Spemann and Mangold’s Discovery of the

Organizer

  Claudio Stern *This
essay is followed by a glossary, including a figure, which explains the
relevant terms and developmental events.

Hans Spemann and Hilde Mangold’s demonstration (published in 1924) of the

"organizer", a region of the early (gastrula stage) embryo that directs the development
of other parts of the embryo, is generally regarded as one of the most major landmarks
in the history of developmental biology. The simple experiment reported in this paper,
with only five cases described, was the first to prove the reality of the concept of
"induction" (an interaction between two groups of cells, by which one group influences
the developmental fate of the other). The idea of induction had been proposed by
several of Spemann’s predecessors, but had eluded definitive demonstration.

For almost seven decades following the publication of this seminal paper, Spemann and
his students, as well as a gradually increasing number of other laboratories, struggled to
understand the physical basis of the action of the organizer: what are the signals
emitted, and what governs the responses of the tissue that receives them? Are there
many different signals, each responsible for one of the distinct activities of the organizer
that were defined in the original paper, or is a single molecule capable of performing all
these tasks? Almost 80 years later, the answers to these profound questions are only
just starting to emerge.

Background 

Without a doubt, the most heated and long-lasting debate in the history of
developmental biology must be that between the doctrines of Preformation and
Epigenesis, which lasted from the 17th century until the 20th (and could be argued to
continue today, although in a somewhat milder form). At their most extreme,
Preformationists believed that the adult is already present but in miniature in one of the
gametes (egg or sperm), and that subsequent development of the embryo merely
increases the size of the "homunculus". Those that favored Epigenesis, on the other
hand, contended that the embryo generates new complexity and builds its organs as it
develops. Until near the end of the 19th century, the prevalent method for studying
embryonic development was simple observation and histology. By the 1880s, the
German embryologist Wilhelm Roux, trying to find an answer to the Preformation-
Epigenesis debate, had introduced the concept that experimental manipulation of the
embryo could be used to uncover the rules that govern normal development. He killed
one of the blastomeres of a two-cell-stage amphibian embryo with a hot needle, and
reported that the cell that had remained alive gave rise to half an embryo (Roux, 1888).
Roux interpreted this result as favoring Preformation: even at the two-cell stage, each
cell already "knew" what it should become, and simply continued along its normal
developmental pathway. A few years later, Hans Driesch performed similar experiments
on sea urchin embryos, but instead of killing one of the cells, he shook the embryos so
as to separate the blastomeres from each other. To his surprise, the isolated
blastomeres gave rise not to half-embryos, but to completely formed (albeit smaller)
embryos (Driesch, 1892), a result clearly more in line with the Epigenetic view. For lack
of a better explanation, Driesch postulated that embryos are made up of more than just
cells, bringing back the Aristotelian concept of "Entelechy" (an intangible soul-like
property).

Was Driesch’s result different from Roux’s because of the different species used, or
because of the way in which the blastomeres were separated? Over the following
decade, Thomas Hunt Morgan, Oscar Hertwig, H. Endres and A. Herlitzka among
others attempted to separate blastomeres in amphibians by various methods, but either
did not succeed or did not obtain enough cases of duplication. Enter Hans Spemann, an
energetic, imaginative and technically highly skilled man in his early 30s who was then a
Privatdozent (Assistant Professor) in Würzburg. In 1901-1903 he used hairs from his
baby daughter Margrette (chosen for their fineness) to tie a knot between the first two
blastomeres of salamander embryos. If the knot was tightened completely and left in
place this manipulation produced a deep constriction between the two cells and
effectively separated them for the rest of development. In some cases, he obtained two
complete embryos, which were sometimes fused to each other when the constriction
had not been complete (Spemann, 1901; Spemann, 1902; Spemann, 1903). This result
delivered a serious blow to the Preformationists. But Spemann also noticed that the
outcome of the experiment depended on the plane along which the constriction was
placed. The first plane of cleavage of salamander embryos is variable, in some cases
separating the future dorsal and ventral sides, while in other cases being at right angles
to this or in other orientations. Spemann observed that when the first cleavage (and
therefore the constriction he made with the hair) separated the dorsal side from the
ventral side, only the dorsal side gave rise to a complete embryo, the ventral half
producing only a Bauchstück ("belly-piece"). It was this observation that eventually led
to Spemann’s discovery of the Organizer, and to the experiment for which he is best
known.

The finding that an isolated dorsal side of the embryo developed more or less
autonomously, while a ventral side did not, suggested to Spemann that the two regions
are somehow different. He then (1914-1919) extended his constriction experiments to
embryos at later stages, and found that he could obtain similar results even up to the
late gastrula stage, in embryos that already had a blastopore. At this stage, isolated
dorsal portions of the embryo which contained the lip of the blastopore formed much
more complete axes than other parts. Dorsal portions which included the blastopore lip
generally gave rise to complex, spatially organized structures such as a notochord,
somites, neural tissue and sometimes parts of the head, while ventral portions often
formed only skin, undifferentiated mesenchyme, kidney tubules and blood, none of
which are parts of the embryonic axis. These observations led Spemann to describe the
lip of the blastopore as a "differentiation center" (Spemann, 1918), and later as an
"organization center" (Spemann, 1919). Intimately linked to the concept of a special
"differentiation/organization center" in the embryo is the realization that this center may
act in part by influencing the differentiation or organization of other cells not situated in
this region. This interaction is what embryologists refer to as "induction" (see note). 

The obvious next step was to use transplantation strategies to test directly whether the
dorsal lip of the blastopore could change the behavior of other cells (or, more
specifically, their developmental fate). This is what Spemann now embarked on doing,
although he was not the first to perform transplantation experiments with this region of
the embryo. Several years earlier (1907), Warren H. Lewis had transplanted the dorsal
lip of the blastopore from a gastrula stage embryo to the ventral side of a similarly
staged host, and reported the formation of a double axis. However, Lewis had
interpreted this as indicating self-differentiation of the graft (in other words, he believed
that the transplanted cells and their descendents had formed the entirety of the second
axis themselves). To demonstrate induction, and to deliver a further blow to the
Preformationists, it was essential to prove that the cells of the dorsal lip of the
blastopore emitted an influence capable of instructing other cells to change their
direction of differentiation so as to give rise to new structures.

The Experiment 

Spemann repeated Lewis’s grafts in 1916, but the next year he realized that the
experiment would not be complete without an unambiguous way to distinguish graft
from host cells. Some years earlier (1903), Ross Harrison had used interspecific grafts
between embryos of two different species of frog, Rana palustris and R. sylvatica, which
are differently pigmented. Spemann now took advantage of this approach, but used
three differently pigmented species of newt, the white Triton (Triturus) cristatus and the
dark T. taeniatus or T. alpestris. In 1917, Spemann started transplantation experiments
with these species, moving pieces from a variety of sites on donor embryos to different
locations on embryos of lighter or darker coloration. His paper reporting the results
appeared in 1921, but contained only a preliminary account of the critical "organizer
experiment" (grafts of the dorsal lip of the blastopore). Included as a postscript, the
account is based on only a single case (Spemann, 1921), achieved by Spemann’s new
graduate student, Hilde Proescholdt (later Hilde Mangold), as he was writing the paper.
After that initial success, Proescholdt, to whom Spemann had entrusted the execution of
this critical experiment, struggled to repeat the experiment successfully, with very few of
the manipulated embryos surviving. Of several hundred chimeric embryos, only five
survived, and these formed the basis for the famous paper that was published in 1924
(Spemann and Mangold, 1924). 

The experiment itself is very simple, as shown in Figure 1. The dorsal lip of the
blastopore from one species was transplanted to one of various ectopic sites in a host
embryo from another species. In a few (of which only one, Um83, survived), the lip was
taken from T. cristatus and grafted into the very darkly pigmented T. alpestris host.
Figure 2 and Figure 3 show that the grafted cells (or at least, those close to the surface)
could be distinguished even in the whole, living embryo. Most of the embryos died at
early neural plate stages, and only one (Um 132) survived to a much later stage ( Figure
3). Histological sections through the 5 surviving embryos showed that the donor and
host cells could also be distinguished in the interior of the embryo ( Figure 4).
It was the histological analysis that clearly revealed the important features of the result.
In all cases, the graft had contributed mostly to axial mesodermal organs (the notochord
and part of the somites) and only very slightly to the neural tube (and exclusively to a
wedge on its ventral side). The bulk of the neural tissue was derived from the host
( Figure 4). This experiment proved, without any doubt, that the dorsal lip of the
blastopore exerts an "organizing" influence on adjacent cells of the host, most likely by
inducing them from a non-neural prospective fate (epidermis) to a neural fate. [I say
"most likely" because Spemann and Mangold did not formally prove that the host neural
cells present in the ectopic axis were not recruited from the host’s own neural plate;
however, many subsequent experiments confirmed that the organizer does indeed act
as a source of neural inducing signals].

A simple experiment, but so much to learn from it. Starting in the original (1924) paper
and extending over many subsequent publications, Spemann recognized many of the
implications:

 The organizer acts nonautonomously (i.e. it acts upon other cells), influencing the fates of cells in both the
ectoderm and the mesoderm of the host (see below);
 In the ectoderm, the graft organizes a neural plate/neural tube from cells of the host ("neural induction");
 In the mesoderm, a region fated to give rise only to ventral structures (mesenchyme, blood) in the absence
of a graft is converted to a somite fate (note the presence of host cells as well as graft cells in one of the
ectopic somites in Figure 4) ("dorsalization");
 Self-differentiation of the organizer. Confirming previous observations by himself and by Lewis, the grafted
organizer has a strong-tendency to give rise to structures similar to those that derive from it in normal
development, namely notochord. In this sense, the organizer differs from other regions of the embryo at this
stage, which do not appear to be committed;
 Polarization of the ectopic axis. The ectopic axis is coherent, and organized both along the head-tail and the
dorso-ventral axis;
 Regulation. The microcautery experiments of Roux (see above) had been interpreted to mean that even
from the earliest stages of development, embryos are "Mosaic": cells cannot deviate from their normal fates.
By contrast, the experiments of Driesch in sea urchin (see above) and Spemann’s own with constricted eggs
(see above) indicated a "Regulative" mode of development: at least some cells develop according to their
position relative to other cells, rather than entirely according to their lineage history. Spemann and
Mangold’s experiment provide a further clear demonstration of Regulative development, since cells were
able to adopt different fates when their positions relative to other cells were changed;
 A hierarchical chain of inductions. Spemann and Mangold discuss the possibility (which had already arisen
in some of Spemann’s earlier papers) that development can be viewed as a series of inductive events that
follow each other in time, and that there may be other "organizers" at other stages of development and at
various positions in the embryo;
 Signals travel through the embryo. "The concept of the organization center is based on the idea that
determination proceeds from cell to cell in the embryo. Such an assumption suggests itself whenever
differentiation, that is, the visible consequence of determination, does not start in all parts simultaneously,
but, beginning at one place, progresses thence in a definite direction" (Spemann and Mangold, 1924);
 Epigenesis versus preformation. Although the 1924 paper does not make a big point of this, the results
obtained clearly argue against preformationist ideas.

Hilde Mangold’s experimental embryos did not survive very well. This turned out to be
mostly due to bacterial infections and the fact that opened embryos do not develop well
in pond water, which differs radically in ionic composition from the intraembryonic fluid.
The introduction by Johannes Holtfreter, a few years later (Holtfreter, 1931), of a simple
saline mix and his innovation of sterilizing the surgical instruments solved both
problems. This made it possible to grow the grafted embryos for long enough to
recognize head and tail portions ( Figure 5), which confirmed Spemann’s and Mangold’s
conclusions concerning head-tail patterning.

The Legacy 

Spemann and Mangold’s paper initiated a flurry of investigations, which were to last
more than three decades, aimed at identifying the "active principle" of the organizer.
Initially, it was not clear that the active principle was chemical in nature, and Spemann
himself leaned towards a vitalistic explanation. Two events changed this perception -
first, the discovery by several investigators (including Johannes Holtfreter, Otto
Mangold, Joseph and Dorothy Needham, C. H. Waddington and many others) that dead
organizers (killed by boiling, fixing or freezing) could also induce a secondary axis, and
second, efforts largely by Joseph Needham, Jean Brachet, C. H. Waddington and
others to start the discipline of "Chemical Embryology", and to try to purify molecules
from tissues that could replace the organizer. By the 1950s, many laboratories
throughout the world (in addition to those above, Saxén and Toivonen in Finland, and
Tiedemann in Germany; for reviews see Spemann, 1938; Sax 閚 and Toivonen, 1962;
Nakamura and Toivonen, 1978; Hamburger, 1988; Sander, 2001), were working hard to
identify the endogenous inducing and patterning substances. This effort gradually died
away, mostly when it was realized that many different chemical insults (including high or
low pH, histological dyes, extracts of calf testicle RNA, steroids, etc.) could all neuralize
and/or generate a partial axis in Urodele embryos.

Further substantial progress on this topic had to await the more universal introduction of
Xenopus as the amphibian experimental species of choice, because Xenopus embryos
cannot be neuralized as easily as can Urodeles. The three decades after the mid-1960s
were relatively fallow, and it was not until the mid-1990s that significant advances were
made at the molecular level, in part because the introduction of molecular biology
offered new approaches for finding and testing candidate molecules for their signalling
properties within the embryo. These came from several laboratories (particularly Eddy
De Robertis, Richard Harland, Ali Hemmati-Brivanlou and Doug Melton) who
independently discovered that the property of "dorsalization" first defined by Spemann
and Mangold can be attributed to antagonists of BMP signalling (such as Chordin,
Noggin, Follistatin and members of the DAN/Cerberus family) emitted by the organizer.
The molecular basis for other functions of the organizer as defined by Spemann and his
followers still remains to be elucidated, but very good progress is currently being made
in many laboratories.

Note

The concept of induction was not invented by Spemann; rather, it came from several of
his predecessors. Wilhelm Pfeffer was the first to use the word, in 1871, and it was later
defined more clearly, notably by Curt Herbst. However, even as early as 1828 von Baer
suggested that the optic vesicle could elicit lens formation in the adjacent ectoderm (see
Oppenheimer, 1991). And even Spemann himself proposed as early as 1903: "it is
conceivable that the neural plate is induced by the archenteron" (quoted from
Hamburger, 1988, p. 31).

The original authors 

Hans Spemann (1869-1941) (These biographical notes

were compiled based on Spemann, 1943; Mangold, 1953;
Hamburger, 1988). Hans Spemann was born on 27 June
1869 in Stuttgart (Swabia, Germany), as the first child of a
well-known bookseller/publisher (Johann Wilhelm
Spemann). He started as an apprentice to his father in
1888/89, which was interrupted by Military service, before
returning to study bookmanship in Hamburg. But his
passion lay in the Life Sciences, and in 1891 he registered
to study pre-clinical Medicine in Heidelberg, where he was
taught by Otto Bütschli and Karl Gegenbaur. In 1892 he
married Klara Binder, and then moved to Munich for his fifth
semester, the start of the clinical portion of the course. But
he did not enjoy clinical Medicine, and decided to move to the Zoology Department of
the University of Würzburg, of which Theodor Boveri was director. Under Boveri,
Spemann started with a study of cell lineage in the parasitic worm Strongylus paradoxus
(which became his Ph.D. thesis) and he then embarked on a description of the
development of the middle ear in the frog, which he presented for his Habilitation (a
qualification required to teach in the University). After completion of this training phase,
he become a Privatdozent (non-tenured faculty) in Würzburg. After contracting
Lungenspitzenkatarh (probably tuberculosis), he retired for a Winter (1896-97) to the
Swiss and Italian Alps, taking along only one book: August Weissmann’s famous book
"The germ plasm: a theory of heredity" (1892), where he presents his theory of the
continuity of germinal cytoplasm. It was this book and the leisurely time he had to think
about the concepts of reproduction and development that heralded Spemann’s entry
into experimental Embryology. He wrote in his autobiography (Spemann, 1943): "I found
here a theory of heredity and development elaborated with uncommon perspicacity to
its ultimate consequences; but also in the same book some experimental results of the
recent past which actually already disproved the theory [of unequal nuclear division].
This stimulated experimental work of my own" (quoted from Hamburger, 1988 page 9).
The experimental work on which he embarked was the attempt to separate the
blastomeres of the early amphibian embryo discussed above, the results of which were
published in 1901-1903. At the same time, Spemann was also conducting his seminal
investigations on induction of the lens in amphibians. In 1908, after 14 years at
Würzburg, he obtained his first independent position as director of the Zoological
Institute in Rostock, where he remained until 1914 when he was appointed co-director
(with Carl Correns) of the Division of Entwicklungsmechanik ("experimental
embryology") at the Kaiser Wilhelm Institute in Dahlem (the outskirts of Berlin). He
continued to work on experimental embryology throughout the war years, and in 1919
moved again, to Freiburg, to assume the directorship of the Zoological Institute. It was
probably this move that delayed the publication of the 1921 paper, since almost all of
the experiments were performed in Dahlem.
In 1935, Spemann received the Nobel Prize for Medicine for his studies on the
organizer, and he remained in his position in Freiburg until he was relieved of his post in
1937 and was succeeded in the directorship by one of his first students, Otto Mangold
(who was a supporter of the Nazi regime). Throughout his time in Freiburg, Spemann
continued to work on many different aspects of induction and patterning by the
organizer. He died of heart failure on 12 September 1941. Figure reproduced from
Hamburger (1988).

Hilde Mangold (née Proescholdt) (1898-1924). Hilde

Proescholdt was born into a relatively well-off merchant
family from Gotha (Thüringen, Germany) in 1898, but there
is relatively little information available about her life (these
brief notes are taken from an Appendix in Hamburger,
1988). She was an avid reader of Philosophy, and the
Classics, an admirer of both Eastern and Western Art and a
lover of Nature, who started her University studies in
Frankfurt, where she had attended a lecture by Spemann
that prompted her to try to join his laboratory, which she did
in 1920, just one year after Spemann’s move to Freiburg.
Hilde’s initial project was to repeat the classical (XVIII
century) regeneration experiments of Abraham Tremblay,
but she was not very successful. After a year or so,
Spemann changed her project and assigned her the task of
exploiting the inter-species (Triton taeniatus/cristatus) graft of the dorsal lip in the first
breeding season in May 1921. Almost immediately she obtained her first positive result
with one embryo, which Spemann quickly included as a postscript to his 1921 paper
(see above), where the concept of the "organizer" was first defined (Spemann, 1921). It
took a further two breeding seasons for Hilde to attempt the experiment several hundred
times and to obtain the five surviving cases that were to form the basis for the famous
paper that was submitted in the Summer of 1923, appearing in Volume 100 of Roux’s
Archiv in 1924 (Spemann and Mangold, 1924). By this time, Hilde had married the most
senior student in Spemann’s lab, Otto Mangold, and had borne a child. Sadly, she died
when a gasoline heater in her kitchen exploded in September 1924. Mangold is shown
with her child in 1924, the year that marked both the publication of the famous paper
and her untimely death. Figure reproduced from Hamburger (1988).

The author 

Claudio Stern works on the cellular and molecular

processes that control formation of the embryonic axis,
neural induction and early embryonic patterning in higher
vertebrate embryos. He is the J. Z. Young Professor and
Head of the Department of Anatomy and Developmental
Biology at University College London.