Browsing by Subject "Neurulation"

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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.
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 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.