Notochord is essential for oligodendrocyte development in Xenopus spinal cord.

Oligodendrocyte precursors originate in the ventral ventricular zone of the developing spinal cord. To examine whether the notochord is essential for the development of oligodendrocytes in Xenopus spinal cord the notochord was prevented from forming, ablated, or transplanted during early stages of development. Differentiated oligodendrocytes did not appear in spinal cord regions lacking a notochord in animals in which notochord failed to develop after UV irradiation at the one-cell stage. Similarly, differentiated oligodendrocytes were not detected in the spinal cord adjacent to the site of segmental notochord ablation at embryonic or larval stages. Transplantation of an additional notochord dorsal to the spinal cord induced the premature appearance of differentiated oligodendrocytes in adjacent lateral and dorsal spinal cord white matter. These results indicate that the development of Xenopus spinal cord oligodendrocytes is dependent on local influences from the notochord and suggest that the notochord is essential for oligodendrocyte development in Xenopus spinal cord.

Development of glial cytoarchitecture in the frog spinal cord.

The origin of astrocytes and oligodendrocytes in the frog spinal cord, and the factors that regulate their differentiation are currently unknown. To determine the timing and pattern of glial cell differentiation, spinal cord sections from Xenopus at different developmental stages were labeled with antibodies specific for astrocytes and oligodendrocytes. Initially, radially oriented glial cells are present in all spinal cord quadrants and their processes contain similar levels of GFAP+ intermediate filaments in both gray and white matter. These cells then appear to differentiate directly into regionally specialized radially oriented astrocytes. Oligodendrocytes labeled with the 'Olig' antibody, however, are first detectable in the ventral quadrant. Differentiation subsequently occurs in a ventral-to-dorsal sequence beginning at the interface between the gray and white matte. These data suggest that the origin and regulation of Xenopus astrocyte and oligodendrocyte differentiation are distinct.

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.

Increased slow transport in axons of regenerating newt limbs after a nerve conditioning lesion.

We have previously shown that a nerve conditioning lesion (CL) made 2 weeks prior to amputation results in an earlier onset of limb regeneration in newts. Studies in fish and mammals demonstrate that when a CL precedes a nerve testing lesion, slow component b (SCb) of axonal transport is increased compared to axons that had not received a CL. We wanted to know whether the earlier initiation of limb regeneration after a CL was associated with an increase in SCb transport. The transport of [35S]methionine labeled SCb proteins was measured by using SDS-PAGE, fluorography, and scintillation counting. The rate of transport and quantity of SCb proteins was determined at 7, 14, 21, and 28 days after injection of [35S]methionine into the motor columns of normal; single lesioned (i.e., transection axotomy, amputation axotomy, or sham CL followed by amputation); and double-lesioned limb axons (i.e., nerve transection CL followed 2 weeks later by amputation axotomy). The rate of SCb transport in axons of unamputated newt limbs was 0.19 mm/day. There was an increase in the amount of labeled SCb proteins transported in axons regenerating as the result of a single lesion but no acceleration in the rate of SCb transport, which was 0.21 mm/day in axons that received a sham CL followed by limb amputation. The rate of SCb transport doubled (0.40 mm/day) and the amount of labeled SCb proteins being transported was increased when amputation was preceded by a CL. This study demonstrates that the earlier onset of limb regrowth, seen when amputation follows a CL, is associated with an increased transport of SCb proteins. This suggests that limb regeneration is, in part, regulated by axonal regrowth. We propose that the blastema requires a minimum quantity of innervation before progressing to the next stage of limb regeneration, and that the transport of SCb proteins determines when that quantity will be available.

Open finger tip healing and replacement after distal amputation in rhesus monkey with comparison to limb regeneration in lower vertebrates.

The left thumbs and great toes of three 8 1/2 month old Rhesus monkeys (Macaca mulatta) were amputated in guillotine fashion one millimeter distal to the base of the nail and allowed to heal by the conservative open wound method. Healing occurred in seven to ten days in these small digits. Each of the thumbs and toes grew back with some blunting and shortening of the digit tips, but were functional. The new structures were cosmetically pleasing as in the human instances. The nails grew essentially to normal size and shape supported by the remaining portions of the distal phalanges. Histological studies showed no evidence of blastema formation such as is observed in the regenerating limb of the Urodele (newt) taken as the comparative representative. The possibility of improving the regrowth is discussed against the background of our knowledge of the importance of nerve during limb regeneration in lower vertebrates.

Expression and function of neural cell adhesion molecule during limb regeneration.

The neural cell adhesion molecule (NCAM) has been detected in regenerating limb bud of adult newts in addition to brain and peripheral nerves. In the regenerating tissue, NCAM was found primarily on mesenchymal cells and also in wound epidermis. Infusion of Fab fragments of antibodies to NCAM into limb buds at the early blastema stage delayed the regenerative process. Previous studies have indicated that NCAM serves as a homophilic ligand for adhesion among cells that express this molecule and, in doing so, can influence the interaction of nerves with their environment. The expression of NCAM in regenerating limb and the effects of antibody infusion are therefore consistent with the observation that limb regeneration requires interactions among axons and mesenchymal cells.

Gangliosides stimulate protein synthesis, growth, and axon number of regenerating limb buds.

When a newt limb is amputated and begins to regrow, regenerating axons exert a neurotrophic influence on the limb regeneration process. Previous studies have shown that direct manipulation of limb nerves, by electrical stimulation or a conditioning lesion, elevates protein synthesis, increases neurotization and accelerates growth of the limb bud. Since exogenously supplied gangliosides accelerate axonal sprouting in regenerating nerves, we wanted to know whether gangliosides would similarly affect limb regeneration. To test this, regrowing limb buds were either infused with or immersed into gangliosides, or animals were injected intraperitoneally with gangliosides. Infused gangliosides elevated protein synthesis in limb buds 15% and increased the number of axons in limb buds 45% by 6 hours after infusion. Regenerating limb bud morphogenesis was initiated 3-4 days earlier in animals receiving i.p. injections of gangliosides every 12 hours. Similarly, limbs immersed daily in gangliosides began regrowth sooner than contralateral controls and this advantage was maintained throughout the period of observation. These findings indicate that treatment with gangliosides has a salutary effect on limb regeneration.

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.

Neurotrophic and neuronotrophic effects in the regenerating newt limb bud after electrical stimulation of brachiospinal nerves.

An earlier work demonstrated that electrical stimulation of newt brachiospinal nerves produces a 20% increase in protein synthesis in the regenerating limb bud at 6 h post-stimulation. The present study shows that if stimulation of nerve cell bodies is prevented by placing procaine between the cell bodies and the stimulating electrode, there is no increase in limb bud protein synthesis compared to the non-stimulated, contralateral control limb bud. Similarly, if colchicine is applied to the brachiospinal nerves at the site of and prior to stimulation, there is no increase in limb bud protein synthesis after stimulation. Colchicine applied to brachiospinal nerves in the absence of stimulation results in a reduction of limb bud protein synthesis that is of the same magnitude as the increase seen with stimulation. The results suggest that the neurotrophic increase in limb bud protein synthesis after stimulation is under the control of the cell body and that this control is mediated by changes in fast axonal transport. A neuronotrophic increase in axonal density in the stimulated side limb bud is seen at the same time as the increase in protein synthesis after stimulation.

Parent and daughter axon density in the limb stump and regenerating limb bud of the newt.

Axonal density in the amputated limb stump and regenerating limb bud of the newt was determined by axon counts and planimetry of silver-stained sections at various distances from the amputation level. Traumatic degeneration occurred for at least 250 micron proximal to the amputation level. Axonal density at 250 micron distal to the amputation was less than half that at 100 micron. Attempts to increase in situ axonal density in limbs of animals naturally incapable of limb regeneration can be based on specific knowledge of natural traumatic degeneration and sprouting prior to axonal manipulation.

The effect of limiting light to the pineal on the rate of forelimb regeneration in the newt.

Newts with bilaterally amputated forelimbs were exposed to either continuous light (LL), total darkness (DD), or continuous light with the dorsal head epithelium painted with an India ink and Nile blue sulfate mixture (LL-II-NBS) that limited light penetration through the skull to the pineal. The LL-II-NBS animals regenerated their forelimbs more slowly then their counterparts in LL.

The effect of prolactin on the rate of forelimb regeneration in newts exposed to photoperiod extremes.

Four groups of bilaterally amputated newts were exposed to (1) continuous light, LL; (2) continuous light with daily prolactin injections, LL-PRL; (3) total darkness, DD; or (4) total darkness with daily prolactin injections, DD-PRL. The results confirmed that LL animals regenerate their limbs more rapidly than DD. The results also showed that LL-PRL animals do not respond to the exogenous prolactin but that DD-PRL animals do respond and in fact regenerate at a rate which exceeds not only DD but also LL and LL-PRL. The possible synergism between prolactin and photoperiod is discussed.

The effect of light on forelimb regeneration in the newt.

The forelimb of the newt, Triturus (Notophthalamus) viridescens, was bilaterally amputated and the animals subjected to different photoperiods during the period of regeneration, namely (1) continuous light, (2) 15 hours of light/day and (3) total darkness. Animals exposed to continuous light reached the palette regeneration stage four to five days before those kept in total darkness with the 15 hours of light/day animals being intermediate. The difference in regeneration rate was first evident in the moderate early stage (blastema accumulation stage); it then increased during subsequent stages and the difference persisted during the observation period. In an attempt to determine whether the retina was the receptor of light affected, some animals were blinded at the time of amputation and subjected to continuous light. Their rate of regeneration was similar to sighted animals subjected to continuous light but greater than sighted animals in total darkness. The pineal is discussed as the likely mediator of the photo effect.

Neurotrophic activity of brain extracts in forelimb regeneration of the urodele, Triturus.

The loss in protein synthesis which the regenerating forelimb of the newt suffers after denervation can be recovered by infusing into it an extract of newt soluble brain protein. Moreover, the synthesis of basic protein shows a greater response to the active brain principle than does that of acidic protein. The active agent of the nervous tissue is destroyed by heat and trypsin digestion. Extracts of liver and spleen, similarly prepared, do not evoke recovery of lost protein synthesis. Synaptosomal extracts of the frog brain also cause recovery of protein synthesis in the denervated regenerate, demonstrating the likelihood that the active agent is not species-specific within these amphibians, that it is a constituent of the neuronal fraction of nervous tissue, and that it is present in axonal terminals. Additional experiments showed that the nervous agent is likely a basic protein, and that the amount of protein infused is of the order of only 1.0% of the total regenerate protein. The significance of the findings is discussed in relation to the nature of the effect on protein synthesis and the nature of the active principle.