Frizzled3 inhibits Vangl2-Prickle3 association to establish planar cell polarity in the vertebrate neural plate.

The orientation of epithelial cells in the plane of the tissue, known as planar cell polarity (PCP), is regulated by interactions of asymmetrically localized PCP protein complexes. In the Xenopus neural plate, Van Gogh-like2 (Vangl2) and Prickle3 (Pk3) proteins form a complex at the anterior cell boundaries, but how this complex is regulated in vivo remains largely unknown. Here, we use proximity biotinylation and crosslinking approaches to show that Vangl2-Pk3 association is inhibited by Frizzled3 (Fz3, also known as Fzd3), a core PCP protein that is specifically expressed in the neuroectoderm and is essential for the establishment of PCP in this tissue. This inhibition required Fz3-dependent Vangl2 phosphorylaton. Consistent with our observations, the complex of Pk3 with nonphosphorylatable Vangl2 did not polarize in the neural plate. These findings provide evidence for in vivo regulation of Vangl2-Pk3 complex formation and localization by a Frizzled receptor.

Pinhead signaling regulates mesoderm heterogeneity via the FGF receptor-dependent pathway.

Among the three embryonic germ layers, the mesoderm plays a central role in the establishment of the vertebrate body plan. The mesoderm is specified by secreted signaling proteins from the FGF, Nodal, BMP and Wnt families. No new classes of extracellular mesoderm-inducing factors have been identified in more than two decades. Here, we show that the pinhead (pnhd) gene encodes a secreted protein that is essential for the activation of a subset of mesodermal markers in the Xenopus embryo. RNA sequencing revealed that many transcriptional targets of Pnhd are shared with those of the FGF pathway. Pnhd activity was accompanied by Erk phosphorylation and required FGF and Nodal but not Wnt signaling. We propose that during gastrulation Pnhd acts in the marginal zone to contribute to mesoderm heterogeneity via an FGF receptor-dependent positive feedback mechanism.

The involvement of PCP proteins in radial cell intercalations during Xenopus embryonic development.

The planar cell polarity (PCP) pathway orients cells in diverse epithelial tissues in Drosophila and vertebrate embryos and has been implicated in many human congenital defects and diseases, such as ciliopathies, polycystic kidney disease and malignant cancers. During vertebrate gastrulation and neurulation, PCP signaling is required for convergent extension movements, which are primarily driven by mediolateral cell intercalations, whereas the role for PCP signaling in radial cell intercalations has been unclear. In this study, we examine the function of the core PCP proteins Vangl2, Prickle3 (Pk3) and Disheveled in the ectodermal cells, which undergo radial intercalations during Xenopus gastrulation and neurulation. In the epidermis, multiciliated cell (MCC) progenitors originate in the inner layer, but subsequently migrate to the embryo surface during neurulation. We find that the Vangl2/Pk protein complexes are enriched at the apical domain of intercalating MCCs and are essential for the MCC intercalatory behavior. Addressing the underlying mechanism, we identified KIF13B, as a motor protein that binds Disheveled. KIF13B is required for MCC intercalation and acts synergistically with Vangl2 and Disheveled, indicating that it may mediate microtubule-dependent trafficking of PCP proteins necessary for cell shape regulation. In the neural plate, the Vangl2/Pk complexes were also concentrated near the outermost surface of deep layer cells, suggesting a general role for PCP in radial intercalation. Consistent with this hypothesis, the ectodermal tissues deficient in Vangl2 or Disheveled functions contained more cell layers than normal tissues. We propose that PCP signaling is essential for both mediolateral and radial cell intercalations during vertebrate morphogenesis. These expanded roles underscore the significance of vertebrate PCP proteins as factors contributing to a number of diseases, including neural tube defects, tumor metastases, and various genetic syndromes characterized by abnormal migratory cell behaviors.

GEF-H1 functions in apical constriction and cell intercalations and is essential for vertebrate neural tube closure.

Rho family GTPases regulate many morphogenetic processes during vertebrate development including neural tube closure. Here we report a function for GEF-H1/Lfc/ArhGEF2, a RhoA-specific guanine nucleotide exchange factor that functions in neurulation in Xenopus embryos. Morpholino-mediated depletion of GEF-H1 resulted in severe neural tube defects, which were rescued by GEF-H1 RNA. Lineage tracing of GEF-H1 morphants at different developmental stages revealed abnormal cell intercalation and apical constriction, suggesting that GEF-H1 regulates these cell behaviors. Molecular marker analysis documented defects in myosin II light chain (MLC) phosphorylation, Rab11 and F-actin accumulation in GEF-H1-depleted cells. In gain-of-function studies, overexpressed GEF-H1 induced Rho-associated kinase-dependent ectopic apical constriction - marked by apical accumulation of phosphorylated MLC, γ-tubulin and F-actin in superficial ectoderm - and stimulated apical protrusive activity of deep ectoderm cells. Taken together, our observations newly identify functions of GEF-H1 in morphogenetic movements that lead to neural tube closure.

Myocardin-related transcription factors regulate morphogenetic events in vertebrate embryos by controlling F-actin organization and apical constriction.

Myocardin-related transcription factors (Mrtfa and Mrtfb), also known as megakaryoblastic leukemia proteins (Mkl1/MAL and Mkl2), associate with serum response factor (Srf) to regulate transcription in response to actin dynamics, however, the functions of Mrtfs in early vertebrate embryos remain largely unknown. Here we document the requirement of Mrtfs for blastopore closure at gastrulation and neural plate folding in Xenopus early embryos. Both stimulation and inhibition of Mrtf activity caused similar gross morphological phenotypes, yet the effects on F-actin distribution and cell behavior were different. Suppressing Mrtf-dependent transcription reduced overall F-actin levels and inhibited apical constriction during gastrulation and neurulation. By contrast, constitutively active Mrtf caused tricellular junction remodeling and induced apical constriction in superficial ectoderm. The underlying mechanism appeared distinct from the one utilized by known apical constriction inducers. We propose that the regulation of apical constriction is among the primary cellular responses to Mrtf. Our findings highlight a dedicated role of specific transcription factors, Mrtfs, in early morphogenetic processes.

Centrosomal localization of Diversin and its relevance to Wnt signaling.

Wnt pathways regulate many developmental processes, including cell-fate specification, cell polarity, and cell movements during morphogenesis. The subcellular distribution of pathway mediators in specific cellular compartments might be crucial for the selection of pathway targets and signaling specificity. We find that the ankyrin-repeat protein Diversin, which functions in different Wnt signaling branches, localizes to the centrosome in Xenopus ectoderm and mammalian cells. Upon stimulation with Wnt ligands, the centrosomal distribution of Diversin is transformed into punctate cortical localization. Also, Diversin was recruited by Frizzled receptors to non-homogeneous Dishevelled-containing cortical patches. Importantly, Diversin deletion constructs, which did not localize to the centrosome, failed to efficiently antagonize Wnt signaling. Furthermore, a C-terminal construct that interfered with Diversin localization inhibited Diversin-mediated beta-catenin degradation. These observations suggest that the centrosomal localization of Diversin is crucial for its function in Wnt signaling.

Pinhead antagonizes Admp to promote notochord formation.

Dorsoventral patterning of a vertebrate embryo critically depends on the activity of Smad1 that mediates signaling by BMP proteins, anti-dorsalizing morphogenetic protein (Admp), and their antagonists. Pinhead (Pnhd), a cystine-knot-containing secreted protein, is expressed in the ventrolateral mesoderm during Xenopus gastrulation; however, its molecular targets and signaling mechanisms have not been fully elucidated. Our mass spectrometry-based screen of the gastrula secretome identified Admp as Pnhd-associated protein. We show that Pnhd binds Admp and inhibits its ventralizing activity by reducing Smad1 phosphorylation and its transcriptional targets. Importantly, Pnhd depletion further increased phospho-Smad1 levels in the presence of Admp. Furthermore, Pnhd synergized with Chordin and a truncated BMP4 receptor in the induction of notochord markers in ectoderm cells, and Pnhd-depleted embryos displayed notochord defects. Our findings suggest that Pnhd binds and inactivates Admp to promote notochord development. We propose that the interaction between Admp and Pnhd refines Smad1 activity gradients during vertebrate gastrulation.

Identification of the centrosomal maturation factor SSX2IP as a Wtip-binding partner by targeted proximity biotinylation.

Wilms tumor-1-interacting protein (Wtip) is a LIM-domain-containing adaptor that links cell junctions with actomyosin complexes and modulates actomyosin contractility and ciliogenesis in Xenopus embryos. The Wtip C-terminus with three LIM domains associates with the actin-binding protein Shroom3 and modulates Shroom3-induced apical constriction in ectoderm cells. By contrast, the N-terminal domain localizes to apical junctions in the ectoderm and basal bodies in skin multiciliated cells, but its interacting partners remain largely unknown. Targeted proximity biotinylation (TPB) using anti-GFP antibody fused to the biotin ligase BirA identified SSX2IP as a candidate protein that binds GFP-WtipN. SSX2IP, also known as Msd1 or ADIP, is a component of cell junctions, centriolar satellite protein and a targeting factor for ciliary membrane proteins. WtipN physically associated with SSX2IP and the two proteins readily formed mixed aggregates in overexpressing cells. By contrast, we observed only partial colocalization of full length Wtip and SSX2IP, suggesting that Wtip adopts a 'closed' conformation in the cell. Furthermore, the double depletion of Wtip and SSX2IP in early embryos uncovered the functional interaction of the two proteins during neural tube closure. Our results suggest that the association of SSX2IP and Wtip is essential for cell junction remodeling and morphogenetic processes that accompany neurulation. We propose that TPB can be a general approach that is applicable to other GFP-tagged proteins.

Oxidative Synthesis of Acid Blue 7 Dye Catalyzed by CuO/Silicotungstic Acid in Water-Phase.

A catalytic oxidation reaction for Acid Blue 7 dye synthesis was evaluated in water. Without lead oxide or manganese oxide derivatives as oxidants, polyoxometalate catalysts were investigated to reduce the usage of harmful heavy metal. A catalyst was prepared by mixing silicotungstic acid with copper oxide, and aqueous hydrogen peroxide (30%) was used as an oxidizing agent. This reaction proceeded to produce Acid Blue 7 from the corresponding leuco acid after 45 min at 95 °C and was viable for a 10 g-scale synthesis.

Both the RGS domain and the six C-terminal amino acids of mouse Axin are required for normal embryogenesis.

Axin is a negative regulator of canonical Wnt signaling, which promotes the degradation of beta-catenin, the major effector in this signaling cascade. While many protein-binding domains of Axin have been identified, their significance has not been evaluated in vivo. Here, we report the generation and analysis of mice carrying modified Axin alleles in which either the RGS domain or the six C-terminal amino acids (C6 motif) were deleted. The RGS domain is required for APC-binding, while the C6 motif has been implicated in the activation of c-Jun N-terminal kinase, but is not required for the effects of Axin on the Wnt/beta-catenin pathway, in vitro. Both mutant Axin alleles caused recessive embryonic lethality at E9.5-E10.5, with defects indistinguishable from those caused by a null allele. As Axin-DeltaRGS protein was produced at normal levels, its inability to support embryogenesis confirms the importance of interactions between Axin and APC. In contrast, Axin-DeltaC6 protein was expressed at only 25-30% of the normal level, which may account for the recessive lethality of this allele. Furthermore, many Axin(DeltaC6/DeltaC6) embryos that were heterozygous for a beta-catenin null mutation survived to term, demonstrating that early lethality was due to failure to negatively regulate beta-catenin.

SAXS and SANS observations of abnormal aggregation of human alpha-crystallin.

Aggregation states of human alpha-crystallins are observed complementarily using small-angle X-ray and small-angle neutron scatterings (SAXS and SANS). Infant alpha-crystallin is almost a monodispersed system of the aggregates with gyration radius of ca. 60 A, which is a normal aggregate. On the other hand, the aged and cataract alpha-crystallins have not only the normal but also the larger aggregates. In the aged alpha-crystallin, the normal aggregate is a major component, but in the cataract alpha-crystallin the larger ones are dominant. Both alpha A- and alpha B-crystallins, which are subunits of alpha-crystallin, also form an aggregate with the size close to the normal aggregate. Under UV irradiation, only aggregates of alpha B-crystallin undergo further aggregation. Therefore, considering increase of ratio of alpha B-crystallin in the aggregate of alpha-crystallin as aging, the abnormal aggregation (formation of the huge aggregates) mainly results in the further aggregation of alpha B-crystallin caused by external stresses.

Isolation of nanobodies against Xenopus embryonic antigens using immune and non-immune phage display libraries.

The use of Xenopus laevis as a model for vertebrate developmental biology is limited by a lack of antibodies specific for embryonic antigens. This study evaluated the use of immune and non-immune phage display libraries for the isolation of single domain antibodies, or nanobodies, with specificities for Xenopus embryonic antigens. The immune nanobody library was derived from peripheral blood lymphocyte RNA obtained from a llama immunized with Xenopus gastrula homogenates. Screening this library by immunostaining of embryonic tissues with pooled periplasmic material and sib-selection led to the isolation of several monoclonal phages reactive with the cytoplasm and nuclei of gastrula cells. One antigen recognized by a group of nanobodies was identified using a reverse proteomics approach as nucleoplasmin, an abundant histone chaperone. As an alternative strategy, a semi-synthetic non-immune llama nanobody phage display library was panned on highly purified Xenopus proteins. This proof-of-principle approach isolated monoclonal nanobodies that specifically bind Nuclear distribution element-like 1 (Ndel1) in multiple immunoassays. Our results suggest that immune and non-immune phage display screens on crude and purified embryonic antigens can efficiently identify nanobodies useful to the Xenopus developmental biology community.

Partial pair correlation functions of low-density supercritical water determined by neutron diffraction with the H/D isotopic substitution method.

Neutron diffraction measurements were carried out on H/ D isotopically substituted water in the low-density supercritical condition (T = 673 K, P = 26.3 MPa, and rho = 0.0068 molecules.A-3) in order to obtain direct information on the intermolecular partial structure functions, gHH inter(r), gOH inter(r), and gOO inter(r). In correspondence to the high-density supercritical water previously reported, the intermolecular nearest neighbor peaks in gHH inter(r), gOH inter(r), and gOO inter(r) are diffuse compared with the ambient ones. The nearest neighbor O...O distance (3.3 A) and its coordination number (2.6) were determined from the observed gOO inter(r). These results indicate that the orientational correlation between neighboring water molecules is considerably lost in low-density supercritical water. Small clusters involving a few water molecules are preferentially formed in low-density supercritical water, which is consistent with results obtained by previous IR and NMR studies.

Structural evolution of human recombinant alpha B-crystallin under UV irradiation.

External stresses cause certain proteins to lose their regular structure and aggregate. In order to clarify this abnormal aggregation process, a structural evolution of human recombinant alphaB-crystallin under UV irradiation was observed with in situ small-angle neutron scattering. The abnormal aggregation process was identified to fall in three time zones: incubation, aggregation, and saturation. During the incubation time, the size of aggregates was almost unchanged but a deformation in the local structure was developing. After the incubation time, abnormal aggregation proceed. When the volume of the aggregates reached around twice the size as that of the initial aggregates, the aggregation rate slowed down, which marked the onset of saturation.

Structural difference between liquidlike and gaslike phases in supercritical fluid.

Density fluctuation structures of supercritical carbon dioxide along the isothermal and isochoric lines were observed with small-angle neutron scattering (SANS). All the scattering intensities in the low-Q range were well described with the Ornstein-Zernike (OZ) equation. It was confirmed that there exists a locus where the OZ correlation length and scattering intensity at Q=0 exhibit extrema on the isothermal lines: this locus, named the ridge, was interpreted as the boundary by which the supercritical state is divided into liquidlike and gaslike phases. In order to clarify the difference of the fluctuation structure between the liquidlike and the gaslike phases, a real-space molecular distribution was obtained with a reverse Monte Carlo (RMC) method. Number density distributions of CO2 molecules at all measured states were calculated with the real-space molecular distributions obtained. In addition, the statistical parameters of the number density distributions, the standard deviations, and the skewnesses, were examined. The standard deviations of the number density distributions almost coincide with the results of the OZ analysis. On the other hand, the skewnesses, which describe the asymmetric nature of the number density distribution, clearly showed a difference between the two phases: the skewness became negative in the liquidlike phase, positive in the gaslike phase, and almost zero at the nearest state to the ridge in all isotherms. It was proved with simple equations of statistical mechanics that the skewness is described as the first differential of the magnitude of the density fluctuation with respect to the pressure. We conclude that the skewness, obtained with a RMC analysis for SANS data, is an important structural parameter distinguishing between the liquidlike and gaslike phases.

Frizzled receptors activate a novel JNK-dependent pathway that may lead to apoptosis.

Extracellular Wnt ligands and their receptors of the Frizzled family control cell fate, proliferation, and polarity during metazoan development. Frizzled signaling modulates target gene expression through a beta-catenin-dependent pathway, functions to establish planar cell polarity in Drosophila epithelia, and activates convergent extension movements and intracellular Ca(2+) signaling in frog and fish embryos. Here, we report that a Frizzled receptor, Xenopus Frizzled 8 (Xfz8), activates c-Jun N-terminal kinases (JNK) and triggers rapid apoptotic cell death in gastrulating Xenopus embryos. This activity of Xfz8 required the cytoplasmic tail of the receptor and was blocked by a dominant inhibitor of JNK. Moreover, the cytoplasmic tail of Xfz8 targeted to the membrane was sufficient for activation of JNK and apoptosis. The apoptotic signaling was shared by a specific subset of Frizzled receptors, was inhibited by Wnt5a, and occurred in a Dishevelled- and T cell factor (TCF)-independent manner. Thus, our experiments identify a novel Frizzled-dependent signaling pathway, which involves JNK and differs from the beta-catenin-dependent and convergent extension pathways.

Structural analysis of polyelectrolyte film absorbing metal ion by SAXS utilizing with X-ray anomalous dispersion effect.

A distribution of Cu ions in polyelectrolyte film (Nafion) is directly observed with a small-angle X-ray scattering (SAXS) method utilizing an X-ray anomalous dispersion effect. A partial structure factor of the Cu ions, GAA(q), can be derived from the SAXS profiles obtained by scanning the incident X-ray energy around the Cu K absorption edge. GAA(q) has two peaks, indicating that the Cu ions hierarchically distribute in Nafion film. In addition, a standard SAXS also shows that Nafion film has a hierarchical structure. These results mean that the Cu ions locate in the domain where the hydrophilic bases aggregate.

Expression of Neural Antigens in Normal Xenopus Embryos and Induced Explants: (Xenopus laevis/neural antigens/monoclonal antibodies/neural induction/neural differentiation).

Monoclonal antibodies were raised against neural tissues of Xenopus larvae. Three monoclonal antibodies, named NEU-1, NEU-3, and NEU-4, were specific for neural tissue and first bound to neural cells at stage 25 after neural tube formation (NEU-1 and NEU-3) or at stage 31 (NEU-4). These antibodies bound to differentiating neural cells, but not to germinal neuroepithelial cells. NEU-1 and NEU-3 recognized antigens in cell bodies as well as neural fibers of neural cells, and these antigens were distributed throughout the central nervous system. NEU-4 bound to antigens in granular materials in neural cells, and these antigens were present in head and trunk regions but not in the tail region. These three antibodies were used as neural markers in two types of induction experiments, in which 1) the animal pole region and the dorsal blastopore lip from stage-10 gastrulae were combined, or 2) the animal pole region and the vegetal pole region from stage-8 blastulae were combined. In both experiments, most conjugated explants expressed the NEU-1, NEU-3, and NEU-4 antigens, although the expression of NEU-4 antigen was delayed compared with those of the NEU-1 and NEU-3 antigens. These results show that these antibodies are useful as markers in neural induction experiments.

Homoiogenetic Neural Induction in Xenopus Chimeric Explants: (Xenopus laevis/Xenopus borealis/chimeric explants/homoiogenetic neural induction/tissue-specific monoclonal antibodies).

We previously raised monoclonal antibodies specific for epidermis (7) and neural tissue (8) of Xenopus for use as markers of tissue differentiation in induction experiments (8). Here we have used these monoclonal antibodies to examine homoiogenetic neural induction, by which cells induced to differentiate to neural tissues can in turn induce competent ectoderm to do the same. Presumptive anterior neural plate excised from late gastrulae of Xenopus laevis was conjugated with competent ectoderm from the initial gastrula of Xenopus borealis, either side by side or with their inner surfaces together. The chimeric explants enabled us to distinguish induced neural tissues from inducing neural tissues. In both types of explant, neural tissues identified by the neural tissue-specific antibody, NEU-1, were induced in the competent ectoderm by the presumptive anterior neural plate. The results suggest that homoiogenetic neural induction does occur in Xenopus embryos.

Immuno-Localization of DEAD Family Proteins in Germ Line Cells of Xenopus Embryos.

In order to investigate whether a vasa-like protein is present in germ line cells of Xenopus, antibodies were produced which react specifically with synthetic oligopeptides of sequences from near the N- or C-termini or with one including the DEAD box of the Drosophila vasa protein. Only the antibody against the oligopeptide including the DEAD box reacted strongly with germ plasm (GP) or with cytoplasm of germ line cells of Xenopus embryos by immunofluorescence microscopy. By immunoelectron microscopy, the antibody was demonstrated to react with the GP-specific structure, germinal granules, in cleaving embryos, and with their derivatives in the germ line cells of embryos at stages extending from gastrula to feeding tadpole. It also reacted with mitochondria not only in the GP and the germ line cells but also in somatic cells, and with myofibrils in muscle cells. By Western blotting, the antibody was shown to react with several bands of Mr 42-69 ± 103 in protein samples from Xenopus embryos. In samples from Drosophila ovaries, it reacted with a Mr 71 ± 103 band which was probably the vasa protein. This indicates the possibility that Xenopus embryos contain several DEAD family proteins. One of these is present on germinal granules, resembling the vasa protein on polar granules of Drosophila.