Browsing by Subject "Embryogenesis"

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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.
The role of serotonin and gap junctions in left-right development of Xenopus laevis
(2011) Beyer, Tina; Blum, Martin
In vertebrates, the correct determination of the left-right (LR) axis is essential for accurate placement of the inner organs, such that the heart points to the left, lung lobation differs between left and right side, spleen and stomach are located on the left,liver on the right body side and the gut coils asymmetrically. Disturbance of this organization can lead to severe impairments of organ function. In the African clawed frog Xenopus laevis, already in four-day old tadpoles asymmetric organ arrangement is visible. This coordinated organ development strictly requires prior Nodal cascade activity in the left lateral plate mesoderm (LPM) in all model organisms examined so far. The initial symmetry breaking event necessary for unilateral induction of Nodal signaling is still under debate. In X. laevis, two models, namely 'ion-flux' and 'cilia-driven leftward fluid flow', were discussed in this context. Leftward flow was first described in the mouse model and later on in fish and rabbit, whereas the 'ion-flux' hypothesis is supported by data derived from chick development. In the present work it was intended to enlighten this putative discrepancy by re-investigating the function of two 'ion-flux' components in context of leftward flow in the model organism X. laevis. First, a link between cell-cell communication via gap junctional communication (GJC)and LR axis establishment was analyzed by using heptanol for general inhibition of channel conductance on the one hand, and single knock-down (KD) of specific subunits on the other hand. Both treatments resulted in absence of the left-sided Nodal cascade. The KD led to shorter GRP cilia when compared to wildtype embryos and loss of bilateral Nodal expression at the GRP margin, respectively. Furthermore, heptanol treatments of stages in which the GRP already has been fully developed also resulted in laterality defects, thus implying a second function of GJC most likely for the signal transfer to the left side. These results indicated a role of GJC in leftward flow establishment and/or post-flow in neurula stages rather than a function in early cleavage stages for LR determination. Second, the early signaling function of the neurotransmitter serotonin (5-HT) was inhibited by over-expression of either a frog or a human receptor ligand binding domain(LBD). In addition, specific KD of a receptor class 3 subunit was performed. Both applications resulted in impaired left-sided marker gene expression and disturbed GRP morphogenesis. Remarkably, marker gene expression of the superficial mesoderm(SM) which gives rise to the GRP during development, was reduced in 5-HT signaling impaired embryos. Very importantly, receptor 3 specific 5-HT signaling was shown to represent a necessary competence factor required for Wnt-dependent axis development in the frog double axis induction assay. Besides the new function of 5-HT during early development, it was further shown that the expression of the SM marker Foxj1 (a master regulator of motile cilia) depended on maternal factors. Based on the work presented here, the following model is proposed: (1) Foxj1 expression is induced maternally, followed by (2) zygotic refinement in post-MBT stages, i.e. inhibition on the ventral and maintenance on the dorsal side. In the organizer region, an interplay of Wnt and 5-HT signaling is required for dorsal development.(3) Cilia driven leftward flow initiate an unknown downstream signal which is transferred to the left LPM. Both events, leftward flow and transfer, require active GJC. (4) A to date unknown signal gets transferred towards the left LPM in a GJC-dependent process and induces the Nodal cascade activity, a prerequisite for proper organ arrangement. Taken together, the data presented in this study indicate that the directed fluid flow in neurula embryos represent the decisive step for symmetry breakage with the 'ion-flux' components being involved in correct flow function.