Within the organism cells do not lead individual lives. They differentiate, proliferate, and die as part of groups of hundreds or thousands of cells called morphogenetic fields, which have the remarkable property of self-regulating pattern after perturbations (see Figure 1). Experimental embryology started in 1891 when Hans Driesch separated the first two cells of an embryo and obtained identical twins. The early embryo is considered the primary morphogenetic field. At later stages, secondary fields determine the formation of organs and body regions such as limbs, heart, and central nervous system (CNS), as first described by Ross Harrison in 1918. The aim of our research is to discover the molecular machinery through which self-regulation works.
Spemann's Dorsal Organizer
The embryo of the frog Xenopus provides an excellent system for unraveling how cells communicate with each other. Large numbers of embryos can be obtained and subjected to microsurgical manipulations before any histotypic differentiations occur. A rich heritage of experimental embryology exists, and classical transplantation techniques can now be combined with knockdowns of individual or multiple genes.
A foundation for understanding self-regulation was provided by an experiment carried out by Spemann and Mangold more than 80 years ago involving grafting of the dorsal lip region of the amphibian embryo (see Movie 1). They found that a small group of cells, called the organizer, is able to induce Siamese twins, including a complete CNS, in neighboring cells. Hans Spemann received the 1935 Nobel Prize in Physiology or Medicine for this discovery of embryonic induction. Isolating the molecules involved in these cell-cell inductions has been the Holy Grail of embryology. We have isolated multiple genes that encode secreted proteins expressed specifically in Spemann's organizer. Studies on Chordin, Cerberus, Frzb-1, and Crescent have contributed to the current realization that growth factor antagonists secreted into the extracellular space mediate the formation of embryonic signaling gradients.
The Ventral Center
On the opposite side of the dorsal organizer, in what we call the ventral center, BMP4/7 (bone morphogenetic proteins, a type of growth factor) are expressed at midgastrula. On the dorsal side, other BMPs—called BMP2 and ADMP—are secreted, but only when BMP levels are low. Ventral center gene expression is driven by high BMP signaling, which phosphorylates and activates the transcription factor Smad1. The ventral center secretes a cocktail of proteins that participate in the extracellular biochemical pathway that mediates self-regulation (Figure 2). Ventral center cells secrete several proteins in addition to BMP4/7. (1) Tolloid is a zinc metalloproteinase that we found cleaves Chordin-BMP complexes flowing from the dorsal side, liberating active BMPs produced in more dorsal regions. (2) Sizzled (a secreted Frizzled-related protein similar to Crescent) functions as a competitive inhibitor of the Tolloid enzyme. (3) Twisted-gastrulation is a protein that binds to BMP (facilitating its solubility and signaling) and to Chordin (making it a better BMP antagonist). (4) Crossveinless-2 is a Chordin-like secreted BMP-binding protein that remains attached to ventral cell surfaces and binds and concentrates diffusing Chordin-BMP complexes on the ventral side, where BMPs can then be released by Tolloid.
Self-Regulation
The rate-limiting step in this novel biochemical pathway is the enzyme Tolloid, which provides, together with Crossveinless-2, a type of ventral "sink" toward which BMP ligands flow. Self-regulation results from the dorsal and ventral centers being under opposite transcriptional control: if BMP levels are lowered, production of dorsal ADMP and BMP2 is increased; at high BMP levels, feedback inhibitors such as Sizzled and Crossveinless-2 dampen the signal.
When the four main BMPs are knocked down simultaneously, self-regulation collapses and the entire ectoderm becomes CNS. By transplanting wild-type tissue into these BMP-depleted embryos, I was able to show that both the dorsal and ventral centers serve as sources of BMPs that diffuse over long distances in the embryo, triggering changes in cell differentiation. This double gradient of BMP signals flowing from opposite poles of the embryo helps explain the resilience of the embryo.
Integrating the D-V and A-P Axes
The biochemical pathway described above explains cell differentiation along the dorsal-ventral (D-V) axis. However, when twins are produced the antero-posterior (A-P) and D-V axes are seamlessly integrated. How is this achieved? Christof Niehrs (German Cancer Research Center, Heidelberg) has discovered that the A-P axis is regulated by a gradient of Wnt signals, which are maximal in the posterior. Wnt signals by regulating the activity of a protein kinase called GSK3. We have recently found that the degradation of Smad1/Mad after activation by BMP requires its phosphorylation by GSK3, which triggers polyubiquitinylation and degradation in proteasomes located in the centrosomes. Because GSK3 is inhibited by Wnt signaling, Wnt causes the duration of the BMP signaling to increase.
In this view of self-regulation, the D-V (BMP) and A-P (Wnt) gradients are integrated at the level of the phosphorylations of Smad1 (see Figure 3). The BMP gradient determines the intensity and the Wnt gradient the duration of Smad1, a transcription factor that in turn regulates the activity of promoters and enhancers of hundreds of downstream genes coordinately. Cells are known to distinguish between duration and intensity of signals. Such a hardwired system of signaling integration might provide robustness to embryonic development, which must form perfect babies time after time.
Movie 1: Reenactment of the Spemann-Mangold experiment: The movie opens with photographs of Hans Spemann and Hilde Mangold (circa 1924) [from International Journal of Developmental Biology: Special Issue, The Spemann-Mangold Organizer (E.M. De Robertis and J. Aréchaga, Eds.), 2001, volume 45.]. Eddy De Robertis at the dissection microscope. In one of two embryos, the dorsal blastopore lip, the organizer, is clearly visible as a crescent. A square of organizer tissue is excised, with the help of a tungsten needle and forceps, in a freehand operation. The organizer is pushed into the ventral side of a recipient gastrula. One hour after transplantation, the graft has healed in the host embryo. Two days later, a Siamese twin with two perfect body axes is seen swimming. The organizer graft induced complete central nervous system and mesodermal somites in tissues of the host that would otherwise have become dorsal tissue. Movie by Edward De Robertis and Hiroki Kuroda.
New Research Avenues
The discovery of this novel branch of the Wnt pathway signaling through Smad1 has opened new and exciting research directions. We generated phosphospecific antibodies that recognize Smad1, or its Drosophila homolog Mad, targeted for degradation. These proteins accumulate in the pericentrosomal region. We found that many other proteins targeted for degradation are similarly localized.
Surprisingly, proteins destined for degradation are inherited asymmetrically when cells divide. The peripheral centrosomal material remains in one cell when the centrioles migrate to opposite poles, so that the other daughter remains pristine. Thus, many somatic cell mitoses, perhaps most, are asymmetrical rather than equal as previously thought. We are now studying the role of Wnt signaling in regulating these remarkable asymmetries of proteins destined for degradation.
The other new direction we are pursuing is to use the power of Drosophila molecular genetics to investigate the extent to which the Mad signaling pathway participates in cell differentiation decisions triggered by Wingless signaling. The results so far indicate that Mad is required, in cooperation with other factors, in a surprising number of developmental choices mediated by Wingless. The fact that vertebrate Smad and invertebrate Mad serve as integrators of BMP and Wnt signals may have profound biological implications for the evolution of animal body plans from a common ancestor, a central problem of the young science of Evo-Devo.
In conclusion, efforts to uncover the molecular basis of an embryological experiment carried out more than 80 years ago have shown that the differentiation of embryonic tissue types is regulated by secreted inhibitory proteins originating from dorsal and ventral organizing centers. These studies in Xenopus embryos have led to new insights on how a network of extracellular proteins controlled by the proteolysis of Chordin mediates pattern self-regulation and is integrated with the Wnt signaling pathway.
A grant from the National Institutes of Health provided support for some of these projects.
As of January 28, 2010
