Browsing by Subject "Neural crest cells"

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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.
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.