Transcriptional regulation of Drosophila gonad formation

The formation of the Drosophila embryonic gonad, involving the fusion of clusters of somatic gonadal precursor cells (SGPs) and their ensheathment of germ cells, provides a simple and genetically tractable model for the interplay between cells during organ formation. In a screen for mutants affecting gonad formation we identified a SGP cell autonomous role for Midline (Mid) and Longitudinals lacking (Lola). These transcriptional factors are required for multiple aspects of SGP behaviour including SGP cluster fusion, germ cell ensheathment and gonad compaction. The lola locus encodes more than 25 differentially spliced isoforms and we have identified an isoform specific requirement for lola in the gonad which is distinct from that in nervous system development. Mid and Lola work in parallel in gonad formation and surprisingly Mid overexpression in a lola background leads to additional SGPs at the expense of fat body cells. Our findings support the idea that although the transcription factors required by SGPs can ostensibly be assigned to those being required for either SGP specification or behaviour, they can also interact to impinge on both processes

Identifizierung und funktionale Analyse von Genen, welche die Keimzellmigration und Gonadenentwicklung kontrollieren

Gonad formation in Drosophila involves the intricate movements of two cell types, the germ cells and the somatic gonadal precursor cells (SGPs), to generate a simple three-dimensional structure. The germ cells must firstly migrate to SGP clusters, which in turn fuse and ensheath the germ cells and the two cell types ultimately coalesce. Underlying this seemingly simple process is an array of genes required to guide germ cells to the SGPs. Additional complex transcriptional networks in SGPs are essential for their specification, maintenance and to initiate their ensheathment behavior. This system represents a good model to study organogenesis using essentially two cell types. In this thesis I have examined aspects of both germ cell migration and SGP behavior. Firstly I examined the role of the Drosophila lipid phosphate phosphatases, Wun and Wun2 (referred to collectively as Wunens), in guiding the migration of germ cells. These multi-pass transmembrane enzymes, expressed both in the germ cells and the embryonic somatic cells, are thought to create an extracellular gradient of lipid phosphate, which is required for both migration and survival of the germ cells. However in order to form this extracellular lipid gradient, Wunens would be expected to act as ecto-enzymes. To investigate the site of requirement of Wun2 activity, I examined the localization of endogenous and different tagged versions of Wun2. Although both the GFP and the Myc tagged Wun2 were functional, the GFP version replicated the endogenous Wun2 plasma membrane localization. In parallel I made a construct in which a TEV protease cleavage site was inserted in a predicted extracellular loop of Wun2. I demonstrated that secretion of TEV protease by neighboring cells reduced the ability of this construct, to rescue wunen loss of function in germ cells. Taken together these data support the notion that the cell surface localization and the ecto-phosphatase activity of Wunens is critical for germ cell migration and survival. Secondly I have characterized the role of three mutants in which gonad formation is defective. In these mutants, although early germ cell migration is unaffected, in later embryos germ cells are scattered and SGP clusters do not fuse or compact. Whole genome sequencing coupled to deficiency complementation was performed to determine the causative mutations in two of the mutants. I identified mutations in two transcription factors, Midline (Mid) and Longitudinals lacking (Lola) and showed that both genes are expressed by SGPs and are required cell autonomously. The mutation in lola affects only one (lola-R) of the 30 predicted lola isoforms. Whilst the SGP defects resulting from mutation of Lola-R could be rescued by expression of this Lola isoform, another functional Lola isoform (Lola-BC) was unable to do so. Therefore, this study identifies the first lola-R specific allele, and describes a role in gonad formation that cannot be substituted by other Lola isoforms. Additionally, I have shown that robust gonad expression of another transcription factor Traffic jam is lost in mid mutants, placing Mid into a SGP transcriptional cascade. Lastly, mid and lola interact genetically suggesting that they could have one or more overlapping downstream targets.Die Morphogenese der Gonaden in Drosophila beinhaltet die verworrene Bewegung zweier Zelltypen, der Keimzellen und der somatischen Gonadenvorläuferzellen (somatic gonadal precursor cells, SGPs), um schließlich eine einfache dreidimensionale Struktur zu bilden. Die Keimzellen müssen als erstes zu den SGP-Clustern migrieren, die wiederum die Keimzellen umschließen und sich mit ihnen zu einer Gonade verdichten. Diesem scheinbar einfachen Prozess liegen eine Reihe von Genen zugrunde, die notwendig sind, um die Keimzellen zu den SGPs zu leiten. Außerdem sind komplexe Transkriptionsnetzwerke in den SGPs notwendig, um deren Spezifizierung und ihre Erhaltung zu gewährleisten, sowie um die Umhüllung der Keimzellen zu initiieren. Dieses System stellt ein gutes Model dar, um die Organogenese mit nur zwei Zelltypen zu untersuchen. In dieser Arbeit habe ich sowohl Aspekte der Keimzellwanderung, als auch das Verhalten der SGPs untersucht. Als erstes habe ich die Rolle der Drosophila Lipid-Phosphat-Phosphatasen Wun und Wun2 (zusammen bezeichnet als Wunen) bei der Steuerung der Keimzellmigration untersucht. Diese Transmembranenzyme, welche die Membran mehrfach durchspannenden, die sowohl in den Keimzellen, als auch in den somatischen Zellen des Embryos exprimiert werden, wird angenommen, dass sie einen extrazellulären Lipid-Phosphat-Gradienten aufbauen, der sowohl für die Migration, als auch für das Überleben der Keimzellen notwendig ist. Um jedoch diesen extrazellulären Gradienten zu erzeugen, würde erwartet werden, dass Wunen als Ektoenzyme agieren. Um zu ermitteln, an welchem Ort die Wun2-Aktivität benötigt wird, habe ich die Lokalisation von endogenen und unterschiedlich markierten Versionen von Wun2 untersucht. Obwohl sowohl GFP- als auch Myc-markiertes Wun2 funktional waren, hat nur die GFP-Version die endogene Wun2 Plasmamembran-Lokalisation gezeigt. Außerdem habe ich ein Konstrukt hergestellt, in welches eine von der TEV-Protease erkannte Schnittstelle in eine prognostizierte extrazelluläre Schleife von Wun2 eingefügt wurde. Ich habe dabei gezeigt, dass die Sekretion von TEV-Protease durch benachbarte Zellen, die Fähigkeit dieses Konstrukts den wunen Funktionsverlust in den Keimzellen zu retten, verringert. Zusammengefasst unterstützen diese Daten die Auffassung, dass die Lokalisation an der Zelloberfläche und die Ekto-Phosphataseaktivität von Wunen entscheidend für die Keimzellmigration und deren Überleben sind. Als zweites habe ich die Rolle von drei Mutanten charakterisiert, in welchen die Gonadenentwicklung fehlerhaft ist. Obwohl in diesen Mutanten die frühe Keimzellmigration unbeeinträchtigt ist, sind die Keimzellen in älteren Embryonen verstreut und die SGP-Cluster fusionieren oder komprimieren sich nicht. Mittels whole-genome-sequencing und Defizienz-Komplementation wurden die zugrundeliegenden Mutationen in zwei der Mutanten untersucht. Dabei habe ich Mutationen in zwei Transkriptionsfaktoren, Midline (Mid) und Longitudinals lacking (Lola), identifiziert und gezeigt, dass beide Gene von SGPs exprimiert werden und zellautonom erforderlich sind. Die Mutation in lola beeinflusst lediglich eine (lola-R) der 30 prognostizierten lola-Isoformen. Während die SGP-Defekte, die aus der Mutation von Lola-R resultieren, durch die Expression dieser Lola-Isoform gerettet werden konnten , war eine andere funktionale Lola-Isoform (Lola-BC) nicht dazu in der Lage. Demzufolge identifiziert diese Studie das erste lola-R spezifische Allel und beschreibt eine Rolle in der Gonadenentwicklung, die durch andere Lola-Isoformen nicht ersetzt werden kann. Darüberhinaus habe ich gezeigt, dass die stabile gonadische Expression eines anderen Transkriptionsfaktors, Traffic jam, in mid Mutanten verloren geht, was demzufolge Mid in eine SGP-spezifischen Transkriptionskaskade einfügt. Zuletzt interagieren mid und lola genetisch, was suggeriert, dass sie ein oder mehrere gemeinsame stromabwärts –liegende Ziele haben könnten

Wunen, a Drosophila lipid phosphate phosphatase, is required for septate junction-mediated barrier function

Lipid phosphate phosphatases (LPPs) are integral membrane enzymes that regulate the levels of bioactive lipids such as sphingosine 1-phosphate and lysophosphatidic acid. The Drosophila LPPs Wunen (Wun) and Wunen-2 (Wun2) have a well-established role in regulating the survival and migration of germ cells. We now show that wun has an essential tissue-autonomous role in development of the trachea: the catalytic activity of Wun is required to maintain septate junction (SJ) paracellular barrier function, loss of which causes failure to accumulate crucial luminal components, suggesting a role for phospholipids in SJ function. We find that the integrity of the blood-brain barrier is also lost in wun mutants, indicating that loss of SJ function is not restricted to the tracheal system. Furthermore, by comparing the rescue ability of different LPP homologs we show that wun function in the trachea is distinct from its role in germ cell migration

The molecular connection of histopathological heterogeneity in hepatocellular carcinoma: A role of Wnt and Hedgehog signaling pathways.

BackgroundHepatocellular carcinoma (HCC) is leading cause of cancer-related mortality and is categorized among the most common malignancies around the world. It is a heterogeneous tumor, which shows significant degree of histopathological heterogeneity. Despite the apparent histopathological diversity there has been very little distinct correlation between histopathological features and molecular aberrations particularly when it comes to the expression level of Wnt and Hh pathway molecules. The role of Wnt and Hh pathways in relation to HCC behavior viz. histopathological heterogeneity and aggressiveness is not known. Determining the sequential molecular changes and associated histopathological characteristic during HCC initiation, promotion, and progression would probably lead to a better treatment and prognosis.MethodsN-Nitrosodiethylamine (DEN) induced HCC model in male Wistar rats were established to study the expression level of Wnt and Hh pathway molecules during different stages of hepatocarcinogenesis. Their expression levels were checked at mRNA and protein levels at initiation, promotion, and progression stages of HCC. The expression levels of Wnt and Hh pathway molecules were correlated with biospecimens of HCC patients of different stages.ResultsIn the present study we identified the comprehensive change in the expression pattern of Wnt and Hh pathway molecules in DEN induced rodent hepatocarcinogenesis model. Our results demonstrate that β-catenin /CTNNB1 plays important role in tumor initiation and promotion by stimulating tumor cell proliferation. The activated Wnt signaling in early stage of HCC is associated with well-differentiated histological pattern. The Hh activity although activated during the initiation stage but is significantly increased during the early promotion stage of hepatocarcinogenesis. The increased activity of both Wnt & Hh pathways during promotion stage is associated with moderately-differentiated histological pattern and was simultaneously linked with an increased expression of MMP9. Furthermore, our data demonstrated that during the progression stage Wnt pathway is modestly down-regulated but the Hh pathway activity sustained which in turn is associated with aggressive and invasive phenotype and poorly-differentiated histopathology.ConclusionOur data uncovers the grade related expression of Wnt and Hh pathway molecules and the potential utility of these molecular signatures in daily clinical practice is to decide best therapy according to patients characteristic. Additionally, our data offer insight into the interaction between Wnt and Hh pathways which triggers HCC development and progression

Mutations in MAST1 Cause Mega-Corpus-Callosum Syndrome with Cerebellar Hypoplasia and Cortical Malformations

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