Anthony J. Robertson

The evolution of Runx genes I. A comparative study of sequences from phylogenetically diverse model organisms

BackgroundRunx genes encode proteins defined by the highly conserved Runt DNA-binding domain. Studies of Runx genes and proteins in model organisms indicate that they are key transcriptional regulators of animal development. However, little is known about Runx gene evolution.ResultsA phylogenetically broad sampling of publicly available Runx gene sequences was collected. In addition to the published sequences from mouse, sea urchin, Drosophila melanogaster and Caenorhabditis elegans, we collected several previously uncharacterised Runx sequences from public genome sequence databases. Among deuterostomes, mouse and pufferfish each contain three Runx genes, while the tunicate Ciona intestinalis and the sea urchin Strongylocentrotus purpuratus were each found to have only one Runx gene. Among protostomes, C. elegans has a single Runx gene, while Anopheles gambiae has three and D. melanogaster has four, including two genes that have not been previously described. Comparative sequence analysis reveals two highly conserved introns, one within and one just downstream of the Runt domain. All vertebrate Runx genes utilize two alternative promoters.ConclusionsIn the current public sequence database, the Runt domain is found only in bilaterians, suggesting that it may be a metazoan invention. Bilaterians appear to ancestrally contain a single Runx gene, suggesting that the multiple Runx genes in vertebrates and insects arose by independent duplication events within those respective lineages. At least two introns were present in the primordial bilaterian Runx gene. Alternative promoter usage arose prior to the duplication events that gave rise to three Runx genes in vertebrates.

Oral-aboral axis specification in the sea urchin embryo III. Role of mitochondrial redox signaling via H2O2

In sea urchin embryos, specification of the secondary (oral-aboral) axis occurs via nodal, expression of which is entirely zygotic and localized to prospective oral ectoderm at blastula stage. The initial source of this spatial anisotropy is not known. Previous studies have shown that oral-aboral (OA) polarity correlates with a mitochondrial gradient, and that nodal activity is dependent both on mitochondrial respiration and p38 stress-activated protein kinase. Here we show that the spatial pattern of nodal activity also correlates with the mitochondrial gradient, and that the latter correlates with inhomogeneous levels of intracellular reactive oxygen species. To test whether mitochondrial H(2)O(2) functions as a redox signal to activate nodal, zygotes were injected with mRNA encoding either mitochondrially-targeted catalase, which quenches mitochondrial H(2)O(2) and down-regulates p38, or superoxide dismutase, which augments mitochondrial H(2)O(2) and up-regulates p38. Whereas the former treatment inhibits the initial activation of nodal and entrains OA polarity toward aboral when confined to half of the embryo via 2-cell stage blastomere injections, the latter does not produce the opposite effects. We conclude that mitochondrial H(2)O(2) is rate-limiting for the initial activation of nodal, but that additional rate-limiting factors, likely also involving mitochondria, contribute to the asymmetry in nodal expression.

The expression of SpRunt during sea urchin embryogenesis.

The runt box (runx) is a highly conserved DNA binding and protein-protein interaction domain that defines a family of heterodimeric transcription factors that regulate development in metazoans. The three mammalian runx genes are oncogenes with essential functions in normal development: Runx1 is required for hematopoiesis and is frequently mutated in human and murine leukemias; Runx2 is required for bone development and is associated with human cleidocranial dysplasia and murine leukemias; and Runx3 (the evolutionarily basal member of the mammalian family) regulates growth of the gut and functions as a tumor suppressor in the gastric epithelium (Westendorf and Hiebert, 1999; Li et al., 2002). The sea urchin Strongylocentrotus purpuratus contains a single runx gene, SpRunt. We present here the initial structural characterization of SpRunt, and its pattern of expression during embryogenesis. SpRunt contains two introns, the locations of which are identical to those of the second and third introns from promoter P2 of the mammalian runx genes. A approximately 6 kb transcript begins to accumulate during cleavage. At mesenchyme blastula stage, SpRunt transcripts are found throughout the embryo, but specifically enriched in the vegetal plate, skeletogenic mesenchyme, and part of the ectoderm. By late gastrula stage expression is localized to the endomesoderm and oral ectoderm. In the pluteus larva SpRunt transcripts remain confined to the endomesoderm and oral ectoderm, with highest levels of accumulation in the foregut and in the ciliary band. These data suggest that SpRunt expression is enhanced in proliferating cells.

Evaluation of developmental phenotypes produced by morpholino antisense targeting of a sea urchin Runx gene

BackgroundRunx transcription factors are important regulators of metazoan development. The sea urchin Runx gene SpRunt was previously identified as a trans-activator of the CyIIIa actin gene, a differentiation marker of larval aboral ectoderm. Here we extend the functional analysis of SpRunt, using morpholino antisense oligonucleotides (morpholinos) to interfere with SpRunt expression in the embryo.ResultsThe developmental effects of four different SpRunt-specific morpholinos were evaluated. The two morpholinos most effective at knocking down SpRunt produce an identical mitotic catastrophe phenotype at late cleavage stage that is an artifact of coincidental mis-targeting to histone mRNA, providing a cautionary example of the insufficiency of two different morpholinos as a control for specificity. The other two morpholinos produce gastrula stage proliferation and differentiation defects that are rescued by exogenous SpRunt mRNA. The expression of 22 genes involved in cell proliferation and differentiation was analyzed in the latter embryos by quantitative polymerase chain reaction. Knockdown of SpRunt was found to perturb the expression of differentiation markers in all of the major tissue territories as well as the expression of cell cycle control genes, including cyclin B and cyclin D.ConclusionsSpRunt is essential for embryonic development, and is required globally to coordinate cell proliferation and differentiation.

Runx-dependent expression of PKC is critical for cell survival in the sea urchin embryo

BackgroundRunx transcription factors play critical roles in the developmental control of cell fate and contribute variously as oncoproteins and tumor suppressors to leukemia and other cancers. To discover fundamental Runx functions in the cell biology of animal development, we have employed morpholino antisense-mediated knockdown of the sea urchin Runx protein SpRunt-1. Previously we showed that embryos depleted of SpRunt-1 arrest development at early gastrula stage and underexpress the conventional protein kinase C SpPKC1.ResultsWe report here that SpRunt-1 deficiency leads to ectopic cell proliferation and extensive apoptosis. Suppression of the apoptosis by pharmacological inhibition of caspase-3 prevents the ectopic proliferation and rescues gastrulation, indicating that many of the overt defects obtained by knockdown of SpRunt-1 are secondary to the apoptosis. Inhibition or knockdown of SpPKC1 also causes apoptosis, while cell survival is rescued in SpRunt-1 morphant embryos coinjected with SpPKC1 mRNA, suggesting that the apoptosis associated with SpRunt-1 deficiency is caused by the deficit in SpPKC1 expression. Chromatin immunoprecipitation indicates that SpRunt-1 interacts physically with SpPKC1 in vivo, and cis-regulatory analysis shows that this interaction activates SpPKC1 transcription.ConclusionsOur results show that Runx-dependent activation of SpPKC1 is essential for maintaining protein kinase C activity at levels conducive to cell survival during embryogenesis.

Genome Sequence of Haloplasma contractile, an Unusual Contractile Bacterium from a Deep-Sea Anoxic Brine Lake

We present the draft genome of Haloplasma contractile, isolated from a deep-sea brine and representing a new order between Firmicutes and Mollicutes. Its complex morphology with contractile protrusions might be strongly influenced by the presence of seven MreB/Mbl homologs, which appears to be the highest copy number ever reported.

Runx Expression Is Mitogenic and Mutually Linked to Wnt Activity in Blastula-Stage Sea Urchin Embryos

Background The Runt homology domain (Runx) defines a metazoan family of sequence-specific transcriptional regulatory proteins that are critical for animal development and causally associated with a variety of mammalian cancers. The sea urchin Runx gene SpRunt-1 is expressed throughout the blastula stage embryo, and is required globally during embryogenesis for cell survival and differentiation. Methodology/Principal Findings Depletion of SpRunt-1 by morpholino antisense-mediated knockdown causes a blastula stage deficit in cell proliferation, as shown by bromodeoxyuridine (BrdU) incorporation and direct cell counts. Reverse transcription coupled polymerase chain reaction (RT-PCR) studies show that the cell proliferation deficit is presaged by a deficit in the expression of several zygotic wnt genes, including wnt8, a key regulator of endomesoderm development. In addition, SpRunt-1-depleted blastulae underexpress cyclinD, an effector of mitogenic Wnt signaling. Blastula stage cell proliferation is also impeded by knockdown of either wnt8 or cyclinD. Chromatin immunoprecipitation (ChIP) indicates that Runx target sites within 5′ sequences flanking cyclinD, wnt6 and wnt8 are directly bound by SpRunt-1 protein at late blastula stage. Furthermore, experiments using a green fluorescent protein (GFP) reporter transgene show that the blastula-stage operation of a cis-regulatory module previously shown to be required for wnt8 expression (Minokawa et al., Dev. Biol. 288: 545–558, 2005) is dependent on its direct sequence-specific interaction with SpRunt-1. Finally, inhibitor studies and immunoblot analysis show that SpRunt-1 protein levels are negatively regulated by glycogen synthase kinase (GSK)-3. Conclusions/Significance These results suggest that Runx expression and Wnt signaling are mutually linked in a feedback circuit that controls cell proliferation during development.

Sea urchin vault structure, composition, and differential localization during development

BackgroundVaults are intriguing ribonucleoprotein assemblies with an unknown function that are conserved among higher eukaryotes. The Pacific coast sea urchin, Strongylocentrotus purpuratus, is an invertebrate model organism that is evolutionarily closer to humans than Drosophila and C. elegans, neither of which possesses vaults. Here we compare the structures of sea urchin and mammalian vaults and analyze the subcellular distribution of vaults during sea urchin embryogenesis.ResultsThe sequence of the sea urchin major vault protein (MVP) was assembled from expressed sequence tags and genome traces, and the predicted protein was found to have 64% identity and 81% similarity to rat MVP. Sea urchin MVP includes seven ~50 residue repeats in the N-terminal half of the protein and a predicted coiled coil domain in the C-terminus, as does rat MVP. A cryoelectron microscopy (cryoEM) reconstruction of isolated sea urchin vaults reveals the assembly to have a barrel-shaped external structure that is nearly identical to the rat vault structure. Analysis of the molecular composition of the sea urchin vault indicates that it contains components that may be homologs of the mammalian vault RNA component (vRNA) and protein components (VPARP and TEP1). The sea urchin vault appears to have additional protein components in the molecular weight range of 14–55 kDa that might correspond to molecular contents. Confocal experiments indicate a dramatic relocalization of MVP from the cytoplasm to the nucleus during sea urchin embryogenesis.ConclusionsThese results are suggestive of a role for the vault in delivering macromolecules to the nucleus during development.

CBFβ is a facultative Runx partner in the sea urchin embryo

BackgroundRunx proteins are developmentally important metazoan transcription factors that form a heterodimeric complex with the non-homologous protein Core Binding Factor β (CBFβ). CBFβ allosterically enhances Runx DNA binding but does not bind DNA itself. We report the initial characterization of SpCBFβ, the heterodimeric partner of SpRunt-1 from the sea urchin Stronylocentrotus purpuratus.ResultsSpCBFβ is remarkably similar to its mammalian homologues, and like them it enhances the DNA binding of the Runt domain. SpCBFβ is entirely of zygotic provenance and its expression is similar that of SpRunt-1, accumulating globally at late blastula stage then later localizing to endoderm and oral ectoderm. Unlike SpRunt-1, however, SpCBFβ is enriched in the endodermal mid- and hindgut of the pluteus larva, and is not highly expressed in the foregut and ciliated band. We showed previously that morpholino antisense-mediated knockdown of SpRunt-1 leads to differentiation defects, as well as to extensive post-blastula stage apoptosis caused by under-expression of the Runx target gene SpPKC1. In contrast, we show here that knockdown of SpCBFβ does not negatively impact cell survival or SpPKC1 expression, although it does lead to differentiation defects similar to those associated with SpRunt-1 deficiency. Moreover, SpRunt-1 containing a single amino acid substitution that abolishes its ability to interact with SpCBFβ retains the ability to rescue cell survival in SpRunt-1 morphant embryos. Chromatin immunoprecipitation shows that while the CyIIIa promoter engages both proteins, the SpPKC1 promoter only engages SpRunt-1.ConclusionSpCBFβ is a facultative Runx partner that appears to be required specifically for cell differentiation.

The evolution of Runx genes II. The C-terminal Groucho recruitment motif is present in both eumetazoans and homoscleromorphs but absent in a haplosclerid demosponge

BackgroundThe Runt DNA binding domain (Runx) defines a metazoan family of sequence-specific transcription factors with essential roles in animal ontogeny and stem cell based development. Depending on cis-regulatory context, Runx proteins mediate either transcriptional activation or repression. In many contexts Runx-mediated repression is carried out by Groucho/TLE, recruited to the transcriptional complex via a C-terminal WRPY sequence motif that is found encoded in all heretofore known Runx genes.FindingsFull-length Runx genes were identified in the recently sequenced genomes of phylogenetically diverse metazoans, including placozoans and sponges, the most basally branching members of that clade. No sequences with significant similarity to the Runt domain were found in the genome of the choanoflagellate Monosiga brevicollis, confirming that Runx is a metazoan apomorphy. A contig assembled from genomic sequences of the haplosclerid demosponge Amphimedon queenslandica was used to construct a model of the single Runx gene from that species, AmqRunx, the veracity of which was confirmed by expressed sequences. The encoded sequence of the Runx protein OscRunx from the homoscleromorph sponge Oscarella carmella was also obtained from assembled ESTs. Remarkably, a syntenic linkage between Runx and Supt3h, previously reported in vertebrates, is conserved in A. queenslandica. Whereas OscRunx encodes a C-terminal Groucho-recruitment motif, AmqRunx does not, although a Groucho homologue is found in the A. queenslandica genome.ConclusionOur results are consistent with the hypothesis that sponges are paraphyletic, and suggest that Runx-WRPY mediated recruitment of Groucho to cis-regulatory sequences originated in the ancestors of eumetazoans following their divergence from demosponges.