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.     

Autonomic control of heart rate in the adult, aquatic Notophthalmus viridescens viridescens

1. We investigated the role of the autonomic nervous system in the control of the heart rate using an isolated heart preparation. 2. Addition of the parasympathetic blocker, atropine, to the organ bath resulted in an increase in heart rate as expected. 3. Addition of the sympathetic blocker, ergotamine, to the organ bath showed no change in the heart rate. 4. Addition of the sympathetic blocker, propranolol, to the organ bath resulted in the expected decrease in heart rate. 5. Both the sympathetic and parasympathetic nervous systems appear to play a role in the control of the heart rate.     

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     

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.     

Tail regeneration and ependymal outgrowth in the adult newt, Notophthalmus viridescens, are adversely affected by experimentally produced ischemia

Spinal axons of the adult newt will regenerate when the spinal cord is severed or when the tail is amputated. Ischemia and associated hypoxia have been correlated with poor central nervous system regeneration in mammals. To test the effects of ischemia on newt spinal cord regeneration, the spinal cord and major blood vessels of the newt tail were severed 2 cm caudal to the cloaca as a primary injury. This primary injury severely reduced circulation in the caudal direction for 7 days; by day 8, circulation was largely restored. After various periods of time after primary injury, tails were amputated 1 cm caudal to the primary injury (in the area of ischemia) and tested for regeneration. If the tail was amputated within 5 days of the primary injury, regeneration did not occur. If amputation was 7 days or longer after the primary injury, a regenerative response occurred. Histology showed that in the non-regenerating tails the spinal cord and associated ependyma, known to be important to tail regeneration, had degenerated in the rostral direction. Such degeneration was prevented when tails were first amputated and allowed to form blastemas before the primary injury. The data indicate that the first 5-7 days of blastema formation are particularly sensitive to compromised blood flow (ischemia/hypoxia). It follows that mechanisms must be present in the adult newt to reduce ischemia to a minimum and thus allow ependymal outgrowth and tail regeneration.     

Regenerative abnormalities in Notophthalmus viridescens induced by repeated amputations.

The fidelity of the regenerative response in the adult newt, Notophthalmus viridescens, was examined following repeated amputations at the level of the distal one-third of humerus. Three to four months following amputation, all regenerates were scored for gross morphology, reamputated, and stained with methylene blue for skeletal elements. The occurrence of abnormal regeneration with respect both to gross morphology and to skeletal structure was found to increase directly with the number of times the limb stumps were required to initiate dedifferentiation and repair. The initial amputation-regeneration process produced structurally normal replacement limbs in 91% of the cases examined. Reamputations of 4-digit regenerates (3--4 months after the previous transection) resulted in structurally abnormal regenerates in 28% of the cases following two amputations; 50% of the cases following three amputations; 65% of the cases following four amputations; and 81% of the cases following five amputations. The relationships between repeated dedifferentiation, proliferation, and redifferentiation and normal limb development are discussed.     

Effects of amiloride analogues on adult Notophthalmus viridescens limb stump currents

We have previously investigated the relevance to limb regeneration of epidermally driven, Na+-dependent limb stump currents by blocking epidermal Na+ channels with amiloride, 3,5-diamino-6-chloro-N-(diaminomethylene)pyrazinecarboxamide. In order to reduce Notophthalmus viridescens stump currents more effectively than with amiloride, we have examined six amiloride analogues. Of these, only benzamil, 3,5-diamino-6-chloro-N-[(benzylamino)aminomethylene]pyrazinecarboxamide, was more effective than amiloride. The concentration of benzamil that reduced stump currents to half their initial value was 0.034 microM, while this concentration for amiloride was 0.42 microM. We also found a contribution of calcium ions to these currents. When immersed in water whose Ca2+ concentration decreased stepwise from 1 to 0 mM, stump currents decreased significantly, although to a variable extent, depending on the batch of newts. With 30 microM benzamil and 0.5 mM calcium (in water that also contained 1.5 mM NaCl and 0.06 mM KCl) stump currents could be reduced to very low levels and, in many cases, changed to incurrents.     

Pattern-deficient forelimb regeneration in adult bullfrogs

This study was designed to test the ability of adult bullfrogs (Rana catesbeiana) to regenerate forelimbs, both with and without various experimental treatments. Distal humerus-level forelimb amputations provided with additional deviated (sciatic) nerve and/or repeated soft-tissue injury exhibited considerable outgrowth. However, control sham-operated forelimbs also produced regenerates with comparable frequency, size, and morphological complexity. The lengths of the regenerates ranged from 0.4 to 2.6 cm, representing an outgrowth of 10-65% of the portion removed by the distal humerus amputation plane; some regenerates exhibited an external morphology indicative of digitlike structures. Some outgrowths were flexible but only one was capable of independent movement. Victoria Blue staining of whole regenerates revealed a variety of internal cartilage elements. Staining showed a single solid mass of cartilage in some regenerates while others had several individual and variably shaped cartilages projecting distally. Histological analysis also revealed the presence of connective tissue, striated muscle, and abundant nerve fibers in addition to the individual cartilage elements. We have tentatively termed these responses pattern-deficient regeneration.     

Scanning electron microscopy of epidermal cell migration in wound healing during limb regeneration in the adult newt, Notophthalmus viridescens

The epidermal cells which migrate over the wound surface of the amputated limb of the adult newt were examined using the scanning electron microscope. Specimens were prepared routinely for scanning electron microscopy or were embedded in Epon 812 for light microscopic observations. A cuff of epidermal cells was seen at the edge of the wound, from which cells appeared to migrate over the wound surface. As early as five hours after transection of the limb, the basal layers of this cuff appeared to send out pseudopodial projections. These seemed to establish a physical contact with a fibrin-like substratum, which apparently served as a means of support for the migrating cells. Subsequently, the epidermal cells became elongate and had the appearance of streaming toward the center of the wound. Between 10 and 13 hours post-amputation, the cells in the central region of the stump were rounded up and some possessed microappendages resembling microplicae and microvilli. Throughout the entire period of wound coverage, the cells seemed to maintain contact with the fibrin network, which appeared to be the first structural element of wound architecture. As a result of these observations, the mechanism by which the epidermal cells migrate has been clarified.     

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     

Bioengineered human growth hormone supports limb regeneration in the hypophysectomized newt Notophthalmus viridescens

It is well documented that growth hormone (GH) replacement therapy will restore normal limb regeneration to hypophysectomized adult newts. However, it is also known that the GH preparations used in previous reports were contaminated by other pituitary hormones shown to support regeneration when administered free of GH. The recent availability of bioengineered human GH was studied for its ability to restore the regenerative capacity to hypophysectomized newts. Five days posthypophysectomy adult newts were subject to forelimb amputation distal to the elbow. Animals were divided into three groups (n greater than 20). Each received one of three GH preparations: pituitary-derived bovine GH, pituitary-derived human GH, or bioengineered human GH. GH was administered via intraperitoneal injection (0.029 IU/50 microliters) on alternate days for either the first 5 days (total of 3 injections) or for 35 days (total of 18 injections). Pituitary-intact and hypophysectomized control newts were subjected to forelimb amputation and injected with hormone diluent. All newts that received GH demonstrated normal limb regeneration to the early digitiform stage by 35 days postamputation. None of the hypophysectomized control newts showed any evidence of regeneration. We conclude that GH alone can restore the ability to undergo normal limb regeneration to hypophysectomized newts.     

Expression profiles of elastase1 (NvElastaseI) and secretory leukocyte protease inhibitor (NvSLPI) during forelimb regeneration in adult Notophthalmus viridescens suggest a role in epithelial remodeling and delamination

Extracellular proteases and their inhibitors may regulate a number of important processes involved in forelimb regeneration in the adult newt, including epithelial remodeling, breakdown of extracellular matrix, and dedifferentiation. We have identified a newt homologue of human ElastaseI (NvElastaseI) and its potential inhibitor, SLPI (NvSLPI), and evaluated their spatial and temporal expression during limb regeneration. NvElastaseI is upregulated early in regeneration and is associated with subdermal and wound epithelial cells, suggesting an involvement in wound healing and the generation of the wound epithelium. Up until 15 days post-amputation, NvElastaseI is also scattered throughout the developing blastema and may have a role in the dedifferentiation of stump tissues. NvSLPI is found at the interface between the intact skin and the wound epithelium, and may limit NvElastaseI activity. NvSLPI is also expressed in dermal glands, and is likely involved in anti-microbial activity or function. Quite apart from regeneration, complementary patterns of expression of NvElastaseI and NvSLPI are associated with newt epithelial sloughing.     

Ultrastructural studies on migrating epidermal cells during the wound healing stage of regeneration in the adult newt, Notophthalmus viridescens

The ultrastructure of the epidermal cells which migrate over the wound surface of the amputated limb of the adult newt, Notophthalmus viridescens, was observed with transmission (TEM) and scanning (SEM) electron microscopy. In order to aid in the visualization of polyanionic surface materials on the wound epithelium and wound surface with TEM, the basic dye, ruthenium red, was introduced into the fixatives and buffer. Control limbs were processed without ruthenium red. Shortly after amputation, basal cells at the wound margin possessed elongated, flattened profiles with long pseudopodial projections (lamellipodia and filopodia) that appeared to make contact with the fibrin exudate covering the stump tissues. Epidermal cells proximal to the site of amputation were also in a state of mobilization. Large intercellular spaces and a reduction in the number of desmosomes were observed in the migrating cells. Epidermal cell nuclei became characteristically euchromatic with well-developed nucleoli. Microfilaments were seen within the cytoplasm, extending toward the plasma membrane of cellular processes. Phagocytosed material was also present in the migrating cells. By approximately 9 hours post-amputation, wound closure was complete, and the wound epithelium consisted of three to four cell layers of a non-cornified epidermis. Generally, the amount of extracellular material present on the surface and in the enlarged intercellular spaces of migrating epidermal cells remained the same throughout the period of wound closure. A layer of polyanionic material was observed consistently over the fibrin meshwork covering the wound surface with TEM.