Browsing by Subject "Embryologie"

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Funktionelle Analyse der Histondeacetylase 6 sowie experimentelle Modellierung von Lateralitätsdefekten während der Links-Rechts-Achsenentwicklung von Xenopus laevis und Paracentrotus lividus
(2017) Tisler, Matthias; Blum, Martin
Vertebrates display an asymmetric positioning of the visceral organs, which is also denominated as left-right body axis. During embryogenesis, an asymmetric gene expression is detectable that is initiated by an evolutionary conserved mechanism of symmetry breakage, which is conserved among deuterostomes. During neurula stages, rotating motile mono-cilia at the so called left-right organizer (LRO) generate an asymmetric stimulus known as extracellular leftward fluid flow that is essential for the unilateral left asymmetric gene expression of the Nodal cascade. Spontaneous mutations or the experimentally induced loss of function of genes influencing ciliogenesis at the LRO, the induction of the Nodal cascade or its propagation lead to left-right defects. Left-right defects are frequently observed in human conjoined twins. Thoracopagous, dicephalic conjoined twins display defects in the arrangement of the inner organs, that are solely reported from the twin located to the right side. While left twins orient the inner organs wildtypically, right twins show a randomization of the left-right axis. The functional cause of the inverted arrangement regarding the right twin has remained enigmatic. It has been hypothesized that the observed laterality determination in conjoined twins, like in wildtype embryos, was dependent on leftward flow. In the course of this thesis, the known unilaterlal left-sided induction of the Nodal cascade in the left conjoined twin, as in singelton embryos, can be linked to leftward flow. The artificial induction of a second body axis leads to a subsequent duplication of the LRO during development. During flow stages endogenous and induced LROs locate in close proximity and display a partial fusion of cell populations. Anti-sense Morpholino Oligomeres or methylcelluose mediated loss of cilia motility lead to a loss of markergene expression in the left-lateral plate mesoderm of the left twin. By combining differential gain- and loss-of-function strategies, it was possible to link the establishment of laterality in conjoined twins to the leftward flow and, moreover, to manipulate it an a predictable manner. The cause of this hitherto enigmatic laterality defects in conjoined twins can therefore be explained by the evolutionary conserved mechanism of left-right establishment. Although the general mechanism of symmetry breakage has been characterized, novel candidate genes are continously beeing identified that act at a specific sequence of this process. The candidate gene histonedeacetylase 6 (hdac6) was shown to impact on left-right development. Anti-sense Morpholino Oligomere induced loss-of-function experiments led to left-right defects in a dose dependent manner regarding, the induction of the genes of the Nodal cascade, indicating a function of hdac6 before fluid flow induced regulation of dand5 mRNA. Taken together: histonedeacetylase 6 acts as modulator of canonical Wnt-signaling in the transcriptional induction of the Wnt-dependent transcription of foxj1, a master control gene of the biogenesis of motile cilia. Loss of Hdac6 leads to defects regarding the ciliogenesis of motile cilia at the LRO as well as the multiciliated epidermis of the embryo. The here presented results represent the first developmental hdac6 loss-of-function phenotype, which was so far not know from Hdac6-/- mice. These experiments shed a new light on the differential in vivo function of this unique histondeacetylase during development. Even though the asymmetric positioning of the inner organs is restricted to vertebrates, the asymmetric expression of the Nodal cascade turns out to be evolutionary conserved among deuterostomes. Comparable to vertebrate species, larvae of the sea urchin (Paracentrotus lividus, Echinodermata) display an asymmetric expression of the Nodal cascade in the ectoderm an during gastrula stages. Experiments from this work could demonstrate that also in sea urchin embryos the asymmetric gene expression depends on motile cilia. The archenteron of gastrula stage embryos was identified and described as homologous structure to vertebrate LROs. Deciliation experiments at different time points of development induce laterality defects and point towards a symmetry breakage during early gastrulation. By this experiments, the cilia dependent establishment of left-right asymmetry is described as a common synapomorphy of the deuterostomes beeing conserved from sea urchin to vertebrates, shedding a new light on the establishment of asymmetric gene expression.
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.
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.
The role of the actin binding protein Calponin2 during embryonic development of Xenopus laevis
(2021) Mantino, Sabrina Maria; Feistel, Kerstin
Despite the abundant variability among adult vertebrate body plans, the developmental steps transforming the single zygote into a multicellular organism of remarkable complexity, are evolutionary highly conserved. Morphogenetic processes such as gastrulation, neural tube closure, body axis extension, neural crest cell migration and organogenesis are thereby at the heart of embryogenesis. Especially the formation of a closed neural tube, which gives rise to the central nervous system, constitutes a fundamental event. Neural tube closure is achieved by convergent extension movements and by apical constriction of neuroepithelial cells. Along with proceeding neurulation, cranial neural cells start to delaminate from the neuroepithelial border. In order to initiate directed migration movements, neural crest cells require polarised cell protrusions and mediate mechanical forces. Changes in cell shape and motility underlying neural tube closure and neural crest cell migration are controlled by specific regulation of the actin cytoskeleton. How these actin dynamics and the myosin-mediated contraction of actin networks are precisely coordinated is not fully understood. In this context, actin filament-associated proteins play an important role for the structural organisation of different actin network types. Calponins constitute an evolutionary highly conserved family of F-actin binding proteins, which are able to influence actin-myosin dynamics and to stabilise actin filaments. Previous studies already demonstrated a role of Calponin proteins in smooth muscle contraction, cell motility and phagocytosis. Vertebrates possess three Calponin isoforms, each displaying specific expression patterns and functions. Calponin2 is expressed in a variety of cell types and several studies performed in vitro indicated that Calponin2 is important for mechanical tension mediation during the course of cell migration. In the early embryo of Xenopus laevis, calponin2 is expressed in tissues that undergo extensive morphogenetic movements and cell migration. This implies an elemental role of Calponin2 for respective morphogenetic steps during embryonic development of this well-established model organism. Within the scope of the present work, the specific function of Calponin2 for dynamic regulation of the actin cytoskeleton was analysed more closely. Localisation of the protein, by utilising a tagged construct, was shown in neural plate cells as well as in migrating neural crest cells. In both cell types, regulated protein degradation occurred, which led to specific expression restricted to the apex of constricting neural plate cells or to forming lamellipodia. Thus, tagged Calponin2 localised to regions of the actin cortex. Loss of Calponin2 function led to defects in neural crest cell specification and migration as well as in convergent extension and apical constriction within the neural plate. All induced phenotypes were rescued by additional calponin2 mRNA injection. In summary, these data demonstrated a specific function of Calponin2 for correct formation of the neural crest as well as for neural tube closure. Furthermore, the precise regulation of protein expression levels, which directly correlated with correct Calponin2 function, was dependent on specific domains that potentially mediate actin-binding. Clik1, Clik2 and the C-terminus were identified as a critical unit regulating protein degradation, both in neural crest cells and neural plate cells. Additionally, it was shown that Calponin2 function for neural apical constriction depends on each of these domains as well. Overall, the degradation of Calponin2, regulated via its F-actin binding, implies a filament stabilising function. Thus, a temporospatial coordination of protein degradation would be necessary to enable dynamic changes of the actin cytoskeleton by a regulated release of actin filaments and to allow the association of other structural effectors during morphogenetic processes of early vertebrate development.