Summary Comparative analysis of BACarray data can provide important insights into complex biological systems. As demonstrated in the accompanying paper, BACarray translational profiling permits comprehensive studies of translated mRNAs in genetically defined cell populations following physiological perturbations. To establish the generality of this approach, we present BACarray translational profiles for twenty four CNS cell populations, and identify known cell-specific and enriched transcripts for each population. We report thousands of cell-specific mRNAs that were not detected in whole tissue microarray studies, and provide examples that demonstrate the benefits deriving from comparative analysis. To provide a foundation for further biological and in silico studies, we provide a resource of sixteen transgenic mouse lines, their corresponding anatomic characterization, and BACarray translational profiles for cell types from a variety of CNS structures. This resource will enable a wide spectrum of molecular and mechanistic studies of both well known and previously uncharacterized neural cell populations.

In the above article, Figure 2A is stated to summarize data from Figures 1A and 1B; however, we inadvertently displayed a plot of a different data set that was collected with a similar but slightly different experimental design. The data in Figures 1A and 1B are from an experiment in which one group of flies underwent mock conditioning and an independent group was conditioned with electric shock, whereas the data in Figure 2A were from an experiment in which the same population of flies sequentially underwent mock conditioning and actual conditioning.We provide here a corrected graph for Figure 2A plotting the data from Figure 1. The new plot does not affect the description of the results in the paper or the conclusions drawn. We apologize for any inconvenience caused by this error.

We have recently developed a novel method for the affinity purification of the complete suite of translating mRNA from genetically labeled cell populations. This method permits comprehensive quantitative comparisons of the genes employed by each specific cell type. We provide a detailed description of tools for analysis of data generated with this and related methodologies. An essential question that arises from these data is how to identify those genes that are enriched in each cell type relative to all others. Genes relatively specifically employed by a cell type may contribute to the unique functions of that cell, and thus may become useful targets for development of pharmacological tools for cell-specific manipulations. We describe here a novel statistic, the specificity index, which can be used for comparative quantitative analysis to identify genes enriched in specific cell populations across a large number of profiles. This measure correctly predicts in situ hybridization patterns for many cell types. We apply this measure to a large survey of CNS cell-specific microarray data to identify those genes that are significantly enriched in each population Data and algorithms are available online (www.bactrap.org).

A vertebrate homologue of the Fox-1 protein from C. elegans was recently shown to bind to the element GCAUG and to act as an inhibitor of alternative splicing patterns in muscle. The element UGCAUG is a splicing enhancer element found downstream of numerous neuron-specific exons. We show here that mouse Fox-1 (mFox-1) and another homologue, Fox-2, are both specifically expressed in neurons in addition to muscle and heart. The mammalian Fox genes are very complex transcription units that generate transcripts from multiple promoters and with multiple internal exons whose inclusion is regulated. These genes produce a large family of proteins with variable N and C termini and internal deletions. We show that the overexpression of both Fox-1 and Fox-2 isoforms specifically activates splicing of neuronally regulated exons. This splicing activation requires UGCAUG enhancer elements. Conversely, RNA interference-mediated knockdown of Fox protein expression inhibits splicing of UGCAUG-dependent exons. These experiments show that this large family of proteins regulates splicing in the nervous system. They do this through a splicing enhancer function, in addition to their apparent negative effects on splicing in vertebrate muscle and in worms.Alternative splicing allows the production of multiple mRNAs from a single pre-mRNA via selection of different splice sites. Regulated exons are controlled by splicing enhancer and silencer elements within the exon or in the adjacent introns. These RNA sequences bind to specific regulatory proteins that contribute to the tissue specificity of splicing. Most exons are controlled by combinations of both positive and negative regulators, and how tissue specificity of splicing is achieved is poorly understood (5, 44).The N1 exon of the c-src gene serves as a model for an exon under both positive and negative control. In nonneuronal cells, the exon is repressed by the polypyrimidine tract binding protein (PTB) that binds to intronic splicing silencer elements flanking the N1 exon (1, 7, 9). In neurons, PTB-mediated repression is absent, and the exon is activated for splicing by an intronic splicing enhancer (4, 38). The enhancer region downstream of the N1 exon is complex, with binding sites for many proteins. However, the element most critical for enhancer activity is the sequence UGCAUG, which is flanked by PTB binding elements (4,37,38). Several proteins, including the hnRNPs F and H, the neuronal homologue of PTB, and the KH-type splicing regulatory protein, assemble onto this region in splicing extracts (8,30,34,35). Immunodepletion and antibody inhibition experiments have indicated a role for these proteins in the splicing of N1 in vitro. However, none of these proteins specifically recognizes the UGCAUG element, and they do not positively affect an exon controlled by just a UG CAUG element in vivo (J. G. Underwood and D. L. Black, unpublished observations). Thus, they do not seem to mediate the function of the strongest enhancer element. Their function may be related to preventing P...

We combined de novo mutation (DNM) data from 10,927 cases of developmental delay and autism to identify 253 candidate neurodevelopmental disease genes with an excess of missense and/or likely gene-disruptive mutations. Of these genes, 124 reach exome-wide significance (p < 5 × 10 −7 ) for DNM. Intersecting these results with copy number variation morbidity data shows an enrichment for genomic disorder regions (30/253, LR+ 1.85, p = 0.0017). We identify genes with an excess of missense DNMs overlapping deletion syndromes (e.g., KIF1A and the 2q37 deletion) as well as duplication syndromes, such as recurrent MAPK3 missense mutations within the chromosome 16p11.2 duplication, recurrent CHD4 missense DNMs in the 12p13 duplication region, and recurrent WDFY4 missense DNMs in the 10q11.23 duplication region. Network analyses of genes showing an excess of DNMs highlights functional networks, including cell-specific enrichments in the D1+ and D2+ spiny neurons of the striatum.

Recent advances have substantially increased the number of genes that are statistically associated with complex genetic disorders of the CNS such as autism and schizophrenia. It is now clear that there will likely be hundreds of distinct loci contributing to these disorders, underscoring a remarkable genetic heterogeneity. It is unclear whether this genetic heterogeneity indicates an equal heterogeneity of cellular mechanisms for these diseases. The commonality of symptoms across patients suggests there could be a functional convergence downstream of these loci upon a limited number of cell types or circuits that mediate the affected behaviors. One possible mechanism for this convergence would be the selective expression of at least a subset of these genes in the cell types that comprise these circuits. Using profiling data from mice and humans, we have developed and validated an approach, cell type-specific expression analysis, for identifying candidate cell populations likely to be disrupted across sets of patients with distinct genetic lesions. Using human genetics data and postmortem gene expression data, our approach can correctly identify the cell types for disorders of known cellular etiology, including narcolepsy and retinopathies. Applying this approach to autism, a disease where the cellular mechanism is unclear, indicates there may be multiple cellular routes to this disorder. Our approach may be useful for identifying common cellular mechanisms arising from distinct genetic lesions.

Previous studies have demonstrated that a small subpopulation of brain tumor cells share key characteristics with neural stem͞ progenitor cells in terms of phenotype and behavior. These findings suggest that brain tumors might contain ''cancer stem cells'' that are critical for tumor growth. However, the molecular pathways governing such stem cell-like behavior remain largely elusive. Our previous study suggests that the phosphatase and tensin homologue deleted on chromosome 10 (PTEN) tumor suppressor gene, one of the most frequently mutated genes in glioblastomas, restricts neural stem͞progenitor cell proliferation in vivo. In the present study, we sought to determine the role of PTEN in longterm maintenance of stem cell-like properties, cell cycle entry and progression, and growth factor dependence and gene expression. Our results demonstrate an enhanced self-renewal capacity and G0-G1 cell cycle entry and decreased growth factor dependency of Pten null neural͞stem progenitor cells. Therefore, loss of PTEN leads to cell physiological changes, which collectively are sufficient to increase the pool of self-renewing neural stem cells and promote their escape from the homeostatic mechanisms of proliferation control.tumor suppressor ͉ conditional knockout mouse model ͉ growth factor dependency ͉ brain tumor T he phosphatase and tensin homologue deleted on chromosome 10 (PTEN) tumor suppressor gene was the first phosphatase identified to be frequently mutated͞deleted somatically in various human cancers, including glioblastoma multiforme (1-3). In addition, germline mutations in the PTEN gene have been associated with Cowden syndrome and related diseases in which patients develop macrocephaly of the brain and hyperplastic lesions in multiple organs with increased risks of malignant transformation (4, 5).PTEN contains a sequence motif that is highly conserved in the members of the protein tyrosine phosphatase family. PTEN possesses phosphatase activity on phosphotyrosyl and phosphothreonyl-containing substrates (3, 6) in vitro and on phosphatidylinositol (3-5) trisphosphate, a product of phosphatidylinositol 3-kinase, both in vitro and in vivo (7-11). PTEN-deficiency leads to accumulation of PIP 3 and activation of signaling molecules that are critical in controlling cell size, cell migration, cell death, cell proliferation, and differentiation, all of which are involved in normal development and tumorigenesis (12).We and other groups have used Cre-loxP technology to conditionally delete Pten in different regions of the brain and at different developmental stages (13-18). By crossing Pten conditional knockout mice with the Nestin-Cre line, we generated mutant mice with a substantially increased brain size and a doubled brain weight (13), similar to macrocephalic phenotypes found in humans with inherited PTEN mutations. Further studies indicated that the enlarged brain resulted from increased cell proliferation, decreased cell death, and enlarged cell size. Because all neural cell types are thought to be derived from ...

Genetic mechanisms regulating CNS progenitor function and differentiation are not well understood. We have used microarrays derived from a representational difference analysis (RDA) subtraction in a heterogeneous stem cell culture system to systematically study the gene expression patterns of CNS progenitors. This analysis identified both known and novel genes enriched in progenitor cultures. In situ hybridization in a subset of clones demonstrated that many of these genes were expressed preferentially in germinal zones, some showing distinct ventricular or subventricular zone labeling. Several genes were also enriched in hematopoietic stem cells, suggesting an overlap of gene expression in neural and hematopoietic progenitors. This combination of methods demonstrates the power of using custom microarrays derived from RDA-subtracted libraries for both gene discovery and gene expression analysis in the central nervous system.