Regeneration and supernumerary limb formation under sparsely innervated conditions

Normally, urodele limb regeneration is nerve-dependent. Reduction in nerve-dependency has been reported for regenerating, transplanted newt limbs (Singer and Mutterperl, '63). Aneurogenic limbs can regenerate without nerves (Yntema, '59). Induction of supernumerary limbs may be obtained from aneurogenic limbs of larval Ambystoma after transplantation orthotopically to innervated larvae and with normal nerve ingrowth to the limb transplant prevented by repeated section of brachial nerves. Of the 13 (of 43) grafts with supernumeraries, nerve counts showed 11 with 0–5; 1 with 5–10; and 1 with 20⁺ fibers. Orthotopically grafted aneurogenic limbs allowed to become innervated showed 14 supernumeraries in 49 grafts. This supernumerary limb induction is thus not nerve-dependent. Normally, innervated larval Ambystoma limbs grafted orthotopically and heteroplastically regenerated in 17 of 37 cases after repeated section of brachial nerves. Of the 17 regenerates nerve counts showed 4 with 0–5; 5 with 5–10; 7 with 10–19; and 1 with 20⁺ fibers. Larval limbs heteroplastically transplanted may require very few or no nerves for regeneration.     

Hybrids between irradiated and unirradiated mammalian cells: Survival and chromosome segregation

We have studied the effect of X or γ irradiation, of one parent of a cell hybrid, on hybrid viability and chromosome segregation. The hybrid types studied were mouse-Chinese hamster (which spontaneously lose a few hamster chromosomes) and Chinese hamster-human (which spontaneously lose most of the human complement). Preirradiation of the segregated and retained cell parent resulted in highly asymmetric hybrid survival curves; survival was greatly reduced when the retained parent was irradiated, especially for hamster-human fusions. Preirradiation of the parents of mouse-hamster hybrids modified both the direction and the extent of chromosome segregation, but no consistent effect on elimination was observed for hamster-human hybrids, and reversal of the direction of loss was never observed. These results are more consistent with the hypothesis that chromosome segregation from hybrids results from an intracellular chromosome selection, than with the hypothesis that cellular selection acts on randomly generated chromosome variants.     

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.     

Postcortisone medullary changes in the irradiated and unirradiated male Wistar rat

Cortisone administration in male Wistar rat induces a depletion of the granulocytic-normoblastic cell population in the bone marrow in parallel with a numerical increase by accumulation of lymphoid and undifferentiated cells. These changes are more intense under irradiation conditions. The reversibility of medullary modifications is shown by the integrity of the reticulinic skeleton of the bone marrow.     

Postovariectomy bone marrow changes in irradiated and unirradiated wistar rats

Bilateral ovariectomy of Wistar rats has per se a stimulative effect on the myeloid tissue, determining a hypercellularity of this organ-tissue. On the other hand, under the conditions of a whole-body irradiation with a single dose of 800 r X-rays, bilateral ovariectomy manifests a protective effect on the myeloid tissue preventing the development of hypoplastic, respectively aplastic modifications.     

Two FGF receptor genes are differentially expressed in epithelial and mesenchymal tissues during limb formation and organogenesis in the mouse.

Fibroblast growth factors (FGFs) can influence the growth and differentiation of cultured cells derived from neuroectoderm, ectoderm or mesenchyme. The FGFs interact with a family of at least four closely related receptor tyrosine kinases that are products of individual genes. To investigate the role of FGFs in the growth and differentiation of embryonic tissues and to determine whether the individual FGF receptor genes might have specific functions, we compared the localization of mRNA for two FGF receptor genes, FGFR1 (the flg gene product) and FGFR2 (the bek gene product), during limb formation and organogenesis in mouse embryos (E9.5-E16.5). Although the two genes were coexpressed in some tissues, the differential expression of FGFR1 and FGFR2 in most embryonic tissues was striking. FGFR1 was expressed diffusely in mesenchyme of limb buds, somites and organ rudiments. In contrast, FGFR2 was expressed predominantly in the epithelial cells of embryonic skin and of developing organs. The differential expression of FGFR1 and FGFR2 in mesenchyme and epithelium respectively, suggests the receptor genes are independently regulated and that they mediate different functions of FGFs during development.     

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.     

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.     

Interactions between jointless and wild-type tomato tissues during development of the pedicel abscission zone and the inflorescence meristem.

The jointless mutation of tomato results in the formation of flower pedicels that lack an abscission zone and inflorescence meristems that revert to vegetative growth. We have analyzed periclinal chimeras and mericlinal sectors of jointless and wild-type tissue to determine how cells in different meristem layers (L1, L2, and L3) and their derivatives interact during these two developmental processes. Cells in the inner meristem layer, L3, alone determined whether the meristem maintained the inflorescence state or reverted to vegetative growth. Moreover, L3 derivatives determined whether a functional pedicel abscission zone formed. Limited and disorganized autonomous development of wild-type L2-derived cells occurred when they overlay mutant tissue. Adjacent mutant and wild-type L3-derived tissues in pedicels developed autonomously, indicating little or no lateral communication. Only the outermost L3-derived cells within the pedicel were capable of orchestrating normal pedicel development in overlying tissues, revealing the special status of those cells as coordinators of development for L1- and L2-derived cells, whereas the innermost L3-derived cells developed autonomously but did not influence the development of other cells.     

Interactions between heterochronic cells in the formation of delayed-type hypersensitivity.

The effect of ageing on lymphocyte and macrophage functional activity in the induction of tuberculin sensitivity was studied in experiments on 440 CBA mice of three age groups. The capacity of old animals to generate delayed-type hypersensitivity following administration of BCG cells was found to be diminished and their lymphocytes caused decreased hypersensitivity on transplantation. The capacity of recipients to reproduce delayed-type hypersensitivity after thymocyte and bone marrow cell transplantation was influenced by the age of transplanted thymocytes. This capacity was markedly suppressed on the recipients of old cells. The antigen presentation function of macrophage did not after significantly as a function of age.     

Effect of caffeine on DNA synthesis in irradiated and unirradiated mammalian cells

Caffeine increased the availability of replication origins, and consequently the number of growing points, in the DNA of Chinese hamster V79 and human (HeLa) cells. Caffeine also prevented the inhibition of replicon initiation normally caused by X-radiation and exposure to low doses of ultraviolet light. When caffeine was removed from the medium after irradiation, replicon initiation was inhibited. Caffeine also reversed the inhibition of replicon initiation caused by novobiocin, which is not a DNA-damaging agent. Because caffeine increases the number of growing points, it also partially reversed the inhibition of total DNA synthesis induced by hydroxyurea. It is proposed that caffeine alters the conformation of intracellular chromatin in such a way that the conformation usually induced by DNA-damaging agents is prevented.     

A nerve-conditioning lesion accelerates limb regeneration in the newt

A nerve-conditioning lesion induced sustained acceleration of limb regeneration. Newt limb nerves were subjected to a conditioning lesion by unilateral axotomy at the elbow 2 weeks prior to amputating both limbs above the elbows. Limbs on the side that had received a conditioning lesion began the regeneration process 3-4 days earlier than contralateral controls and this difference was observed up to recognizable digit formation. Limb buds on the conditioned sides had a twofold greater axonal density than contralateral counterparts at 2 weeks after amputation. Since limb bud formation is dependent on a sufficient quantity of axonal regrowth, accelerated limb regeneration is apparently due to accelerated reinnervation.