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James A Coffman

Publications and source records attributed to James A Coffman.

11 recordsLinked to original sources
PubMed2005

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

BACKGROUND: Runx 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. RESULTS: We 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. CONCLUSIONS: Our results show that Runx-dependent activation of SpPKC1 is essential for maintaining protein kinase C activity at levels conducive to cell survival during embryogenesis.

Animals↗
PubMed2005

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

BACKGROUND: Vaults 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. RESULTS: The 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 approximately 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. CONCLUSIONS: These results are suggestive of a role for the vault in delivering macromolecules to the nucleus during development.

Amino Acid Sequence↗
PubMed2004

Oral-aboral axis specification in the sea urchin embryo II. Mitochondrial distribution and redox state contribute to establishing polarity in Strongylocentrotus purpuratus.

The initial asymmetry that specifies the oral-aboral (OA) axis of the sea urchin embryo has long been a mystery. It was shown previously that OA polarity can be entrained in embryos by imposing a respiratory asymmetry, with the most oxidizing side of the embryo tending to develop as the oral pole. This suggests that one of the earliest observable asymmetries along the incipient OA axis, a redox gradient established by a higher density and/or activity of mitochondria on the prospective oral side of the embryo, might play a causal role in establishing the axis. Here, we examine the origin and functional significance of this early redox gradient. Using MitoTracker Green, we show that mitochondria are asymmetrically distributed in the unfertilized egg of Strongylocentrotus purpuratus, and that the polarity of the maternal asymmetry is maintained in the zygote. Vital staining indicates that the side of the embryo that inherits the highest density of mitochondria tends to develop into the oral pole. This correlation holds when mitochondria are redistributed by centrifugation of eggs or by transfer of purified mitochondria into zygotes, indicating that an asymmetric mitochondrial distribution can entrain OA polarity, possibly through effects on intracellular redox state. In support of this possibility, we find that specification of oral ectoderm is suppressed when embryos are cultured under hypoxic conditions that enforce a relatively reducing redox state. This effect is reversed by overexpression of nodal, an early zygotic marker of oral specification whose localized expression suffices to organize the entire OA axis, indicating that redox state is upstream of nodal expression. We therefore propose that a threshold level of intracellular oxidation is required to effectively activate nodal, and that precocious attainment of this threshold within the blastomeres containing the highest density of mitochondria results in asymmetric nodal activity and consequent specification of the OA axis.

Aldehydes↗
PubMed2004

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

BACKGROUND: Runx 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. RESULTS: The 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. CONCLUSIONS: SpRunt is essential for embryonic development, and is required globally to coordinate cell proliferation and differentiation.

Animals↗
PubMed2004

The sea urchin stem-loop-binding protein: a maternally expressed protein that probably functions in expression of multiple classes of histone mRNA.

Following the completion of oogenesis and oocyte maturation, histone mRNAs are synthesized and stored in the sea urchin egg pronucleus. Histone mRNAs are the only mRNAs that are not polyadenylated but instead end in a stem-loop which has been conserved in evolution. The 3' end binds the stem-loop-binding protein (SLBP), and SLBP is required for histone pre-mRNA processing as well as translation of the histone mRNAs. A cDNA encoding a 59 kDa sea urchin SLBP (suSLBP) has been cloned from an oocyte cDNA library. The suSLBP contains an RNA-binding domain that is similar to the RNA-binding domain found in SLBPs from other species, although there is no similarity between the rest of the suSLBP and other SLBPs. The suSLBP is present at constant levels in eggs and for the first 12 h of development. The levels of suSLBP then decline and remain at a low level for the rest of embryogenesis. The suSLBP is concentrated in the egg pronucleus and is released from the nucleus only when cells enter the first mitosis. SuSLBP expressed by in vitro translation does not bind the stem-loop RNA, suggesting that suSLBP is modified to activate RNA binding in sea urchin embryos.

Amino Acid Sequence↗
PubMed2004

Cell cycle development.

The Keystone Symposium on the Cell Cycle and Development brought together biologists with an interest in how cell cycle control is integrated into the ontogenetic program of multicellular organisms, and showcased research using a wide variety of systems from both animals and plants. A clear indication from the meeting is that this research is changing the conventional wisdom on both cell cycle control and development.

Animals↗
PubMed2003

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

BACKGROUND: Runx 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. RESULTS: A 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. CONCLUSIONS: In 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.

Amino Acid Sequence↗
PubMed2003

Runx transcription factors and the developmental balance between cell proliferation and differentiation.

The runt box (Runx) is a highly conserved DNA binding and protein-protein interaction domain that defines a family of heterodimeric transcription factors with essential roles in metazoan development. The first member of this family to be identified was the Drosophila regulatory gene runt, which was named by virtue of its function in establishing segmentation patterns during embryogenesis, and subsequently discovered to have additional functions in sex determination and neurogenesis. A second Drosophila Runx gene, lozenge, is required for cell patterning in the eye and for hematopoiesis. The genome project has revealed the existence of two additional Drosophila Runx genes, which to date have not been functionally characterized. Other invertebrate species with well-characterized Runx transcription factors include the nematode Caenorhabditis elegans and the sea urchin Strongylocentrotus purpuratus, each of which apparently contains only a single Runx gene. There are three Runx genes in mammals; Runx1 is required for definitive hematopoiesis and is a frequently mutated gene in human leukemia, Runx2 is required for osteogenesis and is associated with cleidocranial dysplasia, and Runx3 controls neurogenesis in the dorsal root ganglia and cell proliferation in the gastric epithelium, and is frequently deleted or silenced in human gastric cancer. Studies using mammalian systems and sea urchins indicate that Runx proteins have essential functions in both cell proliferation and differentiation, and in mammals they are both proto-oncogenes and tumor suppressors. Thus, a central question concerning the cell biology of Runx proteins is how are the opposing functions of this class of transcription factors regulated during development? Here I review current knowledge of Runx protein structure, function and regulation, and outline directions for future research aimed at understanding how Runx protein function is modulated during the transition from cell proliferation to differentiation in animal development.

Amino Acid Sequence↗
PubMed2002

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.

Amino Acid Sequence↗