Browsing by Subject "Entwicklungsbiologie"

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Entstehung und Morphogenese des Vorderhirns - Die Rolle des mit Mikrotubuli assoziierten Proteins Hmmr in Xenopus laevis
(2020) Nickel, Angela; Feistel, Kerstin
The anlage of the central nervous system is formed during early embryonic development. The neuroectoderm establishes the neural plate which folds up to form the neural tube, a process that requires extensive cell rearrangements. During further embryogenesis the anterior part of the neural tube develops into the brain while the posterior part forms the spinal cord. Disturbances during neural tube closure (NTC) lead to severe developmental aberrations. Occurrence of specific neural tube defects indicates a distinct regulation of NTC along the anterior-posterior axis. For example, the severe malformation craniorachischisis is characterized by a failure to close the neural tube from hindbrain levels onwards, while the forebrain region develops normally. This distinct regulation presents itself in the wildtype in a delay between cranial and caudal NTC. While the mechanisms leading to posterior NTC are quite well understood, the morphological processes at the future forebrain level are largely unknown. The aim of this dissertation was to identify cell and tissue morphogenetic processes which are required for the formation and development of the anterior neural tube. As the underlying changes in cell shape as well as cell migration depend on the regulation of the cytoskeleton, the role of the microtubule-associated protein Hmmr was analyzed in the model organism Xenopus laevis. HMMR is a breast cancer susceptibility gene with described roles mainly in the tumor context, regulating cell motility and maintenance of mitotic spindle integrity. In Xenopus, gain as well as loss of function of hmmr delayed NTC and led to defects during further forebrain development. Loss of hmmr impaired separation of telencephalic hemispheres, resembling the human malformation “middle interhemispheric variant of holoprosencephaly”. Failure of ventricle separation could be traced back to disturbed roof plate formation. This was due to impaired NTC resulting from a lack of neural cell convergence. Tissue convergence at the forebrain level is mediated by radial intercalation (RI). During the required regulation of cell polarization and elongation via the microtubule cytoskeleton, hmmr cooperated with the core component of the planar cell polarity (PCP) pathway vangl2, which had been solely characterized as a factor for posterior NTC so far. In addition, experiments with hmmr deletion constructs missing functional domains at the amino- and/or carboxyl-terminus, revealed that elongation and intercalation are distinct processes which are regulated differentially via specific domains of Hmmr. RI required direct binding of Hmmr to microtubules, suggesting that Hmmr influences intercalation movements by regulating dynamic instability of microtubules. RI is essential for mesenchymal to epithelial transition (MET), a physiological morpho- genetic process, which is also involved in establishing tumor metastases in a pathological context. MET is regulated by concerted interaction of canonical Wnt / beta-Catenin and non- canonical Wnt / PCP signaling. Further tissue-specific loss of function experiments uncovered a general role for hmmr in Wnt-modulated RI / MET processes during gastrulation as well as during pronephros and tailbud development in Xenopus. The results suggest that Hmmr regulates microtubule dynamics. Since canonical as well as non-canonical Wnt signaling have been associated with microtubules, hmmr could act as a molecular switch to regulate the activity and interplay of two signaling pathways. This thesis thus identified a new physiological role for the microtubule-binding protein Hmmr, which up to now had been mainly studied in the cancer context. It was shown that Hmmr-mediated RI is a major driving force for anterior NTC. In addition, Hmmr was identified as an essential regulator of microtubule-dependent Wnt signaling in MET processes.
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.
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.
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.