Target genes of ß-catenin in early mouse limb development

Untersuchungen der letzten Jahre führten zum ß-catenin/Wnt-Signalweg als möglichen Schlüssel zur Kontrolle der Differenzierung von mesenchymalen Zellen zu Knorpelzellen. Stabilisierung von ß-catenin in der frühen Gliedmaßenanlage scheint die Expression des Transkriptionsfaktors Sox9 und damit die Differenzierung in Knorpelzellen zu inhibieren. Es ist jedoch unklar wie diese Kontrolle von Sox9 durch ß-catenin/Wnt erfolgt. Diese Diplomarbeit ist Teil eines Projektes das zum Ziel hat den Mechanismus dieser Kontrolle aufzuklären. Dazu sollen ß-catenin/Wnt-regulierte Gene der frühen Gliedmaßenentwicklung identifiziert und auf einen möglichen Einfluss auf die Sox9-Expression getestet werden. Am Beginn dieser Diplomarbeit standen bereits Daten einer Genexpres-sionsstudie zur Verfügung. Nach Analyse dieser Daten wurden 48 mögliche Kanditatengene mittels In-situ-Hybridisierung bzw. RT-PCR validiert. Es konnten so 10 Gene identifiziert werden, welche in der Gliedmaßenanlage von Embryonen, die konditionell stabilisiertes ß-catenin expremieren, hochreguliert sind. Darunter befanden sich die Transkriptionsfaktoren Tcf4 (Itf2), Irx5 und Fra2 (Fosl2). Die Promotoren dieser Gene wurden in Luciferase Reporter Assays auf ihre ß-catenin/Wnt Abhängigkeit untersucht. Weiteres wurden diese Gene in Luciferase Assays mit einem Sox9-Promoter-Reporterkonstrukt getestet. Um Hinweise auf die Funktion der Gene in vivo zu erhalten wurden die kodierenenden Regionen der Gene in einen Vektor kloniert, welcher eine transiente Überexpression in transgenen Embryos erlauben sollte. Für das Gen Tcf4 wurde eine erste Runde von Injektionen durchgeführt. Diese lieferten jedoch nur einen transgenen Embryo, welcher jedoch einen Phenotyp in der Gliedmaßenanlage zeigte. Durch das zeitlich bedingte Ende der Arbeit waren leider keine weiteren Analysen möglich. Zusammenfassend wurden in dieser Arbeit eine Anzahl möglicher Zielgene des ß-catenin/Wnt-Signalweges identifiziert. Für zwei davon Irx5 und Tcf4 ist eine direkte Abhängigkeit vom ß-catenin/Wnt Signalweg sehr wahrscheinlich. Weiters hatten beide Gene das Potential die Sox9-Expression zumindest in vitro zu inhibieren. Jedoch wären weitere Untersuchungen notwendig um eine mögliche Funktion dieser Gene in der Knorpelzellendifferenzierung bzw. Gliedmaßenentwicklung aufzudecken.In recent years Wnt-Signaling has been implicated to play an important role in the control of differentiation of mesenchymal cells to chondrocytes. A key observation has been that stabilization of β-catenin in the early limb bud mesenchyme represses differentiation of mesenchymal cells into skeletal precursors. Probably this is mediated by transcriptional repression of the transcription factor Sox9, which is a master regulator in chondrocyte differentiation. The object of this thesis was to identify putative mediators of this Sox9 repression. Target genes of canonical Wnt signaling in limb bud development were identified and tested for their potential to repress Sox9. To do so I used gene expression data already available. I validated 48 candidate genes suggested by the data analysis using in situ hybridisation and semi-quantitative RT-PCR, respectively. 10 genes could be confirmed as being positively regulated in the limbs of ß-catenin gain of function mice. For three of them, the transcription factors Tcf4 (Itf2), Irx5 and Fosl2 (FRA2), I cloned parts of their promoters into luciferase reporter vectors and tested their response to ß-catenin/Wnt-signaling. Furthermore I cloned their cDNAs and tested their effect on part of the Sox9 promoter in luciferase reporter assays. For Tcf4 and Irx5 I could confirm their positive response to canonical Wnt-signaling, as well as their potential to repress Sox9 in vitro. To gain insight into the in vivo function of these genes, constructs for specific overexpression in the early limb bud mesenchyme have been generated. By the end of the thesis work one round of pronucleus injections has been carried out for the gene Tcf4, which yielded in one transgenic embryo with a strong limb phenotype. Unfortunately further analysis was not carried out due to the fact of the diploma work had ended. In conclusion this work suggests a number of new genes as targets of Wnt/ß-catenin signaling in limb development. For two of them Irx5 and Tcf4 I established a possible direct regulation by the ß-catenin/Wnt-pathway and that they have the potential to inhibit Sox9 at least in vitro. Further studies would now be necessary to examine whether they also excert a negative effect on Sox9 in vivo and to establish their potential functions during limb development

Оценка эффективности комбинированной терапии у больных артериальной гипертензией

Представлены результаты исследования воздействия комбинации антигипертензивного препарата аккупро и психотропного препарата золофта на клинические показатели больных артериальной гипертензией и их психологическое состояние и качество жизни. Показана высокая эффективность комбинированной терапии.The findings of the research of the effect of combination of an antihypertensive drug Accupro and a psychotropic drug Zoloft on clinical parameters in patients with arterial hypertension as well as their mental state and quality of life are presented. A high efficacy of the combined therapy is shown

Entpd5 is essential for skeletal mineralization and regulates phosphate homeostasis in zebrafish

Bone mineralization is an essential step during the embryonic development of vertebrates, and bone serves vital functions in human physiology. To systematically identify unique gene functions essential for osteogenesis, we performed a forward genetic screen in zebrafish and isolated a mutant, no bone (nob), that does not form any mineralized bone. Positional cloning of nob identified the causative gene to encode ectonucleoside triphosphate/diphosphohydrolase 5 (entpd5); analysis of its expression pattern demonstrates that entpd5 is specifically expressed in osteoblasts. An additional mutant, dragonfish (dgf), exhibits ectopic mineralization in the craniofacial and axial skeleton and encodes a loss-of-function allele of ectonucleotide pyrophosphatase phosphodiesterase 1 (enpp1). Intriguingly, generation of double-mutant nob/dgf embryos restored skeletal mineralization in nob mutants, indicating that mechanistically, Entpd5 and Enpp1 act as reciprocal regulators of phosphate/pyrophosphate homeostasis in vivo. Consistent with this, entpd5 mutant embryos can be rescued by high levels of inorganic phosphate, and phosphate-regulating factors, such as fgf23 and npt2a, are significantly affected in entpd5 mutant embryos. Our study demonstrates that Entpd5 represents a previously unappreciated essential player in phosphate homeostasis and skeletal mineralization

Zebrafish enpp1 mutants exhibit pathological mineralization, mimicking features of generalized arterial calcification of infancy (GACI) and pseudoxanthoma elasticum (PXE)

In recent years it has become clear that, mechanistically, biomineralization is a process that has to be actively inhibited as a default state. This inhibition must be released in a rigidly controlled manner in order for mineralization to occur in skeletal elements and teeth. A central aspect of this concept is the tightly controlled balance between phosphate, a constituent of the biomineral hydroxyapatite, and pyrophosphate, a physiochemical inhibitor of mineralization. Here, we provide a detailed analysis of a zebrafish mutant, dragonfish (dgf), which is mutant for ectonucleoside pyrophosphatase/phosphodiesterase 1 (Enpp1), a protein that is crucial for supplying extracellular pyrophosphate. Generalized arterial calcification of infancy (GACI) is a fatal human disease, and the majority of cases are thought to be caused by mutations in ENPP1. Furthermore, some cases of pseudoxanthoma elasticum (PXE) have recently been linked to ENPP1. Similar to humans, we show here that zebrafish enpp1 mutants can develop ectopic calcifications in a variety of soft tissues - most notably in the skin, cartilage elements, the heart, intracranial space and the notochord sheet. Using transgenic reporter lines, we demonstrate that ectopic mineralizations in these tissues occur independently of the expression of typical osteoblast or cartilage markers. Intriguingly, we detect cells expressing the osteoclast markers Trap and CathepsinK at sites of ectopic calcification at time points when osteoclasts are not yet present in wild-type siblings. Treatment with the bisphosphonate etidronate rescues aspects of the dgf phenotype, and we detected deregulated expression of genes that are involved in phosphate homeostasis and mineralization, such as fgf23, npt2a, entpd5 and spp1 (also known as osteopontin). Employing a UAS-GalFF approach, we show that forced expression of enpp1 in blood vessels or the floorplate of mutant embryos is sufficient to rescue the notochord mineralization phenotype. This indicates that enpp1 can exert its function in tissues that are remote from its site of expression

Segmentation of the zebrafish axial skeleton relies on notochord sheath cells and not on the segmentation clock

Contains fulltext : 198218.pdf (publisher's version ) (Open Access