Injection Of Synthetic mRNA Research Articles

β-Catenin is required for the establishment of vegetal embryonic fates in the nemertean, Cerebratulus lacteus

Downstream components of the canonical Wnt signaling pathway that result in the nuclear localization of β-catenin are involved in diverse developmental processes including the formation of the mesendoderm, the regulation of axial properties and asymmetric cell divisions in a wide array of metazoans. The nemertean worm, Cerebratulus lacteus, represents a member of the understudied lophotrochozoan clade that exhibits a highly stereotyped spiral cleavage program in which ectodermal, endodermal, and mesodermal origins are known from intracellular fate mapping studies. Here, the embryonic distribution of β-catenin protein was studied using injection of synthetic mRNA, encoding GFP-tagged β-catenin, into fertilized eggs. During the early cleavage stages β-catenin was destabilized/degraded in animal hemisphere blastomeres and became localized to the nuclei of the four vegetal-most cells at the 64-cell stage, which give rise to definitive larval and adult endoderm. Functional assays indicate that β-catenin plays a key role in the development of the endoderm. Morpholino knockdown of endogenous β-catenin, as confirmed by Western analysis, resulted in the failure to gastrulate, absence of the gut and an animalized phenotype in the resulting larvae, including the formation of ectopic (anterior) apical organ tissue with elongated apical tuft cilia and no indications of dorsoventral polarity. Similarly, over-expression of the cytoplasmic domain of cadherin or a β-catenin-engrailed repressor fusion construct prevented endoderm formation and generated the same animalized phenotype. Injections of mRNA encoding either a stabilized, constitutively activated form of β-catenin or a dominant negative form of GSK3-β converted all or nearly all cells into endodermal fates expressing gut-specific esterase. Thus, β-catenin appears to be both necessary and sufficient to promote endoderm formation in C. lacteus, consistent with its role in endoderm and endomesoderm formation in anthozoan cnidarians, ascidians, and echinoderms. Consistent with the results of other studies, β-catenin may be viewed as playing a role in the development of posterior/vegetal larval fates (i.e., endoderm) in C. lacteus. However, unlike the case found in polychaete annelid and soil nematode embryos, there is no evidence for a role of β-catenin in regulating cell fates and asymmetric cell divisions along the entire anterior–posterior axis.

A Raf/MEK/ERK signaling pathway is required for development of the sea urchin embryo micromere lineage through phosphorylation of the transcription factor Ets.

In the sea urchin embryo, the skeleton of the larva is built from a population of mesenchymal cells known as the primary mesenchyme cells (PMCs). These derive from the large micromeres that originate from the vegetal pole at fourth cleavage. At the blastula stage, the 32 cells of this lineage detach from the epithelium and ingress into the blastocoel by a process of epithelial-mesenchymal transition. We report that shortly before ingression, there is a transient and highly localized activation of the MAP-kinase ERK in the micromere lineage. We show that ingression of the PMCs requires the activity of ERK, MEK and Raf, and depends on the maternal Wnt/beta-catenin pathway. Dissociation experiments and injection of mRNA encoding a dominant-negative form of Ras indicated that this activation is probably cell autonomous. We identified the transcription factors Ets1 and Alx1 as putative targets of the phosphorylation by ERK. Both proteins contain a single consensus site for phosphorylation by the MAP kinase ERK. In addition, the Ets1 protein sequence contains a putative ERK docking site. Overexpression of ets1 by injection of synthetic mRNA in the egg caused a dramatic increase in the number of cells becoming mesenchymal at the blastula stage. This effect could be largely inhibited by treating embryos with the MEK inhibitor U0126. Moreover, mutations in the consensus phosphorylation motif substituting threonine 107 by an aspartic or an alanine residue resulted respectively in a constitutively active form of Ets1 that could not be inhibited by U0126 or in an inactive form of Ets1. These results show that the MAP kinase pathway, working through phosphorylation of Ets1, is required for full specification of the PMCs and their subsequent transition from epithelial to mesenchymal state.

Xenopus bagpipe-related gene, koza, may play a role in regulation of cell proliferation.

The homeobox gene koza is a new member of the vertebrate bagpipe-related gene family. Embryonic expression of koza is observed at highest levels in the muscle layer of the somites and, during later development, is restricted to the lateral somitic cells, which correspond to slow twitch muscle tissue. Expression of koza is also observed in the myocardial layer of the heart and in the cement gland. In each of these tissues, koza transcription commences only after the expression of terminal differentiation markers. By injection of synthetic mRNA, we show that overexpression of koza leads to an apparent decrease in the number of cells in the somites. No reduction in cell number is observed when koza is present in neural tissues, suggesting that koza exhibits some tissue specificity in regulation of cell proliferation. Embryonic manipulations show that restriction of koza expression to the slow twitch muscle layer is independent of axial structures but is, at least partly, regulated by signals arising in ectodermal tissue. Finally, in Drosophila, bagpipe expression is regulated by the hedgehog signaling pathway. By using ectopic expression, we show that koza transcription is positively regulated by banded hedgehog. This result indicates that regulation of bagpipe expression by hedgehog signaling is evolutionarily conserved.

Inducible control of tissue-specific transgene expression in Xenopustropicalis transgenic lines

Analysis of gene function in vertebrates is facilitated by gain-of-function studies, such as injection of synthetic mRNA in amphibian embryos. This approach is hampered by lack of spatial and temporal control of expression of the introduced gene product. An additional level of control is obtained by nuclear-transfer-mediated transgenesis, but functional analyses are complicated by variability and background abnormalities in primary transgenic embryos. The GAL4/UAS system permits establishment of stable lines and elimination of nuclear-transfer-associated abnormalities, through generation of separate UAS-‘effector’ and GAL4 ‘transactivator’ transgenic lines. When the GAL4 DNA-binding domain is combined with a steroid hormone ligand-binding domain, this system allows full temporal regulation of transgene expression by introduction of an exogenous steroid analogue, the progesterone antagonist RU486. We show here that by crossing stable Xenopus tropicalis transgenic lines, one bearing a UAS-enhanced cyan fluorescent protein (ECFP) reporter construct, and the other with a GAL4–progesterone receptor fusion driven by a retina-specific promoter, reporter expression in the resulting embryos can be induced with RU486 in a tissue-specific manner. These results suggest that the inducible binary system, in which the target gene expression can be controlled in a stage- and tissue-specific pattern, should be readily applicable for gene function studies at all stages of development.

The Xenopus homologue of Bicaudal-C is a localized maternal mRNA that can induce endoderm formation.

In Xenopus, zygotic transcription starts 6 hours after fertilization at the midblastula transition and therefore the first steps in embryonic development are regulated by maternally inherited proteins and mRNAs. While animal-vegetal polarity is already present in the oocyte, the dorsoventral axis is only established upon fertilization by the entry of the sperm and the subsequent rotation of the egg cortex. In a screen for maternal mRNAs whose stability is regulated by this cortical rotation, we isolated the Xenopus homologue of the Drosophila gene Bicaudal-C (xBic-C). It encodes a putative RNA-binding molecule expressed maternally and localized predominantly to the vegetal half of the egg. Upon fertilization and cortical rotation, xBic-C mRNA is displaced together with the heavy yolk towards the future dorsal side of the embryo. In UV-ventralized embryos, xBic-C is polyadenylated less than in untreated embryos that undergo cortical rotation. Overexpression of xBic-C by injection of synthetic mRNA in whole embryos or in ectodermal explants leads to ectopic endoderm formation. This endoderm-inducing activity is dependent on the presence of the RNA-binding domain of the protein. In contrast to the two other known maternally encoded endoderm inducers, Vg1 and VegT, xBic-C ectopic expression leads specifically to endoderm formation in the absence of mesoderm induction.

The Xenopus laevis zinc finger protein gene Xfin: developmental expression and in vivo functional studies

Xfin is a Krüppel‐type zinc finger protein characterized by the highest number of zinc finger motives (37) found to date. An interesting feature of this protein is the cytoplasmic location, coupled by in vitro RNA binding activity: both these properties may suggest a role for Xfin in RNA metabolism. As the corresponding mRNA, the protein product of Xfin is part of the maternal store in Xenopus and is present throughout embryogenesis. We used in situ hybridization to establish the expression pattern of Xfin in developmental stages. We show here that it is expressed evenly throughout gastrula and neurula stages embryos, while in later stages its transcripts are mainly enriched in the endoderm. In addition, we performed some in vivo perturbation experiments. We found that overexpression of Xfin by injection of synthetic mRNA in early Xenopus embryos causes disruption of posterior development; also, Xfin overexpression inhibits the normal expression of the pan‐mesodermal marker Xbra.

Analysis of HIV-1 Tat Effects in Xenopus laevis Embryos

Tat is one of the regulatory proteins of the HIV-1 virus. To date, besides the transactivation activity, a myriad of effects exerted by HIV-1 Tat on cellular and viral genes have been observed. The present study investigated the in vivo effects of HIV-1 Tat protein in the Xenopus embryo. We adopted the Xenopus system since expression of putative regulatory factors in the embryo has been widely used as a quick and effective first screen for protein function. Xenopus’ early development is well characterized by stage-specific phenotypes, therefore, an in vivo HIV-1 Tat-mediated aberrant phenotype can easily be detected and analyzed. HIV-1 Tat protein expression through injection of synthetic mRNA into zygotes produced a marked delay in gastrulation leading to altered specification of the anterior-posterior axis and to partial or total loss of anterior structures. HIV-1 Tat effects resulted in a general suppression of gene expression, including that of Xbra and gsc, two early genes whose expression is required for proper gastrulation. The specificity of Tat effects was demonstrated by injecting a ‘loss of function’ mutant (Tat-C37S), lacking a single cysteine residue, which did not yield any effect. Both Tat and Tat-C37S were found to be localized mainly in the nucleus. The importance of subcellular targeting for the effects caused by HIV-1 Tat was demonstrated by injecting a second mutant (Tat-BDM), carrying an altered nuclear localization signal sequence. The Tat-BDM protein localized in the cytoplasm and accumulated at the cell membrane. Embryos injected with Tat-BDM mRNA did not develop beyond gastrulation. The importance of proper protein conformation and subcellular localization in determining Tat effects is discussed.

Analysis of HIV-1 Tat effects in Xenopus laevis embryos.

Tat is one of the regulatory proteins of the HIV-1 virus. To date, besides the transactivation activity, a myriad of effects exerted by HIV-1 Tat on cellular and viral genes have been observed. The present study investigated the in vivo effects of HIV-1 Tat protein in the Xenopus embryo. We adopted the Xenopus system since expression of putative regulatory factors in the embryo has been widely used as a quick and effective first screen for protein function. Xenopus' early development is well characterized by stage-specific phenotypes, therefore, an in vivo HIV-1 Tat-mediated aberrant phenotype can easily be detected and analyzed. HIV-1 Tat protein expression through injection of synthetic mRNA into zygotes produced a marked delay in gastrulation leading to altered specification of the anterior-posterior axis and to partial or total loss of anterior structures. HIV-1 Tat effects resulted in a general suppression of gene expression, including that of Xbra and gsc, two early genes whose expression is required for proper gastrulation. The specificity of Tat effects was demonstrated by injecting a 'loss of function' mutant (Tat-C37S), lacking a single cysteine residue, which did not yield any effect. Both Tat and Tat-C37S were found to be localized mainly in the nucleus. The importance of subcellular targeting for the effects caused by HIV-1 Tat was demonstrated by injecting a second mutant (Tat-BDM), carrying an altered nuclear localization signal sequence. The Tat-BDM protein localized in the cytoplasm and accumulated at the cell membrane. Embryos injected with Tat-BDM mRNA did not develop beyond gastrulation. The importance of proper protein conformation and subcellular localization in determining Tat effects is discussed.

Secretion and mesoderm-inducing activity of the TGF-beta-related domain of Xenopus Vg1.

Vg1 is a maternal mRNA localized to the vegetal hemisphere of Xenopus embryos during blastula stages, a region responsible for the induction of mesoderm in the adjacent marginal zone. Its homology to the transforming growth factor-beta family, which includes several proteins with mesoderm-inducing activity, suggests a role for Vg1 as an endogenous mesoderm-inducing factor. However, expression of Vg1 protein in the animal hemisphere, following injection of synthetic mRNA, has no effect on development, and isolated animal caps are not mesodermalized. It is shown that Vg1 protein fails to form dimers and is not processed to release the putative bioactive domain. Furthermore it is shown that the N-terminal signal peptide of Vg1 is not cleaved following translocation into the ER, which may explain the failure of this protein to dimerize. To explore the role of Vg1 in amphibian development, a fusion protein has been made of the preproregion of Xenopus bone morphogenetic protein-4 and the putative bioactive C-terminal domain of Vg1. This fusion protein forms dimers and the C-terminal domain of Vg1 is secreted. Injection of this construct into Xenopus embryos induces the formation of a second dorsal axis and isolated animal caps are mesodermalized. The results are consistent with a role for Vg1 in mesoderm induction during Xenopus development.