SPALLANZANI'S CONE: the regeneration blastema

--- an Asa Zook dialog about the regrowth of the little salamander's arms and legs

web contact: pietsch@indiana.edu

Asa Zook is a fictional scientist-philosopher who occasionally serves as a resource person for an imaginary popular science writer. Here, the writer engages Zook in a discussion about regeneration of the salamander's limb.

WRITER: Sir, I'd like to ask you a few questions about the blastema....

ZOOK: Before you begin, let me confess a prejudice: I personally, and privately, would prefer the term 'cone'; the term Spallanzani[1] originally gave to what we today call the blastema. 'Blastema' carries hidden implications, some of which may be quite problematic.

WRITER: Gee, don't hit me, Doc. I'm just a poor innocent civilian trying to trade words for bread.

ZOOK: Tea or coffee?

WRITER: Black coffee for me, please.

ZOOK: Forgive my vehemence. But I know of no topic so rife with polemics. Of course, by all means employ the widely used term, blastema. I do myself, in public discourse. It has been in usage to my knowledge since at least1851*. I do urge, though, that we avoid telegraphing connotations with loaded terms, lest we never come to understand the logic -- the abstract governing principles -- of regeneration.

WRITER: Those abstract rules are what made me look you up in the first place. I understand that you think regeneration and memory follow the same logic. ZOOK: In the abstract, of course, at the informational, not the physical, level. The biochemical and physiological processes may be quite dissimilar, regeneration versus remembering. But I believe both processes depend on phase information. Someday, after you know more about regeneration, I promise to elaborate on how phase and fixed point theorems fit into regeneration. But that is for another day, if you please.

You had said on the phone that you want some historical perspective on limb regeneration.

WRITER: Can you help me on that score, Doc?

ZOOK: In a limited way, perhaps.

WRITER: My editor would soil his skivvies if I put this scholar business into the body of my article. We have a very different theory of the ideal reader, the editor and I. To him, reader equals A-hole...

ZOOK: Equals what?

WRITER: Somebody with nads where brains ought to be. Someone who's only interested in sex, pizza, money and the quantity of fuzz generated by his or her own navel.

ZOOK: And your ideal reader?

WRITER: As dumb as I am Doc, I just can't fathom any civilization advancing an inch -- make that a centimeter since I'm a devotee of the metric system -- not a centimeter since the Ice Age if my editor's cynical view of the species is even remotely valid. Look, I know all about literary grab and how to put the verbal hook into a reader. But I'm certain the folks who read my stuff want to know about how a subject got started in the first doggone place. Who found it? What kind of person was he -- I doubt there were any shes in the beginning of regeneration research! With a little history, a writer can often take a topic out of the abstract, make it sort of come alive, jump some... Make it more human than science usually comes off. People, after all, made the discoveries, right Doc? Anyway, whether it gets rejected or not, I'm going to turn in a side bar on this Spallanzani[2] dude along with my main piece. I remember him from Microbe Hunters when I was just a high school kid. I'll never forget how his ingenious experiments really put the poleax to spontaneous generation. He's one of my real heroes, ole Spallanzani. And...

ZOOK: I am very pleased by your respect for the reader's intelligence, and I genuinely applaud your courage. Courage is as essential a personal attribute as intellect in the search and delivery of truth and meaningfulness: in the philosopher's quest, truth and meaningfulness!

WRITER: Gosh Doc, I'm a little embarrassed. All I want to do is report what's there. Philosophy? Cheez! Well, anyway....

ZOOK: I wanted to add, though, that creating heroes and villains can obscure reality and dull truth.

WRITER: Gulp! You're right, Doc. It's one of my shortcomings, good guys and bad guys! Anyway, can you tell me how far back in the record regeneration as a whole goes? I know Spallanzani discovered it in salamanders. But what about in other animals? My own guess is Aristotle?

ZOOK: Aristotle, who lived from 384 to 322 BC, does mention the regrowth of amputated bodily parts -- sometimes erroneously -- in Generations of Animals as well as in the History of Animals. But he is speaking second- and perhaps even third-handedly, and from observations and writings, of others.
Pliny the Elder, circa 77 AD, reported that lizards regrow tails; he also noted lizards with double tails: supernumerary appendage formation externally is akin to regeneration.
[3], [4] Albertus Magnus in the 13th century also mentioned tail regrowth in lizards,[5] although it is possible that he was referring to Aristotle's writings. There were references to what amounts to regeneration in 17th century writings. In 1712, Réaumur reported on the regeneration of the amputated claw of the crayfish.[6] Present day scholars, though, give Abraham Trembley credit for initiating the modern era of research on regeneration:[7] In 1744, he recorded that, the two halves of a bisected hydra, each, regenerate a whole individual.

WRITER: A whole animal from a half piece! That sounds like those shufflebrain experiments. Slicker 'n sheep scheist! if you'll pardon my French. Sorry, sir. Please continue.

ZOOK: Empedocles[8] in the 5th century B. C. is the earliest candidate I am personally able to find. He wrote a poem, of Love and Terror , with the line,

"...domains where headless necks grew..."

WRITER: But Spallanzani[10], [11], [12] is our man when it comes to salamanders' legs and tails, n'est pas? And he discovered our blastema. Cone!

ZOOK: As you said a moment ago, he was a major opponent of spontaneous generation. He was also a very strict preformationist. Unfortunately, his meta-system tainted his investigations of regeneration.

WRITER: Preformationist? You mean he thought a sperm had a little person in it, a homunculus.? I can't believe it, Doc! There's nothing about that in Microbe Hunters. But how does preformation figure into the regenerating limb?

ZOOK: Let me read from an English translation of what he actually wrote:

"The beginning of this reproduction [his term for regeneration] is a cone, which is nothing but the leg in miniature, and only wants to be unfolded. [13]"

ZOOK: No, not initially. Before you leave, I'll let you look at a section of the blastema under the microscope. You will see for yourself that -- initially at least -- it is a mound of mesenchymal cells, capped by epithelium. But I want to make a philosophical point in a moment that, I believe, is especially important in our day. But now let me read a little further:

"At first the cone is a gelatinous substance, endued with the most exquisite feeling;"

"and though the new leg be still very small, the articulations are visible, and the salamander uses them very soon."

WRITER: I'm absolutely crushed, Doc.

ZOOK: By what?

WRITER: How could he be such a friggin' crackpot -- and so damned bigoty about it, too? I mean...He was one of my heroes. De Kruif doesn't even squeak about a little leg in that cone. Why aren't you furious too, Doc?

ZOOK: Bias besets us all, including our magnificent Italian, Spallanzani -- Pasteur, Einstein too! I seriously doubt that I am immune.

WRITER: I guess I ought to know that better than anybody. Maybe that's one of the pitfalls of making scientists --or anybody -- into heroes.

ZOOK: Or A-holes?

WRITER: Eh, Doc! Cool! Coool!

ZOOK: One's system of beliefs can strongly influence the outcome of any experiment or the most discriminating empirical observation, let alone one's conclusions. I do not see how it it can be otherwise. The intelligent approach is not merely to take every conceivable precaution to neutralize bias but to appreciate that bias will probably strike at the most crucial, yet unexpected moments. One must avoid the habit of assigning love or preference to a particular theory or to any system of ideas. Beyond that, science and philosophy should periodically reassess its the most fundamental premises. No belief should ever become charished.

WRITER: Does gravity work this morning?

ZOOK: Exactly. And beyond that, one should continue to find novel predictions in even the most successful theories. Try alternative theories to explain well established bodies of facts. Examine the consequences of new axioms. Often, these exercises extend and enrich a powerful theory. Einstein's relativity immediately comes to mind.

WRITER: I'm forced to say, though, Doc, that I've run across a very few scientists who recognize their limitations, let alone the limits of science, itself. I couldn't agree more with you. But, if you'll forgive my frankness, you're describing heaven, not the world we live in.

ZOOK: Spallanzani's Prodomo , nevertheless, is a masterpiece, and, considering when it was written, a veritable model of how to conduct experimental inquiry. Spallanzani poses very specific experimental questions. When he is looking at tail regeneration, for instance, it is not simply the tail as a whole -- which could have been merely proud flesh or a cancerous growth. He wants to know if the lost vertebrae, per se, are replaced; if muscles and vessels -- specific anatomical parts -- regrow; how the regenerated tissues and organs compare with the parts of the natural appendage they replace; if variables such as species and age factor in.
In one fascinating experiment, involving successive limb re-amputations, he deduced that one particular animal had regenerated a total of 687 individual bones, no less, during three successive amputations over the months of June, July and August. In other words, as he himself observes, repeated amputations failed to deplete the salamander's regenerative capacity. The source of the blastema is not used up by successive bouts of regeneration, which is a crucial point and one of the major attributes of limb regeneration, as we know it today.

WRITER: Is there some seat of blastema cells in the body or do the cells come from all over?

ZOOK: No, which makes Spallanzani's observations all the more intriguing, to me. Tissues at the site of the amputation produced Spallanzani's cones, although this point was not firmly established until the 1940's.

WRITER: How so?

ZOOK: With X-irradiation. A zoologist at Princeton named Elmer Butler[14], [15] had found that relatively high doses of X-irradiation block subsequent limb regeneration. In 1942, he and a postdoctoral fellow, John Patrick O'Brien[16] published a simple but ingenious extension of Butler's earlier findings. Using a radio-opaque lead mask, with a slit cut in it, they protected all but a small zone of the hing limb. X-irradiation, in high dosage, [17] delivered anywhere over the shielded area, including parts of the limb immediately adjacent to the exposed zone, failed to inhibit regeneration.

WRITER: Controls?

ZOOK: Yes. But upon amputation through the exposed part, regeneration utterly failed. Butler and O'Brien bracketed the source of the blastema.

WRITER: Wow! That's a real neat experiment. Say, if the local tissue can keep on producing blastema cells, it's kind of like the Sorcerer's Apprentice, no? I mean, you can keep on cutting and cutting and the regenerate keeps on coming and coming. Right?

ZOOK: That is why Spallanzani's 687 locally generated bones are so intriguing to me.

WRITER: Is the source of the blastema inexhaustible?

ZOOK: I do not know.

WRITER: But back to the preformation thing. The regenerated parts don't just pop up, shake-and-bake, like rehydrated beans, right?

ZOOK: The new skeletal elements differentiate from the blastema cells, even the blastema, isolated from the stump. Muscle is a more complicated matter, which I shall defer until some later time. But the mesenchymal cells of the blastema produce cartilages and, in time, the cartilages ossify.

WRITER: Turn into bone.

ZOOK: Very poorly put! The cartilage does not turn into bone. The cartilages provide a framework for the deposition of bony tissue. Bones, per se, replace the cartilages. But you would mislead your readers to say that cartilage become bone. An analogous situation exists in geology, where a tree, for example, becomes petrified: the wood, as such, does not turn into stone but is replaced by it. Similarly with ossification: the cartilages or membranes or even tendons in some places -- or massive sheets of fibrous connective tissue -- are replaced by, not turned into bone.

WRITER: Membranes? Tendons?

ZOOK: The flat bones of the face and skull start out as mesenchymal membranes. The knee cap ossifies in a tendon -- the tendon of the quadricepts femoris, to be specific. Bone can also occur in a large scar or the heavily exercised belly of an athlete's gastrocnemius muscle.

WRITER: Okay, you win Doc. But let's see. Ah... epigenesis! That's the word I want here, the opposite of preformation. Who originated it, anyway?

ZOOK: William Harvey.[18] His reference, though, was to the fertilized egg, not the limb regenerate.

WRITER: But the blastema is epigenic, right?

ZOOK: You know, until only a few years ago I had doubts that this issue could be settled by experimentally -- versus by polemics. Epigenesis implies a primitive state, one in which the cells are not fixedly determined along one and only one developmental pathway. If you wish, I will describe the experiments that made me a believer in the thesis that limb regeneration in the salamander is, indeed, an epigenic phenomenon.

WRITER: Oh, please do, sir. I love to deliver that kind of inside dope to my reader.

ZOOK: Are you familiar with retinoic acid?

WRITER: The pimple medicine?

ZOOK: Treatment of acne*** is one of the therapeutic, and perhaps best-known -- if potentially dangerous -- uses of retinoic acid, yes. But the research I refer to is in embryonic development, specifically differentiation**** -- the acquisition of particular attributes. In molecular terms, this would mean the opening of previously quiet segments of the genome and the production, eventually, of proteins not previously among the cells' complement; at the cell level, cell types not previously seen; at the organ level, new bodily parts.

WRITER: Development in contrast to what, though?

ZOOK: Growth, per se: the production -- reproduction -- of more of the same. In our day, developmental biologists have regard development as the algebraic sum of the products of growth and differentiation. Retinoic acid may play an important, perhaps even essential, role in starting certain kinds of differentiation, especially -- but not limited to -- cells fated to manufacture skeletal elements.[19], [20]

WRITER: Where does regeneration fit into the picture, sir?

ZOOK: In large doses, and when applied to a regenerating appendage, retinoids -- retinoic acid and other Vitamin A derivatives -- can have bizarre effects, occasionally leading to the formation of supernumerary organs.[21] Regeneration was among the earlier systems investigated in retinoic acid research.

WRITER: And the epigenic thing?

ZOOK: Animals treated with retinoic acid while regeneration is in progress often produce more than one new limb. Sometimes two limbs appear externally to be mirror images of each other.

WRITER: Does that mean that cells of the left stump, say, can make a right limb?

ZOOK: Potentially, yes. But under highly favorable conditions, it has been possible to trace serial sections and actually see that the detailed pattern of the muscles inside the limb are mirror images of each other.

WRITER: Like a Rorschach ink blot, eh?

ZOOK: Those data, which were published just a few years ago,[22] provide an unequivocal case for the thesis that cells of the stump represent what Ross Harrison, referring to the embryonic limb bud, called a self-differentiating harmonious equipotential system.

WRITER: Ugh!

ZOOK: I was never fond of such language, either. But a generation ago, students of development did not have today's choices. To state our proposition in today's lexicon, the blastema cells can, under the right conditions, open the homeoboxes -- genes -- to make organs of the opposite side of the body.

WRITER: Wheph! Doc you've given me a lot to chew on. But I've got a deadline. Also, I'm starting to get punchy. I better get back to my thinking machine before my brain turns into oil. Can I come back and talk to you more about what happens to those blastema cells? I'd still like totake up this regeneration is memory thing.

ZOOK: I will be happy to have you back for a discussion the fate of blastema cells. Good luck with your article. Goodbye.

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web contact: pietsch@indiana.edu