Browsing by Subject "Cilia"

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Determination of Laterality in the Rabbit Embryo: Studies on Ciliation and Asymmetric Signal Transfer
(2007) Feistel, Kerstin; Blum, Martin
The midline of the vertebrate embryo plays a pivotal role in the regulation of left-right (LR) asymmetry. In mammals recent interest has focused on a structure situated at the caudal part of the notochord, the posterior notochord (PNC), which is homologous to Kupffer?s vesicle (KV) in fish and the gastrocoel roof plate (GRP) in frog. Despite highly diverging embryonic architecture, the PNC/KV/GRP is the site where motile monocilia set up a directional fluid flow, an event indispensable for the generation of LR asymmetry. Signals created at the PNC/KV/GRP need to be transferred to the periphery of the embryo, where they initiate the left-specifying program in the left lateral plate mesoderm (LPM). In this study morphogenesis and ciliogenesis of the notochordal plate as well as the signaling processes between midline and LPM were studied in the rabbit embryo. Rabbit development progresses through a flat blastodisc phase and represents the typical mode of mammalian embryogenesis. Transcription of ciliary marker genes, the first sign of beginning ciliogenesis, initiated in Hensen?s node and persisted in the nascent notochord. Cilia emerged on cells leaving Hensen?s node anteriorly to form the notochordal plate. Cilia lengthened to about 5µm and polarized from an initially central position to the posterior pole of cells. Electron microscopic analysis revealed 9+0 and 9+2 cilia and a novel 9+4 axoneme intermingled in a salt-and-pepper-like fashion. These data showed that the ciliogenic gene program essential for laterality determination is conserved at the midline of the rabbit embryo. The present study also provided evidence that initiation as well as repression of the Nodal cascade crucially depended on communication between midline and lateral plate (LP). Separation of LP tissue from the midline before, during and after the 2 somite stage demonstrated that signals from the PNC induced and maintained the competence of LPM to express Nodal. Signals from the midline were necessary after the 2 somite stage to maintain a right-sided identity, i.e. absence of Nodal expression. Gap-junction-dependent intercellular communication (GJC) was shown to play a central role in this process. Previously, GJC had been involved in LR axis determination in cleavage stage frog embryos and early blastodisc stages in chick. This study for the first time demonstrates the role of GJC in mammalian embryos. GJs regulate the signaling between midline and periphery: permeable gap junctions were required specifically at the 2 somite stage to repress Nodal induction in the right LPM, whereas closed GJs were a prerequisite for Nodal signaling on the left side. Establishment of the right-sided fate depended on FGF8, the signaling of which was regulated by the opening status of GJs. A 3-step model is proposed for symmetry breakage and induction of the LR signaling cascade in vertebrates: (1) Nodal protein synthesized at the lateral edges of the PNC diffuses bilaterally and confers competence for the induction of the Nodal cascade to the LPM, (2) at the same time the left-specific cascade is actively repressed by action of the GJC/FGF8 module, and (3) following the onset of leftward flow at the PNC repression gets released specifically on the left side at the 2 somite stage, presumably by transient inhibition of GJC. This model not only is consistent with the presented data, but also with published work in other model organisms.
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.
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.