Perspectives

Nature Reviews Molecular Cell Biology | AOP, published online 1 February 2006; doi:10.1038/nrm1855 PERSPECTIVES Spemann’s organizer) graft with salamander DEVELOPMENTAL CELL BIOLOGY — TIMELINE eggs5 (BOX 1). This experiment established the present view that animal development Spemann’s organizer and self- results from a succession of cell–cell induc- tions in which groups of cells, or organizing centres, signal the differentiation of their regulation in amphibian embryos neighbours. The dorsal-lip graft induced neighbouring cells to adopt the normal pat- Edward M. De Robertis tern of tissue types, so that a Siamese twin was formed. In 1988, a memoir recounting Abstract | In 1924, Spemann and Mangold demonstrated the induction of Siamese the heyday of experimental embryology in twins in transplantation experiments with salamander eggs. Recent work in the Spemann laboratory was published by amphibian embryos has followed their lead and uncovered that cells in signalling Viktor Hamburger6. At least in my case, the centres that are located at the dorsal and ventral poles of the gastrula embryo modern revival of studies into Spemann’s communicate with each other through a network of secreted growth-factor organizer can be traced to this little book, antagonists, a protease that degrades them, a protease inhibitor and bone- which retold the excitement of discover- ing the inductive powers of the organizer. morphogenic-protein signals. Hamburger was 88 years old at the time — clearly, it is never too late to impact the When an embryo is cut in half, it can complete limb, not just half a limb3. The scientific thinking of others. self-regulate to regenerate the missing part part of the embryo where this phenomenon In our own laboratory, studies on (FIG. 1). The field of experimental embryology takes place was called the ‘self-differentiating Spemann’s organizer were approached by originated in 1883 when Roux killed one of morphogenetic field’. cloning its molecular components: cDNA two cells in a frog embryo with a hot needle Self-regulation, as defined by these early libraries were generated from manually and found that the rest gave rise to only experimental embryologists, is one of the dissected dorsal blastopore lips from the part of the embryo, usually a right or a left most interesting and mysterious properties African clawed frog (Xenopus laevis), which half 1 (TIMELINE). However, in 1891, Driesch of embryos. What are the molecular mecha- were first screened for homeobox sequences. separated the two first blastomeres of the sea nisms that explain the intrinsic tendency of Homeobox genes encode DNA-binding urchin embryo and found that each was able the embryo to regulate towards the whole? proteins that are involved in the control to self-regulate and give rise to complete, Here, I recount the story of the birth, decline of development in fruitflies, and we had although smaller, embryos1. In 1895, Thomas and revival of amphibian experimental discovered years earlier that vertebrates, Hunt Morgan — who before becoming a embryology, and how recent studies have namely the African clawed frog, contained geneticist was an experimental embryologist uncovered a molecular pathway of interact- homeobox genes of the Hox type7. In 1991, — repeated Roux’s experiment and showed ing extracellular proteins that explains how our team succeeded in isolating a homeobox that if one of the two blastomeres is gently embryonic self-regulation works. This short gene, goosecoid (Gsc), that was specifically pipetted out of a frog embryo (instead of kill- review focuses on the advances made in expressed in Spemann’s organizer8. The first ing it and leaving it in place), amphibians too amphibians, although great strides have also in situ hybridizations of Gsc mRNA, consti- could self-regulate and give rise to a complete been made in other model systems, such as tuted a truly memorable event, because Gsc embryo from half an egg2. the fruitfly (Drosophila melanogaster), chick mRNA demarcated, very specifically, tissue In 1903, Hans Spemann used a baby-hair (Gallus gallus) and zebrafish (Danio rerio), as belonging to Spemann’s organizer. Since its loop (from his own daughter) to subdivide detailed elsewhere4. Rather than providing discovery almost three-quarters of a century the cleaving amphibian (salamander) egg a comprehensive overview of the entire field earlier, the existence of Spemann’s organizer into two halves. If the half-embryo con- of early development, this short timeline had been deduced from its inductive effects tained part of the future blastopore dorsal presents a personal account of the status after transplantation, but the expression pat- lip (the region where involution of the of experimental embryology, a field that a tern of Gsc now allowed us to visualize, for mesoderm starts), it formed a perfectly well- century ago was at the very front of biologi- the first time, that the Spemann’s organizer proportioned tadpole1 (FIG. 1). In 1918, the cal research. existed as a distinct molecular entity8. great American embryologist Ross Harrison A few months later, the groups of Igor carried out another remarkable experiment: Gene-fishing in Spemann’s organizer Dawid and Milan Jamrich reported that if the forelimb field in the mesoderm of The starting point for understanding self- genes encoding two other transcription a salamander embryo was cut in half and regulation was provided by the most famous factors, Xlim1 and Xfkh1/Hnf3β, were transplanted into the flank of a host embryo, experiment in embryology, the Spemann– also expressed in the organizer region of each half could induce the formation of a Mangold organizer (hereafter referred to as the African clawed frog9,10. Importantly, NATURE REVIEWS | MOLECULAR CELL BIOLOGY ADVANCE ONLINE PUBLICATION | 1 © 2006 Nature Publishing Group PERSPECTIVES In 1992, Richard Harland reported the isolation of the first secreted protein that was expressed in Spemann’s organizer14. He and his colleagues used a different method — the functional screening for molecules that changed embryonic development after overexpression. In this expression-cloning procedure, pools of cDNAs were grown, transcribed in vitro with bacteriophage SP6 RNA polymerase and microinjected into African clawed frog embryos. Those with biological activity were then sub-selected until a single clone was identified. Many interesting genes have been isolated using this powerful method15. Microinjected noggin mRNA was found to induce neural tissue in explants of ectodermal cells16. It was subsequently found that follistatin, yet another secreted molecule that is specific to Spemann’s organizer, was also a neural inducer17. Figure 1 | Embryonic self-regulation. The entire early embryo constitutes a self-differentiating morphogenetic field, in which cells communicate with each other over great distances. This is Spemann’s organizer secretes many demonstrated by experiments such as the one shown here, in which an African clawed frog (Xenopus antagonists. Many secreted proteins that are laevis) embryo was cut into two halves at the blastula stage. If it is ensured that both halves contain specific to Spemann’s organizer have been part of the dorsal organizer region, two perfect identical twins are obtained (an intact sibling is shown isolated from the African clawed frog gas- at the top of the figure). In humans, identical twins are found in 3 out of 1,000 live births, and arise most frequently by the spontaneous separation of the inner cell mass of the mammalian blastula into two, trula through extensive molecular screens. followed by self-regulation60. The ultimate example of self-regulation is provided by another mammal, The expectation was that new growth factors the nine-banded armadillo, in which every blastocyst gives rise to four genetically identical siblings50. would be identified, but, instead, Spemann’s Note that each twin is longer than just half the length of the intact sibling, which represents yet another organizer was discovered to signal mainly effort to regulate towards the normal pattern. Both half-embryos shown here are derived from the by secreting a cocktail of bone morphogenic same blastula. protein (Bmp; these proteins are members of the transforming growth factor (Tgf)β microinjection of Gsc mRNA into ventral expression of secreted signalling proteins superfamily) and Wnt antagonists18,19. The cells caused the formation of twinned axes, that execute the cell-differentiation changes new components and molecular mecha- which indicated that the Gsc gene was part in neighbouring cells. In time this was, in nisms that scientists had hoped to find were of the molecular machinery that leads to the fact, found to be the case11,12. The Gsc gene discovered in abundance, but the fact that activity of Spemann’s organizer8. Because was subsequently isolated in many other these new components were antagonists of Gsc encodes a DNA-binding protein, we species, and became a widely used marker growth factors was a great surprise. Of all the proposed that it might, in turn, activate the for comparative studies of gastrulation13. organizer molecules shown in BOX 2, Timeline | Birth, decline and rebirth of experimental embryology Thomas H. Morgan When Wilhelm removes one of two Reciprocal Roux killed one of blastomeres in a frog Ross Harrison The end of an era: transcriptional two cells in a frog and found that describes what Dead Spemann’s saline solutions The noggin, Tolloid regulation of dorsal egg, the rest gave amphibians could form became known as organizers found to were found to Xlim1 and Xfkh1 metalloproteinase and ventral signals rise to only part of a complete embryo morphogenetic induce neural tissue in induce neural tissue genes are is found to cleave explains embryonic the embryo. from half an egg2. fields3. salamander ectoderm23. in explants25,26. cloned9,10,14. chordin43. self-regulation42,45. 1883 1891 1895 1903 1918 1924 1932 1935 1941 1991 1992 1994 1997 2004 2005 Hans Driesch Hans Spemann Hilde Mangold and Hans Spemann The organizer of the (1994–1996).

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