Injection Of Antisense Morpholino Oligonucleotides Research Articles

Identification of a gene required for de novo DNA methylation of the zebrafish no tail gene

The zebrafish no tail gene (ntl) is indispensable for tail and notochord development. We have shown previously that ntl is de novo methylated during early embryogenesis. To find the gene that de novo methylates ntl and understand the meaning of this methylation, we cloned seven genes that encode the conserved catalytic domain of methyltransferases. We found that injection of antisense morpholino oligonucleotides against one of them, termed dnmt7, into eggs significantly reduced the level of ntl methylation, although no apparent phenotype was induced by the injection. Inhibition of Dnmt7 activity did not change the level of genome-wide methylation nor did it affect de novo methylation of injected plasmid DNA, indicating that Dnmt7 specifically methylates ntl in the genome.

Cdc42 Effector Protein 2 (XCEP2) is required for normal gastrulation and contributes to cellular adhesion in Xenopus laevis.

BackgroundRho GTPases and their downstream effector proteins regulate a diverse array of cellular processes during embryonic development, including reorganization of cytoskeletal architecture, cell adhesion, and transcription. Changes in the activation state of Rho GTPases are converted into changes in cellular behavior by a diversity of effector proteins, which are activated in response to changes in the GTP binding state of Rho GTPases. In this study we characterize the expression and function of one such effector, XCEP2, that is present during gastrulation stages in Xenopus laevis.ResultsIn a search for genes whose expression is regulated during early stages of embryonic development in Xenopus laevis, a gene encoding a Rho GTPase effector protein (Xenopus Cdc42 effector protein 2, or XCEP2) was isolated, and found to be highly homologous, but not identical, to a Xenopus sequence previously submitted to the Genbank database. These two gene sequences are likely pseudoalleles. XCEP2 mRNA is expressed at constant levels until mid- to late- gastrula stages, and then strongly down-regulated at late gastrula/early neurula stages. Injection of antisense morpholino oligonucleotides directed at one or both pseudoalleles resulted in a significant delay in blastopore closure and interfered with normal embryonic elongation, suggesting a role for XCEP2 in regulating gastrulation movements. The morpholino antisense effect could be rescued by co-injection with a morpholino-insensitive version of the XCEP2 mRNA. Antisense morpholino oligonucleotides were found to have no effect on mesodermal induction, suggesting that the observed effects were due to changes in the behavior of involuting cells, rather than alterations in their identity. XCEP2 antisense morpholino oligonucleotides were also observed to cause complete disaggregation of cells composing animal cap explants, suggesting a specific role of XCEP2 in maintenance or regulation of cell-cell adhesion in early embryos. This loss of cell adhesion could be rescued by co-injection with a morpholino-insensitive version of the XCEP2 mRNA.ConclusionsXCEP2 appears to be an essential component in the early developmental program in Xenopus laevis. XCEP2 is involved in maintenance of cell-cell adhesion, and as such may constitute a regulatory component that could help to balance the need for tissue integrity and plasticity during the dynamic cellular rearrangements of gastrulation.

Hermes is a localized factor regulating cleavage of vegetal blastomeres in Xenopus laevis

We have identified the RNA-binding protein Hermes in a screen for vegetally localized RNAs in Xenopus oocytes. The RNA localizes to the vegetal cortex through both the message transport organizer (METRO) and late pathways. Hermes mRNA and protein are both detected at the vegetal cortex of the oocyte; however, the protein is degraded within a several hour period during oocyte maturation. Injection of antisense morpholino oligonucleotides (HE-MO) against Hermes caused a precocious reduction in Hermes protein present during maturation and resulted in a phenotype characterized by cleavage defects in vegetal blastomeres. The phenotype can be partially rescued by injecting Hermes mRNA. These results demonstrate that the localized RNA-binding protein Hermes functions during oocyte maturation to regulate the cleavage of specific vegetally derived cell lineages. Hermes most likely performs its function by regulating the translation or processing of one or more target RNAs. This is an important mechanism by which the embryo can generate unique cell lineages. The regulation of region-specific cell division is a novel function for a localized mRNA.

Lockjaw encodes a zebrafish tfap2a required for early neural crest development.

The neural crest is a uniquely vertebrate cell type that gives rise to much of the craniofacial skeleton, pigment cells and peripheral nervous system, yet its specification and diversification during embryogenesis are poorly understood. Zebrafish homozygous for the lockjaw (low) mutation show defects in all of these derivatives and we show that low (allelic with montblanc) encodes a zebrafish tfap2a, one of a small family of transcription factors implicated in epidermal and neural crest development. A point mutation in low truncates the DNA binding and dimerization domains of tfap2a, causing a loss of function. Consistent with this, injection of antisense morpholino oligonucleotides directed against splice sites in tfap2a into wild-type embryos produces a phenotype identical to low. Analysis of early ectodermal markers revealed that neural crest specification and migration are disrupted in low mutant embryos. TUNEL labeling of dying cells in mutants revealed a transient period of apoptosis in crest cells prior to and during their migration. In the cranial neural crest, gene expression in the mandibular arch is unaffected in low mutants, in contrast to the hyoid arch, which shows severe reductions in dlx2 and hoxa2 expression. Mosaic analysis, using cell transplantation, demonstrated that neural crest defects in low are cell autonomous and secondarily cause disruptions in surrounding mesoderm. These studies demonstrate that low is required for early steps in neural crest development and suggest that tfap2a is essential for the survival of a subset of neural crest derivatives.

The Mix family homeodomain gene bonnie and clyde functions with other components of the Nodal signaling pathway to regulate neural patterning in zebrafish.

Mix family homeodomain proteins, such as Xenopus Mixer and zebrafish Bonnie and clyde (Bon), have been shown to regulate the formation of the endoderm and are likely to be transcriptional mediators of Nodal signaling. Here, we show that, in addition to its previously described role in endoderm formation, Bon also regulates the anteroposterior patterning of the neuroectoderm. bon-mutant embryos exhibit an anterior reduction of the neural plate. By using targeted injection of antisense morpholino oligonucleotides, we demonstrate that Bon is required in the axial mesoderm for anterior neural development. Consistent with these results, bon-mutant embryos show defects in axial mesoderm gene expression starting at mid-gastrulation stages. In addition, genetic analyses demonstrate a functional interaction during neural patterning between bon and two components of the Nodal signaling pathway, the nodal-related gene squint (sqt) and forkhead box H1 [foxh1; mutant locus schmalspur (sur)]. bon-/-;sqt-/- and bon-/-;sur-/- embryos exhibit neural patterning defects that are much more severe than those seen in the single mutants, suggesting that these genes function in parallel in this process. We also show that the severity of the neural patterning defects in the single- and double-mutant embryos correlates with the degree of reduction in expression of the Wnt antagonist gene dickkopf 1. Furthermore, bon-/-;sqt-/- and bon-/-;sur-/- embryos exhibit identical morphological and gene expression defects, suggesting, in part, that bon, sqt and sur (foxh1) play overlapping roles in neural patterning. Taken together, these results provide evidence for a complex genetic network in which bon functions both downstream of, and possibly in parallel to, Nodal signaling to regulate neural patterning via the modulation of mesendodermal gene expression.

Transmembrane Sema4E Guides Branchiomotor Axons to Their Targets in Zebrafish

Class 4 semaphorins are a large class of transmembrane proteins that contain a sema domain and that are expressed in the CNS, but their in vivo neural function is unknown. In zebrafish, the epithelial cells that line the pharyngeal arches express Sema4E. Extension of branchiomotor axons along the mesenchymal cells bounded by these epithelial cells suggests that Sema4E may act as a repulsive guidance molecule to restrict the branchiomotor axons to the mesenchymal cells. To test this hypothesis, Sema4E was misexpressed in hsp70 promoter-regulated transgenic zebrafish in which sema4E was heat-inducible, and Sema4E was knocked down by injection of antisense morpholino oligonucleotides that acted specifically against Sema4E. Ubiquitous induction of Sema4E retarded outgrowth by the facial and gill branchiomotor axons significantly. Furthermore, outgrowth by gill motor axons was specifically inhibited when Sema4E-expressing transgenic cells were transplanted to their pathway in nontransgenic host embryos. Morpholino knockdown of Sema4E caused facial motor axons to defasciculate and follow aberrant pathways. These results show that Sema4E is repulsive for facial and gill motor axons and functions as a barrier for these axons within the pharyngeal arches.

Multifunctional Cyr61 Modulates Gastrulation

Latinkic et al. investigated the role of Cyr61 on early development in Xenopus and discovered that it was required for cell movement during gastrulation, an effect that depended on both cell adhesion and modulation of the Wnt signaling pathway. During gastrulation, cells change their shapes, migrate, and receive fate determination signals. Cyr61, a secreted, heparin-binding, extracellular matrix-associated protein, is a member of a protein family characterized by four domains, each of which is defined by similarities to domains found in other proteins. Latinkic et al. demonstrated that both Cyr61 overexpression (through RNA or protein injection) and depletion (through injection of antisense morpholino oligonucleotides) disrupted cell movement during gastrulation. Morpholino experiments on dissociated cells indicated that the ability to form Ca 2+ -dependent contacts was impaired. The authors grew activin-treated cells on surfaces coated with various substrata, including Cyr61 and a Cyr61 mutant lacking the C-terminal (CT) domain, and demonstrated that this domain was required for Cyr61-mediated cell adhesion. Cyr61 could stimulate the formation of secondary axes, an effect that can be mediated through Wnt activation. Consistent with this, Cyr61 activated a gene reporter responsive to Wnt-β-catenin signaling pathways; the authors used Cyr61 deletion mutants to show that this depended on a different domain, the insulin-like growth factor-binding protein domain. Unexpectedly, Cyr61 inhibited Wnt-mediated secondary axis formation and reporter gene activation. Unlike Wnt pathway activation, Wnt pathway inhibition depended on the CT domain. Thus, Cyr61 appears to regulate gastrulation both through effects on cell adhesion and through activation or inhibition of Wnt signaling mediated through distinct protein domains. B. V. Latinkic, S. Mercurio, B. Bennett, E. M. A. Hirst, Q. Xu, L. F. Lau, T. J. Mohun, J. C. Smith, Xenopus Cyr61 regulates gastrulation movements and modulates Wnt signalling. Development 130 , 2429-2441 (2003). [Abstract] [Full Text]

Multifunctional Cyr61 Modulates Gastrulation

Latinkic et al. investigated the role of Cyr61 on early development in Xenopus and discovered that it was required for cell movement during gastrulation, an effect that depended on both cell adhesion and modulation of the Wnt signaling pathway. During gastrulation, cells change their shapes, migrate, and receive fate determination signals. Cyr61, a secreted, heparin-binding, extracellular matrix-associated protein, is a member of a protein family characterized by four domains, each of which is defined by similarities to domains found in other proteins. Latinkic et al. demonstrated that both Cyr61 overexpression (through RNA or protein injection) and depletion (through injection of antisense morpholino oligonucleotides) disrupted cell movement during gastrulation. Morpholino experiments on dissociated cells indicated that the ability to form Ca2+-dependent contacts was impaired. The authors grew activin-treated cells on surfaces coated with various substrata, including Cyr61 and a Cyr61 mutant lacking the C-terminal (CT) domain, and demonstrated that this domain was required for Cyr61-mediated cell adhesion. Cyr61 could stimulate the formation of secondary axes, an effect that can be mediated through Wnt activation. Consistent with this, Cyr61 activated a gene reporter responsive to Wnt-β-catenin signaling pathways; the authors used Cyr61 deletion mutants to show that this depended on a different domain, the insulin-like growth factor-binding protein domain. Unexpectedly, Cyr61 inhibited Wnt-mediated secondary axis formation and reporter gene activation. Unlike Wnt pathway activation, Wnt pathway inhibition depended on the CT domain. Thus, Cyr61 appears to regulate gastrulation both through effects on cell adhesion and through activation or inhibition of Wnt signaling mediated through distinct protein domains.B. V. Latinkic, S. Mercurio, B. Bennett, E. M. A. Hirst, Q. Xu, L. F. Lau, T. J. Mohun, J. C. Smith, Xenopus Cyr61 regulates gastrulation movements and modulates Wnt signalling. Development 130, 2429-2441 (2003). [Abstract] [Full Text]

Essential Role of the Transcription Factor Ets-2 inXenopus Early Development

The fibroblast growth factor (FGF)/MAPK pathway plays an important role in early Xenopus developmental processes, including mesoderm patterning. The activation of the MAPK pathway leads to induction of Xenopus Brachyury (Xbra), which regulates the transcription of downstream mesoderm-specific genes in mesoderm patterning. However, the link between the FGF/MAPK pathway and the induction of Xbra has not been fully understood. Here we present evidence suggesting that Ets-2 is involved in the induction of Xbra and thus in the development of posterior mesoderm during early embryonic development. Overexpression of Ets-2 caused posteriorized embryos and led to the induction of mesoderm in ectodermal explants. Expression of a dominant-negative form of Ets-2 or injection of antisense morpholino oligonucleotides against Ets-2 inhibited the formation of the trunk and tail structures. Overexpression of Ets-2 resulted in the induction of Xbra, and expression of the dominant-negative Ets-2 inhibited FGF- or constitutively active MEK-induced Xbra expression. Moreover, overexpression of Ets-2 up-regulated the transcription from Xbra promoter reporter gene constructs. Ets-2 bound to the Xbra promoter region in vitro. These results taken together indicate that Xenopus Ets-2 plays an essential role in mesoderm patterning, lying between the FGF/MAPK pathway and the Xbra transcription.

Mezzo, a paired-like homeobox protein is an immediate target of Nodal signalling and regulates endoderm specification in zebrafish.

Endoderm specification in zebrafish is mediated by the zygotic transcription factors Bon/Mixer, Faust/Gata5, Casanova and Sox17, whose expression is induced by Nodal signalling. Bon/Mixer and Gata5 require Casanova in order to promote endoderm formation and all three factors act upstream of sox17, but it is not clear whether Casanova acts downstream of or in parallel to Bon/Mixer and Gata5. An additional factor induced at the margin of the blastoderm by Nodal signalling is thought to be required to induce casanova expression. We show that Mezzo, a novel paired-like homeobox protein, may be this missing transcription factor. The homeobox of Mezzo is mostly related to the homeodomain of the Mix-like and Mixer homeoproteins, but Mezzo is distinct from Bon/Mixer, the product of the bonnie and clyde gene. Like bon/mixer, mezzo is expressed transiently in mesendoderm precursors. By analysing the expression of mezzo in various mutants of Nodal signalling, we show that its expression strictly depends on a functional Nodal signalling pathway. By expressing a constitutively active Nodal receptor in the presence of translation inhibitors, we further demonstrate that mezzo, bonnie and clyde, and casanova are all immediate early targets of Nodal signalling, while sox17 requires post-MBT protein synthesis in order to be induced. Overexpression of mezzo mRNA can induce ectopic expression of casanova and sox17 and can also turn on the pan mesodermal marker gene ntl. We show that the function of mezzo is redundant with that of bonnie and clyde and that mezzo RNA can partially rescue bonnie and clyde mutants. Injection of antisense Morpholino oligonucleotides targeted against mezzo into bonnie and clyde mutant embryos abolishes all sox17 expression and aggravates their mutant phenotype. These results highlight the complexity of the transcriptional network operating during endoderm formation. They place mezzo as a new transcription factor with unique properties acting in parallel with bonnie and clyde, faust and casanova in the Nodal signalling pathway that controls specification of mesoderm and endoderm in zebrafish.

The homeobox genes vox and vent are redundant repressors of dorsal fates in zebrafish.

Ventralizing transcriptional repressors in the Vox/Vent family have been proposed to be important regulators of dorsoventral patterning in the early embryo. While the zebrafish genes vox (vega1) and vent (vega2) both have ventralizing activity in overexpression assays, loss-of-function studies are needed to determine whether these genes have distinct or redundant functions in dorsoventral patterning and to provide critical tests of the proposed regulatory interactions among vox, vent and other genes that act to establish the dorsoventral axis. We show that vox and vent are redundant repressors of dorsal fates in zebrafish. Mutants that lack vox function have little or no dorsoventral patterning defect, and inactivation of either vox or vent by injection of antisense morpholino oligonucleotides has little or no effect on the embryo. In contrast, embryos that lack both vox and vent function have a dorsalized phenotype. Expression of dorsal mesodermal genes, including chordin, goosecoid and bozozok, is strongly expanded in embryos that lack vox and vent function, indicating that the redundant action of vox and vent is required to restrict dorsal genes to their appropriate territories. Our genetic analysis indicates that the dorsalizing transcription factor Bozozok promotes dorsal fates indirectly, by antagonizing the expression of vox and vent. In turn, vox and vent repress chordin expression, restricting its function as an antagonist of ventral fates to the dorsal side of the embryo. Our results support a model in which BMP signaling induces the expression of ventral genes, while vox and vent act redundantly to prevent the expression of chordin, goosecoid and other dorsal genes in the lateral and ventral mesendoderm.

Sprouty4 acts in vivo as a feedback-induced antagonist of FGF signaling in zebrafish.

In looking for novel factors involved in the regulation of the fibroblast growth factor (FGF) signaling pathway, we have isolated a zebrafish sprouty4 gene, based on its extensive similarities with the expression patterns of both fgf8 and fgf3. Through gain- and loss-of-function experiments, we demonstrate that Fgf8 and Fgf3 act in vivo to induce the expression of Spry4, which in turn can inhibit activity of these growth factors. When overexpressed at low doses, Spry4 induces loss of cerebellum and reduction in size of the otic vesicle, thereby mimicking the fgf8/acerebellar mutant phenotype. Injections of high doses of Spry4 cause ventralization of the embryo, an opposite phenotype to the dorsalisation induced by overexpression of Fgf8 or Fgf3. Conversely we have shown that inhibition of Spry4 function through injection of antisense morpholino oligonucleotide leads to a weak dorsalization of the embryo, the phenotype expected for an upregulation of Fgf8 or Fgf3 signaling pathway. Finally, we show that Spry4 interferes with FGF signaling downstream of the FGF receptor 1 (FGFR1). In addition, our analysis reveals that signaling through FGFR1/Ras/mitogen-activated protein kinase pathway is involved, not in mesoderm induction, but in the control of the dorsoventral patterning via the regulation of bone morphogenetic protein (BMP) expression.