Browsing by Subject "Symmetriebruch"

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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.
ATP4 and Wnt-signaling are required for ciliogenesis and left-right axis development of Xenopus
(2012) Walentek, Peter; Blum, Martin
The vertebrate body plan displays left-right (LR) asymmetries of organ placement superimposed on an overt bilaterally symmetrical organization. Symmetry is broken during embryogenesis, and asymmetric gene expression precedes asymmetric organ morphogenesis. The proton/potassium pump ATP4 was shown to play a role in LR-development of the frog Xenopus laevis as well as in other deuterostomes. Two opposing models of symmetry-breakage were proposed, the ?ion-flux? and the ?leftward flow? model. The former proposed that symmetry was broken by LR-asymmetric expression of the a-subunit of ATP4 during cleavage stages. The latter claimed a cilia-based leftward flow at the gastrocoel roof plate (GRP) to take center stage during neurulation, i.e. a day later in development. In the present thesis work, the role of ATP4a in symmetry-breakage was re-addressed and evidence for symmetrical expression and function of ATP4a was gathered. ATP4a was shown to be required for two Wnt-signaling dependent steps during the setup of cilia driven leftward flow at the GRP: (1) Wnt/b-catenin (b-cat) dependent expression of Foxj1 during gastrulation, and (2) Wnt/planar cell polarity (PCP) dependent posterior localization of motile cilia during neurulation. These data challenge the ?ion-flux? hypothesis and argue for a conserved ATP4- and cilia-dependent symmetry-breakage mechanism throughout the vertebrates. Furthermore, the function of Wnt-signaling components was analyzed in the context of GRP-formation: The receptor Frizzled 8 (Fz8) and b-cat were required for Foxj1 expression during gastrulation. Morphogenesis of the GRP, posterior polarization of motile cilia and expression of Xnr1 and Coco in somitic cells were all required for LR-development. Loss of non-canonical Xwnt11b-signaling perturbed these process, suggesting that non-canonical Wnt-signaling branches, in addition to Wnt/PCP, were relevant for LR-development. ATP4-mediated Wnt-signaling was also required for Foxj1 expression and motile cilia in other epithelia during Xenopus development, i.e. the skin, floor plate and the ependymal cell layer. In the floor plate b-cat was required for Foxj1 expression downstream of Hedgehog-signaling. In the skin mucociliary epithelium ATP4a and Wnt/b-cat were required downstream of Notch/Delta-mediated cell-type specification of multiciliated cells. This was also true for a new cell type of serotonergic cells described here, which was characterized morphologically, by analysis of gene expression and response to manipulations of Wnt- and Notch/Delta-signaling. In summary, the data presented in this thesis suggest a conserved function of ATP4a and Wnt-signaling in vertebrate symmetry-breakage and Foxj1-dependent ciliogenesis in Xenopus.