Patrick W. Tank

Pattern Regulation in the Regenerating Limbs of Urodele Amphibians

Pattern formation is the process by which cells in regenerating and developing systesm are spatially organized to form the complex structures of the adult organism. This aspect of morphogenesis has intrigued developmental biologists for over a century. The urodele limb has been used extensively in the study of morphogenesis during regeneration and several models have been proposed to account for a variety of experimental results. The purpose of this paper is to review the experiments that have been published since the turn of the century that deal with patter formation in the regenerating limbs of urodele amphibians and to discuss these results in the context of recent theoretical models.

Regeneration of symmetrical forelimbs in the axolotl, Ambystoma mexicanum☆

Abstract Surgically constructed symmetrical double-anterior and double-posterior upper forelimbs of the axolotl were amputated immediately after surgery. Double-anterior limbs either failed to regenerate or formed single digits or spikes. Double-posterior limbs formed symmetrical double-posterior regenerates in 60% of the cases, thus extending the previous finding that the amount of distal transformation in surgically constructed double-half limbs is inversely proportional to the time between grafting and amputation ( Tank and Holder, 1978 ). When these symmetrical regenerates were amputated through the forearm region, all but one formed a symmetrical secondary regenerate. The majority of the secondary regenerates had a larger number of digits than did their corresponding primary regenerates. Reamputation of the secondary regenerates resulted in symmetrical tertiary regenerates, and the majority of these also had a larger number of digits than did their corresponding primary regenerates. The results are compared to those of Slack and Savage (1978a, b) on embryonically derived double-posterior limbs and they are discussed in terms of a formal model for distal transformation (Bryant and Baca, 1978).

The occurrence of supernumerary limbs following blastemal transplantation in the regenerating forelimb of the axolotl, Ambystoma mexicanum

Abstract Regeneration blastemas at the stages of medium bud and palette were transplanted to contralateral limb stumps so that either their anterior and posterior positions or their dorsal and ventral positions were apposed to those of the stumps. Grafts were shifted from distal levels to proximal levels, or from proximal levels to distal levels, or remained at either a proximal or a distal level. When anterior and posterior positions of graft and stump were apposed, supernumerary limbs were produced at the graft-stump junction in anterior and posterior positions relative to the stump. All analyzable supernumerary limbs were of stump handedness. Apposition of dorsal and ventral positions of graft and stump led to the formation of supernumerary limbs at dorsal and ventral positions relative to stump tissues. All analyzable supernumerary limbs were once again of stump handedness. Shifts from distal levels to proximal levels never resulted in skeletal deletions, as potential deletions in the proximal-distal axis were always filled in. Shifts from proximal levels to distal levels resulted in a low frequency of serial duplications. The results are discussed in view of a recently presented formal model for pattern regulation in epimorphic fields.

The effect of healing time on the proximodistal organization of double-half forelimb regenerates in the axolotl, Ambystoma mexicanum☆

Abstract The effect of healing on the proximodistal organization of regenerates from double-half forelimbs was studied. Double-anterior and double-posterior upper forelimbs were prepared surgically and amputated at 5, 10, 15, 20, 30, and 60 days after grafting. All experimental groups regenerated hypomorphic skeletal patterns. Double-half forelimbs amputated at Days 5 and 10 regenerated more distally complete skeletal patterns than did limbs amputated at Days 30 and 60. The mean numbers of skeletal elements regenerated were seen to decrease as a function of time after grafting, with the maximal suppression of skeletal patterns observed to occur when limbs were amputated 30 days following grafting. There was no appreciable difference between limbs amputated at Days 30 and 60. These results suggest that healing time has a profound effect on the proximodistal organization of limbs regenerated from double-half forelimb stumps.

Morphogenetic interactions occurring between blastemas and stumps after exchanging blastemas between normal and double-half forelimbs in the axolotl, Ambystoma mexicanum

Abstract Regeneration blastemas were exchanged between surgically constructed forelimbs comprised of symmetrical tissues (double-anterior and double-posterior) and normal, unoperated forelimbs. Normal blastemas grafted at the stage of medium bud (MB) onto double-half forelimb stumps regenerated normal skeletal patterns in nearly all cases. Double-half blastemas transplanted at the stage of MB onto normal forelimb stumps did not regenerate complete limb patterns. These results indicate that a double-half blastema cannot be “rescued” by transplantation to a normal stump and that a double-half limb stump does not interfere with the ability of a normal blastema to distally transform. The regeneration blastema possesses sufficient positional information at the stage of MB to permit it to develop autonomously. Supernumerary forelimbs resulted from several types of graft-stump combinations. The location and handedness of these supernumerary limbs are predicted by the rules of a recently presented model for pattern regulation in epimorphic fields [French, V., Bryant, P. J., and Bryant, S. V. (1976). Science 193, 969–981].

Positional information in the forelimb of the axolotl: experiments with double-half tissues.

Abstract It has been demonstrated recently that upper forelimbs of axolotls comprised of symmetrically arranged soft tissues do not regenerate ( P. W. Tank, 1978 , J. Exp. Zool. 204, 325–336). These double-half forelimb stumps contained skin, muscle, and loose connective tissues in symmetrical arrangement. The present study explores the roles of muscle, skin, and epidermis in the regeneration of double-half forelimbs by grafting them separately to create forelimb stumps bearing symmetrical arrangements of these individual tissues. Forelimb stumps bearing symmetrically arranged flexor and extensor muscles and normally arranged skin underwent complete regeneration (96%). Forelimbs comprised of double-half skin overlying normally arranged muscles and deep tissues formed hypomorphic structures and nonregenerates (56%) with some single and multiple regenerates. Limbs with double-half deep tissues and complete epidermis either regenerated distally incomplete patterns (47%), single patterns (33%), or multiple patterns (20%). Those forelimbs comprised of double-half skin and no muscle regenerated incomplete patterns in the majority of cases (56%) but single and multiple limbs also were formed. Based on these results it can be concluded that no single type of tissue is solely responsible for the regenerative failure experienced by double-half forelimbs in the earlier study. The complete failure of forelimb regeneration occurs only when all types of soft tissues tested (skin, muscle, and deep connective tissues) are present in symmetrical arrangement.

Cellular behavior in the anteroposterior axis of the regenerating forelimb of the axolotl, Ambystoma mexicanum

Cellular behavior along the anteroposterior axis of the regenerating axolotl forelimb was studied by use of triploid (3N) tissue grafted into diploid (2N) hosts and three-dimensional computer reconstructions. Asymmetrical upper forelimbs were surgically constructed with one half (anterior or posterior) 3N and the other half 2N. Limbs were amputated immediately after grafting or were permitted to heal for 5 or 30 days prior to amputation. When regenerates had attained the stage of digital outgrowth, the limbs were harvested and sectioned in the transverse axis for histological analysis. When all limbs bearing anterior grafts were considered as a group, 77% of the 3N mesodermal cells were observed in the anterior side of the regenerates and 23% were located in the posterior side of the regenerates. When all limbs bearing posterior grafts were considered as a group, 76% of the 3N mesodermal cells were found in the posterior side of the regenerate and 24% had crossed into the anterior side. Healing times of 0, 5, or 30 days prior to amputation had no effect on the experimental outcome. Three-dimensional computer reconstructions revealed that most 3N cells of mesodermal origin underwent short-distance migration from anterior to posterior or from posterior to anterior and intermixed with diploid mesodermal cells near the midpoint of the regenerated anteroposterior axis. Some 3N cells were observed at greater distances from the graft-host interface. By contrast, labeled epidermal cells from both anterior and posterior grafts exhibited long-distance migration across all surfaces of regenerated limbs. Details of a computer-assisted reconstructive method for studying the three-dimensional distribution of labeled cells in tissues are presented.

How we use standardized patients in gross anatomy: the evolution of a traditional anatomy course

This paper describes a process by which a traditional gross anatomy course has evolved into a clinically oriented anatomy course. Several clinically oriented teaching methods are described which have been introduced into the gross anatomy course in a step-wise fashion. Emphasis is placed on the use of standardized patients in a small group setting to teach the clinical application of traditional gross anatomical information.