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Crohn's disease is a chronic, debilitating, inflammatory bowel disease. Here, we report a critical role of phospholipase C-β3 (PLC-β3) in intestinal homeostasis. In PLC-β3-deficient mice, exposure to oral dextran sodium sulfate induced lethality and severe inflammation in the small intestine. The lethality was due to PLC-β3 deficiency in multiple non-hematopoietic cell types. PLC-β3 deficiency resulted in reduced Wnt/β-catenin signaling, which is essential for homeostasis and the regeneration of the intestinal epithelium. PLC-β3 regulated the Wnt/β-catenin pathway in small intestinal epithelial cells (IECs) at transcriptional, epigenetic, and, potentially, protein–protein interaction levels. PLC-β3-deficient IECs were unable to respond to stimulation by R-spondin 1, an enhancer of Wnt/β-catenin signaling. Reduced expression of PLC-β3 and its signature genes was found in biopsies of patients with ileal Crohn's disease. PLC-β regulation of Wnt signaling was evolutionally conserved in Drosophila. Our data indicate that a reduction in PLC-β3-mediated Wnt/β-catenin signaling contributes to the pathogenesis of ileal Crohn's disease. [ABSTRACT FROM AUTHOR]

In vertebrate embryos, the canonical Wnt ligand primes the formation of dorsal organizers that govern dorsal-ventral patterns by secreting BMP antagonists. In contrast, in Drosophila embryos, Toll-like receptor (Tlr)-mediated NFκB activation initiates dorsal-ventral patterning, wherein Wnt-mediated negative feedback regulation of Tlr/NFκB generates a BMP antagonist-secreting signalling centre to control the dorsal-ventral pattern. Although both Wnt and BMP antagonist are conserved among species, the involvement of Tlr/NFκB and feedback regulation in vertebrate organizer formation remains unclear. By imaging and genetic modification, we reveal that a negative feedback loop between canonical and non-canonical Wnts and Tlr4/NFκB determines the size of zebrafish organizer, and that Tlr/NFκB and Wnts switch initial cue and feedback mediator roles between Drosophila and zebrafish. Here, we show that canonical Wnt signalling stimulates the expression of the non-canonical Wnt5b ligand, activating the Tlr4 receptor to stimulate NFκB-mediated transcription of the Wnt antagonist frzb, restricting Wnt-dependent dorsal organizer formation. Dorsal organizer initiates the dorsal-ventral axis formation in vertebrates. Here, the authors demonstrate that Tlr4/NFκB-mediated negative feedback regulation of Wnt/β-catenin signaling determines the precise size of the zebrafish dorsal organizer. [ABSTRACT FROM AUTHOR]

Wnt2022 was held on November 15th–19th, 2022, in Awaji Yumebutai International Conference Center, Hyogo Prefecture, Japan, as an in‐person meeting for the first time in last 3 years. Wnt signaling is a highly conserved pathway among various species. Since Wnt1 was discovered in 1982, a number of studies using many model animals and human samples have revealed that Wnt signaling plays crucial roles in embryonic development, tissue morphogenesis, and regeneration, as well as many other physiological and pathological processes. Since the year 2022 marks the 40th anniversary of Wnt research, we aimed to look back at our research progress and discuss the future direction of this field. The scientific program consisted of plenary lectures, invited talks, short talks selected from abstracts, and poster sessions. Whereas several different Wnt meetings have been held almost every year in Europe and the United States, this was the first Wnt meeting convened in Asia. Therefore, Wnt2022 was highly anticipated to bring together leaders and young scientists from Europe, the United States, and especially Asia and Oceania. In fact, 148 researchers from 21 countries attended this meeting. Although there were travel and administrative restrictions due to COVID‐19, the meeting was highly successful in enabling face‐to‐face discussions. [ABSTRACT FROM AUTHOR]

The African turquoise killifish Nothobranchius furzeri (N. furzeri) is a useful model organism for studying aging, age-related diseases, and embryonic diapause. CRISPR/Cas9-mediated gene knockout and Tol2 transposon-mediated transgenesis in N. furzeri have been reported previously. However, these methods take time to generate knockout and transgenic fish. In addition, knock-in technology that inserts large DNA fragments as fluorescent reporter constructs into the target gene in N. furzeri has not yet been established. Here, we show that triple-target CRISPR-mediated single gene disruption efficiently produces whole-body biallelic knockout and enables the examination of gene function in the F0 generation. In addition, we developed a method for creating the knock-in reporter N. furzeri without crossing by optimizing the CRISPR/Cas9 system. These methods drastically reduce the duration of experiments, and we think that these advances will accelerate aging and developmental studies using N. furzeri. [ABSTRACT FROM AUTHOR]

Most tumours are thought to arise through oncogenic cell generation followed by additional mutations. How a new oncogenic cell primes tumorigenesis by acquiring additional mutations remains unclear. We show that an additional TP53 mutation stimulates primary tumorigenesis by switching oncogene-induced senescence from a tumour suppressor to a driver. Zebrafish imaging reveals that a newly emerged oncogenic cell with the RasG12V mutation becomes senescent and is eliminated from the epithelia, which is prevented by adding a TP53 gain-of-function mutation (TP53R175H) into RasG12V cells. Surviving RasG12V-TP53R175H double-mutant cells senesce and secrete senescence-associated secretory phenotype (SASP)-related inflammatory molecules that convert neighbouring normal cells into SASP factor-secreting senescent cells, generating a heterogeneous tumour-like cell mass. We identify oncogenic cell behaviours that may control the initial human tumorigenesis step. Ras and TP53 mutations and cellular senescence are frequently detected in human tumours; similar switching may occur during the initial step of human tumorigenesis. It is unclear how a single oncogenic cell primes tumorigenesis. Here the authors visualised this behaviour using a zebrafish larval skin as a model and show that RasG12V oncogenic cell is eliminated through oncogene-senescence while a gain of function mutation in p53 alters this behaviour from tumour suppressive to tumour promoting. [ABSTRACT FROM AUTHOR]

Leucyl-tRNA synthetase (LARS) is an enzyme that catalyses the ligation of leucine with leucine tRNA. LARS is also essential to sensitize the intracellular leucine concentration to the mammalian target of rapamycin complex 1 (mTORC1) activation. Biallelic mutation in the LARS gene causes infantile liver failure syndrome type 1 (ILFS1), which is characterized by acute liver failure, anaemia, and neurological disorders, including microcephaly and seizures. However, the molecular mechanism underlying ILFS1 under LARS deficiency has been elusive. Here, we generated Lars deficient (larsb−/−) zebrafish that showed progressive liver failure and anaemia, resulting in early lethality within 12 days post fertilization. The atg5-morpholino knockdown and bafilomycin treatment partially improved the size of the liver and survival rate in larsb−/− zebrafish. These findings indicate the involvement of autophagy in the pathogenesis of larsb−/− zebrafish. Indeed, excessive autophagy activation was observed in larsb−/− zebrafish. Therefore, our data clarify a mechanistic link between LARS and autophagy in vivo. Furthermore, autophagy regulation by LARS could lead to development of new therapeutics for IFLS1. [ABSTRACT FROM AUTHOR]

The naked mole-rat (NMR; Heterocephalus glaber) exhibits cancer resistance and an exceptionally long lifespan of approximately 30 years, but the mechanism(s) underlying increased longevity in NMRs remains unclear. In the present study, we report unique mechanisms underlying cholesterol metabolism in NMR cells, which may be responsible for their anti-senescent properties. NMR fibroblasts expressed β-catenin abundantly; this high expression was linked to increased accumulation of cholesterol-enriched lipid droplets. Ablation of β-catenin or inhibition of cholesterol synthesis abolished lipid droplet formation and induced senescence-like phenotypes accompanied by increased oxidative stress. β-catenin ablation downregulated apolipoprotein F and the LXR/RXR pathway, which are involved in cholesterol transport and biogenesis. Apolipoprotein F ablation also suppressed lipid droplet accumulation and promoted cellular senescence, indicating that apolipoprotein F mediates β-catenin signaling in NMR cells. Thus, we suggest that β-catenin in NMRs functions to offset senescence by regulating cholesterol metabolism, which may contribute to increased longevity in NMRs. Woei-Yaw Chee et al. explore the molecular mechanisms of β-catenin involvement in cholesterol metabolism suppressing cellular senescence in naked mole-rat cells. The results of this study suggest that β-catenin in NMRs functions to offset senescence by regulating cholesterol metabolism, which may contribute to increased longevity in naked mole rats. [ABSTRACT FROM AUTHOR]

Zebrafish is a useful model to study vertebrate hematopoiesis, but lack of antibodies to zebrafish proteins has limited purification of hematopoietic cells. Here, we purified neutrophils from larval and adult zebrafish using the lectin Phaseolus vulgaris erythroagglutinin (PHA‐E) and DRAQ5, a DNA‐staining fluorescent dye. In adult kidney marrow, we purified neutrophil‐like PHA‐E4low DRAQ5low cells, which neutrophil‐type granules. Specifically, at 96‐hr post‐fertilization, we sorted large‐sized cells from larvae using forward scatter and found that they consisted of PHA‐Elow DRAQ5low populations. These cells had myeloperoxidase activity, were Sudan Black B‐positive and expressed high levels of neutrophil‐specific (csf3r and mpx) mRNAs, all neutrophil characteristics. Using this method, we conducted functional analysis suggesting that zyxin (Zyx) plays a role in neutrophil generation in zebrafish larvae. Overall, PHA‐E and DRAQ5‐based flow cytometry serves as a tool to purify zebrafish neutrophils. [ABSTRACT FROM AUTHOR]

Cyclin-dependent kinase 8 (CDK8) is a member of the CDK/Cyclin module of the mediator complex. A recent study reported that heterozygous missense CDK8 mutations cause a neurodevelopmental disorder in humans. The mechanistic basis of CDK8-related disorder has yet to be delineated. Here, we report 2 patients with de novo missense mutations within the kinase domain of CDK8 along with the results of in vitro and in vivo functional analyses using a zebrafish model. Patient 1 and Patient 2 had intellectual disabilities and congenital anomalies. Exome analyses showed that patient 1 had a heterozygous de novo missense p.G28A variant in the CDK8 (NM_001260.3) gene and patient 2 had a heterozygous de novo missense p.N156S variant in the CDK8 gene. We assessed the pathogenicity of these two variants using cultured-cells and zebrafish model. An in vitro kinase assay of human CDK8 showed that enzymes with a p.G28A or p.N156S substitution showed decreased kinase activity. An in vivo assays of zebrafish overexpression analyses also showed that the p.G28A and p.N156S alleles were hypomorphic alleles. Importantly, the inhibition of CDK8 kinase activity in zebrafish embryos using a specific chemical inhibitor induced craniofacial and heart defects similar to the patients' phenotype. Taken together, zebrafish studies showed that non-synonymous variants in the kinase domain of CDK8 act as hypomorphic alleles causing human congenital disorder. [ABSTRACT FROM AUTHOR]

Formation of the body of vertebrate embryos proceeds sequentially by posterior addition of tissues from the tail bud. Cells of the tail bud and the posterior presomitic mesoderm, which control posterior elongation1, exhibit a high level of aerobic glycolysis that is reminiscent of the metabolic status of cancer cells experiencing the Warburg effect2,3. Glycolytic activity downstream of fibroblast growth factor controls WNT signalling in the tail bud3. In the neuromesodermal precursors of the tail bud4, WNT signalling promotes the mesodermal fate that is required for sustained axial elongation, at the expense of the neural fate3,5. How glycolysis regulates WNT signalling in the tail bud is currently unknown. Here we used chicken embryos and human tail bud-like cells differentiated in vitro from induced pluripotent stem cells to show that these cells exhibit an inverted pH gradient, with the extracellular pH lower than the intracellular pH, as observed in cancer cells6. Our data suggest that glycolysis increases extrusion of lactate coupled to protons via the monocarboxylate symporters. This contributes to elevating the intracellular pH in these cells, which creates a favourable chemical environment for non-enzymatic β-catenin acetylation downstream of WNT signalling. As acetylated β-catenin promotes mesodermal rather than neural fate7, this ultimately leads to activation of mesodermal transcriptional WNT targets and specification of the paraxial mesoderm in tail bud precursors. Our work supports the notion that some tumour cells reactivate a developmental metabolic programme. The authors show that metabolic activity leads to an increase in the intracellular pH of neuromesodermal precursors, and that this increase in pH, by allowing post-translational modification of β-catenin, is required for the activation of WNT signalling and mesodermal fate acquisition. [ABSTRACT FROM AUTHOR]

Unlike humans and other mammals, adult zebrafish have the superior capability to recover from central nervous system (CNS) injury. We previously found that proliferation of radial glia (RG) is induced in response to stab injury in optic tectum and that new neurons are generated from RG after stab injury. However, molecular mechanisms which regulate proliferation and differentiation of RG are not well known. In the present study, we investigated Shh and Notch signaling as potential mechanisms regulating regeneration in the optic tectum of adult zebrafish. We used Shh reporter fish and confirmed that canonical Shh signaling is activated specifically in RG after stab injury. Moreover, we have shown that Shh signaling promotes RG proliferation and suppresses their differentiation into neurons after stab injury. In contrast, Notch signaling was down‐regulated after stab injury, indicated by the decrease in the expression level of her4 and her6, a target gene of Notch signaling. We also found that inhibition of Notch signaling after stab injury induced more proliferative RG, but that inhibition of Notch signaling inhibited generation of newborn neurons from RG after stab injury. These results suggest that high level of Notch signaling keeps RG quiescent and that appropriate level of Notch signaling is required for generation of newborn neurons from RG. Under physiological condition, activation of Shh signaling or inhibition of Notch signaling also induced RG proliferation. In adult optic tectum of zebrafish, canonical Shh signaling and Notch signaling play important roles in proliferation and differentiation of RG in physiological and regenerative conditions. Stab injury in adult zebrafish optic tectum activates Shh signaling and down‐regulates Notch signaling. Inhibition of Shh signaling suppresses proliferation of radial glia and activates production of new neurons. On the other hands, inhibition of Notch signaling promotes proliferation of radial glia and suppresses production of new born neurons. [ABSTRACT FROM AUTHOR]

Summary: Zebrafish embryos are useful to study haematopoietic gene function in vertebrates, although lack of antibodies to zebrafish proteins has limited the purification of specific cell populations. Here, we purified primitive zebrafish erythrocytes using 1, 5‐bis{[2‐(di‐methylamino)ethyl]amino}‐4, 8‐dihydroxyanthracene‐9, 10‐dione (DRAQ5TM), a DNA‐staining fluorescent dye. At 48‐h post‐fertilization, we sorted small‐sized cells from embryos using forward scatter and found that they consisted of DRAQ5high and DRAQ5low populations. DRAQ5high cells contained haemoglobin, lacked myeloperoxidase activity and expressed high levels of embryonic globin (hbae3 and hbbe1.1) mRNA, all characteristics of primitive erythrocytes. Following DRAQ5TM analysis of gata1:dsRed transgenic embryos, we purified primitive DRAQ5high dsRed+ erythrocytes from haematopoietic progenitor cells. Using this method, we identified docking protein 2 (Dok2) as functioning in differentiation of primitive erythrocytes. We conclude that DRAQ5TM‐based flow cytometry enables purification of primitive zebrafish erythrocytes. [ABSTRACT FROM AUTHOR]

The leukotriene B4 (LTB4) receptor 1 (BLT1) is a high affinity receptor for LTB4, a chemotactic and inflammatory eicosanoid. The LTB4 receptor 2 (BLT2) was originally identified as a low affinity receptor for LTB4, and, more recently, as a high affinity receptor for 12-hydroxyheptadecatrienoic acid (12-HHT). The zebrafish BLT receptors have not been previously identified and the in vivo functions of these receptors have been unknown. In this paper, we describe one zebrafish BLT1-like receptor, Blt1, and two zebrafish BLT2-like receptors, Blt2a and Blt2b. Cells expressing Blt1 exhibited LTB4-induced intracellular [Ca2+] increases, inhibition of cAMP production, ligand-dependent [35S]GTPγS binding, and transforming growth factor-α (TGFα) shedding activity in a dose-dependent manner, similar to human BLT1. Cells expressing Blt2a and Blt2b exhibited 12-HHT- and LTB4-induced intracellular [Ca2+] increases, inhibition of cAMP production, [35S]GTPγS binding, and TGFα shedding activity, with a dose-dependency similar to human BLT2. Reverse transcription (RT)-PCR analysis and whole-mount in situ hybridization revealed that blt1, blt2a, blt2b, zebrafish LTA4 hydrolase (lta4h), and zebrafish 5-lipoxiganase (5lo) are expressed in zebrafish embryos. Knockdown of blt1 by morpholino antisense oligonucleotides resulted in delayed epiboly at gastrulation. Consistently, knockdown of lta4h, an enzyme mediating LTB4 production, induced a phenotype similar to knockdown of blt1. These results suggest that the LTB4-BLT1 axis is involved in epiboly in zebrafish development. [ABSTRACT FROM AUTHOR]

In vertebrates, newly emerging transformed cells are often apically extruded from epithelial layers through cell competition with surrounding normal epithelial cells. However, the underlying molecular mechanism remains elusive. Here, using phospho-SILAC screening, we show that phosphorylation of AHNAK2 is elevated in normal cells neighboring RasV12 cells soon after the induction of RasV12 expression, which is mediated by calcium-dependent protein kinase C. In addition, transient upsurges of intracellular calcium, which we call calcium sparks, frequently occur in normal cells neighboring RasV12 cells, which are mediated by mechanosensitive calcium channel TRPC1 upon membrane stretching. Calcium sparks then enhance cell movements of both normal and RasV12 cells through phosphorylation of AHNAK2 and promote apical extrusion. Moreover, comparable calcium sparks positively regulate apical extrusion of RasV12-transformed cells in zebrafish larvae as well. Hence, calcium sparks play a crucial role in the elimination of transformed cells at the early phase of cell competition. [Display omitted] • Phosphorylation of AHNAK2 is elevated in normal cells neighboring transformed cells • TRPC1-mediated calcium sparks in normal cells mediate the AHNAK2 phosphorylation • Calcium sparks promote tissue fluidity and apical extrusion of transformed cells • Comparable calcium sparks regulate apical extrusion in cultured cells and zebrafish Kuromiya et al. use mammalian cell culture and zebrafish systems to show that calcium sparks frequently occur in normal epithelial cells neighboring RasV12-transformed cells at the early phase of cell competition. Calcium sparks then promote epithelial tissue fluidity and apical extrusion of transformed cells through the phosphorylation of AHNAK2. [ABSTRACT FROM AUTHOR]

Summary The phosphoprotein Dishevelled (Dvl) is a common essential component of Wnt/β-catenin and Wnt/planar cell polarity (PCP) signaling pathways. However, the regulation and significance of Dvl phosphorylation are not fully understood. Here, we show that homeodomain-interacting protein kinase 2 (Hipk2) facilitates protein phosphatase 1 catalytic subunit (PP1c)-mediated dephosphorylation of Dvl via its C-terminal domain and that this dephosphorylation blocks ubiquitination and consequent degradation mediated by the E3 ubiquitin ligase Itch, which targets the phosphorylated form of Dvl proteins. Inhibition of Hipk2 or PP1c function reduces Dvl protein levels and suppresses Wnt/β-catenin and Wnt/PCP pathway-dependent events in mammalian cells and zebrafish embryos, suggesting that Hipk2 and PP1c are essential for maintaining Dvl protein levels that are sufficient to activate Wnt signaling. We also show that Wnt-3a, a Wnt/β-catenin ligand, induces dissociation of the Dvl-Hipk2-PP1c complex and Dvl degradation under high-cell-density conditions. This regulation may be a negative feedback mechanism that fine-tunes Wnt/β-catenin signaling. [ABSTRACT FROM AUTHOR]

DP1, a dimerization partner protein of the transcription factor E2F, is known to inhibit Wnt/?-catenin signalling along with E2F, although the function of DP1 itself was not well characterized. Here, we present a novel dual regulatory mechanism of Wnt/?-catenin signalling by DP1 independent from E2F. DP1 negatively regulates Wnt/?-catenin signalling by inhibiting Dvl-Axin interaction and by enhancing poly-ubiquitination of ?-catenin. In contrast, DP1 positively modulates the signalling upon Wnt stimulation, via increasing cytosolic ?-catenin and antagonizing the kinase activity of NLK. In Xenopus embryos, DP1 exerts both positive and negative roles in Wnt/?-catenin signalling during anteroposterior neural patterning. From subcellular localization analyses, we suggest that the dual roles of DP1 in Wnt/?-catenin signalling are endowed by differential nucleocytoplasmic localizations. We propose that these dual functions of DP1 can promote and stabilize biphasic Wnt-on and Wnt-off states in response to a gradual gradient of Wnt/?-catenin signalling to determine differential cell fates. [ABSTRACT FROM AUTHOR]

Nemo-like kinase (NLK/Nlk) is an evolutionarily conserved protein kinase involved in Wnt/?-catenin signalling. However, the roles of NLK in Wnt/?-catenin signalling in vertebrates remain unclear. Here, we show that inhibition of Nlk2 function in zebrafish results in decreased Lymphoid enhancer factor-1 (Lef1)-mediated gene expression and cell proliferation in the presumptive midbrain, resulting in a reduction of midbrain tectum size. These defects are related to phosphorylation of Lef1 by Nlk2. Thus, Nlk2 is essential for the phosphorylation and activation of Lef1 transcriptional activity in neural progenitor cells (NPCs). In NPC-like mammalian cells, NLK is also required for the phosphorylation and activation of LEF1 transcriptional activity. Phosphorylation of LEF1 induces its dissociation from histone deacetylase, thereby allowing transcription activation. Furthermore, we demonstrate that NLK functions downstream of Dishevelled (Dvl) in the Wnt/?-catenin signalling pathway. Our findings reveal a novel role of NLK in the activation of the Wnt/?-catenin signalling pathway. [ABSTRACT FROM AUTHOR]

Spatially and temporally controlled gene expression, including transcription, several mRNA processing steps, and the export of mature mRNA to the cytoplasm, is essential for developmental processes. It is well known that RNA helicases of the DExD/H-box protein family are involved in these gene expression processes, including transcription, pre-mRNA splicing, and rRNA biogenesis. Although one DExD/H-box protein, Prp5, a homologue of vertebrate Ddx46, has been shown to play important roles in pre-mRNA splicing in yeast, the in vivo function of Ddx46 remains to be fully elucidated in metazoans. In this study, we isolated zebrafish morendo (mor), a mutant that shows developmental defects in the digestive organs and brain, and found that it encodes Ddx46. The Ddx46 transcript is maternally supplied, and as development proceeds in zebrafish larvae, its ubiquitous expression gradually becomes restricted to those organs. The results of whole-mount in situ hybridization showed that the expression of various molecular markers in these organs is considerably reduced in the Ddx46 mutant. Furthermore, splicing status analysis with RT-PCR revealed unspliced forms of mRNAs in the digestive organ and brain tissues of the Ddx46 mutant, suggesting that Ddx46 may be required for pre-mRNA splicing during zebrafish development. Therefore, our results suggest a model in which zebrafish Ddx46 is required for the development of the digestive organs and brain, possibly through the control of pre-mRNA splicing. [ABSTRACT FROM AUTHOR]

Although recent findings showed that some Drosophila doublesex and Caenorhabditis elegans mab-3 related genes are expressed in neural tissues during development, their functions have not been fully elucidated. Here, we isolated a zebrafish mutant, ha2, that shows defects in telencephalic neurogenesis and found that ha2 encodes Doublesex and MAB-3 related transcription factor like family A2 (Dmrta2). dmrta2 expression is restricted to the telencephalon, diencephalon and olfactory placode during somitogenesis. We found that the expression of the proneural gene, neurogenin1, in the posterior and dorsal region of telencephalon (posterior-dorsal telencephalon) is markedly reduced in this mutant at the 14-somite stage without any defects in cell proliferation or cell death. In contrast, the telencephalic expression of her6, a Hes-related gene that is known to encode a negative regulator of neurogenin1, expands dramatically in the ha2 mutant. Based on over-expression experiments and epistatic analyses, we propose that zebrafish Dmrta2 controls neurogenin1 expression by repressing her6 in the posterior-dorsal telencephalon. Furthermore, the expression domains of the telencephalic marker genes, foxg1 and emx3, and the neuronal differentiation gene, neurod, are downregulated in the ha2 posterior-dorsal telencephalon during somitogenesis. These results suggest that Dmrta2 plays important roles in the specification of the posterior-dorsal telencephalic cell fate during somitogenesis. [ABSTRACT FROM AUTHOR]

Vertebrates use adaptive mechanisms when exposed to physiologic stresses. However, the mechanisms of pigmentation regulation in response to physiologic stresses largely remain unclear. To address this issue, we developed a novel pigmentation model in adult zebrafish using coldwater exposure (cold zebrafish). When zebrafish were maintained at 17 °C, the pigmentation of their pigment stripes was reduced compared with zebrafish at 26.5 °C (normal zebrafish). In cold zebrafish, gene expression levels of tyrosinase and dopachrome tautomerase, which encode enzymes involved in melanogenesis, were down-regulated, suggesting that either down-regulation of melanin synthesis occurred or the number of melanophores decreased. Both regular and electron microscopic observation of zebrafish skin showed that the number of melanophores decreased, whereas aggregation of melanosomes was not changed in cold zebrafish compared with normal zebrafish. Taken together, we here show that cold exposure down-regulated adult zebrafish pigmentation through decreasing the number of melanophores and propose that the cold zebrafish model is a powerful tool for pigmentation research. [ABSTRACT FROM AUTHOR]

The Notch signalling pathway has a crucial function in determining cell fates in multiple tissues within metazoan organisms. On binding to ligands, the Notch receptor is cleaved proteolytically and releases its intracellular domain (NotchICD). The NotchICD enters the nucleus and acts cooperatively with other factors to stimulate the transcription of target genes. High levels of Notch-mediated transcriptional activation require the formation of a ternary complex consisting of NotchICD, CSL (CBF-1, suppressor of hairless, LAG-1) and a Mastermind family member. However, it is still not clear how the formation of the ternary complex is regulated. Here we show that Nemo-like kinase (NLK) negatively regulates Notch-dependent transcriptional activation by decreasing the formation of this ternary complex. Using a biochemical screen, we identified Notch as a new substrate of NLK. NLK-phosphorylated Notch1ICD is impaired in its ability to form a transcriptionally active ternary complex. Furthermore, knockdown of NLK leads to hyperactivation of Notch signalling and consequently decreases neurogenesis in zebrafish. Our results both define a new function for NLK and reveal a previously unidentified mode of regulation in the Notch signalling pathway. [ABSTRACT FROM AUTHOR]

Nerve growth factor (NGF) promotes neurite outgrowth through regulating cytoskeletal organization and cell adhesion. These activities are modulated by protein phosphorylation. Nemo-like kinase (NLK) is an evolutionarily conserved MAP kinase-like kinase that phosphorylates several transcription factors. Although NLK is known to be expressed at relatively high levels in the nervous system, its function is not well understood. We found that NGF promotes the translocation of NLK to PC12 cells’ leading edges, and triggers NLK kinase activity in them. Activated NLK directly phosphorylates microtubule-associated protein-1B (MAP1B) and the focal adhesion adaptor protein, paxillin. Knockdown of NLK attenuates the phosphorylation of both paxillin and MAP1B and inhibits both the NGF-induced re-distribution of F-actin and neurite outgrowth. We also discovered that NLK is a LiCl-sensitive kinase. LiCl is known to block NGF-induced neurite outgrowth and the phosphorylation of MAP1B and paxillin in PC12 cells. Therefore, the effects of LiCl are mediated in part by blocking NLK activity. These results suggest that NLK controls the dynamics of the cytoskeleton downstream of NGF signaling. [ABSTRACT FROM AUTHOR]

The contrast-to-gradient (CG) resolution has been defined as a weighted harmonic mean of the local resolution, i.e. the length defining the object's local fine structure at each pixel position. The newly defined resolution Res is defined as , where 2σ is a sharpness factor of the image pattern obtained by the conversion 2σ = ARCG + B using default constants A and B. In the present study, we have extended the algorithm to change the constants from default values to calibrated ones using standard images that are same in both pattern and contrast-to-noise ratio (CNR) as the original SEM image to be evaluated. The calibrated image resolution with a weak noise dependency is evaluated with the CNR as a given parameter. [ABSTRACT FROM PUBLISHER]

The contrast-to-gradient (CG) method has been proposed previously for the evaluation of image resolution in scanning electron microscopy (SEM). In the CG algorithm, the image to be evaluated is gradually reduced to form the 1/r-reduced image (r = 1, 2, 3, …) until the representative features of local patterns are accurately recognizable after the corresponding r-th noise reduction. We have studied the influence of a combination of random noise and pattern-edge width (in pixels) on the CG resolution R using both PC-made test images and typical SEM images. As the random noise is larger, the value of R becomes larger (or poorer). The increase in R due to the noise influence is more significant for the narrower width of the pattern edge. This satisfactorily agrees with the common idea that the image resolution is to reflect the sharpness of the pattern edge recognized in the image noise. By revising the form of ΔR from the weighted standard fluctuation of R to the differential of R, a significantly weak Ndot-dependency on R has been favorably confirmed, with keeping firmly the noise-dependency on R. Here, Ndot is the number of pattern dots in the image. [ABSTRACT FROM PUBLISHER]

Nrarp (Notch-regulated ankyrin repeat protein) is a small protein that has two ankyrin repeats. Although Nrarp is known to be an inhibitory component of the Notch signalling pathway that operates in different developmental processes, the in vivo roles of Nrarp have not been fully characterized. Here, we show that Nrarp is a positive regulator in the Wnt signalling pathway. In zebrafish, knockdown of Nrarp-a expression by an antisense morpholino oligonucleotide (MO) results in altered Wnt-signalling-dependent neural-crest-cell development. Nrarp stabilizes LEF1 protein, a pivotal transcription factor in the Wnt signalling cascade, by blocking LEF1 ubiquitination. In accordance with this, the knockdown phenotype of lef1 is similar to that of nrarp-a, at least in part, in its effect on the development of multiple tissues in zebrafish. Furthermore, activation of LEF1 does not affect Notch activity or vice versa. These findings reveal that Nrarp independently regulates canonical Wnt and Notch signalling by modulating LEF1 and Notch protein turnover, respectively. [ABSTRACT FROM AUTHOR]

Signal transducer and activator of transcription 3 (STAT3) is activated by the IL-6 family of cytokines and growth factors. STAT3 requires phosphorylation on Ser-727, in addition to tyrosine phosphorylation on Tyr-70S, to be transcriptionally active. In IL-6 signaling, the two major pathways that derive from the YXXQ and the YSTV motifs of gp130 cause Ser-727 phosphorylation. Here, we show that TGF-β-activated kinase 1 (TAK1) interacts with STAT3, that the TAK1-Nemo-like kinase (NLK) pathway is efficiently activated by IL-6 through the YXXQ motif, and that this is the YXXQ-mediated H7-sensitive pathway that leads to STAT3 Ser-727 phosphorylation. Because NLK was recently shown to interact with STAT3, we explored the role of STAT3 in activating this pathway. Depletion of STAT3 diminished the IL-6-induced NLK activation by >80% without inhibiting IL-6-induced TAK1 activation or its nu- clear entry. We found that STAT3 functioned as a scaffold for TAK1 and NLK in vivo through a region in its carboxyl terminus. Further- more, the expression of the STAT3534-770 region in the nuclei of STAT3-knockdown cells enhanced the IL-6-induced NLK activation in a dose-dependent manner but not the TGFβ-induced NLK activation. TGFβ did not cause STAT3 Ser-727 phosphorylation, even when the carboxyl region of STAT3 was expressed in the nuclei. Together, these results indicate that STAT3 enhances the efficiency of its own Ser-727 phosphorylation by acting as a scaffold for the TAK1-NLK kinases, specifically in the YXXQ motif-derived pathway. [ABSTRACT FROM AUTHOR]

The Wnt/β-catenin signaling pathway regulates many developmental processes by modulating gene expression. Writ signaling induces the stabilization of cytosolic β-catenin, which then associates with lymphoid enhancer factor and T-cell factor (LEF-1/TCF) to form a transcription complex that activates Wnt target genes. Previously, we have shown that a specific mitogen-activated protein (MAP) kinase pathway involving the MAP kinase TAK1 and MAP kinase-related Nemo-like kinase (NLK) suppresses Wnt signaling. In this study, we investigated the relationships among NLK, β-catenin, and LEF-1/TCF. We found that NLK interacts directly with LEF-1/TCF and indirectly with β-catenin via LEF-1/TCF to form a complex. NLK phosphorylates LEF-1/TCF on two serine/threonine residues located in its central region. Mutation of both residues to alanine enhanced LEF-1 transcriptional activity and rendered it resistant to inhibition by NLK. Phosphorylation of TCF-4 by NLK inhibited DNA binding by the β-catenin-TCF-4 complex. However, this inhibition was abrogated when a mutant form of TCF-4 was used in which both threonines were replaced with valines. These results suggest that NLK phosphorylation on these sites contributes to the down-regulation of LEF-1/TCF transcriptional activity. [ABSTRACT FROM AUTHOR]

Wnt signaling controls a variety of developmental processes. The canonical Wnt/β-catenin pathway functions to stabilize β-catenin, and the noncanonical Wnt/Ca[sup 2+] pathway activates Ca[sup 2+]/calmodulin-dependent protein kinase II (CaMKII). In addition, the Wnt/Ca[sup 2+] pathway activated by Wnt-5a antagonizes the Wnt/β-catenin pathway via an unknown mechanism. The mitogen-activated protein kinase (MAPK) pathway composed of TAK1 MAPK kinase kinase and NLK MAPK also negatively regulates the canonical Wnt/β-catenin signaling pathway. Here we show that activation of CaMKII induces stimulation of the TAK1-NLK pathway. Overexpression of Wnt-5a in HEK293 cells activates NLK through TAK1. Furthermore, by using a chimeric receptor (β[sub 2]AR-Rfz-2) containing the ligand-binding and transmembrane segments from the β[sub 2]-adrenergic receptor (β[sub 2]AR) and the cytoplasmic domains from rat Frizzled-2 (Rfz-2), stimulation with the β-adrenergic agonist isoproterenol activates activities of endogenous CaMKII, TAK1, and NLK and inhibits β-catenin-induced transcriptional activation. These results suggest that the TAK1-NLK MAPK cascade is activated by the noncanonical Wnt-5a/Ca[sup 2+] pathway and antagonizes canonical Wnt/β-catenin signaling. [ABSTRACT FROM AUTHOR]

In a previous study, we proposed the contrast‐to‐gradient (CG) method for evaluating image resolution. Here, the CG resolution is defined as a weighted harmonic mean of the local resolution, which is proportional to the quotient of the threshold contrast divided by the local gradient. The local gradient is calculated from the quadratic function that best fits the local pixel intensities over the region of interest (ROI) of 3 × 3 or 5 × 5 pixels in size. To refine the CG method, some modifications are carried out in the present study. Directional resolutions are employed to evaluate images, including astigmatism or strongly directional patterns as well as isotropic patterns. Here, CG resolution is redefined so as to keep the same value even for the image reversed in black‐and‐white contrast, because of no difference in the image information during contrast reversing. Besides, CG resolution is characterized to be independent of the brightness/contrast change unless these changes do not bring about both cut‐off and saturation in the pixel intensities. Dependencies of the denoising effect and the resolution accuracy on ROI size are demonstrated as a function of image noise. [ABSTRACT FROM PUBLISHER]

A Monte Carlo simulation of ion‐induced kinetic electron emission (KE) was carried out to study the material contrast in scanning ion microscope (SIM) images, i.e. secondary electron (SE) yields decreasing with atomic number Z2 of the target, which is opposite to that for scanning electron microscope (SEM) images. The simulations show that SE yields decrease with increasing Z2 for the targets of Al (Z2 = 13), Cu (Z2 = 29) and Au (Z2 = 79) bombarded by 10∼40 keV gallium (Ga) ions. Details of the SE yield according to the collision partners (i.e. Ga ion, recoiled target‐atom and excited electron) clarify the origins of material (or Z2) contrast in the Ga‐SIM images. Cause and effect on the material contrast are as follows: the heavier (or slower) collision partner transfers less energy to the excited electrons and leads to a poorer multiplication of other excited electrons in the cascade process. The simulation also predicts that the Ga‐SIM images are more sensitive to the outermost target surface than the SEM images. [ABSTRACT FROM PUBLISHER]

Presents research which showed that TAK1 activation stimulates NEMO-like kinase activity (NLK) and downregulates transcriptional activation mediated by beta-catenin and T-cell factor (TCF). Role of the Wnt signaling pathway in animal development; Signaling in Drosophila and in Caenorhabditis elegans; Effects of injecting NLK; Negative regulation by the TAK1-NLK-1-MAPK-like pathway of the Wnt pathway.

Presents research which showed that the genes mom-4 and lit-1 are required to downregulate POP-1 in cell fate activity during animal development. Role of the signaling protein Wnt in animal development; Activity of the HMG-domain-containing repressor POP-1 in Caenorhabditis elegans; Traits of the mutant phenotypes of mom-4 and lit-1; Binding by mom-4 and tak-1; Cooperation of a MAPK-related pathway with Wnt signal transduction to downregulate POP-1 activity; Functions in vertebrates.

Local temperature rise Θ in focused-ion-beam (FIB) sample preparation has been calculated using a formalism for laser beam heating by Nissim et al. Typical calculations have been carried out for Si, GaAs, and SiO samples bombarded by the usual 30 keV FIBs. It is figured that an FIB-heated region is like a hemisphere with double the radius of the FIB, and the hemisphere closely follows the scanning FIB. Θ values of less than 100 K are predicted for Si and GaAs samples having their shapes of bulk, semi-bulk, and sheet (sustained with the substrate) under the usual FIB conditions of /=20 nA/μm at maximum. For SiO sample, on the other hand, the Θ values are larger by about 100 times than those for Si sample because of its low thermal conductivity and diflusivity of heat. As to a pillar-shaped sample, the Θ value is rather enhanced due to its poor heat conduction. [ABSTRACT FROM PUBLISHER]

The focused ion beam (FIB) technique has been applied for digging damageable biological specimens of human hair and the housefly eye. Sharp cross-sections have been formed for these specimens and their cross-sectional structures have been observed in scanning ion microscope (SIM) images. The applicability of FIB has been confirmed for these biological specimens [ABSTRACT FROM PUBLISHER]

Frequent mutation of the tumour suppressor RNF43 is observed in many cancers, particularly colon malignancies. RNF43, an E3 ubiquitin ligase, negatively regulates Wnt signalling by inducing degradation of the Wnt receptor Frizzled. In this study, we discover that RNF43 activity requires phosphorylation at a triplet of conserved serines. This phospho-regulation of RNF43 is required for zebrafish development and growth of mouse intestinal organoids. Cancer-associated mutations that abrogate RNF43 phosphorylation cooperate with active Ras to promote tumorigenesis by abolishing the inhibitory function of RNF43 in Wnt signalling while maintaining its inhibitory function in p53 signalling. Our data suggest that RNF43 mutations cooperate with KRAS mutations to promote multi-step tumorigenesis via the Wnt-Ras-p53 axis in human colon cancers. Lastly, phosphomimetic substitutions of the serine trio restored the tumour suppressive activity of extracellular oncogenic mutants. Therefore, harnessing phospho-regulation of RNF43 might be a potential therapeutic strategy for tumours with RNF43 mutations. RNF43 is frequently mutated in cancers and negatively regulates Wnt signalling. Here, the authors report that RNF43 phosphorylation at a serine triplet is required for the negative regulation of Wnt signalling and that the phosphorylation of RNF43 suppresses cancer-associated oncogenic RNF43 mutants. [ABSTRACT FROM AUTHOR]

Morphogen signalling forms an activity gradient and instructs cell identities in a signalling strength-dependent manner to pattern developing tissues. However, developing tissues also undergo dynamic morphogenesis, which may produce cells with unfit morphogen signalling and consequent noisy morphogen gradients. Here we show that a cell competition-related system corrects such noisy morphogen gradients. Zebrafish imaging analyses of the Wnt/β-catenin signalling gradient, which acts as a morphogen to establish embryonic anterior-posterior patterning, identify that unfit cells with abnormal Wnt/β-catenin activity spontaneously appear and produce noise in the gradient. Communication between unfit and neighbouring fit cells via cadherin proteins stimulates apoptosis of the unfit cells by activating Smad signalling and reactive oxygen species production. This unfit cell elimination is required for proper Wnt/β-catenin gradient formation and consequent anterior-posterior patterning. Because this gradient controls patterning not only in the embryo but also in adult tissues, this system may support tissue robustness and disease prevention. Gradients of morphogens such as Wnt provide instructive cues for cell identities during development. Here, the authors report that in the developing zebrafish embryo, cell competition and elimination of unfit cells are required for proper Wnt gradient formation. [ABSTRACT FROM AUTHOR]