Browsing by Subject "Embryonalentwicklung"

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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.
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.
Delimitation of the organizer from the posterior notochord : descriptive and functional studies in mouse and African clawed frog
(2009) Andre, Philipp; Blum, Martin
During vertebrate development, gastrulation is probably the most important phase, as the future body plan is established. Thereby the three body axes anterior-posterior, dorsal-ventral and left-right are determined as well. A central role thereby is taken by the Spemann organizer, as this part of the embryo governs the above mentioned processes. The left-right axis is specified by an extracellular leftward fluid-flow, which results in asymmetric gene expression of the TGFβ factor Nodal. In mice the ciliated epithelium responsible for the fluid-flow as well as the organizer are denominated as ?node?. In contrast to that two distinct entities are thought to be responsible for organizer function and fluid-flow in zebrafish, Xenopus and rabbit embryos. In the present study, it could be shown that this also applies for mouse embryos. In order to prevent further confusion the ciliated epithelium responsible for the fluid-flow was denominated as posterior notochord (PNC) as it is in continuity with the notochord but located anterior to the organizer (?node?). The latter is characterized by the expression of the homeobox gene Goosecoid (Gsc). Gsc possesses, like the tissue of the organizer, the potential to induce an almost complete axis and became therefore famous as ?the organizer gene?. However upon knockout of Gsc in the mouse, surprisingly no gastrulation defects could be detected. Therefore the function of Gsc during gastrulation was investigated using a gain-of-function approach. The analysis of this, in the present and previous studies, indicated that Gsc acts as a switch between two modes of cell movement. Accordingly, Gsc promotes active cell migration and inhibits convergent extension movements. Furthermore it was investigated whether the monoamines adrenaline and serotonin have an influence on the cilia and thus on the leftward fluid-flow, as it was reported in rat and Xenopus experiments. Thereby it could be detected that the addition of adrenaline led to a reduction of the ciliary beat frequency (CBF) and therefore the fluid-flow was attenuated. In contrast to that the addition of serotonin or its antagonists resulted only in minor changes of CBF and thus had no measurable effect on the fluid-flow. The consequences of a malformed PNC were analyzed using embryos mutant for Brachyury (T). Thereby, it was shown that embryos homozygous for this mutation did not develop a functional PNC and thus lacked the fluid-flow. Furthermore a possible cause for the absence of asymmetric Nodal in these embryos was brought into context of an attenuated expression of Fgf8. This indicated that T possesses two distinct roles in left-right development. On the one hand it is necessary for the correct formation of a PNC and on the other hand it is probably needed to maintain the expression of Fgf8, which is a prerequisite for the transcription of Nodal. Finally it was investigated whether these functions were conserved from the African clawed frog Xenopus. Thereby, it could be shown, that Xbra, the homologous gene of T in Xenopus, was also necessary for the formation of the gastrocoel roof plate, the homologous structure of the PNC. Additionally it was observed that the absence of Xbra led to an attenuation of Nodal expression in the midline of Xenopus embryos. This implied that not only the function of Brachyury, but also the process of laterality determination is highly conserved between mammals and amphibians.
Funktionelle Analyse der Gene Brachyury, Goosecoid und Myosin1d für die frühe Musterbildung und Etablierung der Körperachsen während der Embryogenese des Krallenfroschs Xenopus laevis
(2019) Kurz, Sabrina; Blum, Martin
During a fundamental phase of vertebrate embryonic development, gastrulation, the reorganization of the future body plan, is determined. This process controls the embryonic patterning of the antero-posterior (AP) and dorso-ventral (DV) axes, as well as the internal left-right (LR) axis. The establishment of the body axes is initially controlled by the dorsally localized Spemann organizer (SO), whereby the determination of the LR axis takes place the latest. The formation of laterality during subsequent neurulation is executed by a highly conserved mechanism of symmetry breakage within fishes, amphibians and mammals. An extracellular, monocilia-driven fluid-flow („Flow") eventually defines the laterality of the embryo, by left-asymmetric gene expression of the morphogen Nodal. Responsible for the Flow is a ciliated epithelium in the postero-dorsal region of the embryo, which undergoes morphological development and correct positioning during gastrulation and is termed GRP („gastrocoel roof plate") in the frog. Ultimately, this conserved tissue describes the left-right organizer (LRO) and guarantees proper organ situs. The origin and function of the LRO can be traced back to an epithelial organized cell structure on the dorsal surface of the gastrula, characterized by the expression of the canonical Wnt target gene and cilia marker foxj1. Following the specification of this Superficial Mesoderm (SM), the future cells of the LRO invaginate in a directed fashion during gastrulation. The patterns of cellular movement require a restructuring of the actin-cytoskeleton. These migrations, executing the future body plan, are ensured by the PCP- („planar cell polarity”) signaling pathway. This non-canonical Wnt pathway guarantees polarized alignment and migration of cells along the body axes, namely convergent extension (CE). As an organizer gene, the expression of Goosecoid (Gsc) characterizes SO and its’ properties. Contrary to the assumption that a Gsc loss of function thus inhibits gastrulation, no visible impairment was detected in the Knock-Out mouse and Knock-Down in Xenopus. Gain-of-function in the frog, which analyzed the role of Gsc during gastrulation finally demonstrated the homeobox-gene exerting a function in regulating cell movements. Overexpression resulted in impaired CE of dorsal tissue due to defective localization of nuclear proteins of the PCP signaling pathway. Gsc-induced malformations could be compensated by co-injections of associated components. In conclusion, a new function as an inhibitor of PCP-dependent CE during gastrulation was suggested. The morphogenetic movements of CE are responsible for the AP-elongation and LRO-positioning. Both, mechanical forces and cilia-based Flow functionally interact for that matter. In the invertebrate Drosophila, where neither Nodal nor cilia are expressed, organ asymmetry is ensured by internal chirality conducted via motor proteins, such as myosin1d, in a PCP-dependent manner. Similarly, in vertebrates such as Xenopus, myo1d mediates interactions of the actin cytoskeleton asymmetrically, that guarantee laterality of the organ system. The functional preservation of the non-canonical Wnt pathway could be demonstrated by co-injections of PCP core proteins, being able to restore disturbed LRO morphology. The obtained data clearly demonstrated the evolutionary, interspecific, regulation of axis asymmetry by myo1d. Since the strict spatio-temporal regulation of cell movements is fundamental for patterning, CE is propagated by another transcription factor: Brachyury (Tbxt in Xenopus). Its expression induces and controls differentiation of mesodermal cell populations, such as the notochordal cells of the dorsal midline, hence LRO. The necessity specifying progenitor cells in the SM could also be highlighted, showing to be mediated non-cell autonomously to guarantee the induction of foxj1. Additionally, the determination of both tissues is exerted by functional interactions of Tbxt with either PCP-, as well as ß-catenin-dependent Wnt signaling pathway. Loss of Brachyury affects laterality in mutants and morphants. The species-spanning Brachyury-FGF „feedback-loop" for the induction of Nodal and Foxj1 could already act in the SM and showed that function as well as the process of LR development, as suggested between Fgf8 and Brachyury, to be functionally conserved. In Xenopus, signal transduction for SM and foxj1 induction was extended by the function of the Wnt receptor frizzled-8, the ligand and Tbxt target gene wnt11b, and the ventrally acting wnt8a in Whole-Mount embryos and explant co-cultures.
Die Rolle von hmmr während Neurulation und Hirnentwicklung im Afrikanischen Krallenfrosch Xenopus laevis
(2016) Hagenlocher, Cathrin; Schweickert, Axel
The cerebrospinal fluid (CSF) fills the entire ventricular system of the brain, the spinal cavity and the subarachnoid space. CSF mechanically buffers the brain, transports signaling molecules and eliminates waste products. It is produced by the choroid plexus (CP) and transported throughout the ventricular system via motile cilia. Excessive production, diminished transport or reduced absorption of CSF lead to hydrocephalus, a pathological dilatation of the brain ventricles. Mutations in humans and mice showed that dysfunctional and immotile cilia also induce hydrocephalus. The underlying mechanism through which disturbed ciliary motility leads to formation of hydrocephalus is not resolved. In the present thesis the model organism Xenopus laevis was used to analyze the occurrence of hydrocephalus upon on ciliary dysmotility. Biogenesis of motile cilia was described in the Xenopus laevis brain up to metamorphosis. Gene expression of foxj1, the superior regulator of the biogenesis of motile cilia, correlated with development of elongated monocilia and the switch to multiciliated ependymal cells. Cilia on foxj1-positive cells were motile and produced a directional flow of CSF. foxj1 loss-of-function led to impaired or absent motile cilia and resulted in hydrocephalus. The development of the hydrocephalic dilatation correlated with reduced velocity of the cilia-driven CSF-flow below 300 µm/s. In cilia of the airway epithelium regulation of ciliary beat frequency via HMMR has been described with HMMR loss-of-function resulting in reduced ciliary beat frequency. In line with these results, hmmr loss-of-function in Xenopus laevis resulted in reduced velocity of CSF-flow and hydrocephalus. This suggests that especially in the fourth ventricle CSF-flow velocities above 300 µm/s are necessary to maintain a homeostatic fluid pressure in the entire ventricular system. The loss-of-function of foxj1 as well as hmmr further led to severe malformations in the dorsal midline of the brain, especially of the CP and the subcommissural organ. These ciliated structures have already been connected to development of hydrocephalus. Brain defects after loss-of-function of hmmr reflected the human disorder of holoprosencephaly (HPE) which often results from mutations in the Shh-signaling pathway and leads to hydrocephalus. Interestingly after hmmr loss-of-function induced HPE was independent of the Shh-signaling pathway. Forebrain development was disturbed because hmmr was necessary for microtubule-mediated cell adhesion during the morphogenetic movements of neurulation. This study shows for the first time, that CSF in Xenopus laevis is transported via motile cilia and confirmes that dysfunction or absent motile cilia lead to congenital hydrocephalus. Furthermore a novel role for motile cilia during fore- and midbrain morphogenesis was demonstrated. Development of hydrocephalus together with forebrain defects in foxj1 and hmmr morphants implies that cilia-dependent hydrocephalus can result from malformed dorsal midline structures. This study thus provides a basis to establish Xenopus laevis as a model organism to study the development of hydrocephalus caused by primary cilia dyskinesia and by forebrain defects.
The role of serotonin and gap junctions in left-right development of Xenopus laevis
(2011) Beyer, Tina; Blum, Martin
In vertebrates, the correct determination of the left-right (LR) axis is essential for accurate placement of the inner organs, such that the heart points to the left, lung lobation differs between left and right side, spleen and stomach are located on the left,liver on the right body side and the gut coils asymmetrically. Disturbance of this organization can lead to severe impairments of organ function. In the African clawed frog Xenopus laevis, already in four-day old tadpoles asymmetric organ arrangement is visible. This coordinated organ development strictly requires prior Nodal cascade activity in the left lateral plate mesoderm (LPM) in all model organisms examined so far. The initial symmetry breaking event necessary for unilateral induction of Nodal signaling is still under debate. In X. laevis, two models, namely 'ion-flux' and 'cilia-driven leftward fluid flow', were discussed in this context. Leftward flow was first described in the mouse model and later on in fish and rabbit, whereas the 'ion-flux' hypothesis is supported by data derived from chick development. In the present work it was intended to enlighten this putative discrepancy by re-investigating the function of two 'ion-flux' components in context of leftward flow in the model organism X. laevis. First, a link between cell-cell communication via gap junctional communication (GJC)and LR axis establishment was analyzed by using heptanol for general inhibition of channel conductance on the one hand, and single knock-down (KD) of specific subunits on the other hand. Both treatments resulted in absence of the left-sided Nodal cascade. The KD led to shorter GRP cilia when compared to wildtype embryos and loss of bilateral Nodal expression at the GRP margin, respectively. Furthermore, heptanol treatments of stages in which the GRP already has been fully developed also resulted in laterality defects, thus implying a second function of GJC most likely for the signal transfer to the left side. These results indicated a role of GJC in leftward flow establishment and/or post-flow in neurula stages rather than a function in early cleavage stages for LR determination. Second, the early signaling function of the neurotransmitter serotonin (5-HT) was inhibited by over-expression of either a frog or a human receptor ligand binding domain(LBD). In addition, specific KD of a receptor class 3 subunit was performed. Both applications resulted in impaired left-sided marker gene expression and disturbed GRP morphogenesis. Remarkably, marker gene expression of the superficial mesoderm(SM) which gives rise to the GRP during development, was reduced in 5-HT signaling impaired embryos. Very importantly, receptor 3 specific 5-HT signaling was shown to represent a necessary competence factor required for Wnt-dependent axis development in the frog double axis induction assay. Besides the new function of 5-HT during early development, it was further shown that the expression of the SM marker Foxj1 (a master regulator of motile cilia) depended on maternal factors. Based on the work presented here, the following model is proposed: (1) Foxj1 expression is induced maternally, followed by (2) zygotic refinement in post-MBT stages, i.e. inhibition on the ventral and maintenance on the dorsal side. In the organizer region, an interplay of Wnt and 5-HT signaling is required for dorsal development.(3) Cilia driven leftward flow initiate an unknown downstream signal which is transferred to the left LPM. Both events, leftward flow and transfer, require active GJC. (4) A to date unknown signal gets transferred towards the left LPM in a GJC-dependent process and induces the Nodal cascade activity, a prerequisite for proper organ arrangement. Taken together, the data presented in this study indicate that the directed fluid flow in neurula embryos represent the decisive step for symmetry breakage with the 'ion-flux' components being involved in correct flow function.