The threonine that carries fucose, but not fucose, is required for Cripto to facilitate Nodal signaling

Cripto is a membrane-bound co-receptor for Nodal, a member of the transforming growth factor-beta superfamily. Mouse embryos lacking either Cripto or Nodal have the same lethal phenotype at embryonic day 7.5. Previous studies suggest that O-fucosylation of the epidermal growth factor-like (EGF) repeat in Cripto is essential for the facilitation of Nodal signaling. Substitution of Ala for the Thr to which O-fucose is attached led to functional inactivation of both human and mouse Cripto. However, embryos null for protein O-fucosyltransferase 1, the enzyme that adds O-fucose to EGF repeats, do not exhibit a Cripto null phenotype and die at about embryonic day 9.5. This suggested that the loss of O-fucose from the EGF repeat may not have led to the inactivation of Cripto in previous studies. Here we investigate this hypothesis and show the following: 1) protein O-fucosyltransferase 1 is indeed the enzyme that adds O-fucose to Cripto; 2) Pofut1(-/-) embryonic stem cells behave the same as Pofut1(+/+) embryonic stem cells in a Nodal signaling assay; 3) Pofut1(-/-) and Pofut1(+/+) embryoid bodies are indistinguishable in their ability to differentiate into cardiomyocytes; and 4) none of 10 amino acid substitutions at Thr(72), including Ser which acquires O-fucose, rescues the activity of mouse Cripto in Nodal signaling assays. Therefore, the Thr to which O-fucose is linked in Cripto plays a key functional role, but O-fucose at Thr(72) is not required for Cripto to function in cell-based signaling assays or in vivo. By contrast, we show that O-fucose, and not the Thr to which it is attached, is required in the ligand-binding domain of Notch1 for Notch1 signaling.     

Cripto is a noncompetitive activin antagonist that forms analogous signaling complexes with activin and nodal

Cripto plays critical roles during embryogenesis and has been implicated in promoting the growth and spread of tumors. Cripto is required for signaling by certain transforming growth factor-beta superfamily members, such as Nodal, but also antagonizes others, such as activin. The opposing effects of Cripto on Nodal and activin signaling seem contradictory, however, because these closely related ligands utilize the same type I (ALK4) and type II (ActRII/IIB) receptors. Here, we have addressed this apparent paradox by demonstrating that Cripto forms analogous receptor complexes with Nodal and activin and functions as a noncompetitive activin antagonist. Our results show that activin-A and Nodal elicit similar maximal signaling responses in the presence of Cripto that are substantially lower than that of activin-A in the absence of Cripto. In addition, we provide biochemical evidence for complexes containing activin-A, Cripto, and both receptor types and show that the assembly of such complexes is competitively inhibited by Nodal. We further demonstrate that Nodal and activin-A share the same binding site on ActRII and that ALK4 has distinct and separable binding sites for activin-A and Cripto. Finally, we show that ALK4 mutants with disrupted activin-A binding retain Cripto binding and prevent the effects of Cripto on both activin-A and Nodal signaling. Together, our data indicate that Cripto facilitates Nodal signaling and inhibits activin signaling by forming receptor complexes with these ligands that are structurally and functionally similar.     

Dual roles of Cripto as a ligand and coreceptor in the nodal signaling pathway

The EGF-CFC gene Cripto encodes an extracellular protein that has been implicated in the signaling pathway for the transforming growth factor beta (TGF beta) ligand Nodal. Although recent findings in frog and fish embryos have suggested that EGF-CFC proteins function as coreceptors for Nodal, studies in cell culture have implicated Cripto as a growth factor-like signaling molecule. Here we reconcile these apparently disparate models of Cripto function by using a mammalian cell culture assay to investigate the signaling activities of Nodal and EGF-CFC proteins. Using a luciferase reporter assay, we found that Cripto has activities consistent with its being a coreceptor for Nodal. However, Cripto can also function as a secreted signaling factor in cell coculture assays, suggesting that it may also act as a coligand for Nodal. Furthermore, we found that the ability of Cripto to bind to Nodal and mediate Nodal signaling requires the addition of an O-linked fucose monosaccharide to a conserved site within EGF-CFC proteins. We propose a model in which Cripto has dual roles as a coreceptor as well as a coligand for Nodal and that this signaling interaction with Nodal is regulated by an unusual form of glycosylation. Our findings highlight the significance of extracellular modulation of ligand activity as an important means of regulating TGF beta signaling pathways during vertebrate development.     

The pitx2 homeobox protein is required early for endoderm formation and nodal signaling.

Nodal and Nodal-related factors play fundamental roles in a number of developmental processes, including mesoderm and endoderm formation, patterning of the anterior neural plate, and determination of bilateral asymmetry in vertebrates. pitx2, a paired-like homeobox gene, has been proposed to act downstream of Nodal in the gene cascade providing left-right cues to the developing organs. Here, we report that pitx2 is required early in the Nodal signaling pathway for specification of the endodermal and mesodermal germ layers. We found that pitx2 is expressed very early during Xenopus and zebrafish development and in many regions where Nodal signaling is required, including the presumptive mesoderm and endoderm at the blastula and gastrula stages and the prechordal mesoderm at later stages. In Xenopus embryos, overexpression of pitx2 caused ectopic expression of goosecoid and sox-17 and interfered with mesoderm formation. Overexpression of pitx2 in Xenopus animal cap explants partially mimics the effects of Nodal overexpression, suggesting that pitx2 is a mediator of Nodal signaling during specification of the endoderm and prechordal plate, but not during mesoderm induction. We further demonstrate that pitx2 is induced by Nodal signaling in Xenopus animal caps and that the early expression of zebrafish pitx2 is absent when the Nodal signaling pathway is inactive. Inhibition of pitx2 function using a chimeric EnR-pitx2 blocked specification of the mesoderm and endoderm and caused severe embryonic defects resembling those seen when Nodal signaling is inhibited. Following inhibition of pitx2 function, the fate of ventral vegetal blastomeres was shifted from an endodermal to a more mesodermal fate, an effect that was reversed by wild-type pitx2. Finally, we show that inhibition of pitx2 function interferes with the response of cells to Nodal signaling. Our results provide direct evidence that pitx2 function is required for normal specification of the endodermal and mesodermal germ layers.     

A molten globule-to-ordered structure transition of Drosophila melanogaster crammer is required for its ability to inhibit cathepsin

Drosophila melanogaster crammer is a novel cathepsin inhibitor that is involved in LTM (long-term memory) formation. The mechanism by which the inhibitory activity is regulated remains unclear. In the present paper we have shown that the oligomeric state of crammer is pH dependent. At neutral pH, crammer is predominantly dimeric in vitro as a result of disulfide bond formation, and is monomeric at acidic pH. Our inhibition assay shows that monomeric crammer, not disulfide-bonded dimer, is a strong competitive inhibitor of cathepsin L. Crammer is a monomeric molten globule in acidic solution, a condition that is similar to the environment in the lysosome where crammer is probably located. Upon binding to cathepsin L, however, crammer undergoes a molten globule-to-ordered structural transition. Using high-resolution NMR spectroscopy, we have shown that a cysteine-to-serine point mutation at position 72 (C72S) renders crammer monomeric at pH 6.0 and that the structure of the C72S variant highly resembles that of wild-type crammer in complex with cathepsin L at pH 4.0. We have determined the first solution structure of propeptide-like protease inhibitor in its active form and examined in detail using a variety of spectroscopic methods the folding properties of crammer in order to delineate its biomolecular recognition of cathepsin.     

The EGF-CFC protein one-eyed pinhead is essential for nodal signaling

The zebrafish EGF-CFC gene one-eyed pinhead (oep) is required zygotically for the formation of the ventral neuroectoderm, endoderm, and prechordal plate. Here we report that embryos lacking both maternal and zygotic Oep activity are defective in germ layer formation, organizer development, and the positioning of the anterior-posterior axis. An identical phenotype is displayed by double mutants for the nodal-related genes squint and cyclops. Mutations in oep eliminate the response to Squint and Cyclops overexpression but are suppressed by expression of Activin and activated forms of the type I receptor ActRIB and Smad2. Expression of the murine EGF-CFC gene cripto rescues oep mutants. These results suggest a conserved role for EGF-CFC proteins as essential extracellular cofactors for Nodal signaling during vertebrate development.     

Gq signaling is required for allergen-induced pulmonary eosinophilia

The complexity and magnitude of interactions leading to the selective infiltration of eosinophils in response to inhaled allergens are formidable obstacles to a larger understanding of the pulmonary pathology associated with allergic asthma. This study uses knockout mice to demonstrate a novel function for the heterotrimeric G protein, G(q), in the regulation of pulmonary eosinophil recruitment. In the absence of G(q) signaling, eosinophils failed to accumulate in the lungs following allergen challenge. These studies demonstrate that the inhibition of eosinophil accumulation in the airways is attributed to the failure of hemopoietically derived cells to elaborate GM-CSF in the airways. The data suggest that activation of a G(q)-coupled receptor(s) on resident leukocytes in the lung elicits expression of GM-CSF, which, in turn, is required for allergen-induced pulmonary eosinophilia, identifying a novel pathway of eosinophil-associated effector functions leading to pulmonary pathology in diseases such as asthma.     

ATRIP oligomerization is required for ATR-dependent checkpoint signaling

The ATM and ATR kinases signal cell cycle checkpoint responses to DNA damage. Inactive ATM is an oligomer that is disrupted to form active monomers in response to ionizing radiation. We examined whether ATR is activated by a similar mechanism. We found that the ATRIP subunit of the ATR kinase and ATR itself exist as homooligomers in cells. We did not detect regulation of ATR or ATRIP oligomerization after DNA damage. The predicted coiled-coil domain of ATRIP is essential for ATRIP oligomerization, stable ATR binding, and accumulation of ATRIP at DNA lesions. Additionally, the ATRIP coiled-coil is also required for ATRIP to support ATR-dependent checkpoint signaling to Chk1. Replacing the ATRIP coiled-coil domain with a heterologous dimerization domain restored stable binding to ATR and localization to damage-induced intranuclear foci. Thus, the ATR-ATRIP complex exists in higher order oligomeric states within cells and ATRIP oligomerization is essential for its function.     

Autocrine VEGF signaling is required for vascular homeostasis

Vascular endothelial growth factor (VEGF) is essential for developmental and pathological angiogenesis. Here we show that in the absence of any pathological insult, autocrine VEGF is required for the homeostasis of blood vessels in the adult. Genetic deletion of vegf specifically in the endothelial lineage leads to progressive endothelial degeneration and sudden death in 55% of mutant mice by 25 weeks of age. The phenotype is manifested without detectable changes in the total levels of VEGF mRNA or protein, indicating that paracrine VEGF could not compensate for the absence of endothelial VEGF. Furthermore, wild-type, but not VEGF null, endothelial cells showed phosphorylation of VEGFR2 in the absence of exogenous VEGF. Activation of the receptor in wild-type cells was suppressed by small molecule antagonists but not by extracellular blockade of VEGF. These results reveal a cell-autonomous VEGF signaling pathway that holds significance for vascular homeostasis but is dispensable for the angiogenic cascade.     

TLR signaling is required for Salmonella typhimurium virulence

Toll-like receptors (TLRs) contribute to host resistance to microbial pathogens and can drive the evolution of virulence mechanisms. We have examined the relationship between host resistance and pathogen virulence using mice with a functional allele of the nramp-1 gene and lacking combinations of TLRs. Mice deficient in both TLR2 and TLR4 were highly susceptible to the intracellular bacterial pathogen Salmonella typhimurium, consistent with reduced innate immune function. However, mice lacking additional TLRs involved in S. typhimurium recognition were less susceptible to infection. In these TLR-deficient cells, bacteria failed to upregulate Salmonella pathogenicity island 2 (SPI-2) genes and did not form a replicative compartment. We demonstrate that TLR signaling enhances the rate of acidification of the Salmonella-containing phagosome, and inhibition of this acidification prevents SPI-2 induction. Our results indicate that S. typhimurium requires cues from the innate immune system to regulate virulence genes necessary for intracellular survival, growth, and systemic infection.     

BMP signaling is required for normal thymus development

The microenvironment of the thymus fosters the generation of a diverse and self-tolerant T cell repertoire from a pool of essentially random specificities. Epithelial as well as mesenchymal cells contribute to the thymic stroma, but little is known about the factors that allow for communication between the two cells types that shape the thymic microenvironment. In this study, we investigated the role of bone morphogenetic protein (BMP) signaling in thymus development. Transgenic expression of the BMP antagonist Noggin in thymic epithelial cells under the control of a Foxn1 promoter in the mouse leads to dysplastic thymic lobes of drastically reduced size that are ectopically located in the neck at the level of the hyoid bone. Interestingly, the small number of thymocytes in these thymic lobes develops with normal kinetics and shows a wild-type phenotype. Organ initiation of the embryonic thymic anlage in these Noggin transgenic mice occurs as in wild-type mice, but the tight temporal and spatial regulation of BMP4 expression is abrogated in subsequent differentiation stages. We show that transgenic Noggin blocks BMP signaling in epithelial as well as mesenchymal cells of the thymic anlage. Our data demonstrate that BMP signaling is crucial for thymus development and that it is the thymic stroma rather than developing thymocytes that depends on BMP signals.     

ERK signaling is required for eye-specific retino-geniculate segregation

In the mammalian visual system, retinal ganglion cell (RGC) projections from each eye, initially intermixed within the dorsal-lateral geniculate nucleus (dLGN), become segregated during the early stages of development, occupying distinct eye-specific layers. Electrical activity has been suggested to play a role in this process; however, the cellular mechanisms underlying eye-specific segregation are not yet defined. It is known that electrical activity is among the strongest activators of the extracellular signal-regulated kinase (ERK) pathway. Moreover, the ERK pathway is involved in the plasticity of neural connections during development. We examine the role of ERK in the segregation of retinal afferents into eye-specific layers in the dLGN. The activation of this signaling cascade was selectively blocked along the retino-thalamic circuitry by specific inhibitors, and the distribution of RGC fibers in the dLGN was studied. Our results demonstrate that the blockade of ERK signaling prevents eye-specific segregation in the dLGN, providing evidence that ERK pathway is required for the proper development of retino-geniculate connections. Of particular interest is the finding that ERK mediates this process both at the retinal and geniculate level.     

Fgf signaling is required for zebrafish tooth development

We have investigated fibroblast growth factor (FGF) signaling during the development of the zebrafish pharyngeal dentition with the goal of uncovering novel roles for FGFs in tooth development as well as phylogenetic and topographic diversity in the tooth developmental pathway. We found that the tooth-related expression of several zebrafish genes is similar to that of their mouse orthologs, including both epithelial and mesenchymal markers. Additionally, significant differences in gene expression between zebrafish and mouse teeth are indicated by the apparent lack of fgf8 and pax9 expression in zebrafish tooth germs. FGF receptor inhibition with SU5402 at 32 h blocked dental epithelial morphogenesis and tooth mineralization. While the pharyngeal epithelium remained intact as judged by normal pitx2 expression, not only was the mesenchymal expression of lhx6 and lhx7 eliminated as expected from mouse studies, but the epithelial expression of dlx2a, dlx2b, fgf3, and fgf4 was as well. This latter result provides novel evidence that the dental epithelium is a target of FGF signaling. However, the failure of SU5402 to block localized expression of pitx2 suggests that the earliest steps of tooth initiation are FGF-independent. Investigations of specific FGF ligands with morpholino antisense oligonucleotides revealed only a mild tooth shape phenotype following fgf4 knockdown, while fgf8 inhibition revealed only a subtle down-regulation of dental dlx2b expression with no apparent effect on tooth morphology. Our results suggest redundant FGF signals target the dental epithelium and together are required for dental morphogenesis. Further work will be required to elucidate the nature of these signals, particularly with respect to their origins and whether they act through the mesenchyme.     

Endothelial Rho signaling is required for monocyte transendothelial migration

Bacterial toxins affecting Rho activity in microvascular endothelial cells were employed to elucidate whether endothelial Rho participates in regulating the migration of monocytes across monolayers of cultured endothelial cells. Inactivation of Rho by the Clostridium C3 exoenzyme resulted in an increased adhesion of peripheral blood monocytes to the endothelium and a decreased rate of transendothelial monocyte migration. Cytotoxic necrotizing factor 1-mediated activation of endothelial Rho also reduced the rate of monocyte transmigration, but did not affect monocyte-endothelium adhesion. Thus, efficient leukocyte extravasation requires Rho signaling not only within the migrating leukocytes but also within the endothelial lining of the vessel wall.     

Palmitoylation is required for efficient Fas cell death signaling

Localization of the death receptor Fas to specialized membrane microdomains is crucial to Fas-mediated cell death signaling. Here, we report that the post-translational modification of Fas by palmitoylation at the membrane proximal cysteine residue in the cytoplasmic region is the targeting signal for Fas localization to lipid rafts, as demonstrated in both cell-free and living cell systems. Palmitoylation is required for the redistribution of Fas to actin cytoskeleton-linked rafts upon Fas stimulation and for the raft-dependent, ezrin-mediated cytoskeleton association, which is necessary for the efficient Fas receptor internalization, death-inducing signaling complex assembly and subsequent caspase cascade leading to cell death.     

Canonical wnt signaling is required for commissural axon guidance

Morphogens have been identified as guidance cues for postcrossing commissural axons in the spinal cord. Shh has a dual effect on postcrossing commissural axons: a direct repellent effect mediated by Hhip as a receptor, and an indirect effect by shaping a Wnt activity gradient. Wnts were shown to be attractants for postcrossing commissural axons in both chicken and mouse embryos. In mouse, the effects of Wnts on axon guidance were concluded to depend on the planar cell polarity (PCP) pathway. Canonical Wnt signaling was excluded based on the absence of axon guidance defects in mice lacking Lrp6 which is an obligatory coreceptor for Fzd in canonical Wnt signaling. In the loss‐of‐function studies reported here, we confirmed a role for the PCP pathway in postcrossing commissural axon guidance also in the chicken embryo. However, taking advantage of the precise temporal control of gene silencing provided by in ovo RNAi, we demonstrate that canonical Wnt signaling is also required for proper guidance of postcrossing commissural axons in the developing spinal cord. Thus, axon guidance does not seem to depend on any one of the classical Wnt signaling pathways but rather involve a network of Wnt receptors and downstream components. © 2015 Wiley Periodicals, Inc. Develop Neurobiol 76: 190–208, 2016