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Timely NODAL Inhibition Driven by Consecutive Lefty1 and Lefty2 Is Required for Epiblast Patterning

ABSTRACT: The mouse genome encodes two Lefty genes, novel TGF-β superfamily members that antagonize the Nodal signaling gradient during symmetry breaking within the gastrulation-stage embryo. Despite their reported roles as modulators of this key morphogen, accurately describing how the Leftys contribute to cell fate determination is hindered due to the challenge of modeling rapid changes in embryonic complexity from static snapshots of internally developing embryos. Here, we generated two high temporal and cellular resolution transcriptional atlases of Lefty1 and Lefty2 mutant embryos and evaluated the effects of these regulators on cell fate specification using a Network Flow Model to describe the processes of gastrulation continuously. Combined with analysis of dynamic Nodal activity in wild-type and mutant embryos, we capture a sequential shift from Anterior Visceral endoderm (AVE)-based Lefty1 to primitive streak (PS)-based Lefty2 control, which our mutant data suggest act to collectively shield the developing embryo from spurious Definitive endoderm (DefEndo) and Extra-embryonic mesodermal (ExM) differentiation. We find that mutation of either Nodal antagonist converges towards a highly similar phenotype, with only the most rostral neural lineages protected in lefty2 mutant embryos as a hallmark of AVE-based patterning. Collectively, our work highlights the potential of single-cell profiling technologies to decode complex differentiation processes, including addressing how classical morphogens temporally coordinate multiple distinct transcriptional states from the same pluripotent field.

ORGANISM(S): Mus musculus

PROVIDER: GSE272574 | GEO | 2026/06/01

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.

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Timely NODAL Inhibition Driven by Consecutive Lefty1 and Lefty2 Is Required for Epiblast Patterning

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