Browsing by Subject "Leftward flow"

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Multiple Funktionen des FGF-Signalwegs regulieren die Lateralitätsentwicklung im Krallenfrosch Xenopus
(2013) Schneider, Isabelle; Blum, Martin
Early embryogenesis governs the formation of the three body axis. Like in a cartesian coordinate system, the LR-axis is defined by the generation of the anterior-posterior and the dorso-ventral axis. In the course of laterality specification, the original LR-symmetry has to be broken to enable the asymmetric arrangement of inner organs in a specific manner. This is mediated by the expression of conserved gene cascade, namely the Nodal gene cascade, which is expressed in the left but not in the right lateral plate mesoderm of the neurula stage embryo. Symmetry breakage, which leads up to this asymmetric Nodal gene cascade, is manifested by a cilia-based leftward fluid flow. The flow generating epithelium is located at the posterior end of the notochord and expresses Nodal in a bilateral symmetrical mode. This early Nodal domain is a prerequisite of the later asymmetric Nodal gene cascade. Despite the conserved nature of Nodal expression and of leftward flow, no conservation of the role of the FGF signaling has been described for mouse, chick, rabbit and zebrafish. In this work the role of FGF signaling in Xenopus laevis LR-development was investigated. Using of a receptor antagonist to inhibit FGF signaling revealed two temporally distinguishable functions. Firstly, FGF signaling in early gastrula stages is required for the proper expression of FoxJ1, the master control gene of motile cilia. Here, FGF signaling acts in the process of ciliogenesis of the symmetry-breaking epithelium, which is represented by the GRP (“gastrocoel roof plate”) in Xenopus. Secondly, FGF acts in a cilia-independent manner on the bilateral Nodal expression. A series of descriptive and functional studies revealed that these cells constitute the somitic part of the GRP and that inhibition of FGF signaling leads to the loss of these cells. Interestingly, the effect on ciliogenesis is consistent with the role of FGF signaling in zebrafish, whereas the loss of bilateral Nodal expression is in line with the hypomorpic Fgf8 mutant mouse. The description of these two successive functions in Xenopus indicates a higher degree of conservation of the role of FGF signaling than suggested so far. The FGF signaling pathway splits into several branches, two of which play important roles in the early development of Xenopus embryos. Activation of MAPK signaling is implicated in the induction of mesoderm, whereas the PLC/PKC/Calcium signaling branch impacts on morphogenetic movements. FGF-mediated control of Foxj1 expression was temporally correlated with FGF signaling that acts on mesoderm specification. As a consequence, mesodermal gene expression and blastopore closure was seriously affected by loss of FGF signaling at early gastrula stages. By starting inhibition experiments during gastrula stages, when mesoderm induction is almost finished, general mesoderm specification defects were avoided but the effect on the somitic GRP cells persisted. To unravel which FGF-induced signaling branch acted on the two different functions of FGF described here, the PLC/PKC/Calcium signaling branch was inhibited using the antagonist Sprouty1. Sprouty1 gain of function experiments had no effect on ciliogenesis, but caused loss of somitic GRP cells comparable to loss of function experiments using the FGF receptor antagonist. This suggests that the FGF-dependent formation of these cells is regulated by the PLC/PKC/Calcium pathway. A specific role of Calcium was supported by experiments using a calcium-permeable channel. Despite this, ciliogenesis was not affected by inhibition of PLC/PKC/Calcium, suggesting a role of MAPK for the early function of FGF. In conclusion, this work demonstrates two functions of FGF signaling in Xenopus LR-development, which furthermore are consistent with a conserved function of FGF signaling in vertebrate LR-axis determination. Novel insights into the role of FGF signalling in the very cells which sense leftward flow at the lateral margin of the GRP will open new approaches to analyse laterality specification in more detail.
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.