Browsing by Subject "Left-right asymmetry"

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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.
Establishment of a new in vitro culture system and functional analysis of sonic hedgehog and FGF8 in the determination of laterality in the rabbit embryo
(2008) Bitzer, Eva; Blum, Martin
Cilia-driven leftward flow plays a pivotal role in the determination of left-right (LR) asymmetry. In mammals, this extracellular fluid flow is produced by motile monocilia situated on the posterior notochordal plate (PNC). The PNC is homologous to superficial mesoderm derived structures of other species like the gastrocoel roof plate (GRP) in frog and Kupffer's vesicle (KV) in fish. Directional fluid flow created at these structures subsequently leads to the initiation of the left-specifying Nodal signalling cascade in the left lateral plate (LPM). The rabbit develops via a flat blastodisc phase representing the archetypical mode of mammalian embryogenesis. These specific advantages of the rabbit were employed in this study to further examine the role of two central determinants of laterality, namely Sonic hedgehog (Shh) and FGF8, and also extended by the design of a new in vitro culture technique. In this new method, the so-called ring culture, a medium-filled plastic ring was placed upon the extraembryonic tissue of the explanted embryo. In contrast to the semi-dry standard culture method, this setting corresponded more to the in vivo conditions of gastrulating/neurulating rabbit embryos in the uterus. It therefore facilitated stable development of laterality in most cases also when presomite stages were taken into culture. Subsequent analysis showed that this was due to improved development of the PNC. Conversely, in standard-cultured embryos showing altered LR marker gene expression, maturation of the PNC was impaired leading to a disturbance of leftward flow. This study also provided evidence that cilia-driven leftward flow is indispensable for the determination of laterality in rabbit embryos. When the flow was blocked during culture by methylcellulose-containing medium embryos displayed altered LR marker gene expression in a very high proportion. The unilateral gain-of-function of Shh revealed important differences between rabbit and chick embryos. In rabbit, Shh induced right-sided marker gene expression only in the 2 somite stage, whereas in chick this inductive effect lasted from stage 4 until up to the 1-2 somite stage. This indicated that in rabbit Shh works in conjunction with the flow, which has not been described up to now in chick. The systemic inhibition of Shh signalling by cyclopamine led to bilateral expression of LR marker genes in rabbit. This was due to disruption of the floor plate and therefore the loss of the restrictive midline barrier function of Lefty expression as described in Shh mutant mice. FGF8 has a right-sided repressive function in the rabbit implicated in the transfer of laterality cues. In the present study it could be shown that this repressive effect is epistatic to cilia-driven leftward flow, because it also functioned when the flow was blocked. The systemic inhibition of FGF8 signalling with SU5402 caused loss of LR marker gene expression prior to the 2 somite stage but did not influence ciliogenesis or the setup of cilia-driven leftward flow. Taken together, this suggested a dual function for FGF8 signalling: First, it is needed to confer competence to the lateral plate and second, during the 2 somite stage, it is needed for the transfer of LR cues.