A/Professor Sally Dunwoodie, BSc, PhDLaboratory Head, Developmental Biology DivisionAssociate Professor, University of New South Wales
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The genetic analysis of development in the mouse has direct relevance to the molecular basis of congenital abnormalities and other pathological conditions, such as neoplasia, in humans. Consequently current research in our laboratory focuses on: Identifying and characterising genes relevant to mammalian embryonic development; Notch signaling and its role in somite and consequently vertebral column formation; and the roles of Cited1 and Cited2 in formation and function of the heart and placenta.
Notch signaling and the role of Delta-like3 (Dll3) in vertebral column formation
The Notch signalling pathway is crucial in numerous developmental processes, which in vertebrates include neurogenesis, vasculogenesis, hematopoiesis and somitogenesis. Mutations of human genes encoding components of this pathway are associated with diverse conditions including degenerative (Alzheimer, CADASIL) and developmental (Alagille) disorders, and cancer (T-cell acute lymphoblastic leukaemia). Notch genes encode for class-I transmembrane receptors, activated by Delta and Serrate/Jagged class transmembrane ligands. The affinity of Notch for its ligands can be modified by differential glycosylation of both receptor and ligand mediated by Fringe proteins. Notch undergoes proteolytic cleavage at the membrane level, by proteases in mammals that include the gamma-secretase Presenilin 1, this leads to the translocation of its intracellular domain into the nucleus, where together with the transcription factor RBPj/CSL it activates transcription in mouse of downstream target genes, such as Hes (hairy and enhancer of split) and Hey/Hesr/Hrt (Hes-related).
Loss of Dll3, a ligand of Notch, results in axial skeletal defects whereby vertebrae and ribs are irregular in size and shape, and many are absent. These features are very similar to those found in spondylocostal dysostosis (SCD), a heterogeneous group of disorders in humans where severe axial skeletal malformation is characterised radiographically by multiple vertebral segmentation defects with rib misalignment and reduction. These diverse SCD phenotypes can be either familial or sporadic and follow either autosomal recessive (ARSCD) or, less commonly, autosomal dominant inheritance. We have shown that ARSCD is due to mutation in DLL3 (ARSCD type1), or in targets of Notch signalling MESP2 (ARSCD type 2) and LFNG (ARSCD type 3). Mutation in DLL3, MESP2, LFNG or HES7 does not account for all cases of SCD and so the genetic cause of SCD in these instances is being sought.
The International Consortium for Vertebral Anomalies and Scoliosis (ICVAS) has been established to better understand the etiology of scoliosis and vertebral anomalies in humans. Visit http://www.icvas.org/ to learn more about the consortium.
The roles of Cited1 and Cited2 in formation and function of the heart and placenta
The CITED (CBP/p300-Interacting Transactivators with glutamic acid (E)/aspartic acid (D)-rich carboxyl terminal domain) gene family is represented by four genes: Cited1 (formerly Msg1), Cited2 (formerly Mrg1), Cited3 and Cited4. These genes encode proteins that interact with the transcriptional co-activators and acetyl transferases CBP (cAMP-responsive element binding protein) and p300. CITEDs bind the cysteine-histidine-rich (CH1) domain of CBP/p300 like many transcription factors and modulators of transcription; these include RXRa, NFkB, Stat2, Mdm2, Ets-1 and HIF1a. Cited2 is a direct target of the transcription factor, HIF1a (hypoxia inducible factor 1a). In addition it acts to negatively regulate HIF1a by competing with HIF1a for an overlapping site on the CH1 of CBP/p300. This may represent a common mechanism of transcription control for factors that bind the CH1 domain of CBP/p300 since Cited2 also inhibit Ets-1 activity by competing with Ets-1 for the CH1 domain. Consequently, the Cited proteins may well represent major regulators of transcription as they have the capacity to affect the expression of numerous genes. In addition to acting as negative regulators of transcription, Cited proteins also interact directly with transcription factors and co-factors (Lhx2, TFAP2, Smad4, estrogen receptor alpha/beta) and in association with CBP/p300 can enhance transcriptional activity.
We have generated null mutant mouse lines for Cited1 and Cited2; each of these genes is required for normal embryonic development. The predominant defect is in the placenta of the Cited1 null conceptus. Cited2 null embryos have severe cardiac defects as well as defects in placental form and function. The heart defects occur due to multiple roles for Cited2, one is in the establishment of the left-right body axis. Using a combination of methods (histology, gene expression profiling, in situ gene expression analysis, resin casting of blood vessels, explant culture, embryo culture, cell culture and conditional mutagenesis) we are characterising the defects in Cited1 and Cited2 mutants. In addition, we are exploring the function of these proteins at a molecular level.
Co-Investigators:
Stanley Artap, BSc(Hons)
Gavin Chapman, PhD
Wendy Chua, BSc(Hons)
Alex James, BSc(Hons)
Kylie Lopes Floro, PhD
Sharon Pursglove, PhD
Duncan Sparrow, PhD
Natalie Wise, BSc
Michelle Woolford, BSc(Hons)
Collaborators:
Mark de Caestecker MBBS, PhD; Vanderbilt University Medical Center, Nashville TN, USA
Diane Fatkin, MD BSc(Med) FRACP; VCCRI, Sydney, Australia
Achim Gossler, PhD; Institut fur Molekularbiologie, Medizinische Hochschule Hannover, Hannover, German
Hiroshi Hamada, PhD; Graduate School of Frontier Biosciences, Osaka University, Japan
Richard Harvey, PhD; VCCRI, Sydney, Australia
Gerard Hoyne, PhD; Australian National University, ACT Australia
Kenro Kusumi, PhD; Univ. of Pennsylvania School of Medicine, Philadelphia PA, USA
David Sillence, MD PhD; Children's Hospital Westmead, Sydney, Australia
Patrick Tam, PhD; The Children's Medical Research Institute, Westmead, Australia
Paul Thomas PhD; University of Adelaide, Adelaide, Australia
Peter Turnpenny, MBChB FRCP FRCPCH; Royal Devon & Exeter Hospital, Exeter, UK
Emma Whitelaw, PhD; University of Sydney, Sydney, Australia
David Winlaw MD PhD; Children's Hospital Westmead, Sydney, Australia
Merridee Wouters, PhD; VCCRI, Sydney, Australia
Yu-Chung Yang, PhD; Case Western Reserve University, Cleveland, USA
Selected Publications:
Harrison SM, Dunwoodie SL, Arkell RM, Lehrach H, Beddington RSP. Isolation of novel tissue-specific genes from cDNA libraries representing the individual tissue constituents of the gastrulating mouse embryo. Development 1995; 121:2479-2489
Dunwoodie SL, Henrique D, Harrison SM, Beddington RSP. Mouse DII3: a novel divergent Delta gene which may complement the function of other Delta homologues during early pattern formation in the mouse embryo. Development 1997; 124:3065-3076
Avner P, Bruls T, Poras I, Eley L, Gas S, Ruiz P, Wiles MV, Sousa-Nunes R, Kettleborough R, Rana A, Morrisette J, Bentley L, Goldsworthy M, Haynes A, Herebt E, Southam L, Taghavi V, Sartory E, Lehrach H, Weissenbach J, Manenti G, Rodriguez-Tome P, Beddington RSP, Dunwoodie SL, Cox R. A radiation hybrid transcript map of the mouse genome. Nature Genetics 2001; 29:194-200
Martinez Barbera JP, Rodriquez TA, Greene N, Weninger WJ, Simeone A, Copp A, Beddington RSP, Dunwoodie SL. Administration of folic acid prevents excencephaly in Cited2 deficient mice. Human Molecular Genetics 2002; 11:283-293
Dunwoodie SL, Clements M, Sparrow DB, Sa X, Conlon RA, Beddington RSP. Axial skeletal defects caused by mutation in the spondylocostal dysostosis/pudgy gene D113 are associated with disruption of the segmentation clock within the presomitic mesoderm. Development 2002; 129:1795-1806.
Turnpenny PD, Whittock N, Duncan J, Dunwoodie SL, Kusumi K, Ellard S. Novel mutations in DLL3, a somitogenesis gene encoding a ligand for the Notch signalling pathway, cause a consistent pattern of abnormal vertebral segmentation in spondylocostal dysostosis. J Med Genet 2003; 40:333-339.
Sousa-Nunes R, Rana A, Kettleborough R, Brickman JM, Clements M, Forrest A, Grimmond S, Avner P, Smith JC, Dunwoodie SL and Beddington RSP. Characterising Embryonic Gene Expression Patterns in the Mouse Using Non-Redundant Sequence-Based Selection. Genome Res 2003; 13:2609-2620
Rodriguez, TA, Sparrow, DB, Scott, AN, Withington, SL, Lynch, D, Beddington, RSP, Dunwoodie, SL. Cited1 is required for embryonic survival and normal placental development. Mol Cell Biol 2004; 24:228-244
Whittock NV, Sparrow DB, Wouters MA, Sillence D, Ellard D, Dunwoodie SL and Peter D. Turnpenny. Mutated MESP2 causes spondylocostal dysostosis in humans. American Journal Human Genetics 2004; 74:1249-54
Weninger W, Lopes Floro K, Bennett MB, Withington SL, Preis JI, Martinez Barbera JP, Mohun TJ and Dunwoodie SL. Cited2 is required both for heart morphogenesis and establishment of the left-right axis in mouse development. Development 2005; 132:1337-1348.
Sparrow DB, Chapman G, Wouters MA, Whittock NV, Ellard S, Fatkin D, Turnpenny PD, Kusumi K, Sillence D, Dunwoodie SL. Mutation of the LUNATIC FRINGE gene in humans causes spondylocostal dysostosis with a severe vertebral phenotype. Am J Hum Genet 2006; 78(1):28-37
Withington SL; Scott A; Saunders D; Lopes Floro K; Preis JI; Michalicek J; Maclean K; Sparrow D; Martinez-Barbera JP; Dunwoodie SL. Loss of Cited2 affects trophoblast formation and vascularization of the mouse placenta. Dev Biology 2006; 294(1):67-82
Geffers I, Serth K, Chapman G, Jaekel R, Schuster-Gossler K, Cordes R, Sparrow DB, Kremmer E, Dunwoodie SL, Klein T, Gossler A. Divergent functions and distinct localization of the Notch ligands DLL1 and DLL3 in vivo. J Cell Biology 2007; 178 (3): 465-476
Sparrow D, Guillen-Navarro E, Fatkin D, Dunwoodie SL. Mutation of HAIRY-AND-ENHANCER-OF-SPLIT-7 in humans causes Spondylocostal Dysostosis. Human Molecular Genetics 2008 (in press)
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