Signaling Pathways Crucial for Craniofacial Development Revealed by Endothelin-A Receptor-Deficient Mice

AbstractMost of the bone and cartilage in the craniofacial region is derived from cephalic neural crest cells, which undergo three primary developmental events: migration from the rhombomeric neuroectoderm to the pharyngeal arches, proliferation as the ectomesenchyme within the arches, and differentiation into terminal structures. Interactions between the ectomesenchymal cells and surrounding cells are required in these processes, in which defects can lead to craniofacial malformation. We have previously shown that the G-protein-coupled endothelin-A receptor (ETA) is expressed in the neural crest-derived ectomesenchyme, whereas the cognate ligand for ETA, endothelin-1 (ET-1), is expressed in arch epithelium and the paraxial mesoderm-derived arch core; absence of either ETA or ET-1 results in numerous craniofacial defects. In this study we have attempted to define the point at which cephalic neural crest development is disrupted in ETA-deficient embryos. We find that, while neural crest cell migration in the head of ETA−/− embryos appears normal, expression of a number of transcription factors in the arch ectomesenchymal cells is either absent or significantly reduced. These ETA-dependent factors include the transcription factorsgoosecoid, Dlx-2, Dlx-3, dHAND, eHAND, and Barx1, but not MHox, Hoxa-2, CRABP1, or Ufd1. In addition, the size of the arches in E10.5 to E11.5 ETA−/− embryos is smaller and an increase in ectomesenchymal apoptosis is observed. Thus, ETA signaling in ectomesenchymal cells appears to coordinate specific aspects of arch development by inducing expression of transcription factors in the postmigratory ectomesenchyme. Absence of these signals results in retarded arch growth, defects in proper differentiation, and, in some mesenchymal cells, apoptosis. In particular, this developmental pathway appears distinct from the pathway that includesUFD1L, implicated as a causative gene in CATCH 22 patients, and suggests parallel complementary pathways mediating craniofacial development

Selective loss of GABAB receptors in orexin/hypocretin-producing neurons results in disrupted sleep/wakefulness architecture

We generated mice with a selective loss of GABAB receptors in orexin neurons. Orexin neurons in these GABAB1<sup>-/-(orexin)</sup> mice showed reduced responsiveness to GABA<sub>A</sub> receptor agonists due to a compensatory increase in GABAA receptor-mediated inhibition. This increased GABA<sub>A</sub> receptor-mediated inhibition of orexin neurons is due to orexin-1 receptor-mediated activation of local GABAergic interneurons. Surprisingly, orexin neurons were also less responsive to glutamate, apparently because the augmented GABA<sub>A</sub> receptor-mediated inhibition increases the membrane conductance and shunts excitatory currents. These observations indicate that absence of GABA<sub>B</sub> receptors decreases the sensitivity of orexin neurons to both excitatory and inhibitory inputs. GABAB1<sup>-/-(orexin)</sup>mice exhibited severe fragmentation of sleep/wake states during both the light and dark periods without affecting total sleep time or inducing cataplexy, indicating that GABA<sub>B</sub> receptors are crucial regulators of orexin neurons and that "fine tuning" of orexin neurons by inhibitory and excitatory inputs is important for the stability of sleep/waking states

Orexin neurons suppress narcolepsy via 2 distinct efferent pathways

The loss of orexin neurons in humans is associated with the sleep disorder narcolepsy, which is characterized by excessive daytime sleepiness and cataplexy. Mice lacking orexin peptides, orexin neurons, or orexin receptors recapitulate human narcolepsy phenotypes, further highlighting a critical role for orexin signaling in the maintenance of wakefulness. Despite the known role of orexin neurons in narcolepsy, the precise neural mechanisms downstream of these neurons remain unknown. We found that targeted restoration of orexin receptor expression in the dorsal raphe (DR) and in the locus coeruleus (LC) of mice lacking orexin receptors inhibited cataplexy-like episodes and pathological fragmentation of wakefulness (i.e., sleepiness), respectively. The suppression of cataplexy-like episodes correlated with the number of serotonergic neurons restored with orexin receptor expression in the DR, while the consolidation of fragmented wakefulness correlated with the number of noradrenergic neurons restored in the LC. Furthermore, pharmacogenetic activation of these neurons using designer receptor exclusively activated by designer drug (DREADD) technology ameliorated narcolepsy in mice lacking orexin neurons. These results suggest that DR serotonergic and LC noradrenergic neurons play differential roles in orexin neuron-dependent regulation of sleep/wakefulness and highlight a pharmacogenetic approach for the amelioration of narcolepsy. © Copyright 2014 American Society for Clinical Investigation

Differential Actions of Orexin Receptors in Brainstem Cholinergic and Monoaminergic Neurons Revealed by Receptor Knockouts: Implications for Orexinergic Signaling in Arousal and Narcolepsy

Orexin neuropeptides influence multiple homeostatic functions and play an essential role in the expression of normal sleep-wake behavior. While their two known receptors (OX1 and OX2) are targets for novel pharmacotherapeutics, the actions mediated by each receptor remain largely unexplored. Using brain slices from mice constitutively lacking either receptor, we used whole-cell and Ca(2+) imaging methods to delineate the cellular actions of each receptor within cholinergic [laterodorsal tegmental nucleus (LDT)] and monoaminergic [dorsal raphe (DR) and locus coeruleus (LC)] brainstem nuclei-where orexins promote arousal and suppress REM sleep. In slices from OX(-/-) 2 mice, orexin-A (300 nM) elicited wild-type responses in LDT, DR, and LC neurons consisting of a depolarizing current and augmented voltage-dependent Ca(2+) transients. In slices from OX(-/-) 1 mice, the depolarizing current was absent in LDT and LC neurons and was attenuated in DR neurons, although Ca(2+)-transients were still augmented. Since orexin-A produced neither of these actions in slices lacking both receptors, our findings suggest that orexin-mediated depolarization is mediated by both receptors in DR, but is exclusively mediated by OX1 in LDT and LC neurons, even though OX2 is present and OX2 mRNA appears elevated in brainstems from OX(-/-) 1 mice. Considering published behavioral data, these findings support a model in which orexin-mediated excitation of mesopontine cholinergic and monoaminergic neurons contributes little to stabilizing spontaneous waking and sleep bouts, but functions in context-dependent arousal and helps restrict muscle atonia to REM sleep. The augmented Ca(2+) transients produced by both receptors appeared mediated by influx via L-type Ca(2+) channels, which is often linked to transcriptional signaling. This could provide an adaptive signal to compensate for receptor loss or prolonged antagonism and may contribute to the reduced severity of narcolepsy in single receptor knockout mice

1006-44 A Prognostic Factor in Coronary Artery Disease (CAD): Platelet-Dependent Thrombin Generation in Patients with CAD

We examined platelet-dependent thrombin generation in patients with coronary artery disease (CAD). Thrombin generation was measured according to the method of Aronson et al (Circulation, 1992). 0.5ml of platelet rich plasma (PRP, 15×104/ml) was prepared, and 40mM of CaCl, was added to start clotting. 0.5mM of S-2238 was added to each sample in a microtiter plate every 10min, and the plate was read kinetically at a wavelength of 405nm on a microtiter plate reader. The patients with CAD devided into 3 groups.Thrombin generation 20 min after CaCI2, additon is:Control (n=12)48±10(mOD)Stable angina (SAP) (n=15)79±27Unstable angina (UAP) (n=15)**562±155Acute myocardial infarct (AMI) (n=43)**440±269**p<0.01 compared to SAPThe patients with UAP and AMI showed marked increase in thrombin generation compared to SAP and control subjects. AMI patients with severe coronary artery disease (Group B) showed higher levels of thrombin generation (Group A, Gensini score<32: 382±248 mOD vs Group B, Gensini score> 31: 578±238, P<0.05). LVEF of group A is significantly higher than that of group B (P < 0.05). These findings indicate that patients with UAP and AMI have an evidence of hypercoagulable states and that platelet-dependent thrombin generation may play an important role in pathophysiology of UAP or AMI, and may be a prognostic factor in CAD

Tie2-Cre Transgenic Mice: A New Model for Endothelial Cell-Lineage Analysis in Vivo

AbstractEndocardial cells are thought to contribute at least in part to the formation of the endocardial cushion mesenchyme. Here, we created Tie2-Cre transgenic mice, in which expression of Cre recombinase is driven by an endothelial-specific promoter/enhancer. To analyze the lineage of Cre expressing cells, we used CAG-CAT-Z transgenic mice, in which expression of lacZ is activated only after Cre-mediated recombination. We detected pan-endothelial expression of the Cre transgene in Tie2-Cre;CAG-CAT-Z double-transgenic mice. This expression pattern is almost identical to Tie2-lacZ transgenic mice. However, interestingly, we observed strong and uniform lacZ expression in mesenchymal cells of the atrioventricular canal of Tie2-Cre;CAG-CAT-Z double-transgenic mice. We also detected lacZ expression in the mesenchymal cells in part of the proximal cardiac outflow tract, but not in the mesenchymal cells of the distal outflow tract and branchial arch arteries. LacZ staining in Tie2-Cre;CAG-CAT-Z embryos is consistent with endocardial–mesenchymal transformation in the atrioventricular canal and outflow tract regions. Our observations are consistent with previously reported results from Cx43-lacZ, Wnt1-Cre;R26R, and Pax3-Cre;R26R transgenic mice, in which lacZ expression in the cardiac outflow tract identified contributions in part from the cardiac neural crest. Tie2-Cre transgenic mice are a new genetic tool for the analyses of endothelial cell-lineage and endothelial cell–specific gene targeting