Two-headed beasts are not just the stuff of mythology. Although extremely rare, reptiles with two heads are well documented. In 2003, a two-headed turtle was found in South Africa; two-headed human babies have been reported; and now, Eric Buffetaut and his colleagues at the National Centre for Scientific Research in Paris, France report the discovery of the fossilized remains of a two-headed reptile (left). The fossil is of a newly-hatched choristodere, extinct, long-necked aquatic reptiles, and has been dated to about 120 million years ago. The malformation is thought to have occurred as a result of a process called axial bifurcation.
In the early years of the 20th century, Hans Spemann, an embryologist at Freiburg University in Germany, performed microsurgical manipulations which resulted in the duplication of the anterior structures of salamander embryos. Spemann used 2-cell-stage embryos; that is, fertilized eggs which had just undergone their first cell division. He used tweezers to tie strands of his baby daughter’s hair around the embryos, thus constricting them without separating the two cells. In many cases, the constriction resulted in a single embryo with a strange “belly piece” attached to it. In some cases, constriction resulted in the development of twin embryos. Both embryos, although smaller than normal, were otherwise normal. In a few cases, the result was an embryo with two heads, the behaviour of which he ponders in this passage:
Such animals came to the feeding stage and it was now most remarkable to see how once the one head and at another time the other caught a small crustacean, how the food moved through the separate foreguts to the joint posterior intestine…It was probably irrelevant for the well-being of this strange double creature which head had caught the food; it was for the benefit of the whole. Nevertheless, one head pushed the other away with its forelegs. Hence, two egotisms in the place of one, called forth by the spatial separation of the anlagen. The interest was heightened by the occasional occurrence of such double monsters in man. Here, too, a similar interference would have the same disquieting consequences. Thus, at last – I was 28 years old – I had found the beginning of my own scientific journey…It was the fascination of the mystery surrounding “partially split individuality,” then the enjoyment of the elegant experimental technique, but then simply the continued commitment which forced me to seclude myself in my room, one spring after another, and, instead of roaming the lovely world, to bend over the binocular microscope and tie hairloops around the slippery eggs of salamanders, until I had constricted about a thousand and a half.
This work would lead Spemann to conclude that the dorsal and ventral (or top and bottom) halves of the early embryo were different, as the top half appeared to contain a discrete group of cells which somehow organize development. This group of cells is actually visible on the dorsal surface of the 2-cell-stage embryo, as a grey crescent-shaped structure. The outcome of Spemann’s constriction experiments depended upon exactly where this structure was in relation to the constriction. If one half of the constriction contained the entire structure, it developed into a viable embryo while the other half developed into the belly-piece; if the structure was evenly split during the constriction, the result was twinned embryos; and if the structure was unevenly split, the result was, in some cases, an embryo with two heads.
Spemann’s work provided evidence for a fundamental principle of developmental biology. From Mendel’s work, he was aware of the units of inheritance that we now call genes. The constriction experiments sometimes produced complete, twinned embryos. This showed that all cells contained the information needed to build an organism, putting an end to the idea, propounded by Wilhelm Roux and August Weismannn, that cells lose half of their genetic material during division, so that each daughter cell contains a different set of genes.
These early experiments naturally led Spemann to investigate the organizing centre further. He tried constricting the fertilized egg just before the first cell division had taken place, thus forcing the nucleus into one half of the cell. The half containing the nucleus started to develop, while the other half remained as an inactive pocket of cytoplasm; this was evidence that the genetic material was likely found in the nucleus. Spemann watched the cell as it divided, and, after a few divisions had taken place, he loosened the constriction around the embryo. He observed a nucleus from the developing half of the embryo slip through the constriction into the undeveloped half, and then tightened the noose again. To his surprise, that half now began to develop, and formed a normal embryo. With this experiment, Spemann had inadvertently developed a cloning technique – nuclear transfer, the process by which Dolly the sheep was cloned.
In 1924, with graduate student Hilde Mangold, Spemann performed a series of classic embryological experiments in which the organizer was characterized. The experiments were performed at a slightly later stage of embryonic development, during a process called gastrulation, in which a complex series of cell migrations leads to the formation of the three layers of cells (the endoderm on the inside, ectoderm on the outside and mesoderm in the middle) found in all multicellular organisms. Also during gastrulation, a rudimentary body plan, including the major axes of the body, is laid down.
The interior cavity of a gastrulating embryo communicates with the exterior via a small opening called the blastopore, which is visible in the early embryo as a small dimple on the dorsal surface. In their experiments, Spemann and Mangold grafted pieces of blastopore tissue from one newt embryo onto the ventral (lower) surface of a second newt embryo. They used embryos of different species; because the cells from each were pigmented differently, it was possible to trace the fate of the grafted cells.
When cells from a specific region of the blastopore were used for the grafts, they formed a notochord, a transient embryonic structure which induces development of the nervous system. This grafted tissue induced adjacent cells in the host embryo to differentiate into nerve cells, and they subsequently formed a second nervous system in the host. As a result, the embryo developed into a tadpole with two heads. Spemann and Mangold used the term induction to describe the effects of one group of cells on another.
A figure from Spemann’s 1903 paper (left) and a two-headed tadpole created using Spemann and Mangold’s method (right, from Robertis, 2006).
In this film clip, Edward M. De Robertis, of the Howard Hughes Medical Institute, uses an embryo of the African clawed toad (Xenopus laevis) to re-enact the Spemann-Mangold experiments:
The experiments of Spemann and Mangold are the best known of all embryology experiments. They showed that the blastopore is crucial in laying down the body axes during development, and led to the currently held view that development proceeds through an intricate cascade of cell-to-cell interactions. The blastopore is now known to consist of two discrete regions; one of these, the dorsal lip of the blastopore, corresponds to what is now called Spemann’s organizer, which has been shown to secrete many signalling molecules.
In 1935, Spemann was awarded the Nobel Prize for Physiology or Medicine. Tragically, Hilde Mangold died in a house fire, aged just 29, before the findings were published, and therefore did not share the prize with Spemann.