ccl19 and ccl21 affect cell movements and differentiation in early Xenopus development.

We characterized Xenopus laevis C-C motif chemokine ligand 19.L (ccl19.L) and C-C motif chemokine ligand 21.L (ccl21.L) during early Xenopus embryogenesis. The temporal and spatial expression patterns of ccl19.L and ccl21.L tended to show an inverse correlation, except that the expression level was higher in the dorsal side at the gastrula stage. For example, even at the dorsal sector of the gastrulae, ccl19.L was expressed in the axial region and ccl21.L was expressed in the paraxial region. Dorsal overexpression of ccl19.L and ccl21.L and knockdown of Ccl19.L and Ccl21.L inhibited gastrulation, but their functions were different in cell behaviors during morphogenesis. Observation of Keller sandwich explants revealed that overexpression of both ccl19.L and ccl21.L and knockdown of Ccl21.L inhibited the convergent extension movements, while knockdown of Ccl19.L did not. ccl19.L-overexpressing explants attracted cells at a distance and ccl21.L-overexpressing explants attracted neighboring cells. Ventral overexpression of ccl19.L and ccl21.L induced secondary axis-like structures and chrd.1 expression at the ventral side. Upregulation of chrd.1 was induced by ligand mRNAs through ccr7.S. Knockdown of Ccl19.L and Ccl21.L inhibited gastrulation and downregulated chrd.1 expression at the dorsal side. The collective findings indicate that ccl19.L and ccl21.L might play important roles in morphogenesis and dorsal-ventral patterning during early embryogenesis in Xenopus.

maea affects head formation through ß-catenin degradation during early Xenopus laevis development.

Canonical Wnt signalling plays important roles in early embryogenesis, such as axis formation due to its activation and head formation due to its inhibition. ß-catenin protein stability is a key factor in canonical Wnt signalling. Several E3 ubiquitin ligases contribute to ß-catenin degradation through the ubiquitin/proteasome system. We characterised an E3 ubiquitin ligase gene, Xenopus laevis macrophage erythroblast attacher (maea), during early development. maea transcripts were ubiquitously detected in early embryos. The expression levels of the Wnt target genes nodal homolog 3, gene 1 (nodal3.1), and siamois homeodomain 1 (sia1), which were induced by injection with ß-catenin mRNA, were reduced by maea.S mRNA co-injection. maea.S overexpression at the anterior dorsal region enlarged head structures, whereas Maea knockdown interfered with head formation in Xenopus embryos. Maea.S decreased and ubiquitinated ß-catenin protein. ß-catenin-4KRs protein, which mutated the four lysine (K) residues known as ubiquitinated sites to arginine (R) residues, was also ubiquitinated and degraded by Maea.S. These findings suggest that Maea contributes to ß-catenin degradation by ubiquitination of unknown lysine residues in early Xenopus development.

ccr7 affects both morphogenesis and differentiation during early Xenopus embryogenesis.

Chemokines play important roles in early embryogenesis, including morphogenesis and cell differentiation, before the immune system is established. We characterized Xenopus laevis CC-type chemokine receptor 7 S (ccr7.S) to clarify its role during early development. ccr7 transcripts were detected ubiquitously in early embryos. Dorsal overexpression of ccr7.S inhibited gastrulation, and ccr7.S mRNA-injected embryos had short axes and widely opened neural folds. Because the Keller sandwich explants of the injected embryos elongated well, ccr7.S might affect cell migration, but not convergent extension movements. Ventral ccr7.S overexpression induced secondary axes and chrd.1 upregulation in gastrula-stage embryos. Animal cap assays showed increased expression of neural and cement gland marker genes at later stages. Ccr7.S knockdown reduced chrd.1 expression and inhibited gastrulation at the dorsal side. Our findings suggest that ccr7.S plays important roles in morphogenetic movement and cell differentiation.

Preparation of three-notochord explants for imaging analysis of the cell movements of convergent extension during early Xenopus morphogenesis.

We describe a method of generating three-notochord explants to analyze the cell movements of convergent extension (CE) during Xenopus laevis gastrulation and neurulation. This method uses standard microsurgical techniques under a fluorescence stereomicroscope to combine notochordal sectors of gastrulae, side by side (lateral surfaces apposed) into a single explant. Three-notochord explants cultured on bovine serum albumin (BSA)-coated glass converged mediolaterally and extended in the anterior-posterior direction. The individual notochordal cells showed the mediolaterally oriented, bipolar tractional motility and the resulting mediolaterally oriented cell intercalation characteristic of CE, thereby reproducing both the in vivo tissue and the cell movements in an explant. Image analysis of three-notochord explants reveals the effects of overexpressions or knockdowns of genes, of manipulation of the extracellular matrix, and of exposure to chemical reagents on morphogenesis during gastrulation and neurulation, compared with control explants. Moreover, since three-notochord explants provide two zones of cell intercalation between notochords, individual cell behaviors between notochords of different characteristics and experimental treatments can be observed at the same time.

WNK regulates Wnt signalling and ß-Catenin levels by interfering with the interaction between ß-Catenin and GID.

ß-Catenin is an important component of the Wnt signalling pathway. As dysregulation or mutation of this pathway causes many diseases, including cancer, the ß-Catenin level is carefully regulated by the destruction complex in the Wnt signalling pathway. However, the mechanisms underlying the regulation of ß-Catenin ubiquitination and degradation remain unclear. Here, we find that WNK (With No Lysine [K]) kinase is a potential regulator of the Wnt signalling pathway. We show that WNK protects the interaction between ß-Catenin and the Glucose-Induced degradation Deficient (GID) complex, which includes an E3 ubiquitin ligase targeting ß-Catenin, and that WNK regulates the ß-Catenin level. Furthermore, we show that WNK inhibitors induced ß-Catenin degradation and that one of these inhibitors suppressed xenograft tumour development in mice. These results suggest that WNK is a previously unrecognized regulator of ß-Catenin and a therapeutic target of cancer.

The Lhx1-Ldb1 complex interacts with Furry to regulate microRNA expression during pronephric kidney development.

The molecular events driving specification of the kidney have been well characterized. However, how the initial kidney field size is established, patterned, and proportioned is not well characterized. Lhx1 is a transcription factor expressed in pronephric progenitors and is required for specification of the kidney, but few Lhx1 interacting proteins or downstream targets have been identified. By tandem-affinity purification, we isolated FRY like transcriptional coactivator (Fryl), one of two paralogous genes, fryl and furry (fry), have been described in vertebrates. Both proteins were found to interact with the Ldb1-Lhx1 complex, but our studies focused on Lhx1/Fry functional roles, as they are expressed in overlapping domains. We found that Xenopus embryos depleted of fry exhibit loss of pronephric mesoderm, phenocopying the Lhx1-depleted animals. In addition, we demonstrated a synergism between Fry and Lhx1, identified candidate microRNAs regulated by the pair, and confirmed these microRNA clusters influence specification of the kidney. Therefore, our data shows that a constitutively-active Ldb1-Lhx1 complex interacts with a broadly expressed microRNA repressor, Fry, to establish the kidney field.

Roles of Xenopus chemokine ligand CXCLh (XCXCLh) in early embryogenesis.

Several chemokine molecules control cell movements during early morphogenesis. However, it is unclear whether chemokine molecules affect cell fate. Here, we identified and characterized the CXC-type chemokine ligand in Xenopus laevis, Xenopus CXCLh (XCXCLh), during early embryogenesis. XCXCLh is expressed in the dorsal vegetal region at the gastrula stage. Both overexpression and knockdown of XCXCLh in the dorsal region inhibited gastrulation. XCXCLh contributed to the attraction of mesendodermal cells and accelerated the reassembly of scratched culture cells. Also, XCXCLh contributed to early endodermal induction. Overexpression of VegTmRNA or high concentrations of calcium ions induced XCXCLh expression. XCXCLh may play roles in both cell movements and differentiation during early Xenopus embryogenesis.

High variability of expression profiles of homeologous genes for Wnt, Hh, Notch, and Hippo signaling pathways in Xenopus laevis.

Cell signaling pathways, such as Wnt, Hedgehog (Hh), Notch, and Hippo, are essential for embryogenesis, organogenesis, and tissue homeostasis. In this study, we analyzed 415 genes involved in these pathways in the allotetraploid frog, Xenopus laevis. Most genes are retained in two subgenomes called L and S (193 homeologous gene pairs and 29 singletons). This conservation rate of homeologs is much higher than that of all genes in the X. laevis genome (86.9% vs 60.2%). Among singletons, 24 genes are retained in the L subgenome, a rate similar to the average for all genes (82.8% vs 74.6%). In addition, as general components of signal transduction, we also analyzed 32 heparan sulfate proteoglycan (HSPG)-related genes and eight TLE/Groucho transcriptional corepressors-related genes. In these gene sets, all homeologous pairs have been retained. Transcriptome analysis using RNA-seq data from developmental stages and adult tissues demonstrated that most homeologous pairs of signaling components have variable expression patterns, in contrast to the conservative expression profiles of homeologs for transcription factors. Our results indicate that homeologous gene pairs for cell signaling regulation have tended to become subfunctionalized after allotetraploidization. Diversification of signaling pathways by subfunctionalization of homeologs may enhance environmental adaptability. These results provide insights into the evolution of signaling pathways after polyploidization.

Selective inhibition of the kinase DYRK1A by targeting its folding process.

Autophosphorylation of amino-acid residues is part of the folding process of various protein kinases. Conventional chemical screening of mature kinases has missed inhibitors that selectively interfere with the folding process. Here we report a cell-based assay that evaluates inhibition of a kinase at a transitional state during the folding process and identify a folding intermediate-selective inhibitor of dual-specificity tyrosine-phosphorylation-regulated kinase 1A (DYRK1A), which we refer to as FINDY. FINDY suppresses intramolecular autophosphorylation of Ser97 in DYRK1A in cultured cells, leading to its degradation, but does not inhibit substrate phosphorylation catalysed by the mature kinase. FINDY also suppresses Ser97 autophosphorylation of recombinant DYRK1A, suggesting direct inhibition, and shows high selectivity for DYRK1A over other DYRK family members. In addition, FINDY rescues DYRK1A-induced developmental malformations in Xenopus laevis embryos. Our study demonstrates that transitional folding intermediates of protein kinases can be targeted by small molecules, and paves the way for developing novel types of kinase inhibitors.

WDR26 is a new partner of Axin1 in the canonical Wnt signaling pathway.

The stability of ß-catenin is very important for canonical Wnt signaling. A protein complex including Axin/APC/GSK3ß phosphorylates ß-catenin to be degraded by ubiquitination with ß-TrCP. In the recent study, we isolated WDR26, a protein that binds to Axin. Here, we found that WDR26 is a negative regulator of the canonical Wnt signaling pathway, and that WDR26 affected ß-catenin levels. In addition, WDR26/Axin binding is involved in the ubiquitination of ß-catenin. These results suggest that WDR26 plays a negative role in ß-catenin degradation in the Wnt signaling pathway.

IQGAP1 protein regulates nuclear localization of ß-catenin via importin-ß5 protein in Wnt signaling.

In the canonical Wnt signaling pathway, the translocation of ß-catenin is important for the activation of target genes in the nucleus. However, the molecular mechanisms underlying its nuclear localization remain unclear. In the present study, we found IQGAP1 to be a regulator of ß-catenin function via importin-ß5. In Xenopus embryos, depletion of IQGAP1 reduced Wnt-induced nuclear accumulation of ß-catenin and expression of Wnt target genes during early embryogenesis. Depletion of endogenous importin-ß5 associated with IQGAP1 also reduced expression of Wnt target genes and the nuclear localization of IQGAP1 and ß-catenin. Moreover, a small GTPase, Ran1, contributes to the nuclear translocation of ß-catenin and the activation of Wnt target genes. These results suggest that IQGAP1 functions as a regulator of translocation of ß-catenin in the canonical Wnt signaling pathway.

IQGAP1 functions as a modulator of dishevelled nuclear localization in Wnt signaling.

Dishevelled (DVL) is a central factor in the Wnt signaling pathway, which is highly conserved among various organisms. DVL plays important roles in transcriptional activation in the nucleus, but the molecular mechanisms underlying their nuclear localization remain unclear. In the present study, we identified IQGAP1 as a regulator of DVL function. In Xenopus embryos, depletion of IQGAP1 reduced Wnt-induced nuclear accumulation of DVL, and expression of Wnt target genes during early embryogenesis. The domains in DVL and IQGAP1 that mediated their interaction are also required for their nuclear localization. Endogenous expression of Wnt target genes was reduced by depletion of IQGAP1 during early embryogenesis, but notably not by depletion of other IQGAP family genes. Moreover, expression of Wnt target genes caused by depletion of endogenous IQGAP1 could be rescued by expression of wild-type IQGAP1, but not IQGAP1 deleting DVL binding region. These results provide the first evidence that IQGAP1 functions as a modulator in the canonical Wnt signaling pathway.

Characterization of CXC-type chemokine molecules in early Xenopus laevis development.

Chemokine molecules play important roles in the immune system. However, several chemokine molecules are expressed during early development before the immune system is established. Using reverse transcription–polymerase chain reaction (RT-PCR) and overexpression of chemokine molecules, we identified and characterized Xenopus laevis CXC-type chemokine ligands (XCXCL13L1, XCXCL13L2, XCXCLa, XCXCLb, XCXCLd, and XCXCLe) and receptors (XCXCR1/2, XCXCR3, XCXCR5, XCXCR6, and XCXCRa) during early development. The CXC-type ligands have low identity with genes for human CXC ligands (CXCL). With the exception of XCXCRa, the CXC receptors (CXCR) identified in the present study had high (40%–65%) identity with human CXCR genes. Although the expression patterns for the CXCL and CXCR genes differed, transcript levels for all genes were very low during early embryogenesis. Overexpression of XCXCL13L1, XCXCL13L2, XCXCLa, XCXCR3, XCXCR6, and XCXCRa interfered with gastrulation and neural fold closure. The results of the present study suggest that several chemokine molecules are related to cell movements during early morphogenesis.

WNK4 is an essential effector of anterior formation in FGF signaling.

With no lysine (K) (WNK) kinase family is conserved among many species and regulates SPAK/OSR1 and ion cotransporters. WNK is also involved in developmental and cellular processes, but the molecular mechanisms underlying its regulation in these processes remain unknown. In this study, we found that WNK4 is involved in fibroblast growth factor (FGF) signaling during Xenopus development. In Xenopus embryos, depletion of WNK4 by antisense morpholino oligonucleotides (MOs) results in a severe defect in anterior development and impaired expression of endogenous anterior markers. Defects in head formation or expression of anterior marker genes caused by suppression of endogenous WNK4 expression could be rescued by expression of wild-type WNK4, but not mutant WNK4 lacking its kinase activity. It is notable that morphants of Xenopus WNK4 inhibited the expression of anterior marker genes and the target genes induced by FGF signaling. Moreover, knockdown of Wnk4 significantly reduced the phosphorylation level of Osr1 induced by FGF. These results provide the first evidence that FGF signaling regulates WNK4 function required for anterior formation in Xenopus development.

Chemokine ligand Xenopus CXCLC (XCXCLC) regulates cell movements during early morphogenesis.

We cloned the gene for the CXC-type chemokine ligand, Xenopus CXCLC (XCXCLC), the transcripts of which were detected at the dorsal midline during the gastrula and neurula stages. XCXCLC overexpression resulted in the attraction of nearby mesodermal cells, and the excess of chemoattractant interfered with convergent and extension movements. The direction of the deep neural plate cells around the notoplate was also controlled by XCXCLC. Fluorescence signals for XCXCLC + enhanced green fluorescent protein derivatives accumulated around the notochord region. These results indicate that XCXCLC attracts adjacent cells to the midline region, so as to ensure accurate lateral-medial directional tissue convergence during gastrulation and neurulation.

Xenopus furry contributes to release of microRNA gene silencing.

A transcriptional corepressor, Xenopus furry (Xfurry), is expressed in the chordamesodermal region and induces secondary dorsal axes when overexpressed on the ventral side of the embryo. The N-terminal furry domain functions as a repressor, and the C-terminal leucine zipper (LZ) motifs /coiled-coil structure, found only in vertebrate homologs, contributes to the nuclear localization. The engrailed repressor (enR)+LZ repressor construct, which has properties similar to Xfurry, induced several chordamesodermal genes. In contrast, an antisense morpholino oligonucleotide, Xfurry-MO, and the activating construct, herpes simplex virus protein (VP16)+LZ, had effects opposite those of Xfurry overexpression. Because blocking protein synthesis with cycloheximide superinduced several Xfurry transcriptional targets, and because expression of enR+LZ induced such genes under cycloheximide treatment, we analyzed the role of an Xfurry transcriptional target, microRNA miR-15. Cycloheximide reduced the expression of primary miR-15 (pri-miR-15), whereas miR-15 reduced the expression of genes superinduced by cycloheximide treatment. These results show that Xfurry regulates chordamesodermal genes by contributing to repression of pretranscriptional gene silencing by miR-15.

Development of a novel selective inhibitor of the Down syndrome-related kinase Dyrk1A.

Dyrk1A (dual-specificity tyrosine-(Y)-phosphorylation-regulated kinase 1A) is a serine/threonine kinase essential for brain development and function, and its excessive activity is considered a pathogenic factor in Down syndrome. The development of potent, selective inhibitors of Dyrk1A would help to elucidate the molecular mechanisms of normal and diseased brains, and may provide a new lead compound for molecular-targeted drug discovery. Here, we report a novel Dyrk1A inhibitor, INDY, a benzothiazole derivative showing a potent ATP-competitive inhibitory effect with IC(50) and K(i) values of 0.24 and 0.18 µM, respectively. X-ray crystallography of the Dyrk1A/INDY complex revealed the binding of INDY in the ATP pocket of the enzyme. INDY effectively reversed the aberrant tau-phosphorylation and rescued the repressed NFAT (nuclear factor of activated T cell) signalling induced by Dyrk1A overexpression. Importantly, proINDY, a prodrug of INDY, effectively recovered Xenopus embryos from head malformation induced by Dyrk1A overexpression, resulting in normally developed embryos and demonstrating the utility of proINDY in vivo.

TMEPAI, a transmembrane TGF-beta-inducible protein, sequesters Smad proteins from active participation in TGF-beta signaling.

Transforming growth factor-beta (TGF-beta) is a multifunctional cytokine of key importance for controlling embryogenesis and tissue homeostasis. How TGF-beta signals are attenuated and terminated is not well understood. Here, we show that TMEPAI, a direct target gene of TGF-beta signaling, antagonizes TGF-beta signaling by interfering with TGF-beta type I receptor (TbetaRI)-induced R-Smad phosphorylation. TMEPAI can directly interact with R-Smads via a Smad interaction motif. TMEPAI competes with Smad anchor for receptor activation for R-Smad binding, thereby sequestering R-Smads from TbetaRI kinase activation. In mammalian cells, ectopic expression of TMEPAI inhibited TGF-beta-dependent regulation of plasminogen activator inhibitor-1, JunB, cyclin-dependent kinase inhibitors, and c-myc expression, whereas specific knockdown of TMEPAI expression prolonged duration of TGF-beta-induced Smad2 and Smad3 phosphorylation and concomitantly potentiated cellular responsiveness to TGF-beta. Consistently, TMEPAI inhibits activin-mediated mesoderm formation in Xenopus embryos. Therefore, TMEPAI participates in a negative feedback loop to control the duration and intensity of TGF-beta/Smad signaling.

Nemo-like kinase, an essential effector of anterior formation, functions downstream of p38 mitogen-activated protein kinase.

Nemo-like kinase (NLK) is known to function as a mitogen-activated protein kinase (MAPK)-like kinase. However, the upstream molecules and molecular mechanisms that regulate NLK activity remain unclear. In the present study, we identified p38 MAPK as an upstream kinase and activator of NLK. p38 regulates the function of NLK via phosphorylation, and this modification can be abrogated by depletion of endogenous p38. In Xenopus laevis embryos, depletion of either p38beta or NLK by antisense morpholino oligonucleotides results in a severe defect in anterior development and impaired expression of endogenous anterior markers. It is notable that morphants of Xenopus p38alpha, another isoform of the p38 MAPK family, exhibited no obvious defects in anterior development. Defects in head formation or in the expression of anterior marker genes caused by suppression of endogenous p38beta expression could be rescued by expression of wild-type NLK but not by expression of mutant NLK lacking the p38beta phosphorylation site. In contrast, defects in head formation or in the expression of anterior marker genes caused by suppression of endogenous NLK expression could not be rescued by expression of p38. These results provide the first evidence that p38 specifically regulates NLK function, which is required for anterior formation in Xenopus development.

Concentrations of TATA box-binding protein (TBP)-type genes affect chordamesodermal gene expression.

The TATA box-binding protein (TBP) is an essential component of transcription initiation complexes of all three eukaryotic RNA polymerases. Recent knockdown studies revealed that basic transcription factors are essential not only for gene transcription but also for regulating specific gene expression. However, the mechanism of and the effect by regulation of TBP expression are unknown during early embryogenesis. Here we show that the alteration of concentration of each TBP-type gene affected mutually one another's expression, suggesting that an optimal ratio of concentrations of TBP-type genes induce expression of specific genes.