Browsing by Subject "Zilien"

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Left-right asymmetry in Xenopus laevis : functional dissection of leftward flow
(2009) Vick, Philipp; Blum, Martin
Despite their external bilateral symmetry, vertebrates have a conserved left right (LR) asymmetry of their inner organs. For all vertebrates, it is well-known that the asymmetric organogenesis is preceded by the left-sided nodal signaling cascade during embryonic development. A question which has not been settled in detail is how the first asymmetrically directed signal arises, which activates nodal only on the left side. In mice and fish embryos an extracellular leftward fluid flow ? generated by rotating cilia ? was shown to be functionally necessary for gene activation. Recently, this process has also been demonstrated in frog embryos and its mechanic inhibition caused laterality defects. This raised the question if this process is also conserved among vertebrates. The aim of this study was to analyze the mechanism of flow in the frog in the context of the known models. Thereby, its prerequisites and the exact mode of activation of the left-sided genes should be assessed. Finally, general conclusions on the symmetry breakage of vertebrates were to be drawn. Loss of function of axonemal dynein heavy chains inhibited ciliary movement, fluid flow and laterality development of the embryos. By showing that flow was only necessary on the left half of the ciliated epithelium (GRP), definite statements could be made concerning origin, identity and possibility of a transported substance. Moreover, a function for GRP morphogenesis and thus for the generation of flow were proven for the serotonin receptor 3 and the calcium channel Pkd2. These results did not confirm the hypothesis that Pkd2 causes a flow-dependent left-sided calcium signal. Consequently, this contradicted the so-called "2-cilia model" in favor of an early morphogenetic function in frog. In the course of a collaboration it could be shown, that the RNA-binding protein xBic-C has a conserved function for cilia polarization and thus for the flow in both Xenopus and mice. Additionally, up to now, a right-sided nodal inhibitory function has been assigned to the protein coco. However, the exact mechanism was unknown. By specific, combined left- and right-sided loss of function experiments with coco, nodal and the above mentioned components, it could be demonstrated that coco but not nodal is directly dependent on leftward flow. With the flow, coco was downregulated on the left side only and could thus no longer inhibit nodal there. Loss of flow or xBic-C function ? but not that of Pkd2 ? could be rescued by coco inhibition; this revealed a clear hierarchy. Taken together a sequence of conditions could be formulated: Pkd2 and the serotonin receptor 3 are obligatory for the formation of the GRP and correct flow before neurulation. xBic-C also precedes the flow and is required for cilia polarization but seemed also to have a further function. coco is downstream of the fluid flow and is downregulated as its direct consequence on the left side. nodal, in turn, is downstream of this order and is only released on the left side where it can thus act as a putative mediator to transfer the generated signal into the lateral plate mesoderm. These results are discussed in terms of evolutionary origin and conservation.
Towards a unifying model of symmetry breakage in Xenopus laevis : serotonin signaling and the cilia-driven leftward flow
(2011) Thumberger, Thomas; Blum, Martin
Orientation of the three vertebrate body axes anterior-posterior (AP), dorso-ventral (DV) and left-right (LR) is specified during early embryogenesis. Whereas the formation of the AP and DV axes is well understood, it is not finally resolved how and when the left and right sides get molecularly distinct. All deuterostomes analyzed so far, however, display an asymmetric left-sided expression of the TGF-β factor Nodal during embryonic development which precedes asymmetric organogenesis. In zebrafish, medaka, mouse and rabbit embryos a cilia-driven extracellular leftward fluid flow was shown to be causal for the left asymmetric induction of the Nodal gene cascade during early neurulation. In X. laevis, leftward flow was also shown to be driven by a mono-ciliated epithelium in the posterior part of the archenteron roof (gastrocoel roof plate, GRP). Mechanical blockage of this current resulted in laterality defects. Despite the apparent evolutionary conservation of flow, an earlier mechanism to specify the LR axis during early cleavage stages has been reported in X. laevis. Based on mostly inhibitor experiments, the so-called 'ion-flux' hypothesis was put forward which proposes an electrogenic transport and asymmetric accumulation of determinants as early as at the 32-64 cell stage. The monoamine serotonin is the core-effector of this hypothesis and was reported to asymmetrically accumulate at the ventral right blastomeres of early cleavage stage embryos. The aim of this study was to investigate putative interactions of the two apparently opposing mechanisms for breaking the initial LR symmetry of the Xenopus zygote. Reinvestigation of serotonin localization could not confirm the initial report. Further, serotonin signaling was shown to be necessary for LR axis formation on the dorsal but not ventral side, more specifically as a competence factor for the canonical Wnt-pathway. Detailed analyses of specimens impaired for serotonin signaling revealed requirement of serotonin signaling for specification of the superficial mesoderm (SM) which gives rise to the GRP and, consequently, to leftward flow. Leftward flow thus indirectly depends on dorsal serotonin signaling. In a further part of the present thesis, a re-examination of laterality in Siamese twins was performed. It has been known since the earliest experimental investigations of laterality that in induced and naturally occurring Siamese twins the left twin consistently displays wildtype orientation of the visceral organs whereas the orientation in the right twin is randomized. In experimentally induced conjoined twins, this observation holds true regardless of which twin is the induced. A model of symmetry breakage, in order to be plausible, thus should also be able to account for this phenomenon. When experimentally induced twins were analyzed for leftward flow, in the majority of cases a continuous leftward flow was observed, i.e. both twins shared one GRP. Thus, laterality cue(s) get translocated towards the far left side, i.e. only the left embryo receives the wildtype asymmetric information, regardless if it is the induced or endogenous twin. In rare case X. laevis conjoined axes developed far apart from one another such that two separate GRPs and individual leftward flows were observed, a condition that enabled both axes to exhibit a left-sided Nodal cascade. These experiments strongly suggest that Spemann's organizer itself is necessary and sufficient to establish all three body axes. In conclusion, the present analysis of laterality determination in the frog Xenopus supports evolutionary conservation of leftward flow as symmetry breaking event, as previously reported for mouse, rabbit and bony fish.