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Background: Alternative transcription start site (TSS) usage plays important roles in transcriptional control of mammalian gene expression. The growing interest in alternative TSSs and their role in genome diversification spawned many single-gene studies on differential usages of tissue-specific or temporal-specific alternative TSSs. However, exploration of the switching usage of alternative TSS usage on a genomic level, especially in the central nervous system, is largely lacking. Results: In this study, We have prepared a unique set of time-course data for the developing cerebellum, as part of the FANTOM5 consortium ( http://fantom.gsc.riken.jp/5/ ) that uses their innovative capturing of 5′ ends of all transcripts followed by Helicos next generation sequencing. We analyzed the usage of all transcription start sites (TSSs) at each time point during cerebellar development that provided information on multiple RNA isoforms that emerged from the same gene. We developed a mathematical method that systematically compares the expression of different TSSs of a gene to identify temporal crossover and non-crossover switching events. We identified 48,489 novel TSS switching events in 5433 genes during cerebellar development. This includes 9767 crossover TSS switching events in 1511 genes, where the dominant TSS shifts over time. Conclusions: We observed a relatively high prevalence of TSS switching in cerebellar development where the resulting temporally-specific gene transcripts and protein products can play important regulatory and functional roles.

BackgroundThe work of the FANTOM5 Consortium has brought forth a new level of understanding of the regulation of gene transcription and the cellular processes involved in creating diversity of cell types. In this study, we extended the analysis of the FANTOM5 Cap Analysis of Gene Expression (CAGE) transcriptome data to focus on understanding the genetic regulators involved in mouse cerebellar development.ResultsWe used the HeliScopeCAGE library sequencing on cerebellar samples over 8 embryonic and 4 early postnatal times. This study showcases temporal expression pattern changes during cerebellar development. Through a bioinformatics analysis that focused on transcription factors, their promoters and binding sites, we identified genes that appear as strong candidates for involvement in cerebellar development. We selected several candidate transcriptional regulators for validation experiments including qRT-PCR and shRNA transcript knockdown. We observed marked and reproducible developmental defects in Atf4, Rfx3, and Scrt2 knockdown embryos, which support the role of these genes in cerebellar development.ConclusionsThe successful identification of these novel gene regulators in cerebellar development demonstrates that the FANTOM5 cerebellum time series is a high-quality transcriptome database for functional investigation of gene regulatory networks in cerebellar development.

Laser-capture microdissection was used to isolate external germinal layer tissue from three developmental periods of mouse cerebellar development: embryonic days 13, 15, and 18. The cerebellar granule cell-enriched mRNA library was generated with next-generation sequencing using the Helicos technology. Our objective was to discover transcriptional regulators that could be important for the development of cerebellar granule cells-the most numerous neuron in the central nervous system. Through differential expression analysis, we have identified 82 differentially expressed transcription factors (TFs) from a total of 1311 differentially expressed genes. In addition, with TF-binding sequence analysis, we have identified 46 TF candidates that could be key regulators responsible for the variation in the granule cell transcriptome between developmental stages. Altogether, we identified 125 potential TFs (82 from differential expression analysis, 46 from motif analysis with 3 overlaps in the two sets). From this gene set, 37 TFs are considered novel due to the lack of previous knowledge about their roles in cerebellar development. The results from transcriptome-wide analyses were validated with existing online databases, qRT-PCR, and in situ hybridization. This study provides an initial insight into the TFs of cerebellar granule cells that might be important for development and provide valuable information for further functional studies on these transcriptional regulators.

Alternative transcription start site (TSS) usage plays important roles in transcriptional control of mammalian gene expression. The growing interest in alternative TSSs and their role in genome diversification spawned many single-gene studies on differential usages of tissue-specific or temporal-specific alternative TSSs. However, exploration of the switching usage of alternative TSS usage on a genomic level, especially in the central nervous system, is largely lacking.In this study, We have prepared a unique set of time-course data for the developing cerebellum, as part of the FANTOM5 consortium ( http://fantom.gsc.riken.jp/5/ ) that uses their innovative capturing of 5' ends of all transcripts followed by Helicos next generation sequencing. We analyzed the usage of all transcription start sites (TSSs) at each time point during cerebellar development that provided information on multiple RNA isoforms that emerged from the same gene. We developed a mathematical method that systematically compares the expression of different TSSs of a gene to identify temporal crossover and non-crossover switching events. We identified 48,489 novel TSS switching events in 5433 genes during cerebellar development. This includes 9767 crossover TSS switching events in 1511 genes, where the dominant TSS shifts over time.We observed a relatively high prevalence of TSS switching in cerebellar development where the resulting temporally-specific gene transcripts and protein products can play important regulatory and functional roles.

Pax6 is a prominent gene in brain development. The deletion of Pax6 results in devastated development of eye, olfactory bulb, and cortex. However, it has been reported that the Pax6 -null Sey cerebellum only has minor defects involving granule cells despite Pax6 being expressed throughout cerebellar development. The present work has uncovered a requirement of Pax6 in the development of all rhombic lip (RL) lineages. A significant downregulation of Tbr1 and Tbr2 expression is found in the Sey cerebellum, these are cell-specific markers of cerebellar nuclear (CN) neurons and unipolar brush cells (UBCs), respectively. The examination of Tbr1 and Lmx1a immunolabeling and Nissl staining confirmed the loss of CN neurons from the Sey cerebellum. CN neuron progenitors are produced in the mutant but there is an enhanced death of these neurons as shown by increased presence of caspase-3-positive cells. These data indicate that Pax6 regulates the survival of CN neuron progenitors. Furthermore, the analysis of experimental mouse chimeras suggests a cell-extrinsic role of Pax6 in CN neuron survival. For UBCs, using Tbr2 immunolabeling, these cells are significantly reduced in the Sey cerebellum. The loss of UBCs in the mutant is due partly to cell death in the RL and also to the reduced production of progenitors from the RL. These results demonstrate a critical role for Pax6 in regulating the generation and survival of UBCs. This and previous work from our laboratory demonstrate a seminal role of Pax6 in the development of all cerebellar glutamatergic neurons. SIGNIFICANCE STATEMENT Pax6 is a key molecule in development. Pax6 is best known as the master control gene in eye development with mutations causing aniridia in humans. Pax6 also plays important developmental roles in the cortex and olfactory bulb. During cerebellar development, Pax6 is robustly expressed in the germinal zone of all glutamatergic neurons [cerebellar nuclear (CN) neurons, granule cells, and unipolar brush cells (UBCs)]. Past work has not found abnormalities in the CN and UBC populations. Our study reveals that the Pax6 -null mutation dramatically affects these cells and identifies Pax6 as a key regulator of cell survival in CN neurons and of cell production in UBCs. The present study shows how Pax6 is key to the development of glutamatergic cells in the cerebellum.

The Pax6 transcription factor is expressed in cerebellar granule cells and when mutated, as in the Sey/Sey mouse, produces granule cells with disturbed survival and migration and with defects in neurite extension. The impact of Pax6 on other genes in the context of cerebellar development has not been identified. In this study, we performed transcriptome comparisons between wildtype and Pax6-null whole cerebellar tissue at embryonic day (E) 13.5, 15.5 and 18.5 using Affymetrix arrays (U74Av2). Statistical analyses identified 136 differentially regulated transcripts (FDR 0.05, 1.2-fold change cutoff) over time in Pax6-null cerebellar tissue. In parallel we examined the Math1-null granuloprival cerebellum and identified 228 down-regulated transcripts (FDR 0.05, 1.2-fold change cutoff). The intersection of these two microarray datasets produced a total of 21 differentially regulated transcripts. For a subset of the identified transcripts, we used qRT-PCR to validate the microarray data and demonstrated the expression in the rhombic lip lineage and differential expression in Pax6-null cerebellum with in situ hybridisation analysis. The candidate genes identified in this way represent direct or indirect Pax6-downstream genes involved in cerebellar development.

This research deals with six renewalist evangelical Christian ministries that were subject to a three-year U.S. Senate inquiry for alleged failure to account for donations and spending. This research was conducted within the framework of social order theory. Using a content analysis of ministry Web sites, it illustrates how targeted ministries portrayed their relationships with believers and addressed financial accountability issues raised during the Senate inquiry. Findings show that individual ministries responded with positive compliance, hostile resistance, or strategic silence. Four of the six ministries' online responses violated professionally accepted guidelines for public relations crisis response. The ministries' responses to Senator Grassley's inquiry may suggest how the organizations might respond in future crisis situations.

Millions of listeners tune in to Internet-based audio-on-demand programming (commonly known as audio podcasts). College students represent a large and influential group of consumers but little is known about this primary audience's podcast listening choices and related opinions. This research was organized within the uses and gratifications perspective and involved an online survey of college students who identified as podcast listeners. Results showed most collegiate listeners spent no more than an hour a month listening to podcasts, and fewer than half reported listening to all the episodes they downloaded. Most listeners found podcasts fun and entertaining and said they enjoy sharing what they have heard with peers. Respondents reported clearly identified program genre preferences, and reported ‘multitasking’ in a variety of specific ways while listening. This research does not attempt to answer all questions about the college student podcast audience, but it is a starting point for further study into this important user group. Recommendations for future research are offered.

A student-run public relations firm can offer a variety of benefits for an undergraduate program in which there are limited resources for professional involvement by students. The article is a case study that profiles a student-run firm launched in conjunction with a capstone course in a journalism department. Specific learning and professional development goals were established for students, the department, and for clients served by the firm. The article presents a brief summary of successes, along with a discussion of opportunities and challenges. General recommendations are offered for establishing a student-run public relations firm.

ROR-alpha is an orphan nuclear receptor, inactivation of which cell-autonomously blocks differentiation of cerebellar Purkinje cells with a secondary loss of granule neurons. As part of our ENU mutagenesis screen we isolated the recessive tmgc26 mouse mutant, characterized by early-onset progressive ataxia, cerebellar degeneration and juvenile lethality. Detailed analysis of the tmgc26-/- cerebella revealed Purkinje cell and granule cell abnormalities, and defects in molecular layer interneurons and radial glia. Chimera studies suggested a cell-autonomous effect of the tmgc26 mutation in Purkinje cells and molecular layer interneurons, and a non-cell-autonomous effect in granule cells. The mutation was mapped to a 13-Mb interval on chromosome 9, a region that contains the ROR-alpha gene. Sequencing of genomic DNA revealed a T-to-A transition in exon 5 of the ROR-alpha gene, resulting in a nonsense mutation C257X and severe truncation of the ROR-alpha protein. Together, our data identify new roles for ROR-alpha in molecular layer interneurons and radial glia development and suggest tmgc26 as a novel ROR-alpha allele that may be used to further delineate the molecular mechanisms of ROR-alpha action.

In the United States alone, millions of listeners tune in to Internet-based audio-on-demand (commonly known as podcast programming). Religious programming is among the fastest-growing podcast areas. Since studies show that evangelicals are known to be quick to adopt new technology, this research involved a content analysis of 67 podcast episodes produced by five well-known renewalist evangelical ministries. The study found most podcasts were not original in content and were not consistently available for listeners to download. Also, the podcasts had poorer production values than would be expected from broadcast programming. A frame analysis identified ministries' justification of religious messages, listener responses sought, and rewards promised to listeners. More research is needed to answer questions about the impact on listeners and compare the content of renewalist program offerings to other Christian podcasts.

Renewalist Christian ministries espouse a “Health and Wealth” theology that is embraced by increasing numbers of believers. This study investigated leading renewalist ministries’ World Wide Web sites to assess their application of visual, operational, and informational enhancements to communicate social order. A content analysis revealed a communication of social order that is consistent with prosperity theology. Much emphasis is placed on the appearance and personality of the ministry leader. Almost no attention is paid to traditional Christian symbolism. Web sites claimed the value of inclusiveness, but offered little visual representation of children, senior adults, the poor, and people of color. Sites communicated the importance of regular financial donations and product purchases, but offered no encouragement for local church attendance. This research provides limited insight into some key unanswered scholarly questions about use of the Web by religious organizations, but more work is needed to investigate these issues in a broader context.

Kruppel-like factor 4 (Klf4) is a transcription factor that regulates many important cellular processes in stem cell biology, cancer, and development. We used histological and molecular methods to study the expression of Klf4 in embryonic development of the normal and Klf4 knockout cerebellum. We find that Klf4 is expressed strongly in early granule cell progenitor development but tails-off considerably by the end of embryonic development. Klf4 is also co-expressed with Pax6 in these cells. In the Klf4-null mouse, which is perinatal lethal, Klf4 positively regulates Pax6 expression and regulates the proliferation of neuronal progenitors in the rhombic lip, external granular layer and the neuroepithelium. This paper is the first to describe a role for Klf4 in the cerebellum and provides insight into this gene’s function in neuronal development.

Many institutions of higher education are struggling with the problem of excessive alcohol consumption by students. Colleges and universities want to be ‘good neighbors’ in their communities and must limit legal and social risks that result from excessive alcohol consumption by students. At the same time, colleges and universities operate in an increasingly challenging marketplace where many prospective students seek out institutions with a ‘party school’ reputation. Thus, higher education institutions are finding it difficult to define and carry out alcohol reduction measures that satisfy all constituents. This article discusses the approach taken at the University of Wisconsin—La Crosse. UW-L is an institution that identified a serious alcohol consumption problem and had to address the problem in a community where high levels of alcohol consumption are socially and culturally condoned. The article profiles the UW-L community and discusses the campaign model and strategy chosen. Quantitative and qualitative measures of success are discussed, along with lingering issues of resistance. The article ends with a summary of current issues and future directions being taken by the campaign.

This research examines the reporting of negative economic impact resulting from September 11, 2001 terrorism incidents. Student researchers who coordinated the project began with MSNBC’s World Wide Web-based “Layoff List” containing the names of corporate entities that claimed economic harm from the terrorist attacks. Students identified business problems and categorized resulting responses (taken or proposed) reported by MSNBC. Then, a content analysis was conducted of affected firms’ institutional Web sites for corroboration and/or explanation of the claimed economic injury. Although 99% of the business entities subjected to study had institutional Web sites, 84% of the firms made no mention of injury claim(s) on their institutional site. Among entities which did make online corroboration/explanation of media reports, fewer than half did so with detailed financial reports and most used rhetorical strategies that made business issues sound ambiguous while focusing blame for problems on variables outside the organization’s control.

This article is a preliminary effort to make conclusions about how apostate religious organizations use the World Wide Web for information dissemination, evangelization, and proselytization. Ten Christian apostate group Web sites were subjected to a frame analysis that focused on perceived intent, protest, expression, justification, response options, and rewards offered. The analysis revealed most sites were perceived to use an information dissemination rather than evangelization or proselytization frame. Response options and rewards were mostly consistent with the expression of, and justification for, apostasy. However, only 2 of the sites framed apostasy at its highest level. Most did not address the critical issue of worship service attendance, and most may put followers in a potentially awkward and ethically inconsistent position by not asking followers to disaffiliate from the opposed religious group while actively seeking followers' disengagement from that group's theology.

Math1 is the defining molecule of the cerebellar rhombic lip and Pax6 is downstream in the Math1 pathway. In the present study, we discover that Wntless (Wls) is a novel molecular marker of the cells in the interior face of the rhombic lip throughout normal mouse cerebellar development. Wls expression is found complementary to the expression of Math1 and Pax6, which are localized to the exterior face of the rhombic lip. To determine the interaction between these molecules, we examine the loss-of-Math1 or loss-of-Pax6 in the cerebellum, i.e., theMath1-null andPax6-null (Sey) mutant cerebella. The presence of Wls-positive cells in theMath1-null rhombic lip indicates that Wls expression is independent of Math1. In theSeymutant cerebellum, there is an expansion of Wls-expressing cells into regions that are normally colonized by Pax6-expressing cells. The ectopic expression of Wls in thePax6-null cerebellum suggests a negative interaction between Wls-expressing cells and Pax6-positive cells. These findings suggest that the rhombic lip is dynamically patterned by the expression of Wls, Math1, and Pax6. We also examine five rhombic lip cell markers (Wls, Math1, Pax6, Lmx1a, and Tbr2) to identify four molecularly distinct compartments in the rhombic lip during cerebellar development. The existence of spatial compartmentation in the rhombic lip and the interplay between Wls, Math1, and Pax6 in the rhombic lip provides novel views of early cerebellar development.

The mammalian CNS is one of the most complex biological systems to understand at the molecular level. The temporal information from time series transcriptome analysis can serve as a potent source of associative information between developmental processes and regulatory genes. Here, we introduce a new transcriptome database called, Cerebellar Gene Regulation in Time and Space (CbGRiTS). This dataset is populated with transcriptome data across embryonic and postnatal development from two standard mouse strains, C57BL/6J and DBA/2J, several recombinant inbred lines and cerebellar mutant strains. Users can evaluate expression profiles across cerebellar development in a deep time series with graphical interfaces for data exploration and link-out to anatomical expression databases. We present three analytical approaches that take advantage of specific aspects of the time series for transcriptome analysis. We demonstrate the use of CbGRiTS dataset as a community resource to explore patterns of gene expression and develop hypotheses concerning gene regulatory networks in brain development.

The differentiation and maintenance of a neurotransmitter phenotype is guided by the interaction of exogenous cues with intrinsic genetic machinery. For the noradrenergic phenotype, these influences combine to activate the expression of the catecholaminergic biosynthetic enzymes tyrosine hydroxylase and dopamine beta-hydroxylase (DBH). In this study, we evaluate the molecular mechanisms by which the transcription factor Arix/Phox2a contributes to DBH gene transcription. We have evaluated the contribution of individual homeodomain binding sites in the rat DBH promoter region and find that all are essential for both basal and cAMP-dependent protein kinase A (PKA)-stimulated transcription. Using mammalian one-hybrid and two-hybrid systems, we demonstrate that recruitment of Arix to the positions of homeodomain core recognition sites 1 and 2 at -153 to -166 of the DBH gene restores complete responsiveness of the promoter to PKA in SHSY-5Y neuroblastoma and HepG2 hepatoma cells. Intracellular Arix-Arix interactions are evident and may contribute to the interdependence of homeodomain binding sites. Analysis of functional domains of Arix reveals an N-terminal activation domain and a C-terminal repression domain. The N terminus of Arix contains an amino acid motif similar to a region in Brachyury and Pax9 transcription factors. The N-terminal activation domain of Arix interacts with the transcriptional co-activator, cAMP-response element-binding protein-binding protein, which potentiates transcription from the DBH promoter in a PKA-dependent manner. The present study supports the hypothesis that the paired-like homeodomain protein, Arix, acts as a critical phenotype-specific regulator of the DBH promoter by serving as an integrator of signal-dependent transcription activators within the network of the general transcription machinery.

Transcription of the neurotransmitter biosynthetic genes tyrosine hydroxylase and dopamine beta-hydroxylase (DBH) is regulated by cell type-specific transcription factors, including the homeoprotein Arix, and second messengers, including cyclic AMP. The cis-acting regulatory sites of the DBH gene which respond to Arix and cAMP lie adjacent to each other, between bases -180 and -150, in a regulatory element named DB1. Neither Arix nor cyclic AMP analogs alone effectively stimulate transcription from the DBH promoter in non-neuronal cell cultures. However, when Arix is present together with cAMP, transcription is substantially activated. Synergistic transcription from the DBH promoter can also be elicited by cotransfection of Arix with an expression vector encoding the catalytic subunit of protein kinase A. Nuclear extracts from PC12 cells display a cAMP-induced complex binding to the DB1 element, and antisera to transcription factors CREB, CREM, Fos, and Jun indicate that these proteins, or closely related family members, interact with DB1. A dominant negative construct of CREB inhibits the response of the DBH promoter to protein kinase A. These results demonstrate a synergistic interaction between a homeodomain protein and the cAMP signal transduction system and suggest that similar interactions may regulate the tissue-specific expression of neuroendocrine genes.

This study characterized the influence of full-term gestational ethanol exposure on choline acetyltransferase (ChAT)-immunoreactive neurons that project to the hippocampus, within the medial septal (MS) nucleus and the vertical limb of the diagonal band of Broca (DBv). On gestation days 1-22, pregnant dams were fed either a vitamin fortified ethanol-containing liquid diet, pair fed a calorically equivalent sucrose-containing diet, or given rat chow ad libitum. In a previous study, we found that chronic prenatal exposure to ethanol, in this manner, resulted in a significant decline in the ontogenetic upregulation of ChAT activity in the septal area during the second postnatal week, but was followed by recovery to control levels by adulthood. On postnatal days 14 and 60 (P14 and P60) the brains were prepared for ChAT immunocytochemistry. Ethanol exposure had little influence on the number of ChAT-positive neurons in the MS nucleus of animals at either age. Ethanol exposure had no effect on neuronal size or ChAT staining intensity of MS or DBv neurons when compared to chow-fed offspring. Although age-related increases in cholinergic neuronal numbers and decreases in neuronal size were observed between juvenile and adult animals, prenatal ethanol exposure did not appear to influence these postnatal changes in the population as a whole. Overall, these findings suggest that the anatomical maturation of septal cholinergic neurons may be relatively insensitive to prenatal ethanol exposure under conditions of a vitamin-rich dietary supplementation, while biochemical development within this region may be more susceptible to early ethanol influences.

This study investigated the influence of ethanol exposure throughout gestation on cholinergic development within the rat striatal region. Pregnant Long-Evans rats were maintained on three diets throughout gestation: A liquid diet in which ethanol accounted for 3539% of the total calories, a similar diet with the isocaloric substitution of sucrose for ethanol, and a lab chow control diet. At postnatal days 14 and 60 (P14 and P60), the striatal regions of the offspring were analyzed for the number of cholinergic neurons, via choline acetyltransferase (ChAT) immunostaining. The area of the striatum was also measured in these animals. At P14, P21, and P60, ChAT activity was assessed in the same region. These analyses revealed a significant increase in the number of cholinergic striatal neurons at P14 in the animals which had been exposed prenatally to ethanol. This increase was transient, however, with equal numbers of ChAT-positive cells found in all three groups by adulthood (P60). The brain weights of the ethanol-exposed animals were significantly reduced at P14 and P21, but were comparable to controls by P60. There were no significant differences in the striatal area or the overall volume of the region assessed, however, at either P14 or P60. Although there were some increases in ChAT activity across the ages viewed (most notably between P14 and P21), there were no effects of diet on ChAT activity at any age assessed. It is proposed that the increased numbers of cholinergic neurons could be a function of errors in migration, enhanced neurogenesis, diminished cell death, alterations in gene expression, or increased cell survival as a result of alterations in neurotrophic factor production or availability. 1996 ~l~ey-~lss, Inc.

In animal models of fetal alcohol syndrome (FAS), the hippocampus has been shown to be especially sensitive to the effects of prenatal ethanol exposure, exhibiting neuronal loss and alterations in neuritic process elaboration. We have characterized the influence of chronic prenatal ethanol treatment (CPET) on the postnatal expression of choline acetyltransferase (ChAT) In the hippocampus and the septal area that contains neurons that provide the primary cholinergic innervation to the hippocampus. On gestation days 1–22, pregnant rats were either fed an ethanol-containing liquid diet, pair-fed a calorically equivalent sucrose-containing diet, or given rat chow ad libitum. In Chow control animals, the ontogenetic progression of ChAT activity in the septal area and hippocampus was characterized by a significant period of upregulation during the 2nd and 3rd postnatal weeks, exhibiting an approximate 5-fold increase (septal area) and 7-fold increase (hippocampus) by postnatal day 21 (P21). At P14, ethanol exposure reduced septal and hippocampal ChAT activity levels, compared with those of pair-fed offspring. ChAT activity reached control levels by P21 in ethanol-exposed pups, suggesting that the earlier decline in activity may reflect a delay in the ontogenetic upregulation. In addition, there was a trend toward increased septal and hippocampal ChAT activities at P1 and P7 in both liquid diet groups. This liquid diet-stimulated increase may mask the effects of ethanol on early postnatal ChAT expression in the septohippocampal system. The results suggest that prenatal ethanol exposure may influence factors that regulate the developmental expression of ChAT in the septohippocampal system. In addition, we found a close relationship between the effects of CPET on litter birthweight and subsequent postnatal body and brain weight measures, but not between litter birthweight and ChAT activity from individual animals. This may imply that the influence of ethanol exposure on ChAT activity is independent of its effects on postnatal brain or body growth. Such an observation of neurochemical dysfunction in the absence of morphological indices frequently used to diagnose alcohol-related deficits could have potential clinical significance, both in the diagnostic and therapeutic realms.

The Pax6 transcription factor is expressed in cerebellar granule cells and when mutated, as in the Sey/Sey mouse, produces granule cells with disturbed survival, migration, and defects in neurite extension. The impact of Pax6 on other genes in the context of cerebellar development has not been identified. In this study, we performed transcriptome comparisons between wildtype and Pax6-null whole cerebellar tissue at embryonic day (E) 13.5, 15.5 and 18.5 using Affymetrix arrays (U74Av2). Statistical analyses identified 136 differentially regulated transcripts (FDR 0.05, 1.2 fold change cut-off) over time in Pax6-null cerebellar tissue. In parallel we examined the Math1-null granuloprival cerebellum and identified 228 differentially regulated transcripts (FDR 0.05, 1.2 fold change cut-off). The intersection of these two microarray datasets produced a total of 21 differentially regulated transcripts. For a subset of the identified transcripts, we used qRT-PCR to validate the microarray data and demonstrated the expression in the rhombic lip lineage and differential expression in Pax6-null cerebellum with in situ hybridization analysis. The candidate genes identified in this way represent direct or indirect Pax6-downstream genes involved in cerebellar development.

Dissociated septal and hippocampal neurons from E18 fetal rats were cultured with varying concentrations of ethanol (0.6–2.4 g/dl) and in cultures containing ethanol plus nerve growth factor (NGF) or basic fibroblast growth factor (bFGF). These substances have been shown to provide neurotrophic support for these populations and to afford neuroprotection against certain toxic substances or conditions applied to some neuronal populations. Both the septal and hippocampal neurons responded to ethanol in a dosedependent manner. Survival of septal neurons was generally unaffected by initial ethanol concentrations of 0.6 and 1.2 g/dl but was considerably impaired by higher concentrations (1.8 and 2.4 g/dl), while neurite outgrowth was compromised by all ethanol concentrations except the lowest one applied. The hippocampal neurons survived ethanol concentrations up to 2.4 g/dl, although process extension was decreased in concentrations of 1.2 g/dl and higher. NGF or bFGF in the culture medium (in cultures without ethanol) did not affect neuronal survival or process outgrowth in either population, probably owing to the relatively high plating densities of the cultures. NGF did tend to have a moderate ameliorative effect on the ethanol neurotoxicity in the septal cultures, however, and was slightly effective in this regard in hippocampal cultures at intermediate ethanol concentrations (1.8 g/dl). High concentrations of ethanol (2.4 g/dl) reduced the proportion of cholinergic cells in the septal preparations by approximately 50%. This neuronal loss could be reversed by inclusion of high concentrations of NGF in the culture medium (100 ng/ml) but not by a lower concentration (20 ng/ml). bFGF provided some protection against ethanol cytotoxicity with respect to both populations. The implications of these results for studies of fetal alcohol effects are discussed, as well as their relation to prior reports of trophic factor neuroprotection. © 1994 Wiley-Liss, Inc.

Cultures of septal and hippocampal neurons from fetal rat and dorsal root ganglion neurons from embryonic chick were pretreated with GM1 ganglioside or cycloheximide and then supplemented with toxic concentrations of ethanol. GM1 provided significant protection against ethanol neurotoxicity in each population. The inhibition of protein synthesis by cycloheximide, however, which protects against cell death resulting from withdrawal of neurotrophic factor support, did not ameliorate ethanol-induced neuronal loss.

The public relations workplace is changing rapidly. The introduction of new communication technologies, particularly those involving new forms of Internet-based media, is putting many new demands on the industry and those who work within it. In fact, some experts are advising public relations professionals to focus most of their attention on new and developing social media channels rather than on traditional communication media and channels (Zuk, 2009). Public relations professionals are being warned of the dangers of being “slow to catch on” when many of their clients will already be familiar with social marketing technology and request its utilization in communication efforts (Embrace online learning…, 2010, p. 11). Not all public relations professionals are prepared for new technological demands at work. As a result, some public relations people turn to younger, more technologically adept individuals for training to bring the experienced professionals “up to speed.” This kind of training relationship is among the most common variants of what has come to be known as reverse mentoring. Reverse mentoring is not a new concept; it has been applied and studied in the business community for years (Scandura & Viator, 1994). But the use of reverse mentoring has not gotten much attention in public relations, even though it is recognized and generally known to be used by public relations professionals (Guiniven, 2008). Reverse mentoring, defined as a structured or unstructured workplace relationship between senior practitioners and younger less experienced workers, continues to gain in popularity due to rapidly changing technology. Little is known about the extent to which public relations professionals recognize and use reverse mentoring, and the literature in the discipline offers no significant studies on this topic. For that reason, a study was undertaken as an initial effort to learn more about the ways reverse mentoring is used in the public relations workplace. To begin exploration into the prevalence and success of reverse mentoring in public relations, in-depth telephone interviews were conducted with 23 public relations professionals who work in the western United States. Those surveyed were all members of the Public Relations Society of America and the majority (65%) were senior-level professionals working for a for-profit entity (70%). Close to three-fourths of respondents (74%) reported that their organization did not practice reverse mentoring, and , of those that did, only one claimed the existence of a formal, structured program. Only one respondent identified the use of any training for mentors or mentees. Reverse mentoring that did exist was described as “voluntary mentorship” or an “internship program.” Technology was the clear favorite subject area for reverse mentoring, particularly social media. The practice was considered successful by those surveyed but no formal measures of success were utilized. Success in reverse mentoring is defined as having tangible success, cooperation and respect, and the sharing of knowledge and new ideas. Benefits of a reverse mentoring program included improvement in social media skills and improvement in social media vocabulary.

Enhancing Canadian capacity in the research and treatment for neurodevelopmental disorders is central to NeuroDevNet's mission. Building on the notion that it takes a network of scientists, clinicians, and educators to train the next generation researchers, NeuroDevNet brings together the diversity of expertise from across Canada to provide multifaceted, cross-disciplinary training opportunities for our trainees. Our Program provides for a diverse training experience that fosters the development of active young researchers with a collaborative focus and an eye toward the bidirectional translation of knowledge between the bench and the bedside. With funding from the NCE of Canada, as well as public and private partnerships, we offer fellowship and internship opportunities to trainees that encourage collaborative interactions, interdisciplinary exchanges and knowledge translation. This program will enhance the development and integration of NeuroDevNet as well as the Canadian community caring for the health and wellbeing of its citizens with neurodevelopmental disorders.

Dorsal root ganglion (DRG) neurons were cultured with varying concentrations of ethanol and NGF. At low concentrations of NGF (0.1 ng/ml) moderate initial ethanol levels (250 mg/dl) significantly suppressed neurite outgrowth. Higher NGF concentrations (5 ng/ml) protected against this neurotoxicity. At this higher NGF concentration, neuronal survival was not significantly affected by exposure to 0.25-4 g/dl ethanol, although survival was significantly diminished at 5 and 6 g/dl. Neurite outgrowth was a more sensitive indicator of ethanol neurotoxicity in this population, with significant decreases in process extension seen with 1 g/dl ethanol. When cultures were supplemented with 10 ng/ml NGF, however, process elaboration was significantly greater at 1 g/dl ethanol than that measured with 5 ng/ml NGF, and in fact did not differ from NGF controls. These studies indicate that NGF can provide neuroprotective effects against ethanol toxicity under these conditions. The results are discussed in relation to other recent reports of trophic factor neuroprotection.

Homogenate extracts were prepared from various regions of the embryonic chick brain from E6 to E19. The extracts were applied to cultured dorsal root ganglia (DRGs), sympathetic ganglion cells, PC12 cells and E5 spinal cord neurons. Potent neurotrophic activity with respect to DRGs was found in an extract from the forebrain (FBX) from the earliest stage assayed (E6). This activity was comparable to that of nerve growth factor (NGF), and it was not appreciably altered during later stages. By E8, extracts prepared from the optic lobe also induced responsiveness, although to a lesser extent than the FBX and NGF. At E12-19, extracts from the hippocampus (HCX) and cerebellum (CBX) were also tested. Activity found in the HCX resembled that in the FBX, but responsiveness to the CBX was similar to that in control (unsupplemented) cultures. The responsiveness of sympathetic neurons in the FBX was comparable to that with NGF. The FBX induced only moderate differentiation of PC12 cells, and spinal cord neurons were not responsive. Antibodies to NGF and basic fibroblast growth factor did not diminish the influence of the FBX, and the FBX and NGF when combined had synergistic effects. The results demonstrate potent neurotrophin-like activity in the embryonic brain at much earlier stages than had previously been seen, with differential regional and temporal distributions.

The Pleiades Promoter Project integrates genomewide bioinformatics with large-scale knockin mouse production and histological examination of expression patterns to develop MiniPromoters and related tools designed to study and treat the brain by directed gene expression. Genes with brain expression patterns of interest are subjected to bioinformatic analysis to delineate candidate regulatory regions, which are then incorporated into a panel of compact human MiniPromoters to drive expression to brain regions and cell types of interest. Using single-copy, homologous-recombination “knockins” in embryonic stem cells, each MiniPromoter reporter is integrated immediately 5′ of the Hprt locus in the mouse genome. MiniPromoter expression profiles are characterized in differentiation assays of the transgenic cells or in mouse brains following transgenic mouse production. Histological examination of adult brains, eyes, and spinal cords for reporter gene activity is coupled to costaining with cell-type–specific markers to define expression. The publicly available Pleiades MiniPromoter Project is a key resource to facilitate research on brain development and therapies.

Chick embryos were chronically exposed to either ethanol (approximately 30 mg/d) or saline, from E4-E13. Homogenate extract was prepared from forebrain tissue from E16 experimental and control embryos and was applied to cultured dorsal root ganglia (DRG). Neurotrophic activity in the forebrain extract (FBX) was significantly reduced in the ethanol-treated embryos compared to saline controls, both in terms of influences on neuronal survival and process elaboration. In addition, E8-9 DRGs from embryos exposed to ethanol from E4 were less viable in the presence of NGF than were those from controls. DRG survival in the presence of E16 FBX (from untreated embryos) was not different following ethanol treatment, but neurite production was significantly reduced. These results suggest that neurotrophic factor content and responsiveness may be appreciably altered following chronic prenatal ethanol exposure. Such alteration could underlie certain CNS anomalies seen in the fetal alcohol syndrome.

We have engineered a set of useful tools that facilitate targeted single copy knock-in (KI) at the hypoxanthine guanine phosphoribosyl transferase 1 (Hprt1) locus. We employed fine scale mapping to delineate the precise breakpoint location at the Hprt1b-m3 locus allowing allele specific PCR assays to be established. Our suite of tools contains four targeting expression vectors and a complementing series of embryonic stem cell lines. Two of these vectors encode enhanced green fluorescent protein (EGFP) driven by the human cytomegalovirus immediate-early enhancer/modified chicken beta-actin (CAG) promoter, whereas the other two permit flexible combinations of a chosen promoter combined with a reporter and/or gene of choice. We have validated our tools as part of the Pleiades Promoter Project (http://www.pleiades.org), with the generation of brain-specific EGFP positive germline mouse strains.

Background NOGO Receptor 1 (RTN4R) regulates axonal growth, as well as axon regeneration after injury. The gene maps to the 22q11.2 schizophrenia susceptibility locus and is thus a strong functional and positional candidate gene. Methodology/Principal Findings We evaluate evidence for genetic association between common RTN4R polymorphisms and schizophrenia in a large family sample of Afrikaner origin and screen the exonic sequence of RTN4R for rare variants in an independent sample from the U.S. We also employ animal model studies to assay a panel of schizophrenia-related behavioral tasks in an Rtn4r-deficient mouse model. We found weak sex-specific evidence for association between common RTN4R polymorphisms and schizophrenia in the Afrikaner patients. In the U.S. sample, we identified two novel non-conservative RTN4R coding variants in two patients with schizophrenia that were absent in 600 control chromosomes. In our complementary mouse model studies, we identified a haploinsufficient effect of Rtn4r on locomotor activity, but normal performance in schizophrenia-related behavioral tasks. We also provide evidence that Rtn4r deficiency can modulate the long-term behavioral effects of transient postnatal N-methyl-D-aspartate (NMDA) receptor hypofunction. Conclusions Our results do not support a major role of RTN4R in susceptibility to schizophrenia or the cognitive and behavioral deficits observed in individuals with 22q11 microdeletions. However, they suggest that RTN4R may modulate the genetic risk or clinical expression of schizophrenia in a subset of patients and identify additional studies that will be necessary to clarify the role of RTN4R in psychiatric phenotypes. In addition, our results raise interesting issues about evaluating the significance of rare genetic variants in disease and their role in causation.

The Tennessee Mouse Genome Consortium (TMGC) employed an N-ethyl-N-nitrosourea (ENU)-mutagenesis scheme to identify mouse recessive mutants with hearing phenotypes. We employed auditory brainstem responses (ABR) to click and 8, 16, and 32 kHz stimuli and screened 285 pedigrees (1819 mice of 8-11 weeks old in various mixed genetic backgrounds) each bred to carry a homozygous ENU-induced mutation. To define mutant pedigrees, we measured P12 mice per pedigree in P2 generations and used a criterion where the mean ABR threshold per pedigree was two standard deviations above the mean of all offspring from the same parental strain. We thus identified 17 mutant pedigrees (6%), all exhibiting hearing loss at high frequencies (P16 kHz) with an average threshold elevation of 30-35 dB SPL. Interestingly, four mutants showed sex-biased hearing loss and six mutants displayed wide range frequency hearing loss. Temporal bone histology revealed that six of the first nine mutants displayed cochlear morphological defects: degeneration of spiral ganglia, spiral ligament fibrocytes or inner hair cells (but not outer hair cells) mostly in basal turns. In contrast to other ENU-mutagenesis auditory screens, our screen identified high-frequency, mild and sex-biased hearing defects. Further characterization of these 17 mouse models will advance our understanding ofmore » presbycusis and noise-induced hearing loss in humans.« less

N-ethyl-N-nitrosourea (ENU) mutagenesis is presented as a powerful approach to developing models for human disease. The efforts of three NIH Mutagenesis Centers established for the detection of neuroscience-related phenotypes are described. Each center has developed an extensive panel of phenotype screens that assess nervous system structure and function. In particular, these screens focus on complex behavioral traits from drug and alcohol responses to circadian rhythms to epilepsy. Each of these centers has developed a bioinformatics infrastructure to track the extensive number of transactions that are inherent in these large-scale projects. Over 100 new mouse mutant lines have been defined through the efforts of these three mutagenesis centers and are presented to the research community via the centralized Web presence of the Neuromice.org consortium (http://www.neuromice.org). This community resource provides visitors with the ability to search for specific mutant phenotypes, to view the genetic and phenotypic details of mutant mouse lines, and to order these mice for use in their own research program.

Neurotransmitter biosynthesis is regulated by environmental stimuli, which transmit intracellular signals via second messengers and protein kinase pathways. For the catecholamine biosynthetic enzymes, dopamine beta-hydroxylase and tyrosine hydroxylase, regulation of gene expression by cyclic AMP, diacyl glycerol, and Ca2+ leads to increased neurotransmitter biosynthesis. In this report, we demonstrate that the cAMP-mediated regulation of transcription from the dopamine beta-hydroxylase promoter is mediated by the AP1 proteins c-Fos, c-Jun, and JunD. Following treatment of cultured cells with cAMP, protein complexes bound to the dopamine beta-hydroxylase AP1/cAMP response element element change from consisting of c-Jun and JunD to include c-Fos, c-Jun, and JunD. The homeodomain protein Arix is also a component of this DNA-protein complex, binding to the adjacent homeodomain recognition sites. Transfection of a dominant negative JunD expression plasmid inhibits cAMP-mediated expression of the dopamine beta-hydroxylase promoter construct in PC12 and CATH.a cells. In addition to the role of c-Fos in regulating dopamine beta-hydroxylase gene expression in response to cAMP, a second pathway, involving Rap1/B-Raf is involved. These experiments illustrate an unusual divergence of cAMP-dependent protein kinase signaling through multiple pathways that then reconverge on a single element in the dopamine beta-hydroxylase promoter to elicit activation of gene expression.

Extract made from hippocampus of rat pups exposed prenatally to an ethanol-supplemented diet was found to contain more neurotrophic activity at postnatal day 21 than that from animals exposed to control diets, when quantified in a dorsal root ganglion bioassay. This apparent upregulation was specific to hippocampal extract (cerebellar and forebrain/midbrain extracts were also assessed), and to this age (P1, P7, P14 and P60 extracts were also tested). It was suggested that this upregulation may be indicative of, or secondary to, trauma resulting from fetal ethanol exposure. It is speculated that such departures from the normal developmental timetable could contribute to anomalies seen in the fetal alcohol syndrome.

Pregnant Long-Evans rats were maintained on three diets: a liquid diet in which ethanol accounted for 35–39% of the total calories, a similar diet with the isocaloric substitution of sucrose for ethanol, and a lab chow control diet. At gestation day 18, the fetuses were taken and cultures of septal and hippocampal neurons prepared. Neuronal survival and neurite outgrowth were compared in cultures from the three diet groups, using the following media supplements: ethanol (1.2, 1.8 or 2.4 g/dl), neurotrophic factors (nerve growth factor [NGF] with the septal cultures, basic fibroblast growth factor [bFGF] with the hippocampal cultures), or ethanol plus neurotrophic factors. Both the septal and hippocampal neurons responded to ethanol in a dose-dependent manner. The neurons from both populations from fetuses which had been exposed prenatally to ethanol, however, tolerated considerably higher ethanol concentrations before decreases in survival or outgrowth were seen. These ethanol-exposed neuronal populations were also less responsive to neurotrophic factors: in hippocampal cultures, process outgrowth was significantly enhanced by bFGF in control but not ethanol-derived cultures, and in septal and hippocampal cultures, the neurotrophic factors significantly ameliorated ethanol neurotoxicity in control cultures, but not in those from the ethanol-exposed fetuses. The possible relevance of these observations to the fetal alcohol syndrome is discussed.

Chick embryos were given daily injections of ethanol (approximately 30 mg/day) either chronically from embryonic days 4 to 15 (E4-E15) or E18, or acutely from E15 to E18. Untreated and saline-injected embryos served as controls. Although morphological indicators of developmental age were not different among groups, chronic ethanol reduced weights of several brain regions. Similar to rodent models of prenatal ethanol exposure, chronic ethanol treatment reduced cerebellar Purkinje cell numbers compared to controls. Chronic but not acute ethanol exposure resulted in a significant reduction in choline acetyltransferase activity in the optic tectum (OT) and forebrain (FB) compared with controls. This study demonstrates that the chick embryo is a viable model to investigate the effects of ethanol exposure on CNS development. Unlike the mammalian fetus, the avian embryo is isolated from maternal interactions and may prove more useful in investigating the mechanisms by which ethanol directly influences brain development.

background and methods: The incidence and risk factors for erythromycin-induced ototoxicity are unknown. We conducted a prospective, nested case-control study of assessment of auditory function in patients receiving erythromycin versus other antibiotics (control group) for community-acquired pneumonia. Sequential audiograms were performed during antibiotic therapy for both cases and controls by an audiologist unaware of the identity of the therapy administered. Erythromycin serum concentrations were obtained for all patients receiving erythromycin. results: Symptomatic ototoxicity (tinnitus or hearing loss) confirmed by audiograms was documented in five of 30 patients receiving erythromycin and none of 15 receiving other antibiotics. Ototoxicity was significantly related to high peak concentration and high AUC 0-infinity as a function of decreased total systemic clearance. Ototoxicity occurred only in those patients who received 4 g/day versus 2 g/day or no erythromycin (p = 0.05). Ototoxicity resolved in all patients within 6 to 14 days after discontinuation of therapy. conclusions: Erythromycin ototoxicity is dose- and serum concentration-dependent. Patients receiving erythromycin, especially at a total daily dose of 4 g, should be monitored regularly for subjective evidence of sensorineural hearing dysfunction. Ototoxicity is reversible if the diagnosis is made early in the course.

Dissociates from the metencephalic basal plate of early chick embryo neural tubes containing the trigeminal (V) motor nucleus were cultured on substrates conditioned with appropriate target-derived muscle conditioned medium (MCM), laminin (LAM), MCM with nerve growth factor (NGF) in the medium, and fibronectin (FN). Comparisons were made of neuronal survival, the number of neurons with processes, and the length of processes elaborated. It was found that both MCM and LAM significantly enhanced survival and neuritic production from this population when compared to controls grown on a collagen-polyornithine substrate, but MCM surpassed LAM in these measures. When the neurons were grown on an MCM-conditioned substrate with an NGF-supplemented medium, no improvements were produced over the MCM or the NGF conditions alone. Therefore, the two do not appear to act in synergy, as NGF and LAM have been shown to do. FN produced no enhancement of any of the measures taken from this population. An ELISA analysis revealed no detectable LAM in the early target MCM. These results indicate that the specific responsiveness of this early neural tube population to its target MCM is not mediated by LAM, but the growth-enhancing component acts in a similar manner, although its influence is more potent.