A ryanodine receptor-dependent Ca(i)(2+) asymmetry at Hensen's node mediates avian lateral identity.

In mouse, the establishment of left-right (LR) asymmetry requires intracellular calcium (Ca(i)(2+)) enrichment on the left of the node. The use of Ca(i)(2+) asymmetry by other vertebrates, and its origins and relationship to other laterality effectors are largely unknown. Additionally, the architecture of Hensen's node raises doubts as to whether Ca(i)(2+) asymmetry is a broadly conserved mechanism to achieve laterality. We report here that the avian embryo uses a left-side enriched Ca(i)(2+) asymmetry across Hensen's node to govern its lateral identity. Elevated Ca(i)(2+) was first detected along the anterior node at early HH4, and its emergence and left-side enrichment by HH5 required both ryanodine receptor (RyR) activity and extracellular calcium, implicating calcium-induced calcium release (CICR) as the novel source of the Ca(i)(2+). Targeted manipulation of node Ca(i)(2+) randomized heart laterality and affected nodal expression. Bifurcation of the Ca(i)(2+) field by the emerging prechordal plate may permit the independent regulation of LR Ca(i)(2+) levels. To the left of the node, RyR/CICR and H(+)V-ATPase activity sustained elevated Ca(i)(2+). On the right, Ca(i)(2+) levels were actively repressed through the activities of H(+)K(+) ATPase and serotonin-dependent signaling, thus identifying a novel mechanism for the known effects of serotonin on laterality. Vitamin A-deficient quail have a high incidence of situs inversus hearts and had a reversed calcium asymmetry. Thus, Ca(i)(2+) asymmetry across the node represents a more broadly conserved mechanism for laterality among amniotes than had been previously believed.

Structural constraints for alcohol-stimulated Ca2+ release in neural crest, and dual agonist/antagonist properties of n-octanol.

BACKGROUND: Prenatal ethanol exposure is a leading cause of mental retardation. Alcohol damages susceptible neuronal populations through its alteration of signaling pathways that direct cellular activity and survival. In early neural crest cells, ethanol elicits an intracellular Ca2+ transient that is necessary and sufficient to cause apoptosis. We tested the hypothesis that ethanol's activity represents a saturable and selective effect of alcohols upon this pathway. METHODS: Fura-2-loaded chick embryos, at the 3-somite stage, were exposed to n-alcohols ranging in size from ethanol (C2) to decanol (C10). Thereafter, Ca2+ mobilization was measured using Fura-2 and ratiometric imaging. Apoptosis was assessed using acridine orange uptake. RESULTS: Ethanol caused the dose-dependent mobilization of intracellular Ca2+ within neural crest populations, with an EC50 of 52.0 mM. n-Alcohols displayed increasing potency for Ca2+ mobilization through pentanol. Hexanol and heptanol were inactive. Unexpectedly, micromolar n-octanol concentrations triggered significant Ca2+ release and apoptosis in a G-protein-dependent manner. Decanol was inactive. Coaddition of either octanol or decanol antagonized the ability of ethanol to stimulate Ca2+ release. CONCLUSIONS: The selective, saturable effect of n-alcohols upon Ca2+ mobilization in neural crest is consistent with a hypothesis that ethanol stimulates these signals through specific interaction with one or more alcohol-binding sites on a target protein. Octanol may overcome structural constraints imposed upon C6 and C7 in interacting with this protein target; alternatively, it may interact through a unique binding site.

Ethanol triggers neural crest apoptosis through the selective activation of a pertussis toxin-sensitive G protein and a phospholipase Cbeta-dependent Ca2+ transient.

BACKGROUND: Alcohol is a potent neurotoxin that triggers the selective apoptosis of neuronal populations in the developing fetus. For neural crest cells, clinically relevant ethanol levels (0.3%) rapidly elicit a phospholipase C (PLC)-dependent intracellular Ca2+ transient that is sufficient to activate apoptosis. We investigated the biochemical origins of this Ca2+ transient. METHODS: Three somite chick embryos (stage 8-) were pretreated with agonists and antagonists of PLC signaling pathways before ethanol challenge. The resulting intracellular Ca2+ release was quantified using Fluo-3; apoptosis was assessed using vital dyes. RESULTS: Pretreatment of embryos with PLC antagonists U73122 or ET-18-OCH3 confirmed that a phosphoinositide-specific PLC was required for both the ethanol-dependent Ca2+ transient and subsequent cell death. Ethanol rapidly elevated intracellular inositol-1,4,5-trisphosphate [Ins(1,4,5)P3] levels in the rostral portion of the embryo that contains neural crest progenitors. The Ins(1,4,5)P3 receptor antagonist xestospongin C blocked the appearance of the ethanol-dependent Ca2+ transient. Pretreatment with the pan-Galpha protein antagonist GDPbetaS, but not with the tyrosine kinase antagonist genistein, suppressed ethanol's ability to elicit the Ca2+ transient, suggesting that a rise in PLC activity and Ins(1,4,5)P3 concentration originates from stimulation of heterotrimeric G proteins. To probe the identity of this G protein, embryos were treated with G protein antagonists. Pertussis toxin and NF023 suppressed the ethanol-induced Ca2+ transient and subsequent neural crest apoptosis, whereas suramin was weakly inhibitory. C3 exoenzyme was embryolethal over a wide concentration range, consistent with suggestions that Rho family GTPases participate in neural crest development. Galphai2 was identified by immunostaining in the neural crest cells. CONCLUSION: We propose a role for Galphai/o protein activation and subsequent interaction of Gbetagamma with PLCbeta in mediating the proapoptotic effects of ethanol upon the developing neural crest.

A whole-genome shotgun optical map of Yersinia pestis strain KIM.

Yersinia pestis is the causative agent of the bubonic, septicemic, and pneumonic plagues (also known as black death) and has been responsible for recurrent devastating pandemics throughout history. To further understand this virulent bacterium and to accelerate an ongoing sequencing project, two whole-genome restriction maps (XhoI and PvuII) of Y. pestis strain KIM were constructed using shotgun optical mapping. This approach constructs ordered restriction maps from randomly sheared individual DNA molecules directly extracted from cells. The two maps served different purposes; the XhoI map facilitated sequence assembly by providing a scaffold for high-resolution alignment, while the PvuII map verified genome sequence assembly. Our results show that such maps facilitated the closure of sequence gaps and, most importantly, provided a purely independent means for sequence validation. Given the recent advancements to the optical mapping system, increased resolution and throughput are enabling such maps to guide sequence assembly at a very early stage of a microbial sequencing project.