Browsing by Person "Getwan, Maike"

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Analyse des Flow-abhängigen Symmetriebruchs im Frosch Xenopus : die Funktion des Nodal-Inhibitors Coco
(2015) Getwan, Maike; Blum, Martin
The bilaterally symmetrical vertebrate body plan is characterized by the three body axes, anterior-posterior (AP), dorsal-ventral (DV) and the left-right (LR). The LR-axis is the last one to be specified during embryonic development. Its impact on the morphology of the developing organism is visible after a few days in Xenopus laevis, because of the orientation of the visceral organs, such as the heart, gut and the gall bladder. The first molecular differences between the left and right side can already be detected after one day during early neurulation. It is found at the gastrocoel-roof-plate (GRP), a ciliated epithelium which is essential for symmetry breakage. Cilia rotate to produce a leftward fluid movement, which represses the Cerberus/DAN gene Coco in the lateral cells of the epithelium. As Coco acts as an inhibitor of the coexpressed TGFß-type growth factor Nodal (Xnr1), Xnr1 is flow-dependently released from repression on the left side. Xnr1 is capable to induce a unilateral gene-cascade in the left lateral plate mesoderm (LPM) consisting of Nodal itself, its antagonist Lefty/ antivin and the homeobox gene Pitx2c. A central question in this setting concerns the mechanism by which flow results in the repression of Coco. The analysis of Coco transcription gave a first hint, indicating that Coco mRNA is post-transcriptionally degraded and/ or that its translation is blocked. Gene regulation at the level of mRNA usually occurs through the untranslated regions (UTR), in most cases via the 3UTR. To examine the role of the Coco 3UTR for its regulation, protector-RNAs were used which should protect endogenous Coco mRNA from potential inhibitors. Injections led to the interruption of the flow-dependent Coco repression, verifying regulation of Coco via the 3UTR. As 3UTRs are target sites for microRNAs, loss of function experiments of the processing enzyme Dicer were performed. These experiments verified the involvement of miRNAs in the regulation of Coco. Further analyses identified miR-15a as a central player. The interruption of its synthesis or the specific protection of its binding site within the Coco 3UTR prevented flow-dependent down-regulation of Coco. Epistatic experiments demonstrated that the LR-axis of embryos with inhibited flow could be rescued by addition of the miR-15a precursor on the left side. In summary this thesis work revealed miRNAs as a primary target of leftward flow, upstream of the Nodal inhibitor Coco.