|
|
|
|
|
|
|
||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | ||||
First published online September 26, 2008
doi: 10.1242/10.1242/dev.021196
Jeem Classic |
Department of Molecular and Cell Biology and Center for Integrative Genomics, University of California, Berkeley, CA 94720-3200, USA.
e-mail: harland{at}berkeley.edu
SUMMARY
The grafting experiments of Spemann and Mangold have been a textbook classic for years, but as with many conclusions from experimental embryology, the idea that the dorsal lip of the blastopore `organized' the early patterning of the embryo has sometimes come under question. In their 1983 paper in JEEM, Smith and Slack extended these classical experiments in newts to the now-standard amphibian model Xenopus laevis. By using injected lineage tracers, they distinguished the fates of graft and host, and showed unambiguously that the organizer is responsible for neural induction and that it dorsalizes the mesoderm.
Introduction
How do vertebrate embryos generate a dorsal neural plate, notochord and somites on one side, and ventral blood and gut on the other? Two extreme possibilities are that: (1) the egg is endowed with determinants for each tissue that segregate into different regions during cleavage (a so-called mosaic mode of development); or (2) asymmetries build up progressively, and that devoted signaling centers organize the rest of the embryo by cell-cell interactions. According to our current understanding of amphibian embryogenesis, the egg starts with localized determinants, and after some cytoplasmic rearrangement, these determinants dictate the identities of the dorsal and ventral mesoderm, as well as the ectoderm and endoderm. But once these asymmetries are established, induction - a process by which a cell or tissue directs the development of a neighboring tissue or cell - takes over. In Xenopus, for example, it is known that the dorsal mesoderm is an organizing center, and that from the late blastula stage onwards, the tissues of the tadpole are elaborated by cell-to-cell signaling.
The organizer graft is a famous and influential experiment because it
showed that one part of the embryo is endowed with special signaling
properties that dictate the patterning of the neurulating embryo. In the early
part of the 20th century, and prior to the organizer experiment, work by Hans
Spemann and his colleagues had suggested that much of the amphibian embryo was
regulative, such that if a piece of tissue was grafted from a donor embryo to
a different location in a new host embryo, then the graft would develop
according to its new surroundings. However, experiments by Warren Harmon
Lewis, and later by Spemann, showed that the dorsal or upper lip of the
blastopore was an exception to this general rule: in grafting experiments, it
would not adopt a new fate (reviewed by
Sander and Faessler, 2001
).
This led Spemann to address the extent to which this `determined' fate was
exclusive to the graft or determined by some response of the host. To tackle
this definitively, Spemann and his student Hilde Pröscholdt (later Hilde
Mangold) used newts with differently pigmented eggs to track the contributions
of host and graft in their organizer experiments (see
Fig. 1)
(Spemann and Mangold, 1924;
Spemann and Mangold, 2001).
The use of the differences in pigmentation provided the crucial marking which
revealed that the secondary neural plate was induced from the host tissue and
did not self-differentiate from the graft, as both Spemann and Lewis had
erroneously concluded from their earlier trials that lacked a tracer. The
clarity as well as the limitations of using natural pigmentation to mark donor
and host tissue can be seen in photographs of some of the original organizer
grafts (Sander and Faessler,
2001). In addition to the limitations of the pigment as a clear
lineage tracer, the experiments by Spemann and Mangold were few in number, and
not all of them gave the clean results reproduced in the textbooks. In light
of the limitations of these classical experiments, there was a need,
therefore, to examine the activities of the organizer in greater numbers of
embryos and with more attention paid to precisely where the graft was taken
from and to the results obtained. This was indeed what Jim Smith and Jonathan
Slack achieved in their 1983 paper in the Journal of Experimental
Embryology and Morphology (JEEM)
(Smith and Slack, 1983).
New lineage tracers
Most classical lineage tracing studies had used vital dyes, such as Nile
Blue, that could be applied to the outside of tissues and would stain
subcellular structures, such as yolk platelets. Although these tracers
provided a great deal of information about lineage that is still relied upon
today, the possibility that the dye might leach from a graft diminished the
value of such lineage tracers in transplantation experiments. In the 1970s,
new injectable lineage tracers were introduced that were deployed in repeats
of classical experiments, such as those by Spemann and Mangold. However, not
everyone reproduced these experiments' findings; indeed, Marcus Jacobson
concluded that amphibian development was considerably more mosaic, and
criticized Spemann and Mangold's earlier conclusions
(Jacobson, 1982). Thus, in the
early 1980s, the stage was set for more authoritative repeats of the organizer
experiments to be performed using the new and improved lineage tracers.
To be useful, a lineage tracer must be cell autonomous, so that the fate of
adjacent cells is not conflated with that of the marked cell. The tracer must
also be non-toxic and developmentally neutral. Horseradish peroxidase (HRP)
had been used through the 1970s as a retrograde tracer that was taken up by
neurons, then transported back to cell bodies; subsequent staining for the
enzyme revealed the fine structure of these neurons. In 1976, Kenneth Muller
and Jack McMahan (Muller and McMahan,
1976) used the direct injection of HRP into the large neurons of
leech ganglia to describe the fine structure of these cells; from there, David
Weisblat and Gunther Stent (Weisblat et
al., 1978) extended the use of HRP injection to trace the progeny
of the early blastomeres of the leech embryo. Other tracers quickly followed,
including the fixable fluorescent dextran developed by Bob Gimlich and Jochen
Braun (Gimlich and Braun,
1985); this fluorescent tracer was used by Gimlich and Jonathan
Cooke (Gimlich and Cooke, 1983)
for a series of experiments that, like Smith and Slack's work, reinforced the
idea that the organizer acted through induction.
Tracing normal Xenopus development
In their 1983 study, Smith and Slack decided to repeat the organizer graft experiments of Spemann and Mangold in Xenopus laevis, rather than in newts, using HRP as the lineage tracer. It had previously been established that HRP rapidly fills the cell it is injected into, so all of the progeny of the cell are labeled; at the same time, the tracer remains confined to that cell. Smith and Slack also established that cells do not take up HRP from the surrounding medium (where it might be released by dying cells). Thus, by all criteria, this tracer was ideal for the organizer grafting experiments they wanted to perform.
|
). Moreover, the clarity of the histochemical stain
illustrated beautifully that the dorsal marginal zone populates a narrow strip
of dorsal mesoderm - the prechordal plate and notochord - over the entire
craniocaudal extent of the embryo, in addition to the anterior endoderm.
Importantly for the experiments that followed, the dorsal marginal zone was
not seen to contribute to the nervous system.
In contrast to the fate of the dorsal marginal zone, the small piece of
orthotopically grafted ventral marginal zone spread considerably and populated
the posterior lateral plate and endoderm. The latter point has been revisited
lately, with some authors arguing that the prospective posterior fate of the
`ventral' marginal zone should prompt a different term to be used for this
region of the embryo, and, together with the findings of other experiments,
for the axes of the blastula to be renamed (reviewed by
Lane and Sheets, 2006).
However, there is little question that the dorsal marginal zone is both
dorsally specified and dorsally fated, so there also remains a good rationale
to adhere to the nomenclature used by Smith and Slack (reviewed by
Harland, 2004). In any case,
the main motivation of Smith and Slack's fate-mapping experiments was to rule
out the possibility that a grafted dorsal marginal zone might contain any
neural tissue, and, although they may not have provided a comprehensive fate
map of the whole gastrula, this important point was resolved.
Signaling from the organizer
Fate mapping aside, the most important experiments in the Smith and Slack
JEEM paper addressed the signaling activities of the organizer, and
the response of the ventral marginal zone to an organizer graft. Indeed, the
results of the dorsal marginal zone graft showed that neural induction had
occurred, such that the neural tube of the secondary axis was composed of host
cells, and not of self-differentiating cells of the graft. Therefore, the
neural tissue of the host's secondary axis must have been derived from an
inductive interaction. The results presented were extremely clear, and,
together with those of Gimlich and Cooke, published in the same year
(Gimlich and Cooke, 1983),
reinforced the importance of the dorsal marginal zone as an organizing center
that can recruit ectoderm into a secondary neural tube. The idea that the
nervous system was already fully specified in the blastula
(Jacobson, 1982) was
effectively laid to rest.
After disposing of the controversy related to neural induction, the paper
then focused on dorsalization of the mesoderm: the process that respecifies
prospective ventral tissue, such as blood and mesenchyme, to more dorsal
fates, such as muscle. This phenomenon had previously been recognized, but
because so much attention had been devoted to neural induction, it had
received less attention. Furthermore, experiments on mesoderm induction by
Nieuwkoop had suggested that the pattern of the mesoderm was already induced
by graded signals from the vegetal endoderm
(Boterenbrood and Nieuwkoop,
1973). The ability of organizers, or indeed of chemicals
(Yamada, 1950), to dorsalize
mesoderm had been described, but one of the strengths of Smith and Slack's
paper is that it clearly states the distinction between the organizer's role
in dorsalizing the mesoderm and the process of mesoderm induction. Thus, the
paper laid out a clear sequential signaling process: mesoderm induction in the
blastula is followed by dorsalization of the mesoderm by the organizer during
gastrulation. These experiments laid the groundwork for the further dissection
of dorsalization and its molecular basis.
To address whether the ventral marginal zone has signaling activity that is analogous to that of the organizer, Smith and Slack implanted pieces of ventral marginal zone into a slit in the organizer region. The result of this manipulation was a split in the notochord, where the original notochord territory maintained its fate, while the ventral graft stayed in the middle without influencing the identity of surrounding tissue. In contrast to any ventralizing effect of the graft, the graft was itself dorsalized to develop into muscle. So the experimental embryology in the Smith and Slack paper tells us that, instead of dorsal and ventral marginal zones carrying equal weight, the signals from the organizer are dominant signals, and any signals from the ventral mesoderm are neither potent nor long range.
Mesodermal pattern: graded action of mesoderm inducers or dorsalization by the organizer?
Shortly after Smith and Slack's
1983 JEEM study, Smith made the seminal finding that a
soluble mesoderm inducer was made by a cell line, observations that were
published as the very first paper in JEEM's successor:
Development (Smith,
1987). These and subsequent experiments showed that graded doses
of the mesoderm inducer could induce progressively more dorsal structures from
sensitive ectoderm. Therefore, the mesoderm inducer might, in principle, act
as a classical morphogen, dictating different fates, such as notochord,
muscle, kidney and blood, at different threshold concentrations. With the
arrival of a molecular approach to studying mesoderm induction, and the
possibility that mesoderm inducers act as morphogens, the phenomenon of
mesoderm dorsalization, as supported only by experimental embryology, shifted
into the background. However, despite the elegance of the idea that a mesoderm
inducer might act as a morphogen to specify the pattern of the mesoderm, other
experiments in experimental embryology argued that this mechanism was
insufficient to account for mesoderm patterning. One of the clearest
approaches was to assess the state of mesoderm immediately next to the
organizer in the late blastula stage. The first approach used explants
(Dale and Slack, 1987), and, in
another study, two hemispheres cut at different angles from the dorsal midline
were grafted together (Stewart and
Gerhart, 1990). Both of these approaches showed that during the
phase of mesoderm induction, the marginal zone adjacent to the organizer has
not yet received signals to differentiate into muscle. Thus, the proposal that
a graded mesoderm-inducing signal might induce muscle during the blastula
stage was inadequate. These embryological `loss-of-function' experiments
showed that organizer signaling is necessary in normal development for
patterning the mesoderm, and complemented the earlier `gain-of-function'
experiments, which showed that an organizer graft is sufficient to induce
dorsal mesoderm in a secondary axis (Dale
and Slack, 1987; Gimlich and
Cooke, 1983; Smith and Slack,
1983; Stewart and Gerhart,
1990). In modern terms, we understand that the late blastula and
gastrula-stage dorsalizing signals are molecular pathways that are distinct
from those involved in mesoderm induction, and are mediated by dorsalizing
molecules (Noggin, Chordin, Follistatin, Xnr3 and Cerberus) that antagonize
the ventralizing bone morphogenetic proteins (BMPs). In this respect, a
continuing relevance of Smith and Slack's
1983 paper is in the experimental embryology, which tells us that
the source of BMP antagonists is dominant and presumably must produce a molar
excess above the concentration of BMPs that are secreted from a ventral
marginal zone graft.
Conclusion
Needless to say, in the 25 years that have elapsed since the paper was
published, we have reached a much more sophisticated understanding of the
various molecular players that are active in patterning the Xenopus
embryo. Grafting experiments are inherently somewhat variable in their results
and limited in their implications; for example, it was only with the advent of
molecular assays that it became clear that head induction was not a
quantitative or temporal effect of the organizer, but rather due to a
combination of different molecular signals
(Glinka et al., 1997). In
retrospect, it would have been useful to know more about how different types
of organizer graft behaved with respect to the anterior extent of the
secondary axis produced, but in the early 1980s, researchers' frustrations
with the limitations of experimental embryology was driving many to the
genetic and molecular approaches that still dominate developmental biology.
However, it is also still important to know what the embryo tells us through
well-designed `cut and paste' experiments, so we still refer to the initial
grafting experiments in Xenopus that extended the paradigm of the
organizer that was established by Spemann and Mangold.
ACKNOWLEDGMENTS
Thanks to John Gerhart and David Weisblat for discussing the historical background, and Andrea Wills and Jane Alfred for comments on the manuscript. R.M.H. is supported by the NIH.
REFERENCES
Boterenbrood, E. C. and Nieuwkoop, P. D.
(1973). The formation of the mesoderm in Urodelan amphibians: V.
Its regional induction by the endoderm. Wilhelm Roux'
Archiv. 173,319
-332.[CrossRef]
Dale, L. and Slack, J. M. (1987). Regional
specification within the mesoderm of early embryos of Xenopus laevis.
Development 100,279
-295.
Gimlich, R. L. and Braun, J. (1985). Improved
fluorescent compounds for tracing cell lineage. Dev
Biol. 109,509
-514.[CrossRef][Medline]
Gimlich, R. L. and Cooke, J. (1983). Cell
lineage and the induction of second nervous systems in amphibian development.
Nature 306,471
-473.[CrossRef][Medline]
Glinka, A., Wu, W., Onichtchouk, D., Blumenstock, C. and Niehrs,
C. (1997). Head induction by simultaneous repression of Bmp
and Wnt signalling in Xenopus. Nature
389,517
-519.[CrossRef][Medline]
Harland, R. M. (2004). Dorsoventral patterning
of the mesoderm. In Gastrulation: From Cells to Embryo
(ed. C. D. Stern), pp. 373-388. Cold Spring Harbor,
NY: Cold Spring Harbor Laboratory Press.
Jacobson, M. (1982). Origins of the nervous
system in amphibians. In Neuronal Development (ed. N.
C. Spitzer), pp. 45-99. New York: Plenum.
Keller, R. E. (1976). Vital dye mapping of the
gastrula and neurula of Xenopus laevis. II. Prospective areas and
morphogenetic movements of the deep layer. Dev. Biol.
51,118
-137.[CrossRef][Medline]
Lane, M. C. and Sheets, M. D. (2006). Heading
in a new direction: implications of the revised fate map for understanding
Xenopus laevis development. Dev. Biol.
296, 12-28.[CrossRef][Medline]
Muller, K. J. and McMahan, U. J. (1976). The
shapes of sensory and motor neurones and the distribution of their synapses in
ganglia of the leech: a study using intracellular injection of horseradish
peroxidase. Proc. R. Soc. Lond. B Biol. Sci.
194,481
-499.[Medline]
Sander, K. and Faessler, P. E. (2001).
Introducing the Spemann-Mangold organizer: experiments and insights that
generated a key concept in developmental biology. Int. J. Dev.
Biol. 45,1
-11.[Medline]
Smith, J. C. (1987). A mesoderm-inducing factor
is produced by Xenopus cell line. Development
99, 3-14.
Smith, J. C. and Slack, J. M. (1983).
Dorsalization and neural induction: properties of the organizer in Xenopus
laevis. J. Embryol. Exp. Morphol.
78,299
-317.[Medline]
Spemann, H. and Mangold, H. (1924). Über
Induktion von Embryonalanlagen durch, Implantation artfremder Organisatoren.
Arch. mikrosk. Anat. EntwMech.
100,599
-638.
Spemann, H. and Mangold, H. (2001). Induction
of embryonic primordia by implantation of organizers from a different species.
1923. Int. J. Dev. Biol.
45, 13-38.[Medline]
Stewart, R. M. and Gerhart, J. C. (1990). The
anterior extent of dorsal development of the Xenopus embryonic axis depends on
the quantity of organizer in the late blastula.
Development 109,363
-372.[Abstract]
Weisblat, D. A., Sawyer, R. T. and Stent, G. S.
(1978). Cell lineage analysis by intracellular injection of a
tracer enzyme. Science
202,1295
-1298.
Yamada, T. (1950). Dorsalization of the ventral
marginal zone of the triturus gastrula. I. Ammonia-treatment of the
medioventral marginal zone. Biol. Bull.
98, 98-121.
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati 
Twitter What's this?
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
