Treatment of brachial nerves with colchicine inhibits limb regeneration in the newt Notophthalmus viridescens

In urodele amphibians, limb regeneration is dependent on innervation and is blocked by the administration of colchicine. The objective of this experiment was to determine if colchicine blocks limb regeneration by a direct action on the blastema cells or by an indirect action on the nerves, specifically, if colchicine treatment of the brachial nerves would inhibit limb regeneration in the newt Notophthalmus viridescens. Colchicine was applied to the nerves by implanting a colchicine-loaded silastin block adjacent to the brachial nerves of an amputated newt limb. With appropriate dose levels of colchicine, limb regeneration was completely inhibited. Contralateral control limbs, carrying unloaded silastin blocks, and control limbs with colchicine-loaded blocks implanted equidistant from the blastema, but not adjacent to the brachial nerves, regenerated normally. Thus, the results indicate that the colchicine inhibition of limb regeneration is mediated by colchicine effects on the nerves. The possible mechanism of colchicine action on nerves may involve either wallerian degeneration, or inhibition of axoplasmic transport, or both.     

Changes in cyclic GMP levels during forelimb regeneration in adult Notophthalmus viridescens.

Cyclic 3', 5'-guanosine monophosphate (cGMP) was measured at eight stages of forelimb regeneration in adult newts and compared with the cGMP levels of non-regenerating control limbs. There was a significant increase in cGMP content during dedifferentiation followed by a sharp decrease to minimal levels at the cone stage. A second smaller increase in cGMP occurred between the cone stage and mid-differentiation, followed by a decrease to relatively constant levels approaching control values as differentiation progressed. The changes in cGMP during dedifferentiation and during the period of highest cell proliferation indicate that cGMP may play a role in these processes. The smaller increases in cGMP levels during differentiation may reflect a reduced rate of cell division in the differentiating tissues.     

Hox C6 expression during development and regeneration of forelimbs in larval Notophthalmus viridescens

 A central theme concerning the epimorphic regenerative potential of urodele amphibian appendages is that limb regeneration in the adult parallels larval limb development. Results of previous research have led to the suggestion that homeobox containing genes are ”re-expressed” during the epimorphic regeneration of forelimbs of adult Notophthalmus viridescens in patterns which retrace larval limb development. However, to date no literature exists concerning expression patterns of any homeobox containing genes during larval development of this species. The lack of such information has been a hindrance in exploring the similarities as well as differences which exist between limb regeneration in adults and limb development in larvae. Here we report the first such results of the localization of Hox C6 (formerly, NvHBox-1) in developing and regenerating forelimbs of N. viridescens larvae as demonstrated by whole-mount in situ hybridization. Inasmuch as the pattern of Hox C6 expression is similar in developing forelimb buds of larvae and epimorphically regenerating forelimb blastemata of both adults and larvae, our results support the paradigm that epimorphic regeneration in adult newts parallels larval forelimb development. However, in contrast with observations which document the presence of Hox C6 in both intact, as well as regenerating hindlimbs and tails of adult newts, our results reveal no such Hox C6 expression during larval development of hindlimbs or the tail. As such, our findings indicate that critical differences in larval hindlimb and tail development versus adult expression patterns of this gene in these two appendages may be due primarily to differences in gene regulation as opposed to gene function. Thus, the apparent ability of urodeles to regulate genes in such a highly co-ordinated fashion so as to replace lost, differentiated, appendicular structures in adult animals may assist, at least in part, in better elucidating the phenomenon of epimorphic regeneration. Received: 6 November 1998 / Accepted: 12 December 1998     

Stress and changes in the blood of newts,Notophthalmus viridescens,during early regeneration

Summary During the summers of 1984 and 1985, adult red-spotted newts,Notophthalmus viridescens, were maintained in the laboratory at 23°±0.5°C under natural photoperiods. From each of the experimental animals, the right forelimb was amputated just proximal to the elbow. Control newts were not manipulated surgically. Eight, 15, and 22 days after the time of amputation, equal numbers of regenerating and control animals were sacrificed, and blood smears of each individual were prepared with Wright's stain.Mean differential counts of leukocytes of the two groups of newts indicated that the relative number of neutrophils increased and the relative number of lymphocytes decreased in the regenerating animals as compared to their controls (Fig. 1 and Fig. 2). Earlier studies had shown that lymphopenia and neutrophilia occur in red-spotted newts treated with hydrocortisone or with ACTH or subjected to environmental stress (Bennett and Daigle 1983). Consequently, it is suggested that amputation and/or early regeneration may stimulate the increased production of hormones associated with stress in vertebrates, which may, in turn, influence regeneration, itself, and that the detailed study of the distribution of leukocytes inNotophthalmus viridescens may provide an assay with which to study the regulation of regeneration in this species.     

Disruption of normal forelimb regeneration in adult Notophthalmus viridescens by sublethal concentrations of trypan blue.

The effects of the vital dye trypan blue (TpB) on the regeneration of amputated newt forelimbs were examined. Administration of the dye (10 mug/g body weight) via IP injection during the early wound healing and dedifferentiation phases of regeneration inhibited the normal regenerative response. The accumulation phases of regeneration are similarly halted but only by greater concentrations of TpB (50 mug/g body weight) while redifferentiation and morphogenesis are only affected by still greater concentrations of the dye (100 mug/g body weight). In addition to abolishing the regenerative response, low levels of TpB were also capable of inducing skeletal abnormalities in the regenerates as might be expected from previous reports on the teratogenicity of the dye. The in vitro action of newt hyaluronidase (as well as purified testicular hyaluronidase) on hyaluronate was diminished by TpB, with virtually complete inhibition observed at initial reaction mixture concentrations of 100 mug/ml. The results of this study suggest that TpB acts to disrupt the normal regenerative response by preventing dedifferentiation and remodeling, perhaps by inhibition of various necessary lytic enzyme functions or by interference with normal intercellular communications.     

Role of the neural retina in newt (Notophthalmus viridescens) lens regeneration in vitro

Removal of the lens from the eye of an adult newt (Notophthalmus viridescens) is followed by regeneration of a new lens from the dorsal iris epithelial cells at the pupillary margin. This process is dependent upon the neural retina for its normal completion in vivo and in vitro. To examine the relationship between the retina and lens regeneration, we have conducted experiments that delimit the time period during which the retinal presence is critical (in vivo) and have investigated the influence of extracts of the retina on the progress of regeneration (in vitro). In vivo, removal of the retina at day 11 seriously retards further progression of regeneration while removal of the retina at day 15 does not retard regeneration significantly. This defines a "critical period" in regeneration of the lens during which the retina is required. Explantation of regenerates 11 or 12 days after lentectomy to organ culture medium enriched with either crude retinal homogenate or extracts prepared from chick or bovine retinas according to Courty et al. ('85, Biochimie, 67:265-269) reveals that the progress of regeneration can be supported in culture by the crude extract. This is the first demonstration of complete iris-lens transformation in culture in the presence of retinal extract. It is possible that the retina acts indirectly by promoting passage of the iris epithelial cells through the critical number of mitoses required before redifferentiation into lens cells can occur (as proposed by Yamada, '77, Monogr. Dev. Biol., 13:126). It is also possible that the retina acts by directly instructing the iris cells to redifferentiate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Early stages of spinal ganglion formation during tail regeneration in the newt, Notophthalmus viridescens

Stages in the development of sensory ganglia in the regenerating newt tail after amputation are described by taking advantage of the rostrocaudal developmental gradient of the regenerating tail. A series of ganglia, beginning at the tip of the regenerate and progressing rostrally, were examined. Eight-week regenerates were used because they showed the most complete array of stages. The first recognizable ganglia appear as small clusters of cells sitting dorsally on the already established ventral roots. The cluster of ganglionic cells steadily expands with the addition of many new cells. Signs of cell differentiation within the ganglion precede the formation of the dorsal root rudiment, which assumes several different configurations but most commonly enters the cord close to the ventral root. Our material suggests that ganglion precursor cells originate in the ventral region of the developing spinal cord and migrate out of the cord by travelling along the ventral root until, at a suitable distance from the cord, they halt, proliferate, and eventually differentiate. In the regenerate, we saw no evidence of neural crest cells--such as those that give rise to ganglia in the trunk region during development--forming at the dorsal region of the regenerated neural tube. Nor was there any morphological evidence of mesenchymal contribution to the ganglion cell clusters.     

Limb regeneration of the adult newt (Notophthalmus viridescens) in the absence of the spleen

Summary It has been suggested that the immune system might figure prominently in the regulation of forelimb regeneration. However, neither the nature of this influence nor the aspect(s) of regeneration influenced are clearly known. The determination of which components of the immune system are indispensable for regeneration would be a logical first step in attempting to address such questions. This investigation, therefore, examined the effects of removing the spleen, a major lymphoid organ in the newt, upon the progress of regeneration. Splenectomies performed concomitantly with or after forelimb amputation failed to alter the time course of regeneration. Splenectomies, but not sham-splenectomies, performed prior to amputation reduced the time required to achieve successive stages of regeneration under some, but not all conditions, i.e., when performed 10–20 days before amputation, during the late fall and winter. Up until 35 days after amputation, no gross morphological distortions were observed as a result of splenectomy. It was concluded that the spleen is not required for regeneration to occur.Portions of this work constitute part of the thesis submitted by M.E. Fini in partial fulfillment of the requirements for the M.S. degree in Biology at Boston College     

A cytochemical study of myeloid bodies in the retinal pigment epithelium of the newt Notophthalmus viridescens

Summary It has been suggested (Yorke and Dickson 1984) that myeloid bodies (MBs) in the retinal pigment epithelium (RPE) of the newt, Notophthalmus viridescens, may represent areas of endoplasmic reticulum where lipids, such as 11-cis retinal derived from phagocytized outer segment tips, accumulate prior to esterification. Experiments in which an artificial ester substrate was added during in-vitro incubations have shown that esterase activity is represented in all areas of the newt RPE endoplasmic reticulum, including sites adjacent to all MBs. In related tests in which the localization of enzyme activity was restricted to areas of the cell where there had been accumulations of naturally-occurring (endogenous) esters, the products of ester hydrolysis were restricted to profiles of endoplasmic reticulum associated with lipid droplets, and with the interior of about 20% of those MBs that appeared completely circular in sections. This enzyme activity was not associated with other MB configurations. Results from endogenous-ester hydrolysis were identical to those obtained after staining with ZIO. This ZIO-reactivity was not affected by pre-incubation with agents that blocked or protected sulphydryl groups, and ZIO-reactive sites associated with MBs did not form complexes with digitonin. These observations suggest that MBs are a site of lipid-ester formation, but that they do not represent unique intracellular areas for this activity.     

Temporal analysis of the role of growth hormone in the initiation and maintenance of limb regeneration in the hypophysectomized newt Notophthalmus viridescens

This study was designed to investigate and determine for how long, after either hypophysectomy or the third (last) growth hormone injection (to previously hypophysectomized newts), the circulating and now declining titers of endogenous or exogenous hormone remained at a sufficient concentration to permit a morphologically normal forelimb regeneration response in the adult newt Notophthalmus viridescens. To examine the declining levels of endogenous hormone (hormone withdrawal series [HW]), left forelimbs were amputated at specific times following hypophysectomy. Right forelimbs were amputated 5 days prior to hypophysectomy. The declining levels of exogenous hormone (hormone replacement series [HR] were examined in newts whose left forelimbs were amputated at specific times following the last of three consecutive alternate-day growth hormone injections that were initiated 5 days post hypophysectomy. Right forelimbs were amputated immediately following the first hormone injection. All experimental animals were sacrificed when their right forelimbs regenerated to an advanced digitiform regenerate. In both series right forelimbs regenerated normally. In the HW series normal regeneration resulted only when forelimbs were amputated within 48 hours post hypophysectomy, whereas in the HR series normal regeneration occurred in only those newts whose forelimbs were amputated within 12 hours of the last hormone injection. The regeneration response of left forelimbs in both series gradually declined with the time interval between either hypophysectomy or hormone injection and forelimb amputation. As the hormone titer declined, fewer limbs initiated a normal response; they became progressively more hypomorphic and eventually failed to undergo typical regeneration.     

Limb regeneration following the discontinuation of growth hormone therapy in the hypophysectomized newt,Notophthalmus viridescens

Untreated adult newts do not undergo normal limb regeneration following hypohysectomy. A fibrocellular dermal barrier (cicatrix) atypically forms between the apical epithelium and the underlying mesenchymal tissues. Historically, continuous administration of growth hormone or of prolactin in combination with thyroxine restored regenerative capacity to these newts. In a previous investigation, we demonstrated that the initial effect of these two hormone treatments, when administered on alternate days to hypophysectomized newts beginning eight days post-amputation, was to facilitate the erosion of the fibrocellular barrier and establish the epithelial mesenchymal interface that is observed in a regenerating limb. The present investigation was designed to evaluate the necessity of continuous hormone therapy to maintain limb regeneration in hypophysectomized newts. One, two, or three injections of growth hormone or of prolactin in combination with thyroxine was administered on successive alternate days to hypophysectomized newts either immediately following limb amputation (ID) or beginning eight days post-amputation (DD). The ID and DD newts receiving one, two, or three injections of growth hormone showed evidence of regeneration to the digitiform stage by day 30 post-amputation, while those receiving prolactin and thyroxine underwent wound healing. While both hormone treatments initially promoted a dermis-free apical epithelium, only hypophysectomized newts that had received growth hormone were able to continue regenerating. We have, therefore, concluded that discontinuous growth hormone therapy is sufficient to initiate and maintain the conducive environment for limb regeneration to advanced stages in the hypophysectomized newt. While initiating this process, prolactin and thyroxine therapy on a discontinuous regime does not maintain regeneration. The direct and indirect role of growth hormone in supporting limb regeneration in normal and hypophysectomized newts is discussed.     

A scanning electron microscopic and quantitative histologic description of lens regeneration in the newt,Notophthalmus viridescens

Adult newts (Notophthalmus viridescens) were lentectomized and at intervals from 4 to 21 days after lentectomy iridocorneal complexes from these animals were examined by scanning electron microscopy to allow a full appreciation for the shape of the regenerating lens. Until around day 12 after lentectomy the posterior surface of the iris is covered by a dense mat of fibrous material which cannot be removed without damage to the iris and which obscures the events of cytoplasmic shedding. The regenerate becomes visible first around stage IV (day 12). A small but clear groove demarcates the regenerate from the rest of the iris. As regeneration progresses there is a marked reduction in debris on the iris surface and the regenerate appears as a U-shaped thickening occupying about one-third of the dorsal half of the iris. During later stages (VI–X) the regenerate protrudes into the pupil inferiorly and posteriorly towards the retina, but does not encroach laterally on the remaining pigmented iris tissue. Prior to secretion of the lens capsule the outline of individual cells is visible on the surface of the regenerate and some regenerates exhibit a prominent dimple on their posterior aspects. Following secretion of the capsule the surface of the regenerate becomes smooth. Quantitative studies show that volume and maximum section area of the regenerate are both more strongly correlated with developmental stage of regeneration than with time after lentectomy.     

DNA synthesis in the thymus of the adult newt Notophthalmus viridescens.

Lymphocytic activity was examined in thymuses of adult newts by studying the number, location, morphology and fate of cells within thymuses which had been processed for autoradiography 15 min, 2 and 4 h, and 2, 4, and 10 days subsequent to the injection of tritiated thymidine. Results of this study indicate (1) that the adult thymus is a highly proliferative organ, (2) that large and medium-sized lymphocytes present in the peripheral parenchyma give rise to smaller lymphocytes which move centrally and emigrate from the thymus, and (3) that many thymocytes leave the thymus within 2-4 days after they have been produced. The significance of these findings is discussed.     

Neural and hormonal stimulation of DNA and protein synthesis in cultured regeneration blastemata in the newt Notophthalmus viridescens

Distal portions of cone-stage newt forelimb blastemata were cultured transfilter to spinal ganglia for 36 or 72 hr. Addition of insulin to the medium consistently resulted in a significant increase (250% in ganglionated and 238% in nonganglionated blastemata) in the incorporation of [³H]thymidine into DNA, as compared to nontreated controls. When blastemata were cultured without ganglia for 36 or 72 hr, DNA synthesis decreased to 73 and 71%, respectively, of that achieved by ganglionated explants. When insulin was excluded from the medium, DNA synthesis decreased to 40% of insulin-treated explants, and, in the absence of both nerves and insulin, it declined to 31% of insulin-treated, innervated explants. The presence of insulin in the medium also resulted in an augmentation of (¹⁴C)-labeled amino acid incorporation into proteins; the average increase was 168%, as compared to untreated controls. l-thyroxine, growth hormone and hydrocortisone in combination with insulin, did not enhance the effects on DNA or protein synthesis of insulin alone. Also, exogenous cyclic nucleotides (cAMP, cGMP) and alterations of their endogenous levels with acetylcholine, sodium azide, theophylline or prostaglandins failed to elicit significant changes in DNA or protein synthesis. The existence of a synergistic action on DNA synthesis between nerves and insulin is suggested.     

An autoradiographic study of the origin of intestinal blastemal cells in the newt, Notophthalmus viridescens.

Fifty adult newts were used in this investigation; in 44 animals, the intestine was transected perpendicular to its longitudinal axis approximately midway between pylorus and rectum. The free ends of the intestine were held in apposition with a single suture and replaced into the coelom. The animals were injected intraperitoneally with [³H]thymidine from 0 to 35 days after transection of the intestine and killed 6 hr later. In nontransected, control intestines, the only tissue that incorporated [³H]thymidine was the mucosal epithelium. In transected intestines, only the mucosal epithelium labeled in animals which had been injected with [³H]thymidine from 0 to 4 days after the intestine was incised. Later on, serosal cells and smooth muscle cells of the intestinal stump underwent morphological alteration, initiated the incorporation of [³H]thymidine into DNA, and began replication. At 6 days after transection, serosal cells adjacent to the plane of transection were incorporating [³H]thymidine and, at 12 days, smooth muscle cells at the transected surface were labeling. It seems probable that they both furnished cells to the intestinal blastema; the lining epithelium of the mucosa, however, did not appear to contribute to the blastema proper.     

Transfilter mitogenic effect of dorsal root ganglia on cultured regeneration blastemata,in the newt,Notophthalmus viridescens

Cone stage forelimb blastemata from adult newts were separated into proximal and distal regions and cultured along with dorsal root ganglia in both transfilter and cis (same side) configurations, for a period of 96 h in modified Parker's medium (CMRL-1415) containing insulin and l-thyroxin. Mitotic index and differentiation of cartilage were assessed in ganglionated and nonganglionated, proximal and distal explants after 4 days in vitro. The results show that the nerve influence on the regeneration blastema appears to be mediated by a chemical substance capable of transmission through thin filters of low porosity Moreover the neurotrophic substance has mitogenic properties. The ganglia stimulated blastema cell proliferation transfilter, increasing it from a basal level (mitotic index = 0.339), observed in noninnervated explants to almost threefold values (M.I. = 1.124) in corresponding distal innervated explants. In addition, this transfilter mitogenic effect was manifested in the form of a proximodistal gradient with the highest mitotic index close to the neurons, which diminished with distance from the nerve source. When blastema explants were grown in physical contact with ganglionic neurons (cis configuration), they transcended the proliferation phase within the 4 days of culture and differentiation of cartilage whorls resulted. Presumably, a critical mass of blastema cells is achieved earlier in the presence of a higher concentration of neurotrophic factor.