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Asa Zook is a fictional scientist-philosopher who occasionally serves as a resource person for an imaginary popular science writer. In this dialog, the writer engages Zook in a discussion about the salamander's regenerating limb. The writer dropped by, ostensibly, to find out what would make a conservative old fart like Zook think that regeneration and memory depend on the same fundamental principles?WRITER: This regeneration stuff is getting like catnip for me, Doc! Especially that blastema. Do you think we'll ever see the day when people can grow new arms and legs like the salamanders do? Or maybe even spinal cords or damaged areas of the brain?
ZOOK: I do not know!
WRITER: I mean, if a salamander can do it, why not us? We both use DNA for our genes, right; RNA to carry the genes' messages around the cell; and proteins to run cell life, that is, convert the genetic code's destiny from information to Real McCoy living! It's theoretically possible, n'est pas, sir?
ZOOK: Your question is much more complicated than might seem at first blush, even if we tame down your language....
WRITER: Hey don't hit me, Doc! Anyway, I know you don't like this kind of
loose, flap-jaw talk. So let me change the subject back to what I came over to gab about
in the first place.... But I'm absolutely fascinated by that blastema -- ole
Spallanzani's cone. And I've got to confess to you in advance, sir -- à lá "This above all to thine own self be true...." and all that that in the
back of my mind down-to-earth head there's this thought. Maybe the genes for regeneration can
be isolated from the blastema's chromosomes; then cloned; then produced by the tons -- so they'll be as cheap as asprin -- and maybe
end up being used in gene therapy on people.
But wait! Wait! Don't toss me out on my A-hole! I withdraw the question. I just wanted to come clean with you, Doc, about
what's on my mind, is all. I'll stick to what I talked about on the phone. But
you can understand the human side of all this, can't you, sir -- Where I'm
Callng From, to borrow from Raymond Carver...
ZOOK: You borrowed from him on a previous occasion. No, I am not at all unsympathetic. I simply shall not, wittingly, contribute misleading information to an already beclouded intellectual issue. You have a right to speculate. I do not, except in the privacy of my thoughts.
WRITER: Understood, Doc! Anyhow, the last time we chewed the fat you went into the evidence for the local origin of the blastema cells. That is, they don't come from some regeneration pool in the bowels or big toes. And they're not drafted from tissues distributed around the body. You're also pretty sure, you in effect said, that if you tweak them just right the blastema cells -- or the cells they come directly from -- can actually make limb parts that normally belong to the opposite side of the body. And that suggests to you that blastema cells are primitive. And that limb regeneration is actually epigenetic development, ugh! -- something brand-spanking new, in other words, like the in the embryo. Not just stuff made directly like toothpaste from a squeezed tube. Not budded off each old tissues of the stump, right?
ZOOK: Partially. In the limb regenerate, the muscle and the cartilage seem not to develop by a morphallactic regrowth from the musculature and skeleton of the stump...
WRITER: Morpha...who?
ZOOK: Morphallaxis -- the obverse of epigenesis -- the direct growth from the preexisting tissue.
WRITER: Does anything morphallaxize ?
ZOOK: Yes, although I would not draw an analogy, as you did, to extruded toothpaste. Morphallaxis is much more complicated than might appear from the surface. The genetic apparatus of the cells is activated, de novo, to produce new polypeptides -- proteins -- even when no new cells, per se, are being produced. When morphallaxis does involve the addition of cells the process is not merely a direct budding, in the manner of soap bubbles, but rather a complex act of differentiation. It is an efficient way of regrowing tissues in wound repair, and much of the regeneration observed in mammals is morphallaxis.
WRITER: Examples?
ZOOK: Nerves, skin, blood vessels.
WRITER: Thank you. Now before I get you too far off course let me ask the big question. You've said on several occasions that you believe the Hologramic Theory of memory encompasses regeneration. Maybe it's my pea-brain, but I find that to be mighty potent language. And, as I mull it over, it sounds pretty important, if it's true. So let me get rid og my chewing gum and ask you straight out, sir, do you believe this: For a salamander is the program for regenerating a hand like what lets the little beast remembering that a juicy worm is something good to eat?
ZOOK: In abstract principle, yes. The two are transforms of each other -- analogs, if you will, with a common logic, despite their obvious physical differences differences.
WRITER: What ever led you to think that? Phase information? The fixed point theorem? Holologic?
ZOOK: Not directly. I also confess that I first appreciated the connection intuitively, not by deduction or induction or any conscious intervention of intellect, but, as you once termed it: in the nads!
WRITER: I can't believe my ears, Doc! ZOOK: Do you remember, from Hologramic Theory, the Principle of Independence?
WRITER: You mean where shuffling the parts of the salamander's brain didn't scramble the animal's mind? And because a whole code for a memory in one piece is independent of the codes stored on another piece? And you can't cut the code with a knife? Yeah, I do. And didn't Independence account for those wild experiments where a guy transplanted fish brains into salamanders? And made salamanders think like fish?
ZOOK: You state the observation crudely, but basically, yes, providing that...
WRITER: Say wait a minute! Excuse me, sir. But now I remember that fish experiment, vividly. That was the one where the donors were tropical fish. They quit feeding at low temperatures, like in the rooms where the guy kept his salamanders. The fish -- from Trinidad -- quit feeding in the cool because their worm-digesting enzymes stop working when it's chilly. Otherwise, out in the wilds, with a live worm in his belly, he'd become the pièce de résistance; . And with Natural Selection there'd quickly be no more of his kind of fish -- out in the wilds! Now the host salamanders -- which should have kept on feeding -- instead did the same thing as the brain-donating fish: they quit going after worms in the cold -- or at the same temperature where the fish quit. But then they'd go after worms when the water was warmed back up. Same temperature! Just like the fish.
Yes, I remember Independence all right. There were some other experiments in
there, too, as I remember. But that fish thing really got to me. Buster! That was the name of one of those
little stinkpots, -- the salamander, I mean, with fish brains in his head.
But.... But, please forgive my stupidity, Doc. How in the name of cheezen'crackers does all this fit into
regeneration?
ZOOK: First of all, limb regeneration is an epigenic event. Yet morphogenesis among the constituent tissues and organs of the regenerate obeys critical predictions of the Principle of Independence. Taken together, these are the features Karl Lashley ascribed to memory. [8]
WRITER: Morphogenesis?
ZOOK: Differentiation at the organ level, as revealed in the spatial patterns assumed by the tissues, expecially muscle and cartilage.
WRITER: You mean like phase in space rather than time?
ZOOK: Space-time, actually, although the argument requires more background than I believe you are prepared to hear today. Thus let us focus on your principal question, or else I will never get to an answer. WRITER: Why just muscle and cartilage, though? ZOOK: We might be able to reason nerve patterns into the Independence scheme, as well as the new blood vessels, and also the dermis -- deeper layers of the skin. But the evidence for muscle and cartilage is compelling. Therefore, let me focus on the musculature and the skeleton.
WRITER: Cartilage because the limb skeleton starts out as gristle instead of bone, right? And what you're saying is that the cells involved in making the muscles will dance to a different tune than those slated to for cartilage, right?
ZOOK: Dance! I actually like your metaphor.
WRITER: That's a real switch, you liking a metaphor, Doc.
ZOOK: Dancing implies the complex, dynamic use of phase information, which is at the heart of Hologramic Theory and which, I believe, regeneration and memory both entail. We have already discussed hologramic theory memory at considerable length. But as to how I personally came to the belief in question, let me focus, as I said, on Independence as revealed in the muscle and cartilage.
WRITER: It's your Tickie Wrench, Doc.
ZOOK: My what?
WRITER: Private joke, sir, from a Steve McQueen movie. An alternative way of my saying, you're the Boss! Stick with muscle and cartilage! ZOOK: The research had its origins in the 1950's when a group from the University of Pennsylvania's Anatomy Department isolated the blastema from the stump and cultivated....[1]
WRITER: Isolated? Cultivated?
ZOOK: They removed the blastema from the stump and placed it in a miniature tunnel they had reamed into the gelatinous connective tissue of the salamander larva's dorsal tail fin. Now, tissue culture methods for amphibians, available today, did not exist then. The investigators were using a quasi-tissue culture technique called deplantation by its principal developer, the late and distinguished embryologist, Paul Weiss.[2]
WRITER: And?
ZOOK: The blastema produced cartilage but not muscle. According to the dogma of the times, it should have produced either both or nothing!
WRITER: Wait! What about the fin's jelly? Maybe it skrewed up muscle.
ZOOK: Paul Weiss had previously demonstrated that the tissues of the fin support but not influence development of embryonic tissues.
WRITER: Embryonic? Not regenerative? Is that cricket, sir?
ZOOK: Good observation! Your implied question was virtually the point of departure of a graduate student who extended the blastema deplantation experiments. He believed that the first task was to produce adequate controls -- controls for the deplant operation as well as the fin environment. Could myogenesis -- muscle differentiation -- occur under the conditions of the deplant experiment? He was able to accomplish both major objectives simultaneously with series in which he left a portion of the stump attached to the deplanted blastema.[3]
WRITER: Let me see if I can guess what he got. Muscle, right?
ZOOK: More than that! Much more, in fact.When only a narrow band of stump remained at the base of the blastema, the deplant not only produced muscle, but it was normally patterned for the limb.
WRITER: Perfectly?
ZOOK: As perfectly as one can get in a living system -- normal down to exquisitely minute morphological details.
WRITER: Even though the muscle grew in the fin?
ZOOK: Even though it grew in the fin! Even though, by itself, the blastema, for practical purposes, produced only cartilages.
WRITER: That's sort of what Spallanzani said about his cone! The blastema can't make muscle?
ZOOK: It is not quite that simple.
WRITER: For you guys, nothing ever is!
ZOOK: When deplanted at the cone stage, blastemas sometimes produced very small amounts of muscle: an occasional cell here and there, with a lone myofibril -- a contractile organelle -- but not the well-patterned, typically limb musculatures produced in the controls with the stump attached.
WRITER: The stumpies controlled for no nerves, too, right?
ZOOK: Yes. At any rate, the incidence of
muscle increased with age at deplantation; and from 15 days post-amputation on -- when muscle is detectable in the blastema -- the dependency on stump disappeared, suggesting that cells programmed for muscle
were growing into the blastema in large numbers after the cone stage.
But -- vis-à-vis nerves -- the
investigator conducted another interesting experiment. He concurrently deplanted spinal cord along with the blastema. Then, despite the absence of stump,
huge masses of skeletal muscle tissue did develop in the fin from deplanted blastema cells.[4]
WRITER: How was that muscle organized?
ZOOK: As clumps, whorls and solid masses around and next to the cartilages, but unfasciculated and never in a limb-like pattern.
WRITER: What was that word you slipped by me -- faz....pastafasu?
ZOOK: A fasciculus -- or fascicle -- is bundle of muscle and nerve fibers. Fasciculation -- divided into bundles -- is what bestows pattern upon a given musculature. 'Unfasciulated' is tantamount to being without a basis for a morphological organization among the muscle fibers.
WRITER: And that's what the muscle looked like when spinal cord was part of the deplant?
ZOOK: Yes.
WRITER: I'm not sure I get it. What did spinal cord do, specifically?
ZOOK: The investigator believed it stimulated extensive proliferation among rests of myogenically competent cells in the deplanted blastemas. Other investigators had found evidence that spinal cord can enhance muscle development, presumably by inducing replication among the myogenically capable but otherwise dormant cells.
WRITER: Presumably?
ZOOK: The assumption was subsequently validated at the molecular level. But I am getting off track now. Let me defer this line of inquiry.
WRITER: Sure! But this replication and muscle business sounds like it could be important. Can we come back to it one of these days, sir?
ZOOK: Yes, of course. Indeed, the replication-myogenesis issue is an essential part of the regeneration-memory connection.
WRITER: But Independence first, right?
ZOOK: That the memory-regeneration hypothesis is even worthy of serious consideration, I believe, is the purpose of your visit.
WRITER: Fair enough!
ZOOK: Excellent data had already existed to support the thesis that, in fact, a causal relationship exists in the salamander tail between preexisting stump muscle and the musculature of the regenerate.[5] The student who followed up on the original deplant experiments wondered if the limb regenerate was like the tail in the latter regard and, if so, how he could go about proving or disproving the underlying proposition.
WRITER: What did he do?
ZOOK: He transplanted blastemas to the orbit in lieu of the eye and in contact with the frehsly cut ends of the host's extraocular muscles.
WRITER: The orbit is the eye socket, right? And the extraocular muscles are just plain old eye muscles, true?
ZOOK: The important consideration is that the blastema would be exposed to muscle but muscle of a non-limb source.
WRITER: I bet I know what happened! The regenerate did sport muscles. But they were eye muscles, right.
ZOOK: The regenerates did have muscle. And the pattern was not that of the limb. But, without the eye, it was impossible to say just what an orbital pattern would be, and....
WRITER: Godalmighty, Doc! Isn't that carrying conservatism a wee wee bit far? I mean....
ZOOK: The correct word is circumspection, without which science is as unreliable as journalism.
WRITER: Ouch! You're probably right. You know, I bet I'd end up in a fist fight with my editor if I put 'circumspect' into my article. He'd say, 'No. The reader will think you're talking about circumcising salamanders and we'd have the animal rights people picketing outside our headquarters for writing about it.
ZOOK: Please!
WRITER: Sorry, Doc....
ZOOK: The investigator went on to validate, indirectly, the thesis that the musculature in the orbitally transplanted regenerates did indeed reflect the extraocular muscles.
WRITER: How?
ZOOK: He had found that extraocular muscles...
WRITER: Can't I just say 'eye muscle?'
ZOOK: There are also muscles inside the eyeball --intraocular muscles. But, providing you make the distinction, I suppose you could call them simply the eye muscles, at least in the present discourse.
WRITER: Jez! I win one! Eye muscles! I'll have to celebrate...
ZOOK: First he demonstrated that cut extraocular muscles would indeed grow into a transplanted limb -- or other organ, such as spleen. And the in-growing orbital muscle altered the limb's musculature for about a millimeter out on the shank.* Distally, the limb muscle retained its normal pattern. With this in mind, he transplanted 11-day blastemas with about a millimeter of stump attached. He predicted, a priori, that the part of the regenerate near the union with eye muscles would be abnormal while that situated distally would, as it developed, acquire limb-specific morphology.
WRITER: What'd he get?
ZOOK: Examined thirty days later, the muscle of the transplanted stump had lost its limb pattern. In the regenerated segment, proximally -- nearing the union with the host -- the muscle, like that in the stump, was completely altered. At the distal extreme, the patterns were normal for the limb. In the intermediate zone, the muscular pattern was an admixture of typical and atypical muscles. The experiments closely followed the predictions of the hypothesis.
WRITER: Let me get this straight. If the guy left a stump on the transplanted blastema, the regenerate ended up with three zones of muscle: abnormal close to the eye socket; normal far out -- in the hand and wrist; but mixed in the middle. Is that right?
ZOOK: Yes.
WRITER: Let me write that down. And what was his conclusion?
ZOOK: Muscle of the stump determined the pattern of the musculature of the limb regenerate.
WRITER: Isn't that morphallaxis?
ZOOK: Not necessarily. But...
WRITER: What about the skeleton?
ZOOK: Normal! Indeed it was the very question you asked that led the investigator to perform additional investigations -- experiments in which he demonstrated that, even prior to amputation, changes in the stump that would drastically change the muscle, would not concomitantly affect the skeleton.
WRITER: Hey! That's Independence, right?
ZOOK: Yes, as relates to chondrogenesis[6]. These experiments showed that chondrogenesis is resistant to whatever mediates the changes in myogenesis. That is to say, differentiation of the skeleton is independent of the musculature.
WRITER: Can you give me a rundown on some of the details of these experiments, Doc.
ZOOK: That preexisting muscles affected the patterns displayed by regenerated muscles had been well documented in the experiments involving transplantation to the orbit of the blastema. As I have already said, the investigator realized that the critical period is prior to the formation of a blastema, as such. In fact, he realized, that to find out if the cone-stage blastema cells really were committed to limb chondrogenesis, and not myogenesis, he would have to alter the internal environment prior even to amputation.
WRITER: Couldn't he just do that with the knife? I mean, with a little plastic surgery job he could change the limb muscles, nein? Maybe transplant some muscle?
ZOOK: The injured muscle would merely regenerate in a few days, and there was no practicable way of knowing with any certitude that any grafted muscles would survive. In fact, transplanted muscle fibers would probably undergo breakdown and release potential myoblasts. Tracking their fate would have been impossible. In short, the outcome of your plastic surgery experiments would have been sloppy, to call upon your usages.
WRITER: Heeh! But what about just transplanting the limb to the eye socket and....
ZOOK: That is precisely the tact he took. He transplanted limbs, waited until the muscular changes had occurred and then amputated, in some cases, proximally, through changed regions and in other, distally, where the musculature retained its limb-like pattern.
WRITER: I'll bet I know what happened here, too! The long guys regenerated normal muscles; the Shorties' muscles took wack-ball patterns; but both Longies and Shorties alike made normal skeletons, right?
ZOOK: Basically, yes.
WRITER: Wait! How did he know the muscles in the Longies were normal? And that they'd changed in the Shorties?
ZOOK: He examined each amputated piece, microscopically, in serial sections.
WRITER: I hate to be picky, Doc but did he control for the level amputation? I mean, the Longies got amputated through the forearm and the Shorties through the upper arm, right? And...
ZOOK: He did control for the level of amputation. He ran a parallel series in which the transplant was removed at the forearm instead of the upper arm. After healing, these were amputated virtually at the rim of the orbit.
WRITER: They were the real short guys -- the Runts. I'll bet the Runts regenerated abnormal muscles.
ZOOK: Yes.
WRITER: But our Runts made normal skeletons, n'est pas?
ZOOK: Yes.
WRITER: So the orbit experiments made you a believer in the regeneration as memory, eh!
ZOOK: Not at the time. I had reservations about epigenesis in the regeneration of the musculature and was leaning toward morphallaxis. But, while I did accept that chondrogensis proceeded independently of myogenesis, an unambiguous case for the converse remained open -- and insolvable -- for a quarter of a century.
WRITER: You lost me on that one, Doc.
ZOOK: Within the very same system -- the regenerating salamander larva's limb -- could myogenesis reach a demonstrably normal outcome in the face of altered chondrogenesis?
WRITER: And?
ZOOK: Retinoic acid can profoundly distort skeletal development
-- and thus chondrogenesis -- in the limb
regenerate. But twisted, gnarled, reduplicated and distorted though the skeletons may be as a result of retinoic acid treatment, the
muscles within them are still identifiable as organs of the limb.[7]
WRITER: Wow! And this proof of the flip-side of the proposition made you a true believer in Independence?
ZOOK: Yes.
WRITER: And the idea that regeneration and memory are...what's the word I'm looking for here, sir?
ZOOK: Analog: regeneration and memory are analogs of each other.
WRITER: Meaning that if I know the abstract rules of the one, I know what makes the other tick -- in the abstract of course!
ZOOK: Yes.