ATR promotes cilia signalling: links to developmental impacts

Mutations in ATR (ataxia telangiectasia and RAD3-related) cause Seckel syndrome (ATR-SS), a microcephalic primordial dwarfism disorder. Hitherto, the clinical manifestation of ATR deficiency has been attributed to its canonical role in DNA damage response signalling following replication fork stalling/collapse. Here, we show that ATR regulates cilia-dependent signalling in a manner that can be uncoupled from its function during replication. ATR-depleted or patient-derived ATR-SS cells form cilia of slightly reduced length but are dramatically impaired in cilia-dependent signalling functions, including growth factor and Sonic hedgehog signalling. To better understand the developmental impact of ATR loss of function, we also used zebrafish as a model. Zebrafish embryos depleted of Atr resembled ATR-SS morphology, showed a modest but statistically significant reduction in cilia length and other morphological features indicative of cilia dysfunction. Additionally, they displayed defects in left-right asymmetry including ambiguous expression of southpaw, incorrectly looped hearts and randomized localization of internal organs including the pancreas, features typically conferred by cilia dysfunction. Our findings reveal a novel role for ATR in cilia signalling distinct from its canonical function during replication and strengthen emerging links between cilia function and development.

[1]  K. Anderson,et al.  Cilia and developmental signaling. , 2007, Annual review of cell and developmental biology.

[2]  F. Alkuraya,et al.  POC1A truncation mutation causes a ciliopathy in humans characterized by primordial dwarfism. , 2012, American journal of human genetics.

[3]  Randall W King,et al.  Small molecules that delay S phase suppress a zebrafish bmyb mutant , 2005, Nature chemical biology.

[4]  William C Earnshaw,et al.  Mutations in pericentrin cause Seckel syndrome with defective ATR-dependent DNA damage signaling , 2008, Nature Genetics.

[5]  S. Matsuura,et al.  Two unrelated patients with MRE11A mutations and Nijmegen breakage syndrome-like severe microcephaly. , 2011, DNA repair.

[6]  Wallace F. Marshall,et al.  Chapter 1 Basal Bodies , 2008 .

[7]  D. Pellman,et al.  Centrosomes and cilia in human disease. , 2011, Trends in genetics : TIG.

[8]  P. Jeggo,et al.  Deficiency in Origin Licensing Proteins Impairs Cilia Formation: Implications for the Aetiology of Meier-Gorlin Syndrome , 2013, PLoS genetics.

[9]  Judith A. Goodship,et al.  A splicing mutation affecting expression of ataxia–telangiectasia and Rad3–related protein (ATR) results in Seckel syndrome , 2003, Nature Genetics.

[10]  P. Jeggo,et al.  Identification of the First ATRIP–Deficient Patient and Novel Mutations in ATR Define a Clinical Spectrum for ATR–ATRIP Seckel Syndrome , 2012, PLoS genetics.

[11]  M. Brueckner,et al.  Intraciliary calcium oscillations initiate vertebrate left-right asymmetry , 2015, Current Biology.

[12]  Sergey V. Prykhozhij In the Absence of Sonic Hedgehog, p53 Induces Apoptosis and Inhibits Retinal Cell Proliferation, Cell-Cycle Exit and Differentiation in Zebrafish , 2010, PloS one.

[13]  E. Nam,et al.  ATR signalling: more than meeting at the fork. , 2011, The Biochemical journal.

[14]  Kevin W Eliceiri,et al.  NIH Image to ImageJ: 25 years of image analysis , 2012, Nature Methods.

[15]  R. Medema,et al.  Polo-like kinase-1 is a target of the DNA damage checkpoint , 2000, Nature Cell Biology.

[16]  J. García-Verdugo,et al.  Primary cilia are required for cerebellar development and Shh-dependent expansion of progenitor pool. , 2008, Developmental biology.

[17]  M. Caron,et al.  Grk5l controls heart development by limiting mTOR signaling during symmetry breaking. , 2013, Cell reports.

[18]  A. Schier,et al.  Dampened Hedgehog signaling but normal Wnt signaling in zebrafish without cilia , 2009, Development.

[19]  F. Alkuraya,et al.  A founder CEP120 mutation in Jeune asphyxiating thoracic dystrophy expands the role of centriolar proteins in skeletal ciliopathies , 2014, Human molecular genetics.

[20]  M. Asanuma,et al.  Embryonic expression of pericentrin suggests universal roles in ciliogenesis , 2006, Development Genes and Evolution.

[21]  G. Pazour,et al.  Pericentrin forms a complex with intraflagellar transport proteins and polycystin-2 and is required for primary cilia assembly , 2004, The Journal of cell biology.

[22]  Jussi Taipale,et al.  Small molecule modulation of Smoothened activity , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[23]  F. Mulero,et al.  A mouse model of ATR-Seckel shows embryonic replicative stress and accelerated aging , 2009, Nature Genetics.

[24]  S. Burgess,et al.  Analysis of the zebrafish smoothened mutant reveals conserved and divergent functions of hedgehog activity. , 2001, Development.

[25]  M. O’Driscoll,et al.  Congenital microcephaly , 2014, American journal of medical genetics. Part C, Seminars in medical genetics.

[26]  H. Yost,et al.  Kupffer's vesicle is a ciliated organ of asymmetry in the zebrafish embryo that initiates left-right development of the brain, heart and gut , 2005, Development.

[27]  C. Woods,et al.  Primary microcephaly: do all roads lead to Rome? , 2009, Trends in genetics : TIG.

[28]  Peter Nürnberg,et al.  Mutations in PLK4, encoding a master regulator of centriole biogenesis, cause microcephaly, growth failure and retinopathy , 2014, Nature Genetics.

[29]  A. Wright,et al.  ATR localizes to the photoreceptor connecting cilium and deficiency leads to severe photoreceptor degeneration in mice. , 2013, Human molecular genetics.

[30]  L. Karnitz,et al.  The Rad9-Hus1-Rad1 (9-1-1) clamp activates checkpoint signaling via TopBP1. , 2007, Genes & development.

[31]  Hiroshi Hamada,et al.  Establishment of vertebrate left–right asymmetry , 2002, Nature Reviews Genetics.

[32]  Olivia Barton,et al.  Role of ATM and the Damage Response Mediator Proteins 53BP1 and MDC1 in the Maintenance of G2/M Checkpoint Arrest , 2010, Molecular and Cellular Biology.

[33]  Corinne Stoetzel,et al.  Exome Capture Reveals ZNF423 and CEP164 Mutations, Linking Renal Ciliopathies to DNA Damage Response Signaling , 2012, Cell.

[34]  S. Thiberge,et al.  Imaging cilia in zebrafish. , 2010, Methods in cell biology.

[35]  P. Jeggo,et al.  ATR‐dependent phosphorylation and activation of ATM in response to UV treatment or replication fork stalling , 2006, The EMBO journal.

[36]  Hussain Jafri,et al.  A centrosomal mechanism involving CDK5RAP2 and CENPJ controls brain size , 2005, Nature Genetics.

[37]  M. Tohyama,et al.  Pericentrin, a centrosomal protein related to microcephalic primordial dwarfism, is required for olfactory cilia assembly in mice , 2009, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[38]  Peter Satir,et al.  PDGFRalphaalpha signaling is regulated through the primary cilium in fibroblasts. , 2005, Current biology : CB.

[39]  Stephen J. Elledge,et al.  Sensing DNA Damage Through ATRIP Recognition of RPA-ssDNA Complexes , 2003, Science.

[40]  M. Rebagliati,et al.  The zebrafish nodal-related gene southpaw is required for visceral and diencephalic left-right asymmetry , 2003, Development.

[41]  K. Devriendt,et al.  Mutations in the Pericentrin (PCNT) Gene Cause Primordial Dwarfism , 2008, Science.

[42]  D. McClay,et al.  Hedgehog signaling requires motile cilia in the sea urchin. , 2014, Molecular biology and evolution.

[43]  A. Carr,et al.  Targeted disruption of the cell-cycle checkpoint gene ATR leads to early embryonic lethality in mice , 2000, Current Biology.

[44]  Cheol‐Hee Kim,et al.  IFT46 plays an essential role in cilia development. , 2015, Developmental biology.

[45]  Peter Satir,et al.  PDGFRαα Signaling Is Regulated through the Primary Cilium in Fibroblasts , 2005, Current Biology.

[46]  C. Zhao,et al.  Analysis of cilia structure and function in zebrafish. , 2016, Methods in cell biology.

[47]  O. A. Cabello,et al.  Cilia Proteins Control Cerebellar Morphogenesis by Promoting Expansion of the Granule Progenitor Pool , 2007, The Journal of Neuroscience.

[48]  Gemma K. Alderton,et al.  Regulation of mitotic entry by microcephalin and its overlap with ATR signalling , 2006, Nature Cell Biology.

[49]  W. Martin,et al.  AGONISTS AND ANTAGONISTS , 2006 .

[50]  W. Marshall,et al.  Basal bodies platforms for building cilia. , 2008, Current topics in developmental biology.

[51]  A. Álvarez-Buylla,et al.  Role of primary cilia in brain development and cancer , 2010, Current Opinion in Neurobiology.

[52]  J. Goodship,et al.  The ciliary Evc/Evc2 complex interacts with Smo and controls Hedgehog pathway activity in chondrocytes by regulating Sufu/Gli3 dissociation and Gli3 trafficking in primary cilia. , 2013, Human molecular genetics.

[53]  K. Anderson,et al.  The primary cilium: a signalling centre during vertebrate development , 2010, Nature Reviews Genetics.

[54]  J. Opitz,et al.  Studies of microcephalic primordial dwarfism I: approach to a delineation of the Seckel syndrome. , 1982, American journal of medical genetics.

[55]  Pierre Gressens,et al.  Many roads lead to primary autosomal recessive microcephaly , 2010, Progress in Neurobiology.

[56]  J. Wallingford,et al.  Strange as it may seem: the many links between Wnt signaling, planar cell polarity, and cilia. , 2011, Genes & development.

[57]  D. Stern,et al.  Phosphorylation of Plk1 at S137 and T210 is Inhibited in Response to DNA Damage , 2005, Cell cycle.

[58]  Rappold,et al.  Human Molecular Genetics , 1996, Nature Medicine.

[59]  H. Wichterle,et al.  Small-molecule modulators of Hedgehog signaling: identification and characterization of Smoothened agonists and antagonists , 2002, Journal of biology.

[60]  B. Yoder,et al.  Polaris, a protein involved in left-right axis patterning, localizes to basal bodies and cilia. , 2001, Molecular biology of the cell.

[61]  D. Baltimore,et al.  Essential and dispensable roles of ATR in cell cycle arrest and genome maintenance. , 2003, Genes & development.

[62]  N. Katsanis,et al.  The Vertebrate Primary Cilium in Development, Homeostasis, and Disease , 2009, Cell.

[63]  B. Thisse,et al.  High-resolution in situ hybridization to whole-mount zebrafish embryos , 2007, Nature Protocols.

[64]  C. Stiles,et al.  Dual control of cell growth by somatomedins and platelet-derived growth factor. , 1979, Proceedings of the National Academy of Sciences of the United States of America.

[65]  Á. Raya,et al.  Left–right asymmetry in the vertebrate embryo: from early information to higher-level integration , 2006, Nature Reviews Genetics.

[66]  R. Medema,et al.  Inhibition of Polo-like Kinase-1 by DNA Damage Occurs in an ATM- or ATR-dependent Fashion* , 2001, The Journal of Biological Chemistry.

[67]  Mark S. Miller,et al.  A genetic screen in zebrafish identifies cilia genes as a principal cause of cystic kidney , 2004, Development.

[68]  F. Wilson,et al.  Cdc42 deficiency causes ciliary abnormalities and cystic kidneys. , 2013, Journal of the American Society of Nephrology : JASN.

[69]  G. Shivashankar,et al.  ATR Mediates a Checkpoint at the Nuclear Envelope in Response to Mechanical Stress , 2014, Cell.

[70]  Robert A. Bloodgood Sensory reception is an attribute of both primary cilia and motile cilia , 2010, Journal of Cell Science.