The mammalian central nervous system (CNS) contains a remarkable array of neural cells, each with a complex pattern of connections that together generate perceptions and higher brain functions. Here we describe a large-scale screen to create an atlas of CNS gene expression at the cellular level, and to provide a library of verified bacterial artificial chromosome (BAC) vectors and transgenic mouse lines that offer experimental access to CNS regions, cell classes and pathways. We illustrate the use of this atlas to derive novel insights into gene function in neural cells, and into principal steps of CNS development. The atlas, library of BAC vectors and BAC transgenic mice generated in this screen provide a rich resource that allows a broad array of investigations not previously available to the neuroscience community.

Odorant receptors (ORs) mediate the interaction of odorous compounds with olfactory sensory neurons (OSNs) and influence the guidance of OSN axons to synaptic targets in the olfactory bulb (OB). OSNs expressing the same OR send convergent axonal projections to defined glomeruli in the OB and are thought to share the same odorant response properties. This expectation of functional similarity has not been tested experimentally, because it has not been possible to determine reproducibly the response properties of OSNs that express defined ORs. Here, we applied calcium imaging to characterize the odorant response properties of single neurons from gene-targeted mice in which the green fluorescent protein is coexpressed with a particular OR. We show that the odorants acetophenone and benzaldehyde are agonists for the M71 OR and that M71-expressing neurons are functionally similar in their response properties across concentration. Replacing the M71 coding sequence with that of the rat I7 OR changes the stimulus response profiles of this genetically defined OSN population and concomitantly results in the formation of novel glomeruli in the OB. We further show that the mouse I7 OR imparts a particular response profile to OSNs regardless of the epithelial zone of expression. Our data provide evidence that ORs determine both odorant specificity and axonal convergence and thus direct functionally similar afferents to form particular glomeruli. They confirm and extend the notion that OR expression provides a molecular basis for the formation and arrangement of glomerular functional units.

Olfactory sensory neurons (OSNs) expressing a given odorant receptor (OR) gene project their axons to a few specific glomeruli that reside at recognizable locations in the olfactory bulb. Connecting approximately 1000 populations of OSNs to the approximately 1800 glomeruli of the mouse bulb poses a formidable wiring problem. Additional progress in understanding the mechanisms of neuronal connectivity is dependent on knowing how these axonal pathways are organized and how they form during development. Here we have applied a genetic approach to this problem. We have constructed by gene targeting novel strains of mice in which either all OSNs or those that express a specific OR gene, M72 or M71, also produce green fluorescent protein (GFP) or a fusion of tau with GFP. We visualized OSNs and their axons in whole mounts with two-photon laser scanning microscopy. The main conclusion we draw from the three-dimensional reconstructions is the high degree of morphological variability of mature glomeruli receiving axonal input from OR-expressing OSNs and of the pathways taken by the axons to those glomeruli. We also observe that axons of OR-expressing OSNs do not innervate nearby glomeruli in mature mice. Postnatally, a tangle of axons from M72-expressing OSNs occupies a large surface area of the bulb and coalesces abruptly into a protoglomerulus at a reproducible stage of development. These results differ in several aspects from those reported for the development of glomeruli receiving input from OSNs expressing the P2 OR, suggesting the need for a more systematic examination of OR-specific glomeruli.

Axons of olfactory sensory neurons expressing a given odorant receptor converge to a few glomeruli in the olfactory bulb. We have generated mice with unresponsive olfactory sensory neurons by targeted mutagenesis of a cyclic nucleotide-gated channel subunit gene, OCNC1. When these anosmic mice were crossed with mice in which neurons expressing a given odorant receptor can be visualized by coexpression of an axonal marker, the pattern of convergence was affected for one but not another receptor. In a novel paradigm, termed monoallelic deprivation, axons from channel positive or negative neurons that express the same odorant receptor segregate into distinct glomeruli within the same bulb. Thus, the peripheral olfactory projections are in part influenced by mechanisms that depend on neuronal activity.

BackgroundMany eukaryotic genomes encode cis-natural antisense transcripts (cis-NATs). Sense and antisense transcripts may form double-stranded RNAs that are processed by the RNA interference machinery into small interfering RNAs (siRNAs). A few so-called nat-siRNAs have been reported in plants, mammals, Drosophila, and yeasts. However, many questions remain regarding the features and biogenesis of nat-siRNAs.ResultsThrough deep sequencing, we identified more than 17,000 unique siRNAs corresponding to cis-NATs from biotic and abiotic stress-challenged Arabidopsis thaliana and 56,000 from abiotic stress-treated rice. These siRNAs were enriched in the overlapping regions of NATs and exhibited either site-specific or distributed patterns, often with strand bias. Out of 1,439 and 767 cis-NAT pairs identified in Arabidopsis and rice, respectively, 84 and 119 could generate at least 10 siRNAs per million reads from the overlapping regions. Among them, 16 cis-NAT pairs from Arabidopsis and 34 from rice gave rise to nat-siRNAs exclusively in the overlap regions. Genetic analysis showed that the overlapping double-stranded RNAs could be processed by Dicer-like 1 (DCL1) and/or DCL3. The DCL3-dependent nat-siRNAs were also dependent on RNA-dependent RNA polymerase 2 (RDR2) and plant-specific RNA polymerase IV (PolIV), whereas only a fraction of DCL1-dependent nat-siRNAs was RDR- and PolIV-dependent. Furthermore, the levels of some nat-siRNAs were regulated by specific biotic or abiotic stress conditions in Arabidopsis and rice.ConclusionsOur results suggest that nat-siRNAs display distinct distribution patterns and are generated by DCL1 and/or DCL3. Our analysis further supported the existence of nat-siRNAs in plants and advanced our understanding of their characteristics.

Vertebrate central nervous system (CNS) histogenesis depends on glia-guided migration of postmitotic neurons to form neuronal laminae. Previous studies have established that the neuronal protein astrotactin functions in murine cerebellar granule cell migration in vitro. The gene encoding astrotactin predicts a protein with three epidermal growth factor repeats and two fibronectin type III repeats. Astrotactin messenger RNA is expressed in postmitotic neuronal precursors in the cerebellum, hippocampus, cerebrum, and olfactory bulb, where migration establishes laminar structures. Fab fragments of antibodies to a recombinant astrotactin peptide blocked migration of cerebellar granule neurons in vitro along astroglial fibers. Transfection of astrotactin complementary DNA into 3T3 cells indicated that astrotactin acts as a ligand for neuron-glia binding during neuronal migration.

Abstract. Long non-coding RNAs (lncRNAs) are emerging as key molecules in human cancer. Homeobox (HOX) transcript antisense intergenic RNA (HOTAIR), a long non-coding RNA (lncRNA), is associated with a variety of human cancers, such as breast, liver and lung cancer. However, whether HOTAIR can function as a molecular marker in endometrial carcinoma (EC) remains unknown. In the present study, the expression of HOTAIR in 66 EC tissues from patients with EC and 30 normal tissues from healthy age-matched control subjects was determined using quantitative reverse transcription PCR. Furthermore, using in situ hybridization, we measured HOTAIR expression in 129 formalin-fixed paraffin-embedded (FFPE) tissue sections, which included 96 tissues that matched the frozen cases, 21 other EC tissues and 12 atypical hyperplasia tissues. Correlations between HOTAIR expression and the clinicopathological characteristics of patients were analyzed. Our results revealed that HOTAIR expression in the EC tissues was significantly upregulated compared with normal tissues (p<0.001). In addition, we observed a significant association between HOTAIR expression and the EC grade (p<0.05) and lymph node metastasis (p<0.05). Moreover, in the FFPE tissues, but not the frozen tissues, we found that a higher HOTAIR expression also correlated with the depth of myometrial invasion (p=0.019) and lymphovascular space invasion (p=0.015). More importantly, patients with a higher HOTAIR expression showed significantly poorer overall survival than those with lower HOTAIR expression (p<0.05). In conclusion, our results suggest that a high expression of HOTAIR is involved in the progression of cancer and may be a novel biomarker of poor prognosis in patients with EC. IntroductionEndometrial carcinoma (EC) is one of the most common malignancies of the female reproductive system in Western countries. In 2013, an estimated 49,500 new cases and 8,200 deaths due to EC are expected in the USA (1). With the increase in obesity and the decrease in physical activity, the incidence of EC is rising and shows a trend in younger women (2). EC is usually classified into two types to determine the risk of metastasis and recurrence (3). Generally, type I endometrioid endometrial carcinomas (EECs) have a good prognosis and account for 80-85% of the total cases of EC. By contrast, type II non-EECs are often associated with a worse outcome (3,4). However, the prognostic value of this classification is unsatisfactory, as approximately 20% of type I tumors recur, whereas 50% of type II tumors recur (5). A number of previous studies have demonstrated the utility of molecular alterations as prognostic markers, including p53 (6), phosphatase and tensin homolog (PTEN) (7) and ; however, their value is limited (9). Thus, a deeper understanding of the molecular mechanisms responsible for EC is required for risk stratification and a clinical decision regarding individualized treatment strategies.Recent studies have indicated that only 2% of transcripts are protein-coding RNAs, and ...

Pancreatic cancer is an aggressive malignancy with an extremely poor prognosis. The human ether-a-go-go-related potassium channel (HERG1) is a human rapid delayed rectifier, which is involved in many crucial cellular events. In this article, we find that HERG1 expression is dramatically increased both in pancreatic cancer tissues and cell lines, and that increased HERG1 expression is significantly related to the development of pancreatic cancer. HERG1 silencing in pancreatic cancer-derived cell lines PANC-1 and CFPAC-1 strongly inhibits their malignant capacity in vitro as well as tumorigenicity and metastasis in nude mice. In addition, HERG1 is identified as a direct target of miR-96, which is downregulated in pancreatic cancer tissues and cell lines. Ectopic expression of miR-96 represses the HERG1 expression in pancreatic cancer and significantly inhibits malignant behavior of pancreatic cancer cells in vitro and in vivo.Collectively, our findings suggest that miR-96 acts as a tumor suppressor in pancreatic cancer and may therefore serve as a useful therapeutic target for the development of new anticancer therapy.