Browsing by Subject "Wnt-Signalweg"

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    ATP4 and Wnt-signaling are required for ciliogenesis and left-right axis development of Xenopus
    (2012) Walentek, Peter; Blum, Martin
    The vertebrate body plan displays left-right (LR) asymmetries of organ placement superimposed on an overt bilaterally symmetrical organization. Symmetry is broken during embryogenesis, and asymmetric gene expression precedes asymmetric organ morphogenesis. The proton/potassium pump ATP4 was shown to play a role in LR-development of the frog Xenopus laevis as well as in other deuterostomes. Two opposing models of symmetry-breakage were proposed, the ?ion-flux? and the ?leftward flow? model. The former proposed that symmetry was broken by LR-asymmetric expression of the a-subunit of ATP4 during cleavage stages. The latter claimed a cilia-based leftward flow at the gastrocoel roof plate (GRP) to take center stage during neurulation, i.e. a day later in development. In the present thesis work, the role of ATP4a in symmetry-breakage was re-addressed and evidence for symmetrical expression and function of ATP4a was gathered. ATP4a was shown to be required for two Wnt-signaling dependent steps during the setup of cilia driven leftward flow at the GRP: (1) Wnt/b-catenin (b-cat) dependent expression of Foxj1 during gastrulation, and (2) Wnt/planar cell polarity (PCP) dependent posterior localization of motile cilia during neurulation. These data challenge the ?ion-flux? hypothesis and argue for a conserved ATP4- and cilia-dependent symmetry-breakage mechanism throughout the vertebrates. Furthermore, the function of Wnt-signaling components was analyzed in the context of GRP-formation: The receptor Frizzled 8 (Fz8) and b-cat were required for Foxj1 expression during gastrulation. Morphogenesis of the GRP, posterior polarization of motile cilia and expression of Xnr1 and Coco in somitic cells were all required for LR-development. Loss of non-canonical Xwnt11b-signaling perturbed these process, suggesting that non-canonical Wnt-signaling branches, in addition to Wnt/PCP, were relevant for LR-development. ATP4-mediated Wnt-signaling was also required for Foxj1 expression and motile cilia in other epithelia during Xenopus development, i.e. the skin, floor plate and the ependymal cell layer. In the floor plate b-cat was required for Foxj1 expression downstream of Hedgehog-signaling. In the skin mucociliary epithelium ATP4a and Wnt/b-cat were required downstream of Notch/Delta-mediated cell-type specification of multiciliated cells. This was also true for a new cell type of serotonergic cells described here, which was characterized morphologically, by analysis of gene expression and response to manipulations of Wnt- and Notch/Delta-signaling. In summary, the data presented in this thesis suggest a conserved function of ATP4a and Wnt-signaling in vertebrate symmetry-breakage and Foxj1-dependent ciliogenesis in Xenopus.
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    Deskriptive und funktionelle Analyse der Mitglieder der Calponin-Genfamilie Xclp1, Xclp2 und Xclp3 während der Embryonalentwicklung von Xenopus laevis
    (2008) Schmalholz, Silke; Blum, Martin
    The embryonic development of vertebrates is characterized by controlled cell movements. During gastrulation and neurulation cells of the presumptive heart tissue and the neural crest after neural tube closure migrate towards their final position in the embryo. Cell intercalations, which drive the convergent extension (CE) movements to elongate the embryo also depend on active cell migration. The inhibition of CE leads to shortened body axis and neural tube closure defects (NTD). The motility of eukaryotic cells is finally based on the dynamic interaction of cytoskeletal components, which act on the actin filament. Secreted growthfactors of the Wnt family can regulate embryonic cell movement via the non canonical Wnt signaling pathways. The planar cell polarity (PCP) and the Wnt/Ca2+ pathway are thought to be crucial for the process of CE. Up to now there is a lack of knowledge about the cytoskeletal effectors of these signaling cascades. In the presented work, members of the calponin gene family (clp1 to 3) were analysed in this context. Calponins are actin binding proteins, which have been shown to inhibit actin-myosin-interactions and/or to stabilize the actin filament. Expression patterns provided first insights in the transcriptional activity of Xclp1, Xclp2 and Xclp3 during embryonic development. Two Xclp genes (Xclp2 and Xclp3) were already expressed broadly at the onset of gastrulation. Transcription, however, was not detected in the involuted cells, which form the mesodermal germlayer. At neurula stages Xclp2 mRNA was specifically found in the notochord, whereas Xclp3 was expressed in the neuroectoderm. Additionally the migrating cells of the embryonic heart and neural crest were positive for calponin expression. In summary the embryonic calponin pattern correlated with tissues in which cell movements occur. Over- or misexpression experiments were performed to manipulate embryonic calponin function in Xenopus laevis. Gain of function experiments however did not interfere with embryonic development. Probably calponin function was posttranslational negatively regulated in these experiments.The overexpression of calponin proteins, in which specific phosphorylation sites were mutated or known regulatory calponin domains deleted, again didn´t result in altered phenotypes. However, the misexpression of calponin actin binding domaine 2 (ABD2) inhibited the migration of Krox 20 positive neural crest cells, suggesting that in this tissue the Xclp ABD2 acts dominant negative. The presented data are not able to proof or disproof the hypothesis, that calponin proteins are effectors of the non canonical Wnt pathways.
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    Goosecoid und Calponin : zwei neue Regulatoren des PCP-Signalwegs
    (2012) Ulmer, Bärbel Maria; Blum, Martin
    Vertebrate embryogenesis relies on morphogenetic movements such as cell migration and convergent extension (CE). The planar cell polarity (PCP) branch of non-canonical Wnt signaling governs the orientation of cells along embryonic axes. PCP-signaling leads to intracellular polarization of proteins such as Dishevelled, Prickle and Vangl2, resulting in activation of small GTPases such as Rho and Rac, and consequently oriented alignment of the cytoskeleton. This polarity is required for CE, namely for the intercalation of bipolar cells, during gastrulation and neurulation. CE promotes elongation of the notochord and the neural plate, which is a prerequisite of neural tube closure. Previous work had shown that misexpression of the transcription factor Goosecoid (Gsc) in the primitive streak of the mouse and in the dorsal marginal zone of the frog led to neural tube closure defects. The present work demonstrates that misexpression of Gsc inhibits CE in vivo and ex vivo. Gsc gain-of-function (Gsc-GOF) prevented the membrane localization of Dishevelled in the frog animal cap assay, suggesting a disturbance of the PCP pathway. The Gsc-induced phenotypes could be rescued by co-injection of core components of the PCP pathway, Vangl2 and Prickle. Overexpression of RhoA and the non-canonical Wnt11, rescued the effect of Gsc-GOF. Brachyury, a transcriptional activator of Wnt11 and known target of Gsc, was also able to rescue the effect of Gsc-GOF. Gsc thus acted as a repressor of PCP-mediated CE. Furthermore, loss of function experiments in Xenopus were conducted to reveal the endogenous function of Gsc. Due to the conserved and distinct expression of Gsc in Spemann's organizer and the induction of double axes upon injection of Gsc into the ventral marginal zone in Xenopus, a function of Gsc in the specification of dorsal tissue was predicted. The lack of gastrulation defects in the Gsc knock-out mouse, however, questioned an early role of Gsc. The repression of the PCP pathway by Gsc-GOF suggested a novel role of Gsc in the regulation of cell movements. Interestingly, Gsc is expressed in a distinct population of cells in the early organizer, which migrate out of the organizer during early gastrulation to form the prechordal mesoderm. In contrast, the subsequent involuting cells of the notochord undergo CE. Gsc knock-down in the frog reduced the prechordal plate resulting in a narrowing of eye distance. Furthermore, activin-induced CE in animal cap explants was enhanced by Gsc loss-of-function. These findings are consistent with a novel function of the organizer gene Gsc in the regulation of cell movements during early gastrulation, namely the repression of PCP-mediated CE as a prerequisite of active migration of the prechordal mesoderm. The directed migration of neural crest cells represents another embryological process which depends on PCP-signaling. Previous work showed expression of Calponin2 in neural crest cells. Moreover, inhibition of Calponin1 by the Rho-Kinase has been described. In Xenopus, Calponin2 localized to cell protrusion of delaminating and migrating neural crest cells. Loss of function of Calponin2 prevented the polarized outgrowth of cell extensions in neural crest explants and thus migration of neural crest cells. Moreover, additional stress fibers were formed in the central area of neural crest cells at the expense of the peripheral, cortical actin cytoskeleton. The PCP pathway directs migration via the activation of RhoA and inhibition of Rac in the cell compartment opposed to the leading edge. This suggested an interaction of PCP-signaling and Calponin2 during the migration of neural crest cells, which was examined by rescue experiments in vivo and in neural crest explants. Calponin2 knock-down rescued Wnt11 and Rho-Kinase loss-of-function, strongly suggesting that the actin-binding protein Calponin2 acts as an effector of the PCP pathway and directs the polarization of the actin cytoskeleton in migrating neural crest cells. In summary the present work involved two novel regulators of PCP-mediated CE, Gsc at the transcriptional level and Calponin2 as an effector of the actin cytoskeleton.