Mechanisms of radial glia progenitor cell lineage progression

The mammalian cerebral cortex is responsible for higher cognitive functions such as perception, consciousness, and acquiring and processing information. The neocortex is organized into six distinct laminae, each composed of a rich diversity of cell types which assemble into highly complex cortical circuits. Radial glia progenitors (RGPs) are responsible for producing all neocortical neurons and certain glia lineages. Here, we discuss recent discoveries emerging from clonal lineage analysis at the single RGP cell level that provide us with an inaugural quantitative framework of RGP lineage progression. We further discuss the importance of the relative contribution of intrinsic gene functions and non-cell-autonomous or community effects in regulating RGP proliferation behavior and lineage progression

Tumor Suppressor Genes

Functional evidence obtained from somatic cell fusion studies indicated that a group of genes from normal cells might replace or correct a defective function of cancer cells. Tumorigenesis that could be initiated by two mutations was established by the analysis of hereditary retinoblastoma, which led to the eventual cloning of RB1 gene. The two-hit hypothesis helped isolate many tumor suppressor genes (TSG) since then. More recently, the roles of haploinsufficiency, epigenetic control, and gene dosage effects in some TSGs, such as P53, P16 and PTEN, have been studied extensively. It is now widely recognized that deregulation of growth control is one of the major hallmarks of cancer biological capabilities, and TSGs play critical roles in many cellular activities through signaling transduction networks. This book is an excellent review of current understanding of TSGs, and indicates that the accumulated TSG knowledge has opened a new frontier for cancer therapies

The Role of Merlin and Apicobasal Polarity in Endometrial Development and Homeostasis

Apicobasal polarity and cell adhesion are necessary for the proper formation and organization of epithelial tissues. Merlin couples cell polarity and adhesion through correct localization of the polarity protein Par3 and maturation of apical junctions. Merlin and Par3 are necessary for the development and homeostasis of highly regenerative tissues like the epidermis. The continual repopulation of the endometrium after each menstrual cycle requires a constant reorganization of cell polarity and adhesion. The endometrium consists of a luminal epithelium that postnatally gives rise to the distinct glandular epithelium. Endometrial glands are necessary to secrete nutrients for the pre-implantation embryo. In addition, the endometrial gland is thought to be where endometrial cancer originates. While the endometrium is important for female fertility, relatively little is understood about how glands develop or how endometrial cancer forms. We examine the role of Merlin and apicobasal polarity in endometrial development and homeostasis. We determine that Merlin regulation of apicobasal polarity is necessary for proper endometrial gland formation. Apicobasal polarity is disrupted in low-grade endometrial cancer and mediates Notch regulated proliferation and migration in endometrial cancer cells. This dissertation reveals a critical role for Merlin and cell polarity in endometrial gland development, mammalian fertility, and endometrial cancer

Functional Genomics of Nervous System Development and Disease

xiii, 145 p. : ill. (some col.)The goal of functional genomics is to elucidate the relationship between an organism's genotype and phenotype. A key characteristic of functional genomics is the use of genome-wide approaches as opposed to more traditional single-gene approaches. Genome-wide expression profiling is used to investigate the dynamic properties of transcriptomes, provides insights into how biological functions are encoded in genomes, and is an important technique in functional genomics. This dissertation describes the use of genome-wide expression profiling and other functional genomics techniques to address a variety of biological questions related to development and disease of the nervous system. Our results reveal novel and important insights into nervous system development and disease and demonstrate the power of functional genomics approaches for the study of nervous system biology. This dissertation also describes a novel technique called TUtagging that facilitates cell type-specific RNA isolation from intact complex tissues. The isolation of RNA from specific cell types within a complex tissue is a major limiting factor in the application of genome-wide expression profiling, and TU-tagging can be used to address a wide array of interesting and important biological questions. This dissertation includes previously published and unpublished co-authored material.Committee in charge: Dr. John Postlethwait， Chair； Dr. Chris Doe， Advisor； Dr. Bruce Bowerman， Member； Dr. Patrick Phillips， Member； Dr. Tom Stevens， Outside Membe

Functional genomics of brain development and developmentally related brain disease in "Drosophila"

One of the fundamental challenges in basic neuroscience is to understand the molecular genetic networks associated with building the brain. As malfunction in these genetic pathways can lead to disorders like cancer, brain development is also a crucial research area for clinical neuroscience. In the course of this thesis, different molecular aspects of Drosophila brain development and related neoplastic disease were analyzed using high-density oligonucleotide arrays. The homeotic selector gene labial (lab) plays an important role in specification of neuronal identity in the embryonic brain of Drosophila. In labial mutants presumptive neurons in the posterior tritocerebrum fail to differentiate. This leads to severe defects in tritocerebral axon pathways. Using high density oligonucleotide arrays we identified downstream target genes of Labial and showed that only a limited and distinct set of genes expressed in the embryo is regulated by this homeoprotein. Furthermore, we performed genetic rescue experiments to analyze the functional equivalence of Drosophila Hox gene products in specification of the tritocerebral neuromere. Surprisingly, all tested homeotic proteins, with the exception of Abd-B, were able to rescue the labial mutant phenotype in the tritocerebrum. These results indicate that the specificity of homeotic gene action in embryonic brain development has to be modulated by cis-acting regulatory elements. Another study circled around the homeobox transcription factor otd and its human homolog Otx2. Cross-phylum rescue experiments have shown that these genes are functionally equivalent. We used quantitative transcript imaging to analyze otd and Otx gene action in the Drosophila embryo at a genomic level. Our experiments suggest that about one third of the Otd-regulated transcripts in Drosophila can also be controlled by the human Otx2. These common otd/Otx2 downstream genes are likely to represent the molecular basis for the functional equivalence of otd and Otx2 gene action in Drosophila. glial cells missing (gcm) is a key control gene of gliogenesis. gcm loss-of-function leads to a transformation of glial cells into neurons and, conversely, when gcm is ectopically misexpressed, presumptive neurons become glia. Since gcm encodes a transcription factor it is supposed that a set of downstream genes are regulated by GCM that in turn execute the glial differentiation program. Again, a set of full-genome transcript profiling experiments was conducted to identify gcm downstream genes in a comprehensive manner. A set of several hundred candidate gcm target genes were identified in this screen, giving new insights into neuroglial fate specification in Drosophila. Brain tumors have been extensively studied by looking at genetic alterations and mutations that lead to malignant growth. Still, the causes of brain tumorigenesis are largely unknown. Model systems like Drosophila can be of great help to shed light on altered transcriptional activity in brain tumor phenotypes. To investigate the in vivo transcriptional activity associated with a brain tumor, we conducted genome-wide microarray expression analyses of an adult brain tumor in Drosophila caused by homozygous mutation in the tumor suppressor gene brain tumor (brat). Two independent gene expression studies using two different oligonucleotide microarray platforms were used to compare the transcriptome of adult wildtype flies with mutants displaying the adult bratk06028 mutant brain tumor. Cross-validation and stringent statistical criteria identified a core transcriptional signature of bratk06028 neoplastic tissue. We found highly significant expression level changes for 321 annotated genes associated with the adult neoplastic bratk06028 tissue indicating elevated and aberrant metabolic and cell cycle activity, upregulation of the basal transcriptional machinery, as well as elevated and aberrant activity of ribosome synthesis and translation control. One fifth of these genes show homology to known mammalian genes involved in cancer formation. These results identify for the first time the genome-wide transcriptional alterations associated with an adult brain tumor in Drosophila and reveal insights into the possible mechanisms of tumor formation caused by homozygous mutation of the translational repressor brat

Asymmetric cell division intersects with cell geometry : a method to extrapolate and quantify geometrical parameters of sensory organ precursors

La division cellulaire asymétrique (DCA) consiste en une division pendant laquelle des déterminants cellulaires sont distribués préférentiellement dans une des deux cellules filles. Par l’action de ces déterminants, la DCA générera donc deux cellules filles différentes. Ainsi, la DCA est importante pour générer la diversité cellulaire et pour maintenir l’homéostasie de certaines cellules souches. Pour induire une répartition asymétrique des déterminants cellulaires, le positionnement du fuseau mitotique doit être très bien contrôlé. Fréquemment ceci génère deux cellules filles de tailles différentes, car le fuseau mitotique n’est pas centré pendant la mitose, ce qui induit un positionnement asymétrique du sillon de clivage. Bien qu’un complexe impliquant des GTPases hétérotrimériques et des protéines liant les microtubules au cortex ait été impliqué directement dans le positionnement du fuseau mitotique, le mécanisme exact induisant le positionnement asymétrique du fuseau durant la DCA n'est pas encore compris. Des études récentes suggèrent qu’une régulation asymétrique du cytosquelette d’actine pourrait être responsable de ce positionnement asymétrique du faisceau mitotique. Donc, nous émettons l'hypothèse que des contractions asymétriques d’actine pendant la division cellulaire pourraient déplacer le fuseau mitotique et le sillon de clivage pour créer une asymétrie cellulaire. Nos résultats préliminaires ont démontré que le blebbing cortical, qui est une indication de tension corticale et de contraction, se produit préférentiellement dans la moitié antérieure de cellule précurseur d’organes sensoriels (SOP) pendant le stage de télophase. Nos données soutiennent l'idée que les petites GTPases de la famille Rho pourraient être impliqués dans la régulation du fuseau mitotique et ainsi contrôler la DCA des SOP. Les paramètres expérimentaux développés pour cette thèse, pour étudier la régulation de l’orientation et le positionnement du fuseau mitotique, ouvrirons de nouvelles avenues pour contrôler ce processus, ce qui pourrait être utile pour freiner la progression de cellules cancéreuses. Les résultats préliminaires de ce projet proposeront une manière dont les petites GTPases de la famille Rho peuvent être impliqués dans le contrôle de la division cellulaire asymétrique in vivo dans les SOP. Les modèles théoriques qui sont expliqués dans cette étude pourront servir à améliorer les méthodes quantitatives de biologie cellulaire de la DCA.Asymmetric cell division (ACD) consists in a cellular division during which specific cell fate determinants are distributed preferentially in one daughter cell, which then differentiate from its sibling. Hence, ACD is important to generate cell diversity and is used to regulate stem cells homeostasis. For proper asymmetric distribution of cell fate determinants, the positioning of the mitotic spindle has to be tightly controlled. Frequently, this induces a cell size asymmetry, since the spindle is then not centered during mitosis, leading to an asymmetric positioning of the cleavage furrow. Although small small GTPases have been shown to act directly on the spindle, the exact mechanism controlling spindle positioning during ACD is not understood. Recent studies suggest that an independent, yet uncharacterized pathway is involved in spindle positioning, which is likely to involve an asymmetric regulation of the actin cytoskeleton. Indeed, actin enables spindle anchoring to the cortex. Hence we hypothesize that asymmetric actin contractions during cytokinesis might displace the mitotic spindle and the cleavage furrow, leading to cell size asymmetry. Interestingly, from our preliminary results we observed that cortical blebbing, which is a read-out of cortical tension/contraction, preferentially occurs on the anterior side of the dividing sensory organ precursor (SOP) cells at telophase. Our preliminary data support the idea that Rho small GTPases might be implicated in regulation of the mitotic spindle hence controlling asymmetric cell division of SOP cells. The experimental settings developed for this thesis, for studying regulation of the mitotic spindle orientation and positioning will serve as proof of concept of how geneticist and biochemist experts could design ways to control such process by different means in cancerous cells. The preliminary results from this project open novel insights on how the Rho small GTPases might be implicated in controlling asymmetric cell division hence their dynamics in vivo of such process during SOP development. Furthermore, the assays and the theoretical model developed in this study can be used as background that could serve to design improved quantitative experimental methods for cell biology synchronizing sub-networks of ACD mechanism

Mechanisms of Mitotic Spindle Orientation by Plexin-B2

Cells show certain asymmetries in morphology and molecular organization, a characteristic that is known as cell polarity. Polarity is generally regulated by protein complexes and Rho GTPases like Cdc42, Rho or Rac. Epithelial cells are polarized along the apicobasal axis, and this apicobasal polarity influences many cellular processes, including cell division. Polarized mitosis in epithelia is controlled by the orientation of the mitotic spindle. This is crucial in epithelial cells during development for a correct tissue morphogenesis, but also in the adult for maintenance of tissue homeostasis or damage repair. The orientation of the spindle is controlled by a protein complex that includes NuMA, which mediates pulling from the spindle poles and LGN, which links NuMA to the correct regions of the cell cortex. Semaphorin-Plexin signaling is a cell-cell communication pathway involved in many tissues and processes mainly through regulation of the cytoskeleton and adhesion. It has been shown that Plexin-B2 regulates mitotic spindle orientation in kidney epithelial cells and that this regulation is relevant for kidney morphogenesis and repair. However, the molecular mechanisms through which Plexin-B2 controls spindle orientation are still largely unclear. In this work, I demonstrate that Plexin-B2 localizes to cell-cell contacts in the kidney epithelium and in epithelial cell lines in 2D and 3D. Furthermore, I show that Plexin-B2 remains polarized during mitosis. I add that the basolateral localization of Plexin-B2 in the kidney is independent of its ligands, but depends on its intracellular juxtamembrane domain in 3D cultures. Furthermore, I show that this region contains a unique basolateral targeting motif present in all murine class B plexins and conserved in human Plexin-B2. Using CRISPR-Cas genome editing, I confirm that the deletion of Plexin-B2 impairs correct lumen formation in epithelial cell cysts. Additionally, I show that the lack of Plexin-B2 does not influence growth of these cells, but increases the proportion of cells in the S and G2/M phases of the cell cycle and aneuploidy. Importantly, I demonstrate that the deletion of Plexin-B2 does not have an effect on the normal localization of the spindle regulators LGN and NuMA in 3D cell cultures. Therefore, the role of the polarized expression of Plexin-B2 in its control of mitotic spindle orientation and its possible connection with mitotic spindle regulators need to be further investigate

A genome wide RNAi screen in D. melanogaster for the identification of genes involved in border cell migration

Im Verlauf von normalen Entwicklungsprozessen sowie während abnormer Metastasenausbildung weisen Zellen eine bemerkenswerte Umstellung von "unbeweglich" auf "beweglich" auf. Grosses Interesse gilt daher der Erforschung des Expressionsprofils notwendiger Gene während der Zellwanderung selbst. Die Oogenese von der Fruchtfliege Drosophila melanogaster weist programmierte invasive Zellwanderung von sogenannten "Border Cells" auf. Diese spezialisierten Zellen führen einen Übergang von endothelial (stationär) zu mesenchymal (beweglich) durch und wandern zwischen anderen Zellen durch die gesamte Eikammer. Border cell Wanderung ist ein interessantes Modellsystem für in vivo Zellmigration, da viele Parallelen zu sowohl normaler wie auch abnormer Zellmigration vorhanden sind. In Vorversuchen zeige ich, dass durch die RNAi knock down Technik "Border cell" spezifische Gene ausgeschaltet werden können und bereits publizierte Phänotypen reproduziert und phänokopiert werden können. Ich habe die einzigarte RNAi Fliegenbibliothek, (von der Gruppe von Dr. B. Dickson hergestellt) genutzt, um nach Genen zu suchen, die für Zellbewegung speziell während Border Cell Wanderung nötig sind. Zum ersten Mal in Drosophila Genetik war es möglich, einen systematischen genomweiten RNA interference (RNAi) Screen für Border Cell Wanderung durchzuführen. Im Zuge dieses Screens habe ich 52 neue Gene identifiziert, die bisher noch nicht mit Border Cell Wanderung in Verbindung gebracht worden sind. In der vorliegenden Dissertation präsentiere ich die Realisierung und Durchführung eines genomweiten RNAi Screens, der Border Cell Wanderung während Drosophila Oogenese als Modellsystem verwendet. Darüberhinaus stelle ich ein neu identifiziertes Gen CG34139 vor, das bisher noch nicht charakterisiert wurde und benenne es wanderlust. Wanderlust ist ein Vertreter der Neuroligine und reguliert Border Cell Wanderung.The transition of a cell from a sessile to a migratory state is a feature shared by normal cells during development and abnormal cells during metastasis. Much interest thus centers on the profile of gene expression required for programmed and uncontrolled cell migration. Border cells in the fruit fly Drosophila melanogaster undergo invasive and programmed cluster migration during oogenesis and represent an attractive model system for the analysis of cell migration in vivo. The goal of this thesis was to identify novel genes required for cell migration in order to better understand migration processes. A comprehensive knowledge of genes crucial for migration could potentially lead to the assignment of drug targets to specifically block deleterious migration such as occurs in metastasis. I show that RNAi (RNA interference) can be employed to study border cell migration. Background RNAi studies encouraged me to perform a systematic genome wide analysis of border cell migration using the transgenic RNAi collection generated by the group of B.Dickson. The RNAi screen enabled me to identify 52 novel genes, previously not implicated in border cell migration. Here, I present the realization and description of the first genome wide RNAi screen for genes involved in migration using D. melanogaster border cell migration. Furthermore, I present the initiated characterization of one of the genes identified in the screen, designated as wanderlust, which belongs to the neuroligin familiy of proteins thought to be restricted in expression to neuronal cells, but now identified as a regulator of border cell migration