REVIEWS–A PEER REVIEWED FORUM

Regeneration According to SpallanzaniPanagiotis A. Tsonis* and Timothy P. Fox

In this report, we elaborate on a letter that Spallanzani wrote to Bonnet reporting his findings on regenerationin worms, snails, tadpoles, and salamanders. The letter (original in French and translated in English; seeSupplementary Material, which is available online) was written to discuss whether or not regeneration in theseanimals supports Bonnet’s theory on germs. The letter includes several drawings by Spallanzani, which werenot published in the Prodromo, his book on Animal Reproduction. Spallanzani made important observations,which he described with considerable detail, but overall he was unable to confidently support Bonnet’s theory.This letter reflects the way of thinking in the 18th century that shaped the important scientific fields ofregeneration and reproduction. Developmental Dynamics 238:2357–2363, 2009. © 2009 Wiley-Liss, Inc.

Key words: Spallanzani; regeneration; animal reproduction

Accepted 4 June 2009

INTRODUCTION

Rooted in Aristotelian philosophy, thebelief that lower animals were gener-ated spontaneously from decay pre-vailed until the 17th century whenRedi in 1668 carried out well-con-trolled experiments that provided thefirst proof against it (Redi, 1668,1671). A similar view, epigenesis, de-veloped by Harvey, stated that neworganisms appear from undifferenti-ated matter. These ideas were sup-ported later in the 18th century byNeedhan and Buffon who believedthat organic molecules organized byinternal molds were responsible foranimal generation (Benson, 1991).The rival position to epigenesis, whichprevailed at the end of the 17th cen-tury, was preformation. According tothis, the organisms were preformed inthe embryo. Preformation was also infavor when it came to religious beliefsbecause it fit the notion that all gen-erations were established at the time

of Creation. On the contrary, epigene-sis allowed space for questioning therole of God. As expected when the firstexperiments in the 18th century re-vealed the regenerative power of ani-mals, these two competing theorieswere called upon to explain this newproperty of animals.

The 18th century could very well beconsidered as the golden era in regen-eration research. Many studies duringthe previous century had led the wayto understand how reproduction oc-curs and many theories and thoughtswere developed to explain how ani-mals (and humans) reproduce (Cobb,2006). Naturally, scientists experi-mented with regeneration in animals(which then was called actually repro-duction). Pivotal discoveries by Reau-mur on appendage regeneration in in-sects in 1712 (Wheeler, 1926), byTremblay on Hydra regeneration in1744 (Baker, 1952; Dawson, 1987),and by Bonnet on parthenogenesis in

1740 and on worm regeneration in1744 (Savioz, 1948; Dinsmore, 1991)shocked the scientific world. Reaumurand Bonnet were preformationistsand, in fact, Reaumur believed thatgerms were contained within parts re-sponsible for regeneration. In his writ-ings, Bonnet argued that Trembley’sexperiments with Hydra and his withworms supported the pre-existence ofgerms and became a leading propo-nent of the preformation theory.

Among these intellectual giants,Lazzaro Spallanzani is also creditedas being one of the pioneers in regen-eration research. In 1768, Spallanzanipublished the Prodromo, his historicalbook on Animal Reproduction. At thattime, the word “reproduction” wasused to denote “regeneration” as well.In his book, Spallanzani describedseveral types of regeneration withmention of regeneration of the frogtail and salamander limbs. The ac-counts were surprisingly quite short

Additional Supporting Information may be found in the online version of this article.Department of Biology and Center for Tissue Regeneration and Engineering at Dayton, University of Dayton, Dayton, Ohio*Correspondence to: Panagiotis A. Tsonis, Department of Biology and Center for Tissue Regeneration and Engineering atDayton, University of Dayton, Dayton, OH 45469-2320. E-mail: panagiotis.tsonis@notes.udayton.edu

DOI 10.1002/dvdy.22057Published online 3 August 2009 in Wiley InterScience (www.interscience.wiley.com).

DEVELOPMENTAL DYNAMICS 238:2357–2363, 2009

© 2009 Wiley-Liss, Inc.

with not much detail or any drawingsthat were common in publications ofthat time. However, in a long letter toBonnet written on September 21,1766, Spallanzani outlined in greatdetail regeneration in many organ-isms, including earthworm, snail,frog, and salamander (Biagi, 1958;Dinsmore, 1991). The letter, whichreads as a research paper, is also dec-orated with many drawings by Spal-lanzani’s hand (original in French andtranslated in English can be found inSupplementary Material). One of themajor reasons Spallanzani wrote thisletter was to discuss Bonnet’s theoryon germs, according to which repro-duction and regeneration were medi-ated by germs that existed in the or-ganism. Spallanzani initiatedcorrespondence with Bonnet on July18, 1765 by sending him copies of twoof his publications. On August 24,1765, Spallanzani wrote again to Bon-net critically commenting on Need-ham and Buffon’s ideas. Bonnet re-

sponded on September 14 and wasquite happy that Spallanzani sharedhis views and that he was also work-ing on earthworm regeneration. Bon-net in a sense invited Spallanzani topursue the work on earthworm regen-eration. An exchange of letters ensuedin which Spallanzani outlined a seriesof experiments that were inspired byBonnet’s influence. Bonnet mentionedthat regeneration research should beable to resolve the germ concept, thusinviting Spallanzani to elaborate onthis (Biagi, 1958; Dinsmore, 1991).

In his letter of September 21, Spal-lanzani casts doubts on the existence ofgerms for some types of regenerationand obviously he does this with greatcare not to offend Bonnet. Thus, thisletter bears enormous significanceabout how scientists were thinking atthat time and how they dealt with theseobstacles imposed by their social/reli-gious status as well as by the prevailingway of thinking of the time (for exam-ple, it questioned the Cartesian mecha-

nistic view of animal biology or how theissue of soul fits in a regenerating ani-mal). In addition, this letter also reflectsthe clarity of thought that Spallanzanipossessed in order to explain his find-ings. We, therefore, strongly believethat the contents of this letter should beof great historical value to scholars indevelopmental biology and regenera-tion research. Here we will highlight hismajor experiments and points. Also, wehave reproduced the drawings and wepresent them with added color as well.The color is in accordance with the de-scription that Spallanzani provides inhis letter. Figures 1–17 have been set inthe order in which they appear in theletter, but have been grouped accordingto the species. We will end this reviewby reflecting on how these ideas shapedscientific thought in the 18th centuryand how they relate to our thoughts inthe present day. Text in italics containscomments by us related to Spallanza-ni’s observations.

Figs. 1–6. Anatomy and regeneration in worms. Fig. 1: The gross artery (red) attached to the intestines. Fig. 2: The artery branches into five vessels(red) as it reaches the head. Spallanzani describes the vessels as small bags composed of one or many constrictions. Fig. 3: The vein that runs fromhead to tail is winding. It is intersected by another vein, which is straightened. Fig. 4: The “boat” worm, a fresh water worm, whose tail is on the surfaceof the water (light blue), while the head is hidden in the mud (dark blue). Fig. 5: Regeneration in fresh water worm. A cone is produced. The reddishcolor at the base of the cone is the anus. Fig. 6: At the posterior part of the anus, a red line appears in the same direction as the artery of the animal(o). Spallanzani describes that at this stage he could not see circulation in the red line but later blood flows into the artery.

2358 TSONIS AND FOX

REGENERATION INEARTHWORMS ANDFRESHWATER WORMS

After a short salutation and com-ments on how some scientists viewBonnet’s research, Spallanzani setsthe stage by describing certain exper-iments he performed to learn aboutthe anatomy and physiology of theearthworm. Spallanzani providesthree drawings of the anatomy of ar-teries and veins (Figs. 1–3). He thenproceeds by outlining six different ex-periments to study regeneration inearthworm. Drawings outlining theseexperiments and the results areshown in Supplementary Material.

Experiment 1

Transversal section of the earthworminto three parts in a way that theovary, and the small bags (he meansvessels; Fig. 2) remain in the anteriorpart, or in the part where the head islocated. Results: After a few months,the posterior parts, or tails, and themiddle parts all died, with the excep-tion of 5 or 6 of them. The anteriorpart, which contained the ovary andthe small bags, after 16 to 20 daysstarted to generate a very small bud;this small bud stopped growing duringwinter, but continued its developmentduring spring.

Experiment 2

Divide the worm transversally into 3parts in a way that the ovary and thesmall bags belong to the middle part.Results: All anterior parts or the endsof the head died. The tails (posteriorparts) also died with the exception ofthree of them. Many of the middleparts generated a head at their ante-rior extremity, and a tail at their pos-terior extremity.

Spallanzani attempts to providesome explanation of the few survivorsthat did not contain the reproductivesystem in experiments 1 and 2. Obvi-ously, the pieces survived for severalmonths and regeneration of the tailwas more obvious.

Experiment 3

Remove from the worm the part of thehead that remains above the ovaryand the small bags. Results: All the

ends of the head are dead but all theposterior parts containing the ovaryand the small bags generated a head,which in the end was equal to the orig-inal one.

Experiment 4

Divide longitudinally the body intotwo parts from the head to two thirdsof its length and then remove the pos-terior third (tail). Results: The twolongitudinally divided parts and thetail part died.

Experiment 5

Divide the worm longitudinally intotwo parts from the tail to two thirds ofits length and then remove the ante-rior part containing the head. Results:The two parts, which were separated,died, and the third part, which re-mained and contained the head, gener-ated the tail (obviously the part con-tained part of or the whole reproductivesystem).

Experiment 6

Divide longitudinally the whole wormfrom the head to tail. Results: All theworms died.

Spallanzani then proceeds to dis-cuss how earthworms grow. The ques-tion is: Is growth mediated by the ad-dition of new rings or by the expansionof old ones? This question is related topreformation notions. In order to an-swer this question, Spallanzani de-cided to count rings in 11 worms thathatched from eggs and in 11 fullygrown ones. He observed 84, 100, 96,90, 102, 104, 88, 93, 96, 104, 90 in theformer and 88, 94, 73, 101, 87, 83, 96,80, 89, 95, 101 in the latter. Based onthis, he concluded that growth is theresult of expansion of old rings.

Spallanzani then turns to describeregeneration in another species, thefresh water worm. As for its appear-ance, he states that the part that con-tains the head is hidden in the mud,while the tail is pushed at the surfaceof the water forming a structure thatresembles a groove or a boat (Fig. 4).He also describes the anatomy of thecirculatory system for this worm. Aswith earthworms, Spallanzani experi-mented with regeneration. In one ex-periment, he cut 50 worms into two

more or less equal parts. Almost allthe anterior parts regenerated (obvi-ously these parts contained the repro-ductive system) while most of the pos-terior parts (36/50) died. In the secondexperiment, he cut the worm intothree parts in a way that the anteriorpart contained 3–6 rings and no re-productive system, and the middlepart contained the reproductive sys-tem and the posterior part. At the endof the experiment, all the anteriorparts were dead, the middle parts re-generated and many of the posteriorparts were alive but without signs ofregeneration. Spallanzani also men-tions that at the level of the tail, the“boat” structure never failed to form,and he also concludes that theseworms regenerate naturally becauseartificial regeneration (when he cutthe worms) was identical with naturalregeneration (when he found them innature).

Having established that worms canregenerate, Spallanzani then at-tempts to explain the mechanism.Most of his observations are throughthe circulatory system, because thiscan be easily observed. First, he iden-tifies a red dot by the edges of theanus. This shape of the anus stretchesout. A cone starts to form, but theanus he claims is not at the tip of thecone but more at the rear of the cone(Fig. 5). At the posterior end of theanimal (stump), a red line appears inthe same direction as the artery of theanimal (denoted as O artery in Fig. 6).In the beginning, the line does notshow any circulation but later it does,and the blood flows into the O artery.Thus, the red line is the regeneratedartery. The cone grows and the edgesof the anus acquire more redness. Asthe cone grows, rings become obvious.Spallanzani also attempted repeatedamputations, which were successful.He finally concludes that the shape(boat) is always formed and that therepeated amputations lead to shorten-ing of the animal. Spallanzani doesnot elaborate here how germs couldaccount for regeneration in earth-worms, but he compares this later withhead regeneration in snails. At the endof this section, Spallanzani developssome ideas about the respiratory sys-tem, but his observations are ratherinconclusive. Despite the interestingway that earthworms regenerate and

REGENERATION ACCORDING TO SPALLANZANI 2359

the intriguing necessity of the repro-ductive system, which implies depen-dence on some specific factor, not muchis known today at the molecular level.Nevertheless, after Spallanzani, manyscientists experimented with earth-worm regeneration. The necessity ofcertain segments, which include the re-productive system, has been verified(Morgan, 1901). In the earthworm,Eisenia foetida, which has 100 seg-ments, amputation between segments20–35 results in the failure of regener-ation (Supp. Fig. 1). In this worm, re-generation is mediated by the forma-tion of blastema, and ectoderm andendoderm maintain their identities. Italso seems that annelids in generalrely on dedifferentiation of older tis-sues, rather than pluripotent reservecells (Goss, 1969). Nevertheless, stud-ies on asexual reproduction in anne-lids have indicated the regeneration ofprimordial germ cells from parts of thebody devoid of gonads, arising frompiwi-expressing germile stem cells(Weisblat, 2006).

REGENERATION INGARDEN SNAILS (SLUGS)

The next part of the letter deals withregeneration of head structures insnails. He first describes regenerationof the long horns (stalks), which “ bearat their extremity a nice bud adornedby a blackhead.”

Following partial sectioning of a

horn, the rest of the horn graduallybecomes rounded into a small bud,which grows and its color darkens. Fi-nally, a dark spot appears at the tip ofthe bud. The bud grows, becomeslonger, and after some time the muti-lated horn acquires its normal length.Spallanzani notes that at the bound-ary between the old and the regener-ated piece, there is a whitish color.When he cut both horns, both regen-erated as well. Next, Spallanzani dis-cusses the nature of the dark spots atthe tip of the long horns. He disagreeswith other authors that they are eyes.Rather, he believes that they are likeantennas, which direct the snail dur-ing motion. Obviously Spallanzaniwas wrong about this because it is nowwell known that the spots are eyes andtheir regeneration has been well docu-mented (Eakin and Ferlatte, 1973).But most amazingly, Spallanzani ex-perimented with regeneration of thesnail’s head! The severed pieces con-tained two lips, mouth, part of theesophagus, a tooth, muscles, and fourhorns (two long and two small ones).He observed regeneration in many ofthem (he used 200) after 26 days. Hethen attempted to compare these re-sults with regeneration in earth-worms. He attests that regenerationof the head from a sectioned posteriorpart of the earthworm is mediated bya cone, which is a whole organ, a min-iature of a head waiting to fully de-velop. However, in the snail the head

emerged in parts, which appeared oneafter another and reconstituted thehead with time. Spallanzani describesone case where regeneration consistedof a bud made of two lips of the mouthand two small horns. The bud was“implanted” in the piece (the stump)(Fig. 7). The long horns were the nextpart that was regenerated. Spallan-zani makes the case that not all partsare regenerating during the sametime frame. He then proceeded to dis-sect the regenerated head and fol-lowed the direction of the four nervesthat are inserted in the horns. Heclaims that he could not find wherethe old part united with the new one,and this was the case for the skin(outer surface of the horns) and esoph-agus. With these observations and thecomparison he mentions with theearthworm, Spallanzani sets the stagefor discussing Bonnet’s theory ongerms.

Next, Spallanzani describes regen-eration of the tail. He noticed that af-ter amputation, the piece left behindretracts (Figs. 8 and 9). In the middleof the tissue (denoted as O in Fig. 9),there is formation of a “beak,” its colorbeing white. That was in the center ofthe tail’s musculature and it contin-ued to grow in all directions until itbecame equal to the removed part.The regenerated part maintained itswhite color and it was also recognizedfor having a non-stressed gross skinthat is seen in the remainder of the

Figs. 7–9. Regeneration in snails. Fig. 7: Head regeneration. A bud with two lips and two small horns. Spallanzani claims that at this stage the longhorns are not visible. Figs. 8-9: Tail regeneration. After amputation (8) the stump retracts and in the middle (o) a whitish beak appears in the centerof the musculature.

2360 TSONIS AND FOX

tail (maybe he is observing here a kindof wound epithelium, which has notbecome full-thickness skin). Evenwhen the amputation was on anoblique surface, it was completed. Ex-periments with other snails, which hecalls Limaconi Ignudi, were not asgood in that they could regenerate thehorns but not their heads.

Spallanzani then embarks on ex-plaining regeneration based on Bon-net’s germ theory. As briefly men-tioned above for earthworm and freshwater worms, Spallanzani is unable toexplain it as “a simple extension”;therefore, he strongly believes thatpre-existing germs must be involved.He based this on his observation thatthe cone at the site of amputation re-sembles the part that was removed.Even though Spallanzani tries toplease Bonnet by supporting his the-ory, he cannot be quite sure thatgerms are responsible for the wholeregeneration: “let me have a smalldoubt to your sound judgment.”.

The doubt is cast from his observa-tions that the gross artery, the twoveins, intestines, and the musculature

are so perfectly united in the repro-duction (regenerated part), so that theparts are in continuity with the onesin the old animal. He discusses that hehas difficulty seeing how germs canaccount for this. Likewise, he con-cludes the same for snail head regen-eration because, as mentioned above,the parts regenerated separately andthe regenerated nerves looked like ex-tensions of the old ones. In order togain more insight and information onthis, Spallanzani turns next to his ex-periments on salamander and tadpoletail regeneration.

TAIL REGENERATION INSALAMANDERS

First, Spallanzani describes tail regen-eration in adult salamanders. After am-putation, he observed a lot of blood flowand retraction of the skin at the edges ofthe stump. Then he observed that akind of thin surface of reddish whitecolor appear and the edges of the skinseem implanted on this surface. Spal-lanzani, in fact, describes here the for-mation of a wound epithelium (the thin

surface). He then goes on to describe thebeginning of a formation from the thinsurface, which could be round or elliptic(what is known now as blastema). Heobserved that along the longer axis ofthe elliptic surface appears a thin crestof flesh, which is the beginning of thereproduction (regenerating tail). Foranyone who has studied tail or limb re-generation, this is the early cone forma-tion, which is most obvious at the centerof the stump. Spallanzani in fact noticesthe different colors (a thin yellow line)between the old tail and the regenerat-ing one (Fig. 10). He then describes howthat central crest of flesh grows in alldirections (clearly the formation ofblastema) (Fig. 11). From the center ofthe regenerate, two small bands appearand they go up to the tip of the crest.These bands are brownish and the re-mainder of the regenerating tail iswhite (Fig. 12). In fact, Spallanzaninotes that these two bands belong totwo different planes in the regenerate.The bands that Spallanzani refers to arethe regenerating spinal cord and noto-chord. After 3 months, the color haschanged to become darker. Spallanzani

Figs. 10–14. Tail regeneration in salamanders. Fig. 10: Beginning of regeneration with the thin crest of tissue denoted as rst. Fig. 11: Growth of theblastema (reproduction). Fig. 12: Appearance of the spinal cord and notochord (Spallanzani refers to them as bands). Fig. 13: Regeneration after thewhole tail was cut. Two small mounts of flesh (ab and bc) were elevated on each side of the spine (b). Fig. 14: Progression of regeneration from thecase shown in Figure 13. Growth only occurred at the b side to form an elevation.

REGENERATION ACCORDING TO SPALLANZANI 2361

notes here that when he dissected theregenerating tail, it was very soft andhe could not tell that the spine wasmade up of small bones. For him, this isan important observation because sincehe cannot see the small bones and thespine develops with time and is quitesimilar to the spine of the old tail, heconcludes that simple extension cannotexplain regeneration. Thus, this exper-iment would rather support develop-ment of germs. He then describes exper-iments where the whole tail was cut. Heexplains that for 26 days he saw no signof regeneration. Eventually, the thinsurface appeared. Then at each side ofthe spine two mounts of flesh appeared(ab and bc), which covered the spine(Fig. 13). There was some growth at thejunction of these two mounts (b in Fig.13) and not much more (Fig. 14). Spal-lanzani correctly concluded that if toomuch of the tail is removed, regenera-tion is greatly retarded or inhibited. Hethen reports that in young salamanders(one month old), regeneration followsthe same steps but is much faster. Healso suggests that some bony smallgrains seen along the small band makethe spine.

REGENERATION OFTADPOLES’ TAIL

Spallanzani begins by describing thegross macroscopic anatomy of the tad-pole’s tail. He divides the tail in two

parts, a major pointed one, which hecalls “tongue,” and two lateral mem-branes (transparent tissue) (Fig. 15).In Figure 15, the tongue (light browncolor) is demarcated by o, m, u, whilethe two membranes (yellowish-whitecolor) are denoted by v, o, m and u, m,n. Inside the tongue is a kind of mar-row (c, m; dark brown color) that runsacross. This must be the cartilaginousspine because he even points out that itis not bony but is made up of a sub-stance more consistent than the mus-cle. Because the tongue is still opaque,he cannot see circulation in it, but hecan observe many small vessels run-ning from the tongue to the lateraledges of the membranes and back. Hethen goes on to describe the circula-tory organization in the tail and hepoints out that there are two mainvessels. Here he disagrees with Leeu-wenhoek who claimed to have discov-ered four vessels. After tail amputa-tion, he describes two main events.One is the regeneration of the vesselsand the other accumulation of “fila-ments” at the edges of the tongue. Hepoints out that there are very remark-able differences between circulation inthe natural part and the regeneratedone (Fig. 16). In the former, the arteryand vein make one vessel and run par-allel to the length of the tail, but in thelatter when the artery enters the re-generating part, it divides into manybranches, which go on winding on

each side toward the extremity of thetail, and then they turn upward, stillbeing divided from each other. The di-vision remains until they reach backat the amputation plane where theyall unite into a vein. Older tadpolesalso regenerated the tail but Spallan-zani was unable to observe circula-tion. The same conclusions were madefor the continuity between the old andnew filaments. Also the skin that en-velops the muscles of the regeneratingtail was continuous with the old part,but had a different color (Fig. 17). Hebriefly mentions that regeneration oflimbs in old tadpoles was never seen(obviously, he amputated them after aparticular metamorphosis stage inwhich regeneration is not permitted).

The differences in regeneration(morphology) of the vessels allowSpallanzani to declare that tail regen-eration in tadpoles originates from alengthening (extension) of the oldparts and not from development ofgerms. He also thinks that the waythat the membranes regeneratedgives more ground to his conclusion.

Spallanzani then tries very tactfullyto elicit Bonnet’s opinion based on hisresults: “On which side is your incli-nation Dear Sir? In your very kindletter (the one Bonnet had sent toSpallanzani on August 8) you arenicely asking me if my observationsconfirm your principles. Now you can

Figs. 15–17. Tail regeneration in tadpoles. Fig. 15: Anatomy of the tail. Spallanzani proposes that the tail is made of two parts. An elevated “tongue”(light brown; omu) and two lateral membranes (vom and umn). Spallanzani denotes the spine as a kind of marrow (dark brown; cm) and ae “smallparallel ribs,” obviously referring to the musculature. Fig. 16: The circulatory system in intact and regenerated tail. In intact tail, it forms a big red thread.In the regenerated part, the red thread is not present. Fig. 17: The regenerated part, consisting of skin muscles and spinal cord (lighter colors), isdescribed as a continuation of the old (intact) part.

2362 TSONIS AND FOX

see with your own eyes and judge athousand times better than I can.”

Spallanzani then states that Bonnetassigns two sources of repair. Onethat proceeds through the develop-ment of small filaments and explainsregeneration of animal skin or plantpeel and a second that involves thedevelopment of germs to explain re-generation of a whole organ or bodyparts. In a sense, Spallanzani at-tempts to tell Bonnet that germs can-not account for regeneration in snailsand the tadpole’s tail. Spallanzaniends his letter confessing that to becertain he needs to do more experi-ments, especially to identify if hecould observe the union between thegerm (which he now calls an animalgraft) and the stump. Spallanzanispeculates that for circulation to beexplained by germs (the regeneratedpart uniting with the old part, whichresults in common circulation), then afossil of the graft should be found as inthe case of the plant shunt. It is ourimpression that Spallanzani tries toleave room to support Bonnet’s theory.

Bonnet was a scientist who hadlearned not to succumb to dogmatismand thus he knew how to accept criti-cism. On January 17, 1771 he wrote toSpallanzani: “I have always sincerelysought the truth and I have warned ahundred times that I never flatter my-self with the thought that I have al-ways found it. So don’t spare me myerrors, and criticize me whenever youjudge it necessary. When nature pro-nounces against me you must not in-dulge in the language of friendship;and I will be the first to submit to itsdecisions.” (quoted in Savioz, 1948,and translated by Anderson, 1982).

THE PRODROMO

Two years later, in 1768, Spallanzanipublished the Prodromo, his book onAnimal reproduction (Regeneration).Surprisingly, the book was quite shortwith not much detail or illustrationsas he had suggested he would do. Nev-ertheless, it is considered to be a sem-inal publication for regeneration re-search. In the book, Spallanzani alsodescribes regeneration of the limbs insalamanders. He noted that circula-

tion in the regenerating limb is differ-ent than the original and that some-times there would be cases with moreor less bones. Even though he does notspeculate, that observation also goesagainst germ theory.

CONCLUSIONS

It is obvious from studying this letterthat Spallanzani based his conclu-sions on macroscopic observationsmade with different criteria. For ex-ample, several conclusions are basedon the circulatory system, which couldbe hindered by the transparency of thetissue. To scientists in our time, it iseasy to see where Spallanzani failedor succeeded in supporting Bonnet’sgerm theory. Going against the prefor-mation theory was a real moral prob-lem for these scientists, because it haddominated their scientific beliefs andwas rooted in their social status. Thereader has only to consider that eventoday, when it comes to regenerativemedicine and stem cells, opinions varybased on social status and religion.Notwithstanding this great problem,Spallanzani and the other regenera-tion pioneers made outstanding con-tributions to science and many of theirobservations were correct. Theoriesalways are there to explain results.Even today, the mechanisms of regen-eration are still debated. Is there truetransdifferentiation or is regenerationmediated by undifferentiated stemcells? The newt has again been provenan indispensable tool. Studies con-ducted from the 19th century on haveshown that dedifferentiation of so-matic cells accounts for tissue repairduring limb regeneration (Morgan,1901). Thus, muscle, nervous tissue,and bone dedifferentiate to give rise tothe regenerated muscle, nerves, andbone (Tsonis, 1996). During lens re-generation, the iris pigment epithelialcells from the dorsal iris form a con-tinuous vesicle that then transdiffer-entiates to lens (Del Rio-Tsonis andTsonis, 2003). If Spallanzani had seenhistological preparations from theseevents, he would have concluded thatregeneration is most likely explainedthrough simple extension and hewould have discounted germs. Like-

wise, if he knew all these marvelousexamples of reserved stem cells oreven the powerful potential of embry-onic stem cells that can in principlebuild an organism, he might havewritten to Bonnet with joy that thegerm theory is valid!

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Savioz R. 1948. The philosophy of CharlesBonnet of Geneva. Paris: Libraire Philos-ophique J. Vrin (trans. from French).

Spallanzani L. 1768. Prodromo di un operada imprimersi sopra la riproduzioni ana-mali. Giovanni Montanari, Modena.Translated in English by Maty M. 1769.An essay on animal reproduction. Lon-don: T. Becket & DeHondt.

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