Polychrome labeling reveals skeletal triradiate and elongation dynamics and abnormalities in patterning cue-perturbed embryos

IOS-1656752 - National Science FoundationFirst author draf

H+/K+ ATPase activity is required for biomineralization in sea urchin embryos

AbstractThe bioelectrical signatures associated with regeneration, wound healing, development, and cancer are changes in the polarization state of the cell that persist over long durations, and are mediated by ion channel activity. To identify physiologically relevant bioelectrical changes that occur during normal development of the sea urchin Lytechinus variegatus, we tested a range of ion channel inhibitors, and thereby identified SCH28080, a chemical inhibitor of the H+/K+ ATPase (HKA), as an inhibitor of skeletogenesis. In sea urchin embryos, the primary mesodermal lineage, the PMCs, produce biomineral in response to signals from the ectoderm. However, in SCH28080-treated embryos, aside from randomization of the left-right axis, the ectoderm is normally specified and differentiated, indicating that the block to skeletogenesis observed in SCH28080-treated embryos is PMC-specific. HKA inhibition did not interfere with PMC specification, and was sufficient to block continuing biomineralization when embryos were treated with SCH28080 after the initiation of skeletogenesis, indicating that HKA activity is continuously required during biomineralization. Ion concentrations and voltage potential were abnormal in the PMCs in SCH28080-treated embryos, suggesting that these bioelectrical abnormalities prevent biomineralization. Our results indicate that this effect is due to the inhibition of amorphous calcium carbonate precipitation within PMC vesicles

The genomic repertoire for cell cycle control and DNA metabolism in S. purpuratus

A search of the Strongylocentrotus purpuratus genome for genes associated with cell cycle control and DNA metabolism shows that the known repertoire of these genes is conserved in the sea urchin, although with fewer family members represented than in vertebrates, and with some cases of echinoderm-specific gene diversifications. For example, while homologues of the known cyclins are mostly encoded by single genes in S. purpuratus (unlike vertebrates, which have multiple isoforms), there are additional genes encoding novel cyclins of the B and K/L types. Almost all known cyclin-dependent kinases (CDKs) or CDK-like proteins have an orthologue in S. purpuratus; CDK3 is one exception, whereas CDK4 and 6 are represented by a single homologue, referred to as CDK4. While the complexity of the two families of mitotic kinases, Polo and Aurora, is close to that found in the nematode, the diversity of the NIMA-related kinases (NEK proteins) approaches that of vertebrates. Among the nine NEK proteins found in S. purpuratus, eight could be assigned orthologues in vertebrates, whereas the ninth is unique to sea urchins. Most known DNA replication, DNA repair and mitotic checkpoint genes are also present, as are homologues of the pRB (two) and p53 (one) tumor suppressors. Interestingly, the p21/p27 family of CDK inhibitors is represented by one homologue, whereas the INK4 and ARF families of tumor suppressors appear to be absent, suggesting that these evolved only in vertebrates. Our results suggest that, while the cell cycle control mechanisms known from other animals are generally conserved in sea urchin, parts of the machinery have diversified within the echinoderm lineage. The set of genes uncovered in this analysis of the S. purpuratus genome should enhance future research on cell cycle control and developmental regulation in this model

Voltage-gated sodium channel activity mediates sea urchin larval skeletal patterning through spatial regulation of Wnt5 expression

IOS-1656752 - National Science FoundationFirst author draf

The mitochondrial permeability transition in cell death: a common mechanism in necrosis, apoptosis and autophagy

AbstractUsing confocal microscopy, onset of the mitochondrial permeability transition (MPT) in individual mitochondria within living cells can be visualized by the redistribution of the cytosolic fluorophore, calcein, into mitochondria. Simultaneously, mitochondria release membrane potential-indicating fluorophores like tetramethylrhodamine methylester. The MPT occurs in several forms of necrotic cell death, including oxidative stress, pH-dependent ischemia/reperfusion injury and Ca2+ ionophore toxicity. Cyclosporin A (CsA) and trifluoperazine block the MPT in these models and prevent cell killing, showing that the MPT is a causative factor in necrotic cell death. During oxidative injury induced by t-butylhydroperoxide, onset of the MPT is preceded by pyridine nucleotide oxidation, mitochondrial generation of reactive oxygen species, and an increase of mitochondrial free Ca2+, all changes that promote the MPT. During tissue ischemia, acidosis develops. Because of acidotic pH, anoxic cell death is substantially delayed. However, when pH is restored to normal after reperfusion (reoxygenation at pH 7.4), cell death occurs rapidly (pH paradox). This killing is caused by pH-dependent onset of the MPT, which is blocked by reperfusion at acidotic pH or with CsA. In isolated mitochondria, toxicants causing Reye’s syndrome, such as salicylate and valproate, induce the MPT. Similarly, salicylate induces a CsA-sensitive MPT and killing of cultured hepatocytes. These in vitro findings suggest that the MPT is the pathophysiological mechanism underlying Reye’s syndrome in vivo. Kroemer and coworkers proposed that the MPT is a critical event in the progression of apoptotic cell death. Using confocal microscopy, the MPT can be directly documented during tumor necrosis factor-α induced apoptosis in hepatocytes. CsA blocks this MPT and prevents apoptosis. The MPT does not occur uniformly during apoptosis. Initially, a small proportion of mitochondria undergo the MPT, which increases to nearly 100% over 1–3 h. A technique based on fluorescence resonance energy transfer can selectively reveal mitochondrial depolarization. After nutrient deprivation, a small fraction of mitochondria spontaneously depolarize and enter an acidic lysosomal compartment, suggesting that the MPT precedes the normal process of mitochondrial autophagy. A model is proposed in which onset of the MPT to increasing numbers of mitochondria within a cell leads progressively to autophagy, apoptosis and necrotic cell death

RNA-Seq identifies SPGs as a ventral skeletal patterning cue in sea urchins

The sea urchin larval skeleton offers a simple model for formation of developmental patterns. The calcium carbonate skeleton is secreted by primary mesenchyme cells (PMCs) in response to largely unknown patterning cues expressed by the ectoderm. To discover novel ectodermal cues, we performed an unbiased RNA-Seq-based screen and functionally tested candidates; we thereby identified several novel skeletal patterning cues. Among these, we show that SLC26a2/7 is a ventrally expressed sulfate transporter that promotes a ventral accumulation of sulfated proteoglycans, which is required for ventral PMC positioning and skeletal patterning. We show that the effects of SLC perturbation are mimicked by manipulation of either external sulfate levels or proteoglycan sulfation. These results identify novel skeletal patterning genes and demonstrate that ventral proteoglycan sulfation serves as a positional cue for sea urchin skeletal patterning

Ethanol exposure perturbs sea urchin development and disrupts developmental timing

Ethanol is a known vertebrate teratogen that causes craniofacial defects as a component of fetal alcohol syndrome (FAS). Our results show that sea urchin embryos treated with ethanol similarly show broad skeletal patterning defects, potentially analogous to the defects associated with FAS. The sea urchin larval skeleton is a simple patterning system that involves only two cell types: the primary mesenchymal cells (PMCs) that secrete the calcium carbonate skeleton and the ectodermal cells that provide migratory, positional, and differentiation cues for the PMCs. Perturbations in RA biosynthesis and Hh signaling pathways are thought to be causal for the FAS phenotype in vertebrates. Surprisingly, our results indicate that these pathways are not functionally relevant for the teratogenic effects of ethanol in developing sea urchins. We found that developmental morphology as well as the expression of ectodermal and PMC genes was delayed by ethanol exposure. Temporal transcriptome analysis revealed significant impacts of ethanol on signaling and metabolic gene expression, and a disruption in the timing of GRN gene expression that includes both delayed and precocious gene expression throughout the specification network. We conclude that the skeletal patterning perturbations in ethanol-treated embryos likely arise from a loss of temporal synchrony within and between the instructive and responsive tissues.IOS-1656752 - National Science Foundation; National Science FoundationFirst author draf

The Focal Adhesion Kinase Suppresses Transformation-associated, Anchorage-independent Apoptosis in Human Breast Cancer Cells: INVOLVEMENT OF DEATH RECEPTOR-RELATED SIGNALING PATHWAYS

The focal adhesion kinase (FAK) is a mediator of cell-extracellular matrix signaling events and is overexpressed in tumor cells. In order to rapidly down-regulate FAK function in normal and transformed mammary cells, we have used adenoviral gene transduction of the carboxyl-terminal domain of FAK (FAK-CD). Transduction of adenovirus containing FAK-CD in breast cancer cells caused loss of adhesion, degradation of p125(FAK), and induced apoptosis. Furthermore, breast tumor cells that were viable without matrix attachment also underwent apoptosis upon interruption of FAK function, demonstrating that FAK is a survival signal in breast tumor cells even in the absence of matrix signaling. In addition, both anchorage-dependent and anchorage-independent apoptotic signaling required Fas-associated death domain and caspase-8, suggesting that a death receptor-mediated apoptotic pathway is involved. Finally, FAK-CD had no effect on adhesion or viability in normal mammary cells, despite the loss of tyrosine phosphorylation of p125(FAK). These results indicate that FAK-mediated signaling is required for both cell adhesion and anchorage-independent survival and the disruption of FAK function involves the Fas-associated death domain and caspase-8 apoptotic pathway

A functional genomic and proteomic perspective of sea urchin calcium signaling and egg activation

AbstractThe sea urchin egg has a rich history of contributions to our understanding of fundamental questions of egg activation at fertilization. Within seconds of sperm–egg interaction, calcium is released from the egg endoplasmic reticulum, launching the zygote into the mitotic cell cycle and the developmental program. The sequence of the Strongylocentrotus purpuratus genome offers unique opportunities to apply functional genomic and proteomic approaches to investigate the repertoire and regulation of Ca2+ signaling and homeostasis modules present in the egg and zygote. The sea urchin “calcium toolkit” as predicted by the genome is described. Emphasis is on the Ca2+ signaling modules operating during egg activation, but the Ca2+ signaling repertoire has ramifications for later developmental events and adult physiology as well. Presented here are the mechanisms that control the initial release of Ca2+ at fertilization and additional signaling components predicted by the genome and found to be expressed and operating in eggs at fertilization. The initial release of Ca2+ serves to coordinate egg activation, which is largely a phenomenon of post-translational modifications, especially dynamic protein phosphorylation. Functional proteomics can now be used to identify the phosphoproteome in general and specific kinase targets in particular. This approach is described along with findings to date. Key outstanding questions regarding the activation of the developmental program are framed in the context of what has been learned from the genome and how this knowledge can be applied to functional studies