Browsing by Subject "Gene Expression Regulation, Developmental"
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Item Apoptosis Determinants in Drosophila melanogaster(2007-12-17) Chew, Su Kit; Abrams, JohnApoptosis is a form of programmed cell death (PCD) that is governed by a core set of genes conserved across diverse metazoan phyla. Cells dying by apoptosis exhibit a characteristic series of morphological and biochemical changes that is also conserved. This form of PCD plays pivotal roles in homeostatic regulation of cell numbers, developmental sculpting of organs, damage and infection responses; conversely, its disregulation has profound implications in diseases such as cancers, immune disorders infertility and dystrophies. Common parallels in the regulation of the core apoptosis machinery have been elucidated in human and experimental model organisms, though many fundamental questions in our understanding of its regulation remain. A conserved node in the apoptosis pathway is the apoptosome, comprising the apical caspase and its adaptor protein. To understand the functions of this node, I generated a null allele of the apical caspase Dronc in the experimental model organism Drosophila melanogaster. Dronc is required for developmentally regulated apoptosis in multiple tissues during embryogenesis and larval development. Failure of apoptosis correlated with tissue hyperplasia. Notably, the removal of Dronc eliminated the cellular apopototic response to stresses in cells. In some of the stress contexts tested, Dronc depletion partially rescued cell viability to the same levels as pan-caspase inhibition by small peptide inhibitors, suggesting that Dronc functions map specifically to caspase activation and apoptosis. These and similar observations in its adaptor protein Dark point to the apoptosome as a key node for apoptosis in Drosophila. From these observations, I sought to use the induced apoptosis cellular response as a means to identify novel components and regulators in the apoptosis pathway. I optimized a cell culture system for high-throughput cell-based screening using RNA interference (RNAi) mediated gene silencing and a synthetic antagonist of inhibitors of apoptosis proteins (IAPs). From a genome-wide Drosophila RNAi library, I identified 42 potential genes required for apoptosis, of which I characterized 13 highly validated targets for their requirements in multiple stress contexts. One of these hits, Tango7, regulates pro-Dronc protein and represents an unprecedented point of apoptosis regulation. Collectively, my studies bolster the model for the crucial requirement of the apoptosome in apoptosis and identify new regulation entry-points into the apoptosis pathway.Item GATA Like Protein-1: A Somatic Cell Factor Required for Normal Ovarian Development and Function(2010-11-02T18:19:55Z) Strauss, Tamara Joy; Hammes, StephenOogenesis and follicular maturation are processes that require organized and precisely timed communication through paracrine and endocrine signals of neighboring tissues. Deviations in the cross talk between ovarian cells, or aberrant gene expression within one of the cell populations, can lead to germ cell loss and infertility in the adult female. Expression of Glp-1 in the somatic cells of the ovary is required for normal fertility in female mice, as a deficiency for Glp-1 leads to the absence of oocytes at birth and ovarian tubular formation in the adult. However, the nature of germ cell loss and tubular adenoma formation, in the setting of a somatic cell protein deficiency, is not well understood. In this report, I characterize the embryonic germ cell loss phenotype in Glp-1LacZ null mice. Immunohistochemical analyses of Glp-1LacZ null mouse ovaries show that germ cells are appropriately specified and migrate to the nascent gonad similarly to wild type. After their arrival at the gonad, precocious loss of the germ cells begins at or around E13.5. This loss is completed by birth and is accompanied by defects in the expression of oocyte-specific genes associated with meiotic entry. Interestingly, somatic pregranulosa cells retain their ability to secrete paracrine signaling molecules to the oocyte and are still able to form the basement membrane surrounding the germline cysts. In the adult, the structure of the germline cyst persists, albeit without germ cells, and there is loss of HPG axis communication. The loss in HPG communication in Glp-1LacZ null mice can be accounted for by loss of regulated steroidogenesis through the GATA4-dependent transcriptional activation of StAR. These data imply that the somatic cell protein Glp-1 regulates 1) germ cell survival early in embryogenesis and 2) steroidogenesis through StAR promoter activation.Item Mash1 Defines Lineage Restricted Neuronal and Oligodendrocytic Precursor Cells in Spinal Cord Development(2007-05-21) Battiste, James Douglas; Johnson, Jane E.Recent advances have defined distinct neural progenitor and early interneuron pools in the developing spinal cord and the molecular events that influence progenitor cell fate. However, these early neurons have not been traced to adult neuron types. The transcription factor Mash1 is transiently expressed in a subset of neural progenitors and possesses a pro-neural function. The transient nature of its expression limits the ability to trace Mash1+ progenitors. To study the developing neural tube from progenitor to adult neuron, transgenic mouse strains were generated that express GFP, Cre recombinase, and tamoxifen-inducible Cre recombinase. The M1-GIC mouse line, showed faithful Mash1 expression recapitulation and traces Mash1+ progenitors mainly to dI3 and dI5 interneurons. This supports data from the Mash1 null mutant where these populations are decreased or absent. Using M1-GIC;R26R-lacZ mice, I was able to trace Mash1 expressing cells to neurons and oligodendrocytes in the adult mouse, but tracing to astrocytes was never observed. These data refute the conventional understanding that Mash1 is purely pro-neuronal, and is consistent with recent findings of Mash1 descendents in the early postnatal subventricular zone. Using M1-CRE-ER™;R26RlacZ and M1-CRE-ER™;R26R-YFP, Mash1+ cells trace into adulthood in a temporally-dependant manner. Cells expressing Mash1 at E10.5 become neurons of the dorsal horn in lamina I-IV while cells expressing Mash1 at E15.5 become oligodendrocytes spread over both gray and white matter. As a control, Nestin- CreERT2;R26R-lacZ and Nestin-CreERT2;R26R-YFP mice were used to confirm that common progenitors of all neural cell types can be traced from E10.5 to P21. This data provides evidence that Mash1 defines lineage restricted precursors that exit the cell cycle rapidly, and Mash1 is necessary for efficient loss of common progenitor characteristics as seen in the Mash1 null mutant. This data refines our understanding of progenitor characteristics and Mash1 function in the developing spinal cord.Item Multifunctional Regulators of Cardiac Disease and Development(2008-09-12) Kim, Yuri; Olson, EricEmbryogenesis requires delicate regulatory mechanisms. A single cell embryo divides into millions of daughter cells to form an organism comprised of various organs with different shapes and function. Organogenesis is mainly controlled by genes that are expressed in a tissue-specific manner. Thus, regulation of gene expression is a critical step in development. In this thesis, I present my findings on two cardiac transcription factors MEF2 and Yap that play multiple roles in development. First, I show a novel function of myocyte enhancer factor 2 (MEF2) transcription factors in development of endochondral bone. MEF2 proteins are widely known as essential regulators of development of various tissues such as striated muscle and brain. Based on expression patterns of Mef2 genes and skeletal defects present in Mef2c +/-; Mef2d +/- mice, I hypothesized that MEF2is an important regulator of skeletogenesis and generated mice lacking MEF2C and MEF2D in chondrocytes using Mef2c and Mef2d conditional mutant alleles. From this study, I demonstrated that MEF2 proteins are also critical regulators of chondrocyte hypertrophy at least partly through their regulation of procollagen, type X, alpha 1 (Col10a1). I also explored another function of MEF2 protein, which is to mediate stress-dependent cardiac remodeling. Mef2d null mice show impaired response to cardiac remodeling stresses such as pressure overload and chronic Β- adrenergic stimulation; hypertrophy, chamber dilation, fibrosis, and fetal gene activation were blunted in the absence of MEF2D. Conversely, overexpression of MEF2D is sufficient to drive pathological remodeling of the heart. These findings reveal an important role of MEF2D in stress-dependent cardiac growth and reprogramming of gene expression in the adult heart. Finally, I demonstrate that yes-associated protein (Yap) serves as a critical regulator of cardiac function and angiogenesis by generating a Yap conditional mutant allele. Deletion of Yap in cardiomyoctes leads to lethal cardiomyopathy resulting from compromised cardiac angiogenesis and ischemia. I also identify Yap as a coactivator of GATA4, a trascription factor that functions as a regulator of angiogenesis in the heart. Moreover, my studies on deletion of Yap in other tissues suggest the possible role of Yap as a global angiogenic factor. Collectively, these studies show that key transcriptional regulators of cardiogenesis play a significant role not only in heart development, but also in development of other organs. These findings imply that combinatorial actions of transcriptional regulators in a tissue-specific manner are critical in embryogenesis.Item Nuclear Receptors in Lung Cancer(2007-05-22) Jeong, Yangsik; Mangelsdorf, David J.Lung Cancer is a fatal disease with new diagnoses of more than 150,000 Americans every year. Although it has a relatively well-known etiology (e.g. smoking) and has been widely researched, clinical tools and markers for early diagnosis, prognostic prediction, and therapeutic interventions remain limited. Here, for the first time, I propose a novel translational approach for providing diagnostic, prognostic, mechanistic, and therapeutic information by studying of the expression of the nuclear receptor (NR) superfamily in lung cancer. Using quantitative real-time PCR, mRNA expression levels for the 48 members of the NR superfamily were profiled in 56 lung cell lines. Based on the resulting dataset, further analysis was performed to show the diagnostic and therapeutic potential of the NR profile using both an in vitro cell response assay and an in vivo mouse xenograft model with cognate ligand treatment for selected nuclear receptors. In addition, the NR profiles of 30 microdissected and pair-matched patient tissue samples provided a subset of NRs showing dramatic differences in expression and subgroupings that demonstrate individual variations between the normal and corresponding tumor. Furthermore, I identified several individual NRs as well as a subgroup of NRs with prognostic power. The relevance of NRs to disease pathogenesis was then studied in genetically manipulated human bronchial epithelial cells (HBEC3) and in transgenic K-rasV12 mice, a well-known genetic model for lung adenocarcinoma. In the HBEC3 panel, the induced expression of peroxisome proliferator activating receptor gamma (PPARγ) in the parental HBEC3 introduced by oncogenic K-rasV12 is decreased in a subset of tumorigenic clones derived from the parental cells. It appears to be strongly correlated to the expression of cylooxygenase 2 (COX2), which is shown to be decreased with PPARγ ligand treatment. In the transgenic model, I demonstrated that expression of a subgroup of NRs in wild type mice becomes altered in histologically normal tissues that harbor the K-ras mutation, and become further altered in tumor tissues of the mutant. This observation suggests that NR profiling also provides a valuable tool for understanding disease pathogenesis in lung cancer.Item Transcriptional and Translational Regulation of Cardiac Progenitors in the Mouse and Zebrafish(2009-01-09) Cordes, Kimberly Rene; Srivastava, DeepakIn vertebrates, the heart is the first organ to function and cardiac progenitors are among the first cell lineages to be established. Transcriptional networks control the specification of cardiac progenitors, however, it is not fully understood how some transcription factors function in particular cardiac progenitor populations. The basic helix-loop-helix, bHLH, transcription factor, Hand2 has been discovered over a decade ago, and has a severe loss-of-function cardiac phenotype in vivo, yet its function is still not completely known. It is expressed in the early cardiac progenitors of the neural crest cells and second heart field lineages. The first part of my thesis touches on the beginnings to understand the role of Hand2 in the cardiac neural crest progenitors. Generally, expression levels in vertebrates reflect the combined transcription of both alleles of the gene being transcribed. Although there are notable exceptions (i.e., X chromosome genes), the presence of only one functional copy or more than two copies of a gene can have detrimental effects on the development of the organism. Many of the genetic examples of congenital heart disease, which affects 1% of live births, are a result of a haploinsufficient gene dose. Like Hand2, which acts in a dosage-sensitive manner to regulate ventricular formation, the precise dose of proteins can be very important in regulating cardiac development. One way to fine-tune the activity of genes is through the newly identified class of small RNAs, microRNAs (miRNAs), which translationally repress the production of proteins by binding to target sites on messenger RNA (mRNA). miRNAs provide a sophisticated way to adjust protein levels in a spatiotemporal manner. One miRNA may control several mRNAs, including transcription factors, which are the 'master switches' that regulate gene expression. And cooperatively, cell type-specific transcription factors can regulate the tissue-specificity of miRNA expression. Together with transcription factors, miRNAs function in cell fate determination, cell differentiation, proliferation and disease progression. Similar to transcription factors, which activate or repress a set of genes in a particular cell type, miRNAs create an environment, tailored for each cell type, allowing translation of some genes to occur, while repressing others. To date, less than a handful of miRNAs have been identified that function during heart development. The latter half of my thesis represents efforts to identify cardiac progenitor miRNAs and understand their function during development. I found that miRNA function is important in the cardiac mesodermal progenitors. In addition, I present a family of miRNAs, miR-143 and miR-145, that is specific to cardiac and smooth muscle progenitors, and I discuss their function in regulating their respective environments during cardiovascular development and disease.Item Understanding the Role of SCL in Early Mammalian Development Using Mouse Embryonic Stem Cell Differentiation as a Model(2010-05-14) Ismailoglu, Ismail; Kyba, MichaelHow a complete organism develops from a single cell is among the most complicated questions in life sciences. Early experimental studies on the development of animals were performed on amphibians and birds due to the size and accessibility of their embryos, while studies in placental mammals have been limited by the difficulty posed by in utero development. In vitro differentiation of ES cells provides a convenient model for the study of the mammalian development. Since ES cells can be grown and maintained in a pluripotent state virtually forever, ample amount of research material for molecularbiological studies can be produced; differentiating ES cells are easily accessible and they can also be manipulated genetically. I have used the ES cell differentiation model to study the bHLH factor SCL, a critical regulator of the formation of the hematopoietic lineage in the early embryo and the maturation of erythrocytes and megakaryocytes later on. The latter function of the protein has been studied extensively, but a complete molecular analysis of the former function has been lacking. My work shows that SCL can skew the patterning of the mesoderm towards the hematopoietic lineage. This function required the interaction of SCL with LMO2. Transcriptional profiling revealed organizer genes FoxA2 and Chordin as novel downregulated targets of SCL during this time. Differentiation of human pluripotent cells to be used in cellular therapy or to generate replacement tissues; is considered to be one of the most promising branches of medical research. Considering the importance of SCL in hematopoiesis, we hypothesized that SCL can direct differentiation of pluripotent cells to this lineage in a simple culture system. Ectopic expression of SCL induced hematopoiesis at low levels. Co-expression of LMO2 and GATA2 increased efficiency of the programming significantly.