The sudanophilic lipid of normal and regenerating limb tissues of the adult newt,Diemictylus viridescens

The most intense and widely distributed sudanophilic responses of cryostat-sectioned newt limb tissues were obtained with a simultaneous fix and stain procedure of 1:1 10% formal-calcium and sudan black B. Droplets and globules of lipid mixtures and rodlets (mitochondria) were typical responses distributed within the epidermis, subcutaneous glands, dermis and other connective tissues, striated muscle (also with positive fibrils), tunics of blood vessels, and blood cells. A prominent droplet response was located subjacent to the adepidermal basement membrane. The myelin of brachial nerve stained intensely. In regenerating limbs, the wound epithelium response was comparable to that of epidermis. Post-amputational lipophanerosis of injured muscle and brachial nerves was observed. The retrograde degeneration of nerve myelin was extensive, and continued into the early differentiative phase of regeneration. Lipid-engorged macrophages were prominent among the injured tissues, distal to these, and within the wound epithelium. The regeneration blastema revealed a large quantity of sudanophilic lipid. Prominent droplet and rodlet responses were typical of the myelinating regenerating nerves. The response of regenerating muscle equaled that of the mature stump fibers. The cells of the regenerating chondroskeleton contained sudanophilic lipid. Organic solvents such as acetone, ether, chloroform and chloroform:methanol reduced or prevented the sudanophilic responses. Sudan red 7B revealed less lipid than did sudan black B. A fixation effect was demonstrated with post-chromated formalcalcium, and chromic-formalin fixed sections. In the latter preparations, swollen-bodies, identified as mitochondria, stained intensely.     

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.     

Differentiation of liquor contacting neurons in the regenerating spinal cord of the newt Triturus

This paper reports a detailed electron microscopic study of the ventricular cells in the growing spinal cord during the first two months of tail regeneration in Triturus vulgaris and T. cristatus. In the proliferating ependyma some cells undergo a neural differentiation in order to produce "Cerebro-Spinal-Fluid-Contacting-Neurons (CSFCNs). These round or pear-like cells store dense core vesicles, 80-100 nm in diameter, and show less electron-density than ependymal elements. The stereociliar apparatus is the last cytological differentiation of these elements and after two months of regeneration, synapse-like endings were occasionally found.     

Retinoic acid-dependent attraction of adult spinal cord axons towards regenerating newt limb blastemas in vitro

Adult urodele amphibians possess the unique ability to regenerate amputated limbs and to re-innervate these regenerating structures; however, the factors involved in mediating this re-innervation are largely unknown. Here, we investigated the role of retinoic acid (RA) and one of its receptors, RARbeta, in the reciprocal neurotropic interactions between regenerating limb blastemas and spinal cord explants from the adult newt Notophthalmus viridescens. First, we showed that retinoic acid induced directed axonal outgrowth from cultured spinal cord tissue. This RA-induced outgrowth was significantly reduced when spinal cord explants were pre-treated with either the synthetic RAR pan antagonist, LE540, or the specific RARbeta antagonist, LE135. The role of RARbeta was also investigated using co-cultured regenerating limb blastemas and spinal cord explants. Blastemas induced significantly more axonal outgrowth from the near side of co-cultured explants, than from the far side (when cultured less than 1 mm apart). This blastema-induced directed outgrowth from co-cultured spinal cord explants was also abolished in the presence of the RARbeta antagonist, LE135. These data strongly suggest that endogenous retinoic acid is one of the tropic factors produced by the blastema and that it may be capable of guiding re-innervating axons to their targets. Moreover, this interaction is likely mediated by the retinoic acid beta nuclear receptor.     

Assay of cyclic 3'-5'-monophosphate in the regenerating forelimb of the newt, Triturus.

The cyclic adenosine 3′-5′-monophosphate content of four regenerate stages in the forelimb of the newt, Triturus viridescens, was assayed using the Gilman method and compared to the content in the normal, unamputated, forelimb. The concentration was found to be highest in the earliest stages of regeneration, followed by a sharp drop and then a rise to a plateau approximately that of the unamputated limb. The possibility that cyclic AMP acts as a second messenger for nervous and hormonal influences on regeneration is discussed.     

The influence of the nerve on lower jaw regeneration in the adult newt, Triturus viridescens

The present investigation was undertaken in an attempt to determine the role played by the nerve in the regeneration of the lower jaw of the adult newt, Triturus viridescens. The results indicated that the number of nerve fibers normally available at the amputation surface was very low compared with that of the newt forelimb. Furthermore, denervation of the lower jaw reduced the number of nerve fibers available to an extremely low level and maintained the number at a low level for up to four weeks without intervening redenervations. The regenerative events in the denervated and amputated lower jaws were indistinguishable histologically from those in amputated jaws having normal innervation. This presented an apparent exception to the general rule that regeneration of external body parts is dependent on the nerve. Several possible explanations are proposed by which this apparent exception might be explained. The process following amputation might be an exaggerated form of wound healing and tissue regeneration which can occur in the absence of nerves. The tissues of the lower jaw might be more sensitive to the influence of those nerve fibers present. The nerve fibers themselves might be qualitatively different and thus exert a greater influence on the tissues.     

The fine structure of limb tissues of the adult newt, Diemictylus viridescens

The limb tissues of the adult newt investigated for their fine structure include epidermis, subcutaneous glands, dermis, striated muscle, peripheral nerves and blood vessels. This survey complements and extends previous observations, emphasizing intercellular junctions, and the ubiquitous “glycocalyx” (= polysaccharide-protein lamella, around cells and adjacent to epithelia). Our survey touches on the characteristic tonofilaments, intercellular desmosomes and basal hemidesmosomes of the epidermis. The subcutaneous glands consist of secretory cells with a granular product, and myoepithelial cells; intercellular desmosomes are present. The adepidermal reticulum of collagen fibrils reveals periodic regions of intersecting fibrils ( = nodules), and fibril continuity with the underlying dermis: a striking feature is the adipose tissue closely applied to the adepidermal reticulum. The limb striated muscle displays typical banded myofibrils, and a triad system with centrotubules in the I-band close to the Z-band: terminal sacs of sarcoplasmic reticulum complete the triad system. A particularly prominent glycocalyx is applied to the surface of the sarcolemma. The peripheral nerves of the limb possess connective tissue sheaths with prominent vesiculation of the cell membranes, and an occasional intercellular desmosomal junction. Blood vessels typically have endothelial cells with prominently vesiculated plasma membranes. This investigation serves as the basis for recognizing the fine structure of tissue responses to trauma, their repair, and regeneration.     

The evolution of the adult body form of the crested newt (Triturus cristatus superspecies,Caudata, Salamandridae)

We characterized the adult body form of the crested newt (Triturus cristatus superspecies) and explored its evolution. From seven morphometric traits, we determined that body size, interlimb distance and head width define the body form. None of the morphometric traits showed a phylogenetic signal. Three body‐shape morphotypes (Triturus dobrogicus + T. cristatus, Triturus carnifex + Triturus macedonicus and Triturus karelinii + Triturus arntzeni) and three body‐size morphotypes (T. dobrogicus, T. cristatus and all other crested newts) could be recognized. The ancestral phenotype (a large body with a short trunk and a wide head) characterized T. karelinii and T. arntzeni. Triturus carnifex and T. macedonicus had a somewhat different phenotype (large body and wide head, accompanied by mild body elongation). The most derived phenotype included body size reduction and more pronounced body elongation in T. cristatus and, especially, in T. dobrogicus. Body elongation occurred by trunk lengthening but not head and tail lengthening. Additionally, contrary to other tetrapods, evolutionary axis elongation in crested newts was followed by a decrease in body size. We advocate the hypothesis that ecology drives the evolution of body form in crested newts.     

Lipid of normal and regenerating limb tissues of the adult newt, Diemictylus viridescens: acidic and non-acidic lipids, phospholipids, and cholesterol

Cryostat-cut sections of unamputated and amputated-regenerating limbs of the adult newt were examined following the Nile blue test for acidic and non-acidic lipids, the acid hematein and plasmal tests for phospholipids, and a Schultz test for cholesterol. Triglycerides (Nile blue test) are prominent in dermis and macrophages: triglyceride droplets are scattered in epidermis, wound epithelium, and regeneration blastema. Fatty acids (Nile blue test) are present in all tissues of the normal and regenerating limb: nerve myelin contains relatively little free fatty acid, while macrophages appear to contain the least amount of this lipidic substance. Plasmalogens (plasmal test) are prominent in the myelin of nerves, and macrophages: a weak cytoplasmic reaction obtains in the epidermis, subcutaneous glands, striated muscle, tunics of blood vessels, wound epithelium, blastema cells, chondrocytes, perichondrium and periosteum. Mitochondria responding for cephalin, lecithin, and sphingomyelin (acid hematein test) are ubiquitously distributed among the cells and tissues of the normal and regenerating limb. These phosphatides are prominent in nerve myelin, macrophages, and in dermal droplets: a variable response obtains from the myofibrils of striated muscle. Cholesterol (Schultz test) was demonstrated only in nerve myelin and in macrophages associated with injured nerves. Particular attention was paid to the lipid responses of the regeneration blastema, and the conclusion was reached that not all of the lipid previously demonstrated with sudan dyes was characterized by the current series of lipid tests. A modified Nile blue sulfate test that promises greater specificity in distinguishing between acidic and non-acidic lipids is introduced.     

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.