In comparison to mammals, zebrafish are able to regenerate many organs and tissues, including the central nervous system (CNS). Within the CNS-derived neural retina, light lesions result in a loss of photoreceptors and the subsequent activation of Müller glia, the retinal stem cells. Müller glia-derived progenitors differentiate and eventually restore the anatomical tissue architecture within 4 weeks. However, little is known about how light lesions impair vision functionally, as well as how and to what extent visual function is restored during the course of regeneration, in particular in adult animals. Here, we applied quantitative behavioral assays to assess restoration of visual function during homeostasis and regeneration in adult zebrafish. We developed a novel vision-dependent social preference test, and show that vision is massively impaired early after lesion, but is restored to pre-lesion levels within 7 days after lesion. Furthermore, we employed a quantitative optokinetic response assay with different degrees of difficulty, similar to vision tests in humans. We found that vision for easy conditions with high contrast and low level of detail, as well as color vision, was restored around 7–10 days post lesion. Vision under more demanding conditions, with low contrast and high level of detail, was regained only later from 14 days post lesion onwards. Taken together, we conclude that vision based on contrast sensitivity, spatial resolution and the perception of colors is restored after light lesion in adult zebrafish in a gradual manner.

The neurodegenerative disorder Parkinson's Disease (PD) represents both a major socioeco-nomic challenge and an individual burden for many patients. Despite major efforts, neither satisfactory explanations of the pathogenesis of PD, nor disease-modifying drugs have been developed to date. Mutations of the multidomain kinase LRRK2 represent the overall most common cause of he-reditary PD. Furthermore, LRRK2 mutations have been linked to dysregulations of the immune system such as inflammatory bowel disease, cancer, or the susceptibility towards mycobacte-rial infections. Several pathogenic point mutations have been identified that – directly or indi-rectly – lead to a pathological gain-of-function of the protein’s kinase domain. Despite recent advances, the physiological functions of LRRK2, as well as the underlying processes of LRRK2-mediated pathologies, remain largely unknown. Much research effort has aimed at generating reliable animal models for the study of LRRK2. Nevertheless, neither loss-of-function of the gene, nor overexpression of normal or mutant LRRK2, has yielded definitive results. Previous work from our research group on zebrafish (Danio rerio) has generated two genetic lrrk2 knock-out lines using different mutagenesis strat-egies: lrrk2TILLING and lrrk2CRISPR (Ahrendt, 2011; Suzzi, 2017). These studies’ results were par-tially contradictory, and the described phenotypes were not stable. Whilst this previous work investigated lrrk2 loss-of-function, so far, no genetic knock-in line carrying one of the multiple known pathogenic lrrk2 mutations, has been reported in zebrafish. Therefore, this work aimed to further investigate the effects of Lrrk2-deficiency in zebrafish and to establish a genetic knock-in of the common pathogenic G2019S substitution to model the genotype of PD patients more accurately. A variety of methods was applied to achieve these aims. Immunohistochemistry and conventional histology studies were performed on zebrafish brains and kidneys at different developmental stages, both under physiological conditions and following the induction of brain regeneration. Since the zebrafish’s neuroregenerative capabil-ity is closely linked to an initial neuroinflammation, and previous studies on lrrk2 knock-out zebrafish have suggested an impaired immune response and reduced brain regeneration, the neuroinflammation and neuroregeneration of adult lrrk2TILLING zebrafish were investigated by inducing a telencephalic stab-lesion and a subsequent BrdU-pulse-chase analysis. To investi-gate functional effects of the gene knock-out, a set of behavioral experiments was performed. Using CRISPR/Cas9 genome editing, the basis for a knock-in of the G2019S substitution was established. Immunohistochemistry analyses of larval and adult zebrafish brains were performed in a set of experiments. By quantifying all mitotic cells in larval brains at different time points, the basal brain proliferation levels during development were analyzed, as well as the levels of constitutive neurogenesis during adulthood. Under physiological conditions, basal brain prolif-eration was found unimpaired in Lrrk2-deficient zebrafish. Similarly, the number of microglia found in the telencephalon of Lrrk2-deficient zebrafish was not reduced under physiological conditions, although one experimental group showed signs of neuroinflammation. Upon induc-tion of a traumatic brain injury in adult fish, neither the trauma-induced proliferation of leuko-cytes, nor the number of regenerated neurons were altered in Lrrk2-deficient animals. A multi-dimensional behavioral analysis of Lrrk2-deficient zebrafish revealed no significant constraints. The total swimming distance, average velocity and ratio of mobility states were unimpaired upon lrrk2-knock-out, as was the fish’s exploratory behavior in an anxiety model using a light-dark-box. In a test for social preference, Lrrk2-deficient and wild-type zebrafish showed the same tendency to join a group of conspecific animals, suggesting no major deficits in overall social interaction. In contrast to these preserved functions, adult Lrrk2-deficient kidneys revealed a pronounced accumulation of vacuole-like particles in the proximal renal tubules, a finding that may indicate disruptions in the endolysosomal pathway and that is in line with phenotypes described in LRRK2-deficient rodents as well as with the side effects induced by pharmacological LRRK2 inhibitors. These findings represent a promising lead for future exploration. During this work a CRISPR/Cas9 target site with high cleavage efficiency was established within the Lrrk2 kinase domain of freshly spawned zebrafish eggs. In combination with recent advances in CRISPR methodology, these results provide an opportunity for the generation of a genetic Lrrk2-G2019S knock-in line in zebrafish. In summary, this work found Lrrk2-deficient zebrafish unimpaired regarding various physiolog-ical functions. While in line with previously reported results, a satisfactory explanation for Lrrk2-mediatied pathogenesis is still lacking. Morphological alterations of Lrrk2-deficient kidneys hint towards perturbations in the lysosomal homeostasis, and a promising target for future re-search. Modelling human LRRK2 genotypes more precisely will hopefully provide further in-sights into the enigma of LRRK2 and its link to neurodegeneration. / Die neurodegenerative Erkrankung Morbus Parkinson (Idiopathisches Parkinson-Syndrom, IPS) stellt sowohl eine individuelle Belastung für betroffene Menschen als auch eine große sozio-ökonomische Herausforderung für die Gesellschaft dar. Trotz großer Anstrengungen konnten bisher weder zufriedenstellende pathophysiologische Erklärungen des IPS, noch krankheits-modulierende Medikamente entwickelt werden. Mutationen der Kinase LRRK2 sind die insgesamt häufigste Ursache für erbliche Parkinson-Syndrome. Darüber hinaus wurden LRRK2-Mutationen mit immundysregulatorischen Syndro-men wie chronisch-entzündlichen Darmerkrankungen, Malignomen oder der Anfälligkeit ge-genüber Mykobakterien-Infektionen in Verbindung gebracht. Verschiedene pathogene Punktmutationen von LRRK2 sind bekannt. Diese führen – direkt oder indirekt – zu einer pa-thologischen Überaktivierung seiner Kinasedomäne. Trotz jüngster Fortschritte in der For-schung sind die Funktionen von LRRK2 und die Prozesse, die zu den LRRK2-vermittelten Pathologien führen, weiterhin weitgehend unbekannt. Viele Studien haben sich um die Entwicklung zuverlässiger Tiermodelle für die Untersuchung von LRRK2 bemüht. Dennoch haben weder die Untersuchung eines Gen-Funktionsverlusts noch die Überexpression von normalem oder mutiertem LRRK2 bislang zu eindeutigen Ergeb-nissen geführt. Frühere Arbeiten unserer Arbeitsgruppe haben in Zebrafischen (Zebrabärbling, Danio rerio) zwei genetische lrrk2-Knockout-Linien mit unterschiedlichen Mutagenesestrate-gien erzeugt: lrrk2TILLING und lrrk2CRISPR (Ahrendt, 2011; Suzzi, 2017). Die Ergebnisse dieser Studien widersprachen sich teilweise, und die beschriebenen Phänotypen waren nicht stabil reproduzierbar. Während alle bisherigen Arbeiten einen Funktionsverlust von Lrrk2 untersuch-ten, wurde bisher noch keine genetische Knock-in-Linie im Zebrafisch publiziert, die eine der zahlreichen bekannten pathogen-überaktivierenden LRRK2-Mutationen trägt. Ziel dieser Arbeit war es daher zum einen, die Auswirkungen eines Lrrk2-Funktionsverlusts in Zebrafischen weiter zu untersuchen, und zum anderen eine genetische Knock-in-Linie der häufigen pathogenen G2019S-Mutation zu etablieren, um den Genotyp menschlicher Parkin-son-Patienten präziser zu modellieren. Um diese Ziele zu erreichen, wurde eine Vielzahl von Methoden angewandt. Es wurden im-munhistochemische und konventionelle histologische Untersuchungen an Gehirnen und Nie-ren von Zebrafischen in verschiedenen Entwicklungsstadien durchgeführt, sowohl unter phy-siologischen Bedingungen als auch nach der Induktion einer Gehirnregeneration. Da die Fä-higkeit des Zebrafischs zur umfassenden Neuroregeneration durch eine initiale Neuroinflam-mation vermittelt wird und frühere Studien an lrrk2-Knockout-Zebrafischen in Folge traumati-scher Hirnverletzungen eine beeinträchtigte Immunreaktion und eine verringerte Neurorege-neration feststellen konnten, wurden die posttraumatische Neuroinflammation und die Neuroregeneration von adulten lrrk2TILLING-Zebrafischen untersucht, indem eine Stichverlet-zung des Großhirns induziert und eine anschließende BrdU-Pulse-Chase-Analyse durchge-führt wurde. Um die funktionellen Auswirkungen des Gen-Knockouts zu untersuchen, wurde eine Reihe von Verhaltensexperimenten durchgeführt. Mit Hilfe von CRISPR/Cas9-Genom-Editierung wurde die Grundlage für den Knock-in der G2019S-Mutation geschaffen. In einer ersten Reihe von Experimenten wurden larvale und adulte Zebrafischgehirne immun-histochemisch analysiert. Durch die Quantifizierung aller zerebraler mitotischer Zellen zu ver-schiedenen Zeitpunkten wurden die basale Hirnproliferation während der larvalen Entwicklung sowie die konstitutive Neurogenese im Erwachsenenalter analysiert. Unter physiologischen Bedingungen war die basale Hirnproliferation bei Lrrk2-defizienten Zebrafischen nicht beein-trächtigt. Auch die Anzahl der Mikroglia im Telenzephalon der Lrrk2-defizienten Zebrafische war unter physiologischen Bedingungen nicht verringert, obwohl eine Versuchsgruppe Anzei-chen einer Neuroinflammation zeigte. Infolge einer gezielten Verletzung einer Großhirnhemi-sphäre waren bei Lrrk2-defizienten Tieren weder die traumabedingte Proliferation von Leuko-zyten noch die Anzahl der anschließend regenerierten Neuronen verändert. Eine Verhaltensanalyse von Lrrk2-defizienten Zebrafischen ergab keine signifikanten Ein-schränkungen. Die Gesamtschwimmdistanz, die Durchschnittsgeschwindigkeit und das Ver-hältnis verschiedener Mobilitätszustände waren durch den Lrrk2-Knock-out unbeeinträchtigt, ebenso wie das Erkundungsverhalten der Fische in einem Angstmodell mit einer Hell-Dunkel-Kammer. In einem Test auf soziale Präferenz zeigten Lrrk2-defiziente und Wildtyp-Zebrafische die gleiche Tendenz, sich einer Gruppe von Artgenossen anzuschließen, was auf keine größeren Defizite in der allgemeinen sozialen Interaktion hindeutet. Im Gegensatz zu diesen unauffälligen Ergebnissen zeigten erwachsene Lrrk2-defiziente Nie-ren eine ausgeprägte Anhäufung vakuolenartiger Partikel in den proximalen Tubuli. Dieser Befund könnte auf Störungen im endolysosomalen Weg hinweisen und ist konsistent zu den bei LRRK2-defizienten Nagetieren beschriebenen Phänotypen, sowie den durch pharmakolo-gische LRRK2-Inhibitoren hervorgerufenen Nebenwirkungen. Diese Ergebnisse sind ein viel-versprechender Ansatzpunkt für künftige Experimente. Im Rahmen dieser Arbeit wurde eine CRISPR/Cas9-target-site mit hoher Schnitteffizienz in-nerhalb der LRRK2-Kinasedomäne von Zebrafisch-Embryonen etabliert. In Kombination mit Fortschritten in der CRISPR-Methodik bilden diese Ergebnisse eine Grundlage zur Erzeugung einer lrrk2-G2019S Knock-in-Linie. Zusammenfassend zeigt sich in dieser Arbeit, dass Lrrk2-defiziente Zebrafische in Hinblick auf verschiedene physiologische Funktionen nicht beeinträchtigt zu sein scheinen. Obwohl dies im Einklang mit früher berichteten Ergebnissen steht, bleibt eine zufriedenstellende Erklärung für die Lrrk2-vermittelte Pathogenese weiterhin aus. Morphologische Veränderungen in Lrrk2-defizienten Nieren deuten auf Störungen in der Homöostase des Lysosoms hin und bieten ein vielversprechendes Forschungsziel. Eine präzisere Modellierung des menschlichen LRRK2-Genotyps in fortschrittlichen Tiermodellen könnte zukünftig mehr Einblick in das Rätsel von LRRK2 und seiner Rolle in der Neurodegeneration bieten.

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.

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.

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"

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)

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.

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.

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 regenerate. / Einleitung: 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.

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.