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.

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.

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.

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.

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 FCS. / Fluoreszenz-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.

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.

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.

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.

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.

Humans can, if they survived a cardiac injury such as heart infarction, heal this cardiac injury only by scarring and with minimal regeneration of some cardiac cells. Zebrafish, however, can fully regenerate cardiac tissue after surgical resection of up to 20% of the ventricle. Regenerating tissue includes cells of the three cardiac layers, i.e. myocardium, epicardium and endocardium. Thus, zebrafish, with its ability to regenerate damaged heart and as a model enabling genetic manipulations, provides the possibility to study cellular and molecular mechanisms of heart regeneration. Understanding these mechanisms may help develop new therapeutic approaches to improve the situation after a heart injury in humans. Since molecular mechanisms regulating heart regeneration are so far largely unknown, I aimed to identify and analyze molecular signals that are important for cardiac regeneration in zebrafish. Molecular signals that are crucial during heart development have been suggested to be reactivated during cardiac regeneration. Since Wnt/β-catenin signaling is crucial for vertebrate heart development, it is likely to be important for zebrafish cardiac regeneration as well. First, I focused on the functional role of Wnt/β-catenin signaling in cardiac regeneration mainly by using transgenic fish lines that allow inducible activation or inhibition of the pathway. By using in situ hybridization and expression profiling, I tested whether endogenous Wnt/β-catenin target genes are detectable in regenerating hearts and screened for activity of the β-catenin responsive reporter in TOPdGFP transgenic fish (Tg(TOP:GFP)w25) after ventricular resection. I could not identify endogenous Wnt/β-catenin targets during the early phase of regeneration up to 7 days post amputation (dpa) using oligoexpression microarrays or in situ hybridization. An injury specific activation of the β-catenin responsive TOPdGFP reporter was not detectable either, suggesting that Wnt/β-catenin signaling is not active during this early phase of regeneration. The manipulation of Wnt/β-catenin signaling using transgenic fish lines did not influence cell proliferation or the overall extent of zebrafish heart regeneration. These results suggested that Wnt/β-catenin signaling has no functional role during entire zebrafish heart regeneration. Second, I found the transcription factor Sox9a to be upregulated after ventricular resection during the early phase of heart regeneration. Using transgenic reporter fish lines, I detected Sox9a expression in cardiomyocytes and endothelial cells, part of which were proliferative. Furthermore Sox9a was expressed in some cells of the epicardial layer that activated the expression of developmental genes in the entire heart in response to injury. These results indicated that Sox9a is expressed in cells that were actively involved in the regenerative response. To gain insight into the functional role of Sox9a, I generated a transgenic fish line where a repressor construct is inducibly expressed, which then interferes with Sox9a target gene transcription. I detected a significant reduction in myocardial and endothelial regeneration after induction of the repressor. These results suggested that Sox9a function is important for regeneration of endothelial and myocardial cells after heart injury. Third, using oligoexpression microarrays, I performed systematic gene expression profiling of the zebrafish heart regeneration within the first 2 weeks following amputation. I found that known genes, which have previously been shown to be strongly expressed during heart regeneration, as well as novel genes were upregulated after ventricular resection. Some of these genes have been implicated in vertebrate heart development, supporting the idea that cardiac developmental genes are reactivated during heart regeneration. Hence, these results reveal a good starting point for further analysis of the cellular and molecular events occurring within the first days after cardiac injury. Finally, I developed a cryoinjury method that more closely resembled the injured tissue after human heart infarction. I induced tissue death by exposing the ventricle to dry ice and detected that the zebrafish heart can regenerate upon this cardiac injury similarly as in response to a ventricular resection injury. After cryoinjury, the entire epicardium activated the expression of developmental genes and started to proliferate. I detected also proliferating cardiomyocytes, indicating that similar cellular mechanisms are induced in the epicardium and the myocardium after cryoinjury and ventricular resection. Furthermore, activation of Sox9 expression early after cryoinjury suggested that molecular mechanisms of regeneration are also similar in both injury methods. Thus, cryoinjury provides a useful tool for future studies of zebrafish heart regeneration with more relevance to human cardiac infarction. I discuss all results with reference to vertebrate heart development and to the response after mammalian heart infarction. Furthermore, the results were put into the context of cellular mechanisms that are present in the process of zebrafish heart regeneration.