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2
Fett, Mareike. "Funktionelle Analyse von Parkin im Zebrafisch." Diss., lmu, 2010. http://nbn-resolving.de/urn:nbn:de:bvb:19-123597.
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Krawitz, Peter. "Funktionelle Analyse der Signalpeptidpeptidase Familie im Zebrafisch." Diss., lmu, 2009. http://nbn-resolving.de/urn:nbn:de:bvb:19-96745.
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Wiest, Waltraud. "Genetischer Screen nach Thymusmutanten im Zebrafisch (Danio rerio)." [S.l. : s.n.], 2001. http://www.bsz-bw.de/cgi-bin/xvms.cgi?SWB9624821.
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Klier, Ina. "Die Untersuchung der menschlichen Kaliumkanalfamilie KCNQ im Modelltier Zebrafisch." Diss., lmu, 2010. http://nbn-resolving.de/urn:nbn:de:bvb:19-117370.
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Hans, Stefan. "Eine Charakterisierung cis-regulatorischer Elemente des Zebrafisch deltaD-Gens." [S.l. : s.n.], 2001. http://deposit.ddb.de/cgi-bin/dokserv?idn=962759805.
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Lourenco, da Conceicao Luz Marta. "Cellular mechanisms involved in Wnt8 distribution and function in zebrafish neurectoderm patterning." Doctoral thesis, Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2008. http://nbn-resolving.de/urn:nbn:de:bsz:14-ds-1228815553128-55176.
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Wnt proteins have key roles in patterning of multicellular animals, acting at a distance from their sites of production. However, it is not well understood how these molecules propagate. This question has become even more puzzling by the discovery that Wnts harbour post-translational lipid-modifications, which enhance association with membranes and may therefore limit propagation by simple diffusion in an aqueous environment. The cellular mechanisms involved in Wnt propagation are largely unknown for vertebrate organisms. Here, I discuss my findings on the cellular localization of zebrafish Wnt8, as an example of a vertebrate Wnt. Wnt8 is a key signal for positioning the midbrain-hindbrain brain boundary (MHB) organizer along the anterior-posterior axis of the developing brain in vertebrates. However, it is not clear how this protein propagates from its source, the blastoderm margin, to the target cells, in the prospective neural plate. For this purpose, I have analysed a biologically active, fluorescently tagged Wnt8 in live zebrafish embryos. Wnt8 was present in live tissue in membrane associated punctate structures. In Wnt8 expressing cells these puncta localise to filopodial cellular processes, from which the protein is released to neighbouring cells. This filopodial release requires posttranslational palmitoylation. Although palmitoylation-defective Wnt8 retains auto- and juxtacrine signaling activity, it fails to signal over a long-range. Additionally, this Wnt8 palmitoylation is necessary for regulation of its neural plate target genes. These results suggest that vertebrate Wnt proteins use cell-to-cell contact through filopodia as a shortrange propagation mechanism while released palmitoylated Wnt is required for longrange signaling activity. Furthermore, I show that a Wnt8 receptor, Frizzled9 can negatively influence Wnt8 propagation and signaling range. Finally, I was able to determine the presence of an endogenous Wnt8 gradient in the neurectoderm. I discuss these findings in the context of Wnt8 signaling function in mediating anterior-posterior patterning during early brain development.
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Weber, Michael. "All-Optical 4D In Vivo Monitoring And Manipulation Of Zebrafish Cardiac Conduction." Doctoral thesis, Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2015. http://nbn-resolving.de/urn:nbn:de:bsz:14-qucosa-166647.
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The cardiac conduction system is vital for the initiation and maintenance of the heartbeat. Over the recent years, the zebrafish (Danio rerio) has emerged as a promising model organism to study this specialized system. The embryonic zebrafish heart’s unique accessibility for light microscopy has put it in the focus of many cardiac researchers.
However, imaging cardiac conduction in vivo remained a challenge. Typically, hearts had to be removed from the animal to make them accessible for fluorescent dyes and electrophysiology. Furthermore, no technique provided enough spatial and temporal resolution to study the importance of individual cells in the myocardial network.
With the advent of light sheet microscopy, better camera technology, new fluorescent reporters and advanced image analysis tools, all-optical in vivo mapping of cardiac conduction is now within reach. In the course of this thesis, I developed new methods to image and manipulate cardiac conduction in 4D with cellular resolution in the unperturbed zebrafish heart.
Using my newly developed methods, I could detect the first calcium sparks and reveal the onset of cardiac automaticity in the early heart tube. Furthermore, I could visualize the 4D cardiac conduction pattern in the embryonic heart and use it to study component-specific calcium transients. In addition, I could test the robustness of embryonic cardiac conduction under aggravated conditions, and found new evidence for the presence of an early ventricular pacemaker system. My results lay the foundation for novel, non-invasive in vivo studies of cardiac function and performance.
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Brand, Michael, Jan Kaslin, Stefan Hans, Julia Ganz, and Oliviera-Carlos Vanessa de. "Notch Receptor Expression in Neurogenic Regions of the Adult Zebrafish Brain." Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2015. http://nbn-resolving.de/urn:nbn:de:bsz:14-qucosa-189160.
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The adult zebrash brain has a remarkable constitutive neurogenic capacity. The regulation and maintenance of its adult neurogenic niches are poorly understood. In mammals, Notch signaling is involved in stem cell maintenance both in embryonic and adult CNS. To better understand how Notch signaling is involved in stem cell maintenance during adult neurogenesis in zebrafish we analysed Notch receptor expression in five neurogenic zones of the adult zebrafish brain. Combining proliferation and glial markers we identified several subsets of Notch receptor expressing cells. We found that 90 [Formula: see text] of proliferating radial glia express notch1a, notch1b and notch3. In contrast, the proliferating non-glial populations of the dorsal telencephalon and hypothalamus rarely express notch3 and about half express notch1a/1b. In the non-proliferating radial glia notch3 is the predominant receptor throughout the brain. In the ventral telencephalon and in the mitotic area of the optic tectum, where cells have neuroepithelial properties, notch1a/1b/3 are expressed in most proliferating cells. However, in the cerebellar niche, although progenitors also have neuroepithelial properties, only notch1a/1b are expressed in a high number of PCNA [Formula: see text] cells. In this region notch3 expression is mostly in Bergmann glia and at low levels in few PCNA [Formula: see text] cells. Additionally, we found that in the proliferation zone of the ventral telencephalon, Notch receptors display an apical high to basal low gradient of expression. Notch receptors are also expressed in subpopulations of oligodendrocytes, neurons and endothelial cells. We suggest that the partial regional heterogeneity observed for Notch expression in progenitor cells might be related to the cellular diversity present in each of these neurogenic niches.
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Gundlach, Jule [Verfasser]. "Charakterisierung von Olfactomedin im in vivo Modellorganismus Zebrafisch / Jule Gundlach." Ulm : Universität Ulm, 2019. http://d-nb.info/1201603269/34.
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Quesada, Hernandez Elena. "The Role of Cell Division Orientation during Zebrafish Early Development." Doctoral thesis, Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2011. http://nbn-resolving.de/urn:nbn:de:bsz:14-qucosa-64735.
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The development of multicellular organisms is dependent on the tight coordination between tissue growth and morphogenesis. The stereotypical orientation of cell divisions has been proposed to be a fundamental mechanism by which proliferating and growing tissues take shape.
However, the actual contribution of stereotypical cell division orientation (SDO) to tissue morphogenesis is unclear. In zebrafish, cell divisions with stereotypical orientation have been implicated in both body axis elongation and neural rod formation, although there is little direct evidence for a critical function of SDO in either of these processes.
Making use of extended time-lapse, multi-photon microscopy and a careful three-dimensional analysis of cell division orientation, we show that SDO is required for neural rod midline formation during neurulation, but dispensable for body axis elongation during gastrulation. Our data indicate that SDO during both gastrulation and neurulation is dependent on the non-canonical Wnt receptor Frizzled 7 (Fz7), and that interfering with cell division orientation leads to severe defects in neural rod midline formation, but not body axis elongation.
These findings suggest a novel function for Fz7 controlled cell division orientation in neural rod midline formation during neurulation. They also shed new light on the field of cell division orientation by uncoupling it from tissue elongation.
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Burkhardt, Markus. "Electron multiplying CCD – based detection in Fluorescence Correlation Spectroscopy and measurements in living zebrafish embryos." Doctoral thesis, Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2010. http://nbn-resolving.de/urn:nbn:de:bsz:14-qucosa-61021.
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Fluorescence correlation spectroscopy (FCS) is an ultra-sensitive optical technique to investigate the dynamic properties of ensembles of single fluorescent molecules in solution. It is in particular suited for measurements in biological samples. High sensitivity is obtained by employing confocal microscopy setups with diffraction limited small detection volumes, and by using single-photon sensitive detectors, for example avalanche photo diodes (APD). However, fluorescence signal is hence typically collected from a single focus position in the sample only, and several measurements at different positions have to be performed successively. To overcome the time-consuming successive FCS measurements, we introduce electron multiplying CCD (EMCCD) camera-based spatially resolved detection for FCS. With this new detection method, multiplexed FCS measurements become feasible. Towards this goal, we perform FCS measurements with two focal volumes. As an application, we demonstrate spatial cross-correlation measurements between the two detection volumes, which allow to measure calibration-free diffusion coefficients and direction-sensitive processes like molecular flow in microfluidic channels. FCS is furthermore applied to living zebrafish embryos, to investigate the concentration gradient of the morphogen fibroblast growth factor 8 (Fgf8). It is shown by one-focus APD-based and two-focus EMCCD-based FCS, that Fgf8 propagates largely by random diffusion through the extracellular space in developing tissue. The stable concentration gradient is shown to arise from the equilibrium between a local morphogen production and the sink function of the receiving cells by receptor-mediated removal from the extracellular space. The study shows the applicability of FCS to whole model organisms. Especially in such dynamically changing systems in vivo, the perspective of fast parallel FCS measurements is of great importance. In this work, we exemplify parallel, spatially resolved FCS by utilizing an EMCCD camera. The approach, however, can be easily adapted to any other class of two-dimensional array detector. Novel generations of array detectors might become available in the near future, so that multiplexed spatial FCS could then emerge as a standard extension to classical one-focus FCSFluoreszenz-Korrelations-Spektroskopie (FCS) ist eine hochempfindliche optische Methode, um die dynamischen Eigenschaften eines Ensembles von einzelnen, fluoreszierenden Molekülen in Lösung zu erforschen. Sie ist insbesondere geeignet für Messungen in biologischen Proben. Die hohe Empfindlichkeit wird erreicht durch Verwendung konfokaler Mikroskop-Aufbauten mit beugungsbegrenztem Detektionsvolumen, und durch Messung der Fluoreszenz mit Einzelphotonen-empfindlichen Detektoren, zum Beispiel Avalanche-Photodioden (APD). Dadurch wird das Fluoreszenzsignal allerdings nur von einer einzelnen Fokusposition in der Probe eingesammelt, und mehrfache Messungen an verschiedenen Positionen in der Probe müssen nacheinander durchgeführt werden. Um die zeitaufwendigen, aufeinanderfolgenden FCS-Einzelmessungen zu überwinden, entwickeln wir in dieser Arbeit Elektronenvervielfachungs-CCD (EMCCD) Kamera-basierte räumlich aufgelöste Detektion für FCS. Mit dieser neuartigen Detektionsmethode werden Multiplex-FCS Messungen möglich. Darauf abzielend führen wir FCS Messungen mit zwei Detektionsvolumina durch. Als Anwendung nutzen wir die räumliche Kreuzkorrelation zwischen dem Signal beider Fokalvolumina. Sie ermöglicht die kalibrationsfreie Bestimmung von Diffusionskoeffizienten und die Messung von gerichteter Bewegung, wie zum Beispiel laminarem Fluss in mikrostrukturierten Kanälen. FCS wird darüber hinaus angewendet auf Messungen in lebenden Zebrafischembryonen, um den Konzentrationsgradienten des Morphogens Fibroblasten-Wachstumsfaktor 8 (Fgf8) zu untersuchen. Mit Hilfe von APD-basierter ein-Fokus FCS und EMCCD-basierter zwei-Fokus FCS zeigen wir, dass Fgf8 hauptsächlich frei diffffundiert im extrazellulären Raum des sich entwickelnden Embryos. Der stabile Konzentrationsgradient entsteht durch ein Gleichgewicht von lokaler Morphogenproduktion und globalem Morphogenabbau durch Rezeptor vermittelte Entfernung aus dem extrazellulären Raum. Die Studie zeigt die Anwendbarkeit von FCS in ganzen Modell-Organismen. Gerade in diesen sich dynamisch ändernden Systemen in vivo ist die Perspektive schneller, paralleler FCS-Messungen von großer Bedeutung. In dieser Arbeit wird räumlich aufgelöste FCS am Beispiel einer EMCCD Kamera durchgeführt. Die Herangehensweise ist jedoch einfach übertragbar auf jede andere Art von zwei-dimensionalem Flächendetektor. Neuartige Flächendetektoren könnten in naher Zukunft verfügbar sein. Dann könnte räumlich aufgelöste Multiplex-FCS eine standardisierte Erweiterung zur klassischen ein-Fokus FCS werden
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Brand, Michael, Jan Kaslin, Stefan Hans, Julia Ganz, and Oliviera-Carlos Vanessa de. "Notch Receptor Expression in Neurogenic Regions of the Adult Zebrafish Brain." PLoS, 2013. https://tud.qucosa.de/id/qucosa%3A29059.
Full textAbstract:
The adult zebrash brain has a remarkable constitutive neurogenic capacity. The regulation and maintenance of its adult neurogenic niches are poorly understood. In mammals, Notch signaling is involved in stem cell maintenance both in embryonic and adult CNS. To better understand how Notch signaling is involved in stem cell maintenance during adult neurogenesis in zebrafish we analysed Notch receptor expression in five neurogenic zones of the adult zebrafish brain. Combining proliferation and glial markers we identified several subsets of Notch receptor expressing cells. We found that 90 [Formula: see text] of proliferating radial glia express notch1a, notch1b and notch3. In contrast, the proliferating non-glial populations of the dorsal telencephalon and hypothalamus rarely express notch3 and about half express notch1a/1b. In the non-proliferating radial glia notch3 is the predominant receptor throughout the brain. In the ventral telencephalon and in the mitotic area of the optic tectum, where cells have neuroepithelial properties, notch1a/1b/3 are expressed in most proliferating cells. However, in the cerebellar niche, although progenitors also have neuroepithelial properties, only notch1a/1b are expressed in a high number of PCNA [Formula: see text] cells. In this region notch3 expression is mostly in Bergmann glia and at low levels in few PCNA [Formula: see text] cells. Additionally, we found that in the proliferation zone of the ventral telencephalon, Notch receptors display an apical high to basal low gradient of expression. Notch receptors are also expressed in subpopulations of oligodendrocytes, neurons and endothelial cells. We suggest that the partial regional heterogeneity observed for Notch expression in progenitor cells might be related to the cellular diversity present in each of these neurogenic niches.
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Carvalho, Lara. "The role of yolk syncytial layer and blastoderm movements during gastrulation in zebrafish." Doctoral thesis, Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2008. http://nbn-resolving.de/urn:nbn:de:bsz:14-ds-1200566640735-93186.
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During gastrulation, a set of highly coordinated morphogenetic movements creates the shape and internal organization of the embryo. In teleostean fishes, these morphogenetic movements involve not only the embryonic progenitor cells (deep cells) but also two extra-embryonic tissues: an outer sheet of epithelial cells (EVL) and a yolk syncytial layer (YSL). Epiboly is characterized by the spreading of the blastoderm (deep cells and EVL) to cover the large yolk cell, whereas convergence and extension leads, respectively, to mediolateral narrowing and anteroposterior elongation of the embryo. Recent studies have shown that the nuclei of the YSL undergo epiboly and convergence and extension movements similarly to the overlying deep cells, suggesting that these tissues interact during gastrulation. However, it is so far not clear whether and how the movements of YSL nuclei and deep cells influence each other. In the first part of this thesis, the convergence and extension movement of YSL nuclei was quantitatively compared to the movement of the overlying mesendodermal progenitor (or “hypoblast)” cells. This revealed that, besides the similarity in the overall direction of movement, YSL nuclei and hypoblast cell movements display differences in speed and directionality. Next, the interaction between YSL and hypoblast was addressed. The movement of the blastoderm was analyzed when YSL nuclei movement was impaired by interfering with the YSL microtubule cytoskeleton. We found that YSL and blastoderm epiboly were strongly reduced, while convergence and extension were only mildly affected, suggesting that YSL microtubules and YSL nuclei movement are required for epiboly, but not essential for convergence and extension of the blastoderm. We also addressed whether blastodermal cells can influence YSL nuclei movement. In maternal-zygotic one-eyed pinhead (MZoep) mutant embryos, which lack hypoblast cells, YSL nuclei do not undergo proper convergence movement. Moreover, transplantation of wild type hypoblast cells into these mutants locally rescued the YSL nuclei convergence phenotype, indicating that hypoblast cells can control the movement of YSL nuclei. Finally, we propose that the hypoblast influences YSL nuclei movement as a result of shape changes caused by the collective movement of cells, and that this process requires the adhesion molecule E-cadherin.
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Lourenco, da Conceicao Luz Marta. "Cellular mechanisms involved in Wnt8 distribution and function in zebrafish neurectoderm patterning." Doctoral thesis, Technische Universität Dresden, 2007. https://tud.qucosa.de/id/qucosa%3A23716.
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Wnt proteins have key roles in patterning of multicellular animals, acting at a distance from their sites of production. However, it is not well understood how these molecules propagate. This question has become even more puzzling by the discovery that Wnts harbour post-translational lipid-modifications, which enhance association with membranes and may therefore limit propagation by simple diffusion in an aqueous environment. The cellular mechanisms involved in Wnt propagation are largely unknown for vertebrate organisms. Here, I discuss my findings on the cellular localization of zebrafish Wnt8, as an example of a vertebrate Wnt. Wnt8 is a key signal for positioning the midbrain-hindbrain brain boundary (MHB) organizer along the anterior-posterior axis of the developing brain in vertebrates. However, it is not clear how this protein propagates from its source, the blastoderm margin, to the target cells, in the prospective neural plate. For this purpose, I have analysed a biologically active, fluorescently tagged Wnt8 in live zebrafish embryos. Wnt8 was present in live tissue in membrane associated punctate structures. In Wnt8 expressing cells these puncta localise to filopodial cellular processes, from which the protein is released to neighbouring cells. This filopodial release requires posttranslational palmitoylation. Although palmitoylation-defective Wnt8 retains auto- and juxtacrine signaling activity, it fails to signal over a long-range. Additionally, this Wnt8 palmitoylation is necessary for regulation of its neural plate target genes. These results suggest that vertebrate Wnt proteins use cell-to-cell contact through filopodia as a shortrange propagation mechanism while released palmitoylated Wnt is required for longrange signaling activity. Furthermore, I show that a Wnt8 receptor, Frizzled9 can negatively influence Wnt8 propagation and signaling range. Finally, I was able to determine the presence of an endogenous Wnt8 gradient in the neurectoderm. I discuss these findings in the context of Wnt8 signaling function in mediating anterior-posterior patterning during early brain development.
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Steiner, Rahel Elisabeth [Verfasser], and Martin [Akademischer Betreuer] Moser. "Der Einfluss von BMPER und Tsg auf die arteriovenöse Differenzierung beim Zebrafisch." Freiburg : Universität, 2020. http://d-nb.info/1222436353/34.
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Ludwig, Tim Alexander [Verfasser]. "Der Zebrafisch als Modellorganismus für den Knockdown von Palladin / Tim Alexander Ludwig." Greifswald : Universitätsbibliothek Greifswald, 2014. http://d-nb.info/1059782014/34.
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Zeller, René. "Whole-mount-in-situ-Hybridisierungsscreen im Zebrafisch von homologen krankheitsinvolvierter Gene im Menschen." [S.l.] : [s.n.], 2003. http://www.diss.fu-berlin.de/2003/76/index.html.
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Renz, Marc Andreas. "The role of CCM proteins inβ1 Integrin-Klf2-Egfl7-mediated angiogenesis." Doctoral thesis, Humboldt-Universität zu Berlin, Lebenswissenschaftliche Fakultät, 2015. http://dx.doi.org/10.18452/17386.
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Angiogenese ist entscheidend für die meisten physiologische Prozesse und viele pathologische Umstände. Dabei wird Angiogenese durch die Interaktion zwischen der extrazellulären Matrix (ECM) und endothelialen Zellen reguliert. Während der kardiovaskulären Entwicklung im Zebrafisch fördert Klf2, ein blutstrom-sensitiver Transkriptionsfaktor, die VEGF-abhängige Angiogenese. Der Mechanismus, bei dem biophysikalische Reize die Klf2 Expression regulieren und Angiogenese kontrollieren, ist größtenteils unbekannt. In meiner Studie zeige ich, dass erhöhte klf2 mRNA Expression den molekularen und morphogenetischen kardiovaskulären Defekten in Zebrafisch ccm Mutanten zugrundeliegen. Desweiteren zeige ich, dass diese Defekte durch verstärkte egfl7-Expression und Angiogenese vermittelt werden. Meine Studie zeigt ausserdem, dass die Klf2-Expression unabhängig vom Blutstrom durch den Extrazellularmatrix-bindenden Rezeptor beta1 Integrin reguliert wird. Der CCM-Protein-Komplex, zusammen mit dem ihm verbundenden Integrin-regulierenden Protein ICAP-1 verhindert ein verstärktes Angiogenese-Signal in endothelialen Zellen, indem es die beta1 Integrin-abhängige Klf2 Expression begrenzt. Zusammenfassend zeigt meine Arbeit einen neuen beta1 Integrin-Klf2-Egfl7 Signalweg, der durch zerebrale kavernöse malformations (CCM) Proteine reguliert wirdAngiogenesis is critical to most physiological processes and many pathological conditions. This process is controlled by physical interactions between the extracellular matrix (ECM) and endothelial cells. Klf2, a blood flow–sensitive transcription factor, promotes VEGF-dependent angiogenesis during zebrafish cardiovascular development. However, the mechanism by which biophysical stimuli regulate Klf2 expression and control angiogenesis remains largely unknown. In my study, I show that elevated klf2 mRNA levels underlie the molecular and morphogenetic cardiovascular defects in zebrafish ccm mutants. Furthermore, I demonstrate that these defects are mediated by enhanced egfl7 expression and angiogenesis signaling. My study also revealed that Klf2 expression is regulated by the extracellular matrix-binding receptor beta1 integrin in the absence of blood flow. The CCM protein complex and its associated beta1 integrin-regulatory protein ICAP-1 prevents increased angiogenesis signaling in endothelial cells by limiting beta1 integrin-mediated expression of Klf2. ln sum, my work uncovered a novel beta1 integrin-Klf2-Egfl7 signaling pathway, which is regulated by the cerebral cavernous malformations (CCM) proteins.
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Gerstner, Norman. "Endocytic Modulation of Developmental Signaling during Zebrafish Gastrulation." Doctoral thesis, Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2014. http://nbn-resolving.de/urn:nbn:de:bsz:14-qucosa-156636.
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Biological information processing in living systems like cells, tissues and organs critically depends on the physical interactions of molecular signaling components in time and space. How endocytic transport of signaling molecules contributes to the regulation of developmental signaling in the complex in vivo environment of a developing organism is not well understood.
In a previously performed genome-wide screen on endocytosis, several genes have been identified, that selectively regulate transport of signaling molecules to different types of endosomes, without disrupting endocytosis. My PhD thesis work provides the first functional in vivo characterization of one of these candidate genes, the novel, highly conserved Rab5 effector protein P95 (PPP1R21). Cell culture studies suggest that P95 is a novel endocytic protein important to maintain the balance of distinct endosomal sub-populations and potentially regulates the sorting of signaling molecules between them (unpublished work, Zerial lab).
The scientific evidence presented in this study demonstrates that zebrafish P95 is essential for early zebrafish embryogenesis. Both, knockdown and overexpression of zebrafish P95 compromise accurate morphogenetic movements and patterning of the zebrafish gastrula, showing that P95 functions during zebrafish gastrulation. P95 is functionally required to maintain signaling activity of signaling pathways that control embryonic patterning, in particular for WNT/β-catenin signaling activity. Knockdown of zebrafish P95 amplifies the recruitment of β-catenin to early endosomes, which correlates with the limitation of β-catenin to translocate to the nucleus and function as transcriptional activator.
The obtained results suggest that zebrafish P95 modulates the cytoplasmic pools of β-catenin in vivo, via endosomal transport of β-catenin. In conclusion, the data presented in this thesis work provides evidence that the cytoplasm-to-nucleus shuttling of β-catenin is modulated by endocytic trafficking of β-catenin in vivo. We propose the endocytic modulation of β-catenin cytoplasm-to-nucleus trafficking as potential new mechanism to fine-tune the functional output of WNT/β-catenin signaling during vertebrate gastrulation.
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Suzzi, Stefano. "Loss of lrrk2 impairs dopamine catabolism, cell proliferation, and neuronal regeneration in the zebrafish brain." Doctoral thesis, Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2017. http://nbn-resolving.de/urn:nbn:de:bsz:14-qucosa-229200.
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Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are a major cause of Parkinson’s disease (PD), which is why modelling PD by replicating effects in animal models attracts great interest. However, the exact mechanisms of pathogenesis are still unclear. While a gain-of-function hypothesis generally receives consensus, there is evidence supporting an alternative loss-of-function explanation. Yet, neither overexpression of the human wild-type LRRK2 protein or its pathogenic variants, nor Lrrk2 knockout recapitulates key aspects of human PD in rodent models. Furthermore, there is conflicting evidence from morpholino knockdown studies in zebrafish regarding the extent of zygotic developmental abnormalities.
Because reliable null mutants may be useful to infer gene function, and because the zebrafish is a more tractable laboratory vertebrate system than rodents to study disease mechanisms in vivo, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein-9 nuclease (Cas9) genomic editing was used to delete the ~60-kbp-long zebrafish lrrk2 locus containing the entire open reading frame. Constitutive removal of both the maternal and the zygotic lrrk2 function (mzLrrk2 individuals) causes a pleomorphic phenotype in the larval brain at 5 days post-fertilisation (dpf), including increased cell death, delayed myelination, and reduced and morphologically abnormal microglia/leukocytes. However, the phenotype is transient, spontaneously attenuating or resolving by 10 dpf, and the mutants are viable and fertile as adults. These observations are mirrored by whole-larva transcriptome data, revealing a more than eighteen-fold drop in the number of differentially expressed genes in mzLrrk2 larvae from 5 to 10 dpf.
Additionally, analysis of spontaneous swimming activity shows hypokinesia as a predictor of Lrrk2 protein deficiency in larvae, but not in adult fish.
Because the catecholaminergic (CA) neurons are the main clinically relevant target of PD in humans, the CA system of larvae and adult fish was analysed on both cellular and metabolic level. Despite an initial developmental delay at 5 dpf, the CA system is structurally intact at 10 dpf and later on in adult fish aged 6 and 11 months. However, monoamine oxidase (Mao)-dependent degradation of biogenic amines, including dopamine, is increased in older fish, possibly suggesting impaired synaptic transmission or a leading cause of cell damage in the long term.
Furthermore, decreased mitosis rate in the larval brain was found, in the anterior portion only at 5 dpf, strongly and throughout the whole organ at 10 dpf. Conceivably, lrrk2 may have a more general role in the control of cell proliferation during early development and a more specialised one in the adult stage, possibly conditional, for example upon brain damage. Because the zebrafish can regenerate lost neurons, it represents a unique opportunity to elucidate the endogenous processes that may counteract neurodegeneration in a predisposing genetic background. To this aim, the regenerative potential of the adult telencephalon upon stab injury was tested in mzLrrk2 fish. Indeed, neuronal proliferation was reduced, suggesting that a complete understanding of Lrrk2 biology may not be fully appreciated without recreating challenging scenarios.
To summarise, the present results demonstrate that loss of lrrk2 has an early effect on zebrafish brain development that is later often compensated. Nonetheless, perturbed aminergic catabolism, and specifically increased Mao-dependent aminergic degradation, is reported for the first time in a LRRK2 knockout model. Furthermore, a link between Lrrk2 and the control of basal cell proliferation in the brain, which may become critical under challenging circumstances such as brain injury, is proposed. Future directions should aim at exploring which brain cell types are specifically affected by the mzLrrk2 hypoproliferative phenotype and the resulting consequences on a circuitry level, particularly in very old fish (i.e., over 2 years of age).
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Kolanczyk, Maria Elzbieta. "Signaling mechanisms and developmental function of fibroblast growth factor receptors in zebrafish." Doctoral thesis, Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2009. http://nbn-resolving.de/urn:nbn:de:bsz:14-ds-1242722157657-12154.
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Fibroblast growth factor (Fgf) signaling plays multiple inductive roles during development of vertebrates (Itoh 2007). Some Fgfs, such as Fgf8, are locally secreted and signal over a long range to provide positional information in the target tissue (Scholpp and Brand 2004). Fgf ligands signal in a receptor-dependent manner via tyrosine kinase receptors, four of which have been so far identified. Fgf8 signaling was shown to depend both on receptor activation as well as endocytosis. The specificity of Fgf ligands and receptors as well as the function of receptors in the control of the Fgf signaling range have been, however, largely unclear. In this study, we show that the putative Fgf8 receptor Fgfr1 is duplicated in zebrafish and that it acts redundantly in the formation of the posterior mesoderm. Also, in overexpression studies we confirm the notion that receptor endocytosis influences Fgf8 signaling range. Through TILLING mutant recovery and morpholino knockdown studies we also show that Fgfr2 is required for growth and skeletal development in zebrafish, whereas Fgfr4 is required for pectoral fin specification and growth.
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Carvalho, Lara. "The role of yolk syncytial layer and blastoderm movements during gastrulation in zebrafish." Doctoral thesis, Technische Universität Dresden, 2007. https://tud.qucosa.de/id/qucosa%3A25041.
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During gastrulation, a set of highly coordinated morphogenetic movements creates the shape and internal organization of the embryo. In teleostean fishes, these morphogenetic movements involve not only the embryonic progenitor cells (deep cells) but also two extra-embryonic tissues: an outer sheet of epithelial cells (EVL) and a yolk syncytial layer (YSL). Epiboly is characterized by the spreading of the blastoderm (deep cells and EVL) to cover the large yolk cell, whereas convergence and extension leads, respectively, to mediolateral narrowing and anteroposterior elongation of the embryo. Recent studies have shown that the nuclei of the YSL undergo epiboly and convergence and extension movements similarly to the overlying deep cells, suggesting that these tissues interact during gastrulation. However, it is so far not clear whether and how the movements of YSL nuclei and deep cells influence each other. In the first part of this thesis, the convergence and extension movement of YSL nuclei was quantitatively compared to the movement of the overlying mesendodermal progenitor (or “hypoblast)” cells. This revealed that, besides the similarity in the overall direction of movement, YSL nuclei and hypoblast cell movements display differences in speed and directionality. Next, the interaction between YSL and hypoblast was addressed. The movement of the blastoderm was analyzed when YSL nuclei movement was impaired by interfering with the YSL microtubule cytoskeleton. We found that YSL and blastoderm epiboly were strongly reduced, while convergence and extension were only mildly affected, suggesting that YSL microtubules and YSL nuclei movement are required for epiboly, but not essential for convergence and extension of the blastoderm. We also addressed whether blastodermal cells can influence YSL nuclei movement. In maternal-zygotic one-eyed pinhead (MZoep) mutant embryos, which lack hypoblast cells, YSL nuclei do not undergo proper convergence movement. Moreover, transplantation of wild type hypoblast cells into these mutants locally rescued the YSL nuclei convergence phenotype, indicating that hypoblast cells can control the movement of YSL nuclei. Finally, we propose that the hypoblast influences YSL nuclei movement as a result of shape changes caused by the collective movement of cells, and that this process requires the adhesion molecule E-cadherin.
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Hu, Bo. "Analysis of cellular drivers of zebrafish heart regeneration by single-cell RNA sequencing and high-throughput lineage tracing." Doctoral thesis, Humboldt-Universität zu Berlin, 2021. http://dx.doi.org/10.18452/23324.
Full textAbstract:
Das Herz eines Zebrafishs ist bemerkenswert, da es sich nach einer Verletzung vollständig regenerieren kann. Der Regenerationsprozess wird von Fibrose begleitet - der Bildung von überschüssigem Gewebe der extrazellulären Matrix (ECM). Anders als bei Säugetieren ist die Fibrose im Zebrafish nur transient. Viele Signalwege wurden identifiziert, die an der Herzregeneration beteiligt sind. Allerdings sind die Zelltypen, insbesondere Nicht-Kardiomyozyten, die für die Regulation des Regenerationsprozesses verantwortlich sind, weitgehend unbekannt. In dieser Arbeit haben wir systematisch alle Zelltypen des gesunden und des verletzten Zebrafischherzens mithilfe einer auf Mikrofluidik basierenden Hoch-Durchsatz- Einzelzell-RNA-Sequenzierung bestimmt. Wir fanden eine große Heterogenität von ECM-produzierenden Zellen, einschließlich einer Reihe neuer Fibroblasten, die nach einer Verletzung mit unterschiedlicher Dynamik auftreten. Wir konnten aktivierte Fibroblasten beschreiben und Fibroblasten-Subtypen mit einer pro-regenerativen Funktion identifizieren.
Darüber hinaus haben wir eine Methode entwickelt, um die Transkriptomanalyse und die Rekonstruktion von Zell-Verwandtschaften auf Einzelzellebene zu kombinieren. Unter Verwendung der CRISPR-Cas9-Technologie führten wir zufällige Mutationen in bekannte und ubiquitär transkribierte DNA-Loci während der Embryonalentwicklung von Zebrafischen ein. Diese Mutationen dienten als zellspezifische, permanente und vererbbare “Barcodes”, die zu einem späteren Zeitpunkt erfasst werden konnten. Mit maßgeschneiderten Analysealgorithmen konnten wir dann Stammbäume der sequenzierten Einzelzellen erstellen. Mit dieser neuen Methode haben wir gezeigt, dass im sich regenerierenden Zebrafischherz ECM-produzierende Zellpopulationen entweder mit dem Epi- oder mit dem Endokardium verwandt sind. Zusätzlich entdeckten wir, dass vom Endokardium abgeleitete Zelltypen vom Wnt-Signalweg abhängig sind.The zebrafish heart has the remarkable capacity to fully regenerate after injury. The regeneration process is accompanied by fibrosis - the formation of excess extracellular matrix (ECM) tissue, at the injury site. Unlike in mammals, the fibrosis of the zebrafish heart is only transient. While many pathways involved in heart regeneration have been identified, the cell types, especially non-myocytes, responsible for the regulation of the regenerative process have largely remained elusive. Here, we systematically determined all different cell types of both the healthy and cryo-injured zebrafish heart in its regeneration process using microfluidics based high-throughput single-cell RNA sequencing. We found a considerable heterogeneity of ECM producing cells, including a number of novel fibroblast cell types which appear with different dynamics after injury. We could describe activated fibroblasts that extensively switch on gene modules for ECM production and identify fibroblast sub- types with a pro-regenerative function. Furthermore, we developed a method that is capable of combining transcriptome analysis with lineage tracing on the single-cell level. Using CRISPR-Cas9 technology, we introduced random mutations into known and ubiquitously transcribed DNA loci during the zebrafish embryonic development. These mutations served as cell-unique, permanent, and heritable barcodes that could be captured at a later stage simultaneously with the transcriptome by high-throughput single-cell RNA sequencing. With custom tailored analysis algorithms, we were then able to build a developmental lineage tree of the sequenced single cells. Using this new method, we revealed that in the regenerating zebrafish heart, ECM contributing cell populations derive either from the epi- or the endocardium. Additionally, we discovered in a functional experiment that endocardial derived cell types are Wnt signaling dependent.
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Bock, Sarah [Verfasser]. "Natrium-Kalium-ATPase (atp1a1a.1) : ein essentieller Regulator des kardialen Rhythmus im Zebrafisch / Sarah Bock." Ulm : Universität Ulm, 2018. http://d-nb.info/115193822X/34.
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Kleinert, Henning [Verfasser]. "Charakterisierung von gpr17 und dessen Einfluss auf die Myelinisierung im Zebrafisch (Danio rerio) / Henning Kleinert." Bonn : Universitäts- und Landesbibliothek Bonn, 2018. http://d-nb.info/118872570X/34.
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Herrgen, Leah. "The Regulation of Segmentation Clock Period in Zebrafish." Doctoral thesis, Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2008. http://nbn-resolving.de/urn:nbn:de:bsz:14-ds-1228749843309-64183.
Full textAbstract:
Oscillations are present at many different levels of biological organization. The cell cycle that directs the division of individual cells, the regular depolarization of neurons in the sinu-atrial node which underlies the regular beating of the heart, the circadian rhythms that govern the daily activity cycles of virtually all organisms, and the clocks that make entire populations of fireflies flash on and off in unison feature as prominent examples of biological clocks. During development, biological clocks regulate the patterning of growing tissues, as is the case in vertebrate somitogenesis, and potentially also in vertebrate limb outgrowth and axial segmentation of invertebrate embryos. During vertebrate segmentation, the embryonic axis is subdivided along its anterior-posterior axis into epithelial spheres of cells called somites. This rhythmic process is thought to be driven by a multicellular oscillatory gene network, the so-called segmentation clock. Oscillations of hairy and enhancer of split gene products have been proposed to constitute the core clockwork in individual cells, and these oscillators are coupled to each other by Delta-Notch intercellular signaling. The interaction of the segmentation clock with a posteriorly-moving arrest wavefront then translates the temporal information encoded by the clock into a spatial pattern of segments. In the framework of this Clock and Wavefront model, segment length is determined by both clock period and arrest wavefront velocity. How the period of the segmentation clock is regulated is presently unknown, and understanding the mechanism of period setting might yield insight into the nature and function of the segmentation clock. In this study, two different but complementary approaches were pursued to investigate how period is regulated in the zebrafish segmentation clock. First, it has been reported that zebrafish mind bomb (mib) mutant embryos form somites more slowly than their wt siblings, suggesting that Mib might be implicated in period setting. Mib is an E3 ubiquitin ligase required for ubiquitination and endocytosis of the Notch ligand Delta, and Notch signaling is impaired in mutants with defective Mib. It has been suggested that the mechanistic basis for the requirement of Delta endocytosis in Notch signaling is a need for Delta to enter a particular endocytic compartment, potentially a recycling endosome, in a ubiquitin-dependent manner, where its signaling ability might be established or amplified by an as yet unknown posttranslational modification. In the present study, Delta trafficking through the endocytic pathway was analyzed in the PSM of wt and mib embryos through colocalization studies with endocytic markers. The rationale of this approach was that if Delta gained access to a particular endocytic compartment through Mib-dependent endocytosis, the presence of Delta in this compartment would be expected to be reduced in mutants with defective Mib, thereby revealing the compartment’s identity. However, no qualitative changes in colocalization with different endocytic markers could be detected in mib mutants, and the methods available did not allow for quantification of colocalization in wt or mutant backgrounds. However, Delta colocalized with 13 markers of recycling endosomes, consistent with the hypothesis that these are functionally important in Notch signaling. More refined techniques will be necessary for a quantitative analysis of normal as compared to impaired Delta trafficking. A genetic approach to period regulation proved to be successful for the Drosophila circadian clock, where the identification of period mutants advanced the understanding of the clock’s genetic circuitry. This motivated a screen for period mutants of the segmentation clock, which was carried out by measuring somitogenesis period, segment length and arrest wavefront velocity in a pool of candidate mutants. A subset of Delta-Notch mutants, and embryos treated with a small-molecule inhibitor that impairs Notch signaling, displayed correlated increases in somitogenesis period and segment length, while there was no detectable change in arrest wavefront velocity. Combined, these findings suggested that segmentation clock period is increased in experimental conditions with impaired Delta-Notch signaling. Using a theoretical description of the segmentation clock as an array of coupled phase oscillators, the delay in the coupling and the autonomous frequency of individual cells were estimated from the direction and magnitude of the period changes. The mutants presented here are the first candidates for segmentation clock period mutants in any vertebrate. The nature of the molecular lesions in these mutants, all of which affect genes implicated in intercellular Delta-Notch signaling, suggests that communication between oscillating PSM cells is a key factor responsible for setting the period of the segmentation clock.
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Schötz, Eva-Maria. "Dynamics and Mechanics of Zebrafish Embryonic Tissues." Doctoral thesis, Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2008. http://nbn-resolving.de/urn:nbn:de:swb:14-1191291301268-96071.
Full textAbstract:
Developmental biologists try to elucidate how it is possible for cells, all originating from the same egg, to develop into a variety of highly specialized structures, such as muscles, skin, brain and limbs. What organizes the behavior of these cells, and how can the information encoded in the DNA account for the observed patterns and developmental processes? Cell movements and tissue flow during embryogenesis constitute a beautiful problem of bridging scales: On the microscopic scale, cells are expressing particular genes which determine their identities and also their fate during morphogenesis. These molecular determinants then lead to the macroscopic phenomena of cell movements and tissue arrangements, for which one needs a continuum description in terms of active fluids. Taking into account that the number of cells is fairly small, a complete coarse graining is not possible, and a characterization of both mesoscopic (individual cell motion) and macroscopic (flow) behavior is required for a full description. In the here presented work, a set of different experimental methods was applied to investigate the mechanical and dynamical properties of zebrafish embryonic cells and tissues. This thesis is structured as follows: In chapter 2, we introduce the fundamental concepts that are important for the study of cell motion during zebrafish embryonic development. In chapter 3, the materials and methods applied in this work are described. The experimental results of my thesis-work are presented in chapters 4-8: Chapter 4 concentrates on the physical properties of whole tissues. It is shown that tissues are viscoelastic materials. Tissue viscoelasticity is not a new concept, but this study is the first one to quantify the mechanical properties of tissues that are in actual contact in a developing embryo. In chapter 5, cell rearrangements in culture, such as cell sorting and tissue wetting are discussed. These experiments show that tissue interactions are largely determined by tissue surface and interfacial tensions. In chapter 6, an optical stretcher device is applied to measure, solely by means of laser light, the material properties of individual cells. Hereby it is shown that single cells from the two investigated tissue types differ in their mechano-physical properties. After the study of cell and tissue mechanics, the dynamics of cell migration in three dimensions in tissue aggregates and in developing zebrafish embryos is addressed: In chapter 7, 3D-cell migration in multicellular aggregates is analyzed quantitatively by studying the mean square displacement, cell velocity distribution and velocity autocorrelation. In chapter 8, we study the cell motion within the developing zebrafish embryo. By following the motion of many cells in four dimensions, we are able to generate a velocity flow profile for this cell-flow. Chapter 9 gives a brief summary of the obtained results and an outlook to future projects motivated by the presented study. The final part of this thesis are four appendices. Appendix A contains protocols and additional methods. Appendix B contains several calculations, whose results were used in the main part of this work. Appendix C contains additional data and discussions, which were excluded from the main part due to space limitations. Finally, Appendix D consists of a compact disc with 11 movies and a movie description, which serves as supplemental material to the presented data. (Die Druckexemplare enthalten jeweils eine CD-ROM als Anlagenteil: 650 MB: Movies - Nutzung: Referat Informationsservice der SLUB)
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Kalbhen, Karl Pascal [Verfasser], Dietmar [Gutachter] Fischer, and Olga [Gutachter] Sergeeva. "Molekulare Mechanismen axonaler Regeneration retinaler Ganglienzellen im Zebrafisch / Karl Pascal Kalbhen ; Gutachter: Dietmar Fischer, Olga Sergeeva." Düsseldorf : Universitäts- und Landesbibliothek der Heinrich-Heine-Universität Düsseldorf, 2018. http://d-nb.info/115600778X/34.
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Heß, Isabell. "Zum regulatorischen Code Chorda-spezifischer Enhancer Analyse des E1-Enhancers von sox9a und sox9b im Zebrafisch /." [S.l. : s.n.], 2007. http://nbn-resolving.de/urn:nbn:de:bsz:25-opus-43665.
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Witzel, Sabine. "Local Wnt11 Signalling and its role in coordinating cell behaviour in zebrafish embryos." Doctoral thesis, Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2006. http://nbn-resolving.de/urn:nbn:de:swb:14-1162424627109-87779.
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Wnt11 is a key signalling molecule that regulates cell polarity/migration during vertebrate development and also promotes the invasive behaviour of adult cancer cells. It is therefore essential to understand the mechanisms by which Wnt11 signalling regulates cell behaviour. The process of vertebrate gastrulation provides an excellent developmental system to study Wnt11 function in vivo. It is known that Wnt11 mediates coordinated cell migration during gastrulation via the non-canonical Wnt pathway that shares several components with a the planar cell polarity pathway (PCP) in Drosophila. However, the mechanisms by which these PCP components facilitate Wnt11 function in vertebrates is still unclear. While in Drosophila, the asymmetric localization of PCP components is crucial for the establishment of cell polarity, no asymmetric localization of Wnt11 pathway components have so far been observed in vertebrates. To shed light on the cellular and molecular mechanisms underlying Wnt11 signalling, I developed an assay to visualize Wnt11 activity in vivo using live imaging of Wnt11 pathway components tagged to fluorescent proteins. This allowed me to determine the sub-cellular distribution of these components and to correlate the effect of Wnt11 activity with the behaviour of living embryonic cells. I found that Wnt11 locally accumulates together with its receptor Frizzled7 (Fz7) at sites of cell-cell contacts and locally recruits the intra-cellular signalling mediator Dishevelled (Dsh) to those sites. Monitoring these apparent Wnt11 signalling centres through time-lapse confocal microscopy revealed, that Wnt11 activity locally increases the persistency of cell-cell contacts. In addition, I found that the atypical cadherin Flamingo (Fmi) is required for this process. Fmi accumulates together with Wnt11/Fz7 at sites of cell-cell contact and locally increased cell adhesion, via a mechanism that appears to be independent of known downstream effectors of Wnt11 signalling such as RhoA and Rok2. This study indicates that Wnt11 locally interacts with Fmi and Fz7 to control cell-contact persistency and to facilitate coherent and coordinated cell migration. This provides a novel mechanism of non-canonical Wnt signalling in mediating cell behaviour, which is likely relevant to other developmental systems. (Die Druckexemplare enthalten jeweils eine CD-ROM als Anlagenteil: 50 MB: Movies - Nutzung: Referat Informationsvermittlung der SLUB)
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Scheer, Nico. "Die GAL4-UAS-Methode zur gezielten Genexpression eine kritische Betrachtung ihrer funktionellen Anwendung beim Zebrafisch, Danio rerio /." [S.l. : s.n.], 2000. http://deposit.ddb.de/cgi-bin/dokserv?idn=960755691.
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Kadner, Diana. "Mechanism of cell adhesion at the midbrain-hindbrain neural plate in the teleost Danio rerio." Doctoral thesis, Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2009. http://nbn-resolving.de/urn:nbn:de:bsz:14-qucosa-23142.
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The correct development of multicellular organisms is tightly regulated by intrinsic and extrinsic factors at specific time points. Disturbance on any level of these multiple processes may result in drastic phenotypes or eventually death of the organism.
The midbrain-hindbrain boundary (also termed isthmic organizer) is a region of high interest as well in early as also in later development. The isthmic region carries organizer identity by the expression and subsequent release of FGF8. False patterning events of this region in early developmental stages would therefore display dramatic results over time. As it has been shown that the midbrain-hindbrain boundary (mhb) in the zebrafish is a compartment (or lineage restriction) boundary I tried to understand the underlying molecular mechanism for its correct establishment.
In this work I focused both on embryological, molecular and genetic means to characterize involved molecules and mechanisms. In the first part of the thesis I followed in vivo cell transplantation assays, having started with an unbiased one. Cells of either side the mhb were challenged with this boundary by bringing them into direct cell contact with their ectopic counterpart. In a biased approach, cells overexpressing mRNA of specific candidate genes were transplanted and their clonal distribution in host embryos was analyzed.
In the second part of the thesis I started interfering with specific candidate genes by transiently knocking down their protein translation. The adhesion molecules of the Eph/ephrin class had been shown to restrict cell mixing and thereby creating compartment boundaries in other tissues, such as the hindbrain, in the zebrafish and other organisms. Additionally, we generated several stable genetic mutant lines in cooperation with the Tilling facility at the Max-Planck-Institute. The only acquired potential null mutant ephrinB2bhu2971 was analyzed and characterized further. I observed that a knock down or knock out of only one of the ephrinB2 ligands does not seem to be sufficient for a loss of compartment boundary formation. The combinatory approach of blocking translation of EphrinB2a in ephrinB2bhu2971 mutants gave very complex and interesting phenotypes, which need to be investigated further.
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Martin, Milena Christina [Verfasser]. "Untersuchung von Spen – einer Komponente des Notch Repressorkomplexes – mit Schwerpunkt Herzentwicklung und Kardiomyozytenproliferation im Zebrafisch / Milena Christina Martin." Ulm : Universität Ulm, 2020. http://d-nb.info/1205001859/34.
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Frese, Karen Sonia [Verfasser], and Thomas [Akademischer Betreuer] Wieland. "A2BP1-vermitteltes RNA-Spleißen ist essentiell für die kardiale Funktion im Zebrafisch / Karen Sonia Frese ; Betreuer: Thomas Wieland." Heidelberg : Universitätsbibliothek Heidelberg, 2013. http://d-nb.info/1177809818/34.
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Frese, Karen S. [Verfasser], and Thomas [Akademischer Betreuer] Wieland. "A2BP1-vermitteltes RNA-Spleißen ist essentiell für die kardiale Funktion im Zebrafisch / Karen Sonia Frese ; Betreuer: Thomas Wieland." Heidelberg : Universitätsbibliothek Heidelberg, 2013. http://nbn-resolving.de/urn:nbn:de:bsz:16-heidok-157835.
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Schötz, Eva-Maria. "Dynamics and Mechanics of Zebrafish Embryonic Tissues." Doctoral thesis, Technische Universität Dresden, 2007. https://tud.qucosa.de/id/qucosa%3A23924.
Full textAbstract:
Developmental biologists try to elucidate how it is possible for cells, all originating from the same egg, to develop into a variety of highly specialized structures, such as muscles, skin, brain and limbs. What organizes the behavior of these cells, and how can the information encoded in the DNA account for the observed patterns and developmental processes? Cell movements and tissue flow during embryogenesis constitute a beautiful problem of bridging scales: On the microscopic scale, cells are expressing particular genes which determine their identities and also their fate during morphogenesis. These molecular determinants then lead to the macroscopic phenomena of cell movements and tissue arrangements, for which one needs a continuum description in terms of active fluids. Taking into account that the number of cells is fairly small, a complete coarse graining is not possible, and a characterization of both mesoscopic (individual cell motion) and macroscopic (flow) behavior is required for a full description. In the here presented work, a set of different experimental methods was applied to investigate the mechanical and dynamical properties of zebrafish embryonic cells and tissues. This thesis is structured as follows: In chapter 2, we introduce the fundamental concepts that are important for the study of cell motion during zebrafish embryonic development. In chapter 3, the materials and methods applied in this work are described. The experimental results of my thesis-work are presented in chapters 4-8: Chapter 4 concentrates on the physical properties of whole tissues. It is shown that tissues are viscoelastic materials. Tissue viscoelasticity is not a new concept, but this study is the first one to quantify the mechanical properties of tissues that are in actual contact in a developing embryo. In chapter 5, cell rearrangements in culture, such as cell sorting and tissue wetting are discussed. These experiments show that tissue interactions are largely determined by tissue surface and interfacial tensions. In chapter 6, an optical stretcher device is applied to measure, solely by means of laser light, the material properties of individual cells. Hereby it is shown that single cells from the two investigated tissue types differ in their mechano-physical properties. After the study of cell and tissue mechanics, the dynamics of cell migration in three dimensions in tissue aggregates and in developing zebrafish embryos is addressed: In chapter 7, 3D-cell migration in multicellular aggregates is analyzed quantitatively by studying the mean square displacement, cell velocity distribution and velocity autocorrelation. In chapter 8, we study the cell motion within the developing zebrafish embryo. By following the motion of many cells in four dimensions, we are able to generate a velocity flow profile for this cell-flow. Chapter 9 gives a brief summary of the obtained results and an outlook to future projects motivated by the presented study. The final part of this thesis are four appendices. Appendix A contains protocols and additional methods. Appendix B contains several calculations, whose results were used in the main part of this work. Appendix C contains additional data and discussions, which were excluded from the main part due to space limitations. Finally, Appendix D consists of a compact disc with 11 movies and a movie description, which serves as supplemental material to the presented data. (Die Druckexemplare enthalten jeweils eine CD-ROM als Anlagenteil: 650 MB: Movies - Nutzung: Referat Informationsservice der SLUB)
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Linder, Bastian [Verfasser], and Utz [Akademischer Betreuer] Fischer. "Systemischer Spleißfaktormangel im Zebrafisch Danio rerio – Etablierung und Charakterisierung eines Tiermodells für Retinitis pigmentosa / Bastian Linder. Betreuer: Utz Fischer." Würzburg : Universitätsbibliothek der Universität Würzburg, 2013. http://d-nb.info/1035370956/34.
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Rattka, Manuel [Verfasser]. "Das SPlit ENds (SPEN) Gen – in vivo Analyse eines neuen Kandidatengens der dilatativen Kardiomyopathie im Modellorganismus Zebrafisch / Manuel Rattka." Ulm : Universität Ulm, 2019. http://d-nb.info/1176022296/34.
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Benz, Alexander [Verfasser], and David [Akademischer Betreuer] Hassel. "miR-19b - ein essentieller Regulator der Länge und Form des ventrikulären Aktionspotentials im Zebrafisch / Alexander Benz ; Betreuer: David Hassel." Heidelberg : Universitätsbibliothek Heidelberg, 2016. http://d-nb.info/1180615476/34.
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Janßen, Julia Annika. "In vivo FLIM-FRET as a novel technique to assess cAMP and cGMP in the intact zebrafish heart." Doctoral thesis, Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2018. http://nbn-resolving.de/urn:nbn:de:bsz:14-qucosa-232452.
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Introduction: 23 million patients worldwide suffer from heart failure. These patients depend on cardiac research, because cardiac research enables the development of new therapeutic strategies and –targets. In cardiomyocytes, the compartmentalization of cAMP and cGMP depends on many factors. T-tubuli and PDEs are responsible for the division of cells in microdomains in which localized and specific cAMP and cGMP-signaling occurs. The aim of this thesis was to develop a method to answer the open questions that remain about the physiological and pathophysiological significance of cAMP/cGMP compartmentalization. Methods: I used the zebrafish as a model, because the transparency of zebrafish larvae enabled non-invasive fluorescent imaging in cardiomyocytes in the living animal. I cloned the Fluorescence Resonance Energy Transfer (FRET) sensors EPAC1-camps for cAMP and cGi500 for cGMP and injected them into zebrafish fertilized embryos. Then I used the F0 generation for Fluorescence Lifetime Imaging (FLIM) -FRET-measurements of cAMP and cGMP. Ca2+ is an important downstream mediator of cAMP and cGMP, because Ca2+ regulates cardiac contraction. Therefore, I also cloned the Ca2+ sensor GCaMP6 and used the dye Fluo-4 AM to include intracellular Ca2+ in the imaging. Results: The cloned sensors for cAMP, cGMP and Ca2+ were successfully injected into the zebrafish and showed expression in individual cardiomyocytes. I developed a protocol to mount the living zebrafish embryos and to measure intracellular cAMP and cGMP with FLIM-FRET in vivo with high spatial resolution. I characterized the sensors in their functionality by showing that the sensors react to changes in intracellular concentrations of cAMP and cGMP. The results of this study include evidence that zebrafish have mechanisms that lead to cAMP/cGMP compartmentalization in the absence of T-tubuli, and these mechanisms keep compartmentalization constant even under extreme cAMP or cGMP increasing drug treatment. Furthermore, I imaged intracellular Ca2+ by confocal microscopy and developed a protocol to use Fluo-4 AM for Ca2+ imaging. Conclusion: The method used in this thesis should allow the investigation of subcellular cAMP/cGMP compartmentalization and Ca2+ and to subsequently answer open questions in the field, for example whether a change of cAMP compartmentalization leads to the pathological phenotypes of cardiac disease or if a changed compartmentalization of cAMP in cardiac disease influences Ca2+ concentrations and therefore contraction. Additionally, this method can be used to learn more about cAMP, cGMP und Ca2+ during regeneration in the heart, because the zebrafish cardiomyocytes can regenerateEinleitung: Weltweit sind mehr als 23 Millionen unter Herzinsuffizienz leidende Patienten auf die kardiologische Grundlagenforschung angewiesen, da diese die Voraussetzung für eine bessere Versorgung durch adaptierte und neue Behandlungswege schafft. In Kardiomyozyten hängt die Kompartimentierung von cAMP und cGMP von vielen Faktoren ab. T-Tubuli und PDEs werden unter anderem für die Aufteilung der Zellen in Mikrodomänen, in denen lokalisierte und spezifische cAMP- und cGMP-Signalgebung stattfinden kann, verantwortlich gemacht. Das Ziel dieser Arbeit war die Etablierung einer Methode, mithilfe derer offene Fragen bezüglich der physiologischen und insbesondere der pathophysiologischen Relevanz der cAMP- und cGMP Kompartimentierung beantwortet werden können. Methode: Als Modell diente der Zebrafisch, da die Transparenz von Zebrafisch Embryonen eine nicht-invasive Bildgebung von Fluoreszenz in Kardiomyozyten im lebenden Tier ermöglicht. Dafür klonierte ich die Förster Resonance Energy Transfer (FRET) -Sensoren EPAC1-camps als cAMP-Sensor und cGi500 als cGMP-Sensor und injizierte diese in befruchtete Zebrafisch Embryonen. Anschließend benutzte ich die F0-Generation für Fluorescence Lifetime Imaging (FLIM) -FRET-Messungen von cAMP und cGMP. Da Ca2+ als wichtiger downstream Mediator von cAMP und cGMP die kardiale Kontraktion reguliert, klonierte ich außerdem den Ca2+-Sensor GCaMP6 und benutzte den Farbstoff Fluo-4 AM, um intrazelluläres Ca2+ darzustellen. Ergebnisse: Die klonierten Sensoren für cAMP, cGMP und Ca2+ konnten erfolgreich in den Zebrafisch injiziert werden und zeigten alle Expression in einzelnen Kardiomyozyten. Ich entwickelte ein Protokoll, dass die Fixierung von lebenden Zebrafisch Embryonen und nachfolgender Bildgebung von cAMP und cGMP mit hoher zellulärer Auflösung mit FLIM-FRET in vivo erlaubte. Ich konnte eine funktionelle Charakterisierung der Sensoren durchführen, indem ich zeigte, dass sie auf Konzentrationsänderungen von intrazellulärem cAMP und cGMP reagieren sowie zeigen, dass Zebrafische trotz fehlender T-Tubuli eine signifikante cAMP- und cGMP Kompartimentierung aufweisen, auch unter extremen Bedingungen nach Gabe von cAMP/cGMP stimulierenden Substanzen in hoher Dosierung. Ich konnte zudem subzelluläres Ca2+ durch konfokale Mikroskopie bildgebend darstellen und entwickelte ein Protokoll, um mit Fluo-4 AM eine schnelle Möglichkeit zu haben, Ca2+ mit in die Messungen einzubeziehen. Ausblick: Die in dieser Arbeit benutzte Methode bietet eine gute Möglichkeit, subzelluläre cAMP- und cGMP-Kompartimentierung und Ca2+ zu untersuchen und damit zum Beispiel die Fragen zu beantworten, ob eine veränderte cAMP/cGMP Kompartimentierung zu Herzkrankheiten wie Hypertrophie führt oder ob eine veränderte cAMP Kompartimentierung den zellulären Ca2+ Haushalt und damit die kardiale Kontraktion beeinflusst. Darüber hinaus kann das von mir etablierte Protokoll dazu genutzt werden, mehr über cAMP, cGMP und Ca2+ während der Regeneration im Herzen zu lernen, da der Zebrafisch über ausgeprägte Regenerationsfähigkeiten verfügt
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Lautwein, Tobias [Verfasser]. "Analyse der Präsenz von 'stk33' im Reich 'Animalia' und Versuche zur Herstellung einer Zebrafisch KO-Linie mittels CRISPR/Cas9 / Tobias Lautwein." Mainz : Universitätsbibliothek Mainz, 2017. http://d-nb.info/1135983216/34.
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Dries, Ramona [Verfasser], and M. [Akademischer Betreuer] Bastmeyer. "Neuronale Zelladhäsionsmoleküle im Zebrafisch: Divergente Funktionen und Wechselwirkungen während der Entwicklung und Regeneration des posterioren Seitenlinienorgans / Ramona Dries ; Betreuer: M. Bastmeyer." Karlsruhe : KIT-Bibliothek, 2019. http://d-nb.info/1196208697/34.
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Weidinger, Gilbert, Kristin Schnabel, Chi-Chung Wu, and Thomas Kurth. "Regeneration of Cryoinjury Induced Necrotic Heart Lesions in Zebrafish Is Associated with Epicardial Activation and Cardiomyocyte Proliferation." Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2016. http://nbn-resolving.de/urn:nbn:de:bsz:14-qucosa-191530.
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In mammals, myocardial cell death due to infarction results in scar formation and little regenerative response. In contrast, zebrafish have a high capacity to regenerate the heart after surgical resection of myocardial tissue. However, whether zebrafish can also regenerate lesions caused by cell death has not been tested. Here, we present a simple method for induction of necrotic lesions in the adult zebrafish heart based on cryoinjury. Despite widespread tissue death and loss of cardiomyocytes caused by these lesions, zebrafish display a robust regenerative response, which results in substantial clearing of the necrotic tissue and little scar formation. The cellular mechanisms underlying regeneration appear to be similar to those activated in response to ventricular resection. In particular, the epicardium activates a developmental gene program, proliferates and covers the lesion. Concomitantly, mature uninjured cardiomyocytes become proliferative and invade the lesion. Our injury model will be a useful tool to study the molecular mechanisms of natural heart regeneration in response to necrotic cell death.
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Schweitzer, Jörn. "Klonierung und funktionelle Analyse der Zellerkennungsmoleküle Tenascin-R, Tenascin-C und P0 in Entwicklung und Regeneration im Zebrafisch Danio rerio (Hamilton 1822)." [S.l. : s.n.], 2003. http://deposit.ddb.de/cgi-bin/dokserv?idn=968468764.
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Fuchs, Alexander [Verfasser]. "Charakterisierung der Mutation FHL 1 C22W im Zebrafisch, Generierung von transgenen Zebrafischlinien aus FHL 1 wt, H123Y, C132F sowie C224W / Alexander Fuchs." Ulm : Universität Ulm, 2017. http://d-nb.info/1145037097/34.
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Fuchs, Alexander Sebastian [Verfasser]. "Charakterisierung der Mutation FHL 1 C22W im Zebrafisch, Generierung von transgenen Zebrafischlinien aus FHL 1 wt, H123Y, C132F sowie C224W / Alexander Fuchs." Ulm : Universität Ulm, 2017. http://d-nb.info/1145037097/34.
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Ulrich, Florian. "Regulation of Zebrafish Gastrulation Movements by slb/wnt11." Doctoral thesis, Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2005. http://nbn-resolving.de/urn:nbn:de:swb:14-1125651469323-78929.
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During zebrafish gastrulation, highly coordinated cellular rearrangements lead to the formation of the three germ layers, ectoderm, mesoderm and endoderm. Recent studies have identified silberblick (slb/wnt11) as a key molecule that regulates gastrulation movement through a conserved pathway, which shares significant similarity with a signalling pathway that establishes epithelial planar cell polarity (PCP) in Drosophila (Heisenberg et al., 2000; Veeman et al., 2003), suggesting a role for cell polarity in regulating gastrulation movements. However, the cellular and molecular mechanisms by which slb/wnt11 functions during zebrafish gastrulation are still not fully understood. In the first part of the thesis, the three-dimensional movement and morphology of individual cells in living embryos during the course of gastrulation were recorded and analysed using high resolution confocal microscopy. It was shown that in slb/wnt11 mutant embryos, hypoblast cells within the forming germ ring display slower, less directed migratory movements at the onset of gastrulation, which are accompanied by defects in the orientation of cellular processes along the individual movement directions of these cells. The net movement direction of the cells is not changed, suggesting that slb/wnt11-mediated orientation of cellular processes serves to facilitate and stabilize cell movements during gastrulation. By using an in vitro reaggregation assay on mesendodermal cells, combined with an analysis of the endogenous expression levels and distribution of E-cadherin in zebrafish embryos at the onset of gastrulation, E-cadherin mediated adhesion was found to be a downstream mechanism regulating slb/wnt11 function during gastrulation. Interestingly, the effects of slb/wnt11 on cell adhesion appear to be dependent on Rab5-mediated endocytosis, suggesting endocytic turnover of cell-cell contacts as one possible mechanism through which slb/wnt11 mediates its effects on gastrulation movements. - Die Druckexemplare enthalten jeweils eine CD-ROM als Anlagenteil: QuickTimeMovies (ca. 23 MB)- Übersicht über Inhalte siehe Dissertation S. 92 - 93"
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Ranjan, Ashish. "Targeted differentiation of ES cell into serotonergic neurons." Doctoral thesis, Humboldt-Universität zu Berlin, Mathematisch-Naturwissenschaftliche Fakultät I, 2015. http://dx.doi.org/10.18452/17258.
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Serotonin ist ein Neurotransmitter im zentralen Nervensystem (ZNS), die eine Vielzahl von Funktionen in der menschlichen Physiologie hat. Serotonergen Neuronen in der Raphe-Kerne des Gehirns Unser Ziel war die Leitung der Differenzierung von embryonalen Stammzellen (ES-Zellen) eingeengt und pluripotenten Stammzellen (iPS) Zellen in eine angereicherte Population von Serotonin-produzierenden Zellen, neuartige Gene, die wesentlich für die Entwicklung zu identifizieren und die Funktion des serotonergen Systems. Zu diesem Zweck haben wir differenzierten ES-Zellen in Serotonin-produzierenden Neuronen. Verwendung von RNA zu verschiedenen Zeitpunkten im Verlauf der ES-Zelldifferenzierung wir Gene spezifisch in serotonergen Linie von Affymetrix Genarray angereichert identifiziert isoliert. Um Kandidatengene bewerten wir neu programmiert Maus und Ratte embryonale Fibroblasten zu iPS-Zellen und anschließend differenziert sie serotonergen Neuronen. Wir haben uns für Cacna2d1, für eine alpha2 / delta-Untereinheit von spannungsabhängigen Calciumkanäle als prominentesten Kandidaten unter diesen Genen kodiert. Zur Analyse der Rolle des Proteins Cacna2d1 wir verwendet Cacna2d1 Knockout-Mäusen und Morpholino-Knockdown im Zebrafisch. Wir versäumt, direkte Beteiligung der Cacna2d1 mit serotonergen Systems sehen. Allerdings Immunfärbung für Cacna2d1 in Zebrafisch zeigte zeitabhängige Muster während der frühen Entwicklung. Cacna2d1 Expression wurde in seitlichen Mittellinie Stamm gesehen; vermutlich in Neuromasten Zellen. Übereinstimmend mit ihrer Charakterisierung als Neuromasten werden diese Cacna2d1-positiven Zellen in Richtung der Schwanz der Migration. Darüber hinaus zeigte Zebrafisch gestörten Migrationsverhalten der Neuromasten nach Morpholino-Knockdown von Cacna2d1. So ist diese Studie stellte klar, dass Cacna2d1 ist für Zebrafisch Seitenlinie Entwicklung aber keinen Einfluss auf die Einrichtung des serotonergen Systems.Serotonin is a neurotransmitter in the central nervous system (CNS), which has a wide range of functions in human physiology. Serotonergic neurons are concentrated in the raphe nuclei of the brain We aimed at directing the differentiation of embryonic stem (ES) cells and induced pluripotent stem (iPS) cells into an enriched population of serotonin producing cells to identify novel genes that are essential for the development and function of serotonergic system. To this purpose we differentiated ES cells into serotonin producing neurons. Using RNA isolated at different time points during the course of ES cell differentiation we identified genes specifically enriched in the serotonergic lineage by Affymetrix gene array. To evaluate candidate genes we reprogrammed mouse and rat embryonic fibroblast to iPS cells and subsequently differentiated them to serotonergic neurons. We selected Cacna2d1, coding for an alpha2/delta subunit of voltage dependent calcium channels as a most prominent candidate among these genes. To analyse the role of the Cacna2d1 protein we used Cacna2d1 knockout mice and morpholino-knockdown in zebrafish. We failed to see direct involvement of Cacna2d1 with serotonergic system. However immunostaining for Cacna2d1 in zebrafish revealed time-dependent pattern during early development. Cacna2d1 expression was seen in lateral midline trunk; presumably in neuromast cells. Concordantly with their characterization as neuromasts, these Cacna2d1-positive cells are migrating towards the tail. Moreover, zebrafish showed disturbed migration behaviours of neuromasts after morpholino-knockdown of Cacna2d1. Thus, this study clarified that Cacna2d1 is essential for zebrafish lateral line development but does not affect the establishment of the serotonergic system.
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Arboleda-Estudillo, Yoana. "Mechanical cell properties in germ layer progenitor migration during zebrafish gastrulation." Doctoral thesis, Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2010. http://nbn-resolving.de/urn:nbn:de:bsz:14-qucosa-27725.
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Gastrulation leads to the formation of the embryonic germ layers, ectoderm, mesoderm and endoderm, and is the first key morphogenetic process that occurs in development. Gastrulation provides a unique developmental assay system in which to study cellular movements and rearrangements in vivo.
The different cell movements occurring during gastrulation take place in a highly coordinated spatial and temporal manner, indicating that they must be controlled by a complex interplay of morphogenetic and inductive events. Generally, cell movement constitutes a highly integrated program of different cellular behaviors including sensing, polarization, cytoskeletal reorganization, and changes in adhesion and cell shape. During migration, these different behaviors require a continuous regulation and feedback control to direct and coordinate them.
In this work, we analyze the cellular and molecular mechanisms underlying the different types of cell behaviors during gastrulation in zebrafish. Specifically, we focus on the role of the adhesive and mechanical properties of germ layer progenitors in the regulation of gastrulation movements. In the first part of the project, we investigated the role of the adhesive and mechanical properties of the different germ layer progenitor cell types for germ layer separation and stratification. In the second part of this study, we applied the same methodology to determine the function of germ layer progenitor cell adhesion in collective cell migration.
Tissue organization is thought to depend on the adhesive and mechanical properties of the constituent cells. However, it has been difficult to determine the precise contribution of these different properties due to the lack of tools to measure them. Here we use atomic force microscopy (AFM) to quantify the adhesive and mechanical properties of the different germ layer progenitor cell types. Applying this methodology, we demonstrate that mesoderm and endoderm progenitors are more adhesive than ectoderm cells and that E-cadherin is the main adhesion molecule regulating this differential adhesion. In contrast, ectoderm progenitors exhibit a higher actomyosin-dependent cell cortex tension than mesoderm and endoderm progenitors. Combining these data with tissue self-assembly in vitro and in vivo, we provide evidence that the combinatorial activities of cell adhesion and cell cortex tension direct germ layer separation and stratification.
It has been hypothesized that the directionality of cell movement during collective migration results from a collective property. Using a single cell transplantation assay, we show that individual progenitor cells are capable of normal directed migration when moving as single cells, but require cell-cell adhesion to participate in coordinated and directed migration when moving collectively.
These findings contribute to the understanding of the gastrulation process. Cell-cell adhesion is required for collective germ layer progenitor cell migration, and cell cortex tension is critical for germ layer separation and stratification. However, many questions still have to be solved. Future studies will have to explore the interaction between the adhesive and mechanical progenitor cell properties, as well as the role of these properties for cell protrusion formation, cell polarization, interaction with extracellular matrix, and their regulation by different signaling pathways.