Oxygen, pH, and oral-aboral axis specification in the sea urchin embryo.
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ABSTRACT:
SUBMITTER: Coluccio AE
PROVIDER: S-EPMC4187349 | biostudies-literature | 2011 Feb
REPOSITORIES: biostudies-literature
Publications
Oxygen, pH, and oral-aboral axis specification in the sea urchin embryo.
Coluccio Alison E AE LaCasse Taylor J TJ Coffman James A JA
Molecular reproduction and development 20110120 2
Similar Datasets
Project description: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.
| S-EPMC2748885 | biostudies-literature
Project description:The oral-aboral axis of the sea urchin embryo is specified conditionally via a regulated feedback circuit involving the signaling gene nodal and its antagonist lefty. In normal development nodal activity becomes localized to the prospective oral side of the blastula stage embryo, a process that requires lefty. In embryos of Strongylocentrotus purpuratus, a redox gradient established by asymmetrically distributed mitochondria provides an initial spatial input that positions the localized domain of nodal expression. This expression is perturbed by hypoxia, leading to development of radialized embryos lacking an oral-aboral axis. Here we show that this radialization is not caused by a failure to express nodal, but rather by a failure to localize nodal activity to one side of the embryo. This occurs even when embryos are removed from hypoxia at late cleavage stage when nodal is first expressed, indicating that the effect involves the initiation phase of nodal activity, rather than its positive feedback-driven amplification and maintenance. Quantitative fluorescence microscopy of MitoTracker Orange-labeled embryos expressing nodal-GFP reporter gene revealed that hypoxia abolishes the spatial correlation between mitochondrial distribution and nodal expression, suggesting that hypoxia eliminates the initial spatial bias in nodal activity normally established by the redox gradient. We propose that absent this bias, the initiation phase of nodal expression is spatially uniform, such that the ensuing Nodal-mediated community effect is not localized, and hence refractory to Lefty-mediated enforcement of localization.
| S-EPMC3929957 | biostudies-literature
Project description:The current gene regulatory network (GRN) for the sea urchin embryo pertains to pregastrular specification functions in the endomesodermal territories. Here we extend gene regulatory network analysis to the adjacent oral and aboral ectoderm territories over the same period. A large fraction of the regulatory genes predicted by the sea urchin genome project and shown in ancillary studies to be expressed in either oral or aboral ectoderm by 24 h are included, though universally expressed and pan-ectodermal regulatory genes are in general not. The loci of expression of these genes have been determined by whole mount in situ hybridization. We have carried out a global perturbation analysis in which expression of each gene was interrupted by introduction of morpholino antisense oligonucleotide, and the effects on all other genes were measured quantitatively, both by QPCR and by a new instrumental technology (NanoString Technologies nCounter Analysis System). At its current stage the network model, built in BioTapestry, includes 22 genes encoding transcription factors, 4 genes encoding known signaling ligands, and 3 genes that are yet unknown but are predicted to perform specific roles. Evidence emerged from the analysis pointing to distinctive subcircuit features observed earlier in other parts of the GRN, including a double negative transcriptional regulatory gate, and dynamic state lockdowns by feedback interactions. While much of the regulatory apparatus is downstream of Nodal signaling, as expected from previous observations, there are also cohorts of independently activated oral and aboral ectoderm regulatory genes, and we predict yet unidentified signaling interactions between oral and aboral territories.
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Project description:Specification of the non-skeletogenic mesoderm (NSM) in sea urchin embryos depends on Delta signaling. Signal reception leads to expression of regulatory genes that later contribute to the aboral NSM regulatory state. In oral NSM, this is replaced by a distinct oral regulatory state in consequence of Nodal signaling. Through regulome wide analysis we identify the homeobox gene not as an immediate Nodal target. not expression in NSM causes extinction of the aboral regulatory state in the oral NSM, and expression of a new suite of regulatory genes. All NSM specific regulatory genes are henceforth expressed exclusively, in oral or aboral domains, presaging the mesodermal cell types that will emerge. We have analyzed the regulatory linkages within the aboral NSM gene regulatory network. A linchpin of this network is gataE which as we show is a direct Gcm target and part of a feedback loop locking down the aboral regulatory state.
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Project description:Canonical Wnt (cWnt) signalling is involved in a plethora of basic developmental processes such as endomesoderm specification, gastrulation and patterning the main body axis. To activate the signal, Wnt ligands form complexes with LRP5/6 and Frizzled receptors, which leads to nuclear translocation of β-catenin and a transcriptional response. In Bilateria, the expression of different Frizzled genes is often partially overlapping, and their functions are known to be redundant in several developmental contexts. Here, we demonstrate that all four Frizzled receptors take part in the cWnt-mediated oral-aboral axis patterning in the cnidarian Nematostella vectensis but show partially redundant functions. However, we do not see evidence for their involvement in the specification of the endoderm - an earlier event likely relying on maternal intracellular β-catenin signalling components. Finally, we demonstrate that the main Wnt ligands crucial for the early oral-aboral patterning are Wnt1, Wnt3 and Wnt4. Comparison of our data with knowledge from other models suggests that distinct but overlapping expression domains and partial functional redundancy of cnidarian and bilaterian Frizzled genes may represent a shared ancestral trait.
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Project description:In both vertebrate bone, containing carbonated hydroxyapatite as the mineral phase, and in invertebrate hard tissue comprised of calcium carbonate, a popular view is that the mineral phase develops from a long-lived amorphous precursor which later transforms into crystal form. Important questions linked to this popular view are: when and where is the crystallized material formed, and is amorphous solid added subsequently to the crystalline substrate? Sea urchin teeth, in which the earliest mineral forms within isolated compartments, in a time and position dependent manner, allow direct investigation of the timing of crystallization of the calcite primary plates. Living teeth of the sea urchin Lytechinus variegatus, in their native coelomic fluid, were examined by high-energy synchrotron X-ray diffraction. The diffraction data show that calcite is present in the most aboral portions of the plumula, representing the very earliest stages of mineralization, and that this calcite has the same crystal orientation as in the more mature adoral portions of the same tooth. Raman spectroscopy of the aboral plumula confirms the initial primary plate mineral material is calcite and does not detect amorphous calcium carbonate; in the more mature adoral incisal flange, it does detect a broader calcite peak, consistent with two or more magnesium compositions. We hypothesize that some portion of each syncytial membrane in the plumula provides the information for nucleation of identically oriented calcite crystals that subsequently develop to form the complex geometry of the single crystal sea urchin tooth.
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Project description:Sea urchin embryos initiate cell specifications at the 16-cell stage by forming the mesomeres, macromeres and micromeres according to the relative position of the cells in the animal-vegetal axis. The most vegetal cells, micromeres, autonomously differentiate into skeletons and induce the neighbouring macromere cells to become mesoendoderm in the β-catenin-dependent Wnt8 signalling pathway. Although the underlying molecular mechanism for this progression is largely unknown, we have previously reported that the initial events might be triggered by the Ca2+ influxes through the egg-originated L-type Ca2+ channels distributed asymmetrically along the animal-vegetal axis and through the stretch-dependent Ca2+channels expressed specifically in the micromere at the 4th cleavage. In this communication, we have examined whether one of the earliest Ca2+ targets, protein kinase C (PKC), plays a role in cell specification upstream of β-catenin. To this end, we surveyed the expression pattern of β-catenin in early embryos in the presence or absence of the specific peptide inhibitor of Hemicentrotus pulcherrimus PKC (HpPKC-I). Unlike previous knowledge, we have found that the initial nuclear entrance of β-catenin does not take place in the micromeres, but in the macromeres at the 16-cell stage. Using the HpPKC-I, we have demonstrated further that PKC not only determines cell-specific nucleation of β-catenin, but also regulates a variety of cell specification events in the early sea urchin embryos by modulating the cell adhesion structures, actin dynamics, intracellular Ca2+ signalling, and the expression of key transcription factors.
| S-EPMC4416383 | biostudies-literature
Project description:Dorsal/ventral patterning of the sea urchin embryo depends upon the establishment of a Nodal-expressing ventral organizer. Recently, we showed that spatial positioning of this organizer relies on the dorsal-specific transcription of the Hbox12 repressor. Building on these findings, we determined the influence of the epigenetic milieu on the expression of hbox12 and nodal genes. We find that Trichostatin-A, a potent and selective histone-deacetylases inhibitor, induces histone hyperacetylation in hbox12 chromatin, evoking broad ectopic expression of the gene. Transcription of nodal concomitantly drops, prejudicing dorsal/ventral polarity of the resulting larvae. Remarkably, impairing hbox12 function, either in a spatially-restricted sector or in the whole embryo, specifically rescues nodal transcription in Trichostatin-A-treated larvae. Beyond strengthen the notion that nodal expression is not allowed in the presence of functional Hbox12 in the same cells, these results highlight a critical role of histone deacetylases in regulating the spatial expression of hbox12.
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Project description:Specification of sea urchin embryo micromeres occurs early in cleavage, with the establishment of a well defined regulatory state. The architecture of the gene regulatory network controlling the specification process indicates that transcription of the initial tier of control genes depends on a double-negative gate. A gene encoding a transcriptional repressor, pmar1, is activated specifically in micromeres, where it represses transcription of a second repressor that is otherwise active globally. Thus, the micromere-specific control genes, which are the target of the second repressor, are expressed exclusively in this lineage. The double-negative specification gate was logically required from the results of numerous prior experiments, but the identity of the gene encoding the second repressor remained elusive. Here we show that hesC is this gene, and we demonstrate experimentally all of its predicted functions, including global repression of micromere-specific regulatory genes. As logically required, blockade of hesC mRNA translation and global overexpression of pmar1 mRNA have the same effect, which is to cause all of the cells of the embryo to express micromere-specific genes.
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Project description:By gastrulation the ectodermal territories of the sea urchin embryo have developed an unexpectedly complex spatial pattern of sharply bounded regulatory states, organized orthogonally with respect to the animal/vegetal and oral/aboral axes of the embryo. Although much is known of the gene regulatory network (GRN) linkages that generate these regulatory states, the principles by which the boundaries between them are positioned and maintained have remained undiscovered. Here we determine the encoded genomic logic responsible for the boundaries of the oral aspect of the embryo that separate endoderm from ectoderm and ectoderm from neurogenic apical plate and that delineate the several further subdivisions into which the oral ectoderm per se is partitioned. Comprehensive regulatory state maps, including all spatially expressed oral ectoderm regulatory genes, were established. The circuitry at each boundary deploys specific repressors of regulatory states across the boundary, identified in this work, plus activation by broadly expressed positive regulators. These network linkages are integrated with previously established interactions on the oral/aboral axis to generate a GRN model encompassing the 2D organization of the regulatory state pattern in the pregastrular oral ectoderm of the embryo.
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