Molecular analysis of a self-organizing signaling pathway for Xenopus axial patterning from egg to tailbud.

Xenopus embryos provide a favorable material to dissect the sequential steps that lead to dorsal-ventral (D-V) and anterior-posterior (A-P) cell differentiation. Here, we analyze the signaling pathways involved in this process using loss-of-function and gain-of-function approaches. The initial step was provided by Hwa, a transmembrane protein that robustly activates early ß-catenin signaling when microinjected into the ventral side of the embryo leading to complete twinned axes. The following step was the activation of Xenopus Nodal-related growth factors, which could rescue the depletion of ß-catenin and were themselves blocked by the extracellular Nodal antagonists Cerberus-Short and Lefty. During gastrulation, the Spemann-Mangold organizer secretes a cocktail of growth factor antagonists, of which the BMP antagonists Chordin and Noggin could rescue simultaneously D-V and A-P tissues in ß-catenin-depleted embryos. Surprisingly, this rescue occurred in the absence of any ß-catenin transcriptional activity as measured by ß-catenin activated Luciferase reporters. The Wnt antagonist Dickkopf (Dkk1) strongly synergized with the early Hwa signal by inhibiting late Wnt signals. Depletion of Sizzled (Szl), an antagonist of the Tolloid chordinase, was epistatic over the Hwa and Dkk1 synergy. BMP4 mRNA injection blocked Hwa-induced ectopic axes, and Dkk1 inhibited BMP signaling late, but not early, during gastrulation. Several unexpected findings were made, e.g., well-patterned complete embryonic axes are induced by Chordin or Nodal in ß-catenin knockdown embryos, dorsalization by Lithium chloride (LiCl) is mediated by Nodals, Dkk1 exerts its anteriorizing and dorsalizing effects by regulating late BMP signaling, and the Dkk1 phenotype requires Szl.

Na,K-ATPase activity promotes macropinocytosis in colon cancer via Wnt signaling.

Recent research has shown that membrane trafficking plays an important role in canonical Wnt signaling through sequestration of the ß-catenin destruction complex inside multivesicular bodies (MVBs) and lysosomes. In this study, we introduce Ouabain, an inhibitor of the Na,K-ATPase pump that establishes electric potentials across membranes, as a potent inhibitor of Wnt signaling. We find that Na,K-ATPase levels are elevated in advanced colon carcinoma, that this enzyme is elevated in cancer cells with constitutively activated Wnt pathway and is activated by GSK3 inhibitors that increase macropinocytosis. Ouabain blocks macropinocytosis, which is an essential step in Wnt signaling, probably explaining the strong effects of Ouabain on this pathway. In Xenopus embryos, brief Ouabain treatment at the 32-cell stage, critical for the earliest Wnt signal in development-inhibited brains, could be reversed by treatment with Lithium chloride, a Wnt mimic. Inhibiting membrane trafficking may provide a way of targeting Wnt-driven cancers.

Celebrating the centennial of the most famous experiment in embryology: Hilde Mangold, Hans Spemann and the organizer.

This Issue of Cells & Development celebrates the centennial of the Spemann-Mangold organizer experiment. This was the most famous experiment in embryology and its reverberations have greatly influenced developmental biology. This historical issue describes the impact of the discovery and is a prelude to the second volume of this Festschrift, which will consist of the proceedings of the international meeting to be held in Freiburg University, at the place where the organizer was discovered.

The early dorsal signal in vertebrate embryos requires endolysosomal membrane trafficking.

Fertilization triggers cytoplasmic movements in the frog egg that lead in mysterious ways to the stabilization of ß-catenin on the dorsal side of the embryo. The novel Huluwa (Hwa) transmembrane protein, identified in China, is translated specifically in the dorsal side, acting as an egg cytoplasmic determinant essential for ß-catenin stabilization. The Wnt signaling pathway requires macropinocytosis and the sequestration inside multivesicular bodies (MVBs, the precursors of endolysosomes) of Axin1 and Glycogen Synthase Kinase 3 (GSK3) that normally destroy ß-catenin. In Xenopus, the Wnt-like activity of GSK3 inhibitors and of Hwa mRNA can be blocked by brief treatment with inhibitors of membrane trafficking or lysosomes at the 32-cell stage. In dorsal blastomeres, lysosomal cathepsin is activated and intriguing MVBs surrounded by electron dense vesicles are formed at the 64-cell stage. We conclude that membrane trafficking and lysosomal activity are critically important for the earliest asymmetries in vertebrate embryonic development.

Strategies to decrease inequalities in cancer therapeutics, care and prevention: Proceedings on a conference organized by the Pontifical Academy of Sciences and the European Academy of Cancer Sciences, Vatican City, February 23-24, 2023.

Analyses of inequalities related to prevention and cancer therapeutics/care show disparities between countries with different economic standing, and within countries with high Gross Domestic Product. The development of basic technological and biological research provides clinical and prevention opportunities that make their implementation into healthcare systems more complex, mainly due to the growth of Personalized/Precision Cancer Medicine (PCM). Initiatives like the USA-Cancer Moonshot and the EU-Mission on Cancer and Europe's Beating Cancer Plan are initiated to boost cancer prevention and therapeutics/care innovation and to mitigate present inequalities. The conference organized by the Pontifical Academy of Sciences in collaboration with the European Academy of Cancer Sciences discussed the inequality problem, dependent on the economic status of a country, the increasing demands for infrastructure supportive of innovative research and its implementation in healthcare and prevention programs. Establishing translational research defined as a coherent cancer research continuum is still a challenge. Research has to cover the entire continuum from basic to outcomes research for clinical and prevention modalities. Comprehensive Cancer Centres (CCCs) are of critical importance for integrating research innovations to preclinical and clinical research, as for ensuring state-of-the-art patient care within healthcare systems. International collaborative networks between CCCs are necessary to reach the critical mass of infrastructures and patients for PCM research, and for introducing prevention modalities and new treatments effectively. Outcomes and health economics research are required to assess the cost-effectiveness of new interventions, currently a missing element in the research portfolio. Data sharing and critical mass are essential for innovative research to develop PCM. Despite advances in cancer research, cancer incidence and prevalence is growing. Making cancer research infrastructures accessible for all patients, considering the increasing inequalities, requires science policy actions incentivizing research aimed at prevention and cancer therapeutics/care with an increased focus on patients' needs and cost-effective healthcare.

Head organizer: Cerberus and IGF cooperate in brain induction in Xenopus embryos.

Neural induction by cell-cell signaling was discovered a century ago by the organizer transplantations of Spemann and Mangold in amphibians. Spemann later found that early dorsal blastopore lips induced heads and late organizers trunk-tail structures. Identifying region-specific organizer signals has been a driving force in the progress of animal biology. Head induction in the absence of trunk is designated archencephalic differentiation. Two specific head inducers, Cerberus and Insulin-like growth factors (IGFs), that induce archencephalic brain but not trunk-tail structures have been described previously. However, whether these two signals interact with each other had not been studied to date and was the purpose of the present investigation. It was found that Cerberus, a multivalent growth factor antagonist that inhibits Nodal, BMP and Wnt signals, strongly cooperated with IGF2, a growth factor that provides a positive signal through tyrosine kinase IGF receptors that activate MAPK and other pathways. The ectopic archencephalic structures induced by the combination of Cerberus and IGF2 are of higher frequency and larger than either one alone. They contain brain, a cyclopic eye and multiple olfactory placodes, without trace of trunk structures such as notochord or somites. A dominant-negative secreted IGF receptor 1 blocked Cerberus activity, indicating that endogenous IGF signals are required for ectopic brain formation. In a sensitized embryonic system, in which embryos were depleted of ß-catenin, IGF2 did not by itself induce neural tissue while in combination with Cerberus it greatly enhanced formation of circular brain structures expressing the anterior markers Otx2 and Rx2a, but not spinal cord or notochord markers. The main conclusion of this work is that IGF provides a positive signal initially uniformly expressed throughout the embryo that potentiates the effect of an organizer-specific negative signal mediated by Cerberus. The results are discussed in the context of the history of neural induction.

Addition of exogenous diacylglycerol enhances Wnt/ß-catenin signaling through stimulation of macropinocytosis.

Activation of Wnt signaling triggers macropinocytosis and drives many tumors. We now report that the exogenous addition of the second messenger lipid sn-1,2 DAG to the culture medium rapidly induces macropinocytosis. This is accompanied by potentiation of the effects of added Wnt3a recombinant protein or the glycogen synthase kinase 3 (GSK3) inhibitor lithium chloride (LiCl, which mimics Wnt signaling) in luciferase transcriptional reporter assays. In a colorectal carcinoma cell line in which mutation of adenomatous polyposis coli (APC) causes constitutive Wnt signaling, DAG addition increased levels of nuclear ß-catenin, and this increase was partially inhibited by an inhibitor of macropinocytosis. DAG also expanded multivesicular bodies marked by the tetraspan protein CD63. In an in vivo situation, microinjection of DAG induced Wnt-like twinned body axes when co-injected with small amounts of LiCl into Xenopus embryos. These results suggest that the DAG second messenger plays a role in Wnt-driven cancer progression.

The PMA phorbol ester tumor promoter increases canonical Wnt signaling via macropinocytosis.

Activation of the Wnt pathway lies at the core of many human cancers. Wnt and macropinocytosis are often active in the same processes, and understanding how Wnt signaling and membrane trafficking cooperate should improve our understanding of embryonic development and cancer. Here, we show that a macropinocytosis activator, the tumor promoter phorbol 12-myristate 13-acetate (PMA), enhances Wnt signaling. Experiments using the Xenopus embryo as an in vivo model showed marked cooperation between the PMA phorbol ester and Wnt signaling, which was blocked by inhibitors of macropinocytosis, Rac1 activity, and lysosome acidification. Human colorectal cancer tissue arrays and xenografts in mice showed a correlation of cancer progression with increased macropinocytosis/multivesicular body/lysosome markers and decreased GSK3 levels. The crosstalk between canonical Wnt, focal adhesions, lysosomes, and macropinocytosis suggests possible therapeutic targets for cancer progression in Wnt-driven cancers.

The PMA Phorbol Ester Tumor Promoter Increases Canonical Wnt Signaling Via Macropinocytosis.

Activation of the Wnt pathway lies at the core of many human cancers. Wnt and macropinocytosis are often active in the same processes, and understanding how Wnt signaling and membrane trafficking cooperate should improve our understanding of embryonic development and cancer. Here we show that a macropinocytosis activator, the tumor promoter Phorbol 12-myristate 13-acetate (PMA), enhances Wnt signaling. Experiments using the Xenopus embryo as an in vivo model showed marked cooperation between the PMA phorbol ester and Wnt signaling, which was blocked by inhibitors of macropinocytosis, Rac1 activity, and lysosome acidification. Human colorectal cancer tissue arrays and xenografts in mice showed a correlation of cancer progression with increased macropinocytosis/multivesicular body/lysosome markers and decreased GSK3 levels. The crosstalk between canonical Wnt, focal adhesions, lysosomes, and macropinocytosis suggests possible therapeutic targets for cancer progression in Wnt-driven cancers.

The developmental gene Chordin is amplified and expressed in human cancers.

Chordin (CHRD) is a secreted protein important in early development, yet a role for CHRD in human disease has not been identified. In this study we investigated CHRD in cancer and normal adult tissues using the wealth of genome-wide data available in public databases. We found that Chordin is amplified in the DNA of specific cancers such as lung squamous cell and others, although copy number variation did not strictly correlate with higher mRNA expression. In some cancers, such as renal and stomach carcinomas, increased CHRD expression significantly correlated with poor survival. In normal adult human tissues, CHRD mRNA was highest in hepatocytes. Crossveinless-2/BMPER, a component of the Chordin morphogenetic pathway expressed at the opposite side in embryos, was expressed in liver stellate cells. This raises the intriguing possibility that a BMP gradient might be established in the extracellular matrix of the space of Disse that surrounds portal sinusoid capillaries.

Protocol for culturing and imaging of ectodermal cells from Xenopus.

The Xenopus embryo provides an advantageous model system where genes can be readily transplanted as DNA or mRNA or depleted with antisense techniques. Here, we present a protocol to culture and image the cell biological properties of explanted Xenopus cap cells in tissue culture. We illustrate how this protocol can be applied to visualize lysosomes, macropinocytosis, focal adhesions, Wnt signaling, and cell migration. For complete details on the use and execution of this protocol, please refer to Tejeda-Muñoz et al. (2022).

Lysosomes are required for early dorsal signaling in the Xenopus embryo.

Lysosomes are the digestive center of the cell and play important roles in human diseases, including cancer. Previous work has suggested that late endosomes, also known as multivesicular bodies (MVBs), and lysosomes are essential for canonical Wnt pathway signaling. Sequestration of Glycogen Synthase 3 (GSK3) and of ß­catenin destruction complex components in MVBs is required for sustained canonical Wnt signaling. Little is known about the role of lysosomes during early development. In the Xenopus egg, a Wnt-like cytoplasmic determinant signal initiates formation of the body axis following a cortical rotation triggered by sperm entry. Here we report that cathepsin D was activated in lysosomes specifically on the dorsal marginal zone of the embryo at the 64-cell stage, long before zygotic transcription starts. Expansion of the MVB compartment with low-dose hydroxychloroquine (HCQ) greatly potentiated the dorsalizing effects of the Wnt agonist lithium chloride (LiCl) in embryos, and this effect required macropinocytosis. Formation of the dorsal axis required lysosomes, as indicated by brief treatments with the vacuolar ATPase (V-ATPase) inhibitors Bafilomycin A1 or Concanamycin A at the 32-cell stage. Inhibiting the MVB-forming machinery with a dominant-negative point mutation in Vacuolar Protein Sorting 4 (Vps4-EQ) interfered with the endogenous dorsal axis. The Wnt-like activity of the dorsal cytoplasmic determinant Huluwa (Hwa), and that of microinjected xWnt8 messenger RNA, also required lysosome acidification and the MVB-forming machinery. We conclude that lysosome function is required for early dorsal axis development in Xenopus. The results highlight the intertwining between membrane trafficking, lysosomes, and vertebrate axis formation.

Evo-Devo of Urbilateria and its larval forms.

Developmental biology has contributed greatly to evolutionary biology in the past century. With the discovery that vertebrates share Hox genes with Drosophila in 1984, it became apparent that all animals evolved from variations of an ancestral embryonic patterning genetic tool-kit. In the dorsal-ventral (D-V) axis, a fundamental experiment was the Spemann-Mangold organizer transplant performed in 1924. Almost a century later, D-V genes have been subjected to saturating molecular screens in Xenopus and extensive genetic screens in zebrafish. A network of secreted growth factor antagonists has emerged, and we review here in detail the Chordin/Tolloid/BMP pathway. Chordin establishes a morphogen gradient spanning the entire embryo that was present even in the cnidarian Nematostella. This ancient system was present in Urbilateria, the last common ancestor of the protostome and deuterostome bilateral animals. We suggest that Urbilateria had a complex life cycle with an adult benthic form on the sea bottom, and also a primary larval pelagic or planktonic phase to disperse the species in the marine milieu. Larvae with two rows of cilia beating in opposite directions to entrap food particles, an apical sensory organ, and a rudimentary eye, are present in many protostome and deuterostome phyla. Although the larval phase has been lost multiple times in evolution, and larvae can adopt traits present in their adult forms, the simplest explanation is that Urbilateria had a pelago-benthic life cycle. The use of conserved developmental patterning systems likely placed evolutionary constraints in the animal forms that evolved by natural selection.

Canonical Wnt signaling induces focal adhesion and Integrin beta-1 endocytosis.

During canonical Wnt signaling, the Wnt receptor complex is sequestered together with glycogen synthase kinase 3 (GSK3) and Axin inside late endosomes, known as multivesicular bodies (MVBs). Here, we present experiments showing that Wnt causes the endocytosis of focal adhesion (FA) proteins and depletion of Integrin ß 1 (ITGß1) from the cell surface. FAs and integrins link the cytoskeleton to the extracellular matrix. Wnt-induced endocytosis caused ITGß1 depletion from the plasma membrane and was accompanied by striking changes in the actin cytoskeleton. In situ protease protection assays in cultured cells showed that ITGß1 was sequestered within membrane-bounded organelles that corresponded to Wnt-induced MVBs containing GSK3 and FA-associated proteins. An in vivo model using Xenopus embryos dorsalized by Wnt8 mRNA showed that ITGß1 depletion decreased Wnt signaling. The finding of a crosstalk between two major signaling pathways, canonical Wnt and focal adhesions, should be relevant to human cancer and cell biology.

Wnt, GSK3, and Macropinocytosis.

Here we review the regulation of macropinocytosis by Wnt growth factor signaling. Canonical Wnt signaling is normally thought of as a regulator of nuclear ß-catenin, but emerging results indicate that there is much more than ß-catenin to the Wnt pathway. Macropinocytosis is transiently regulated by EGF-RTK-Ras-PI3K signaling. Recent studies show that Wnt signaling provides for sustained acquisition of nutrients by macropinocytosis. Endocytosis of Wnt-Lrp6-Fz receptor complexes triggers the sequestration of GSK3 and components of the cytosolic destruction complex such as Axin1 inside multivesicular bodies (MVBs) through the action of the ESCRT machinery. Wnt macropinocytosis can be induced both by the transcriptional loop of stabilized ß-catenin, and by the inhibition of GSK3 even in the absence of new protein synthesis. The cell is poised for macropinocytosis, and all it requires for triggering of Pak1 and the actin machinery is the inhibition of GSK3. Striking lysosomal acidification, which requires macropinocytosis, is induced by GSK3 chemical inhibitors or Wnt protein. Wnt-induced macropinocytosis requires the ESCRT machinery that forms MVBs. In cancer cells, mutations in the tumor suppressors APC and Axin1 result in extensive macropinocytosis, which can be reversed by restoring wild-type protein. In basal cellular conditions, GSK3 functions to constitutively repress macropinocytosis.

Cell Biology of Canonical Wnt Signaling.

Wnt signaling has multiple functions beyond the transcriptional effects of ß-catenin stabilization. We review recent investigations that uncover new cell physiological effects through the regulation of Wnt receptor endocytosis, Wnt-induced stabilization of proteins (Wnt-STOP), macropinocytosis, increase in lysosomal activity, and metabolic changes. Many of these growth-promoting effects of canonical Wnt occur within minutes and are independent of new protein synthesis. A key element is the sequestration of glycogen synthase kinase 3 (GSK3) inside multivesicular bodies and lysosomes. Twenty percent of human proteins contain consecutive GSK3 phosphorylation motifs, which in the absence of Wnt can form phosphodegrons for polyubiquitination and proteasomal degradation. Wnt signaling by either the pharmacological inhibition of GSK3 or the loss of tumor-suppressor proteins, such as adenomatous polyposis coli (APC) and Axin1, increases lysosomal acidification, anabolic metabolites, and macropinocytosis, which is normally repressed by the GSK3-Axin1-APC destruction complex. The combination of these cell physiological effects drives cell growth.

The cytokine FAM3B/PANDER is an FGFR ligand that promotes posterior development in Xenopus.

Fibroblast growth factor (FGF)/extracellular signal-regulated kinase (ERK) signaling plays a crucial role in anterior-posterior (A-P) axial patterning of vertebrate embryos by promoting posterior development. In our screens for novel developmental regulators in Xenopus embryos, we identified Fam3b as a secreted factor regulated in ectodermal explants. Family with sequence similarity 3 member B (FAM3B)/PANDER (pancreatic-derived factor) is a cytokine involved in glucose metabolism, type 2 diabetes, and cancer in mammals. However, the molecular mechanism of FAM3B action in these processes remains poorly understood, largely because its receptor is still unidentified. Here we uncover an unexpected role of FAM3B acting as a FGF receptor (FGFR) ligand in Xenopus embryos. fam3b messenger RNA (mRNA) is initially expressed maternally and uniformly in the early Xenopus embryo and then in the epidermis at neurula stages. Overexpression of Xenopus fam3b mRNA inhibited cephalic structures and induced ectopic tail-like structures. Recombinant human FAM3B protein was purified readily from transfected tissue culture cells and, when injected into the blastocoele cavity, also caused outgrowth of tail-like structures at the expense of anterior structures, indicating FGF-like activity. Depletion of fam3b by specific antisense morpholino oligonucleotides in Xenopus resulted in macrocephaly in tailbud tadpoles, rescuable by FAM3B protein. Mechanistically, FAM3B protein bound to FGFR and activated the downstream ERK signaling in an FGFR-dependent manner. In Xenopus embryos, FGFR activity was required epistatically downstream of Fam3b to mediate its promotion of posterior cell fates. Our findings define a FAM3B/FGFR/ERK-signaling pathway that is required for axial patterning in Xenopus embryos and may provide molecular insights into FAM3B-associated human diseases.

A Brief History of Xenopus in Biology.

Xenopus is one of the premier model systems to study cell and developmental biology in vivo in vertebrates. Here we briefly review how this South African frog came to be favored by a large community of scientists after the explosive growth of molecular biology and examine some of the original discoveries arising from this sturdy frog. Experimental embryology started in Rana but developed in newt embryos for historical reasons. A long lineage of mentorship, starting with Theodor Boveri, Hans Spemann, Fritz Baltzer, Ernst Hadorn, and Michail Fischberg, used newt embryos. In Oxford, Fischberg made the transition to Xenopus laevis because it was widely available for human pregnancy tests and laid eggs year-round, and he fortuitously isolated a one-nucleolus mutant. This mutant allowed nuclear transfer experiments showing that genetic information is not lost during cell differentiation and the demonstration that the nucleolus is the locus of transcription of the large ribosomal RNAs. With the advent of DNA cloning, the great equalizer among all fields of biology, microinjected Xenopus oocytes became an indispensable tool, providing the first living-cell mRNA translation, polymerase II and III transcription, and coupled transcription-translation systems in eukaryotes. Xenopus embryos provide abundant material to study the earliest signaling events during vertebrate development and have been subjected to saturating molecular screens in the genomic era. Many novel principles of development and cell biology owe their origins to this remarkably resilient frog.

Wnt-inducible Lrp6-APEX2 interacting proteins identify ESCRT machinery and Trk-fused gene as components of the Wnt signaling pathway.

The canonical Wnt pathway serves as a hub connecting diverse cellular processes, including ß-catenin signaling, differentiation, growth, protein stability, macropinocytosis, and nutrient acquisition in lysosomes. We have proposed that sequestration of ß-catenin destruction complex components in multivesicular bodies (MVBs) is required for sustained canonical Wnt signaling. In this study, we investigated the events that follow activation of the canonical Wnt receptor Lrp6 using an APEX2-mediated proximity labeling approach. The Wnt co-receptor Lrp6 was fused to APEX2 and used to biotinylate targets that are recruited near the receptor during Wnt signaling at different time periods. Lrp6 proximity targets were identified by mass spectrometry, and revealed that many endosomal proteins interacted with Lrp6 within 5 min of Wnt3a treatment. Interestingly, we found that Trk-fused gene (TFG), previously known to regulate the cell secretory pathway and to be rearranged in thyroid and lung cancers, was strongly enriched in the proximity of Lrp6. TFG depletion with siRNA, or knock-out with CRISPR/Cas9, significantly reduced Wnt/ß-catenin signaling in cell culture. In vivo, studies in the Xenopus system showed that TFG is required for endogenous Wnt-dependent embryonic patterning. The results suggest that the multivesicular endosomal machinery and the novel player TFG have important roles in Wnt signaling.