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Zebrafish 50% epiboly embryos: controls vs. Aplnra/b morphants

ABSTRACT: Transcriptional profiling of 50% epiboly zebrafish embryos comparing wildtype (control) to Aplnra/b morpholino injected embryos. Goal was to determine any gene expression changes in embryos deficient for the Apelin receptor. Publication summary: The Apelin receptor (Aplnr) has been shown to be essential for proper cardiac progenitor migration during gastrulation. Loss of the Aplnr results in a delay in the ingression of lateral marginal cells and the complete absence of the heart and early cardiac gene expression. How the Aplnr is required at a molecular level for cardiac progenitor migration remains completely unknown. We present evidence to suggest that the Aplnr enhances Nodal signaling to initiate the migration program at the correct time in development. Apelin receptor loss of function embryos exhibit a reduction in a wide range of Nodal target genes while activation of the receptor is capable of boosting their expression. Furthermore, Aplnr deficient embryos are exquisitely sensitive to Nodal receptor inhibitors. In order to directly interrogate the functional consequence of reduced Nodal signaling in the Aplnr cardiac phenotype, we raised the level of Nodal activity in aplnr mutants and found that this was able to provide a near complete rescue. We next sought to determine a mechanism by which lower Nodal signaling levels could translate into a delay in migration. Remarkably, we observe a delay in the expression of mesp family members in Aplnr loss of function embryos at the beginning of gastrulation. The mesp family has previously been implicated in mesodermal migration and we show that they are downstream targets of Nodal. We propose that in the absence of the Aplnr the appropriate Nodal threshold required to initiate the downstream cardiac progenitor “specification/migration” program requires a longer length of time to be attained. This translates into a delay in migration and consequently cardiac progenitors are not capable of reaching their final destination. In this study we demonstrate how the fine tuning of a key signaling pathway is critical for the proper development of the early embryo.

ORGANISM(S): Danio rerio

PROVIDER: GSE58683 | GEO | 2016/04/29

SECONDARY ACCESSION(S): PRJNA253177

REPOSITORIES: GEO

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Project description:Mammalian genomes are subjected to epigenetic modifications, including cytosine methylation by DNA methyltransferases (Dnmt) and further oxidation by Ten-eleven-translocation (Tet) family of dioxygenases. Cytosine methylation plays key roles in multiple processes such as genomic imprinting and X-chromosome inactivation. However, the functional significance of cytosine methylation and the further oxidation has remained undetermined in mouse embryogenesis. Here we show that global inactivation of all three Tet genes in mice led to consistent defects in gastrulation. The defects include reduced specification of the axial mesoderm and paraxial mesoderm, mimicking phenotypes in embryos with gain-of-function Nodal signaling, a cardinal cue for gastrulation. Introduction of a single mutant allele of Nodal in the Tet mutant background partially restored patterning, suggesting that hyperactive Nodal signaling is a leading cause for the gastrulation failure of Tet mutants. Increased Nodal signaling is likely due to diminished expression of the Lefty1 and Lefty2 genes, inhibitors of Nodal signaling. Moreover, reduction in the Lefty gene expression can be ascribed to elevated DNA methylation as both Lefty-Nodal signaling and normal morphogenesis are largely restored in Tet-deficient embryos when the Dnmt3a and Dnmt3b genes are disrupted. Additionally, specific inactivation of Tet by point mutations abolishing the dioxygenase activity causes similar molecular and gastrulation abnormalities. Taken together, our results show that Tet-mediated DNA oxidation modulates the Lefty-Nodal signaling by promoting demethylation of the shared target genes with Dnmt3a and Dnmt3b. These findings reveal a fundamental epigenetic mechanism featuring dynamic DNA methylation and demethylation and their role in the regulation of key signaling in body plan formation during early embryogenesis. Examine RNA expression and DNA methylation differences between Tet-null and wild type samples of mouse epiblast in E6.5.

Project description:Gene expression was measured using microarrays in 8 hour postfertilization embryos, comparing control versus ethanol-treated (2 to 8 hours postfertilization) embryos. This experiment was performed to determine the gene expression changes that occur in response to ethanol treatment as a model of fetal alcohol spectrum disorder. Fetal alcohol spectrum disorder (FASD) occurs when pregnant mothers consume alcohol, causing embryonic ethanol exposure and characteristic birth defects that include craniofacial, neural and cardiac defects. Gastrulation is a particularly sensitive developmental stage for teratogen exposure, and zebrafish is an outstanding model to study gastrulation and FASD. Epiboly (spreading blastomere cells over the yolk cell), prechordal plate migration and convergence/extension cell movements are sensitive to early ethanol exposure. Here, experiments are presented to characterize mechanisms of ethanol toxicity on epiboly and gastrulation. Epiboly mechanisms include blastomere radial intercalation cell movements and yolk cell microtubule cytoskeleton pulling the embryo to the vegetal pole. Both of these processes were disrupted by ethanol exposure. Ethanol effects on cell migration also indicated that cell adhesion was affected, which was confirmed by cell aggregation assays. E-cadherin cell adhesion molecule expression and distribution, which control epiboly and gastrulation, were not affected. Gene expression microarray analysis was used to identify potential causative factors for early development defects, and expression of the cell adhesion molecule protocadherin-18a (pcdh18a), which controls epiboly, was significantly reduced in ethanol-exposed embryos. Injecting pcdh18a synthetic mRNA in ethanol-treated embryos partially rescued epiboly cell movements, including enveloping layer cell shape changes. Together, data show that epiboly and gastrulation defects induced by ethanol are multifactorial, and include yolk cell (extraembryonic tissue) microtubule cytoskeleton disruption and blastomere adhesion defects, in part caused by reduced pcdh18a expression. Control vs. ethanol-treated zebrafish embryos that were treated from 2 to 8 hours postfertilization. Total RNA from control and ethanol-treated embryos were harvested at 8 hours postfertilization.

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Zebrafish 50% epiboly embryos: controls vs. Aplnra/b morphants

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