Search

We identified a zebrafish homologue of Dickkopf-1 (Dkk1), which was previously identified in Xenopus as a Wnt inhibitor with potent head-inducing activity. Zebrafish dkk1 is expressed in the dorsal marginal blastoderm and also in the dorsal yolk syncytial layer after mid-blastula transition. At later blastula stages, the expression expands to the entire blastoderm margin. During gastrulation, dkk1-expressing cells are confined to the embryonic shield and later to the anterior axial mesendoderm, prospective prechordal plate. Embryos, in which dkk1 was ectopically expressed, exhibited enlarged forebrain, eyes, and axial rnesendoderm such as prechordal plate and notochord, dkk1 expression in the dorso.anterior mesendoderm during gastrulation was prominently reduced in zebrafish mutants bozozok (boz), squint (sqt), and one-eyed pinhead (oep), which all display abnormalities in the formation and function of the Spemann organizer and axial mesendoderm, dkk1 expression was normal in these embryos during the blastula period, indicating that zygotic functions of these genes are required for maintenance but not establishment of dkk1 expression. Overexpression of dkk1 suppressed defects in the development of forebrain, eyes, and notochord in boz mutants. Overexpression of dkk1 promoted anterior neuroectoderm development in the embryos injected with antivin RNA, which lack most of the mesoderm and endoderm, suggesting that Dkk1 can affect regionalization of neuroectoderm independently of dorso-anterior mesendoderm. These data indicate that Dkk1, expressed in dorsal mesendoderm, functions in the formation of both the anterior nervous system and the axial mesendoderm in zebrafish.

Cell lineages during ascidian embryogenesis are invariant. In a previous study, the developmental fate of each blastomere during embryogenesis of the ascidian Halocynthia roretzi was analyzed in detail by intracellular injection of a tracer molecule, horseradish peroxidase (Nishida, H., Dev. Biol. 121, 526-541, 1987). In the present study, the developmental fates of various larval mesodermal tissues after metamorphosis in H. roretzi were traced by labeling each precursor blastomere of larval tissues. Mesenchyme cells of larvae gave rise to tunic cells of juveniles. Trunk ventral cells gave rise to body-wall (atrial siphon and latitudinal mantle) muscle, heart, and pericardium of juvenile. Trunk lateral cells gave rise to blood (coelomic) cells as well as body-wall (oral siphon and longitudinal mantle) muscle. Larval muscle and notochord cells did not contribute to juvenile tissues. It was revealed that it is predictable to which juvenile tissue a blastomere of the 110-cell embryo will give rise. Therefore, in ascidian development, developmental fates after metamorphosis were almost invariant with regard to cell type of descendants. Key Words: cell lineages; ascidian development; metamorphosis; HRP injection; mesoderm differentiation.

The zebrafish homeobox gene dharma/bozozok (boz) is required for the formation and/or function of the Nieuwkoop center and the subsequent induction of the Spemann organizer, dharma is expressed soon after the midblastula transition in the dorsal blastomeres and the dorsal yolk syncytial layer (YSL). We found that the expression of dharma was upregulated or ectopically induced by misexpression of a Wnt protein and cytoplasmic components of the Wnt signaling pathway and downregulated by the expression of dominant-negative Tcf3. A 1.4-kbp fragment of the dharma promoter region contains consensus sequences for Tcf/Lef binding sites. This promoter region recapitulated the Wnt-dependent and dorsal dharma expression pattern when it was fused to luciferase or GFP. Deletion and point mutant analyses revealed that the Tcf/Lef binding sites were required to drive this expression pattern. These data established that dharma/boz functions between the dorsal determinants-mediated Wnt signals and the formation of the Nieuwkoop center. [C] 2001 Academic Press Key Words: dharma; bozozok; Wnt signal; Nieuwkoop center; [Beta]-catenin; Tcf; Lef.

Kheper is a novel member of the ZFH (zinc-finger and homeodomain protein)/[Delta]EF1 family in zebrafish. kheper transcripts are first detected in the epiblast of the dorsal blastoderm margin at the early gastrula stage and kheper is expressed in nearly all the neuroectoderm at later stages, kheper expression was expanded in noggin RNA-injected embryos and also in swirl mutant embryos and was reduced in bmp4 RNA-injected embryos and chordino mutant embryos, suggesting that kheper acts downstream of the neural inducers Noggin and Chordino. Overexpression of Kheper elicited ectopic expansion of the neuroectoderm-specific genes fkd3, hoxa-1, and eng3, and the ectopic expression of hoxa-1 was not inhibited by BMP4 overexpression. Kheper interacted with the transcriptional corepressors CtBP1 and CtBP2. Overexpression of a Kheper mutant lacking the homeodomain or of a VP16-Kheper fusion protein disturbed the development of the neuroectoderm and head structures. These data underscore the role of Kheper in the development of the neuroectoderm and indicate that Kheper acts as a transcriptional repressor. [C] 2000 Academic Press Key Words: zebrafish; ZFH; [Delta]EF1; zinc-finger; homeodomain; transcription factor; ectoderm; mesoderm; CtBP; repressor.

HuC encodes an RNA binding protein homologous to Drosophila elav that serves as an excellent early marker for differentiating neurons. We have characterized the promoter of the zebrafish HuC gene by examining the ability of 5'-upstream fragments to drive expression of green fluorescent protein (GFP) in live embryos. We determined that 9.8 kb of the 5'-flanking sequence is sufficient to restrict GFP gene expression to neurons. The core promoter spans 251 base pairs and contains a CCAAT box and one SP1 sequence but no TATA box is present near the transcription start site. A putative MyT1 binding site and at least 17 E-box sequences are necessary to maintain the neuronal specificity of HuC expression. Interestingly, sequential removal of the putative MyT1 binding site and 14 distal E boxes does not appear to abolish neuronal expression; rather, it leads to a progressive expansion of GFP expression into muscle cells. Further removal of the three proximal E boxes eliminates neuronal and muscle specificity of GFP expression and leads to ubiquitous expression of GFP in the whole body. Identification of key components of the HuC promoter has led to the establishment of a stable zebrafish transgenic line (HuC-GFP) in which GFP is expressed specifically in neurons. We crossed mind bomb (mib) fish with this line to visualize their neurogenic phenotype in live [mib.sup.-/-] mutant embryos. This cross illustrates how HuC-GFP fish could be used in the future to identify and analyze zebrafish mutants with an aberrant pattern of early neurons. [C] 2000 Academic Press Key Words: HuC; elav; zebrafish; neuron; promoter; Myt1; E box; transgenic; mind bomb.

Summary: The IL-6-triggered positive feedback loop for NFκB signaling (or the IL-6 amplifier/Inflammation amplifier) was originally discovered as a synergistic-activation signal that follows IL-17/IL-6 stimulation in nonimmune cells. Subsequent results from animal models have shown that the amplifier is activated by stimulation of NFκB and STAT3 and induces chemokines and inflammation via an NFκB loop. However, its role in human diseases is unclear. Here, we combined two genome-wide mouse screens with SNP-based disease association studies, revealing 1,700 genes related to the IL-6 amplifier, 202 of which showed 492 indications of association with ailments beyond autoimmune diseases. We followed up on ErbB1 from our list. Blocking ErbB1 signaling suppressed the IL-6 amplifier, whereas the expression of epiregulin, an ErbB1 ligand, was higher in patients with inflammatory diseases. These results indicate that the IL-6 amplifier is indeed associated with human diseases and disorders and that the identified genes may make for potential therapeutic targets. [ABSTRACT FROM AUTHOR]

Interleukin 6 (IL-6), which was originally identified as a B-cell differentiation factor, is now known to be a multifunctional cytokine that regulates the immune response, hematopoiesis, the acute phase response, and inflammation. Deregulation of IL-6 production is implicated in the pathology of several disease processes. The expression of constitutively high levels of IL-6 in transgenic mice results in fatal plasmacytosis, which has been implicated in human multiple myeloma. Increased IL-6 levels are also observed in several diseases, including rheumatoid arthritis (RA), systemic-onset juvenile chronic arthritis (JCA), osteoporosis, and psoriasis. IL-6 is critically involved in experimentally induced autoimmune disease, such as antigen-induced arthritis (AIA), and experimental allergic encephalomyelitis. All these clinical data and animal models suggest that IL-6 plays critical roles in the pathogenesis of autoimmune diseases. Here we review the evidence for the involvement of IL-6 in the pathophysiology of autoimmune diseases and chronic inflammatory proliferative diseases (CIPD) and discuss the possible molecular mechanisms of its involvement. [Copyright &y& Elsevier]

Signaling via suppressors of cytokine signaling (SOCS) is an important negative feedback system for cytokine-mediated signal transduction. Recently in Molecular Cell, described the tertiary structure of SOCS3 in complex with a phosphotyrosine-containing peptide from the IL-6 receptor subunit gp130, and they identified the specific amino acids that are critical for binding. [Copyright &y& Elsevier]

Zinc (Zn) is an essential nutrient, and Zn deficiency causes immunodeficiency and skin disorders. Basophils express FcɛRI on their surface and release multiple mediators after receptor cross-linking, including large amounts of IL-4. However, the mechanisms involved in the FcɛRI-mediated regulation of basophil IL-4 production are currently unclear. Here, we show that the Zn-binding metallothionein (MT) proteins are essential for the FcɛRI-induced basophil production of IL-4. Basophils from MT-I/II(−/−) mice produced significantly less FcɛRI-induced IL-4 than did wild-type basophils. The MTs were involved in maintaining intracellular Zn levels, thereby regulated the calcineurin activity and nuclear factor of activated T-cell (NFAT)-mediated IL-4 production. These results suggest that the MT-dependent control of Zn homeostasis is a novel mechanism for regulating basophil IL-4 production. [ABSTRACT FROM AUTHOR]

The human SLC39A13 gene encodes ZIP13, a member of the LZT (LIV-1 subfamily of ZIP zinc transporters) family. The ZIP13 protein is important for connective tissue development, and its loss of function is causative for the spondylocheiro dysplastic form of Ehlers-Danlos syndrome. However, this protein has not been characterized in detail. Here we report the first detailed biochemical characterization of the human ZIP13 protein using its ectopic expressed and the purified recombinant protein. Protease accessibility, microscopic, and computational analyses demonstrated that ZIP13 contains eight putative transmembrane domains and a unique hydrophilic region and that it resides with both its N and C termini facing the luminal side on the Golgi. Analyses including cross-linking, immunoprecipitation, Blue Native-PAGE, and size-exclusion chromatography experiments indicated that the ZIP13 protein may form a homo-dimer. We also demonstrated that ZIP13 mediates zinc influx, as assessed by monitoring the expression of the metallothionein gene and by detecting the intracellular zinc level with a zinc indicator, FluoZin-3. Our data indicate that ZIP13 is a homo-dimerized zinc transporter that possesses some domains that are not found in other LZT family members. This is the first biochemical characterization of the physiologically important protein ZIP13 and the demonstration of homo-dimerization for a mammalian ZIP zinc transporter family member. This biochemical characterization of the human ZIP13 protein provides important information for further investigations of its structural characteristics and function. [ABSTRACT FROM AUTHOR]

Grb2-associated binder 1 (Gab1) coordinates various receptor tyrosine kinase signaling pathways. Although skeletal muscle differentiation is regulated by some growth factors, it remains elusive whether Gab1 coordinates myogenic signals. Here, we examined the molecular mechanism of insulin-like growth factor-I (IGF-I)-mediated myogenic differentiation, focusing on Gab1 and its downstream signaling. Gab1 underwent tyrosine phosphorylation and subsequent complex formation with protein-tyrosine phosphatase SHP2 upon IGF-I stimulation in C2C12 myoblasts. On the other hand, Gab1 constitutively associated with phosphatidylinositol 3-kinase regulatory subunit p85. To delineate the role of Gab! in IGF-I-dependent signaling, we examined the effect of adenovirus-mediated forced expression of wild-type Gab1 (Gab1WT), mutated Gab1 that is unable to bind SHP2 (Gab1ΔSHP2), or mutated Gab1 that is unable to bind p85 (Gab1Δp85), on the differentiation of C2C12 myoblasts. IGF-I-induced myogenic differentiation was enhanced in myoblasts overexpressing Gab1ΔSHP2, but inhibited in those overexpressing either Gab1WT or Gab1Δp85. Conversely, IGF-I-induced extracellular signal-regulated kinase 1/2 (ERK1/2) activation was significantly repressed in myoblasts overexpressing Gab1ΔSHP2 but enhanced in those overexpressing either Gab1WT or Gab1Δp85. Furthermore, small interference RNA-mediated Gab! knockdown enhanced myogenic differentiation. Overexpression of catalytic-inactive SHP2 modulated IGF-I-induced myogenic differentiation and ERK1/2 activation similarly to that of Gab1ΔSHP2, suggesting that Gab1- SHP2 complex inhibits IGF-I-dependent myogenesis through ERK1/2. Consistently, the blockade of ERK1/2 pathway reversed the inhibitory effect of Gab1WT overexpression on myogenic differentiation, and constitutive activation of the ERK1/2 pathway suppressed the enhanced myogenic differentiation by overexpression of Gab1ΔSHP2. Collectively, these data suggest that the Gab1-SHP2-ERK1/2 signaling pathway comprises an inhibitory axis for IGF-I-dependent myogenic differentiation. [ABSTRACT FROM AUTHOR]

Abstract: Mice having a point mutation of IL-6 signal transducer, gp130, named gp130F759 induced a rheumatoid arthritis-like disease. The disease induction is totally dependent on mature lymphocytes, since no arthritis-like disease induced in a double mutant between gp130F759 and RAG2KO mice. We prepared several other double mutants including gp130F759/IgMKO, gp130F759/CD8KO, gp130F759/CIITAKO and gp130F759/CD4KO and analyzed the development of the arthritis. We found that development of the disease attenuated in gp130F759/CIITAKO and gp130F759/CD4KO, suggesting the MHC class II-restricted CD4+ T cells are important for the disease development. We showed memory/activated phenotype of CD4+ T cells increased in gp130F759 mice. Since activation of CD4+ T cells is mainly controlled by dendritic cells (DC) in vivo, we investigated this feature of DC in gp130F759 mice. We isolated DCs from superficial lymph nodes and observed that IL-6-mediated signaling suppresses maturation of DCs in vivo. However, total number of DCs in vivo significantly increased in gp130F759 mice compared with wild type controls. Thus, we hypothesize that the rheumatoid arthritis-like disease in gp130F759 mice is induced by an interaction between CD4+ T cells and DC under gp130F759 signal regulation. [Copyright &y& Elsevier]

The first cell differentiation event in mammalian embryogenesis segregates inner cell mass lineage from the trophectoderm at the blastocyst stage. Oct-4, a member of the POU family of transcription factors, is necessary for the pluripotency of the inner cell mass lineage. Embryonic stem (ES) cells, which contribute to all of embryonic lineages, express the Oct-4 gene. Trophoblast stem (TS) cells, which have the ability to differentiate into trophoblast lineage in vitro, never contribute to embryonic proper tissues in chimeras and differentiate only into trophoblastic cells in the placenta. Expression of the Oct-4 gene was undetectable and severely repressed in trophoblastic lineage, including the stem cells. We found that the culture of TS cells with 5-aza-2′- deoxycytidine or trichostatin A caused the activation of the Oct-4 gene. Analysis of the DNA methylation status of mouse Oct-4 gene upstream region revealed that Oct.4 enhancer/promoter region was hypomethylated in ES cells but hypermethylated in TS cells. Furthermore, in vitro methylation suppressed Oct-4 enhancer/promoter activity in reporter assay. In the placenta of Dnmt1n/n mutant mice, most of the CpGs in the enhancer/promoter region were unmethylated, and Oct-4 gene expression was aberrantly detected. Chromatin immunoprecipitation assay revealed that Oct-4 enhancer/ promoter region was hyperacetylated in ES cells compared with TS cells, thus demonstrating that DNA methylation status is closely linked to the chromatin structure of the Oct-4 gene. Here we propose that the epigenetic mechanism, consisting of DNA methylation and chromatin remodeling, underlies the developmental stage- and cell type-specific mechanism of Oct-4 gene expression. [ABSTRACT FROM AUTHOR]

In zebrafish, the program for dorsal specification begins soon after fertilization. Dorsal determinants are localized initially to the vegetal pole, then transported to the blastoderm, where they are thought to activate the canonical Wnt pathway, which induces the expression of dorsal-specific genes. We identified a novel maternal-effect recessive mutation, tokkaebi (tkk), that affects formation of the dorsal axis. Severely ventralized phenotypes, including a lack of dorso-anterior structures, were seen in 5–100% of the embryos obtained from tkk homozygous transmitting females. tkk embryos displayed defects in the nuclear accumulation of β-catenin on the dorsal side, and reduced or absent expression of dorsal-specific genes. Mesoderm and endoderm formation outside the dorsal axis was not significantly affected. Injection of RNAs for activated β-catenin, dominant-negative forms of Axin1 and GSK3β, and wild-type Dvl3, into the tkk embryos suppressed the ventralized phenotypes and/or dorsalized the embryos, and restored or induced an ectopic and expanded expression of bozozok/dharma and goosecoid. However, dorsalization by wnt RNAs was affected in the tkk embryos. Inhibition of cytoplasmic calcium release elicited an ectopic and expanded expression of chordin in the wild-type, but did not restore chordin expression efficiently in the tkk embryos. These data indicate that the tkk gene product functions upstream of or parallel to the β-catenin-degradation machinery to control the stability of β-catenin. The tkk locus was mapped to chromosome 16. These data provide genetic evidence that the maternally derived canonical Wnt pathway upstream of β-catenin is involved in dorsal axis formation in zebrafish. [Copyright &y& Elsevier]

Dharma/Bozozok (Dha/Boz) is a homeodomain protein containing an Engrailed homology (Eh) 1 repressor motif. It is important in zebrafish dorsal organizer formation. Dha/Boz interacted with a co-repressor Groucho through the Eh1 motif. Expression of a Dha/Boz fused to the transcriptional activator VP16 repressed dorsal axis formation and the expression of organizer genes but led to the dorsal expansion of expression of the homeobox gene vox/vega1, indicating that Dha/Boz functions as a transcriptional repressor for dorsal axis formation. We also isolated a novel homeobox gene, ved, whose expression was negatively regulated by dha/boz. ved''s sequence and expression profile were similar to those of vox/vega1 and vent/vega2. Like Vox/Vega1 and Vent/Vega2, Ved acted as a transcriptional repressor. The combined inhibition of ved, vox/vega1, and vent/vega2, by antisense morpholino injection, strongly dorsalized the embryos and elicited ventral expansion of organizer gene expression, compared with the effect of inhibiting each of these genes alone. These results suggest that ved is a target for the repressor Dha/Boz. ved functions redundantly with vox/vega1 and vent/vega2 to restrict the organizer domain. [Copyright &y& Elsevier]

Vertebrate axis formation requires both the correct specification of cell fates and the coordination of gastrulation movements. We report that the zebrafish signal transducer and activator of transcription 3 (Stat3) is activated on the dorsal side by the maternal Wnt/β-catenin pathway. Zebrafish embryos lacking Stat3 activity display abnormal cell movements during gastrulation, resulting in a mispositioned head and a shortened anterior-posterior axis, but show no defects in early cell fate specification. Time course analysis, cell tracing, and transplantation experiments revealed that Stat3 activity is required cell autonomously for the anterior migration of dorsal mesendodermal cells and non-cell autonomously for the convergence of neighboring paraxial cells. These results reveal a role for Stat3 in controlling cell movements during gastrulation. [Copyright &y& Elsevier]