Special Research Centre for the Molecular Genetics of Development (CMGD)
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The Centre for the Molecular Genetics of Development is a Special Research Centre of the Australian Research Council with research groups located at the University of Adelaide and the Australian National University.
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Item Metadata only A constitutively active dioxin/aryl hydrocarbon receptor induces stomach tumors(Natl Acad Sciences, 2002) Andersson, P.; McGuire, J.; Rubio, C.; Gradin, K.; Whitelaw, M.; Pettersson, S.; Hanberg, A.; Poellinger, L.; Centre for the Molecular Genetics of DevelopmentThe dioxin/aryl hydrocarbon receptor (AhR) functions as a ligand-activated transcription factor regulating transcription of a battery of genes encoding xenobiotic metabolizing enzymes. Known receptor ligands are environmental pollutants including polycyclic aromatic hydrocarbons and polychlorinated dioxins. Loss-of-function (gene-disruption) studies in mice have demonstrated that the AhR is involved in toxic effects of dioxins but have not yielded unequivocal results concerning the physiological function of the receptor. Gain-of-function studies therefore were performed to unravel the biological functions of the AhR. A constitutively active AhR expressed in transgenic mice reduced the life span of the mice and induced tumors in the glandular part of the stomach, demonstrating the oncogenic potential of the AhR and implicating the receptor in regulation of cell proliferation.Item Metadata only A Drosophila overexpression screen for modifiers of Rho signalling in cytokinesis(Landes Bioscience, 2007) Gregory, S.; Shandala, T.; O'Keefe, L.; Jones, L.; Murray, M.; Saint, R.; Centre for the Molecular Genetics of DevelopmentTo identify genes that modulate Rho signalling during cytokinesis we tested the effect of overexpressing a set of 2190 genes on an eye phenotype caused by defective Rho activation. The resulting 112 modifier loci fell into three main classes: cell cycle genes, signalling effectors and metabolic enzymes. We developed a further series of genetic tests to refine the interactors into those most likely to modify Rho signalling during cytokinesis. In addition to a number of genes previously implicated in the Rho pathway during cytokinesis, we identified four novel primary candidates: cdc14, Pitslre, PDK1 and thread/diap1. cdc14 orthologs have, however, been implicated in cytokinesis in other organisms, as have molecules related to Thread/Diap1. The identification of several modifiers that are genetically redundant paralogs highlights the ability of overexpression screens to identify genes that are refractory to traditional loss-of-function approaches. Overexpression screens and sensitized phenotypes, therefore, may help identify the many factors that are expected to be involved in cytokinesis but have not been discovered by previous genetic screens.Item Metadata only A family of RS domain proteins with novel subcellular localization and trafficking(Oxford Univ Press, 2005) Kavanagh, S.; Schulz, T.; Davey, P.; Claudianos, C.; Russell, C.; Rathjen, P.; Centre for the Molecular Genetics of DevelopmentWe report the sequence, conservation and cell biology of a novel protein, Psc1, which is expressed and regulated within the embryonic pluripotent cell population of the mouse. The Psc1 sequence includes an RS domain and an RNA recognition motif (RRM), and a sequential arrangement of protein motifs that has not been demonstrated for other RS domain proteins. This arrangement was conserved in a second mouse protein (BAC34721. The identification of Psc1 and BAC34721homologues in vertebrates and related proteins, more widely throughout evolution, defines a new family of RS domain proteins termed acidic rich RS (ARRS) domain proteins. Psc1 incorporated into the nuclear speckles, but demonstrated novel aspects of subcellular distribution including localization to speckles proximal to the nuclear periphery and localization to punctate structures in the cytoplasm termed cytospeckles. Integration of Psc1 into cytospeckles was dependent on the RRM. Cytospeckles were dynamic within the cytoplasm and appeared to traffic into the nucleus. These observations suggest a novel role in RNA metabolism for ARRS proteins.Item Metadata only A genetic screen for dominant modifiers of a cyclin E hypomorphic mutation identifies novel regulators of S-Phase entry in Drosophila(Genetics, 2004) Brumby, A.; Secombe, J.; Horsfield, J.; Coombe, M.; Amin, N.; Coates, D.; Saint, R.; Richardson, H.; Centre for the Molecular Genetics of DevelopmentCyclin E together with its kinase partner Cdk2 is a critical regulator of entry into S phase. To identify novel genes that regulate the G1- to S-phase transition within a whole animal we made use of a hypomorphic cyclin E mutation, DmcycEJP, which results in a rough eye phenotype. We screened the X and third chromosome deficiencies, tested candidate genes, and carried out a genetic screen of 55,000 EMS or X-ray-mutagenized flies for second or third chromosome mutations that dominantly modified the DmcycEJP rough eye phenotype. We have focused on the DmcycEJP suppressors, S(DmcycEJP), to identify novel negative regulators of S-phase entry. There are 18 suppressor gene groups with more than one allele and several genes that are represented by only a single allele. All S(DmcycEJP) tested suppress the DmcycEJP rough eye phenotype by increasing the number of S phases in the postmorphogenetic furrow S-phase band. By testing candidates we have identified several modifier genes from the mutagenic screen as well as from the deficiency screen. DmcycEJP suppressor genes fall into the classes of: (1) chromatin remodeling or transcription factors; (2) signaling pathways; and (3) cytoskeletal, (4) cell adhesion, and (5) cytoarchitectural tumor suppressors. The cytoarchitectural tumor suppressors include scribble, lethal-2-giant-larvae (lgl), and discs-large (dlg), loss of function of which leads to neoplastic tumors and disruption of apical-basal cell polarity. We further explored the genetic interactions of scribble with S(DmcycEJP) genes and show that hypomorphic scribble mutants exhibit genetic interactions with lgl, scab (alphaPS3-integrin--cell adhesion), phyllopod (signaling), dEB1 (microtubule-binding protein--cytoskeletal), and moira (chromatin remodeling). These interactions of the cytoarchitectural suppressor gene, scribble, with cell adhesion, signaling, cytoskeletal, and chromatin remodeling genes, suggest that these genes may act in a common pathway to negatively regulate cyclin E or S-phase entry.Item Metadata only A highly conserved novel family of mammalian developmental transcription factors related to Drosophila grainyhead(Elsevier Science BV, 2002) Wilanowski, T.; Tuckfield, A.; Cerruti, L.; O'Connell, S.; Saint, R.; Parekh, V.; Tao, J.; Cunningham, J.; Jane, S.; Centre for the Molecular Genetics of DevelopmentThe Drosophila transcription factor Grainyhead regulates several key developmental processes. Three mammalian genes, CP2, LBP-1a and LBP-9 have been previously identified as homologues of grainyhead. We now report the cloning of two new mammalian genes (Mammalian grainyhead (MGR) and Brother-of-MGR (BOM)) and one new Drosophila gene (dCP2) that rewrite the phylogeny of this family. We demonstrate that MGR and BOM are more closely related to grh, whereas CP2, LBP-1a and LBP-9 are descendants of the dCP2 gene. MGR shares the greatest sequence homology with grh, is expressed in tissue-restricted patterns more comparable to grh and binds to and transactivates the promoter of the human Engrailed-1 gene, the mammalian homologue of the key grainyhead target gene, engrailed. This sequence and functional conservation indicates that the new mammalian members of this family play important developmental roles.Item Metadata only A rapid apoptosis assay measuring relative acridine orange fluorescence in zebrafish embryos(Mary Ann Liebert, Inc. Publishers, 2007) Tucker, B.; Lardelli, M.; Centre for the Molecular Genetics of DevelopmentThe ability to easily analyze apoptosis is important in studies of molecular cell biology and to evaluate the relative toxicity of different treatments or environments. This is particularly the case when substances such as morpholino oligonucleotides are injected into embryos, as such treatments can cause widespread, complex patterns of apoptosis. Zebrafish embryos are well suited for cell biological and environmental toxicity analyses, but the need remains for a simple method that can analyze levels of apoptosis in a statistically significant number of embryos. Here we present a "group fluorescence" method for rapid, large-scale analysis of relative levels of apoptosis based on densitometric techniques.Item Metadata only A redox mechanism controls differential DNA binding activities of hypoxia-inducible factor (HIF) 1a and the HIF-like factor(Amer Soc Biochemistry Molecular Biology Inc, 2000) Lando, D.; Pongratz, I.; Poellinger, L.; Whitelaw, M.; Centre for the Molecular Genetics of DevelopmentHypoxia-inducible factor 1α (HIF-1α) and the HIF-like factor (HLF) are two highly related basic Helix-Loop-Helix/Per-Arnt-Sim (bHLH/PAS) homology transcription factors that undergo dramatically increased function at low oxygen levels. Despite strong similarities in their activation mechanisms (e.g. they both undergo rapid hypoxia-induced protein stabilization, bind identical target DNA sequences, and induce synthetic reporter genes to similar degrees), they are both essential for embryo survival via distinct functions during vascularization (HIF-1α) or catecholamine production (HLF). It is currently unknown how such specificity of action is achieved. We report here that DNA binding by HLF, but not by HIF-1α, is dependent upon reducing redox conditions. In vitro DNA binding and mammalian two-hybrid assays showed that a unique cysteine in the DNA-binding basic region of HLF is a target for the reducing activity of redox factor Ref-1. Although the N-terminal DNA-binding domain of HIF-1α can function in the absence of Ref-1, we found that the C-terminal region containing the transactivation domain requires Ref-1 for full activity. Our data reveal that the hypoxia-inducible factors are subject to complex redox control mechanisms that can target discrete regions of the proteins and are the first to establish a discriminating control mechanism for differential regulation of HIF-1α and HLF activity.Item Metadata only A RhoGEF and Rho family GTPase-activating protein complex links the contractile ring to cortical microtubules at the onset of cytokinesis(Cell Press, 2003) Somers, W.; Saint, R.; Centre for the Molecular Genetics of DevelopmentThe mechanism that positions the cytokinetic contractile ring is unknown, but derives from the spindle midzone. We show that an interaction between the Rho GTP exchange factor, Pebble, and the Rho family GTPase-activating protein, RacGAP50C, connects the contractile ring to cortical microtubules at the site of furrowing in D. melanogaster cells. Pebble regulates actomyosin organization, while RacGAP50C and its binding partner, the Pavarotti kinesin-like protein, regulate microtubule bundling. All three factors are required for cytokinesis. As furrowing begins, these proteins colocalize to a cortical equatorial ring. We propose that RacGAP50C-Pavarotti complexes travel on cortical microtubules to the cell equator, where they associate with the Pebble RhoGEF to position contractile ring formation and coordinate F-actin and microtubule remodeling during cytokinesis.Item Metadata only A role for angiotensin-converting enzyme in the characterization, enrichment, and proliferation potential of adult murine pituitary colony-forming cells(Alphamed Press, 2006) Lepore, D.; Jokubaitis, V.; Simmons, P.; Roeszler, K.; Rossi, R.; Bauer, K.; Thomas, P.; Centre for the Molecular Genetics of DevelopmentRecently, we described a rare cell type within the adult murine pituitary gland with progenitor cell hallmarks (PCFCs). PCFCs are contained exclusively within a subpopulation of cells that import fluorescent ß-Ala-Lys-N-AMCA (7-amino-4-methylcoumarin-3-acetic acid). Herein, we investigate the utility of cell surface molecules angiotensin-converting enzyme (ACE) and stem cell antigen-1 (Sca-1) to further enrich for PCFCs. ACE and Sca-1 were expressed on 61% and 55% of AMCA+CD45–CD31– cells, respectively, and coexpressed on 38%. ACE+Sca-1+AMCA+ cells enriched for PCFCs by 195-fold over unselected cells. ACE+AMCA+ cells enriched for PCFCs by 170-fold, and colonies were twofold larger than for AMCA+ selection alone. Conversely, ACE–-selected cells reduced both colony-forming activity and size. Notably, colonies generated from AMCA+ cells obtained from ACEnull mice were 2.7-fold smaller than for wild-type mice. These data identify ACE as a previously unrecognized marker of PCFCs and suggest that ACE is functionally important for PCFC proliferation. Anatomically, the cells that imported AMCA and expressed ACE were situated in the marginal epithelial cell layer of the pituitary cleft and in the adjacent subluminal zone, thus supporting previous proposals that the luminal zone is a source of precursor cells in the adult pituitary.Item Metadata only A simple plan - cnidarians and the origins of developmental mechanisms(Nature Publishing Group, 2004) Ball, E.; Hayward, D.; Saint, R.; Miller, D.; Centre for the Molecular Genetics of DevelopmentComparisons with cnidarians, long considered to be 'simple' animals, are providing crucial insights into the origins of conserved developmental mechanisms and the nature of the common metazoan ancestor. Traditionally, an extra germ layer and a second axis of body symmetry are the features that distinguish 'higher' Metazoa from lower animals such as cnidarians. Moreover, it was expected that 'lower' animals would have a simple gene set that corresponds to their simple morphology. Now, molecular genetic approaches are blurring the developmental divide between cnidarians and bilateral animals, and cnidarian sequencing projects are showing that the common metazoan ancestor was more genetically complex than was previously assumed.Item Metadata only Activating mutation of the renal epithelial chloride channel ClC-Kb predisposing to hypertension(Lippincott Williams & Wilkins, 2006) Milton, A.; Jannes, J.; Hamilton-Bruce, M.; Koblar, S.; Centre for the Molecular Genetics of DevelopmentItem Metadata only Activity of hypoxia-inducible factor 2α is regulated by association with the NF-κB essential modulator(Amer Soc Biochemistry Molecular Biology Inc, 2005) Bracken, C.; Whitelaw, M.; Peet, D.; Centre for the Molecular Genetics of DevelopmentThe hypoxia-inducible factors 1α (HIF-1α) and 2α (HIF-2α) are key regulators of the transcriptional response to low oxygen and are closely related in domain architecture, DNA binding, and activation mechanisms. Despite these similarities, targeted disruption of the HIF- α genes in mice results in distinctly different phenotypes demonstrating nonredundancy of function, although the underlying mechanisms remain unclear. Here we report on the novel and specific interaction of HIF-2α, but not HIF-1α, with the NF- κB essential modulator (NEMO) using immunoprecipitation, mammalian two-hybrid, and in vitro protein interaction assays. Reporter gene assays demonstrate that this interaction specifically enhances normoxic HIF-2α transcriptional activity, independently of the HIF-2α transactivation domain, consistent with a model by which NEMO aids CBP/p300 recruitment to HIF-2α. In contrast, HIF-2α overexpression does not alter NF- κB signaling, suggesting that the functional consequence of the HIF-2α /NEMO interaction is limited to the HIF pathway. The specificity of NEMO for HIF-2α represents one of the few known differential protein-protein interactions between the HIF-α proteins, which has important implications for the activity of HIF-2α and is also the first postulated NF-κ B-independent role for NEMO.Item Metadata only Alzheimer disease: amyloidogenesis, the presenilins and animal models(Elsevier Science BV, 2007) Newman, M.; Musgrave, I.; Lardelli, M.; Centre for the Molecular Genetics of DevelopmentAlzheimer’s disease is the most prevalent form of dementia. Neuropathogenesis is proposed to be a result of the accumulation of amyloid beta peptides in the brain together with oxidative stress mechanisms and neuroinflammation. The presenilin proteins are central to the gamma-secretase cleavage of the amyloid prescursor protein (APP), releasing the amyloid beta peptide. Point mutations in the presenilin genes lead to cases of familial Alzheimer’s disease by increasing APP cleavage resulting in excess amyloid beta formation. This review discusses the molecular mechanism of Alzheimer’s disease with a focus on the presenilin genes. Alternative splicing of transcripts from these genes and how these may function in several disease states is discussed. There is an emphasis on the importance of animal models in elucidating the molecular mechanisms behind the development of Alzheimer’s disease and how the zebrafish, Danio rerio, can be used as a model organism for analysis of presenilin function and Alzheimer’s disease pathogenesis.Item Metadata only Analysis of Drosophila Cyclin EI and II function during development: Identification of an inhibitory zone within the morphogenetic furrow of the eye imaginal disc that blocks the function of Cyclin EI but not Cyclin EII(Academic Press Inc, 2002) Denton, D.; Secombe, J.; Coombe, M.; Brumby, A.; Saint, R.; Richardson, H.; Centre for the Molecular Genetics of DevelopmentThe Drosophilacyclin E (DmcycE) gene gives rise to two transcripts encoding proteins that differ at their N termini, DmcycEII and DmcycEI. This study presents the first in vivo dissection of Cyclin E function. Ectopic expression studies using N- and C-terminal deletions of DmcycEI revealed that a region of 322 residues surrounding the cyclin box is sufficient to induce entry of G₁-arrested larval eye imaginal disc cells into S phase. Ectopic expression of DmcycEI in the eye disc has been previously shown to drive anterior, but not posterior, G₁-phase cells within the morphogenetic furrow (MF) into S phase. Significantly, ectopic expression of DmcycEII and N-terminal deletions of DmcycEI were able to drive all G₁ cells within the morphogenetic furrow into S phase, while a C-terminal deletion of DmcycEI could not. The p21 homolog Dacapo was shown by yeast two-hybrid, coimmunolocalization, and in vivo functional studies not to be the mediator of the DmcycEI inhibition in posterior part of the MF. Taken together, these results reveal a novel zone within the posterior region of the MF where DmcycEI but not DmcycEII function is inhibited, and suggest that DmcycEII is a more potent inducer of S phase.Item Metadata only Analysis of intracranial volume in Apert Syndrome genotypes(Karger, 2004) Anderson, P.; Netherway, D.; Abbott, A.; Cox, T.; Roscioli, T.; David, D.; Centre for the Molecular Genetics of DevelopmentObjective: Apert syndrome is caused by a mutation of the fibroblastic growth factor type 2 gene and in nearly all of the cases where the mutation has been identified it occurs in one of two adjacent sites of the gene, either position 252 or position 253. There is currently uncertainty whether a worse neurosurgical outcome occurs in association with a particular genotype. We investigated whether there were clinically subtle (but relevant) morphological differences in the craniofacial skeleton, which would result in differences in the intracranial volume, which might account for apparent differences in surgical outcome. Method: Three-dimensional CT scans of pre-operative Apert syndrome whose genotype had been identified had the intracranial volume measured using the Cavalieri estimator with correction for partial voluming effects. The values were compared to age and sex normals and then the two genotypes compared. Results: Intracranial volumes were measured for 22 cases, 16 with the 252 mutation and 6 with the 253 mutation. Conclusions: All cases except two had greater than their sex- and age-adjusted mean normal intracranial volumes. For the 252 and 253 genotypes there were no discernible differences in intracranial volumes between the two genotypes.Item Metadata only Analysis of Meiosis in Fixed and Live Oocytes by Light Microscopy(Cold Spring Harbor Laboratory Press, 2000) Matthies, H.; Clarkson, M.; Saint, R.; Namba, R.; Hawley, R.; Centre for the Molecular Genetics of DevelopmentItem Open Access Animal cell division: a fellowship of the double ring?(Company of Biologists Ltd, 2003) Saint, R.; Somers, W.; Centre for the Molecular Genetics of DevelopmentDespite a century of research into the nature of animal cell division, a molecular explanation for the positioning of the actomyosin contractile ring has remained elusive. The discovery of a novel interaction between regulators of Rho family small GTPases has revealed a link between the mitotic microtubules and the contractile ring during the later stages of mitosis. The properties of the interacting Rho regulators suggest a molecular model for the positioning and initiation of contractile ring furrowing in animal cells. In this ‘double ring’ model, centralspindlin complexes, localized by the action of their kinesin-like protein component, position and activate a cortical equatorial ring of Rho GTPase exchange factors. The resulting ring of activated Rho would then trigger a cascade of events leading to formation and constriction of the contractile ring.Item Metadata only Animal Evolution: The enigmatic phylum placozoa revisited(Elsevier, 2005) Miller, David James; Ball, Eldon E.; Centre for the Molecular Genetics of DevelopmentA recent report of high levels of genetic variation between strains of Trichoplax adhaerens challenges the traditional view that the phylum Placozoa comprises only one species. At the morphological level, placozoans are amongst the simplest extant animals, but molecular evidence suggests that they may have more complex origins.Item Metadata only ARDent about acetylation and deacetylation in hypoxia signalling(Elsevier, 2006) Bilton, Rebecca Louise; Trottier, Eric; Pouyssegur, Jacques; Brahimi-Horn, M. Christiane; Centre for the Molecular Genetics of Development; School of Molecular and Biomedical ScienceGiven the key role that the α subunit of the αβ heterodimeric transcription factor hypoxia-inducible factor-1 (HIF-1) has in tumourigenesis, and in particular in angiogenesis, a full understanding of its regulation is crucial to the development of cancer therapeutics. Posttranslational acetylation and deacetylation of this subunit by an acetyltransferase called Arrest-defective-1 (ARD1) and by different histone deacetylases (HDACs), respectively, has been suggested as a mechanism. However, conflicting data bring into question the foundations of this mechanism and at present it is not clear what the precise role of these proteins is with respect to HIF. Nonetheless, the observation that small-molecule inhibitors of HDACs have anti-angiogenic activity suggests that acetylation and deacetylation of HIF or HIF modifiers represents a potential target in cancer therapy.Item Metadata only ARID proteins come in from the desert(Elsevier Science London, 2000) Kortschak, R.; Tucker, P.; Saint, R.; Centre for the Molecular Genetics of DevelopmentMembers of the recently discovered ARID (AT-rich interaction domain) family of DNA-binding proteins are found in fungi and invertebrate and vertebrate metazoans. ARID-encoding genes are involved in a variety of biological processes including embryonic development, cell lineage gene regulation and cell cycle control. Although the specific roles of this domain and of ARID-containing proteins in transcriptional regulation are yet to be elucidated, they include both positive and negative transcriptional regulation and a likely involvement in the modification of chromatin structure.