Edward De Robertis studies how long-range cell communication between the dorsal and ventral sides of the embryo occurs through the diffusion of growth factor antagonists. The discovery of Chordin, a BMP antagonist, provided a new paradigm in which facilitated diffusion of a morphogen takes place in the narrow extracellular space that separates the ectoderm from the endomesoderm. This gradient is further integrated with Wnt signaling through the sequestration of the enzyme GSK3 inside multivesicular endosomes.
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 such as bisection. The aim of our research is to discover the molecular mechanisms by which self-regulation works.
Spemann's Dorsal Organizer
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. They found that a small group of cells, called the organizer, is able to induce Siamese twins, including a complete central nervous system (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. Using the frog Xenopus, 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 entire embryo participates in the dorsal-ventral (D-V) morphogenetic field. Chordin is produced dorsally and another secreted protein, Sizzled, is expressed in the ventral side. We used molecular cloning and biochemical methods to unravel a network of Chordin-interacting proteins that form a self-regulating gradient of BMP activity. Key components are dorsal and ventral BMPs as well as Tolloid, a protease that we found specifically cleaves Chordin, releasing active BMPs in the ventral side. Sizzled and BMPs bind to Tolloid and inhibit its rate-limiting enzyme activity. Self-regulation results from the dorsal and ventral centers being under opposite transcriptional control: when BMP levels are lowered, production of dorsal ADMP and BMP2 is increased; at high BMP levels, feedback inhibitors such as Sizzled dampen the signal by inhibiting the degradation of Chordin.
The Chordin Gradient
In the ectoderm, BMP inhibition causes differentiation of CNS, and high levels of BMP signaling induce epidermis. In the mesoderm, at low levels of BMP signaling notochord is formed, and at progressively higher levels kidney, lateral plate mesoderm, and blood tissues are induced. Thus, histotypic differentiation in the vertebrate embryo depends on the graded activity of BMP. Remarkably, the three germ layers respond coordinately to changes in BMP signaling. A key question is whether a single signaling gradient or multiple ones are used to pattern the cell differentiation of the different germ layers. We developed a novel immunolocalization method to follow the distribution of endogenous Chordin during Xenopus gastrulation. Chordin protein secreted by the dorsal Spemann organizer was found to diffuse along a narrow region that separates the ectoderm from anterior endoderm and mesoderm. This fibronectin-rich extracellular matrix (ECM), called Brachet's cleft in Xenopus, is present in all vertebrate embryos. Chordin forms a smooth gradient that encircles the embryo, diffusing over a distance of 2 mm in this signaling highway between ectoderm and mesoderm. After embryo bisection or transplantation of an organizer, the gradient self-regulates. Chordin must reach very high concentrations in this narrow space. It appears that as ectoderm and mesoderm undergo morphogenetic movements during gastrulation, cells in both germ layers read their positional information from a common Chordin/BMP gradient in ECM.
Integrating BMP and Wnt Signaling
The Chordin/BMP biochemical pathway explains cell differentiation along the D-V axis. However, embryonic morphology is also regulated by other morphogens, such as Wnt and fibroblast growth factor (FGF). How are these signaling pathways integrated seamlessly in the embryo? Wnt signals by inhibiting a protein kinase called glycogen synthase kinase 3 (GSK3), but the mechanism by which this occurs is unclear. While investigating the BMP transcription factor Smad1 phosphorylation by GSK3, we discovered a novel cellular mechanism for cell signaling. Upon Wnt signaling, cytosolic GSK3 binds to the Wnt receptor complex, which consists of the Frizzled and LRP6 coreceptors, Axin, Dishevelled, and β-catenin. All of these proteins are substrates for GSK3, which is translocated together with them into small intraluminal vesicles located inside multivesicular bodies (MVBs). In this way, GSK3 becomes sequestered from its many cytosolic substrates. MVBs are an obligatory intermediate organelle for the trafficking of activated plasma membrane receptors destined for degradation in lysosomes. The sequestration of GSK3 requires the activity of ESCRT proteins required for the formation of MVB intraluminal vesicles, such as HRS/Vp27 and Vps4, which we found are also essential for canonical Wnt signaling.
The GSK3 sequestration mechanism has predictive value. Interfering with membrane trafficking downstream of MVB formation potentiates Wnt signaling by causing GSK3 to remain sequestered inside MVBs for longer periods. We found that depletion of Presenilin 1 and 2, two intramembrane proteases mutated in early-onset familial Alzheimer's disease and required for membrane trafficking, greatly increased Wnt/GSK3 signaling, suggesting novel pathogenic mechanisms in neurodegenerative disease.
Remarkably, the sequestration of GSK3 extended the half-life of many proteins in addition to the well-known Wnt target beta-catenin. Pulse-chase studies with radioactive amino acids showed that total cellular half-life is extended by Wnt3a treatment. Bioinformatic analyses revealed that 20 percent of human proteins contain three or more putative GSK3 sites in a row. This is a much higher frequency than expected at random. Our ongoing studies indicate that GSK3 sites can be predictors of proteins regulated by Wnt. For example, we find that in the presence of FGF, which promotes a mitogen-activated protein kinase that primes phosphorylation by GSK3, Smad4 activity is potently increased by Wnt. This suggests that the transforming growth factor beta/Nodal morphogen gradient, which is fundamental for mesoderm induction, may be intimately integrated with the FGF and Wnt gradients. Smad4 is a tumor suppressor that is depleted during progression of pancreatic, colorectal, and prostate cancers. The discovery that its activity is not constitutive but rather is regulated by FGF and Wnt may have applications in cancer treatment.
In conclusion, efforts to uncover the molecular basis of embryonic self-regulation have shown that the differentiation of embryonic tissue types is regulated by an extracellular gradient of proteins diffusing between ectoderm and endomesoderm. Studies of basic embryonic patterning mechanism led to the discovery of unexpected connections between endosomal trafficking, growth factor signaling, and protein degradation, which are being actively explored.
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. 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. 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 CenterOn 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 . 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.
The rate-limiting step in this novel biochemical pathway is the enzyme Tolloid, which provides, together with Crossveinless-2, a type of ventral ?sink? towards 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. 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.
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.
Dr. De Robertis is also Norman Sprague Professor of Biological Chemistry at the University of California, Los Angeles, School of Medicine. He received an M.D. from the University of Uruguay and a Ph.D. in Chemistry from the University of Buenos Aires. His postdoctoral training was with Sir John Gurdon at the Medical Research Council Laboratory of Molecular Biology, Cambridge, U.K. Before moving to UCLA, he was professor at the University of Basel. Dr. De Robertis is a member of the Pontifical Academy of Sciences, the National Academy of Sciences, the European Molecular Biology Organization and the Latin American Academy of Sciences, and a Fellow of the American Academy of Arts and Sciences. He served as President of the International Society of Developmental Biologists until 2006.
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