Die Transkriptions-Regulation des EGFR-Zielgens pipe und Analyse der KASH- und der SUN-Domänen-Proteine in der Oogenese von Drosophila melanogaster

Diese Arbeit gliedert sich in zwei Teile. Der erste Teil befasst sich mit der Transkriptionsregulation des Gens pipe, dessen spezifische Expression in ventralen Follikelzellen der Eikammer für die Bildung der dorsoventralen Achse des Drosophila-Embryos entscheidend ist. Die Repression der pipe Expression in dorsalen Follikelzellen hängt von der Aktivierung des EGF-Rezeptors durch den, in der Oocyte asymmetrisch lokalisierten, TGF-alpha-artigen Liganden Gurken ab. Der Einfluss verschiedener Kandidaten der pipe Transkriptionsregulation wurde von uns mittels klonaler Analyse getestet. Dabei zeigte sich, dass alle EGFR-regulierten Transkriptionsfaktoren, für die eine mögliche Rolle bei der Kontrolle der pipe Expression vermutet worden ist, nicht für die EGFR-Signal vermittelte, dorsale Repression von pipe verantwortlich sind. Parallel zu diesen Experimenten wurde die cis-regulatorische Region von pipe außerdem mit Hilfe bioinformatischer Methoden, basierend auf der evolutionären Konservierung funktionaler Elemente (phylogenetic footprinting), sowie durch die Analyse einer Reihe von Promotor-Reporter-Konstrukten untersucht. Dadurch konnten wir ein cis-regulatorisches Element von 31bp identifizieren, welches für die dorsale Repression von pipe eine essentielle Rolle spielt. In Gelretardationsexperimenten konnten wir in vitro eine spezifische Bindung von Proteinen aus Extrakten von Ovarien an dieses Element nachweisen. Der zweite Teil der Arbeit befasst sich mit der Analyse der KASH (Klarsicht/Anc-1/Syne Homologie)- und SUN (Sad1/UNC-84)-Domänen-Proteine in Drosophila. KASH-Domänen-Proteine anderer Organismen lokalisieren an der äußeren Kernmembran und spielen unter anderem eine Rolle bei der Verknüpfung des Zellkerns mit Cytoskelett-Elementen; wir waren daher insbesondere an einer möglichen Funktion dieser Proteine bei der Kernpositionierung in der Oogenese interessiert. In C elegans und in Vertebraten hängt die Membran-Lokalisation der KASH-Proteine von einer direkten Interaktion mit den SUN-Proteinen ab, die in der inneren Kernmembran lokalisiert sind. In Drosophila findet man zwei KASH-Proteine, Msp-300 und Klarsicht (Klar), sowie zwei SUN-Proteine CG18584/CG3287 (auch Klaroid, Koi) und CG6589, von denen CG6589 jedoch nur in Männchen exprimiert wird. Bisher ist nur Klar intensiv untersucht worden, welches unter anderem eine Rolle bei der Kernwanderung während der Augenentwicklung spielt. Wir haben Deletionen von Msp-300 und CG18584/CG3287 generiert. Die Gesamtdeletion von Msp-300 führt zu larvaler Letalität, Teildeletionen deuten darauf hin, dass Msp-300-Protein-Isoformen ohne eine KASH-Domäne in Drosophila eine essentielle Funktion haben. Msp-300 und Klar werden während der Oogenese exprimiert und lokalisieren in den Nährzellen und der Oocyte an der Kernhülle. Der gleichzeitige Funktionsverlust beider KASH- Proteine hat jedoch keine Auswirkungen auf die Oogenese. Damit konnten wir eine frühere Veröffentlichung widerlegen, die eine Funktion von Msp-300 bei der Positionierung der Nährzellkerne aufzeigt. Die Deletion des SUN-Homologs CG18584/CG3287 führt zu einem Verlust der Kernmembran-Lokalisation beider KASH-Proteine. Die Funktion der SUN-Proteine bei der Lokalisation der KASH-Proteine ist somit auch in Drosophila konserviert. Die Deletion des SUN-Domänen-Homologs ist semilethal und homozygote Fliegen zeigen einen klar-identischen Augenphänotyp, die Oogenese ist in homozygoten Weibchen jedoch nicht betroffen. Auch CG18584/CG3287 hat somit keine essentielle Funktion in der Oogenese

Early mortality and loss to follow-up in HIV-infected children starting antiretroviral therapy in Southern Africa.

BACKGROUND: Many HIV-infected children in Southern Africa have been started on antiretroviral therapy (ART), but loss to follow up (LTFU) can be substantial. We analyzed mortality in children retained in care and in all children starting ART, taking LTFU into account. PATIENTS AND METHODS: Children who started ART before the age of 16 years in 10 ART programs in South Africa, Malawi, Mozambique, and Zimbabwe were included. Risk factors for death in the first year of ART were identified in Weibull models. A meta-analytic approach was used to estimate cumulative mortality at 1 year. RESULTS: Eight thousand two hundred twenty-five children (median age 49 months, median CD4 cell percent 11.6%) were included; 391 (4.8%) died and 523 (7.0%) were LTFU in the first year. Mortality at 1 year was 4.5% [95% confidence interval (CI): 2.8% to 7.4%] in children remaining in care, but 8.7% (5.4% to 12.1%) at the program level, after taking mortality in children and LTFU into account. Factors associated with mortality in children remaining in care included age [adjusted hazard ratio (HR) 0.37; 95% CI: 0.25 to 0.54 comparing > or =120 months with <18 months], CD4 cell percent (HR: 0.56; 95% CI: 0.39 to 0.78 comparing > or =20% with <10%), and clinical stage (HR: 0.12; 95% CI: 0.03 to 0.45 comparing World Health Organization stage I with III/IV). CONCLUSIONS: In children starting ART and remaining in care in Southern Africa mortality at 1 year is <5% but almost twice as high at the program level, when taking LTFU into account. Age, CD4 percentage, and clinical stage are important predictors of mortality at the individual level

Nervous systems of the sea anemone Nematostella vectensis are generated by ectoderm and endoderm and shaped by distinct mechanisms

As a sister group to Bilateria, Cnidaria is important for understanding early nervous system evolution. Here we examine neural development in the anthozoan cnidarian Nematostella vectensis in order to better understand whether similar developmental mechanisms are utilized to establish the strikingly different overall organization of bilaterian and cnidarian nervous systems. We generated a neuron-specific transgenic NvElav1 reporter line of N. vectensis and used it in combination with immunohistochemistry against neuropeptides, in situ hybridization and confocal microscopy to analyze nervous system formation in this cnidarian model organism in detail. We show that the development of neurons commences in the ectoderm during gastrulation and involves interkinetic nuclear migration. Transplantation experiments reveal that sensory and ganglion cells are autonomously generated by the ectoderm. In contrast to bilaterians, neurons are also generated throughout the endoderm during planula stages. Morpholino-mediated gene knockdown shows that the development of a subset of ectodermal neurons requires NvElav1, the ortholog to bilaterian neural elav1 genes. The orientation of ectodermal neurites changes during planula development from longitudinal (in early-born neurons) to transverse (in late-born neurons), whereas endodermal neurites can grow in both orientations at any stage. Our findings imply that elav1-dependent ectodermal neurogenesis evolved prior to the divergence of Cnidaria and Bilateria. Moreover, they suggest that, in contrast to bilaterians, almost the entire ectoderm and endoderm of the body column of Nematostella planulae have neurogenic potential and that the establishment of connectivity in its seemingly simple nervous system involves multiple neurite guidance systems

Incorporation of a Horizontally Transferred Gene into an Operon during Cnidarian Evolution

Genome sequencing has revealed examples of horizontally transferred genes, but we still know little about how such genes are incorporated into their host genomes. We have previously reported the identification of a gene (flp) that appears to have entered the Hydra genome through horizontal transfer. Here we provide additional evidence in support of our original hypothesis that the transfer was from a unicellular organism, and we show that the transfer occurred in an ancestor of two medusozoan cnidarian species. In addition we show that the gene is part of a bicistronic operon in the Hydra genome. These findings identify a new animal phylum in which trans-spliced leader addition has led to the formation of operons, and define the requirements for evolution of an operon in Hydra. The identification of operons in Hydra also provides a tool that can be exploited in the construction of transgenic Hydra strains

Diversity, Phylogeny and Expression Patterns of Pou and Six Homeodomain Transcription Factors in Hydrozoan Jellyfish Craspedacusta sowerbyi

Formation of all metazoan bodies is controlled by a group of selector genes including homeobox genes, highly conserved across the entire animal kingdom. The homeobox genes from Pou and Six classes are key members of the regulation cascades determining development of sensory organs, nervous system, gonads and muscles. Besides using common bilaterian models, more attention has recently been targeted at the identification and characterization of these genes within the basal metazoan phyla. Cnidaria as a diploblastic sister group to bilateria with simple and yet specialized organs are suitable models for studies on the sensory organ origin and the associated role of homeobox genes. In this work, Pou and Six homeobox genes, together with a broad range of other sensory-specific transcription factors, were identified in the transcriptome of hydrozoan jellyfish Craspedacusta sowerbyi. Phylogenetic analyses of Pou and Six proteins revealed cnidarian-specific sequence motifs and contributed to the classification of individual factors. The majority of the Craspedacusta sowerbyi Pou and Six homeobox genes are predominantly expressed in statocysts, manubrium and nerve ring, the tissues with sensory and nervous activities. The described diversity and expression patterns of Pou and Six factors in hydrozoan jellyfish highlight their evolutionarily conserved functions. This study extends the knowledge of the cnidarian genome complexity and shows that the transcriptome of hydrozoan jellyfish is generally rich in homeodomain transcription factors employed in the regulation of sensory and nervous functions

Molecular mechanisms of EGF signaling-dependent regulation of pipe, a gene crucial for dorsoventral axis formation in Drosophila

During Drosophila oogenesis the expression of the sulfotransferase Pipe in ventral follicle cells is crucial for dorsoventral axis formation. Pipe modifies proteins that are incorporated in the ventral eggshell and activate Toll signaling which in turn initiates embryonic dorsoventral patterning. Ventral pipe expression is the result of an oocyte-derived EGF signal which down-regulates pipe in dorsal follicle cells. The analysis of mutant follicle cell clones reveals that none of the transcription factors known to act downstream of EGF signaling in Drosophila is required or sufficient for pipe regulation. However, the pipe cis-regulatory region harbors a 31-bp element which is essential for pipe repression, and ovarian extracts contain a protein that binds this element. Thus, EGF signaling does not act by down-regulating an activator of pipe as previously suggested but rather by activating a repressor. Surprisingly, this repressor acts independent of the common co-repressors Groucho or CtBP

An NF-κB and Slug Regulatory Loop Active in Early Vertebrate Mesoderm

BACKGROUND: In both Drosophila and the mouse, the zinc finger transcription factor Snail is required for mesoderm formation; its vertebrate paralog Slug (Snai2) appears to be required for neural crest formation in the chick and the clawed frog Xenopus laevis. Both Slug and Snail act to induce epithelial to mesenchymal transition (EMT) and to suppress apoptosis. METHODOLOGY & PRINCIPLE FINDINGS: Morpholino-based loss of function studies indicate that Slug is required for the normal expression of both mesodermal and neural crest markers in X. laevis. Both phenotypes are rescued by injection of RNA encoding the anti-apoptotic protein Bcl-xL; Bcl-xL's effects are dependent upon IκB kinase-mediated activation of the bipartite transcription factor NF-κB. NF-κB, in turn, directly up-regulates levels of Slug and Snail RNAs. Slug indirectly up-regulates levels of RNAs encoding the NF-κB subunit proteins RelA, Rel2, and Rel3, and directly down-regulates levels of the pro-apopotic Caspase-9 RNA. CONCLUSIONS/SIGNIFICANCE: These studies reveal a Slug/Snail–NF-κB regulatory circuit, analogous to that present in the early Drosophila embryo, active during mesodermal formation in Xenopus. This is a regulatory interaction of significance both in development and in the course of inflammatory and metastatic disease