Browsing by Subject "Developmental biology"

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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.
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.