Injection Of Antisense Morpholino Oligonucleotides Research Articles

KEPI plays a negative role in the repression that accompanies translational inhibition guided by the uORF element of human CHOP transcript during stress response

Translation of the downstream coding sequence of some mRNAs may be repressed by the upstream open reading frame (uORF) at their 5′-end. The mechanism underlying this uORF-mediated translational inhibition (uORF-MTI) is not fully understood in vivo. Recently, it was found that zebrafish Endouc or its human orthologue ENDOU (Endouc/ENDOU) plays a positive role in repressing the uORF-MTI of human CHOP (uORFchop-MTI) during stress by blocking its activity However, the repression of uORFchop-MTI assisted by an as-yet unidentified negative effector remains to be elucidated. Compared to the upregulated CHOP transcript, we herein report that the kepi (kinase-enhanced PP1 inhibitor) transcript was downregulated in the zebrafish embryos treated with both heat shock and hypoxia. Quantitative RT-PCR also revealed that the level of kepi mRNA was noticeably decreased in both heat-shock-treated and hypoxia-exposed embryos. When kepi mRNA was microinjected into the one-celled embryos from transgenic line huORFZ, the translation of downstream GFP reporter controlled by the uORFchop-MTI was reduced in the hypoxia-exposed embryos. In contrast, when kepi was knocked down by injection of antisense Morpholino oligonucleotide, the translation of downstream GFP reporter was induced and expressed in the brain and spinal cord of injected embryos in the absence of stress. During normal condition, overexpression of KEPI increased eIF2α phosphorylation, resulting in inducing the translation of uORF-tag mRNA, such as ATF4 and CHOP mRNAs. However, during stress condition, overexpression of KEPI decreased eIF2α phosphorylation, resulting in reducing the GFP reporter and CHOP proteins. This is the first report to demonstrate that KEPI plays a negative role in uORFchop – mediated translation during ER stress.

ETV6/TEL1 Is Required for HSC Emergence during Embryonic Development.

ETV6/TEL1 (hereinafter referred to as TEL1) is an ets family transcription factor that is frequently involved in chromosomal translocations associated with leukaemia, including t(12:21) which generates the TEL-AML1 fusion gene, the most common genetic abnormality in childhood cancer. The role of TEL1 in normal haemopoiesis has been extensively studied in the mouse where knock-out studies have indicated that TEL1 is not required for development of primitive (ie yolk sac-derived) haemopoiesis. TEL1-knockout mice die at day E10.5 due to a defect in angiogenesis precluding direct analysis of the definitive (ie intraembryonically-derived) haematopoietic system. Analysis of chimaeric mice generated using TEL1−/− ES cells suggest that TEL1 is not required for generation of the definitive haemopoietic system during ontogeny but is required for HSC survival perinatally at bone marrow stages. Conditional knockout studies have subsequently identified a role for TEL1 in the survival of adult bone marrow-derived HSC. We now report the analysis of TEL1 function in amphibian (Xenopus) embryos where haemopoietic development can be studied even in embryos with defective angiogenesis. We characterised the TEL1 expression pattern and knocked down its function using antisense morpholino oligonucleotide injection. Consistent with the mouse knockout, specification of the primitive haemopoietic system was unaffected, however a defect in maturation of primitive erythroid cells, not previously reported in the mouse, was also apparent. We next analysed the development of the definitive haemopoietic system, thought to arise as clusters of HSCs in the floor of the dorsal aorta. But, no clusters of HSCs were found. Formation of the dorsal aorta however, proceeded normally, indicating that TEL1 controls the decision between the arterial and HSC programmes. Given, the critical role for TEL1 in the development of HSC indicated by these studies, a temporal analysis of emergence of intraembryonic HSC in TEL-deficient mice may be warranted.

No Evidence for AID/MBD4-Coupled DNA Demethylation in Zebrafish Embryos

The mechanisms responsible for active DNA demethylation remain elusive in Metazoa. A previous study that utilized zebrafish embryos provided a potent mechanism for active demethylation in which three proteins, AID, MBD4, and GADD45 are involved. We recently found age-dependent DNA hypomethylation in zebrafish, and it prompted us to examine if AID and MBD4 could be involved in the phenomenon. Unexpectedly, however, we found that most of the findings in the previous study were not reproducible. First, the injection of a methylated DNA fragment into zebrafish eggs did not affect either the methylation of genomic DNA, injected methylated DNA itself, or several loci tested or the expression level of aid, which has been shown to play a role in demethylation. Second, aberrant methylation was not observed at certain CpG islands following the injection of antisense morpholino oligonucleotides against aid and mbd4. Furthermore, we demonstrated that zebrafish MBD4 cDNA lacked a coding region for the methyl-CpG binding domain, which was assumed to be necessary for guidance to target regions. Taken together, we concluded that there is currently no evidence to support the proposed roles of AID and MBD4 in active demethylation in zebrafish embryos.

Growth cone dynamics in the zebrafish embryonic forebrain are regulated by Brother of Cdo

During development of the embryonic zebrafish brain, the differential expression of axon guidance molecules directs the growth of axons along defined neuronal tracts. Neurons within the dorsorostral cluster of the presumptive telencephalon project axons ventrally along the supraoptic tract. Brother of Cdo (Boc) is a known axon guidance molecule that is expressed in a broad band lying ventral to the dorsorostral cluster of neurons. Loss of Boc function has previously been shown to perturb the development of the supraoptic tract. We have used live cell imaging of individual growth cones within the living zebrafish embryo to determine how Boc regulates the growth cone dynamics and axon guidance within the supraoptic tract. A plasmid construct encoding elavl3-eGFP was injected into early embryos to selectively label a small number of neurons while the expression of Boc was knocked down by injection of antisense morpholino oligonucleotides. Time-lapse imaging of growth cones within the living embryos revealed that loss of Boc significantly affected the morphology of growth cones in comparison to axons within control embryos. Growth cones navigating along the supraoptic tract in the absence of Boc extended significantly longer filopodia in the rostrocaudal direction. These results indicate that Boc acts to restrict axons and their filopodia within the narrow pathway of the supraoptic tract. The highly selective nature of these pathfinding defects reveal that Boc is likely to be one of many molecules that coordinate the trajectory of axons within the supraoptic tract.

Sphingosine-1-Phosphate Receptor-1 Controls Venous Endothelial Barrier Integrity in Zebrafish

Endothelial sphingosine-1-phosphate (S1P) receptor-1 (S1P(1)) affects different vascular functions, including blood vessel maturation and permeability. Here, we characterized the role of the zS1P(1) ortholog in vascular development in zebrafish. zS1P(1) is expressed in dorsal aorta and posterior cardinal vein of zebrafish embryos at 24 to 30 hours postfertilization. zS1P(1) downregulation by antisense morpholino oligonucleotide injection causes early pericardial edema, lack of blood circulation, alterations of posterior cardinal vein structure, and late generalized edema. Also, zS1P(1) morphants are characterized by downregulation of vascular endothelial cadherin (VE-cadherin) and Eph receptor EphB4a expression and by disorganization of zonula occludens 1 junctions in posterior cardinal vein endothelium, with no alterations of dorsal aorta endothelium. VE-cadherin knockdown results in similar vascular alterations, whereas VE-cadherin overexpression is sufficient to rescue venous vascular integrity defects and EphB4a downregulation in zS1P(1) morphants. Finally, S1P(1) small interfering RNA transfection and the S1P(1) antagonist (R)-3-amino-(3-hexylphenylamino)-4-oxobutylphosphonic acid (W146) cause EPHB4 receptor down-modulation in human umbilical vein endothelial cells and the assembly of zonula occludens 1 intercellular contacts is prevented by the EPHB4 antagonist TNYL-RAW peptide in these cells. The data demonstrate a nonredundant role of zS1P(1) in the regulation of venous endothelial barrier in zebrafish and identify a S1P(1)/VE-cadherin/EphB4a genetic pathway that controls venous vascular integrity.

GLUT10 is required for the development of the cardiovascular system and the notochord and connects mitochondrial function to TGFβ signaling

Growth factor signaling results in dramatic phenotypic changes in cells, which require commensurate alterations in cellular metabolism. Mutations in SLC2A10/GLUT10, a member of the facilitative glucose transporter family, are associated with altered transforming growth factor-β (TGFβ) signaling in patients with arterial tortuosity syndrome (ATS). The objective of this work was to test whether SLC2A10/GLUT10 can serve as a link between TGFβ-related transcriptional regulation and metabolism during development. In zebrafish embryos, knockdown of slc2a10 using antisense morpholino oligonucleotide injection caused a wavy notochord and cardiovascular abnormalities with a reduced heart rate and blood flow, which was coupled with an incomplete and irregular vascular patterning. This was phenocopied by treatment with a small-molecule inhibitor of TGFβ receptor (tgfbr1/alk5). Array hybridization showed that the changes at the transcriptome level caused by the two treatments were highly correlated, revealing that a reduced tgfbr1 signaling is a key feature of ATS in early zebrafish development. Interestingly, a large proportion of the genes, which were specifically dysregulated after glut10 depletion gene and not by tgfbr1 inhibition, play a major role in mitochondrial function. Consistent with these results, slc2a10 morphants showed decreased respiration and reduced TGFβ reporter gene activity. Finally, co-injection of antisense morpholinos targeting slc2a10 and smad7 (a TGFβ inhibitor) resulted in a partial rescue of smad7 morphant phenotypes, suggesting scl2a10/glut10 functions downstream of smads. Taken together, glut10 is essential for cardiovascular development by facilitating both mitochondrial respiration and TGFβ signaling.

The maternal genes Ci-p53/p73-a and Ci-p53/p73-b regulate zygotic ZicL expression and notochord differentiation in Ciona intestinalis embryos

I isolated a Ciona intestinalis homolog of p53, Ci-p53/p73-a, in a microarray screen of rapidly degraded maternal mRNA by comparing the transcriptomes of unfertilized eggs and 32-cell stage embryos. Higher expression of the gene in eggs and lower expression in later embryonic stages were confirmed by whole-mount in situ hybridization (WISH) and quantitative reverse transcription-PCR (qRT-PCR); expression was ubiquitous in eggs and early embryos. Knockdown of Ci-p53/p73-a by injection of antisense morpholino oligonucleotides (MOs) severely perturbed gastrulation cell movements and expression of notochord marker genes. A key regulator of notochord differentiation in Ciona embryos is Brachyury ( Ci-Bra), which is directly activated by a zic-like gene ( Ci-ZicL). The expression of Ci-ZicL and Ci-Bra in A-line notochord precursors was downregulated in Ci-p53/p73-a knockdown embryos. Maternal expression of Ci-p53/p73-b, a homolog of Ci-p53/p73-a, was also detected. In Ci-p53/p73-b knockdown embryos, gastrulation cell movements, expression of Ci-ZicL and Ci-Bra in A-line notochord precursors, and expression of notochord marker gene at later stages were perturbed. The upstream region of Ci-ZicL contains putative p53-binding sites. Cis-regulatory analysis of Ci-ZicL showed that these sites are involved in expression of Ci-ZicL in A-line notochord precursors at the 32-cell and early gastrula stages. These results suggest that p53 genes are maternal factors that play a crucial role in A-line notochord differentiation in C. intestinalis embryos by regulating Ci-ZicL expression.

Wnt16 Is Required for Specification of Vertebrate Hematopoietic Stem Cells through Notch.

AbstractAbstract 2616Hematopoietic stem cells (HSCs) are self-renewing progenitor cells that provide all adult blood lineages over the lifetime of an individual. Understanding the signaling events that regulate specification of these cells during embryonic development is a key precondition to recapitulating those processes in vitro for the purposes of regenerative medicine. Here we report that non-canonical Wnt signaling by the conserved vertebrate ligand, Wnt16 is required for specification of HSCs in zebrafish. WNT16 was originally identified as a gene aberrantly expressed in pre-B acute lymphoblastic leukemia (ALL) cells containing the t(1;19) chromosomal translocation, leading to expression of E2A-PBX1. Wnt16 is expressed in mammals at times when hemogenic endothelial cells first appear in the dorsal aorta. In zebrafish, wnt16 is expressed in somites, which lie adjacent to the dorsal aorta. Knock down of Wnt16 function by injection of antisense morpholino oligonucleotides leads to loss of HSCs and definitive blood lineages, such as T-cells, during development. Non-blood tissues including vasculature appear largely unaffected. Thus, wnt16 is required for HSC specification.To better understand the Wnt16 signal transduction pathway, we examined its ability to activate transcription of target genes through β-catenin/Tcf-dependent “canonical” signaling. Although Wnt16 overexpression causes morphological abnormality, it does not yield ectopic expression of endogenous or transgenic canonical reporter genes at time points relevant to blood specification. Thus, Wnt16 signals independently of β-catenin/Tcf through a “non-canonical” Wnt pathway.Notch signaling is required for specification of HSCs across phyla. To determine whether Notch signaling is disrupted in Wnt16 morphants, we examined the expression of all Notch ligands and receptors in these animals and found that expression of two ligands, deltaC (dlc) and deltaD (dld) are decreased. Zebrafish mutants, homozygous for a null allele of dlc, and embryos injected with a dld morpholino each display decreased numbers of HSCs during development, but recover. Dlc mutants injected with dld morpholino show complete loss of HSCs. To determine whether defects in Notch signaling are responsible for loss of HSCs in Wnt16 morphants, we performed a rescue experiment. Transgenic animals carrying an inducible dominant activator of Notch target genes were injected with Wnt16 morpholino, and Notch activity was either induced or not. Wnt16 morphants with enforced Notch activity recovered HSC marker expression. Taken together, our results indicate that Wnt16 regulates expression of the Notch ligands dlc and dld, and these are redundantly required for HSC specification during development.Chimera experiments in mouse using wild type and Notch1-deficient cells indicate that there is a cell-autonomous requirement for Notch signaling in specification of HSCs. To determine when the first Notch-responsive cells that contribute to the adult hematopoietic system appear, we used animals carrying a transgene encoding a photconvertible, green-to-red Kaede protein under the control of a Notch-responsive promoter. By photoconverting all embryonic cells at various times during development, we determined that cells destined to become HSCs first experience a Notch signal at approximately the time when HSCs first appear, just before 24 hours post fertilization (hpf). At earlier time points, Notch responsive cells were present, but did not contribute to blood, although they did contribute to tissues in or near the dorsal aorta, which contains the hemogenic endothelium that gives rise to HSCs. Surprisingly, HSC rescue in Wnt16 morphants by Notch activation could only be achieved earlier than 16 hpf, long before a cell-autonomous Notch signal is received in pre-HSCs. Moreover, loss of the critical ligands, dlc and dld, in Wnt16 morphant animals was confined to somitic tissue prior to 20 hpf, while ligand expression in or near the dorsal aorta at later times was relatively normal. Together, our results strongly suggest a previously unappreciated, non-cell-autonomous requirement for Notch signaling in the somites. We hypothesize that somitic Notch signaling regulates a morphological process or expression of a relay signal required for HSC specification during development. Disclosures:No relevant conflicts of interest to declare.

Protein O-mannosylation is necessary for normal embryonic development in zebrafish

Two distinct cDNAs corresponding to two zebrafish protein O-mannosyltransferase genes, zPOMT1 and zPOMT2, were cloned from early developmental embryos. Gene expression analysis revealed that zPOMT1 and zPOMT2 were expressed in similar patterns during early embryonic development and in all adult tissues. To study the regulation of zPOMT1 and zPOMT2 mRNA distribution during zebrafish embryogenesis, we injected enhanced green fluorescent protein (EGFP) mRNA fused to the 3'untranslated regions of each zPOMT gene. The distribution of EGFP resulting from the two constructs was similar. Injection of antisense morpholino oligonucleotides of zPOMT1 and zPOMT2 resulted in several severe phenotypes-including bended body, edematous pericaridium and abnormal eye pigmentation. Immunohistochemistry using anti-glycosylated alpha-dystroglycan antibody (IIH6) and morphological analysis revealed that the phenotypes of zPOMT2 knockdown were more severe than those of zPOMT1 knockdown, even though the IIH6 reactivity was lost in both zPOMT1 and zPOMT2 morphants. Finally, only when both zPOMT1 and zPOMT2 were expressed in human embryonic kidney 293T cells were high levels of protein O-mannosyltransferase activity detected, indicating that both zPOMT1 and zPOMT2 were required for full enzymatic activity. Moreover, either heterologous combination, zPOMT1 and human POMT2 (hPOMT2) or hPOMT1 and zPOMT2, resulted in enzymatic activity in cultured cells. These results indicate that the protein O-mannosyltransferase machinery in zebrafish and humans is conserved and suggest that zebrafish may be useful for functional studies of protein O-mannosylation.

Differential expression patterns and developmental roles of duplicated scinderin‐like genes in zebrafish

Scinderin, the closest homologue of the actin-severing protein, gelsolin, has two similar paralogs (Scinla and Scinlb) in zebrafish. Scinla is abundant in the adult cornea; Scinlb comprises considerably less corneal protein. Here, we show that scinla is expressed in the nose, lens, brain, cornea and annular ligament of the iridocorneal angle; by contrast, scinlb is expressed in the hatching gland, floor plate, notochord, otic vesicle, brain, pharynx, cartilage, swim bladder and cornea. Activity of scinla and scinlb promoter fragments driving the EGFP reporter gene in transgenic zebrafish resembled scinla or scinlb expression. Previously, we showed that reduction of scinla by injection of antisense morpholino oligonucleotides ventralized embryos; here, specific reduction of scinlb expression led to subtle brain abnormalities associated with increased cell death, decreased shhb expression in the floor plate, and slightly reduced eye distance. Thus, scinla and scinlb have different expression patterns and developmental roles during zebrafish development.

Spatio-temporal intersection of Lhx3 and Tbx6 defines the cardiac field through synergistic activation of Mesp

Mesp encodes a bHLH transcription factor required for specification of the cardiac mesoderm in Ciona embryos. The activities of Macho-1 and β-catenin, two essential maternal determinants, are required for Mesp expression in the B7.5 blastomeres, which constitute the heart field. The T-box transcription factor Tbx6 functions downstream of Macho-1 as a direct activator of Mesp expression. However, Tbx6 cannot account for the restricted expression of Mesp in the B7.5 lineage since it is expressed throughout the presumptive tail muscles. Here we present evidence that the LIM-homeobox gene Lhx3, a direct target of β-catenin, is essential for localized Mesp expression. Lhx3 is expressed throughout the presumptive endoderm and B7.5 blastomeres. Thus, the B7.5 blastomeres are the only cells to express sustained levels of the Tbx6 and Lhx3 activators. Like mammalian Lhx3 genes, Ci-Lhx3 encodes two isoforms with distinct N-terminal peptides. The Lhx3a isoform appears to be expressed both maternally and zygotically, while the Lhx3b isoform is exclusively zygotic. Misexpression of Lhx3b is sufficient to induce ectopic Mesp activation in cells expressing Tbx6b. Injection of antisense morpholino oligonucleotides showed that the Lhx3b isoform is required for endogenous Mesp expression. Mutations in the Lhx3 half-site of Tbx6/Lhx3 composite elements strongly reduced the activity of a minimal Mesp enhancer. We discuss the delineation of the heart field by the synergistic action of muscle and gut determinants.

Cold-inducible RNA binding protein (CIRP), a novel XTcf-3 specific target gene regulates neural development in Xenopus

BackgroundAs nuclear mediators of wnt/β-catenin signaling, Lef/Tcf transcription factors play important roles in development and disease. Although it is well established, that the four vertebrate Lef/Tcfs have unique functional properties, most studies unite Lef-1, Tcf-1, Tcf-3 and Tcf-4 and reduce their function to uniformly transduce wnt/β-catenin signaling for activating wnt target genes. In order to discriminate target genes regulated by XTcf-3 from those regulated by XTcf-4 or Lef/Tcfs in general, we performed a subtractive screen, using neuralized Xenopus animal cap explants.ResultsWe identified cold-inducible RNA binding protein (CIRP) as novel XTcf-3 specific target gene. Furthermore, we show that knockdown of XTcf-3 by injection of an antisense morpholino oligonucleotide results in a general broadening of the anterior neural tissue. Depletion of XCIRP by antisense morpholino oligonucleotide injection leads to a reduced stability of mRNA and an enlargement of the anterior neural plate similar to the depletion of XTcf-3.ConclusionDistinct steps in neural development are differentially regulated by individual Lef/Tcfs. For proper development of the anterior brain XTcf-3 and the Tcf-subtype specific target XCIRP appear indispensable. Thus, regulation of anterior neural development, at least in part, depends on mRNA stabilization by the novel XTcf-3 target gene XCIRP.

Mammalian Tumor Xenografts Induce Neovascularization in Zebrafish Embryos

The zebrafish (Danio rerio)/tumor xenograft model represents a powerful new model system in cancer. Here, we describe a novel exploitation of the zebrafish model to investigate tumor angiogenesis, a pivotal step in cancer progression and target for antitumor therapies. Human and murine tumor cell lines that express the angiogenic fibroblast growth factor (FGF) 2 and/or vascular endothelial growth factor (VEGF) induce the rapid formation of a new microvasculature when grafted close to the developing subintestinal vessels of zebrafish embryos at 48 h postfertilization. Instead, no angiogenic response was exerted by related cell clones defective in the production of these angiogenic growth factors. The newly formed blood vessels sprout from the subintestinal plexus of the zebrafish embryo, penetrate the tumor graft, and express the transcripts for the zebrafish orthologues of the early endothelial markers Fli-1, VEGF receptor-2 (VEGFR2/KDR), and VE-cadherin. Accordingly, green fluorescent protein-positive neovessels infiltrate the graft when tumor cells are injected in transgenic VEGFR2:G-RCFP zebrafish embryos that express green fluorescent protein under the control of the VEGFR2/KDR promoter. Systemic exposure of zebrafish embryos immediately after tumor cell injection to prototypic antiangiogenic inhibitors, including the FGF receptor tyrosine kinase inhibitor SU5402 and the VEGFR2/KDR tyrosine kinase inhibitor SU5416, suppresses tumor-induced angiogenesis without affecting normal blood vessel development. Accordingly, VE-cadherin gene inactivation by antisense morpholino oligonucleotide injection inhibits tumor neovascularization without affecting the development of intersegmental and subintestinal vessels. These data show that the zebrafish/tumor xenograft model represents a novel tool for investigating the neovascularization process exploitable for drug discovery and gene targeting in tumor angiogenesis.

High-throughput transgenesis in Xenopus using I-SceI meganuclease

In this report we describe an easy, highly efficient transgenesis method for Xenopus. The method is very simple; a commercially available meganuclease, I-SceI, is incubated with a transgene construct carrying its recognition sites, and is subsequently microinjected into fertilized eggs. Approximately 30% (in Xenopus tropicalis) or 20% (in Xenopus laevis) of injected embryos exhibit non-mosaic, promoter-dependent transgene expression, and transgenes from the founder animals are transmitted to offspring. The method is compatible with mRNA or antisense morpholino oligonucleotide injection, and these secondary reagents can be introduced simultaneously or sequentially with a transgene to test their interaction. This high-throughput transgenic technique will be a powerful tool for studying the complex wiring of regulatory networks at the genome-wide level, as well as for facilitating genetic studies in the rapidly breeding diploid frog, X. tropicalis.

Identification of nesfatin-1 as a satiety molecule in the hypothalamus

The brain hypothalamus contains certain secreted molecules that are important in regulating feeding behaviour. Here we show that nesfatin, corresponding to NEFA/nucleobindin2 (NUCB2), a secreted protein of unknown function, is expressed in the appetite-control hypothalamic nuclei in rats. Intracerebroventricular (i.c.v.) injection of NUCB2 reduces feeding. Rat cerebrospinal fluid contains nesfatin-1, an amino-terminal fragment derived from NUCB2, and its expression is decreased in the hypothalamic paraventricular nucleus under starved conditions. I.c.v. injection of nesfatin-1 decreases food intake in a dose-dependent manner, whereas injection of an antibody neutralizing nesfatin-1 stimulates appetite. In contrast, i.c.v. injection of other possible fragments processed from NUCB2 does not promote satiety, and conversion of NUCB2 to nesfatin-1 is necessary to induce feeding suppression. Chronic i.c.v. injection of nesfatin-1 reduces body weight, whereas rats gain body weight after chronic i.c.v. injection of antisense morpholino oligonucleotide against the gene encoding NUCB2. Nesfatin-1-induced anorexia occurs in Zucker rats with a leptin receptor mutation, and an anti-nesfatin-1 antibody does not block leptin-induced anorexia. In contrast, central injection of alpha-melanocyte-stimulating hormone elevates NUCB2 gene expression in the paraventricular nucleus, and satiety by nesfatin-1 is abolished by an antagonist of the melanocortin-3/4 receptor. We identify nesfatin-1 as a satiety molecule that is associated with melanocortin signalling in the hypothalamus.

Visualization in zebrafish larvae of Na+uptake in mitochondria-rich cells whose differentiation is dependent onfoxi3a

Uptake of Na(+) from the environment is an indispensable strategy for the survival of freshwater fish, as they easily lose Na(+) from the plasma to a diluted environment. Nevertheless, the location of and molecules involved in Na(+) uptake remain poorly understood. In this study, we utilized Sodium Green, a Na(+)-dependent fluorescent reagent, to provide direct evidence that Na(+) absorption takes place in a subset of the mitochondria-rich (MR) cells on the yolk sac surface of zebrafish larvae. Combined with immunohistochemistry, we revealed that the Na(+)-absorbing MR cells were exceptionally rich in vacuolar-type H(+)-ATPase (H(+)-ATPase) but moderately rich in Na(+)-K(+)-ATPase. We also addressed the function of foxi3a, a transcription factor that is specifically expressed in the H(+)-ATPase-rich MR cells. When foxi3a was depleted from zebrafish embryos by antisense morpholino oligonucleotide injection, differentiation of the MR cells was completely blocked and Na(+) influx was severely reduced, indicating that MR cells are the primary sites for Na(+) absorption. Additionally, foxi3a expression is initiated at the gastrula stage in the presumptive ectoderm; thus, we propose that foxi3a is a key gene in the control of MR cell differentiation. We also utilized a set of ion transport inhibitors to assess the molecules involved in the process and discuss the observations.

Promoting Epiboly

Although steroids have numerous functions--regulating electrolyte balance; carbohydrate, protein, and fat metabolism; the inflammatory response; and the development and maintenance of secondary sex characteristics--their role during early embryogenesis has been unclear. Hsu et al. used in situ hybridization to investigate the expression of the genes encoding Cyp11a1 (which converts cholesterol to pregnenolone, thereby initiating steroid biosynthesis) and Hsd3b (which converts pregnenolone into progesterone) during zebrafish embryogenesis. Shortly after fertilization, mRNA for both enzymes was present in the cytoplasm of blastomeres located above the yolk. However, during epiboly (a process during which the blastoderm envelops the yolk), cyp11a1 and hsd3b disappeared from the blastomeres and appeared in the yolk syncytial layer, which is composed of yolk syncytial nuclei and located at the junction between the blastomeres and the yolk. Injection of antisense morpholino oligonucleotides to cyp11a1 delayed epiboly without affecting cell fate. Whereas coinjection of cyp11a1 mRNA or incubation with pregnenolone partially rescued the delay in epiboly, progesterone worsened it. Yolk microtubules extend from yolk syncytial nuclei to drive epiboly, and yolk microtubules from embryos treated with morpholinos were more sensitive to disruption by nocodazole than those from wild-type embryos, an effect that was reversed by pregnenolone. Pregnenolone also increased the abundance of polymerized tubulin in taxol-treated extracts from morpholino-treated embryos. Moreover, fluorescently labeled pregnenolone colocalized with polymerized tubulin from morpholino-treated embryo extracts. Thus, pregnenolone appears to stabilize microtubules and thereby promote cell migration during epiboly, a role that likely involves binding to microtubules rather than association with nuclear receptors. H.-J. Hsu, M.-R. Liang, C.-T. Chen, B.-c. Chung, Pregnenolone stabilizes microtubules and promotes zebrafish embryonic cell movement. Nature 439 , 480-483 (2006). [PubMed]

Below the Speed Limit in the Absence of PGE 2

Prostaglandins had been implicated in early development, and in zebrafish loss of cyclooxygenase 1 (COX-1) caused gastrulation defects. Cha et al. now show that signaling mediated by the production of prostaglandin E 2 (PGE 2 ) is responsible for the embryonic defects. Transcripts for PGE 2 synthase (Ptges in zebrafish) and COX-1 were expressed at the same time and places during gastrulation. Furthermore, PGE 2 was detected by mass spectrometry during gastrulation. Injection of antisense morpholino oligonucleotides against Ptges caused gastrulation defects consistent with aberrant cell movement without affecting patterning or cell fate specification (based on marker analysis). Only exogenous PGE 2 , not PGI 2 or PGF 2α , rescued the Ptges-deficient embryos. Sequence searching revealed two candidate zebrafish PGE 2 receptors, EP2 and EP4, of which only EP4 was present during gastrulation. Injection of antisense morpholino oligonucleotides against EP4 caused gastrulation defects like those observed for loss of COX-1 or Ptges. Time-lapse microscopy revealed that the EP4-deficient cells moved more slowly, but in the proper direction. Finally, analysis of the phosphorylation state of Akt and extracellular signal-regulated kinase (ERK) indicated that EP4 was coupled to a phosphoinositide 3-kinase (PI3K) to Akt pathway. Thus, PGE 2 appears to play an essential permissive role in gastrulation through its effects on cell migration. Y. I. Cha, S.-H. Kim, D. Sepich, F. G. Buchanan, L. Solnica-Krezel, R. N. DuBois, Cyclooxygenase-1-derived PGE 2 promotes cell motility via the G-protein-coupled EP4 receptor during vertebrate gastrulation. Genes Dev. 20 , 77-86 (2006). [Abstract] [Full Text]

The zebrafish dorsal axis is apparent at the four-cell stage

A central question in the development of multicellular organisms pertains to the timing and mechanisms of specification of the embryonic axes. In many organisms, specification of the dorsoventral axis requires signalling by proteins of the Transforming growth factor-beta and Wnt families. Here we show that maternal transcripts of the zebrafish Nodal-related morphogen, Squint (Sqt), can localize to two blastomeres at the four-cell stage and predict the dorsal axis. Removal of cells containing sqt transcripts from four-to-eight-cell embryos or injection of antisense morpholino oligonucleotides targeting sqt into oocytes can cause a loss of dorsal structures. Localization of sqt transcripts is independent of maternal Wnt pathway function and requires a highly conserved sequence in the 3' untranslated region. Thus, the dorsoventral axis is apparent by early cleavage stages and may require the maternally encoded morphogen Sqt and its associated factors. Because the 3' untranslated region of the human nodal gene can also localize exogenous sequences to dorsal cells, this mechanism may be evolutionarily conserved.

Inositol Polyphosphates Regulate Zebrafish Left-Right Asymmetry

Vertebrate body plans have a conserved left-right (LR) asymmetry manifested in the position and anatomy of the heart, visceral organs, and brain. Recent studies have suggested that LR asymmetry is established by asymmetric Ca2+ signaling resulting from cilia-driven flow of extracellular fluid across the node. We report here that inositol 1,3,4,5,6-pentakisphosphate 2-kinase (Ipk1), which generates inositol hexakisphosphate, is critical for normal LR axis determination in zebrafish. Zebrafish embryos express ipk1 symmetrically during gastrulation and early segmentation. ipk1 knockdown by antisense morpholino oligonucleotide injection randomized LR-specific gene expression and organ placement, effects that were associated with reduced intracellular Ca2+ flux in cells surrounding the ciliated Kupffer's vesicle, a structure analogous to the mouse node. Our data suggest that the pathway for inositol hexakisphosphate production is a key regulator of asymmetric Ca(2+) flux during LR specification.