Statins impact primary embryonic mouse neural stem cell survival, cell death, and fate through distinct mechanisms.

Statins inhibit HMG-CoA reductase, the rate-limiting enzyme in the cholesterol biosynthesis pathway (CBP), and are used for the prevention of cardiovascular disease. The anti-inflammatory effects of statins may also provide therapeutic benefits and have led to their use in clinical trials for preeclampsia, a pregnancy-associated inflammatory condition, despite their current classification as category X (i.e. contraindicated during pregnancy). In the developing neocortex, products of the CBP play essential roles in proliferation and differentiation of neural stem-progenitor cells (NSPCs). To understand how statins could impact the developing brain, we studied effects of pravastatin and simvastatin on primary embryonic NSPC survival, proliferation, global transcription, and cell fate in vitro. We found that statins dose dependently decrease NSPC expansion by promoting cell death and autophagy of NSPCs progressing through the G1 phase of the cell cycle. Genome-wide transcriptome analysis demonstrates an increase in expression of CBP genes following pravastatin treatment, through activation of the SREBP2 transcription factor. Co-treatment with farnesyl pyrophosphate (FPP), a CBP metabolite downstream of HMG-CoA reductase, reduces SREBP2 activation and pravastatin-induced PARP cleavage. Finally, pravastatin and simvastatin differentially alter NSPC cell fate and mRNA expression during differentiation, through a non-CBP dependent pathway.

Research Resource: The Dexamethasone Transcriptome in Hypothalamic Embryonic Neural Stem Cells.

Exposure to excess glucocorticoids during fetal development has long-lasting physiological and behavioral consequences, although the mechanisms are poorly understood. The impact of prenatal glucocorticoids exposure on stress responses in juvenile and adult offspring implicates the developing hypothalamus as a target of adverse prenatal glucocorticoid action. Therefore, primary cultures of hypothalamic neural-progenitor/stem cells (NPSCs) derived from mouse embryos (embryonic day 14.5) were used to identify the glucocorticoid transcriptome in both males and females. NPSCs were treated with vehicle or the synthetic glucocorticoid dexamethasone (dex; 100nM) for 4 hours and total RNA analyzed using RNA-Sequencing. Bioinformatic analysis demonstrated that primary hypothalamic NPSC cultures expressed relatively high levels of a number of genes regulating stem cell proliferation and hypothalamic progenitor function. Interesting, although these cells express glucocorticoid receptors (GRs), only low levels of sex-steroid receptors are expressed, which suggested that sex-specific differentially regulated genes identified are mediated by genetic and not hormonal influences. We also identified known or novel GR-target coding and noncoding genes that are either regulated equivalently in male and female NPSCs or differential responsiveness in one sex. Using gene ontology analysis, the top functional network identified was cell proliferation and using bromodeoxyuridine (BrdU) incorporation observed a reduction in proliferation of hypothalamic NPSCs after dexamethasone treatment. Our studies provide the first characterization and description of glucocorticoid-regulated pathways in male and female embryonically derived hypothalamic NPSCs and identified GR-target genes during hypothalamic development. These findings may provide insight into potential mechanisms responsible for the long-term consequences of fetal glucocorticoid exposure in adulthood.

Noncoding RNAs that associate with YB-1 alter proliferation in prostate cancer cells.

The highly conserved, multifunctional YB-1 is a powerful breast cancer prognostic indicator. We report on a pervasive role for YB-1 in which it associates with thousands of nonpolyadenylated short RNAs (shyRNAs) that are further processed into small RNAs (smyRNAs). Many of these RNAs have previously been identified as functional noncoding RNAs (http://www.johnlab.org/YB1). We identified a novel, abundant, 3'-modified short RNA antisense to Dicer1 (Shad1) that colocalizes with YB-1 to P-bodies and stress granules. The expression of Shad1 was shown to correlate with that of YB-1 and whose inhibition leads to an increase in cell proliferation. Additionally, Shad1 influences the expression of additional prognostic markers of cancer progression such as DLX2 and IGFBP2. We propose that the examination of these noncoding RNAs could lead to better understanding of prostate cancer progression.

Genome-wide transcript profiling reveals novel breast cancer-associated intronic sense RNAs.

Non-coding RNAs (ncRNAs) play major roles in development and cancer progression. To identify novel ncRNAs that may identify key pathways in breast cancer development, we performed high-throughput transcript profiling of tumor and normal matched-pair tissue samples. Initial transcriptome profiling using high-density genome-wide tiling arrays revealed changes in over 200 novel candidate genomic regions that map to intronic regions. Sixteen genomic loci were identified that map to the long introns of five key protein-coding genes, CRIM1, EPAS1, ZEB2, RBMS1, and RFX2. Consistent with the known role of the tumor suppressor ZEB2 in the cancer-associated epithelial to mesenchymal transition (EMT), in situ hybridization reveals that the intronic regions deriving from ZEB2 as well as those from RFX2 and EPAS1 are down-regulated in cells of epithelial morphology, suggesting that these regions may be important for maintaining normal epithelial cell morphology. Paired-end deep sequencing analysis reveals a large number of distinct genomic clusters with no coding potential within the introns of these genes. These novel transcripts are only transcribed from the coding strand. A comprehensive search for breast cancer associated genes reveals enrichment for transcribed intronic regions from these loci, pointing to an underappreciated role of introns or mechanisms relating to their biology in EMT and breast cancer.

Minireview: the impact of antenatal therapeutic synthetic glucocorticoids on the developing fetal brain.

The life-threatening, emotional, and economic burdens of premature birth have been greatly alleviated by antenatal glucocorticoid (GC) treatment. Antenatal GCs accelerate tissue development reducing respiratory distress syndrome and intraventricular hemorrhage in premature infants. However, they can also alter developmental processes in the brain and trigger adverse behavioral and metabolic outcomes later in life. This review summarizes animal model and clinical studies that examined the impact of antenatal GCs on the developing brain. In addition, we describe studies that assess glucocorticoid receptor (GR) action in neural stem/progenitor cells (NSPCs) in vivo and in vitro. We highlight recent work from our group on two GR pathways that impact NSPC proliferation, ie, a nongenomic GR pathway that regulates gap junction intercellular communication between coupled NSPCs through site-specific phosphorylation of connexin 43 and a genomic pathway driven by differential promoter recruitment of a specific GR phosphoisoform.

Caveolin-1 regulates genomic action of the glucocorticoid receptor in neural stem cells.

While glucocorticoids (GCs) are used clinically to treat many conditions, their neonatal and prenatal usage is increasingly controversial due to reports of delayed adverse outcomes, especially their effects on brain development. Such alterations may reflect the impact of GCs on neural progenitor/stem cell (NPSC) function. We previously demonstrated that the lipid raft protein caveolin-1 (Cav-1) was required for rapid GC signaling in embryonic mouse NPSCs operating through plasma membrane-bound glucocorticoid receptors (GRs). We show here that genomic GR signaling in NPSCs requires Cav-1. Loss of Cav-1 impacts the transcriptional response of many GR target genes (e.g., the serum- and glucocorticoid-regulated kinase 1 gene) that are likely to mediate the antiproliferative effects of GCs. Microarray analysis of wild-type C57 or Cav-1-deficient NPSCs identified approximately 100 genes that are differentially regulated by GC treatment. These changes in hormone responsiveness in Cav-1 knockout NPSCs are associated with the loss of GC-regulated phosphorylation of GR at serine 211 but not at serine 226. Chromatin recruitment of total GR to regulatory regions of target genes such as Fkbp-5, RhoJ, and Sgk-1, as well as p211-GR recruitment to Sgk-1, are compromised in Cav-1 knockout NPSCs. Cav-1 is therefore a multifunctional regulator of GR in NPSCs influencing both rapid and genomic action of the receptor to impact cell proliferation.

An in-depth map of polyadenylation sites in cancer.

We present a comprehensive map of over 1 million polyadenylation sites and quantify their usage in major cancers and tumor cell lines using direct RNA sequencing. We built the Expression and Polyadenylation Database to enable the visualization of the polyadenylation maps in various cancers and to facilitate the discovery of novel genes and gene isoforms that are potentially important to tumorigenesis. Analyses of polyadenylation sites indicate that a large fraction (∼30%) of mRNAs contain alternative polyadenylation sites in their 3' untranslated regions, independent of the cell type. The shortest 3' untranslated region isoforms are preferentially upregulated in cancer tissues, genome-wide. Candidate targets of alternative polyadenylation-mediated upregulation of short isoforms include POLR2K, and signaling cascades of cell-cell and cell-extracellular matrix contact, particularly involving regulators of Rho GTPases. Polyadenylation maps also helped to improve 3' untranslated region annotations and identify candidate regulatory marks such as sequence motifs, H3K36Me3 and Pabpc1 that are isoform dependent and occur in a position-specific manner. In summary, these results highlight the need to go beyond monitoring only the cumulative transcript levels for a gene, to separately analysing the expression of its RNA isoforms.

Sall1 regulates cortical neurogenesis and laminar fate specification in mice: implications for neural abnormalities in Townes-Brocks syndrome.

Progenitor cells in the cerebral cortex undergo dynamic cellular and molecular changes during development. Sall1 is a putative transcription factor that is highly expressed in progenitor cells during development. In humans, the autosomal dominant developmental disorder Townes-Brocks syndrome (TBS) is associated with mutations of the SALL1 gene. TBS is characterized by renal, anal, limb and auditory abnormalities. Although neural deficits have not been recognized as a diagnostic characteristic of the disease, ~10% of patients exhibit neural or behavioral abnormalities. We demonstrate that, in addition to being expressed in peripheral organs, Sall1 is robustly expressed in progenitor cells of the central nervous system in mice. Both classical- and conditional-knockout mouse studies indicate that the cerebral cortex is particularly sensitive to loss of Sall1. In the absence of Sall1, both the surface area and depth of the cerebral cortex were decreased at embryonic day 18.5 (E18.5). These deficiencies are associated with changes in progenitor cell properties during development. In early cortical progenitor cells, Sall1 promotes proliferative over neurogenic division, whereas, at later developmental stages, Sall1 regulates the production and differentiation of intermediate progenitor cells. Furthermore, Sall1 influences the temporal specification of cortical laminae. These findings present novel insights into the function of Sall1 in the developing mouse cortex and provide avenues for future research into potential neural deficits in individuals with TBS.

Comprehensive polyadenylation site maps in yeast and human reveal pervasive alternative polyadenylation.

The emerging discoveries on the link between polyadenylation and disease states underline the need to fully characterize genome-wide polyadenylation states. Here, we report comprehensive maps of global polyadenylation events in human and yeast generated using refinements to the Direct RNA Sequencing technology. This direct approach provides a quantitative view of genome-wide polyadenylation states in a strand-specific manner and requires only attomole RNA quantities. The polyadenylation profiles revealed an abundance of unannotated polyadenylation sites, alternative polyadenylation patterns, and regulatory element-associated poly(A)(+) RNAs. We observed differences in sequence composition surrounding canonical and noncanonical human polyadenylation sites, suggesting novel noncoding RNA-specific polyadenylation mechanisms in humans. Furthermore, we observed the correlation level between sense and antisense transcripts to depend on gene expression levels, supporting the view that overlapping transcription from opposite strands may play a regulatory role. Our data provide a comprehensive view of the polyadenylation state and overlapping transcription.

New class of gene-termini-associated human RNAs suggests a novel RNA copying mechanism.

Small (<200 nucleotide) RNA (sRNA) profiling of human cells using various technologies demonstrates unexpected complexity of sRNAs with hundreds of thousands of sRNA species present. Genetic and in vitro studies show that these RNAs are not merely degradation products of longer transcripts but could indeed have a function. Furthermore, profiling of RNAs, including the sRNAs, can reveal not only novel transcripts, but also make clear predictions about the existence and properties of novel biochemical pathways operating in a cell. For example, sRNA profiling in human cells indicated the existence of an unknown capping mechanism operating on cleaved RNA, a biochemical component of which was later identified. Here we show that human cells contain a novel type of sRNA that has non-genomically encoded 5' poly(U) tails. The presence of these RNAs at the termini of genes, specifically at the very 3' ends of known mRNAs, strongly argues for the presence of a yet uncharacterized endogenous biochemical pathway in cells that can copy RNA. We show that this pathway can operate on multiple genes, with specific enrichment towards transcript-encoding components of the translational machinery. Finally, we show that genes are also flanked by sense, 3' polyadenylated sRNAs that are likely to be capped.

A sensitive non-radioactive northern blot method to detect small RNAs.

The continuing discoveries of potentially active small RNAs at an unprecedented rate using high-throughput sequencing have raised the need for methods that can reliably detect and quantitate the expression levels of small RNAs. Currently, northern blot is the most widely used method for validating small RNAs that are identified by methods such as high-throughput sequencing. We describe a new northern blot-based protocol (LED) for small RNA (approximately 15-40 bases) detection using digoxigenin (DIG)-labeled oligonucleotide probes containing locked nucleic acids (LNA) and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide for cross-linking the RNA to the membrane. LED generates clearly visible signals for RNA amounts as low as 0.05 fmol. This method requires as little as a few seconds of membrane exposure to outperform the signal intensity using overnight exposure of isotope-based methods, corresponding to approximately 1000-fold improvement in exposure-time. In contrast to commonly used radioisotope-based methods, which require freshly prepared and hazardous probes, LED probes can be stored for at least 6 months, facilitate faster and more cost-effective experiments, and are more environmentally friendly. A detailed protocol of LED is provided in the Supplementary Data.

Sall3 is required for the terminal maturation of olfactory glomerular interneurons.

Sall3 is a zinc finger containing putative transcription factor and a member of the Sall gene family. Members of the Sall gene family are highly expressed during development. Sall3-deficient mice die in the perinatal period because of dehydration and display alterations in palate formation and cranial nerve formation (Parrish et al. [2004] Mol Cell Biol 24:7102-7112). We examined the role of Sall3 in the development of the olfactory system. We determined that Sall3 is expressed by cells in the olfactory epithelium and olfactory bulb. Sall3 deficiency specifically alters formation of the glomerular layer. The glomerular layer was hypocellular, because of a decrease in the number of interneurons. The lateral ganglionic eminence and rostral migratory stream developed normally in Sall3-deficient animals, which suggests that Sall3 is not required for the initial specification of olfactory bulb interneurons. Fewer GAD65/67-, Pax6-, calretinin-, and calbindin-positive cells were detected in the glomerular layer, accompanied by an increase in cells positive for these markers in the granule cell layer. In addition, a complete absence of tyrosine hydroxylase expression was observed in the olfactory bulb in the absence of Sall3. However, expression of Nurr1, a marker of dopaminergic precursors, was maintained, indicating that dopaminergic precursors were present. Our data suggest that Sall3 is required for the terminal maturation of neurons destined for the glomerular layer.

Sall1 regulates mitral cell development and olfactory nerve extension in the developing olfactory bulb.

Sall1 is a zinc finger containing transcription factor that is highly expressed during mammalian embryogenesis. In humans, the developmental disorder Townes Brocks Syndrome is associated with mutations in the SALL1 gene. Sall1-deficient animals die at birth due to kidney deficits; however, its function in the nervous system has not been characterized. We examined the role of Sall1 in the developing olfactory system. We demonstrate that Sall1 is expressed by cells in the olfactory epithelium and olfactory bulb (OB). Sall1-deficient OBs are reduced in size and exhibit alterations in neurogenesis and mitral cell production. In addition, the olfactory nerve failed to extend past the ventral-medial region of the OB in Sall1-deficient animals. We observed intrinsic patterns of neurogenesis during olfactory development in control animals. In Sall1-mutant animals, these patterns of neurogenesis were disrupted. These findings suggest a role for Sall1 in regulating neuronal differentiation and maturation in developing neural structures.

Abnormal development of zinc-containing cortical circuits in the absence of the transcription factor Tailless.

Absence of the transcription factor tailless (tlx) leads to premature laminar development and thinning of neocortex. We used zinc autometallography to determine if tailless deletion alters the organization of cortical circuits. In tlx-/- mice, layer 4 barrels, which normally lack synaptic zinc, are densely innervated by zinc-containing terminals. Furthermore, barrels with zinc inputs are constructed, in part, from zinc-sequestering neurons, a phenotype not normally found in layer 4.

The Tlx gene regulates the timing of neurogenesis in the cortex.

The tailless (tlx) gene is a forebrain-restricted transcription factor. Tlx mutant animals exhibit a reduction in the size of the cerebral hemispheres and associated structures (Monaghan et al., 1997). Superficial cortical layers are specifically reduced, whereas deep layers are relatively unaltered (Land and Monaghan, 2003). To determine whether the adult laminar phenotype has a developmental etiology and whether it is associated with a change in proliferation/differentiation decisions, we examined the cell cycle and neurogenesis in the embryonic cortex. We found that there is a temporal and regional requirement for the Tlx protein in progenitor cells (PCs). Neurons prematurely differentiate at all rostrocaudal levels up to mid-neurogenesis in mutant animals. Heterozygote animals have an intermediate phenotype indicating there is a threshold requirement for Tlx in early cortical neurogenesis. Our studies indicate that PCs in the ventricular zone are sensitive to loss of Tlx in caudal regions only; however, PCs in the subventricular zone are altered at all rostrocaudal levels in tlx-deficient animals. Furthermore, we found that the cell cycle is shorter from embryonic day 9.5 in tlx-/- embryos. At mid-neurogenesis, the PC population becomes depleted, and late PCs have a longer cell cycle in tlx-deficient animals. Consequently, later generated structures, such as upper cortical layers, the dentate gyrus, and the olfactory bulbs, are severely reduced. These studies indicate that tlx is an essential intrinsic regulator in the decision to proliferate or differentiate in the developing forebrain.

Activating transcription factor 1 and CREB are important for cell survival during early mouse development.

Activating transcription factor 1 (ATF1), CREB, and the cyclic AMP (cAMP) response element modulatory protein (CREM), which constitute a subfamily of the basic leucine zipper transcription factors, activate gene expression by binding as homo- or heterodimers to the cAMP response element in regulatory regions of target genes. To investigate the function of ATF1 in vivo, we inactivated the corresponding gene by homologous recombination. In contrast to CREB-deficient mice, which suffer from perinatal lethality, mice lacking ATF1 do not exhibit any discernible phenotypic abnormalities. Since ATF1 and CREB but not CREM are strongly coexpressed during early mouse development, we generated mice deficient for both CREB and ATF1. ATF1(-/-) CREB(-/-) embryos die before implantation due to developmental arrest. ATF1(+/-) CREB(-/-) embryos display a phenotype of embryonic lethality around embryonic day 9.5 due to massive apoptosis. These results indicate that CREB and ATF1 act in concert to mediate signals essential for maintaining cell viability during early embryonic development.

Loss of the tailless gene affects forebrain development and emotional behavior.

We are studying the role of the evolutionarily conserved tlx gene in forebrain development in mice. Tlx is expressed in the ventricular zone that gives rise to neurons and glia of the forebrain. We have shown by mutating the tlx gene in mice, that in the absence of this transcription factor, mutant animals survive, but suffer specific anatomical defects in the limbic system. Because of these developmentally induced structural changes, mice with a mutation in the tlx gene can function, but exhibit extreme behavioral pathology. Mice show heightened aggressiveness, excitability, and poor cognition. In this article, we present a summary of our findings on the cellular and behavioral changes in the forebrain of mutant animals. We show that absence of the tlx gene leads to abnormal proliferation and differentiation of progenitor cells (PCs) in the forebrain from embryonic day 9 (E9). These abnormalities lead to hypoplasia of superficial cortical layers and subsets of GABAergic interneurons in the neocortex. We examined the behavior of mutant animals in three tests for anxiety/fear: the open field, the elevated plus maze, and fear conditioning. Mutant animals are less anxious and less fearful when assessed in the elevated plus and open-field paradigm. In addition, mutant animals do not condition to either the tone or the context in the fear-conditioning paradigm. These animals, therefore, provide a genetic tool to delineate structure/function relationships in defined regions of the brain and decipher how their disruption leads to behavioral abnormalities.