[1]Conducted while the author was an assistant professor in the Department of Anatomy, State University of New York at Buffalo.

[2] Originally presented at the Third Annual Meeting of the American Society for Cell Biology, Commodore Hotel, New York City, November, 1963.

[3] Supported by NIH grant GM-09217-02.

[4]A tunnel was reamed into the Jello-like mesenchymal connective tissue of the dorsal fin of A. tigrinum larvae (similar in essence to what is shown in the preparation that may be seen at this site ): A 2 mm segment of brachial-level spinal cord from a sibling donor was transplanted deep into the tunnel; immediately thereafter, the host animal's own right forelimb was removed at the neck of the humerus and the cut end eased into the mouth of the tunnel. With a view to eventual amputation, the distal end of the limb was allowed to protrude to the exterior from beyond the elbow; its proximal, cut end abutted directly against the transplanted segment of spinal cord.
After 2 weeks most transplants were well innervated, as indicated by vigorous, spotaneous or tactually elicited spasms, twitches and contractures. [A parallel series of experiments was carried out to test the efficacy of innervation: an eye was transplanted concomitant with the limb and cord; then the on-off tempo of light used to establish, quantitatively, a causal connection between stimulus and response; i. e., the rate of twitching was directly related to the rate of activation of the light switch.]
Innervated transplants were amputated just below the elbow; each animal's previously intact left forelimb, amputated through the same plane, supplied the control.

[5]Beginning a day after limb amputation, and extending to the day before sacrifice, animals were intracoelomically injected twice daily, with approximately 10 microcuries of thymidine-H³ (6700 mc/mM); injections were carried out with an automated syringe plunger driven by a low speed electrical motor designed specifically for use under the dissecting microscope. (Precision was regulated by time rather than volume.) Uptake of thymidine-H³ was assessed not by absolute amounts but by the differentiatial in specific activity between each transplant and its orthotopic counterpart on the animal's left side (i. e., the control). Larvae were killed 6-15 days after amputation and placed in Carnoy's fluid; regenerates were process for serial microscopic sectioning, and mounted on glass slides for examination under a phase microscope to check on the quality of the tissue. Slides were processed by the methods of Ogur and Rosen (Arch. Biochem. 25:262, 1950) to remove oligonucleotides. Then, under the dissecting microscope, the epithelium and any nonlimb tissues were scraped off the slide leaving only the deep limb tissues; tissues remaining on the slide were sized with an image-shearing eyepiece micrometer mounted on a phase micrscope. After scoring with a diamond knife, slides were broken into small squares and incubated in 2 ml Hyamine hudroxide at 67 degrees C until the tissues dissolved. Radioactivity was determined by liquid scintillation counting in aliquots of the tissue hydrolysates. [Similar experiments were performed with unamputated transplants and the corresponding intact left forelimbs, the animals received the two daily doses for six days. These tisses provided control values.]

[6]No enhancement of DNA synthesis was evident in the transplant/intact ratio of unamputated limbs, nor of 6, 9 and 12 day regenerates by virtue of spinal cord innervation (table 1). The ratios in the uptake of tritiated thymidine for the latter collectively showed a mean of 0.0959 +/- 0.14 s. d. In contrast, 15 day regenerates tritiated thymidine incorporation ratios were 1.831 and 1.505, indicating that the spinal cord had increased DNA synthesis 50-80 percent, transplant versus normal. It should be noted that myogenesis becomes evident in the limb regenerate at 15-16. Thus spinal cord dramatically increased DNA synthesis concomitant with the advent of myogenesis. Additional experiments conducted on the latter point are presented in the next paragraph.

Blastemas younger than 15 postamputation days produce only miniscule amounts of muscle when transplanted to the dorsal fin, unless:

• a piece of stump is left attached to the base; or
• spinal cord is concomitantly transplanted.

Table 2. UPTAKE OF INTRACOELOMICALLY INJECTED THYMIDINE-H³ BY TRANSPLANTED REGENERATING LIMB BLASTEMAS OF AMBLYSTOMA LARVAE

Experiment	Host Site	Spinal Cord?	Dry Weight micrograms	DPM/microgram
I	dorsal fin	absent	48.61	445
	ventral fin	absent	54.12	389
II	dorsal fin	absent	34.13	549
	ventral fin	absent	39.24	500
III	dorsal fin	present	40.03	1171
	ventral fin	absent	40.01	520

pietsch@indiana.edu