates. We also show that Notch, Hairless, and Suppressor of Hairless, three components of this intercellular signaling pathway, exhibit dose-dependent genetic interactions. Finally we address how this intercellular signaling mechanism may be modulated to result in highly reproducible outcomes. © 1996 Wiley-Liss, Inc.     

The histology of inhibition of limb regeneration in the newt, Triturus, by actinomycin D

Adult newts, Triturus viridescens, were treated with from 1.0–10.0 μg/g body weight of actinomycin D one day before amputation of both forelimbs. Mean survival times ranged from over 50 days in newts treated with 1.0 μg/g to 13.2 days in animals given 10.0 μg/g body weight of actinomycin. Low doses little altered the course of regeneration, but animals treated with over 2.0 μg/g never formed blastemas. In another series, animals were given doses of 2.5 μg/g body weight of actinomycin D at intervals from 14 days before to 30 days after amputation. It was found that certain signs of toxicity (loss of equilibrium) are related to the time of administration of the drug whereas others (hemorrhage into the limb stumps) are restricted to a definite phase of the regenerative process. Early administration of actinomycin completely inhibits regeneration whereas later treatment results in a considerably lessened effect. The postamputational stages which are basically destructive in nature are not noticeably affected by actinomycin D, but the phases of dedifferentiation, blastema formation and redifferentiation are strongly inhibited.     

Cell cycle and cell fate in the nervous system

Recently, a number of molecules originally thought to have a primary role in cell determination have been shown to affect the cell cycle at specific check points, while other molecules discovered for their roles in the cell cycle progression are known to affect the determination and differentiation of neurons. These discoveries have led to a more detailed investigation of the complex molecular machinery that co-ordinates proliferation and differentiation.     

Nerve dependent sulphated glycosaminoglycan synthesis in limb regeneration of the newt Pleurodeles waltl

Denervation of the amputated limb of newts stops the regeneration process by decreasing blastema cell proliferation. We investigated the effect of the denervation on each of the two compartments (epidermal cap, mesenchyme) in mid-bud blastemas on the level of sulphated glycosaminoglycans (GAGS). Denervation resulted in an increase of about threefold in the incorporation of [³⁵S] sulphate into mesenchyme GAGs but had no effect on the epidermal cap. The increase of GAG synthesis in the mesenchymal part of the blastema involved both heparan sulphates and chondroitin-dermatan sulphates. Gel filtration showed no change in GAGs size after denervation. These results confirm that the mesenchymal part of the mid-bud blastema is the main target of nerves and, as heparan sulphates are known to store acidic fibroblast growth factor (aFGF), a polypeptide found in the blastema (Boilly et al.. 1991), this suggest that the nerves' effect on glycosaminoglycans turnover could be implicated in the control of bioavailability of this growth factor in the blastema.     

Changes in microtubule structures during the first cell cycle of physiologically polyspermic newt eggs

The unfertilized egg of the newt, Cynops pyrrhogaster, has a second meiotic spindle at the animal pole and numerous cortical cytasters. After physiologically polyspermic fertilization, all sperm nuclei incorporated into the egg develop sperm asters, and the cortical cytasters change into bundles of cortical microtubules. The size of the sperm asters in the animal hemisphere is ∼5.6-fold larger than that in the vegetal hemisphere. Only one sperm nucleus moves toward the center of the animal hemisphere to form a zygote nucleus with the egg nucleus. This movement is inhibited by nocodazole, but not by cytochalasin B. The centrosome in the zygote nucleus divides into two parts to form a bipolar spindle for the first cleavage synchronously with the nuclear cycle, but centrosomes of accessory sperm nuclei in the vegetal hemisphere remained to form monopolar interphase asters and subsequently degenerate around the first cleavage stage. The size of sperm asters in monospermically fertilized Xenopus eggs was ∼37-fold larger than those in Cynops eggs. Since sperm asters that formed in polyspermically fertilized Xenopus eggs exclude each other, the formation of a zygote nucleus is inhibited. Cynops sperm nuclei form larger asters in Xenopus eggs, whereas Xenopus sperm nuclei form smaller asters in Cynops eggs compared with those in homologous eggs. Since there was no significant difference in the concentration of monomeric tubulin between those eggs, the size of sperm asters is probably regulated by a component(s) in egg cytoplasm. Smaller asters in physiologically polyspermic newt eggs might be useful for selecting only one sperm nucleus to move toward the egg nucleus. Mol. Reprod. Dev. 47:210–221, 1997. © 1997 Wiley-Liss, Inc.