Centre for the Molecular Genetics of Development publications
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Item Metadata only Receptor tyrosine kinase signaling regulates different modes of Groucho-dependent control of Dorsal(Dell Press, 2000) Hader, T.; Wainwright, D.; Shandala, T.; Saint, R.; Taubert, H.; Bronner, G.; Jackle, H.; Centre for the Molecular Genetics of DevelopmentTranscriptional control of the Drosophila terminal gap gene huckebein (hkb) depends on Torso (Tor) receptor tyrosine kinase (RTK) signaling and the Rel/NFB homolog Dorsal (Dl) . Dl acts as an intrinsic transcriptional activator in the ventral region of the embryo, but under certain conditions, such as when it is associated with the non-DNA-binding co-repressor Groucho (Gro), it is converted into a repressor . Gro is recruited to the enhancer element in the vicinity of Dl by sequence-specific transcription factors such as Dead Ringer (Dri) . We examined the interplay between Dl, Gro and Dri on the hkb enhancer and show that when acting over a distance, Gro abolishes rather than converts Dl activator function. Reducing the distance between Dl- and Dri-binding sites, however, switches Dl into a Gro-dependent repressor that overrides activation of transcription. Both of the distance-dependent regulatory options of Gro - quenching and silencing of transcription- are inhibited by RTK signaling. These data describe a newly identified mode of function for Gro when acting in concert with Dl. RTK signaling provides a way of modulating Dl function by interfering either with Gro activity or with Dri-dependent recruitment of Gro to the enhancer.Item Metadata only Phylogeny: The continuing classificatory conundrum of chaetognaths(Elsevier, 2006) Ball, Eldon E.; Miller, David J.; Centre for the Molecular Genetics of DevelopmentThe phylogenetic conundrum posed by the Chaetognatha, a cryptic phylum consisting largely of planktonic predators, is the subject of two short papers in this issue of Current Biology. These analyses go some way towards defining the phylogenetic position of the chaetognaths, which possess features apparently spanning the protostome/deuterostome divide.Item Metadata only Components of both major axial patterning systems of the Bilateria are differentially expressed along the primary axis of a 'radiate' animal, the anthozoan cnidarian Acropora millepora(Elsevier, 2006) de Jong, Danielle M.; Hislop, Nikki R.; Hayward, David C.; Reece-Hoyes, John S.; Pontynen, Patricia C.; Ball, Eldon E.; Miller, David J.; Centre for the Molecular Genetics of DevelopmentCnidarians are animals with a single (oral/aboral) overt body axis and with origins that nominally predate bilaterality. To better understand the evolution of axial patterning mechanisms, we characterized genes from the coral, Acropora millepora (Class Anthozoa) that are considered to be unambiguous markers of the bilaterian anterior/posterior and dorsal/ventral axes. Homologs of Otx/otd and Emx/ems, definitive anterior markers across the Bilateria, are expressed at opposite ends of the Acropora larva; otxA-Am initially around the blastopore and later preferentially toward the oral end in the ectoderm, and emx-Am predominantly in putative neurons in the aboral half of the planula larva, in a domain overlapping that of cnox-2Am, a Gsh/ind gene. The Acropora homologs of Pax-3/7, NKX2.1/vnd and Msx/msh are expressed in axially restricted and largely non-overlapping patterns in larval ectoderm. In Acropora, components of both the D/V and A/P patterning systems of bilateral animals are therefore expressed in regionally restricted patterns along the single overt body axis of the planula larva, and two 'anterior' markers are expressed at opposite ends of the axis. Thus, although some specific gene functions appear to be conserved between cnidarians and higher animals, no simple relationship exists between axial patterning systems in the two groups.Item Open Access Loss of Atrx affects trophoblast development and the pattern of x-inactivation in extraembryonic tissues(Public Library of Science, 2006) Garrick, David; Sharpe, Jackie A.; Arkell, Ruth; Dobbie, Lorraine; Smith, Andrew J. H.; Wood, William G.; Higgs, Douglas R.; Gibbons, Richard J.; Centre for the Molecular Genetics of DevelopmentATRX is an X-encoded member of the SNF2 family of ATPase/helicase proteins thought to regulate gene expression by modifying chromatin at target loci. Mutations in ATRX provided the first example of a human genetic disease associated with defects in such proteins. To better understand the role of ATRX in development and the associated abnormalities in the ATR-X (alpha thalassemia mental retardation, X-linked) syndrome, we conditionally inactivated the homolog in mice, Atrx, at the 8- to 16-cell stage of development. The protein, Atrx, was ubiquitously expressed, and male embryos null for Atrx implanted and gastrulated normally but did not survive beyond 9.5 days postcoitus due to a defect in formation of the extraembryonic trophoblast, one of the first terminally differentiated lineages in the developing embryo. Carrier female mice that inherit a maternal null allele should be affected, since the paternal X chromosome is normally inactivated in extraembryonic tissues. Surprisingly, however, some carrier females established a normal placenta and appeared to escape the usual pattern of imprinted X-inactivation in these tissues. Together these findings demonstrate an unexpected, specific, and essential role for Atrx in the development of the murine trophoblast and present an example of escape from imprinted X chromosome inactivation.Item Metadata only Neural plate morphogenesis during mouse neurulation is regulated by antagonism of Bmp signalling(Company of Biologists, 2007) Ybot-Gonzalez, Patricia; Gaston-Massuet, Carles; Girdler, Gemma; Klingensmith, John; Arkell, Ruth; Greene, Nicholas D. E.; Copp, Andrew J.; Centre for the Molecular Genetics of DevelopmentDorsolateral bending of the neural plate, an undifferentiated pseudostratified epithelium, is essential for neural tube closure in the mouse spinal region. If dorsolateral bending fails, spina bifida results. In the present study, we investigated the molecular signals that regulate the formation of dorsolateral hinge points (DLHPs). We show that Bmp2 expression correlates with upper spinal neurulation (in which DLHPs are absent); that Bmp2-null embryos exhibit premature, exaggerated DLHPs; and that the local release of Bmp2 inhibits neural fold bending. Therefore, Bmp signalling is necessary and sufficient to inhibit DLHPs. By contrast, the Bmp antagonist noggin is expressed dorsally in neural folds containing DLHPs, noggin-null embryos show markedly reduced dorsolateral bending and local release of noggin stimulates bending. Hence, Bmp antagonism is both necessary and sufficient to induce dorsolateral bending. The local release of Shh suppresses dorsal noggin expression, explaining the absence of DLHPs at high spinal levels, where notochordal expression of Shh is strong. DLHPs `break through' at low spinal levels, where Shh expression is weaker. Zic2 mutant embryos fail to express Bmp antagonists dorsally and lack DLHPs, developing severe spina bifida. Our findings reveal a molecular mechanism based on antagonism of Bmp signalling that underlies the regulation of DLHP formation during mouse spinal neural tube closure.Item Metadata only Photoactivatable GFP resolves Drosophila mesoderm migration behaviour(Company of Biologists Ltd, 2007) Murray, M.; Saint, R.; Centre for the Molecular Genetics of DevelopmentMesoderm migration is a pivotal event in the early embryonic development of animals. One of the best-studied examples occurs during Drosophila gastrulation. Here, mesodermal cells invaginate, undergo an epithelial-to-mesenchymal transition (EMT), and spread out dorsally over the inner surface of the ectoderm. Although several genes required for spreading have been identified, our inability to visualise mesodermal cells in living embryos has left us to speculate about the cell rearrangements involved. Several mechanisms, such as chemotaxis towards a dorsally expressed attractant, differential affinity between mesodermal cells and the ectoderm, and convergent extension, have been proposed. Here we resolve the behaviour of Drosophila mesodermal cells in live embryos using photoactivatable-GFP fused to -Tubulin (PAGFP-Tub). By photoactivating presumptive mesodermal cells before gastrulation, we could observe their migration over non-fluorescent ectodermal cells. We show that the outermost (outer) cells, which are in contact with the ectoderm, migrate dorsolaterally as a group but can be overtaken by more internal (inner) cells. Using laser-photoactivation of individual cells, we then show that inner cells adjacent to the centre of the furrow migrate dorsolaterally away from the midline to reach dorsal positions, while cells at the centre of the furrow disperse randomly across the mesoderm, before intercalating with outer cells. These movements are dependent on the FGF receptor Heartless. The results indicate that chemotactic movement and differential affinity are the primary drivers of mesodermal cell spreading. These characterisations pave the way for a more detailed analysis of gene function during early mesoderm development.Item Open Access Array comparative genomic hybridisation analysis of boys with X-linked hypopituitarism identifies a 3.9 Mb duplicated critical region at Xq27 containing SOX3(British Med Journal Publ Group, 2007) Solomon, N.; Ross, S.; Forrest, S.; Thomas, P.; Morgan, T.; Belsky, J.; Hol, F.; Karnes, P.; Hopwood, N.; Myers, S.; Tan, A.; Warne, G.; Centre for the Molecular Genetics of DevelopmentItem Open Access The innate immune repertoire in Cnidaria - ancestral complexity and stochastic gene loss(BioMed Central, 2007) Miller, David James; Hemmrich, Georg; Ball, Eldon E.; Hayward, David C.; Khalturin, Konstantin; Funayama, Noriko; Kiyokazu Agata; Bosch, Thomas C. G.; Centre for the Molecular Genetics of DevelopmentBackground: Characterization of the innate immune repertoire of extant cnidarians is of both fundamental and applied interest - it not only provides insights into the basic immunological 'tool kit' of the common ancestor of all animals, but is also likely to be important in understanding the global decline of coral reefs that is presently occurring. Recently, whole genome sequences became available for two cnidarians, Hydra magnipapillata and Nematostella vectensis, and large expressed sequence tag (EST) datasets are available for these and for the coral Acropora millepora. Results: To better understand the basis of innate immunity in cnidarians, we scanned the available EST and genomic resources for some of the key components of the vertebrate innate immune repertoire, focusing on the Toll/Toll-like receptor (TLR) and complement pathways. A canonical Toll/TLR pathway is present in representatives of the basal cnidarian class Anthozoa, but neither a classic Toll/TLR receptor nor a conventional nuclear factor (NF)-κB could be identified in the anthozoan Hydra. Moreover, the detection of complement C3 and several membrane attack complex/perforin domain (MAC/PF) proteins suggests that a prototypic complement effector pathway may exist in anthozoans, but not in hydrozoans. Together with data for several other gene families, this implies that Hydra may have undergone substantial secondary gene loss during evolution. Such losses are not confined to Hydra, however, and at least one MAC/PF gene appears to have been lost from Nematostella. Conclusion: Consideration of these patterns of gene distribution underscores the likely significance of gene loss during animal evolution whilst indicating ancient origins for many components of the vertebrate innate immune system.Item Metadata only Expression and evolution of the mammalian brain gene Ttyh1(Blackwell Publishing Ltd, 2007) Matthews, C.; Shaw, J.; Hooper, J.; Young, I.; Crouch, M.; Campbell, H.; Centre for the Molecular Genetics of DevelopmentHomologues of the Drosophila melanogaster tweety (tty) gene are present in mammals and Caenorhabditis elegans. The encoded proteins have five predicted membrane-spanning regions and recent findings suggest that some family members may be chloride channels. Phylogenetic analysis of the tty family including novel members from slime mould Entamoeba and plants has revealed the occurrence of independent gene duplication events in different lineages. expressed sequence tag data indicate that expression of the mammalian Ttyh1 gene is restricted mainly to neural tissue and is up-regulated in astrocytoma, glioma and several other cancers. In this study, mammalian expression vectors were used to investigate the subcellular localization and the effect of over-expression of Ttyh1 in human epithelial kidney cells. The results confirm that Ttyh1 is a membrane protein and show that it is deposited on the substratum along the migration paths of motile cells above the α5β1-integrin complex. The ectopic expression of Ttyh1 also induced long filopodia, which were branched and dynamic in both stationary and migratory cells. The filopodia contained F-actin and occurred at the ends of microtubules which were polarized towards the membrane. Upon contact with nearby cells some filopodia stabilized and filled with F-actin, whereas Ttyh1 was highly concentrated at the cell-cell interface. Ttyh1 N- and C-terminal antipeptide antibodies detected Ttyh1 along the axons of neurones in primary rat hippocampal cell cultures, and in situ in whole rat brain slices around the hippocampus and occasionally between cells. These data suggest a role for Ttyh1 in process formation, cell adhesion and possibly as a transmembrane receptor.Item Metadata only Tandem organization of independently duplicated homeobox genes in the basal cnidarian Acropora millepora(Springer Berlin, 2005) Hislop, Nikki R.; de Jong, Danielle M.; Hayward, David C.; Ball, Eldon E.; Miller, David James; Centre for the Molecular Genetics of DevelopmentAbstract A number of examples of independently duplicated regulatory genes have been identified in cnidarians, but the extent of this phenomenon and organization of these duplicated genes are unknown. Here we describe the identification of three pairs of independently duplicated homeobox genes in the anthozoan cnidarian, Acropora millepora. In each case, the pairs of paralogous genes are tightly linked, but the extent of sequence divergence implies that these do not reflect recent duplication events. The phenomenon is likely to be more general, as the examples reported here represent most of the limited number of Acropora homeobox genes for which genomic data are yet available.Item Metadata only Characterization of ankyrin repeat-containing proteins as substrates of the asparaginyl hydroxylase factor inhibiting hypoxia-inducible transcription factor(Elsevier Academic Press Inc, 2007) Karttunen, S.; Hampton-Smith, R.; Peet, D.; Sies, H.; Brune, B.; Centre for the Molecular Genetics of DevelopmentThe hypoxia-inducible transcription factors (HIFs) are essential mediators of the genomic response to oxygen deficiency (hypoxia) in multicellular organisms. The HIFs are regulated by four oxygen-sensitive hydroxylases-three prolyl hydroxylases and one asparaginyl hydroxylase. These hydroxylases are all members of the 2-oxoglutarate (2OG)-dependent dioxygenase superfamily and convey changes in cellular oxygen concentration to the HIF-alpha (alpha) subunit, leading to potent accumulation and activity in hypoxia versus degradation and repression in normoxia. HIF-alpha asparaginyl hydroxylation is catalyzed by factor-inhibiting HIF-1 (FIH-1) and directly regulates the transcription activity of the HIF-alpha proteins. Recent work has demonstrated that, in addition to hydroxylating HIF-alpha, FIH-1 can also hydroxylate the ankyrin domains of a wide range of proteins. This paper presents in vitro and cell-based techniques for the preliminary characterization of ankyrin domain-containing proteins as FIH-1 substrates and interacting proteins. Strategies are presented for the expression and purification of FIH-1 from mammalian or bacterial cells. Similar to the HIF-alpha proteins, the ankyrin-containing substrates are examined as purified proteins expressed in bacteria and overexpressed in mammalian cells or in the form of synthetic peptides. Specific conditions for the efficient expression of ankyrin-containing proteins compared with the HIF-alpha substrates in Escherichia coli are detailed. Hydroxylation is rapidly inferred, utilizing the described in vitro CO(2) capture assay. Finally, substrate and non-substrate interactions are examined using in vitro affinity pull-down assays and mammalian cell-based co-immunoprecipitation assays. Together, these methods are rapid and well suited to the preliminary characterization of potential substrates of the therapeutically relevant oxygen-sensing enzyme FIH-1.Item Metadata only Hypoxia Inducible Factor(McGraw-Hill, 2007) Hampton-Smith, R.; Peet, D.; Staff, T.; Centre for the Molecular Genetics of DevelopmentItem Metadata only Genomic and microarray approaches to coral reef conservation biology(Springer, 2007) Foret, S.; Kassahn, K.; Grasso, L.; Hayward, D.; Iguchi, A.; Ball, E.; Miller, D.; Centre for the Molecular Genetics of DevelopmentNew technologies based on DNA microarrays and comparative genomics hold great promise for providing the background biological information necessary for effective coral reef conservation and management. Microarray analysis has been used in a wide range of applications across the biological sciences, most frequently to examine simultaneous changes in the expression of large numbers of genes in response to experimental manipulation or environmental variation. Other applications of microarray methods include the assessment of divergence in gene sequences between species and the identification of fast-evolving genes. Arrays are presently available for only a limited range of species, but with appropriate controls they can be used for related species, thus avoiding the considerable costs associated with development of a system de novo. Arrays are in use or preparation to study stress responses, early development, and symbiosis in Acropora and Montastraea. Ongoing projects on several corals are making available large numbers of expressed gene sequences, enabling the identification of candidate genes for studies on gamete specificity, allorecognition and symbiont interactions. Over the next few years, microarray and comparative genomic approaches are likely to assume increasingly important and widespread use to study many aspects of the biology of coral reef organisms. Application of these genomic approaches to enhance our understanding of genetic and physiological correlates during stress, environmental disturbance and disease bears direct relevance to the conservation of coral reef ecosystems.Item Metadata only Light-responsive cryptochromes from a simple multicellular animal, the coral Acropora millepora(2007) Levy, O.; Appelbaum, L.; Leggat, W.; Gothilf, Y.; Hayward, David C.; Miller, David James; Hoegh-Guldberg, O; Centre for the Molecular Genetics of Development; School of Earth and Environmental SciencesHundreds of species of reef-building corals spawn synchronously over a few nights each year, and moonlight regulates this spawning event. However, the molecular elements underpinning the detection of moonlight remain unknown. Here we report the presence of an ancient family of blue-light–sensing photoreceptors, cryptochromes, in the reef-building coral Acropora millepora. In addition to being cryptochrome genes from one of the earliest-diverging eumetazoan phyla, cry1 and cry2 were expressed preferentially in light. Consistent with potential roles in the synchronization of fundamentally important behaviors such as mass spawning, cry2 expression increased on full moon nights versus new moon nights. Our results demonstrate phylogenetically broad roles of these ancient circadian clock–related molecules in the animal kingdom.Item Metadata only Frontier Technologies for Brain Repair(Control Publications, 2007) Barry, C.; Koblar, S.; O'Carroll, D.; Centre for the Molecular Genetics of DevelopmentItem 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 Cnidarians and ancestral genetic complexity in the animal kingdom(Elsevier, 2005) Miller, David J.; Ball, Eldon E.; Technau, Ulrich; Centre for the Molecular Genetics of DevelopmentEleven of the twelve recognized wingless (Wnt) subfamilies are represented in the sea anemone Nematostella vectensis, indicating that this developmentally important gene family was already fully diversified in the common ancestor of ‘higher’ animals. In deuterostomes, although duplications have occurred, no novel subfamilies of Wnts have evolved. By contrast, the protostomes Drosophila and Caenorhabditis have lost half of the ancestral Wnts. This pattern – loss of genes from an ancestrally complex state – might be more important in animal evolution than previously recognized.Item Metadata only Maintenance of ancestral complexity and non-metazoan genes in two basal cnidarians(Elsevier Science London, 2005) Technau, U.; Rudd, S.; Maxwell, P.; Gordon, P.; Saina, M.; Grasso, L.; Hayward, D.; Sensen, C.; Saint, R.; Holstein, T.; Ball, E.; Miller, D.; Centre for the Molecular Genetics of DevelopmentCnidarians are among the simplest extant animals; however EST analyses reveal that they have a remarkably high level of genetic complexity. In this article, we show that the full diversity of metazoan signaling pathways is represented in this phylum, as are antagonists previously known only in chordates. Many of the cnidarian ESTs match genes previously known only in non-animal kingdoms. At least some of these represent ancient genes lost by all bilaterians examined so far, rather than genes gained by recent lateral gene transfer.Item Metadata only Tum/RacGAP50C provides a critical link between anaphase microtubules and the assembly of the contractile ring in Drosophila melanogaster(Company of Biologists Ltd, 2005) Zavortink, M.; Contreras, N.; Addy, T.; Bejsovec, A.; Saint, R.; Centre for the Molecular Genetics of DevelopmentA central question in understanding cytokinesis is how the cleavage plane is positioned. Although the positioning signal is likely to be transmitted via the anaphase microtubule array to the cell cortex, exactly how the microtubule array determines the site of contractile ring formation remains unresolved. By analysing tum/RacGAP50C mutant Drosophila embryos we show that cells lacking Tum do not form furrows and fail to localise the key cytokinetic components Pebble (a RhoGEF), Aurora B kinase, Diaphanous, Pav-KLP and Anillin. The GAP activity of Tum is required for cytokinesis: in its absence cytokinesis fails early even though Tum is present on microtubules at the cell equator where the furrow should form. Disruption of the Pebble-interacting domain leaves Tum localised to the cell equator on cortically associated microtubules, again with no evidence of furrowing. These data support a model in which Tum/RacGAP, via its interaction with Pbl, provides a critical link between the anaphase microtubule spindle and cytokinetic furrow formation in Drosophila cells.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.