Conditional and transgenic mouse models as tools to study the role of TGFbeta, BMP signaling in skeletal development

Die TGFbeta/BMP Signaltransduktionskaskade ist wichtig für viele Entwicklungsprozesse fast aller embryonaler sowie extraembryonaler Gewebe und sie ist ebenso essentiell bei der Aufrechterhaltung der Homöostase im adulten Organismus. In vielen Mausmodellen und Zellkulturversuchen wurde gezeigt, dass Liganden dieses Signalweges in verschiedene Stadien der Knorpel- und Knochenentwicklung involviert sind. BMPs sind beispielsweise maßgeblich an der frühen Kondensation und Bildung des Knorpels und später an Proliferation und Hypertrophie der Chondrozyten beteiligt. BMPs können ektopisch Knochenbildung auslösen und das Expressionsmuster der Liganden und spezifischen Rezeptoren in der Wachstumsfuge lässt auf eine wichtige Rolle der BMPs in der Wachstumsfuge schließen. Der gezielte knock out der BMP-Rezeptoren Bmpr1a und Bmpr1b in proliferierenden Chondrozyten führt zur Ausbildung einer generellen Chondrodysplasie. Smad1, Smad5 und Smad8 sind die Mediatoren der BMP-Signalkaskade. Im Rahmen der vorliegenden Arbeit sollte die Rolle und Funktion der Smad1- und Smad5-Proteine in der Wachstumsfuge untersucht werden. Hierzu wurden konditionale Smad1-knock out-Mäuse mit einer transgenen Mauslinie gekreuzt, die die Cre-Rekombinase spezifisch in proliferierenden Chondrozyten exprimiert. Diese Mäuse wurden mit und ohne heterozygotem Smad5-Hintergrund charakterisiert. Bei einem knock out von Smad1 allein konnte ein leichte Verkürzung der Wachstumsfuge beobachtet werden, wobei prähypertrophe und hypertrophe Zone gleichermaßen betroffen waren. Dieser Phänotyp war verstärkt in Mäusen mit zusätzlichem heterozygotem Smad5-Hintergrund. Eine Verringerung der Proliferationsrate konnte zusammen mit einer verminderten Ihh-Expression nachgewiesen werden. Zusätzlich konnte anhand von Röntgenaufnahmen eine Dysorganisation der nasalen Region und ein fehlendes nasales Septum beobachtet werden. Produktion und Mineralisation der extrazellulären Matrix waren nicht beeinträchtigt. Um die Rolle der BMP- und TGFbeta-Signalkaskaden während der endochondralen Ossifikation zu vergleichen, wurden transgene Mäuse generiert, in denen die TGFbeta-Signalkaskade spezifisch in proliferierenden Chondrozyten gestört war. Zwei Mauslinien, die ähnliche Phänotypen zeigten, wurden untersucht. Esl1 ist ein TGFbeta-bindendes Protein, von dem man annimmt, dass es die TGFbeta-Signalkaskade inhibieren kann. Esl1-knock out-Mäuse sind kleiner als Wildtypmäuse und die Überexpression von Esl1 in proliferierenden Chondrozyten führt zu einer Verlängerung der Wachstumsfuge und einer verstärkten Proliferationsrate. Knorpelmarker, wie Col2a1 und Sox9 sind in diesen Mäusen herunterreguliert, während Col10a1 und Ihh als Marker für die hypertrophe und prähypertrophe Zone herunterreguliert waren. Dies führt zu der Annahme, dass mehr Zellen in die terminale Differenzierung eintreten. Bei transgenen Mäusen, in denen ein dominant-negativer (dn) TGFbeta-Rezeptor in proliferierenden Chondrozyten überexprimiert wurde, konnte eine verlängerte prähypertrophe Zone, eine erhöhte Ihh-Expression, sowie eine verstärkte Proliferationsrate beobachtet werden. Zusätzlich konnte in homozygoten Tieren ein craniofacialer Phänotyp beschrieben werden, der zu Problemen bei der Nahrungsaufnahme und damit zu einer starken Wachstumsbeeinträchtigung führte. Die BMP- und TGFbeta-Signalkaskaden haben möglicherweise antagonistische Effekte in der Wachstumsfuge. Während der Ausfall von BMP in proliferierenden Chondrozyten aufgrund einer gesunkenen Proliferationsrate zu einer Verkürzung der Wachstumsfuge führte, kann man in Mäusen mit einer Störung der TGFbeta-Signalkaskade eine verstärkte Proliferation in einer daher verlängerten Wachstumsfuge beobachten. Ein weiteres Ziel dieser Arbeit war die Generation einer transgenen Mauslinie, die die Cre-Rekombinase spezifisch in hypertrophen Chondrozyten exprimiert. Promoterstudien mit transgenen Mäusen weisen darauf hin, dass ein putatives AP1-Element, etwa 4 kb vor dem ersten Exon des Col10a1 gelegen, wichtig für die spezifische Expression in hypertrophen Chondrozyten ist. Ein Konstrukt, dass vier Kopien dieses Elements und den basalen Promoter enthält, wurde benutzt, um die Cre-Rekombinase spezifisch zu exprimieren. Diese Mauslinie befindet sich in der Testphase und erste Daten deuten auf eine spezifische Expression der Cre-Rekombinase in hypertrophen Chondrozyten hin.

Geochemistry, petrogenesis and tectonic setting of ophiolites and mafic-ultramafic complexes in the Northeastern Aegean Region

In this study two ophiolites and a mafic-ultramafic complexes of the northeastern Aegean Sea, Greece, have been investigated to re-evaluate their petrogenetic evolution and tectonic setting. These complexes are: the mafic-ultramafic complex of Lesvos Island and the ophiolites of Samothraki Island and the Evros area. In order to examine these complexes in detail whole-rock major- and trace-elements as well as Sr and Nd isotopes, and minerals were analysed and U-Pb SHRIMP ages on zircons were determined. The mafic-ultramafic complex of Lesvos Island consists of mantle peridotite thrusted over a tectonic mélange containing metasediments, metabasalts and a few metagabbros. This succession had previously been interpreted as an ophiolite of Late Jurassic age. The new field and geochemical data allow a reinterpretation of this complex as representing an incipient continental rift setting that led to the subsequent formation of the Meliata-Maliac-Vardar branches of Neotethys in Upper Permian times (253 ± 6 Ma) and the term “Lesvos ophiolite” should be abandoned. With proceeding subduction and closure of the Maliac Ocean in Late Jurassic times (155 Ma) the Lesvos mafic-ultramafic complex was obducted. Zircon ages of 777, 539 and 338 Ma from a gabbro strongly suggest inheritance from the intruded basement and correspond to ages of distinct terranes recently recognized in the Hellenides (e.g. Florina terrane). Geochemical similar complexes which contain rift associations with Permo-Triassic ages can be found elsewhere in Greece and Turkey, namely the Teke Dere Thrust Sheet below the Lycian Nappes (SW Turkey), the Pindos subophiolitic mélange (W Greece), the Volcanosedimentary Complex on Central Evia Island (Greece) and the Karakaya Complex (NW Turkey). This infers that the rift-related rocks from Lesvos belong to an important Permo-Triassic rifting episode in the eastern Mediterranean. The ‘in-situ’ ophiolite of Samothraki Island comprises gabbros, sparse dykes and basalt flows as well as pillows cut by late dolerite dykes and had conventionally been interpreted as having formed in an ensialic back-arc basin. The results of this study revealed that none of the basalts and dolerites resemble mid-ocean ridge or back-arc basin basalts thus suggesting that the Samothraki ophiolite cannot represent mature back-arc basin crust. The age of the complex is regarded to be 160 ± 5 Ma (i.e. Oxfordian; early Upper Jurassic), which precludes any correlation with the Lesvos mafic-ultramafic complex further south (253 ± 6 Ma; Upper Permian). Restoration of the block configuration in NE Greece, before extensional collapse of the Hellenic hinterland and exhumation of the Rhodope Metamorphic Core Complex (mid-Eocene to mid-Miocene), results in a continuous ophiolite belt from Guevgueli in the NW to Samothraki in the SE, thus assigning the latter to the Innermost Hellenic Ophiolite Belt. In view of the data of this study, the Samothraki ophiolite represents a rift propagation of the Sithonia ophiolite spreading ridge into the Chortiatis calc-alkaline arc. The ophiolite of the Evros area consists of a plutonic sequence comprising cumulate and non-cumulate gabbros with plagiogranite veins, and an extrusive sequence of basalt dykes, massive and pillow lavas as well as pyroclastic rocks. Furthermore, in the Rhodope Massif tectonic lenses of harzburgites and dunites can be found. All rocks are spatially separated. The analytical results of this study revealed an intra-oceanic island arc setting for the Evros ophiolitic rocks. During late Middle Jurassic times (169 ± 2 Ma) an intra-oceanic arc has developed above a northwards directed intra-oceanic subduction zone of the Vardar Ocean in front of the Rhodope Massif. The boninitic, island arc tholeiitic and calc-alkaline rocks reflect the evolution of the Evros island arc. The obduction of the ophiolitic rocks onto the Rhodope basement margin took place during closure of the Vardar ocean basins. The harzburgites and dunites of the Rhodope Massif are strongly depleted and resemble harzburgites from recent oceanic island arcs. After melt extraction they underwent enrichment processes by percolating melts and fluids from the subducted slab. The relationship of the peridotites and the Evros ophiolite is still ambiguous, but the stratigraphic positions of the peridotites and the ophiolitic rocks indicate separated origin. The harzburgites and dunites most probably represent remnants of the mantle wedge of the island arc of the Rhodope terrane formed above subducted slab of the Nestos Ocean in late Middle Jurassic times. During collision of the Thracia terrane with the Rhodope terrane thrusting of the Rhodope terrane onto the Thracia terrane took place, whereas the harzburgites and dunites were pushed between the two terranes now cropping out on top of the Thracia terrane of the Rhodope Massif.

Soil biogenic emissions of nitric oxide from arid and semi-arid ecosystems

The biogenic production of NO in the soil accounts for between 10% and 40% of the global total. A large degree of the uncertainty in the estimation of the biogenic emissions stems from a shortage of measurements in arid regions, which comprise 40% of the earth’s land surface area. This study examined the emission of NO from three ecosystems in southern Africa which cover an aridity gradient from semi-arid savannas in South Africa to the hyper-arid Namib Desert in Namibia. A laboratory method was used to determine the release of NO as a function of the soil moisture and the soil temperature. Various methods were used to up-scale the net potential NO emissions determined in the laboratory to the vegetation patch, landscape or regional level. The importance of landscape, vegetation and climatic characteristics is emphasized. The first study occurred in a semi-arid savanna region in South Africa, where soils were sampled from 4 landscape positions in the Kruger National Park. The maximum NO emission occurred at soil moisture contents of 10%-20% water filled pore space (WFPS). The highest net potential NO emissions came from the low lying landscape positions, which have the largest nitrogen (N) stocks and the largest input of N. Net potential NO fluxes obtained in the laboratory were converted in field fluxes for the period 2003-2005, for the four landscape positions, using soil moisture and temperature data obtained in situ at the Kruger National Park Flux Tower Site. The NO emissions ranged from 1.5-8.5 kg ha-1 a-1. The field fluxes were up-scaled to a regional basis using geographic information system (GIS) based techniques, this indicated that the highest NO emissions occurred from the Midslope positions due to their large geographical extent in the research area. Total emissions ranged from 20x103 kg in 2004 to 34x103 kg in 2003 for the 56000 ha Skukuza land type. The second study occurred in an arid savanna ecosystem in the Kalahari, Botswana. In this study I collected soils from four differing vegetation patch types including: Pan, Annual Grassland, Perennial Grassland and Bush Encroached patches. The maximum net potential NO fluxes ranged from 0.27 ng m-2 s-1 in the Pan patches to 2.95 ng m-2 s-1 in the Perennial Grassland patches. The net potential NO emissions were up-scaled for the year December 2005-November 2006. This was done using 1) the net potential NO emissions determined in the laboratory, 2) the vegetation patch distribution obtained from LANDSAT NDVI measurements 3) estimated soil moisture contents obtained from ENVISAT ASAR measurements and 4) soil surface temperature measurements using MODIS 8 day land surface temperature measurements. This up-scaling procedure gave NO fluxes which ranged from 1.8 g ha-1 month-1 in the winter months (June and July) to 323 g ha-1 month-1 in the summer months (January-March). Differences occurred between the vegetation patches where the highest NO fluxes occurred in the Perennial Grassland patches and the lowest in the Pan patches. Over the course of the year the mean up-scaled NO emission for the studied region was 0.54 kg ha-1 a-1 and accounts for a loss of approximately 7.4% of the estimated N input to the region. The third study occurred in the hyper-arid Namib Desert in Namibia. Soils were sampled from three ecosystems; Dunes, Gravel Plains and the Riparian zone of the Kuiseb River. The net potential NO flux measured in the laboratory was used to estimate the NO flux for the Namib Desert for 2006 using modelled soil moisture and temperature data from the European Centre for Medium Range Weather Forecasts (ECMWF) operational model on a 36km x 35km spatial resolution. The maximum net potential NO production occurred at low soil moisture contents (<10%WFPS) and the optimal temperature was 25°C in the Dune and Riparian ecosystems and 35°C in the Gravel Plain Ecosystems. The maximum net potential NO fluxes ranged from 3.0 ng m-2 s-1 in the Riparian ecosystem to 6.2 ng m-2 s-1 in the Gravel Plains ecosystem. Up-scaling the net potential NO flux gave NO fluxes of up to 0.062 kg ha-1 a-1 in the Dune ecosystem and 0.544 kg h-1 a-1 in the Gravel Plain ecosystem. From these studies it is shown that NO is emitted ubiquitously from terrestrial ecosystems, as such the NO emission potential from deserts and scrublands should be taken into account in the global NO models. The emission of NO is influenced by various factors such as landscape, vegetation and climate. This study looks at the potential emissions from certain arid and semi-arid environments in southern Africa and other parts of the world and discusses some of the important factors controlling the emission of NO from the soil.

UHP metamorphic rocks of the Eastern Rhodope Massif, NE Greece: new constraints from petrology, geochemistry and zircon ages

In this PhD thesis, a multidisciplinary study has been carried out on metagranitoids and paragneisses from the Eastern Rhodope Massif, northern Greece, to decipher the pre-Alpine magmatic and geodynamic evolution of the Rhodope Massif and to correlate the eastern part with the western/central parts of the orogen. The Rhodope Massif, which occupies the major part of NE Greece and S Bulgaria, represents the easternmost part of the Internal Hellenides. It is regarded as a nappe stack of high-grade units, which is classically subdivided into an upper unit and a lower unit, separated by a SSE-NNW trending thrust plane, the Nestos thrust. Recent research in the central Greek Rhodope Massif revealed that the two units correspond to two distinct terranes of different age, the Permo-Carboniferous Thracia Terrane, which was overthrusted by the Late Jurassic/Early Cretaceous Rhodope Terrane. These terranes are separated by the Nestos suture, a composite zone comprising metapelites, metabasites, metagranitoids and marbles, which record high-pressure and even ultrahigh-pressure metamorphism in places. Similar characteristic rock associations were investigated during this study along several well-constrained cross sections in vincity to the Ada, Sidiro and Kimi villages in the Greek Eastern Rhodope Massif. Field evidence revealed that the contact zone of the two terranes in the Eastern Rhodope Massif is characterized by a mélange of metapelites, migmatitic amphibolites/eclogites, strongly sheared orthogneisses and marbles. The systematical occurrence of this characteristic rock association between the terranes implies that the Nestos suture is a continuous belt throughout the Greek Rhodope Massif. In this study, a new UHP locality could be established and for the first time in the Greek Rhodope, metamorphic microdiamonds were identified in situ in their host zircons using Laser-Raman spectroscopy. The presence of the diamonds as well as element distribution patterns of the zircons, obtained by TOF-SIMS, indicate metamorphic conditions of T > 1000 °C and P > 4 GPa. The high-pressure and ultrahigh-pressure rocks of the mélange zone are considered to have formed during the subduction of the Nestos Ocean in Jurassic times at ~150 Ma. Melting of metapelitic rocks at UHP conditions facilitated the exhumation to lower crustal levels. To identify major crust forming events, basement granitoids were dated by LA-SF-ICPMS and SHRIMP-II U-Pb analyses of zircons. The geochronological results revealed that the Eastern Rhodope Massif consists of two crustal units, a structurally lower Permo-Carboniferous unit corresponding to the Thracia Terrane and a structurally upper Late Jurassic/Early Cretaceous unit corresponding to the Rhodope Terrane, like it was documented for the Central Rhodope Massif. Inherited zircons in the orthogneisses from the Thracia Terrane of the Eastern Rhodope Massif indicate the presence of a pre-existing Neoproterozoic and Ordovician-Silurian basement in this region. Triassic magmatism is witnessed by the zircons of few orthogneisses from the easternmost Rhodope Massif and is interpreted to be related to rifting processes. Whole-rock major and trace element analyses indicate that the metagranitoids from both terranes originated in a subduction-related magmatic-arc environment. The Sr-Nd isotope data for both terranes of the Eastern and Central Rhodope Massif suggest a mixed crust-mantle source with variable contributions of older crustal material as already indicated by the presence of inherited zircons. Geochemical and isotopic similarity of the basement of the Thracia Terrane and the Pelagonian Zone implies that the Thracia Terrane is a fragment of a formerly unique Permo-Carboniferous basement, separated by rifting and opening of the Meliata-Maliac ocean system in Triassic times. A branch of the Meliata-Maliac ocean system, the Nestos Ocean, subducted northwards in Late Jurassic times leading to the formation of the Late Jurassic/Early Cretaceous Rhodope magmatic arc on remnants of the Thracia Terrane as suggested by inherited Permo-Carboniferous zircons. The ~150 Ma zircon ages of the orthogneisses from the Rhodope Terrane indicate that subduction-related magmatism and HP/UHP metamorphism occurred during the same subduction phase. Subduction ceased due to the closure of the Nestos Ocean in the Late Jurassic/Early Cretaceous. The post-Jurassic evolution of the Rhodope Massif is characterized by the exhumation of the Rhodope core complex in the course of extensional tectonics associated with late granite intrusions in Eocene to Miocene times.