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.

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.

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.

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.

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.

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.

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.

Expression cloning of camelid nanobodies specific for Xenopus embryonic antigens.

Developmental biology relies heavily on the use of conventional antibodies, but their production and maintenance involves significant effort. Here we use an expression cloning approach to identify variable regions of llama single domain antibodies (known as nanobodies), which recognize specific embryonic antigens. A nanobody cDNA library was prepared from lymphocytes of a llama immunized with Xenopus embryo lysates. Pools of bacterially expressed cDNAs were sib-selected for the ability to produce specific staining patterns in gastrula embryos. Three different nanobodies were isolated: NbP1 and NbP3 stained yolk granules, while the reactivity of NbP7 was predominantly restricted to the cytoplasm and the cortex. The isolated nanobodies recognized specific protein bands in immunoblot analysis. A reverse proteomic approach identified NbP1 target antigen as EP45/Seryp, a serine protease inhibitor. Given the unique stability of nanobodies and the ease of their expression in diverse systems, we propose that nanobody cDNA libraries represent a promising resource for molecular markers for developmental biology.

Role of Rab11 in planar cell polarity and apical constriction during vertebrate neural tube closure.

Epithelial folding is a critical process underlying many morphogenetic events including vertebrate neural tube closure, however, its spatial regulation is largely unknown. Here we show that during neural tube formation Rab11-positive recycling endosomes acquire bilaterally symmetric distribution in the Xenopus neural plate, being enriched at medial apical cell junctions. This mediolateral polarization was under the control of planar cell polarity (PCP) signalling, was necessary for neural plate folding and was accompanied by the polarization of the exocyst component Sec15. Our further experiments demonstrate that similar PCP-dependent polarization of Rab11 is essential for ectopic apical constriction driven by the actin-binding protein Shroom and during embryonic wound repair. We propose that anisotropic membrane trafficking has key roles in diverse morphogenetic behaviours of individual cells and propagates in a tissue by a common mechanism that involves PCP.

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.

Distribution dynamics of recombinant Lactobacillus in the gastrointestinal tract of neonatal rats.

One approach to deliver therapeutic agents, especially proteins, to the gastro-intestinal (GI) tract is to use commensal bacteria as a carrier. Genus Lactobacillus is an attractive candidate for use in this approach. However, a system for expressing exogenous proteins at a high level has been lacking in Lactobacillus. Moreover, it will be necessary to introduce the recombinant Lactobacillus into the GI tract, ideally by oral administration. Whether orally administered Lactobacillus can reach and reside in the GI tract has not been explored in neonates. In this study, we have examined these issues in neonatal rats. To achieve a high level of protein expression in Lactobacillus, we tested the impact of three promoters and two backbones on protein expression levels using mRFP1, a red fluorescent protein, as a reporter. We found that a combination of an L-lactate dehydrogenase (ldhL) promoter of Lactobacillus sakei with a backbone from pLEM415 yielded the highest level of reporter expression. When this construct was used to transform Lactobacillus casei, Lactobacillus delbrueckii and Lactobacillus acidophilus, high levels of mRFP1 were detected in all these species and colonies of transformed Lactobacillus appeared pink under visible light. To test whether orally administered Lactobacillus can be retained in the GI tract of neonates, we fed the recombinant Lactobacillus casei to neonatal rats. We found that about 3% of the bacteria were retained in the GI tract of the rats at 24 h after oral feeding with more recombinant Lactobacillus in the stomach and small intestine than in the cecum and colon. No mortality was observed throughout this study with Lactobacillus. In contrast, all neonatal rats died within 24 hours after fed with transformed E. coli. Taken together, our results indicate that Lactobacillus has the potential to be used as a vehicle for the delivery of therapeutic agents to neonates.

The phosphatase-transcription activator EYA1 is targeted by anaphase-promoting complex/Cdh1 for degradation at M-to-G1 transition.

The phosphatase and transactivator EYA family proteins are overexpressed in many cancer cell lines and are abundantly distributed in undifferentiated cells during development. Loss-of-function studies have shown that EYA1 is required for cell proliferation and survival during mammalian organogenesis. However, how EYA1 is regulated during development is unknown. Here, we report that EYA1 is regulated throughout the cell cycle via ubiquitin-mediated proteolysis. The level of EYA1 protein fluctuates in the cell cycle, peaking during mitosis and dropping drastically as cells exit into G(1). We found that EYA1 is efficiently degraded during mitotic exit in a Cdh1-dependent manner and that these two proteins physically interact. Overexpression of Cdh1 reduces the protein levels of ectopically expressed or endogenous EYA1, whereas depletion of Cdh1 by RNA interference stabilizes the EYA1 protein. Together, our results indicate that anaphase-promoting complex/cyclosome (APC/C)-Cdh1 specifically targets EYA1 for degradation during M-to-G(1) transition, failure of which may compromise cell proliferation and survival.

Identification of Nedd4 E3 ubiquitin ligase as a binding partner and regulator of MAK-V protein kinase.

MAK-V/Hunk is a scantily characterized AMPK-like protein kinase. Recent findings identified MAK-V as a pro-survival and anti-apoptotic protein and revealed its role in embryonic development as well as in tumorigenesis and metastasis. However molecular mechanisms of MAK-V action and regulation of its activity remain largely unknown. We identified Nedd4 as an interaction partner for MAK-V protein kinase. However, this HECT-type E3 ubiquitin ligase is not involved in the control of MAK-V degradation by the ubiquitin-proteasome system that regulates MAK-V abundance in cells. However, Nedd4 in an ubiquitin ligase-independent manner rescued developmental defects in Xenopus embryos induced by MAK-V overexpression, suggesting physiological relevance of interaction between MAK-V and Nedd4. This identifies Nedd4 as the first known regulator of MAK-V function.

Regulation of basal body and ciliary functions by Diversin.

The centrosome is essential for the formation of the cilia and has been implicated in cell polarization and signaling during early embryonic development. A number of Wnt pathway components were found to localize at the centrosome, but how this localization relates to their signaling functions is unclear. In this study, we assessed a role for Diversin, a putative Wnt pathway mediator, in developmental processes that involve cilia. We find that Diversin is specifically localized to the basal body compartment near the base of the cilium in Xenopus multi-ciliated skin cells. Overexpression of Diversin RNA disrupted basal body polarization in these cells, suggesting that tightly regulated control of Diversin levels is crucial for this process. In cells depleted of endogenous Diversin, basal body structure appeared abnormal and this was accompanied by disrupted polarity, shortened or absent cilia and defective ciliary flow. These results are consistent with the involvement of Diversin in processes that are related to the acquisition of cell polarity and require ciliary functions.

Polarized translocation of fluorescent proteins in Xenopus ectoderm in response to Wnt signaling.

Cell polarity is a fundamental property of eukaryotic cells that is dynamically regulated by both intrinsic and extrinsic factors during embryonic development. One of the signaling pathways involved in this regulation is the Wnt pathway, which is used many times during embryogenesis and critical for human disease. Multiple molecular components of this pathway coordinately regulate signaling in a spatially-restricted manner, but the underlying mechanisms are not fully understood. Xenopus embryonic epithelial cells is an excellent system to study subcellular localization of various signaling proteins. Fluorescent fusion proteins are expressed in Xenopus embryos by RNA microinjection, ectodermal explants are prepared and protein localization is evaluated by epifluorescence. In this experimental protocol we describe how subcellular localization of Diversin, a cytoplasmic protein that has been implicated in signaling and cell polarity determination is visualized in Xenopus ectodermal cells to study Wnt signal transduction. Coexpression of a Wnt ligand or a Frizzled receptor alters the distribution of Diversin fused with red fluorescent protein, RFP, and recruits it to the cell membrane in a polarized fashion. This ex vivo protocol should be a useful addition to in vitro studies of cultured mammalian cells, in which spatial control of signaling differs from that of the intact tissue and is much more difficult to analyze.

Regulation of TCF3 by Wnt-dependent phosphorylation during vertebrate axis specification.

A commonly accepted model of Wnt/ß-catenin signaling involves target gene activation by a complex of ß-catenin with a T-cell factor (TCF) family member. TCF3 is a transcriptional repressor that has been implicated in Wnt signaling and plays key roles in embryonic axis specification and stem cell differentiation. Here we demonstrate that Wnt proteins stimulate TCF3 phosphorylation in gastrulating Xenopus embryos and mammalian cells. This phosphorylation event involves ß-catenin-mediated recruitment of homeodomain-interacting protein kinase 2 (HIPK2) to TCF3 and culminates in the dissociation of TCF3 from a target gene promoter. Mutated TCF3 proteins resistant to Wnt-dependent phosphorylation function as constitutive inhibitors of Wnt-mediated activation of Vent2 and Cdx4 during anteroposterior axis specification. These findings reveal an alternative in vivo mechanism of Wnt signaling that involves TCF3 phosphorylation and subsequent derepression of target genes and link this molecular event to a specific developmental process.

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.

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.

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.