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Spemann/Mangold Organizer

Introduction

Basic experimental technique practiced by Spemann and Mangold
Hilde Mangold, top, and Hans Spemann, bottom

In 1935, Hans Spemann of the University of Freiburg in Germany received the Nobel Prize in Medicine "for his discovery of the organizer effect in embryonic development." "The organizer" was a small region of tissue in the early amphibian embryo that Spemann and his students showed to be the driving force behind the formation of neural structures as the embryo developed. They conducted a plethora of experiments during the early twentieth century to study this organizer, but the most famous and influential occurred between 1921 and 1922. These transplantation experiments, carried out by PhD student Hilde Mangold, not only provided developmental biology with answers to some of the field's oldest questions, but also opened up many new lines of study into this fascinating phenomenon called embryonic induction.

Background

The famous organizer experiments did not arise in a vacuum. Several previous experiments led the way. The first experiment that inspired this line of research took place in 1903. Using a hair from his infant child, Spemann constricted a Triturus taeniatus newt embryo in the plane of the first cleavage, in order to isolate a single nucleus and ultimately a single cell away from the rest of the developing embryo. Both the primary mass of cells and the isolated cell developed into normal embryos. This was evidence of the concept of nuclear equivalence, the concept that all nuclei contain the instructions to build the entire organism. However, when he performed the same constriction experiment lassoing the embryo longitudinally, only the future dorsal side of the original embryo formed a complete larva. The ventral side developed into a disorganized mass of ventral (belly) tissues. If all nuclei contain the same information, why did the ventral portion not organize properly into a normal embryo? Certain regions of the early embryo must be more important than others for correct morphogenesis (the creation of shape).

Formation of twin newts via Spemann's technique of lassoing and splitting the early embryo

In experiments using the Triton embryo, Spemann and Mangold experimentally found that different sections of the embryo may be exchanged at the beginning of gastrulation, and that these procedures did not prevent normal embryonic development. Even in embryos of different ages and different species, sections of the embryo, such as the presumptive epidermis and the presumptive brain, may be switched without disturbing development. From these experiments, Spemann and Mangold concluded that while these implanted samples partially retain the characteristics of their organism of origin, the fact that they readily adopt the cellular fate of their implantation location indicates two key facts: these samples are relatively re-programmable in terms of cellular fate, and the host embryos must be capable of determining the fate of indifferent cells.

However, samples from the upper blastopore lip displayed markedly different behavior from that of samples retrieved from other embryonic regions; these sections of tissue not only failed to adopt the cellular fate of their implanted location, but also went on to form a second embryonic primordium, including a neural tube, notochord, and somites. From this phenomenon, Spemann and Mangold drew the conclusion that the upper blastopore lip tissue must already have a cellular fate in order to resist determination by its implantation location.

Beyond these general observations, Spemann and Mangold began to develop the concept of an organization center, or an embryonic region that is prematurely fated, thus influencing the developmental fates of surrounding regions. From these concepts and premises, Spemann and Mangold then began further developing the ideas of an organizing region through experiments that followed.

Induction

The Spemann-Mangold organizer experiments are almost always discussed in conjunction with the phenomenon of embryonic induction. Induction is the process by which one cell or group of cells in the embryo determines the developmental direction of another group of cells. It is one of the most fundamental concepts in developmental biology today, but at the time of the organizer experiments it was a little-known idea. Spemann and Mangold were not the first to notice that transplanted tissue sometimes took on the identity of the surrounding host tissue. They were the first, however, to thoroughly examine the reverse--transplanted tissue influencing the host embryo.

The induction of neural tissue by the dorsal blastopore lip organizer, the major finding of their research, has been called the primary embryonic induction. This event is vital for formation of neural structures and the anterior-posterior body axis. Scores of other inductions have since been discovered in many other species. The discovery of induction was a vital step in the forward progress of developmental biology as a whole because it helped clear up one of the great mysteries of the discipline: how can a blob of non-specific cells organize itself into a complex multicellular organism? We now know that position-dependent induction is part of the answer.

Cell Determination

The path by which a cell goes from totipotent (able to become any cell type) to a mature, determined state is complex. Early theories proposed that cells lose any unneeded "determinants" (DNA was not yet understood to be the molecule of inheritance). This would mean each cell in a multicellular organism contained only the portion of the genome which its cell type required. However, experiments in separating blastomeres, including Spemann's constriction experiment, soon showed that this was not the case. Early embryonic cells could sometimes generate an entire organism even when separated from their counterparts. In other species and later in development, however, cells taken from one part of an embryo will continue to develop into the structures or tissues that they would normally have become, regardless of whether or not they are transplanted somewhere else. And, of course, most cells of an adult organism do not change cell types once their fate has been sealed. Between the first few cell divisions and the final product, the number of potential fates a cell may have is progressively reduced. This process is called differentiation, or cell determination.

Spemann and Mangold's experiments illustrated that a) it is possible for transplanted cells to retain their original identity (i.e. positional effects and cytoplasmic determinants alone do not determine cell fate) and b) some cells have the intrinsic ability to tell other cells what to do. The capacity of the organizer to influence and combine with the host cells to form not only organs, but partial secondary embryos, also made it clear that cells do communicate, in both directions.

Methods

Techniques


In order to test their hypotheses, Spemann and Mangold analyzed developing newt embryos of the genus Triton, now known as Triturus. Of the Triton species available, Triton taeniatus achieves the most developmental success even with an absent egg membrane, so these embryos were typically used as the “receiving” embryo within experiments, often being implanted with the organizer from Triton cristatus within its presumptive epidermis. Triton taeniatus, the host species, has darkly pigmented eggs and, therefore, pigmented embryos. Triton cristatus eggs and embryos are unpigmented. The choice of unpigmented transplant tissue and pigmented host embryos made it easy to track the organizer tissue when sections were taken from the experimental embryos. That way, the researchers could distinguish structures formed by the organizer itself (as a result of its predetermined cell fate) from anomalous host structures formed as a result of induction by the organizer.

Microsurgery was a fairly crude undergoing, and the tools were not readily available for purchase. Spemann and his students had to make their own instruments for removing and transplanting tissue grafts. Spemann developed a technique of melting and stretching a glass rod over a burner to draw out a thin needle. This needle with a minute tip was small enough to remove an embryo from its gelatinous outer membrane without destroying the embryo inside. Spemann also invented an early form of micropipette, a simple hollow glass rod with a bit of rubber over the end opposite the working tip. Pressing down the rubber created suction. The micropipettes were what the researchers used to remove a tiny bit of tissue from one embryo, with an unprecedented degree of precision, and then graft that tissue onto another embryo.

The overall experimental procedure used by Spemann and Mangold is called heteroplastic transplantation. Heteroplastic transplantation is simply the act of transplanting tissue between two different species of the same genus. Its counterpart, homoplastic transplantation (between individuals of the same species) had already been in use in embryology for some time. The advantage of heteroplastic transplantation was in the ability to distinguish donor and host cells due to their natural histological differences.

Advanced development of newt embryos formed using the techniques of Spemann and Mangold

Experiment Triton 1921, Um 8b


In Experiment Triton 1921, Um 8b, a piece of Cristatus embryo from just above the U-shaped blastopore, the “organizer,” was exchanged with a sample of presumptive epidermis from Taeniatus. For the most part, Spemann and Mangold focused on the development of the explant “organizer” in Taeniatus, and several hours after implantation, neural folds began appearing within the Taeniatus embryo, even though a primary neural plate already existed. Throughout embryonic development, the progression of the secondary neural plate, originating from the implanted Cristatus tissue, was slightly delayed in comparison to the development of the primary neural plate. While observations of the developing embryo were useful to Spemann and Mangold, much of their key data was the result of embryo fixation and cross-section creation. Once their embryo was fixed and analyzed in cross-sections, Spemann and Mangold were able to come to a key conclusion: not only did the Cristatus explant resist the developmental influence of Triton taeniatus tissue in order to avoid forming epidermis, but also influenced surrounding foreign tissue to aid in the formation of a second neural plate, thus altering their previous potential fates. While these conclusions certainly helped illuminate the role of the organizer, the manner or sequence of events that enables the implanted organizer to influence surrounding tissue was still obscured by lack of knowledge following this experiment.

Experiment Triton 1922, Um 25b


In Experiment Triton 1922, Um 25b, a section of the upper blastoporal lip from a Criastatus embryo was transplanted into a Triton taeniatus embryo of similar age and development, but in a region distant from the primary blastoporal lip. Although the implant seemed to disappear from the embryonic surface, its influence as an organizer led to a second neural plate forming at the implantation site. Unlike the first experiment carried out by Spemann and Mangold, this case involves the formation of a uniform mass, uninterrupted by host mesoderm; although they reached no conclusions regarding this point, Spemann and Mangold noted that this phenomenon may be related to the fact that the embryo sunk beneath the cell surface after implantation.

Experiment Triton 1922, Um 131b


In Experiment Triton 1922, Um 131b, a section of Criastatus from directly above the blastopore was exchanged with a section of Taeniatus of unknown origin; this implantation was performed using advanced gastrulae. With regards to the Taeniatus implant, this region did not participate in invagination, but instead led to the creation of an unusual fission within the embryo. However, the development that took place in the Taeniatus embryo containing the Criastatus implant was even more intriguing. In this case, the Criastatus implant again led to the formation of a secondary neural tube but in this case experienced even more advanced development. By the time the embryo was preserved in order to prevent any disintegration, the two neural tubes, host and implant, had developed sufficiently in order to share a common lumen with some tubal separation near the posterior region. Nevertheless, the Criastatus implant was able to induce the formation of a secondary tail bud, as well as somites composed of primarily Criastatus cells, but with interaction of Taeniatus cells. Unlike past experiments, Um 131b is unique in that somites were formed and that the Criastatus organizer was able to interfere with the Taeniatus organizer, even when placed a significant distance apart.

Experiment Triton 1922, Um 83


In a deviation from previous experiments, Spemann and Mangold chose to use a Triton alpestris embryo rather than a Taeniatus embryo, as the difference in pigmentation between Criastatus and Alpestris is more pronounced. Additionally, in this case no implantation was made upon the Criastatus embryo, as it disintegrated following the removal of the explant. A medial section of the blastopore which contained the organizer was removed from a Criastatus early gastrula, then placed in an indeterminate region of the animal pole of an alpestris embryo of the blastula stage. Following implantation, the Criastatus tissue moved beneath the surface of the embryo briefly, but then became visible again once neural fold formation began, eventually creating a secondary neural tube of Criastatus origin. While primary neural tube development occurred in a normal fashion, interference of secondary neural tube development led the two neural tubes to meet and fuse at an acute angle. Just as in Um 131b, somite formation occurred within the secondary neural tube, and in this case Criastatus cells make up a majority of the notochord, but with distinct Alpestris cells interspersed alongside the implanted embryonic cells. Just as in previous experiments, the embryo was preserved for further analysis and revealed several interesting results: within the Alpestris embryo, the secondary neural tube and somites were characterized by unusually dark pigmentation, although this may be related to their region of origin in the animal half, and that the implanted tissue lies at an acute angle to the organs that it is responsible for inducing.

Experiment Triton 1922, Um 132


Similar to past experiments, Um 132 began with the implantation of an organizer from Criastatus, this time in advanced gastrulation, into a similarly aged Taeniatus embryo. Unfortunately, the Criastatus embryo that received a Taeniatus implant was lost before it could be sectioned. However, in the Taeniatus embryo that received a portion of Criastatus, the organizer formed a shallow indentation before disappearing beneath the surface of the embryo. After allowing the altered Taeniatus embryo to develop, two neural folds have formed, eventually giving rise to two linked embryos, both at different developmental stages. Just before the embryo was preserved, the primary embryo, originating from the Taeniatus organizer, contained otic pits, a significant number of somites, and a distinct tail bud. Unlike past embryos, this one was notable for its almost completely independent normal and induced embryonic primordia. Apart from stronger development in the inner embryo than in the outer embryo, and the fact that certain structures are connected, such as the pronephric ducts, the two embryonic anlagen have developed as expected.

Limitations


Spemann and Mangold acknowledged that their methods, while insightful, could not answer all the questions they would have liked to investigate. Their experiments relied on extremely precise microsurgery, a technique that can be difficult today, but was extremely challenging at the time. The embryos of many species could not tolerate the procedure, putting a damper on further research of this sort with more organisms. It was often not feasible to transplant tissue beneath the surface of an embryo because the ectoderm layer is only one-cell thick in most places.

Another complication, mentioned by Spemann and Mangold in their paper, was the inability to determine the orientation of the transplanted tissue. Their micropipettes punched out circular samples of tissue, making it very difficult or impossible to keep track of the polarity of the transplant when it was transferred to the host. As such, most often the axes of the donor organism in the transplant would mismatch with the axes already established in the host embryo. To what extent this might impact the induction and development processes, they could not be certain.

Relevance

One of the possible key conclusions that Spemann and Mangold drew from these experimental trials was that their mysterious organizer, later to be named the Spemann-Mangold organizer, was able to direct surrounding cells to differentiate and form the essential components of the nervous system. However, the actual mechanism of this organizer has proven much more genetically complex than initially thought; recent discoveries have indicated that the organizer inhibits signaling mechanisms to the ectoderm above, and it wasn’t until 1992 that noggin, a protein molecule essential for neural and head development, as well as cellular induction at the Spemann-Mangold Organizer, was discovered by Richard M. Harland and William C. Smith of the University of California at Berkeley.

Later work by embryologists, also inspired by the foundation laid by Spemann and Mangold, led to the discovery of bone morphogenetic protein-4 (BMP-4). This protein is released by the ventral blastula, creating a diffusion gradient responsible for skill cell induction that is blocked by chordin and noggin released by the organizer, thus allowing tissues closest to the organizer to become CNS cells.

Since the groundbreaking work by Spemann and Mangold occurred in the early 1920s, several other analogous structures have been observed and researched in other organisms, such as the embryonic shield in fish development and Hensen’s node in birds. Aside from these connections with other species, the work completed by Spemann and Mangold effectively allowed the study of embryogenesis to flourish, and introduced the idea that signaling molecules may somehow be responsible for the effect of the organizer region. At the time of Spemann and Mangold's experiments, signaling molecules were relatively unknown. In many cases, the technology to detect them simply did not exist. However, in their landmark paper on the organizer, Spemann and Mangold proposed that some sort of secreted factor(s) must allow the organizer to induce the tissue around it. From then on, the search for the mysterious organizer molecules was underway.

References

Frost, D. (n.d.). Triturus Rafinesque, 1815. Retrieved February 24, 2016, from http://research.amnh.org/vz/herpetology/amphibia/index.php//Amphibia/Caudata/Salamandridae/Pleurodelinae/Triturus

Gilbert, S. F. (2010). Amphibians and Fish: Early Development and Axis Formation. In Developmental Biology, Ninth Edition (pp. 241–286). Sunderland, MA: Sinauer Associates, Inc.

Harland, R. (2008). Induction Into the Hall of Fame: Tracing the Lineage of Spemann's Organizer. Retrieved February 27, 2016, from http://dev.biologists.org/content/135/20/3321.figures-only Fig. 1

King, R. C., Stansfield, W. D., & Mulligan, P. K. (2007). Heteroplastic Transplantation. In A Dictionary of Genetics (7 ed.) (p. 288). Oxford: Oxford University Press. doi:10.1093/acref/9780195307610.001.0001

The Nobel Prize in Physiology or Medicine 1935. (2014). Retrieved from http://www.nobelprize.org/nobel_prizes/medicine/laureates/1935/

Philbrick, S., & O’Neil, E. (2012, January 12). Spemann-Mangold Organizer. Retrieved from http://embryo.asu.edu/pages/spemann-mangold-organizer

Spemann, H., & Mangold, H. (1924). Induction of Embryonic Primordia by Implantation of Organizers from a Different Species. Archiv Für Mikroskopische Anatomie Und Entwicklungsmechanik, 100, 599-638. Retrieved February 24, 2016, from https://courses.ncsu.edu/gn434/lec/001/block_video%20project/original%20papers%20for%20video%20wiki%20references/uber%20induction%20spemann.pdf.