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Carrie Dickey-Sims

Publications and source records attributed to Carrie Dickey-Sims.

5 recordsLinked to original sources

CBFbeta is a facultative Runx partner in the sea urchin embryo.

BACKGROUND: Runx proteins are developmentally important metazoan transcription factors that form a heterodimeric complex with the non-homologous protein Core Binding Factor beta (CBFbeta). CBFbeta allosterically enhances Runx DNA binding but does not bind DNA itself. We report the initial characterization of SpCBFbeta, the heterodimeric partner of SpRunt-1 from the sea urchin Stronylocentrotus purpuratus. RESULTS: SpCBFbeta is remarkably similar to its mammalian homologues, and like them it enhances the DNA binding of the Runt domain. SpCBFbeta 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, SpCBFbeta 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 SpCBFbeta 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 SpCBFbeta 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. CONCLUSION: SpCBFbeta is a facultative Runx partner that appears to be required specifically for cell differentiation.

Amino Acid Substitution↗

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↗

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↗

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↗

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↗