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Primary objective of my MS thesis project is to characterize and develop a B cell ablation model for investigating the pathogenesis of human diseases such as hepatitis and systemic lupus erythematosus (SLE). Conditional and targeted cell ablation is a powerful approach for studying cellular functions in vivo. However, currently available cell ablation models still have some limitations and therefore limit their broader application in biomedical research. For example, two of the most common cell ablation methods currently employed utilize the herpes simplex virus 1 thymidine kinase (HSVtk) or diphtheria toxin (DT) receptor combined with their respective transgenic strategies. The ablation using HSVtk transgenic mice eliminates dividing cells, but does not affect non-dividing cells. In addition, because of its extremely high potency (a single molecule of DT-A, the active cleavage product of DT is sufficient to induce apoptosis), dose dependent responses are difficult to achieve and off-target effects are frequently observed. These facts highlight the unmet need to develop alternative methods of targeted cell ablation, which our model very successfully addresses. Our recently established approach using intermedilysin (ILY)-mediated cell ablation that is specific for human CD59 (hCD59) expressing cells, obviates these problems and provides an excellent and significantly improved alternative approach to the existing cell ablation methodologies. Intermedilysin (ILY), a toxin secreted by Streptococcus intermedius, exclusively binds to the human cell membrane protein CD59 (hCD59) but not to CD59 of any other species. Once bound, ILY rapidly and potently lyses the cells. Using genetic engineering, animal models can be created that express hCD59 in a spatially regulated manner. Administration of ILY will then selectively ablate only those cells in the animals that express hCD59 without any non-specific effect. To expand and facilitate the application of this newly generated model, we recently generated a Cre-inducible floxedSTOP-hCD59 transgenic mouse line (ihCD59), where specific hCD59 expression occurs following Cre-mediated recombination. By crossing ihCD59 mice with specific immune cell (T cells or macrophage) Cre transgenic lines, we obtained double transgenic mice expressing hCD59. ILY administration mediated specific cell ablation in these target cell populations in a dose dependent manner. Based on these results, I wanted to establish a new B cell ablation model for further studying B cell functionality in the pathogenesis of human diseases. CD19-Cre mice expressing the Cre-recombinase in B cell population were crossed with ihCD59 mice to generate the double positive transgenic mice (ihCD59+/-/CD19-Cre+/-). In Aim 1, I have demonstrated that hCD59 is specifically expressed in the B cell populations. In Aim 2, I have documented that 1) ILY has a large pharmacological window, and 2) ILY injection to ihCD59+/-/CD19-Cre+/- mice resulted in a rapid cell ablation of the B cell cells with off-target effect. Further, I have demonstrated that the specific ablation of B cells did not prevent the immune (Con A)-mediated hepatitis. In the future, I will apply this conditional B cell ablation model for investigating the functionality of B cells in the pathogenesis of human disease such as SLE. / Biomedical Sciences

Malkostrake Krebse zeigen im postnauplialen Keimstreif ein invariantes Zellteilungsmuster und eine Zelllinie. Embryonen eines Isopoden (Porcellio scaber) und eines Amphipoden (Orchestia cavimana) wurden bezüglich ihrer Zelllinie vergleichend Untersucht. Mittels immunhistochemischer Färbungen wurde das Expressionsmuster der Gene engrailed und Distal-less in Hinblick auf die Zelllinie und die Morphogenese der Segmente und der Beinentwicklung analysiert. Die Zelllinie wurde bei Porcellio scaber mit der Methode der 4D-Mikroskopie untersucht. Mit Zellablationsexperimenten wurden Abhängigkeiten der Zellen untereinander aufgezeigt. Die Ergebnisse dieser Untersuchungen zeigen: I. Die Genexpression von Distal-less und engrailed wird unabhängig von der Zelllinie reguliert. II. Die Bildung der konvergenten einästigen Beine im Thoraxbereich beider Krebse erfolgt durch unterschiedliche Regulation des Gens Distal-less - bei Orchestia cavimana wird die Expression des Gens in den lateralen Zellen abgeschaltet, während diese Zellen bei Porcellio scaber erst gar nicht Distal-less exprimieren. III. Die Anwendung des Systems der 4D-Mikroskopie zeigte unter anderem, dass in den vorderen Reihen nichtektoteloblastischen Ursprungs bei Porcellio scaber die Zelllinie variabel ist und eine Zellsortierung und Zellimmigration stattfindet. IV. Die Ähnlichkeit der Bildung dieser Reihen bei Porcellio scaber mit der der Tanaidaceen lässt auf ein Schwestergruppenverhältnis der Taxa Isopoda und Tanaidacea schliessen. Die Dissertation enthält fünf Videoaufnahmen als separate AVI-Dateien. / Malacostracan crustaceans show in their postnaupliar germ-band an invariant cleavage pattern and a cell-lineage. A comparative analysis of this cell-lineage in an Isopod (Porcellio scaber) and an amphipod (Orchestia cavimana) was done in this thesis. Immunohistochemical stainings of the gene products Distal-less and Engrailed were used, to show the relation of these genes to the morphogenesis of segments and legs. Further, the cell-lineage of Porcellio scaber was analyzed with a 4D-microscope system. Cell-ablation experiments were used to show regulational networks in the development of the germ-band. The results of this work show: I. The regulation of the genes Distal-less and engrailed is independent of the cell-lineage. II. The morphogenesis of the convergent monoramous limbs in the thorax is reflected by different expression patterns of the gene Distal-less - in Orchestia cavimana the expression of Distal-less is switched off in the lateral cells, in Porcellio scaber these cells do not start the Distal-less expression. III. The 4D-microscopy analysis show, that the cell-lineage in the cellrows wich have a non-ectoteloblastic origin is not invariant. In these rows of cells show cell sorting. IV. The formation of these rows in the isopod Porcellio scaber shows similarity to the formation in tanaidaceans. A sister group relationship of Tanaidacea and Isopoda is strongly supported. This dissertation contains five video recordings as separate AVI files.

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During embryogenesis and post-embryonic patterning, active transport of the phytohormone auxin, reflected in the expression of the Arabidopsis PIN family of auxin efflux mediators, generates local auxin distributions that are crucial for correct organ and tissue specification. Polar auxin transport routes have also long been postulated to regulate vein formation in the leaf. The molecular identification of PIN proteins has made it possible to investigate this hypothesis further by visualizing auxin transport routes in developing leaves. In Arabidopsis leaf primordia, PIN1 is expressed before the earliest known markers of vascular identity, in domains that are gradually restricted to sites of vein formation. PIN1 polarity indicates that auxin is directed towards distinct “convergence points” (CPs) in the marginal epidermis, from which it defines the sites of major vein formation. Within incipient veins, PIN1 polarity indicates drainage of auxin into preexisting veins, such that veins connected at both ends display two divergent polarities. Local auxin application triggers the formation of ectopic CPs and new veins, demonstrating the sufficiency of auxin as a vein-specifying signal. However, not all PIN1-labeled auxin transport routes differentiate as veins: Minor veins are initially unstable, suggesting local competition for auxin transport. Expression of ATHB8, a marker of vascular cell selection, correlates with enhanced PIN1 expression domain (PED) stability and vascular differentiation. Auxin application and auxin transport inhibition reveal that both CP formation in the epidermis and subepidermal PED dynamics are auxin-dependent and self-organizing. Furthermore, normal auxin perception through the ARF-Aux/IAA signaling pathway is required for the restriction of PIN1-mediated auxin transport to narrow subepidermal domains. ARF-Aux/IAA signaling is known to control auxin transport through the regulation of PIN1 dynamics, but the mechanism of this regulation is unclear. It is here shown that two redundantly acting AUXIN RESPONSE FACTOR (ARF) transcription factors, ARF5/MONOPTEROS (MP) and ARF7/NPH4, jointly regulate both PIN1 expression and localization during lateral root patterning in Arabidopsis, in part through the direct transcriptional activation of PIN1 by MP. Taken together, these results indicate that feedback between PIN-mediated auxin transport and ARF-Aux/IAA signaling regulates the patterning of root and shoot organs.

During embryogenesis and post-embryonic patterning, active transport of the phytohormone auxin, reflected in the expression of the Arabidopsis PIN family of auxin efflux mediators, generates local auxin distributions that are crucial for correct organ and tissue specification. Polar auxin transport routes have also long been postulated to regulate vein formation in the leaf. The molecular identification of PIN proteins has made it possible to investigate this hypothesis further by visualizing auxin transport routes in developing leaves. In Arabidopsis leaf primordia, PIN1 is expressed before the earliest known markers of vascular identity, in domains that are gradually restricted to sites of vein formation. PIN1 polarity indicates that auxin is directed towards distinct “convergence points” (CPs) in the marginal epidermis, from which it defines the sites of major vein formation. Within incipient veins, PIN1 polarity indicates drainage of auxin into preexisting veins, such that veins connected at both ends display two divergent polarities. Local auxin application triggers the formation of ectopic CPs and new veins, demonstrating the sufficiency of auxin as a vein-specifying signal. However, not all PIN1-labeled auxin transport routes differentiate as veins: Minor veins are initially unstable, suggesting local competition for auxin transport. Expression of ATHB8, a marker of vascular cell selection, correlates with enhanced PIN1 expression domain (PED) stability and vascular differentiation. Auxin application and auxin transport inhibition reveal that both CP formation in the epidermis and subepidermal PED dynamics are auxin-dependent and self-organizing. Furthermore, normal auxin perception through the ARF-Aux/IAA signaling pathway is required for the restriction of PIN1-mediated auxin transport to narrow subepidermal domains. ARF-Aux/IAA signaling is known to control auxin transport through the regulation of PIN1 dynamics, but the mechanism of this regulation is unclear. It is here shown that two redundantly acting AUXIN RESPONSE FACTOR (ARF) transcription factors, ARF5/MONOPTEROS (MP) and ARF7/NPH4, jointly regulate both PIN1 expression and localization during lateral root patterning in Arabidopsis, in part through the direct transcriptional activation of PIN1 by MP. Taken together, these results indicate that feedback between PIN-mediated auxin transport and ARF-Aux/IAA signaling regulates the patterning of root and shoot organs.