DOCSLIB.ORG

EXAMINING the POST-TRANSCRIPTIONAL REGULATION of LUNATIC FRINGE (Lfng) in the MOUSE SEGMENTATION CLOCK

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
EXAMINING the POST-TRANSCRIPTIONAL REGULATION of LUNATIC FRINGE (Lfng) in the MOUSE SEGMENTATION CLOCK EXAMINING THE POST-TRANSCRIPTIONAL REGULATION OF LUNATIC FRINGE (Lfng) IN THE MOUSE SEGMENTATION CLOCK DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Kanu Wahi Graduate Program in Molecular Genetics The Ohio State University 2016 Dissertation Committee: Dr. Susan Cole, Advisor Dr. Keith Slotkin Dr. Robin Wharton Dr. Dawn Chandler Copyright by Kanu Wahi 2016 Abstract Somitogenesis is a developmental process in vertebrates involving periodic formation of somites that bud from an unsegmented region known as the pre-somitic mesoderm (PSM) and give rise to the axial skeleton and skeletal muscle in the developed organism. The process of somitogenesis is regulated by a "segmentation clock" that times somite formation and is evolutionarily conserved among vertebrates. Genes, such as Lunatic fringe (Lfng), are required for normal clock function in chickens, mice and humans and exhibit rapid cyclic expression in the PSM with a period that matches the rate of somite formation. To maintain rapid oscillations, especially in the posterior PSM, it is hypothesized that the Lfng transcript should be promptly degraded to ensure its clearance of from cells before the next round of oscillation begins. The 3’UTR of Lfng contains number of conserved sequences that could regulate Lfng expression at the post transcriptional level. In this study we explore the post- transcriptional regulation of the Lfng transcript by the Lfng 3’UTR in the context of the segmentation clock. The Lfng 3'UTR has binding sites for microRNAs (miRNAs), such as miR-125a and miR-200b that are known to affect transcript stability via the 3’UTR. We tested the effect of mutating the mir-125a binding sites in the mouse Lfng 3’UTR on mRNA stability ii of Venus reporter constructs in transgenic mice and observed constitutive Venus expression in the posterior PSM, as opposed to the oscillatory Venus expression when the miR-125a binding sites are not mutated. This implies that the miR-125a binding sites in the Lfng 3'UTR influence RNA turnover in the posterior region of the mouse PSM. Interestingly, when we generated mutant mouse lines that do not express miR-125a we found no effect on somitogenesis and skeletal formation. This indicates that either a compensatory mechanism may be in effect or that miR-125a does not play a role in the mouse segmentation clock. We also examined if another highly conserved region of the Lfng 3'UTR, that is 120 bp in length and has a miR-200 binding site within it, can sufficiently destabilize the transcript. We examined this in two contexts. We found that blocking the miR-200b binding site in the Lfng 3’UTR in chicken embryos has no effect on somitogenesis. Similarly, in mouse myoblast cells the 120 bp sequence of the mouse Lfng 3'UTR is not sufficient to destabilize the transcript. Our results indicate that the 120 bp sequence and the miR-200b binding site within it, are not sufficient to destabilize the transcript and it is likely that this regulation operates in a tissue specific manner. Future work in the lab will examine the function of the endogenous miR-125a binding site in the Lfng 3'UTR on mouse segmentation and skeletal formation. Understanding the post transcriptional regulation of clock genes like Lfng would shed light on mechanisms that maintain oscillatory gene expression in the PSM and ensure timely somite formation. iii Dedication This document is dedicated to my husband, Dhrupad Siddhanta, for his endless support and for being my cheerleader for life. iv Acknowledgments I would like to sincerely thank my advisor, Dr. Susan Cole, without whose constant guidance, motivation and support I would not have been able to make it this far. I would like to acknowledge her patience in bearing with my shortcomings and helping me overcome them. Her nurturing mentorship has made my graduate school experience truly enriching and enjoyable. I would like to thank my lab members: Dr. Maurisa Riley, who provided the stepping stone for this project; Sophia Friesen, an extremely dedicated and hardworking undergraduate student, who has contributed immensely to the analysis of the miR-125a mutant mice; Dr. Dustin Williams, Skye Bochter and Kara Braunreiter for always supporting me and for making lab feel like a second home; Undergraduates, Madeline Parker and Ben Schott for their assistance with lab chores. I am grateful to my committee members: Dr. Dawn Chandler, Dr.Keith Slotkin and Dr. Robin Wharton for sharing their expertise and for their valuable advice during committee meetings that has greatly influenced my project. I would like to thank Dr. Jared Talbot and Dr. Sharon Amacher for sharing their expertise and equipment for the High Resolution Melt Analysis (HRMA). Dr. Vincenzo Coppola, Director of the Mouse facility at OSU for helping us generate the Venus v transgenic and miR-125a mice using the CRISPR/Cas9 system. I immensely thank the all members of the Dr. Harold Fisk’s for always being so helpful, especially Dr. Dwitiya Sawant for guiding me with the use of the fluorescent microscope. I would like to thank the Pelotonia Foundation for their financial support from August 2014 to 2016 and the Alumni Grants Graduate Research Scholarships (AGGRS) association at OSU for funding a part of my thesis project. Lastly, I would like to thank my family: my parents for their unconditional love and support that has helped me reach my goals, my brother and sister-in-law for their encouragement throughout graduate school and my husband for always being by my side and motivating me throughout my graduate career. vi Vita 2003 ...............................................................National Public School, INDIA 2006 ...............................................................B.S. Biotechnology, Kasturba Medical College, INDIA 2008 ...............................................................M.S. Medical Biotechnology, Manipal University, INDIA 2010 to present .............................................Graduate Fellow, Department of Molecular Genetics, The Ohio State University Publications 1. Wahi K, Bochter MS, Cole SE. The many roles of Notch signaling during vertebrate somitogenesis. Semin Cell Dev Biol. 2016 Jan; 49:68-75. Epub 2014 Dec 4. 2. Korlimarla A, Prabhu JS, Anupama CE, Remacle J, Wahi K, Sridhar TS. Separate Quality-Control Measures Are Necessary for Estimation of RNA and Methylated DNA from Formalin-Fixed, Paraffin-Embedded Specimens by Quantitative PCR. J Mol Diagn. 2014 Mar; 16(2):253-60. vii 3. Riley MF, Bochter MS, Wahi K, Nuovo GJ, & Cole SE, mir-125a-5p-mediated Regulation of Lfng is Essential for the Avian Segmentation Clock. Dev Cell. 2013 Mar 11; 24(5):554-61. 4. Prabhu JS, Wahi K, Korlimarla A, Correa M, Manjunath S, Raman N, Srinath BS, Sridhar TS. The epigenetic silencing of the estrogen receptor (ER) by hypermethylation of the ESR1 promoter is seen predominantly in triple-negative breast cancers in Indian women. J Tum Bio. Apr 2012; 33(2). Fields of Study Major Field: Molecular Genetics viii Table of Contents Abstract ................................................................................................................................ii Dedication ........................................................................................................................... iv Acknowledgments................................................................................................................ v Vita ..................................................................................................................................... vii List of Tables ..................................................................................................................... xiii List of Figures .................................................................................................................... xiv Chapter 1: Introduction ..................................................................................................... 1 1.1 Segmentation in vertebrates…………………………………………………………………………………. 2 1.2 The clock and wavefront model can explain periodic somite formation …………………6 1.3 The segmentation clock operates in the vertebrate PSM ……………………………………….7 1.4 Notch signaling pathway establishes cell-to-cell communication ………………………….10 1.5 Notch pathway is required for normal segmentation clock function …………………….14 1.5.1 Role of Notch pathway in the zebrafish segmentation clock……………………….. 17 1.5.2 Role of the Notch pathway in the chicken and mouse segmentation clock…..19 1.6 Setting up the wavefront in the vertebrate PSM………………………………………………….. 25 1.7 Somite patterning in the anterior PSM is influenced by the Notch pathway…………..29 1.8 Fine tuning oscillatory Notch signaling in the segmentation clock………………………….33 ix Chapter 2: Regulation of Lunatic fringe by mir-125a in the mouse segmentation clock 2.1 Introduction………………………………………………………………………………………………………….. 37 2.2.1 Segmentation in vertebrates……………………………………………………………………….. 38 2.2.2 Oscillatory Lunatic fringe expression is essential for the mouse segmentation clock………………………………………………………………………………………………………………39 2.2.3 Post-transcriptional regulation of Lfng by the 3’ UTR…………………………………… 40 2.2 Materials and Methods 2.2.1 Construction of Venus reporter plasmids…………………………………………………….. 45 2.2.2 Transient transfections and generation of stable cell lines………………………..…..47 2.2.3 RT-PCR and Quantitative RT-PCR………………………………………………………………….. 49 2.2.4 Venus transgenic mice………………………………………………………………………………….
Load more

Recommended publications
  • Anti-Lunatic Fringe Antibody (ARG40136)
    Product datasheet [email protected] ARG40136 Package: 100 μl anti-Lunatic Fringe antibody Store at: -20°C Summary Product Description Rabbit Polyclonal antibody recognizes Lunatic Fringe Tested Reactivity Hu Tested Application FACS, IHC-P, WB Host Rabbit Clonality Polyclonal Isotype IgG Target Name Lunatic Fringe Antigen Species Human Immunogen KLH-conjugated synthetic peptide corresponding to aa. 86-114 of Human Lunatic Fringe. Conjugation Un-conjugated Alternate Names O-fucosylpeptide 3-beta-N-acetylglucosaminyltransferase; EC 2.4.1.222; SCDO3; Beta-1,3-N- acetylglucosaminyltransferase lunatic fringe Application Instructions Application table Application Dilution FACS 1:10 - 1:50 IHC-P 1:50 - 1:100 WB 1:1000 Application Note * The dilutions indicate recommended starting dilutions and the optimal dilutions or concentrations should be determined by the scientist. Positive Control K562 Calculated Mw 42 kDa Properties Form Liquid Purification Purification with Protein A and immunogen peptide. Buffer PBS and 0.09% (W/V) Sodium azide. Preservative 0.09% (W/V) Sodium azide. Storage instruction For continuous use, store undiluted antibody at 2-8°C for up to a week. For long-term storage, aliquot and store at -20°C or below. Storage in frost free freezers is not recommended. Avoid repeated freeze/thaw cycles. Suggest spin the vial prior to opening. The antibody solution should be gently mixed before use. www.arigobio.com 1/3 Note For laboratory research only, not for drug, diagnostic or other use. Bioinformation Gene Symbol LFNG Gene Full Name LFNG O-fucosylpeptide 3-beta-N-acetylglucosaminyltransferase Background This gene is a member of the fringe gene family which also includes radical and manic fringe genes.
    [Show full text]
  • Segmentation in Vertebrates: Clock and Gradient Finally Joined
    Downloaded from genesdev.cshlp.org on September 24, 2021 - Published by Cold Spring Harbor Laboratory Press REVIEW Segmentation in vertebrates: clock and gradient finally joined Alexander Aulehla1 and Bernhard G. Herrmann2 Max-Planck-Institute for Molecular Genetics, Department of Developmental Genetics, 14195 Berlin, Germany The vertebral column is derived from somites formed by terior (A–P) axis. Somite formation takes place periodi- segmentation of presomitic mesoderm, a fundamental cally in a fixed anterior-to-posterior sequence. process of vertebrate embryogenesis. Models on the In the chick embryo, a new somite is formed approxi- mechanism controlling this process date back some mately every 90 min, whereas in the mouse embryo, the three to four decades. Access to understanding the mo- periodicity varies, dependent on the axial position (Tam lecular control of somitogenesis has been gained only 1981). Classical embryology experiments revealed that recently by the discovery of molecular oscillators (seg- periodicity and directionality of somite formation are mentation clock) and gradients of signaling molecules, controlled by an intrinsic program set off in the cells as as predicted by early models. The Notch signaling path- they are recruited into the psm. For instance, when the way is linked to the oscillator and plays a decisive role in psm is inverted rostro–caudally, somite formation in the inter- and intrasomitic boundary formation. An Fgf8 sig- inverted region proceeds from caudal to rostral, main- naling gradient is involved in somite size control. And taining the original direction (Christ et al. 1974). More- the (canonical) Wnt signaling pathway, driven by Wnt3a, over, neither the transversal bisection nor the isolation appears to integrate clock and gradient in a global of the psm from all surrounding tissues stops the seg- mechanism controlling the segmentation process.
    [Show full text]
  • Autism Multiplex Family with 16P11.2P12.2 Microduplication Syndrome in Monozygotic Twins and Distal 16P11.2 Deletion in Their Brother
    European Journal of Human Genetics (2012) 20, 540–546 & 2012 Macmillan Publishers Limited All rights reserved 1018-4813/12 www.nature.com/ejhg ARTICLE Autism multiplex family with 16p11.2p12.2 microduplication syndrome in monozygotic twins and distal 16p11.2 deletion in their brother Anne-Claude Tabet1,2,3,4, Marion Pilorge2,3,4, Richard Delorme5,6,Fre´de´rique Amsellem5,6, Jean-Marc Pinard7, Marion Leboyer6,8,9, Alain Verloes10, Brigitte Benzacken1,11,12 and Catalina Betancur*,2,3,4 The pericentromeric region of chromosome 16p is rich in segmental duplications that predispose to rearrangements through non-allelic homologous recombination. Several recurrent copy number variations have been described recently in chromosome 16p. 16p11.2 rearrangements (29.5–30.1 Mb) are associated with autism, intellectual disability (ID) and other neurodevelopmental disorders. Another recognizable but less common microdeletion syndrome in 16p11.2p12.2 (21.4 to 28.5–30.1 Mb) has been described in six individuals with ID, whereas apparently reciprocal duplications, studied by standard cytogenetic and fluorescence in situ hybridization techniques, have been reported in three patients with autism spectrum disorders. Here, we report a multiplex family with three boys affected with autism, including two monozygotic twins carrying a de novo 16p11.2p12.2 duplication of 8.95 Mb (21.28–30.23 Mb) characterized by single-nucleotide polymorphism array, encompassing both the 16p11.2 and 16p11.2p12.2 regions. The twins exhibited autism, severe ID, and dysmorphic features, including a triangular face, deep-set eyes, large and prominent nasal bridge, and tall, slender build. The eldest brother presented with autism, mild ID, early-onset obesity and normal craniofacial features, and carried a smaller, overlapping 16p11.2 microdeletion of 847 kb (28.40–29.25 Mb), inherited from his apparently healthy father.
    [Show full text]
  • Product Description SALSA® MLPA® Probemix P463-A2 MRKH to Be Used with the MLPA General Protocol
    MRC-Holland ® Product Description version A2-01; Issued 16 July 2020 MLPA Product Description SALSA® MLPA® Probemix P463-A2 MRKH To be used with the MLPA General Protocol. Version A2. As compared to version A1, five reference probes have been replaced and one probe length has been adjusted. For complete product history see page 7. Catalogue numbers: P463-025R: SALSA MLPA Probemix P463 MRKH, 25 reactions. P463-050R: SALSA MLPA Probemix P463 MRKH, 50 reactions. P463-100R: SALSA MLPA Probemix P463 MRKH, 100 reactions. To be used in combination with a SALSA MLPA reagent kit and Coffalyser.Net data analysis software. MLPA reagent kits are either provided with FAM or Cy5.0 dye-labelled PCR primer, suitable for Applied Biosystems and Beckman/SCIEX capillary sequencers, respectively (see www.mlpa.com). Certificate of Analysis: Information regarding storage conditions, quality tests, and a sample electropherogram from the current sales lot is available at www.mlpa.com. Precautions and warnings: For professional use only. Always consult the most recent product description AND the MLPA General Protocol before use: www.mlpa.com. It is the responsibility of the user to be aware of the latest scientific knowledge of the application before drawing any conclusions from findings generated with this product. General information: The SALSA MLPA Probemix P463 MRKH is a research use only (RUO) assay for the detection of deletions or duplications in the TBX6, LHX1, HNF1B, and TBX1 genes, which are associated with Mayer-Rokitansky-Küster-Hauser syndrome (MRKH). MRKH is characterised by normal physical development of the secondary sexual characteristics and a normal female 46,XX karyotype but with complete aplasia of the uterus, cervix, and superior parts of vagina leading to failure to menstruate and infertility.
    [Show full text]
  • The Regulation of Lunatic Fringe During Somitogenesis
    THE REGULATION OF LUNATIC FRINGE DURING SOMITOGENESIS DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Emily T. Shifley ***** The Ohio State University 2009 Dissertation Committee: Approved by Professor Susan Cole, Advisor Professor Christine Beattie _________________________________ Professor Mark Seeger Advisor Graduate Program in Molecular Genetics Professor Michael Weinstein ABSTRACT Somitogenesis is the morphological hallmark of vertebrate segmentation. Somites bud from the presomitic mesoderm (PSM) in a sequential, periodic fashion and give rise to the rib cage, vertebrae, and dermis and muscles of the back. The regulation of somitogenesis is complex. In the posterior region of the PSM, a segmentation clock operates to organize cohorts of cells into presomites, while in the anterior region of the PSM the presomites are patterned into rostral and caudal compartments (R/C patterning). Both of these stages of somitogenesis are controlled, at least in part, by the Notch pathway and Lunatic fringe (Lfng), a glycosyltransferase that modifies the Notch receptor. To dissect the roles played by Lfng during somitogenesis, we created a novel allele that lacks cyclic Lfng expression within the segmentation clock, but that maintains expression during R/C somite patterning (Lfng∆FCE1). Lfng∆FCE1/∆FCE1 mice have severe defects in their anterior vertebrae and rib cages, but relatively normal sacral and tail vertebrae, unlike Lfng knockouts. Segmentation clock function is differentially affected by the ∆FCE1 deletion; during anterior somitogenesis the expression patterns of many clock genes are disrupted, while during posterior somitogenesis, certain clock components have recovered. R/C patterning occurs relatively normally in Lfng∆FCE1/∆FCE1 embryos, likely contributing to the partial phenotype rescue, and confirming that Lfng ii plays separate roles in the two regions of the PSM.
    [Show full text]
  • Capacity Grants.Gov Application Guide
    7. SF424A Budget 7.1 Enter Budget information on the SF 424a You must complete the required fields on each page of the SF 424a for the specified grant program, which includes attaching a budget justification (See 7.X Budget Justification, of this section). Only list one program (i.e. Smith-Lever, Hatch, etc.) per SF424a. Budget information is for the upcoming Fiscal year. 7.2 Enter information on Section A, Budget Summary, Line 1 (a) Grant Program function of Activity: Enter the capacity program RFA title under which the application is being submitted (e.g. Smith-Lever, Hatch; Hatch Multistate; Section 1444; Section 1445 Evans Allen; McIntire-Stennis; or Animal Health Disease Research) (b) Catalogue of Federal Domestic Assistance Number (CFDA): Enter the CFDA number for the program. Reminder, extension CFDAs changed in FY 2019; see https://nifa.usda.gov/resource/10500- change for more information. (c) Estimated Unobligated Funds – Federal: Enter the amount of Federal carry over funds from the previous FY. For Smith-lever, since funds may be carried over up to 5 years, this will be the cumulative amount of carry-over from previous FY’s. (d) Estimated Unobligated Funds – Non Federal: Enter the amount of matching funds (Non Federal share) being carried over; funds reported are from the corresponding FY to the Federal award. (e) New or Revised Budget – Federal: Enter the amount from Appendix A in the appropriate capacity RFA. (f) New or Revised Budget – Non Federal: Enter the required match, if applicable. If you are submitting a matching waiver request with the application, enter the amount of match that would be required if the waiver is approved.
    [Show full text]
  • Neural Development
    31 October 2007 NEURAL DEVELOPMENT www.neuraldevelopment.com Analysis of Lrrn1 expression and its relationship to neuromeric boundaries during chick neural development Laura C Andreae et al. Neural Development 2007, 2:22 http://www.neuraldevelopment.com/content/2/1/22 Neural Development BioMed Central Research article Open Access Analysis of Lrrn1 expression and its relationship to neuromeric boundaries during chick neural development LauraCAndreae1,2, Daniela Peukert1, Andrew Lumsden1 and Jonathan D Gilthorpe*1 Address: 1MRC Centre for Developmental Neurobiology, King's College London, New Hunt's House, Guy's Campus, London, UK, SE1 1UL and 2Department of Neurophysiology, National Institute for Medical Research, The Ridgeway, Mill Hill, London, UK, NW7 1AA Email: Laura C Andreae - [email protected]; Daniela Peukert - [email protected]; Andrew Lumsden - [email protected]; Jonathan D Gilthorpe* - [email protected] * Corresponding author Published: 31 October 2007 Received: 26 March 2007 Accepted: 31 October 2007 Neural Development 2007, 2:22 doi:10.1186/1749-8104-2-22 This article is available from: http://www.neuraldevelopment.com/content/2/1/22 © 2007 Andreae et al.; licensee BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract Background: The Drosophila leucine-rich repeat proteins Tartan (TRN) and Capricious (CAPS) mediate cell affinity differences during compartition of the wing imaginal disc. This study aims to identify and characterize the expression of a chick orthologue of TRN/CAPS and examine its potential function in relation to compartment boundaries in the vertebrate central nervous system.
    [Show full text]
  • Novel ENU-Induced Mutation in Tbx6 Causes Dominant Spondylocostal Dysostosis-Like Vertebral Malformations in the Rat
    RESEARCH ARTICLE Novel ENU-Induced Mutation in Tbx6 Causes Dominant Spondylocostal Dysostosis-Like Vertebral Malformations in the Rat Koichiro Abe1*, Nobuhiko Takamatsu2, Kumiko Ishikawa1, Toshiko Tsurumi3, Sho Tanimoto1, Yukina Sakurai2, Thomas Lisse4, Kenji Imai1, Tadao Serikawa3, Tomoji Mashimo3¤ 1 Department of Molecular Life Science, Tokai University School of Medicine, Isehara, Kanagawa, Japan, 2 Department of Biosciences, School of Science, Kitasato University, Sagamihara, Kanagawa, Japan, 3 Institute of Laboratory Animals, Graduate School of Medicine, Kyoto University, Kyoto, Japan, 4 MDI Biological Laboratory, Davis Center for Regenerative Biology and Medicine, Bar Harbor, Maine, United States of America ¤ Current address: Institute of Experimental Animal Sciences, Graduate School of Medicine, Osaka University, Osaka, Japan. * [email protected] OPEN ACCESS Abstract Citation: Abe K, Takamatsu N, Ishikawa K, Tsurumi T, Tanimoto S, Sakurai Y, et al. (2015) Novel ENU- Congenital vertebral malformations caused by embryonic segmentation defects are rela- Induced Mutation in Tbx6 Causes Dominant tively common in humans and domestic animals. Although reverse genetics approaches in Spondylocostal Dysostosis-Like Vertebral mice have provided information on the molecular mechanisms of embryonic somite seg- Malformations in the Rat. PLoS ONE 10(6): e0130231. doi:10.1371/journal.pone.0130231 mentation, hypothesis-driven approaches cannot adequately reflect human dysmorphology within the population. In a N-ethyl-N-nitrosourea (ENU) mutagenesis project in Kyoto, the Editor: Tom J. Carney, Institute of Molecular and Cell Oune Biology, SINGAPORE mutant rat strain was isolated due to a short and kinked caudal vertebra phenotype. Skeletal staining of heterozygous rats showed partial loss of the cervical vertebrae as well Received: July 27, 2014 as hemivertebrae and fused vertebral blocks in lumbar and sacral vertebrae.
    [Show full text]
  • Caspase-8, Receptor-Interacting Protein Kinase 1 (RIPK1), and RIPK3 Regulate Retinoic Acid-Induced Cell Differentiation and Necroptosis
    Cell Death & Differentiation (2020) 27:1539–1553 https://doi.org/10.1038/s41418-019-0434-2 ARTICLE Caspase-8, receptor-interacting protein kinase 1 (RIPK1), and RIPK3 regulate retinoic acid-induced cell differentiation and necroptosis 1,2 1,3 4 3 1,2,4 Masataka Someda ● Shunsuke Kuroki ● Hitoshi Miyachi ● Makoto Tachibana ● Shin Yonehara Received: 1 July 2019 / Revised: 4 October 2019 / Accepted: 4 October 2019 / Published online: 28 October 2019 © The Author(s) 2019. This article is published with open access Abstract Among caspase family members, Caspase-8 is unique, with associated critical activities to induce and suppress death receptor-mediated apoptosis and necroptosis, respectively. Caspase-8 inhibits necroptosis by suppressing the function of receptor-interacting protein kinase 1 (RIPK1 or RIP1) and RIPK3 to activate mixed lineage kinase domain-like (MLKL). Disruption of Caspase-8 expression causes embryonic lethality in mice, which is rescued by depletion of either Ripk3 or Mlkl, indicating that the embryonic lethality is caused by activation of necroptosis. Here, we show that knockdown of Caspase-8 expression in embryoid bodies derived from ES cells markedly enhances retinoic acid (RA)-induced cell differentiation and necroptosis, both of which are dependent on Ripk1 and Ripk3; however, the enhancement of RA-induced 1234567890();,: 1234567890();,: cell differentiation is independent of Mlkl and necrosome formation. RA treatment obviously enhanced the expression of RA-specific target genes having the retinoic acid response element (RARE) in their promoter regions to induce cell differentiation, and induced marked expression of RIPK1, RIPK3, and MLKL to stimulate necroptosis. Caspase-8 knockdown induced RIPK1 and RIPK3 to translocate into the nucleus and to form a complex with RA receptor (RAR), and RAR interacting with RIPK1 and RIPK3 showed much stronger binding activity to RARE than RAR without RIPK1 or RIPK3.
    [Show full text]
  • Regulation of Myogenesis by Cardiotrophin-1 and Tgfp Signalling
    Regulation of Myogenesis by Cardiotrophin-1 and TGFp signalling Tetsuaki Miyake A dissertation submitted to the Faculty of Graduate Studies in partial fulfillment of the requirements for the degree of Doctor of Philosophy Graduate Programme in Biology York University Toronto, Ontario, Canada Jan 2010 Library and Archives Bibliotheque et 1*1 Canada Archives Canada Published Heritage Direction du Branch Patrimoine de I'edition 395 Wellington Street 395, rue Wellington OttawaONK1A0N4 OttawaONK1A0N4 Canada Canada Your file Votre reference ISBN: 978-0-494-64946-6 Our file Notre reference ISBN: 978-0-494-64946-6 NOTICE: AVIS: The author has granted a non­ L'auteur a accorde une licence non exclusive exclusive license allowing Library and permettant a la Bibliotheque et Archives Archives Canada to reproduce, Canada de reproduire, publier, archiver, publish, archive, preserve, conserve, sauvegarder, conserver, transmettre au public communicate to the public by par telecommunication ou par I'lnternet, preter, telecommunication or on the Internet, distribuer et vendre des theses partout dans le loan, distribute and sell theses monde, a des fins commerciales ou autres, sur worldwide, for commercial or non­ support microforme, papier, electronique et/ou commercial purposes, in microform, autres formats. paper, electronic and/or any other formats. The author retains copyright L'auteur conserve la propriete du droit d'auteur ownership and moral rights in this et des droits moraux qui protege cette these. Ni thesis. Neither the thesis nor la these ni des extraits substantiels de celle-ci substantial extracts from it may be ne doivent etre imprimes ou autrement printed or otherwise reproduced reproduits sans son autorisation.
    [Show full text]
  • Species-Specific Oscillation Periods of Human and Mouse Segmentation
    bioRxiv preprint doi: https://doi.org/10.1101/650648; this version posted May 26, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 1 Species-specific oscillation periods of human and mouse segmentation clocks 2 are due to cell autonomous differences in biochemical reaction parameters 3 4 Mitsuhiro Matsuda1, 2, Hanako Hayashi1, Jordi Garcia-Ojalvo3, Kumiko Yoshioka- 5 Kobayashi4, Ryoichiro Kageyama4, Yoshihiro Yamanaka5, Makoto Ikeya5, Junya 6 Toguchida4, 5, Cantas Alev5, Miki Ebisuya1, 2* 7 8 1RIKEN Center for Biosystems Dynamics Research (RIKEN BDR) 9 2-2-3 Minatojima-minamimachi, Chuo-ku, 650-0047 Kobe, Japan 10 2European Molecular Biology Laboratory (EMBL) Barcelona 11 Dr. Aiguader 88, 08003 Barcelona, Spain 12 3Department of Experimental and Health Sciences, Universitat Pompeu Fabra 13 Dr. Aiguader 88, 08003 Barcelona, Spain 14 4Institute for Frontier Life and Medical Sciences, Kyoto University 15 Shogoin-Kawahara-cho, Sakyo-ku, 606-8507 Kyoto, Japan 16 5Center for iPS Cell Research and Application (CiRA), Kyoto University 17 53 Shogoin-Kawahara-cho, Sakyo-ku, 606-8507 Kyoto, Japan 18 19 20 *Correspondence to M Ebisuya ([email protected]) 21 1 bioRxiv preprint doi: https://doi.org/10.1101/650648; this version posted May 26, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
    [Show full text]
  • Subtitle 22A-6B
    Regulations of Connecticut State Agencies TITLE 22a. Environmental Protection Agency Department of Environmental Protection Subject Assessment of Civil Penalties Inclusive Sections §§ 22a-6b-1—22a-6b-701 CONTENTS Assessment of Civil Penalties Sec. 22a-6b-100—22a-6b-701. Repealed Administrative Civil Penalty Sec. 22a-6b-1. Authority Sec. 22a-6b-2. Purpose Sec. 22a-6b-3. Definitions Sec. 22a-6b-4. Procedures Sec. 22a-6b-5. Scope of issues at hearing Sec. 22a-6b-6. Burden of proof Sec. 22a-6b-7. Commissioner’s powers Sec. 22a-6b-8. Method and schedule for calculating an administrative civil penalty Sec. 22a-6b-9. Assessment of administrative civil penalty—penalty recalculation Sec. 22a-6b-10. Settlement conferences Sec. 22a-6b-11. Assessment of administrative civil penalty—resolu - tion of penalty notice prior to completion of settle - ment conference Sec. 22a-6b-12. Assessment of administrative civil penalty—resolu - tion of penalty notice after settlement conference but prior to hearing Sec. 22a-6b-13. Assessment of administrative civil penalty—resolu - tion of penalty notice by consent order Sec. 22a-6b-14. Final decision on a penalty notice Sec. 22a-6b-15. Payment of penalties Revised: 2015-3-6 R.C.S.A. §§ 22a-6b-1—22a-6b-701 - I- Regulations of Connecticut State Agencies TITLE 22a. Environmental Protection Department of Environmental Protection §22a-6b-3 Assessment of Civil Penalties Assessment of Civil Penalties Sec. 22a-6b-100—22a-6b-701. Repealed Repealed May 29, 2007. Administrative Civil Penalty Sec. 22a-6b-1. Authority Sections 22a-6b-1 to 22a-6b-15, inclusive, shall be known as the department’s Administrative Civil Penalty Regulations.
    [Show full text]