Neural-specific Gene Expression Research Articles

Carbachol, along with calcium, indicates new strategy in neural differentiation of human adipose tissue-derived mesenchymal stem cells in vitro

Over the past few years, stem cells have represented a promising treatment in neurological disorders due to the well-defined characteristics of their capability to proliferate and differentiate into any cell type, both invitro and invivo. Additionally, previous studies have shown that calcium signaling modulates the proliferation and differentiation of neural progenitor cells. The present study investigated the effect of carbachol (CCh), a cholinergic agonist activating acetylcholine receptors, with and without calcium, on the neural differentiation of human adipose tissue-derived mesenchymal stem cells (hADSCs) in neural media, including forskolin and 3-isobutyl-1-methyl-xanthine and retinoic acid. For this purpose, first, the MTT assay and acridine orange staining were studied to obtain the optimal concentration of CCh. Next, the differentiation tests, such as cellular calcium assay as well as evaluation of qualitative and quantitative expression of neuronal index markers through immunofluorescence staining and gene expression analysis, respectively, were performed on days 7 and 14 of the differentiation period. According to the results, CCh at 1μM concentration had no cytotoxicity on hADSCs and also induced cell proliferation. Furthermore, CCh with and without calcium increased the expression of neural-specific genes (NSE, MAP2, β-III-tubulin, and MAPK3) and proteins (γ-enolase, MAP2, and β-III-tubulin) as well as the amount of calcium in differentiated hADSCs at 7 and 14 days after induction. In conclusion, the findings suggest that CCh acts as an influential therapeutic factor in the field of neural regenerative medicine and research.

The Transcription Factor FEZF1, a Direct Target of EWSR1-FLI1 in Ewing Sarcoma Cells, Regulates the Expression of Neural-Specific Genes.

Simple SummaryEwing sarcoma is a rare pediatric tumor characterized by chromosomal translocations that give rise to aberrant chimeric transcription factors (e.g., EWSR1-FLI1). EWSR1-FLI1 defines a specific transcriptomic profile in Ewing sarcoma cells, which determines the tumorigenesis process. Our study focused on the identification of transcription factors regulated by EWSR1-FLI1. FEZF1 (FEZ family zinc finger protein 1), a transcription factor involved in neural cell identity, was identified as one of the most strongly upregulated genes by EWSR1-FLI1. Functional studies were carried out to characterize the involvement of FEZF1 in Ewing sarcoma pathogenesis. As a result, the inhibition of FEZF1 diminished clonogenicity and cell proliferation in three Ewing sarcoma cell lines. Transcriptomic analysis revealed several neural-specific genes transcriptionally regulated by FEZF1 and concomitantly regulated by EWSR1-FLI1, which could explain the neural-like phenotype observed in several Ewing sarcoma cell lines and tumors.Ewing sarcoma is a rare pediatric tumor characterized by chromosomal translocations that give rise to aberrant chimeric transcription factors (e.g., EWSR1-FLI1). EWSR1-FLI1 promotes a specific cellular transcriptional program. Therefore, the study of EWSR1-FLI1 target genes is important to identify critical pathways involved in Ewing sarcoma tumorigenesis. In this work, we focused on the transcription factors regulated by EWSR1-FLI1 in Ewing sarcoma. Transcriptomic analysis of the Ewing sarcoma cell line A673 indicated that one of the genes more strongly upregulated by EWSR1-FLI1 was FEZF1 (FEZ family zinc finger protein 1), a transcriptional repressor involved in neural cell identity. The functional characterization of FEZF1 was performed in three Ewing sarcoma cell lines (A673, SK-N-MC, SK-ES-1) through an shRNA-directed silencing approach. FEZF1 knockdown inhibited clonogenicity and cell proliferation. Finally, the analysis of the FEZF1-dependent expression profile in A673 cells showed several neural genes regulated by FEZF1 and concomitantly regulated by EWSR1-FLI1. In summary, FEZF1 is transcriptionally regulated by EWSR1-FLI1 in Ewing sarcoma cells and is involved in the regulation of neural-specific genes, which could explain the neural-like phenotype observed in several Ewing sarcoma tumors and cell lines.

Goosecoid Controls Neuroectoderm Specification via Dual Circuits of Direct Repression and Indirect Stimulation in Xenopus Embryos.

Spemann organizer is a center of dorsal mesoderm and itself retains the mesoderm character, but it has a stimulatory role for neighboring ectoderm cells in becoming neuroectoderm in gastrula embryos. Goosecoid (Gsc) overexpression in ventral region promotes secondary axis formation including neural tissues, but the role of gsc in neural specification could be indirect. We examined the neural inhibitory and stimulatory roles of gsc in the same cell and neighboring cells contexts. In the animal cap explant system, Gsc overexpression inhibited expression of neural specific genes including foxd4l1.1, zic3, ncam, and neurod. Genome-wide chromatin immunoprecipitation sequencing (ChIP-seq) and promoter analysis of early neural genes of foxd4l1.1 and zic3 were performed to show that the neural inhibitory mode of gsc was direct. Site-directed mutagenesis and serially deleted construct studies of foxd4l1.1 promoter revealed that Gsc directly binds within the foxd4l1.1 promoter to repress its expression. Conjugation assay of animal cap explants was also performed to demonstrate an indirect neural stimulatory role for gsc. The genes for secretory molecules, Chordin and Noggin, were up-regulated in gsc injected cells with the neural fate only achieved in gsc uninjected neighboring cells. These experiments suggested that gsc regulates neuroectoderm formation negatively when expressed in the same cell and positively in neighboring cells via soluble factors. One is a direct suppressive circuit of neural genes in gsc expressing mesoderm cells and the other is an indirect stimulatory circuit for neurogenesis in neighboring ectoderm cells via secreted BMP antagonizers.

CHD7 promotes neural progenitor differentiation in embryonic stem cells via altered chromatin accessibility and nascent gene expression

CHARGE syndrome, a rare multiple congenital anomaly condition, is caused by haploinsufficiency of the chromatin remodeling protein gene CHD7 (Chromodomain helicase DNA binding protein 7). Brain abnormalities and intellectual disability are commonly observed in individuals with CHARGE, and neuronal differentiation is reduced in CHARGE patient-derived iPSCs and conditional knockout mouse brains. However, the mechanisms of CHD7 function in nervous system development are not well understood. In this study, we asked whether CHD7 promotes gene transcription in neural progenitor cells via changes in chromatin accessibility. We used Chd7 null embryonic stem cells (ESCs) derived from Chd7 mutant mouse blastocysts as a tool to investigate roles of CHD7 in neuronal and glial differentiation. Loss of Chd7 significantly reduced neuronal and glial differentiation. Sholl analysis showed that loss of Chd7 impaired neuronal complexity and neurite length in differentiated neurons. Genome-wide studies demonstrated that loss of Chd7 leads to modified chromatin accessibility (ATAC-seq) and differential nascent expression (Bru-Seq) of neural-specific genes. These results suggest that CHD7 acts preferentially to alter chromatin accessibility of key genes during the transition of NPCs to neurons to promote differentiation. Our results form a basis for understanding the cell stage-specific roles for CHD7-mediated chromatin remodeling during cell lineage acquisition.

YTHDF2 destabilizes m6A-modified neural-specific RNAs to restrain differentiation in induced pluripotent stem cells

N6-methyladenosine (m6A) is an abundant post-transcriptional modification that can impact RNA fate via interactions with m6A-specific RNA binding proteins. Despite accumulating evidence that m6A plays an important role in modulating pluripotency, the influence of m6A reader proteins in pluripotency is less clear. Here, we report that YTHDF2, an m6A reader associated with mRNA degradation, is highly expressed in induced pluripotent stem cells (iPSCs) and down-regulated during neural differentiation. Through RNA sequencing, we identified a group of m6A-modified transcripts associated with neural development that are directly regulated by YTDHF2. Depletion of YTHDF2 in iPSCs leads to stabilization of these transcripts, loss of pluripotency, and induction of neural-specific gene expression. Collectively, our results suggest YTHDF2 functions to restrain expression of neural-specific mRNAs in iPSCs and facilitate their rapid and coordinated up-regulation during neural induction. These effects are both achieved by destabilization of the targeted transcripts.

Cis-regulatory analysis of the Drosophila pdm locus reveals a diversity of neural enhancers.

BackgroundOne of the major challenges in developmental biology is to understand the regulatory events that generate neuronal diversity. During Drosophila embryonic neural lineage development, cellular temporal identity is established in part by a transcription factor (TF) regulatory network that mediates a cascade of cellular identity decisions. Two of the regulators essential to this network are the POU-domain TFs Nubbin and Pdm-2, encoded by adjacent genes collectively known as pdm. The focus of this study is the discovery and characterization of cis-regulatory DNA that governs their expression.ResultsPhylogenetic footprinting analysis of a 125 kb genomic region that spans the pdm locus identified 116 conserved sequence clusters. To determine which of these regions function as cis-regulatory enhancers that regulate the dynamics of pdm gene expression, we tested each for in vivo enhancer activity during embryonic development and postembryonic neurogenesis. Our screen revealed 77 unique enhancers positioned throughout the noncoding region of the pdm locus. Many of these activated neural-specific gene expression during different developmental stages and many drove expression in overlapping patterns. Sequence comparisons of functionally related enhancers that activate overlapping expression patterns revealed that they share conserved elements that can be predictive of enhancer behavior. To facilitate data accessibility, the results of our analysis are catalogued in cisPatterns, an online database of the structure and function of these and other Drosophila enhancers.ConclusionsThese studies reveal a diversity of modular enhancers that most likely regulate pdm gene expression during embryonic and adult development, highlighting a high level of temporal and spatial expression specificity. In addition, we discovered clusters of functionally related enhancers throughout the pdm locus. A subset of these enhancers share conserved elements including sequences that correspond to known TF DNA binding sites. Although comparative analysis of the nubbin and pdm-2 encoding sequences indicate that these two genes most likely arose from a duplication event, we found only partial evidence of sequence duplication between their enhancers, suggesting that after the putative duplication their cis-regulatory DNA diverged at a higher rate than their coding sequences.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1897-2) contains supplementary material, which is available to authorized users.

Modular and coordinated expression of immune system regulatory and signaling components in the developing and adult nervous system.

During development, the nervous system (NS) is assembled and sculpted through a concerted series of neurodevelopmental events orchestrated by a complex genetic programme. While neural-specific gene expression plays a critical part in this process, in recent years, a number of immune-related signaling and regulatory components have also been shown to play key physiological roles in the developing and adult NS. While the involvement of individual immune-related signaling components in neural functions may reflect their ubiquitous character, it may also reflect a much wider, as yet undescribed, genetic network of immune–related molecules acting as an intrinsic component of the neural-specific regulatory machinery that ultimately shapes the NS. In order to gain insights into the scale and wider functional organization of immune-related genetic networks in the NS, we examined the large scale pattern of expression of these genes in the brain. Our results show a highly significant correlated expression and transcriptional clustering among immune-related genes in the developing and adult brain, and this correlation was the highest in the brain when compared to muscle, liver, kidney and endothelial cells. We experimentally tested the regulatory clustering of immune system (IS) genes by using microarray expression profiling in cultures of dissociated neurons stimulated with the pro-inflammatory cytokine TNF-alpha, and found a highly significant enrichment of immune system-related genes among the resulting differentially expressed genes. Our findings strongly suggest a coherent recruitment of entire immune-related genetic regulatory modules by the neural-specific genetic programme that shapes the NS.

Neurotrophine-3 may contribute to neuronal differentiation of mesenchymal stem cells through the activation of the bone morphogenetic protein pathway.

We investigated whether neurotrophin-3 (NT-3) can promote differentiation of mouse bone mesenchymal stem cells (MSCs) into neurons via the bone morphogenetic protein pathway. MSCs were prepared from rat bone marrow and either transfected with pIRES2-EGFP or pIRES2-EGFP-NT-3 or treated with bone morphogenetic protein 4. The pIRES2-EGFP-NT-3-transfected MSCs further underwent noggin treatment or siRNA-mediated knockout of the TrkC gene or were left untreated. Immunofluorescence staining, real-time PCR and Western blot analyses were performed to evaluate the transcription and expression of neural-specific genes and BMP-Smad signaling. MSCs were efficiently transduced by the NT-3 gene via pIRES2-EGFP vectors. pIRES2- EGFP-NT-3 could initiate the transcription and expression of neural-specific genes, including nestin, NSE and MAP-2, and stimulate BMP-Smad signaling. The transcription and expression of neural-specific genes and BMP-Smad signaling were significantly suppressed by siRNA-mediated knockdown of the TrkC gene of MSCs. These findings suggest that the BMP signaling pathway may be a key regulatory point in NT-3-transfected neuronal differentiation of MSCs. The BMP and neurotrophin pathways contribute to a tightly regulated signaling network that directs the precise connections between neuronal differentiation of MSCs and their targets.

Neural differentiation of human umbilical cord mesenchymal stem cells by cerebrospinal fluid.

Wharton's jelly (WJ) is the gelatinous connective tissue from the umbilical cord. It is composed of mesenchymal stem cells, collagen fibers, and proteoglycans. The stem cells in WJ have properties that are interesting for research. For example, they are simple to harvest by noninvasive methods, provide large numbers of cells without risk to the donor, the stem cell population may be expanded in vitro, cryogenically stored, thawed, genetically manipulated, and differentiated in vitro. In our study, we investigated the effect of human cerebrospinal fluid (CSF) on neural differentiation of human WJ stem cells. The cells in passage 2 were induced into neural differentiation with different concentrations of human cerebrospinal fluid. Differentiation along with neural lineage was documented by expression of three neural markers: Nestin, Microtubule-Associated Protein 2 (MAP2), and Glial Fibrillary Astrocytic Protein (GFAP) for 21 days. The expression of the identified genes was confirmed by Reverse Transcriptase PCR (RT-PCR). Treatment with 100 and 200μg/ml CSF resulted in the expression of GFAP and glial cells marker on days 14 and 21. The expression of neural-specific genes following CSF treatment was dose-dependent and time-dependent. Treatment of the cells with a twofold concentration of CSF, led to the expression of MAP2 on day 14 of induction. No expression of GFAP was detected before day 14 or MAP2 before day 21, which shows the importance of the treatment period. In the present study, expression analysis for the known neural markers: Nestin, GFAP, and MAP2 using RT-PCR were performed. The data demonstrated that CSF could play a role as a strong inducer. RT-PCR showed that cerebrospinal fluid promotes the expression of Nestin, MAP2, and GFAP mRNA in a dose-dependent manner, especially at a concentration of 200 μl/ml. In summary, CSF induces neurogenesis of WJ stem cells that encourages tissue engineering applications with these cells for treatments of neurodegenerative defects and traumatic brain injury.

205 ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY ON NEURAL DIFFERENTIATION IN HUMAN EMBRYONIC STEM CELLS

Human embryonic stem cells (hESCs) have a potential for differentiation into neuronal cells. Neural differentiation of hESCs is currently used in many fields of neurological study. Therefore, evaluation of developmental neurotoxicity in hESCs is possible during the embryonic period. In the current study, we investigated the neuronal toxicity of hydroxyurea, cytosine arabinoside, and penicillin G at multiple doses (low, medium, high) for 28 days for neural differentiation. For assessment of neural toxicity, we examined the expression of marker genes that represent neural cell development. The mRNA levels of the marker genes were evaluated by real-time PCR in hESCs. Morphological changes of hESCs during neuronal differentiation were also estimated. The results for low and medium doses of hydroxylurea showed a significant increase of dopaminergic neuron marker gene (NR4A2) and GABA neuron marker gene (GAD2). However, glutamartergic neuron marker gene (SLC1A2) and oligodendrocyte marker gene (CNP) showed a significant decrease. Results for another drug, cytosine arabinoside, showed a significant decrease of glutamartergic neuron marker gene (SLC1A2) and oligodendrocyte marker gene (CNP) at a high dose. In addition, cytosine arabinoside caused a significant decrease of dopaminergic neuron marker gene (NR4A2) without significant change in GAD2. For the control, penicillin G, no significant difference in expression of neural specific-genes was observed at all tested doses. These findings suggest that neural-specific genes are perturbed by hydroxyurea and cytosine arabinoside, which may be involved in abnormal neural development during the embryonic neurogenesis period in hESCs.

TAp63γ demethylation regulates protein stability and cellular distribution during neural stem cell differentiation.

p63 is a close relative of the p53 tumor suppressor and transcription factor that modulates cell fate. The full-length isoform of p63, containing a transactivation (TA) domain (TAp63) is an essential proapoptotic protein in neural development. The role of p63 in epithelial development is also well established; however, its precise function during neural differentiation remains largely controversial. Recently, it has been demonstrated that several conserved elements of apoptosis are also integral components of cellular differentiation; p53 directly interacts with key regulators of neurogenesis. The aim of this study was to evaluate the role of p63 during mouse neural stem cell (NSC) differentiation and test whether the histone H3 lysine 27-specific demethylase JMJD3 interacts with p63 to redirect NSCs to neurogenesis. Our results showed that JMJD3 and TAp63γ are coordinately regulated to establish neural-specific gene expression programs in NSCs undergoing differentiation. JMJD3 overexpression increased TAp63γ levels in a demethylase activity-dependent manner. Importantly, overexpression of TAp63γ increased β-III tubulin whereas downregulation of TAp63γ by specific p63 siRNA decreased β-III tubulin. Immunoprecipitation assays demonstrated direct interaction between TAp63γ and JMJD3, and modulation of TAp63γ methylation status by JMJD3-demethylase activity. Importantly, the demethylase activity of JMJD3 influenced TAp63γ protein stabilization and cellular distribution, as well as TAp63γ-regulated neurogenesis. These findings clarify the role of p63 in adult neural progenitor cells and reveal TAp63γ as a direct target for JMJD3-mediated neuronal commitment.

Effect of neuronal induction on NSE, Tau, and Oct4 promoter methylation in bone marrow mesenchymal stem cells

Cell differentiation involves widespread epigenetic reprogramming, including modulation of DNA methylation patterns. The differentiation potential differences in DNA methylation patterns might function in pluripotency restriction, while tissue-specific differences might work in lineage restriction. To investigate the effects of neuronal induction on promoter methylation pattern in rat bone marrow mesenchymal stem cells (MSCs), we used bisulfite sequencing to analyze the methylation status of the promoter regions in neuron-specific enolase (NSE), microtubule-associated protein Tau, and Oct4 genes in MSCs pre- and post-chemical induction. Neurocytes from the newborn rat brains were used as control. Data showed that NSE and Tau were abundantly expressed in the brain cells and MSC-derived neurocyte-like cells as well but not in the MSCs. However, both NSE promoter (-214~+57 bp) and Tau promoter (-239~+131 bp) were hypomethylated (<4 % CpG methylation). Oct4 was expressed in MSCs, and the Oct4 promoter (-293~-85 bp) was hypermethylated (>79 % CpG methylation). Interestingly, it was found that the methylation of the locus -113 bp upstream of Oct4 transcription start site was specifically enhanced in the process of MSCs' neuronal differentiation. Further experiments in hepatocytes derived from MSCs and hepar tissue proved that the -113 bp locus methylation increased also in non-neurogenic lineages. Tfsitescan prediction showed that AP-2-alpha/gamma and Sp1 might regulate Oct4 transcription upon MSC differentiation by binding the -113 bp locus. So, we conclude that promoter methylation modifies pluripotency-specific gene, rather than regulates the expression of neural-specific genes when MSCs differentiate into neurocyte-like cells.

TAK1 promotes BMP4/Smad1 signaling via inhibition of erk MAPK: A new link in the FGF/BMP regulatory network

FGFs and BMPs act in concert to regulate a wide range of processes in vertebrate development. In most cases, FGFs and BMPs have opposing effects, and specific developmental outcomes arise out of a balance between the two growth factors. We and others have previously demonstrated that signaling pathways activated by FGFs and BMPs interact via inhibitory crosstalk. Here we demonstrate a role for the BMP effector TGF-β Activated Kinase 1 (TAK1) in the maintenance of Smad1 activity in Xenopus embryos, via the inhibition of erk MAPK. Up- or downregulation of TAK1 levels produces an inverse alteration in the amount of activated erk MAPK. The inhibition of erk MAPK by TAK1 is mediated by p38 and a corresponding decrease in phosphorylation of MEK. TAK1 morphant embryos show a decrease in the nuclear accumulation of Smad1. Conversely, reduction of erk MAPK activity via overexpression of MAP Kinase Phosphatase1 (MKP1) leads to an increase in nuclear Smad1. Both TAK1 morphant ectoderm and ectoderm treated with FGF show a decrease in the expression of several Smad1-inducible genes. Neural-specific gene expression is inhibited in isolated ectoderm coexpressing noggin and TAK1, suggesting that TAK1 is sufficient to inhibit neural specification. Introduction of TAK1 morpholino oligonucleotide expands the expression of organizer genes, disrupts formation of the boundary between organizer and non-organizer mesoderm, and increases the spatial range of MAPK activation in response to localized FGF. Our results indicate that inhibitory interactions between FGF and BMP4 effector pathways increase the robustness of BMP signaling via a feed-forward mechanism.

Neural differentiation of mesenchymal-like stem cells from cord blood is mediated by PKA

Mesenchymal stem cells (MSC) are multipotent and give rise to distinctly differentiated cells from all three germ layers. While umbilical cord blood derived mesenchymal-like cells were previously shown to be capable of differentiating into the neural lineage both in vitro and in vivo, the underlying molecular mechanisms and signal transduction pathways remain to be elucidated. In this study, we show that mesenchymal-like cells from umbilical cord blood are capable of neural differentiation and this capability is mediated by the Protein kinase A (PKA) signal transduction pathway. While activation of PKA via experimental cAMP upregulation leads to outgrowth of neurite-like structures as well as expression of neural marker genes, blocking PKA activity completely abolishes all these features. Thus, our results demonstrate that PKA function is sufficient and required for neurite-like outgrowth and regulation of neural specific gene expression in mesenchymal-like stem cells from cord blood.

Novel gene ashwin functions in Xenopus cell survival and anteroposterior patterning

The novel gene ashwin was isolated in a differential display screen for genes activated or up-regulated early in neural specification. ashwin is expressed maternally and zygotically, and it is up-regulated in the neural ectoderm after the midgastrula stage. It is expressed in the neural plate and later in the embryonic brain, eyes, and spinal cord. Overexpression of ashwin in whole embryos leads to anterior truncations and other defects. However, a second Organizer does not form, and the secondary axial structures may result from splitting of the Organizer, rather than axis duplication. Morpholino oligonucleotide-mediated reduction in ashwin expression leads to lethality or abnormalities in gastrulation, as well as significant apoptosis in midgastrula embryos. Apoptosis is also observed in midgastrula embryos overexpressing ashwin. Coexpression of ashwin with the bone morphogenetic protein-4 antagonist noggin has a synergistic effect on neural-specific gene expression in isolated animal cap ectoderm. Ashwin has no previously characterized domains, although two nuclear localization signals can be identified. Orthologues have been identified in the human, mouse, chicken, and pufferfish genomes. Our results suggest that ashwin regulates cell survival and anteroposterior patterning.

Identification of a BMP inhibitor-responsive promoter module required for expression of the early neural gene zic1

Expression of the transcription factor zic1 at the onset of gastrulation is one of the earliest molecular indicators of neural fate determination in Xenopus. Inhibition of bone morphogenetic protein (BMP) signaling is critical for activation of zic1 expression and fundamental for establishing neural identity in both vertebrates and invertebrates. The mechanism by which interruption of BMP signaling activates neural-specific gene expression is not understood. Here, we report identification of a 215 bp genomic module that is both necessary and sufficient to activate Xenopus zic1 transcription upon interruption of BMP signaling. Transgenic analyses demonstrate that this BMP inhibitory response module (BIRM) is required for expression in the whole embryo. Multiple consensus binding sites for specific transcription factor families within the BIRM are required for its activity and some of these regions are phylogenetically conserved between orthologous vertebrate zic1 genes. These data suggest that interruption of BMP signaling facilitates neural determination via a complex mechanism, involving multiple regulatory factors that cooperate to control zic1 expression.

Frimousse – a spontaneous ascidian mutant with anterior ectodermal fate transformation

Among vertebrates, the cellular and molecular mechanisms of neural induction appear largely divergent [1], thus providing little indication of the ancestral chordate mode. Ascidians, which belong to a sub-phylum of the chordates (Urochordata) long separated from the lineage leading to the vertebrates, may provide insight to these questions [2,3]. We have investigated neural specification in ascidians using a genetic approach. Ascidians are hermaphrodites, and the ability of ascidians of the genus Ciona to self-fertilize provides a rapid means for identifying recessive zygotic mutations [4,5]. We screened Ciona intestinalis for naturally occurring mutations, which can be detected in a large percentage of the wild population One such mutant is frimousse (frm). In the ascidian larva, the sensory vesicle, the putative homolog of the vertebrate foreand midbrain [6], is the most prominent feature of the anterior CNS, and contains two pigmented sensory organs, the ocellus and the otolith, which are sensitive to light and gravity, respectively (Figure 1A). The frimousse (frm) mutant was initially identified in larvae of a self-fertilized brood due to the absence of the pigmented sensory structures in ~25% of individuals (Figure 1B). The founder was subsequently crossed to wild-type individuals, and the mutant line has been maintained for over six generations without a change in phenotype. Conservationists have good reason to ring alarm bells about the decline in numbers of many species worldwide. Most are linked to the increasing intrusion from human activity, but one decline has proved a major puzzle: all around the world there appears to be a decline in numbers of amphibians and often in habitats with minimal human influence. A new study has looked at the falling numbers of the mountain yellow-legged frog (Rana muscosa) in the Sierra Nevada in California. Reporting in the Proceedings of the National Academy of Sciences (published online) a researcher has for once got some good news. Not only does he determine one major factor in the frog population's decline, he describes how to reverse it. The key factor was thought by Vance Vredenburg, at the University of California, Berkeley, to be the widespread introduction of both the rainbow trout and brook trout into lakes in these mountains where the frogs live. His study experimentally manipulated the presence and absence of these two fish species. From 1996 to 2003, the introduced trout were removed from five lakes in a remote protected area, and 16 nearby lakes were used as controls, eight with introduced trout and eight without. To determine the vulnerable life stages, rainbow trout were placed in cages in three lakes containing amphibians. The study found that removal of introduced trout resulted in rapid recovery of frog populations and in the caging experiment, tadpoles were found to be vulnerable to trout predation. The results suggest that the widespread introduction of these fish has had a major impact on frog populations in this region of California, but that removal of them can lead to a rapid and dramatic reversal in the amphibians' fortunes. Other observations in Costa Rica, Spain and field experiments in Australia implicate introduced trout in the decline of local frog populations, but this study has shown for this frog species what has actually been happening and what can be done. Trout out

The Whens and Wheres of Neural Expression

For normal organ and tissue development and function, certain genes must be expressed at the appropriate place and time. For example, neural genes must be expressed in neural tissue but shut down in nonneural tissues. Lunyak et al. examined mechanisms by which neural-specific gene expression can be restricted from nonneural tissues. The zinc-finger transcription factor REST/NRSF can mediate extraneural restriction through two different mechanisms, one of which uses active repression via a histone deacetylation complex and one that involves gene silencing via DNA methylation and the recruitment of the corepressor CoREST and silencing machinery. The latter mechanism can mediate gene silencing of specific chromosomal regions, including gene clusters encompassing neuron-specific genes, some of which do not themselves contain REST/NRSF response elements. V. V. Lunyak, R. Burgess, G. G. Prefontaine, C. Nelson, S.-H. Sze, J. Chenoweth, P. Schwartz, P. A. Pevzner, C. Glass, G. Mandel, M. G. Rosenfeld, Corepressor-dependent silencing of chromosomal regions encoding neuronal genes. Science 298 , 1747-1752 (2002). [Abstract] [Full Text]

The expression of neural-specific genes reveals the structural and molecular complexity of the planarian central nervous system

Planarians are attractive animals in which various questions related to the central nervous system (CNS) can be addressed, such as its origin and evolution, its degree of functional conservation among different organisms, and the plasticity and regenerative capabilities of neural cells and networks. However, it is first necessary to characterize at the gene expression level how this CNS is organized in intact animals. Previous studies have shown that the planarian brain can be divided into at least three distinct domains based on the expression of otd/Otx-related genes. In order to further characterize the planarian brain, we have recently isolated a large number of planarian neural-specific genes through DNA microarrays and ESTs projects. Here, we describe new molecular domains within the brain of intact planarians by the expression of 16 planarian neural-specific genes, including the putative homologues of protein tyrosine phosphatase receptor, synaptotagmin VII, slit, G protein and glutamate and acetylcholine receptors, by in situ hybridization in both whole-mount and transverse sections. Our results indicate that planarian otd/Otx-positive domains can be further subdivided into distinct molecular regions according to the expression of different neural genes. We found differences at the gene expression level between the dorsal and ventral sides of the brain, along its antero-posterior axis and also between the proximal and distal parts of the brain lateral branches. This high level of regionalization in the planarian brain contrasts with its apparent simplicity at the morphological level.

Identification and localization of M-CoREST (1A13), a mouse homologue of the human transcriptional co-repressor CoREST, in the developing mouse CNS.

By means of subtractive and differential hybridization techniques we have identified a novel murine gene (1A13) the expression of which is developmentally regulated in the mouse brain. Comparison of the nucleotide and predicted protein sequence revealed closest relationship of 1A13 to human CoREST, a transcriptional co-repressor required for regulation of neural-specific gene expression. Thus, we will refer to 1A13 as M-CoREST. As shown by in situ hybridization and Northern blotting, expression of M-CoREST mRNA is abundant in neural tissue at early embryonic stages but declines significantly towards birth, coincident with the progression of CNS maturation.