Identification of novel cerebellar developmental transcriptional regulators with motif activity analysis.

BACKGROUND: The 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. RESULTS: We 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. CONCLUSIONS: The 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.

An integrated expression atlas of miRNAs and their promoters in human and mouse.

MicroRNAs (miRNAs) are short non-coding RNAs with key roles in cellular regulation. As part of the fifth edition of the Functional Annotation of Mammalian Genome (FANTOM5) project, we created an integrated expression atlas of miRNAs and their promoters by deep-sequencing 492 short RNA (sRNA) libraries, with matching Cap Analysis Gene Expression (CAGE) data, from 396 human and 47 mouse RNA samples. Promoters were identified for 1,357 human and 804 mouse miRNAs and showed strong sequence conservation between species. We also found that primary and mature miRNA expression levels were correlated, allowing us to use the primary miRNA measurements as a proxy for mature miRNA levels in a total of 1,829 human and 1,029 mouse CAGE libraries. We thus provide a broad atlas of miRNA expression and promoters in primary mammalian cells, establishing a foundation for detailed analysis of miRNA expression patterns and transcriptional control regions.

A Novel and Multivalent Role of Pax6 in Cerebellar Development.

UNLABELLED: 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.

Wls provides a new compartmental view of the rhombic lip in mouse cerebellar development.

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., the Math1-null and Pax6-null (Sey) mutant cerebella. The presence of Wls-positive cells in the Math1-null rhombic lip indicates that Wls expression is independent of Math1. In the Sey mutant 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 the Pax6-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.

Genome-wide microarray comparison reveals downstream genes of Pax6 in the developing mouse 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.

Spatial and temporal requirements for huntingtin (Htt) in neuronal migration and survival during brain development.

Huntington's disease (HD), caused by an expanded triplet repeat in the huntingtin (Htt) gene, results in extensive neuropathology, but study of the Htt gene in CNS development through gene knockout is problematic as the knockout leads to embryonic lethality in mice. Here, we report that the knockdown of Htt expression in neuroepithelial cells of neocortex results in disturbed cell migration, reduced proliferation, and increased cell death that is relatively specific to early neural development. In the cerebellum, however, Htt knockdown results in cell death but not perturbed migration. The cell death phenotype in cortex can be partially reversed with co-knockdown of Casp9, indicating that mitochondria-mediated cell apoptotic processes are involved in the neuronal death. The timing of knockdown during early development is also an important variable. These results indicate a spatial and temporal requirement for Htt expression in neural development. Although it is uncertain whether the loss of wild-type huntingtin function contributes to pathogenesis in Huntington's disease, these results clearly contraindicate the use of nonspecific knockdown of Htt as a therapeutic measure in HD, particularly in utero.

Conserved and differential gene interactions in dynamical biological systems.

MOTIVATION: While biological systems operated from a common genome can be conserved in various ways, they can also manifest highly diverse dynamics and functions. This is because the same set of genes can interact differentially across specific molecular contexts. For example, differential gene interactions give rise to various stages of morphogenesis during cerebellar development. However, after over a decade of efforts toward reverse engineering biological networks from high-throughput omic data, gene networks of most organisms remain sketchy. This hindrance has motivated us to develop comparative modeling to highlight conserved and differential gene interactions across experimental conditions, without reconstructing complete gene networks first. RESULTS: We established a comparative dynamical system modeling (CDSM) approach to identify conserved and differential interactions across molecular contexts. In CDSM, interactions are represented by ordinary differential equations and compared across conditions through statistical heterogeneity and homogeneity tests. CDSM demonstrated a consistent superiority over differential correlation and reconstruct-then-compare in simulation studies. We exploited CDSM to elucidate gene interactions important for cellular processes poorly understood during mouse cerebellar development. We generated hypotheses on 66 differential genetic interactions involved in expansion of the external granule layer. These interactions are implicated in cell cycle, differentiation, apoptosis and morphogenesis. Additional 1639 differential interactions among gene clusters were also identified when we compared gene interactions during the presence of Rhombic lip versus the presence of distinct internal granule layer. Moreover, compared with differential correlation and reconstruct-then-compare, CDSM makes fewer assumptions on data and thus is applicable to a wider range of biological assays. AVAILABILITY: Source code in C++ and R is available for non-commercial organizations upon request from the corresponding author. The cerebellum gene expression dataset used in this article is available upon request from the Goldowitz lab (dang@cmmt.ubc.ca, http://grits.dglab.org/). CONTACT: joemsong@cs.nmsu.edu SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Molecular characterization of NMDA-like receptors in Aplysia and Lymnaea: relevance to memory mechanisms.

The N-methyl-D-aspartate (NMDA) receptor belongs to the group of ionotropic glutamate receptors and has been implicated in synaptic plasticity, memory acquisition, and learning in both vertebrates and invertebrates, including molluscs. However, the molecular identity of NMDA-type receptors in molluscs remains unknown. Here, we cloned two NMDA-type receptors from the sea slug Aplysia californica, AcNR1-1 and AcNR1-2, as well as their homologs from the freshwater pulmonate snail Lymnaea stagnalis, LsNR1-1 and LsNR1-2. The cloned receptors contain a signal peptide, two extracellular segments with predicted binding sites for glycine and glutamate, three recognized transmembrane regions, and a fourth hydrophobic domain that makes a hairpin turn to form a pore-like structure. Phylogenetic analysis suggests that both the AcNR1s and LsNR1s belong to the NR1 subgroup of ionotrophic glutamate receptors. Our in situ hybridization data indicate highly abundant, but predominantly neuron-specific expression of molluscan NR1-type receptors in all central ganglia, including identified motor neurons in the buccal and abdominal ganglia as well as groups of mechanosensory cells. AcNR1 transcripts were detected extrasynaptically in the neurites of metacerebral cells of Aplysia. The widespread distribution of AcNR1 and LsNR1 transcripts also implies diverse functions, including their involvement in the organization of feeding, locomotory, and defensive behaviors.

Mapping of nasopharyngeal carcinoma tumor-suppressive activity to a 1.8-megabase region of chromosome band 11q13.

Nasopharyngeal carcinoma (NPC) is a malignancy that is particularly prevalent among populations from Southern China and Southeast Asian countries. Evidence for a genetic contribution to the disease has been documented, although the genetic basis for NPC development is not yet fully understood. Previous functional evidence of tumor-suppressive activity on chromosome band 11q13 in NPC was obtained using a microcell-mediated chromosome-transfer approach with HONE1 NPC cells. In the present study, this region was subjected to a detailed investigation of microcell hybrids and their tumor segregants using microsatellite analysis to narrow down the region of tumor-suppressive activity. Fluorescence in situ hybridization was also performed with BAC and cosmid probes to confirm the microsatellite data. The critical region responsible for tumor suppression was narrowed down to a 1.8-Mb interval, which does not tolerate an additional normal allele by chromosome transfer. One or two alleles from either endogenous or exogenous chromosomes at 11q13 were consistently eliminated during tumor growth. Results of this study suggest that a candidate tumor-suppressor gene, not the MEN1 gene, maps between D11S4907 and GSTP1 in NPC.