X-chromosome inactivation is the process by which female mammals (with two X chromosomes) achieve expression of X-chromosomal genes equivalent to that of males (one X and one Y chromosome). This results in the transcriptional silencing of virtually all genes on one of the X chromosomes in female somatic cells. X-chromosome inactivation has been shown to act in cis and to initiate and spread from a single site on the X chromosome known as the X-inactivation centre (Xic). The Xic has been localized to a 450-kilobase region of the mouse X chromosome. The Xist gene also maps to this region and is expressed exclusively from the inactive X chromosome. Xist is unusual in that it appears not to code for a protein but produces a nuclear RNA which colocalizes with the inactive X chromosome. The creation of a null allele of Xist in embryonic stem cells has demonstrated that this gene is required for X inactivation to occur in cis. Here we show that Xist, introduced onto an autosome, is sufficient by itself for inactivation in cis and that Xist RNA becomes localized close to the autosome into which the gene is integrated. In addition, the presence of autosomal Xist copies leads to activation of the endogeneous Xist gene in some cells, suggesting that elements required for some aspects of chromosome counting are contained within the construct. Thus the Xist gene exhibits properties of the X-inactivation centre.

The alpha- and beta-globin gene clusters have been extensively studied. Regulation of these genes ensures that proteins derived from both loci are produced in balanced amounts, and that expression is tissue-restricted and specific to developmental stages. Here we compare the subnuclear location of the endogenous alpha- and beta-globin loci in primary human cells in which the genes are either actively expressed or silent. In erythroblasts, the alpha- and beta-globin genes are localized in areas of the nucleus that are discrete from alpha-satellite-rich constitutive heterochromatin. However, in cycling lymphocytes, which do not express globin genes, the distribution of alpha- and beta-globin genes was markedly different. beta-globin loci, in common with several inactive genes studied here (human c-fms and SOX-1) and previously (mouse lambda5, CD4, CD8alpha, RAGs, TdT and Sox-1), were associated with pericentric heterochromatin in a high proportion of cycling lymphocytes. In contrast, alpha-globin genes were not associated with centromeric heterochromatin in the nucleus of normal human lymphocytes, in lymphocytes from patients with alpha-thalassaemia lacking the regulatory HS-40 element or entire upstream region of the alpha-globin locus, or in mouse erythroblasts and lymphocytes derived from human alpha-globin transgenic mice. These data show that the normal regulated expression of alpha- and beta-globin gene clusters occurs in different nuclear environments in primary haemopoietic cells.

The master clock driving mammalian circadian rhythms is located in the suprachiasmatic nuclei (SCN) of the hypothalamus and entrained by daily light/dark cycles. SCN lesions abolish circadian rhythms of behavior and result in a loss of synchronized circadian rhythms of clock gene expression in peripheral organs (e.g., the liver) and of hormone secretion (e.g., corticosterone). We examined rhythms of behavior, hepatic clock gene expression, and corticosterone secretion in VPAC 2 receptor-null (Vipr2 Ϫ/Ϫ ) mice, which lack a functional SCN clock. Unexpectedly, although Vipr2 Ϫ/Ϫ mice lacked robust circadian rhythms of wheel-running activity and corticosterone secretion, hepatic clock gene expression was strongly rhythmic, but advanced in phase compared with that in wild-type mice. The timing of food availability is thought to be an important entrainment signal for circadian clocks outside the SCN. Vipr2 Ϫ/Ϫ mice consumed food significantly earlier in the 24 h cycle than wild-type mice, consistent with the observed timing of peripheral rhythms of circadian gene expression. When restricted to feeding only during the daytime (RF), mice develop rhythms of activity and of corticosterone secretion in anticipation of feeding time, thought to be driven by a food-entrainable circadian oscillator, located outside the SCN. Under RF, mice of both genotypes developed food-anticipatory rhythms of activity and corticosterone secretion, and hepatic gene expression rhythms also became synchronized to the RF stimulus. Thus, food intake is an effective zeitgeber capable of coordinating circadian rhythms of behavior, peripheral clock gene expression, and hormone secretion, even in the absence of a functional SCN clock.

Many molecules expressed in the CNS contribute to cognitive functions either by modulating neuronal activity or by mediating neuronal trophic support and/or connectivity. An ongoing discussion is whether signaling of nerve growth factor (NGF) through its high-affinity receptor TrkA contributes to attention behavior and/or learning and memory, based on its expression in relevant regions of the CNS such as the hippocampus, cerebral cortex, amygdala and basal forebrain. Previous animal models carrying either a null allele or transgenic manipulation of Ngf or Trka have proved difficult in addressing this question. To overcome this problem, we conditionally deleted Ngf or Trka from the CNS. Our findings confirm that NGF-TrkA signaling supports survival of only a small proportion of cholinergic neurons during development; however, this signaling is not required for trophic support or connectivity of the remaining basal forebrain cholinergic neurons. Moreover, comprehensive behavioral analysis of young adult and intermediate-aged mice lacking NGF-TrkA signaling demonstrates that this signaling is dispensable for both attention behavior and various aspects of learning and memory.

Adenosine inhibits excitatory neurons widely in the brain through adenosine A 1 receptor, but activation of adenosine A 2A receptor (A 2A R) has an opposite effect promoting discharge in neuronal networks. In the hippocampus A 2A R expression level is low, and the receptor's effect on identified neuronal circuits is unknown. Using optogenetic afferent stimulation and whole-cell recording from identified postsynaptic neurons we show that A 2A R facilitates excitatory glutamatergic Schaffer collateral synapses to CA1 pyramidal cells, but not to GABAergic inhibitory interneurons. In addition, A 2A R enhances GABAergic inhibitory transmission between CA1 area interneurons leading to disinhibition of pyramidal cells. Adenosine A 2A R has no direct modulatory effect on GABAergic synapses to pyramidal cells. As a result adenosine A 2A R activation alters the synaptic excitation -inhibition balance in the CA1 area resulting in increased pyramidal cell discharge to glutamatergic Schaffer collateral stimulation. In line with this, we show that A 2A R promotes synchronous pyramidal cell firing in hyperexcitable conditions where extracellular potassium is elevated or following high-frequency electrical stimulation. Our results revealed selective synapse-and cell type specific adenosine A 2A R effects in hippocampal CA1 area. The uncovered mechanisms help our understanding of A 2A R's facilitatory effect on cortical network activity.

The formation of mouse coat color is a relatively complex developmental process that is affected by a large number of mutations, both naturally occurring and induced. The cloning of the genes in which these mutations occur and the elucidation of the mechanisms by which these mutations disrupt the normal pigmentation pattern is leading to an understanding of the way interactions between gene products lead to a final phenotype.

BackgroundThe vestibular system provides the primary input of our sense of balance and spatial orientation. Dysfunction of the vestibular system can severely affect a person's quality of life. Therefore, understanding the molecular basis of vestibular neuron survival, maintenance, and innervation of the target sensory epithelia is fundamental.ResultsHere we report that a point mutation at the phospholipase Cγ (PLCγ) docking site in the mouse neurotrophin tyrosine kinase receptor TrkB (Ntrk2) specifically impairs fiber guidance inside the vestibular sensory epithelia, but has limited effects on the survival of vestibular sensory neurons and growth of afferent processes toward the sensory epithelia. We also show that expression of the TRPC3 cation calcium channel, whose activity is known to be required for nerve-growth cone guidance induced by brain-derived neurotrophic factor (BDNF), is altered in these animals. In addition, we find that absence of the PLCγ mediated TrkB signalling interferes with the transformation of bouton type afferent terminals of vestibular dendrites into calyces (the largest synaptic contact of dendrites known in the mammalian nervous system) on type I vestibular hair cells; the latter are normally distributed in these mutants as revealed by an unaltered expression pattern of the potassium channel KCNQ4 in these cells.ConclusionsThese results demonstrate a crucial involvement of the TrkB/PLCγ-mediated intracellular signalling in structural aspects of sensory neuron plasticity.

Summary. Background: Circadian rhythms control a vast array of biological processes in a broad spectrum of organisms. The contribution of circadian rhythms to the development of megakaryocytes and the regulation of platelet biology has not been defined. Objectives: This study tested the hypothesis that murine megakaryocytes exhibit hallmarks of circadian control. Methods: Mice expressing a PER2::LUCIFERASE circadian reporter protein and C57BI/6 mice were used to establish if megakaryocytes expressed circadian genes in vitro and in vivo. Mice were also subjected to 3 weeks on a restricted feeding regime to separate food-entrained from light-entrained circadian rhythms. Quantitative real time polymerase chain reaction (PCR), flow cytometry and imunohistochemistry were employed to analyse gene expression, DNA content and cell-cycle behavior in megakaryocytes collected from mice over a 24-h period. Results: Megakaryocytes exhibited rhythmic expression of the clock genes mPer2 and mBmal1 and circadian rhythms in megakaryopoiesis. mPer2 and mBmal1 expression phase advanced 8 h to coincide with the availability of food; however, food availability had a more complex effect on megakaryopoiesis, leading to a significant overall increase in megakaryocyte ploidy levels and cell-cycle activity. Conclusions: Normal megakaryopoiesis requires synchrony between food-and light-entrained circadian oscillators.