Echoes of Spallanzani

To the inspecting eyes of a budding scienziato the Reggian countryside was not simply teeming with life, it also posed myriad of intriguing questions. How do bug wings work and can you stick them back once you have removed them? How far are the stars that can be seen in the sky, and are natural fountains really just the tears of sad, beautiful girls lost in the woods? Armed with a healthy dose of skepticism, the young Lazzaro Spallanzani, one of the brightest minds of his age, was more than happy to tackle these questions. In the end, his curiosity and stubborn adherence to natural philosophy derailed the legal career much wanted by his father, and “l’astrologo”, as his friends knew him, ended up as a teacher of logic, metaphysics and Greek at the University of Reggio Emilia.

Today Spallanzani is most credited for his work debunking the mistaken theory of John Tuberville Needham and Georges Buffon about the origins of microscopic lifeforms. The theory of “spontaneous generation”, as it is remembered, argued that really small lifeforms can originate from inanimate matter: maggots from dead flesh, microscopic “animalcules” (protists and bacteria, effectively) from water and mud. This was the celebrated, dominant theory of the age, yet it took Spallanzani only a few long-necked, easily sealable glass bottles and some freshly boiled broth to lay it to rest for good.

Equally ingenious were, however, his experiments on regeneration, which he conducted in order to test what at the time seemed like the equivalent of spontaneous generation for embryologists, epigenesis. Following his childhood instincts, Spallanzani sectioned everything that crawled or ran in the nearby woods. He cut worms and snails far and wide, removed tails and limbs of tadpoles and salamanders with methodological rigour and watched them to regenerate. In fact, almost single-handedly he put the field of regeneration on a solid scientific footing.

What he saw in these experiments thrilled him. His results, detailed in his seminal work, Prodromo and his letter to Charles Bonnet, show that Spallanzani realized that regeneration was more than simple growth. It could not be simply explained by the expansion of some preexisting “germs” after injury. Yet, if not exactly heresy, such views contradicted the dominant theory of the age, namely preformation.

The adherents of this theory claimed that an organism is preformed from its origins, and development is mere growth. Spallanzani was originally sympathetic to the preformationist ideas and some of his results were in line with these: limb regeneration for the most part was about growth, after all. Yet there were significant observations, especially about the early steps of the regenerative process, that were hard to square with the preformationist ideas. For example, the intricate circulatory system of the regenerating limb stump formed in a stepwise manner, and often was clearly different from the original circulation. And this strongly suggested that growth could not be the whole story, there were also epigenetic forces at work, and preformationists (including Bonnet) were mistaken.

In the absence of better tools and molecular reagents Spallanzani could not delve into the details of regeneration. It took several centuries and generations of researchers till the major points of the regenerative process were uncovered.

Although the presence of a special proliferative tissue at the stump, the blastema, described by Spallanzani as a “cone of gelatinous substance”, was confirmed by later scientists, the origin of these dividing cells was hotly disputed till recently. Some attributed a blood cell origin to these progenitor cells, others thought that they were derived from the wound epithelium. More in line with today’s major schools of thought were those who posited the existence of a dormant, stem-cell-like population of cells in the regenerating tissues, and those who argued for the dedifferentiation of committed, non-dividing cells in the stump, close to the amputation plane.

After years of ambiguity, it turned out that in some species (e.g., planarians) stem cells are responsible for the remarkable regenerative capacity, whereas in vertebrates the “dedifferentiation” hypothesis is correct. Recently a string of research papers also provided strong evidence that the dedifferentiating cells have a “memory” of their earlier life and their progeny will differentiate accordingly.

For example in 2009 Elly Tanaka’s group at the Center of Regenerative Therapies in Dresden, showed that in the regenerating axolotl limb blastemal cells derived from the skeleton contribute almost exclusively to the regenerating skeleton, old epidermis is the source of blastemal cells that give rise to new epidermis, muscle cells form new muscle. Similar properties were shortly described by other research groups for the regenerating zebrafish heart and caudal fin, thus firmly establishing the importance of dedifferentiation during regeneration.

Despite all the excitement, our current knowledge about how dedifferentation occurs is rather limited. We do understand the basics of the underlying biological processes, but we are still in the dark when it comes to the details and the molecular underpinnings of these events.

A fully formed organ is made up of specialized cells that stop replicating their genetic material, express a very specific set of genes (and keep all others silent) and build elaborate cytoplasmic structures to perform specific functions. A heart muscle cell, a cardiomyocyte, is filled with contractile filaments and expresses the genes that are required to build them. A so called osteoblast makes the bone matrix and has an elaborate intercellular architecture that helps its function. But when the signalling events after the injury instruct these differentiated cells to start dividing again, they have to reset their entire genome, shut down the genes that make them what they are and activate those that will make them divide. During dedifferentiation all those specialized structures in the cell suddenly become liabilities that need to be eliminated, so the cell division apparatus can be assembled. How this effective restructuring occurs, both in the nucleus and in the cytoplasm, is still unclear.

Similarly, we have only a vague idea about how dedifferentiating cells keep track of their former identity. The consensus is that during the “reset” of the genome, some modifications of the DNA and associated histone proteins, called epigenetic marks, are kept in particular stretches of the genetic material, forming some sort of memory. A similar epigenetic memory-mark is invoked for the cell’s awareness of their position within the appendage: one of the most intriguing findings of Spallanzani was that during appendage regeneration, regardless where the amputation is made, only the missing part will regrow. Thus the cells “know” where they are, and how much of the organ is still missing. We just still don’t know how they do that.

If we could find the answers to all these questions, perhaps the Holy Grail of regenerative medicine would be within reach, and we could more effectively obtain regeneration in humans, too. Because amazingly, despite staggering progress in molecular biology and developmental genetics, even after almost a quarter of a millenium, Spallanzani’s final words from the end of Prodromo still resonate and reflect the ultimate goal of the field:

“But if the above-mentioned animals, either aquatic or amphibious, recover their legs, even when kept on dry ground, how comes it to pass, that other land animals, at least such as are commonly accounted perfect, and are better known to us, are not endued with the same power? Is it to be hoped they may acquire them by some useful dispositions? and should the flattering expectation of obtaining this advantage for ourselves be considered entirely as chimerical?”

References and further reading:

Tsonis PA, Fox TP (2000) Regeneration according to Spallanzani. Dev Dyn 238(9):2357-63.
Odelberg SJ (2004) Unraveling the molecular basis for regenerative cellular plasticity. PLoS Biol 2(8):E232.
Tanaka EM, Reddien PW (2011) The cellular basis for animal regeneration. Dev Cell 21(1):172-85.
Spallanzani L (1769) An Essay on Animal Reproductions. London: T. Becket. 86 p. In Maty M, translator. Translation of: Prodromo di un opera da imprimersi sopra la riproduzioni animali (Italian).
de Kruif P (1926) Microbe hunters. Harcourt, Brace Co., New York, NY.
 
 

Image source: Wikimedia Commons

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