Stump currents in regenerating salamanders and newts

We report here that a variety of salamanders and newts from differing habitats all drive a steady ionic electric current out of the forelimb stump tip after forelimb amputation. Several hours after amputation the density of this stump current ranges from about 10 to 100 microA/cm2 in most species, and declines with time. In most cases, the magnitude of the stump current is dependent on the concentration of Na+ in the external medium (an artificial pondwater), suggesting that the well-known Na+ -dependent transcutaneous voltage described in amphibia (particularly frogs) is the EMF for this stump current. These measurements add to those previously reported for the North American red spotted newt (Notophthalmus viridescens), and suggest that electrical changes following amputation of urodele limbs are widespread among members of this group.     

Levels of cyclic GMP and cyclic AMP in regenerating forelimbs of adult newts following denervation

The effect of short-term denervation (0, 12, 24, and 72 hours) on the levels of cyclic 3'5'-guanosine monophosphate (cGMP) and cyclic 3'5'-adenosine monophosphate (cAMP) in adult newt (Notophthalmus viridescens) forelimbs at 15, 22, and 35 days of regeneration was investigated. Regenerate blastema and stump cyclic nucleotide levels were compared with those of the contralateral intact forelimb and hindlimb, with levels in the normally regenerating blastema, and with levels measured in the forelimbs of intact, nonoperated animals. Variations in cyclic nucleotide levels occurred according to regeneration stage and tissue type. Changes in level were noted immediately upon denervation and subsequently at other sample times in all regenerate and control series. Parallel fluctuations occurred in regenerate stump and contralateral intact forelimbs. Our results from nonamputated denervated and sham-denervated animals indicate that short-term, denervation-associated cyclic nucleotide fluctuations cannot be attributed solely to the loss of innervation.     

Effects of growth hormone and nutrient on limb regeneration in hypophysectomized adult newts

The effect of hypophysectomy, growth hormone (GH) and an amino acid-glucose mixture on the regenerative ability of the hypophysectomized Triturus pyrrhogaster yielded the following results:

• 1 The survival time of hypophysectomized newts can be prolonged substantially by the sulfamide application.
• 2 Although the limb regeneration in the hypophysectomized newt is retarded as compared with that of the pituitary intact control, it finally completes morphogenetic process under such conditions of prolonged survival.
• 3 The injection of 100 μg of GH restored the speed of regeneration of pituitary-deprived limbs to almost a normal level.
• 4 Injections of the amino acid-glucose mixture also promoted the limb regeneration in hypophysectomized newts. However, initial delay in regeneration to the time of bud appearance was not restored by the nutrients.

     

A monoclonal antibody detects a difference in the cellular composition of developing and regenerating limbs of newts

We have previously described a monoclonal antibody (called 22/18) that reacts with the early blastemal cells of the regenerating limb of the newt (Notophthalmus viridescens). In embryos of two newt species the antibody reacts with the epidermis, glial cells in the neural tube, the lens and cells in a restricted region of the aorta. In the developing limb bud less than 1% of the mesenchymal cells were reactive with 22/18, although most cells stained brightly with an antibody to another cytoskeletal component. When limbs were amputated prior to the arrival of nerves (axons and Schwann cells) at the amputation plane there was no extra reactivity with 22/18 as compared to the contralateral unamputated control, even though the amputated buds regenerated satisfactorily. Limbs amputated after nerves are present at the plane of amputation respond by forming a 22/18-positive blastema. The appearance of the 22/18 responses is a function of the stage of limb development as shown by amputation of forelimb and hindlimb buds at a larval stage where development of the forelimb is greatly advanced relative to the hindlimb. The distribution of the 22/18-positive cells in larval blastemas showed them to be closely associated with axons as detected by double staining with an antiserum to a neurofilament subunit. The clear antigenic difference between development and regeneration may be related to the relationship between embryonic regulation and epimorphic regeneration, and also to the acquisition of nerve-dependent proliferation of blastemal cells.     

The growth and differentiation of the regenerating spinal cord of the lizard, Anolis carolinensis

The present work describes the ultrastructure of the spinal cord in the regenerating tail of the lizard, Anolis. The distal growing region of the tail contains the advancing ependymal tube which is relatively devoid of axons but already contains channels between ependymal cell processes which anticipate their ingrowth. More proximally, fascicles of naked axons having their origin in the stump are present in the ependymal channels. Therefore, the pattern of fiber regeneration in the spinal cord is prescribed by the ependyma and not by the growing axons. Details of the ultrastructure of proximal, intermediate, and distal regions of the regenerate are reported. Particular attention is paid to the structure and differentiation of the ependymal cells and the relation of the ependyma to other glial cells, to nerve fibers, and to meningeal tissues.     

Hepatocyte growth factor affects satellite cell activation and differentiation in regenerating skeletal muscle

Hepatocyte growth factor (HGF) is the onlyknown growth factor that activates quiescent satellite cells inskeletal muscle. We hypothesized that local delivery of HGF may enhanceregeneration after trauma by increasing the number of myoblastsavailable for restoring normal tissue architecture. Injection of HGFinto muscle at the time of injury increases myoblast number but doesnot enhance tissue repair as determined using quantitative histologicalanalyses. Rather, depending on the dose and the timing of HGFadministration relative to the injury, regeneration can be inhibited.The greatest inhibitory effect is observed when HGF is administered onthe day of injury and continued for 3 days, corresponding to the time when satellite cell activation, proliferation, and earlydifferentiation normally occur. To establish a mechanism for thisinhibition, we show that HGF can act directly on primary muscle cellsto block differentiation. These results demonstrate that1) exogenous HGF synergizes withfactors in damaged muscle to increase myoblast number,2) regeneration is not regulatedsolely by myoblast number, and 3)HGF inhibits muscle differentiation both in vitro and in vivo.

     

A radioautographic study of collagen secretion in the dogfish nidamental gland

Optical microscope and ultrastructural autoradiography have been used to localise the sites of collagen secretion in the gland zones, and track its cellular synthesis and mode of cellular secretion. Optical microscope observation has been done after (3)H proline injection into the gland. Untrastructural study has been achieved after applying the incubation method. Optical microscope study has demonstrated that all the gland zones were involved in the secretion of the collageneous protein, though some of them (the D zone and lamellae) secrete more collagen than the others. The ultrastructural study demonstrates that the forms of secretion are numerous: various-looking grains, whose elaboration follows the path of vertebrate collagen produced by fibroblasts, or grains whose only source is rER. The protein periodicity which is observed in the mature capsule (37 nm) is acquired only outside the cells, in the tubular or glandular lumen. It is possible that two types of collagen are secreted by the gland, the first forming the major part of the capsule wall, the other contributing to the plugs sealing the slits of dehiscence.     

Neuronal differentiation in vitro from precursor cells of regenerating spinal cord of the adult teleost Apteronotus albifrons

This study documents neuronal differentiation in vitro from undifferentiated precursor cells of caudalmost regenerating spinal cord of the teleost Apteronotus albifrons. At 11 days in vitro, cells from the caudalmost tip of the regenerating cord are flat and polygonal in shape, lack neuronal processes and do not stain with antibody against neuron-specific filaments. At 15 days in vitro, some of the caudalmost cells have developed short, neurite-like processes; at 18 days in vitro, some cells react positively with antibody against neuron-specific filaments. At 26 days in vitro, many of the caudalmost cells have long branching neurites and react positively with anti-neurofilament antibody. Addition of insulin-like growth factor-I to the medium accelerates the process of neuronal differentiation from the caudalmost precursor cells in vitro. The source of these precursor cells is ultimately cells of the ependymal layer of adult spinal cord. Further investigation of the factors that control production and differentiation of these cells will be important in defining the developmental potential possible for vertebrate spinal cord cells and may aid in creating an optimal environment for regeneration of axons within mammalian spinal cord.     

Neurogenesis during caudal spinal cord regeneration in adult newts

 After tail amputation in urodele amphibians, dramatic changes appear in the spinal cord rostral to the amputation level. Transection induces a proliferation response in cells lining the ependymal canal, giving rise to an ependymal tube in which neurogenesis occurs. Using the thymidine analog bromodeoxyuridine (BrdU) in short- and long-term labeling of cells undergoing DNA synthesis (S phase of the cell cycle), specific cell markers, and cell cultures, we show that neurons derive from the proliferative ependymal layer of the ependymal tube. Received: 30 November 1998 / Accepted: 22 December 1998     

A radioautographic study of protein synthesis in loach blastoderm]

The dynamics of protein synthesis in the loach embryos has been studied by means of autoradiography at the stages of cleavage, blastula and gastrula. During the synchronous cleavage divisions, nuclear proteins are mainly synthesized. From the early blastula stage until the early gastrula stage, the intensity of nuclear protein synthesis increases 2.5 times whereas the intensity of cytoplasmic and total protein synthesis is low and relatively constant. After the onset of gastrulation the intensity of nuclear and cytoplasmic protein synthesis increases 3-4 times and at the late gastrula stage it decreases twice as compared with that at the midgastrula stage. During blastulation, no regional differences in the intensity of nuclear and cytoplasmic protein synthesis were found. With the onset of gastrulation, a vegeto-animal gradient of labeled aminoacid incorporation into nuclear and cytoplasmic proteins appears. During gastrulation, reliable differences were found between the intensity of labeled aminoacid incorporation into proteins of the cells of intact and dissociated blastoderms. During this period, the intensity of protein synthesis in embryonic shield is higher than that in the extraembryonic part of blastoderm.     

Limb regeneration and survival of prolactin treated hypophysectomized adult newts

Hypophysectomized adult newts exhibited 98% survival and limb regeneration at 23 days post-hypophysectomy when injected intraperitoneally every other day with prolactin (0.015 U/newt) and kept continuously in aquaria with 1 × 10^?7 concentration of thyroxine. Thyroxine alone was no more effective than saline injections. Prolactin (1.2 U/newt every other day) alone increased survival and limb regeneration, but less effectively than did the prolactin-thyroxine combination.     

Effect of vitamin A on wound epidermis during forelimb regeneration in adult newts.

The effects of vitamin A on blastemal epidermis were studied during the early postamputational period of forelimb regeneration in Triturus alpestris. Vitamin A was administered through oral intubation at a dose of 250 IU per gram of body weight per day. The results were evaluated by morphometry, histology, and autoradiography. After 7, 11 and 14 days of treatment, several alterations were observed in the wound epidermis: a) reversal of keratinization; fewer keratinized cells were counted in sections from vitamin A-treated limbs; b) decrease in the incorporation of tritiated thymidine, as judged by estimation of labeling indices; c) increased mitotic activity in the cells of the stratum germinativum, and in the middle layer of the epithelial cells, as well. The significance of these cellular effects is discussed against the relevant literature.     

A radioautographic study of collagen synthesis by earthworm epidermis

Secreation by the epidermis of two oligochaetes (Eisenia and Enchytraeus) was investigated radioautographically following administration of 3H-proline, 3H-tryptophan or Na2(35)SO4. Regionally epidermal columnar cells of Enchytraeus synthesize the overlying, probably collagenous, cuticle. Eisenia epidermis does not recordably synthesize the cuticle until after wounding (first eight segments removed). By two days postoperative the epidermal columnar cells of Eisenia synthesize the collagenous cuticle and, later in regeneration, the epidermis may simultaneously synthesize the different collagen of the underlying basement lamella. The epidermis of Enchytraeus, but not of Eisenia, synthesizes some sulfated material associated with the cuticle surface.