Edward M. De Robertis

Wnt Signaling Requires Sequestration of Glycogen Synthase Kinase 3 inside Multivesicular Endosomes

Canonical Wnt signaling requires inhibition of Glycogen Synthase Kinase 3 (GSK3) activity, but the molecular mechanism by which this is achieved remains unclear. Here, we report that Wnt signaling triggers the sequestration of GSK3 from the cytosol into multivesicular bodies (MVBs), so that this enzyme becomes separated from its many cytosolic substrates. Endocytosed Wnt colocalized with GSK3 in acidic vesicles positive for endosomal markers. After Wnt addition, endogenous GSK3 activity decreased in the cytosol, and GSK3 became protected from protease treatment inside membrane-bounded organelles. Cryoimmunoelectron microscopy showed that these corresponded to MVBs. Two proteins essential for MVB formation, HRS/Vps27 and Vps4, were required for Wnt signaling. The sequestration of GSK3 extended the half-life of many other proteins in addition to β-Catenin, including an artificial Wnt-regulated reporter protein containing GSK3 phosphorylation sites. We conclude that multivesicular endosomes are essential components of the Wnt signal-transduction pathway.

Integrating Patterning Signals: Wnt/GSK3 Regulates the Duration of the BMP/Smad1 Signal

BMP receptors determine the intensity of BMP signals via Smad1 C-terminal phosphorylations. Here we show that a finely controlled cell biological pathway terminates this activity. The duration of the activated pSmad1(Cter) signal was regulated by sequential Smad1 linker region phosphorylations at conserved MAPK and GSK3 sites required for its polyubiquitinylation and transport to the centrosome. Proteasomal degradation of activated Smad1 and total polyubiquitinated proteins took place in the centrosome. Inhibitors of the Erk, p38, and JNK MAPKs, as well as GSK3 inhibitors, prolonged the duration of a pulse of BMP7. Wnt signaling decreased pSmad1(GSK3) antigen levels and redistributed it from the centrosome to cytoplasmic LRP6 signalosomes. In Xenopus embryos, it was found that Wnts induce epidermis and that this required an active BMP-Smad pathway. Epistatic experiments suggested that the dorsoventral (BMP) and anteroposterior (Wnt/GSK3) patterning gradients are integrated at the level of Smad1 phosphorylations during embryonic pattern formation.

Depletion of Bmp2, Bmp4, Bmp7 and Spemann organizer signals induces massive brain formation in Xenopus embryos

To address the patterning function of the Bmp2, Bmp4 and Bmp7 growth factors, we designed antisense morpholino oligomers (MO) that block their activity in Xenopus laevis. Bmp4 knockdown was sufficient to rescue the ventralizing effects caused by loss of Chordin activity. Double Bmp4 and Bmp7 knockdown inhibited tail development. Triple Bmp2/Bmp4/Bmp7 depletion further compromised trunk development but did not eliminate dorsoventral patterning. Unexpectedly, we found that blocking Spemann organizer formation by UV treatment or β-Catenin depletion caused BMP inhibition to have much more potent effects, abolishing all ventral development and resulting in embryos having radial central nervous system (CNS) structures. Surprisingly, dorsal signaling molecules such as Chordin, Noggin, Xnr6 and Cerberus were not re-expressed in these embryos. We conclude that BMP inhibition is sufficient for neural induction in vivo, and that in the absence of ventral BMPs, Spemann organizer signals are not required for brain formation.

Morpholinos: Antisense and Sensibility

For over 15 years, antisense morpholino oligonucleotides (MOs) have allowed developmental biologists to make key discoveries regarding developmental mechanisms in numerous model organisms. Recently, serious concerns have been raised as to the specificity of MO effects, and it has been recommended to discontinue their usage, despite the long experience of the scientific community with the MO tool in thousands of studies. Reviewing the many advantages afforded by MOs, we conclude that adequately controlled MOs should continue to be accepted as generic loss-of-function approach, as otherwise progress in developmental biology will greatly suffer.

BMP gradients: A paradigm for morphogen-mediated developmental patterning

BMP mophogens direct growth and fate As shown in classic fate-mapping studies, tissues and organs arise from specific regions of the embryo. Work over the past few decades has identified molecular players directing this choreographed development. Bone morphogenetic proteins (BMPs) and their antagonists establish domains in developing embryos. Bier and De Robertis review historical events for key discoveries in this area. They go on to lay out the current understanding of how diffusible morphogens form gradients to subdivide germ layers into distinct territories and organize body axes, regulate growth, and maintain stem cell niches. Science, this issue 10.1126/science.aaa5838 BACKGROUND Classic embryological studies showed that diffusible factors (morphogens) influence cell fate during dorsal-ventral (DV) axis patterning. Subsequently, mathematical analyses applied reaction-diffusion equations in a theoretical framework to model how stable gradients of morphogenetic factors might be created in developing cell fields, according to the laws of physical chemistry. This work suggested mechanisms by which such gradients form and are read in a threshold-dependent fashion to establish distinct cellular responses. As highlighted in this Review, these pioneering experimental and intellectual insights laid the groundwork for more recent studies that have elucidated the mechanisms by which morphogen gradients are generated and stabilized by molecular feedback circuits. ADVANCES The molecular players involved in early DV patterning uncovered over the past two decades constitute a highly conserved cohort of extracellular factors that regulate bone morphogenetic protein (BMP) signaling. A key insight was the identification of the homologous proteins Short gastrulation (Sog) and Chordin as BMP-binding proteins in Drosophila and Xenopus 20 years ago. Since then, analysis of this patterning system has led to dramatic advances in our understanding of the molecular mechanisms regulating early DV axis specification. Elements of this pathway include secreted BMP ligands and BMP antagonists, as well as extracellular metalloproteinases that cleave and inactivate BMP antagonists. Identification of these and other accessory proteins provided strong support for the proposal that an inversion of the DV axis had occurred between arthropods and vertebrates. Analysis of how these components are deployed in an array of species with divergent developmental strategies has deepened our understanding of this ancestral DV patterning biochemical pathway. These comparative studies have shed light on the broader question of a how a conserved core pathway can be modified during evolution to accommodate different forms of embryogenesis while maintaining common output effector functions. In addition, advances in computational analysis have provided the necessary tools to analyze BMP-mediated signaling in quantitative terms and have provided important insights into how this patterning process is integrated with cell proliferation and tissue growth. One such insight is the identification of expanders (such as Pentagone and Sizzled), which are secreted molecules typically produced at the low end of a gradient that stabilize the ligand, scaling the gradient to the growth of tissues. OUTLOOK An important unanswered question is how morphogen gradients form and function reliably in the face of intrinsic signal-degrading processes to achieve consistent developmental patterning and growth. One testable hypothesis, based on the “wisdom of crowds” concept, that may shed light on this challenging problem is that several independent features of morphogen gradients can be read in parallel by cells and can also serve as inputs to an array of feedback modules that integrate instantaneous levels of signaling, perform time averaging of signals, and act locally to coordinate signaling between neighboring cells. A consensus-based estimate of the relative position of a cell may be reached by deploying multiple parallel feedback modules. In addition, it will be important to determine the roles of mechanisms, such as free or facilitated diffusion in the extracellular space; exosomes; and cytonemes in morphogen gradient function. Understanding the mechanisms by which morphogen-mediated patterning systems evolve to maintain key elements of overall body design while allowing for a marked diversity in the spatial deployment of various subsets of signaling components is another compelling challenge. Such studies should better illuminate the precise nature of highly constrained developmental processes and delineate more fluid features of the networks that permit remodeling of core components to meet the specialized selective needs of particular organisms. These future studies should refine and strengthen one of the best paradigms for understanding development. Conserved BMP-mediated patterning of the DV axis. Gradients of proteins in vertebrates (left: blue Chordin stain) and invertebrates (left: red/yellow Sog stain) initiate patterning along the DV axis. These gradients are then read to establish distinct zones of gene expression within the central nervous system (right: dpp, yellow; msh, red; ind, green; vnd, blue). CREDITS: CHORDIN GRADIENT (LEFT) COLORED AND MODIFIED BY PHOTOSHOP FROM FIG. 3A OF J. L. PLOUHINEC ET AL., PROC. NATL. ACAD. SCI. U.S.A. 110, 20372–20379 (2013); SOG GRADIENT (RIGHT) MODIFIED BY PHOTOSHOP FROM FIG. 1B′′ OF S. SRINIVASAN ET AL., DEV. CELL 2, 91–101 (2002) Bone morphogenetic proteins (BMPs) act in dose-dependent fashion to regulate cell fate choices in a myriad of developmental contexts. In early vertebrate and invertebrate embryos, BMPs and their antagonists establish epidermal versus central nervous system domains. In this highly conserved system, BMP antagonists mediate the neural-inductive activities proposed by Hans Spemann and Hilde Mangold nearly a century ago. BMPs distributed in gradients subsequently function as morphogens to subdivide the three germ layers into distinct territories and act to organize body axes, regulate growth, maintain stem cell niches, or signal inductively across germ layers. In this Review, we summarize the variety of mechanisms that contribute to generating reliable developmental responses to BMP gradients and other morphogen systems.

MITF drives endolysosomal biogenesis and potentiates Wnt signaling in melanoma cells

Significance MITF, a master regulator of melanocytes and a major melanoma oncogene amplified in 30-40% of melanomas, determines proliferative or invasive phenotypes. Previously unrecognized as a driver of lysosomal biogenesis, we found that MITF expression correlates with many lysosomal genes and generates late endosomes that are not functional in proteolysis. This accumulation of incomplete organelles expands the late endosomal compartment, enhancing Wnt signaling by entrapping the Wnt machinery in multivesicular bodies. Wnt signaling can stabilize many proteins besides β-catenin. Our study identifies MITF as an oncogenic protein stabilized by Wnt, and describes three novel glycogen synthase kinase 3-regulated phosphorylation sites in this oncogene. This study deepens our knowledge on proliferative stages of melanoma: MITF, multivesicular bodies, and Wnt may form a feedback loop that drives proliferation. Canonical Wnt signaling plays an important role in development and disease, regulating transcription of target genes and stabilizing many proteins phosphorylated by glycogen synthase kinase 3 (GSK3). We observed that the MiT family of transcription factors, which includes the melanoma oncogene MITF (micropthalmia-associated transcription factor) and the lysosomal master regulator TFEB, had the highest phylogenetic conservation of three consecutive putative GSK3 phosphorylation sites in animal proteomes. This finding prompted us to examine the relationship between MITF, endolysosomal biogenesis, and Wnt signaling. Here we report that MITF expression levels correlated with the expression of a large subset of lysosomal genes in melanoma cell lines. MITF expression in the tetracycline-inducible C32 melanoma model caused a marked increase in vesicular structures, and increased expression of late endosomal proteins, such as Rab7, LAMP1, and CD63. These late endosomes were not functional lysosomes as they were less active in proteolysis, yet were able to concentrate Axin1, phospho-LRP6, phospho-β-catenin, and GSK3 in the presence of Wnt ligands. This relocalization significantly enhanced Wnt signaling by increasing the number of multivesicular bodies into which the Wnt signalosome/destruction complex becomes localized upon Wnt signaling. We also show that the MITF protein was stabilized by Wnt signaling, through the novel C-terminal GSK3 phosphorylations identified here. MITF stabilization caused an increase in multivesicular body biosynthesis, which in turn increased Wnt signaling, generating a positive-feedback loop that may function during the proliferative stages of melanoma. The results underscore the importance of misregulated endolysosomal biogenesis in Wnt signaling and cancer.

Presenilin deficiency or lysosomal inhibition enhances Wnt signaling through relocalization of GSK3 to the late-endosomal compartment.

Sustained canonical Wnt signaling requires the inhibition of glycogen synthase kinase 3 (GSK3) activity by sequestration of GSK3 inside multivesicular endosomes (MVEs). Here, we show that Wnt signaling is increased by the lysosomal inhibitor chloroquine, which causes accumulation of MVEs. A similar MVE expansion and increased Wnt responsiveness was found in cells deficient in presenilin, a protein associated with Alzheimers disease. The Wnt-enhancing effects were entirely dependent on the functional endosomal sorting complex required for transport (ESCRT), which is needed for the formation of intraluminal vesicles in MVEs. We suggest that accumulation of late endosomal structures leads to enhanced canonical Wnt signaling through increased Wnt-receptor/GSK3 sequestration. The decrease in GSK3 cytosolic activity stabilized cytoplasmic GSK3 substrates such as β-catenin, the microtubule-associated protein Tau, and other proteins. These results underscore the importance of the endosomal pathway in canonical Wnt signaling and reveal a mechanism for regulation of Wnt signaling by presenilin deficiency.

Chordin forms a self-organizing morphogen gradient in the extracellular space between ectoderm and mesoderm in the Xenopus embryo

Significance Cell differentiation in the embryo is regulated by diffusible substances called “morphogens,” but these have never been directly visualized as endogenous components of the extracellular space. Chordin is an antagonist of the bone morphogenetic protein (BMP) pathway copiously secreted by a dorsal region of the Xenopus embryo called “Spemann’s organizer” that has potent tissue-inducing activity. We report that Chordin protein forms a dorsal-to-ventral gradient in the embryo. This gradient is located in a narrow space containing extracellular matrix (ECM) that separates the ectoderm from the endomesoderm, which seems to serve as a highway for the diffusion of Chordin–BMP complexes over very long distances (2 mm) in the embryo. All vertebrate embryos have a similar ECM between ectoderm and mesoderm during gastrulation. The vertebrate body plan follows stereotypical dorsal–ventral (D-V) tissue differentiation controlled by bone morphogenetic proteins (BMPs) and secreted BMP antagonists, such as Chordin. The three germ layers—ectoderm, mesoderm, and endoderm—are affected coordinately by the Chordin–BMP morphogen system. However, extracellular morphogen gradients of endogenous proteins have not been directly visualized in vertebrate embryos to date. In this study, we improved immunolocalization methods in Xenopus embryos and analyzed the distribution of endogenous Chordin using a specific antibody. Chordin protein secreted by the dorsal Spemann organizer was found to diffuse along a narrow region that separates the ectoderm from the anterior endoderm and mesoderm. This Fibronectin-rich extracellular matrix is called “Brachet’s cleft” in the Xenopus gastrula and is present in all vertebrate embryos. Chordin protein formed a smooth gradient that encircled the embryo, reaching the ventral-most Brachet cleft. Depletion with morpholino oligos showed that this extracellular gradient was regulated by the Chordin protease Tolloid and its inhibitor Sizzled. The Chordin gradient, as well as the BMP signaling gradient, was self-regulating and, importantly, was able to rescale in dorsal half-embryos. Transplantation of Spemann organizer tissue showed that Chordin diffused over long distances along this signaling highway between the ectoderm and mesoderm. Chordin protein must reach very high concentrations in this narrow region. We suggest that as ectoderm and mesoderm undergo morphogenetic movements during gastrulation, cells in both germ layers read their positional information coordinately from a single morphogen gradient located in Brachet’s cleft.

Systems control of BMP morphogen flow in vertebrate embryos

Embryonic morphogenetic programs coordinate cell behavior to ensure robust pattern formation. Having identified components of those programs by molecular genetics, developmental biology is now borrowing concepts and tools from systems biology to decode their regulatory logic. Dorsal-ventral (D-V) patterning of the frog gastrula by Bone Morphogenetic Proteins (BMPs) is one of the best studied examples of a self-regulating embryonic patterning system. Embryological analyses and mathematical modeling are revealing that the BMP activity gradient is maintained by a directed flow of BMP ligands towards the ventral side. Pattern robustness is ensured through feedback control of the levels of extracellular BMP pathway modulators that adjust the flow to the dimensions of the embryonic field.

Phosphorylation of Mad Controls Competition Between Wingless and BMP Signaling

The transcription factor Mad participates in two different developmentally important signaling pathways depending on its phosphorylation status. Mad for Both Wingless and BMP The Wingless (also known as Wnt) and bone morphogenetic protein (BMP) signaling pathways are critical during development. In the Drosophila BMP signaling pathway, ligand-bound BMP receptors phosphorylate sites in the C terminus of the transcription factor Mad, thereby activating it. The BMP signal is terminated by degradation of Mad, a process that requires phosphorylation of Mad at sites distinct from those that activate it. Eivers et al. now show a role for unphosphorylated Mad in Wingless signaling. Drosophila with a mutant form of Mad with reduced degradation showed developmental defects typical of excess Wingless signaling, and phosphorylation-resistant Mad interacted with two transcriptional effectors of the Wingless pathway. Thus, the phosphorylation state of Mad determines whether it participates in BMP or Wingless signaling. Bone morphogenetic proteins (BMPs) and Wnts are growth factors that provide essential patterning signals for cell proliferation and differentiation. Here, we describe a molecular mechanism by which the phosphorylation state of the Drosophila transcription factor Mad determines its ability to transduce either BMP or Wingless (Wg) signals. Previously, Mad was thought to function in gene transcription only when phosphorylated by BMP receptors. We found that the unphosphorylated form of Mad was required for canonical Wg signaling by interacting with the Pangolin-Armadillo transcriptional complex. Phosphorylation of the carboxyl terminus of Mad by BMP receptor directed Mad toward BMP signaling, thereby preventing Mad from functioning in the Wg pathway. The results show that Mad has distinct signal transduction roles in the BMP and Wnt pathways depending on its phosphorylation state.