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ABSTRACT: Mesencephalic dopaminergic (mesDA) neurons originate from the floor plate of the midbrain, a transient embryonic organizing center located at the ventral-most midline. Since the loss of mesDA leads to Parkinson's disease, the molecular mechanisms controlling the genesis and differentiation of dopaminergic progenitors are extensively studied and the identification and characterization of new genes is of interest. Here, we show that the expression of the basic helix-loop-helix transcription factor Nato3 (Ferd3l) increases in parallel to the differentiation of SN4741 dopaminergic cells in vitro. Nato3 transcription is directly regulated by the transcription factor Foxa2, a target and effector of the Sonic hedgehog (Shh) signaling cascade. Moreover, pharmacological inhibition of Shh signaling downregulated the expression of Nato3, thus defining Nato3 as a novel component of one of the major pathways controlling cell patterning and generation of mesDA. Furthermore, we show that Nato3 regulated Shh and Foxa2 through a novel feed-backward loop. Up- and downregulation of Nato3 further affected the transcription of Nurr1, implicated in the genesis of mesDA, but not of TH. Taken together, these data shed new light on the transcriptional networks controlling the generation of mesDA and may be utilized in the efforts to direct stem cells towards a dopaminergic fate.
Journal of Molecular Neuroscience 12/2012; · 2.50 Impact Factor
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ABSTRACT: The development of the neural tube into a complex central nervous system involves morphological, cellular and molecular changes, all of which are tightly regulated. The floor plate (FP) is a critical organizing center located at the ventral-most midline of the neural tube. FP cells regulate dorsoventral patterning, differentiation and axon guidance by secreting morphogens. Here we show that the bHLH transcription factor Nato3 (Ferd3l) is specifically expressed in the spinal FP of chick and mouse embryos. Using in ovo electroporation to understand the regulation of the FP-specific expression of Nato3, we have identified an evolutionarily conserved 204 bp genomic region, which is necessary and sufficient to drive expression to the chick FP. This promoter contains two Foxa2-binding sites, which are highly conserved among distant phyla. The two sites can bind Foxa2 in vitro, and are necessary for the expression in the FP in vivo. Gain and loss of Foxa2 function in vivo further emphasize its role in Nato3 promoter activity. Thus, our data suggest that Nato3 is a direct target of Foxa2, a transcription activator and effector of Sonic hedgehog, the hallmark regulator of FP induction and spinal cord development. The identification of the FP-specific promoter is an important step towards a better understanding of the molecular mechanisms through which Nato3 transcription is regulated and for uncovering its function during nervous system development. Moreover, the promoter provides us with a powerful tool for conditional genetic manipulations in the FP.
Molecular and Cellular Neuroscience 01/2011; 46(1):187-99. · 3.66 Impact Factor
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Doron Lancet, Nissim Ben-Arie,
Shuki Cohen,
Uri Gat,
Ruth Gross-Isseroff,
Shirley Horn-Saban,
Miriam Khen,
Hans Lehrach,
Michael Natochin,
Michael North,
Eyal Seidemann,
Naomi Walker
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ABSTRACT: The emerging understanding of the molecular basis of olfactory mechanisms allows one to answer some long-standing questions regarding the complex recognition machinery involved. The ability of the olfactory system to detect chemicals at sub-nanomolar concentrations is explained by a plethora of amplification devices, including the coupling of receptors to second messenger generation through GTP-binding proteins. Specificity and selectivity may be understood in terms of a diverse repertoire of olfactory receptors of the seven-transmembrane-domain receptor superfamily, which are probably disposed on olfactory sensory neurons according to a clonal exclusion rule. Signal termination may be related to sets of biotransformation enzymes that process odorant molecules, as well as to receptor desensitization. Many of the underlying molecular components show specific expression in olfactory epithelium, with a well-orchestrated developmental sequence of emergence, possibly related to sensory neuronal function and connectivity requirements. A general model for molecular recognition in biological receptor repertoires allows a prediction of the number of olfactory receptors necessary to achieve efficient detection and sheds light on the analogy between the immune and olfactory systems. The molecular cloning and mapping of a human genomic olfactory receptor cluster on chromosome 17 provides insight into olfactory receptor diversity, polymorphism and evolution. Combined with future genotype-phenotype correlation, with particular reference to specific anosmia, as well as with computer-based molecular modelling, these studies may provide insight into the odorant specificity of olfactory receptors.
09/2007: pages 131 - 149; , ISBN: 9780470514511
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ABSTRACT: The basic helix-loop-helix (bHLH) transcription factor Math1 and its orthologs are fundamental for proper development of various neuronal subpopulations, such as cerebellar granule cells, D1 interneurons in the spinal cord, and inner ear hair cells. Although crucial for neurogenesis, the mechanisms by which Math1 specifically recognizes its direct targets are not fully understood. To search for direct and indirect target genes and signaling pathways controlled by Math1, we analyzed the effect of Math1 knockout on the expression profile of multiple genes in the embryonic cerebellum. Eighteen differentially expressed transcripts were identified and found to belong to a few developmentally-related functional groups, such as transcriptional regulation, proliferation, organogenesis, signal transduction, and apoptosis. Importantly, genomic analysis of E-box motifs has identified a significant enrichment and clustering of MATH1-binding E-boxes only in a subset of differentially expressed genes (Nr2f6, Hras1, and Hes5) in both mouse and man. Moreover, Math1 was shown by chromatin immunoprecipitation (ChIP) to bind, and by a luciferase reporter assay to activate transcription, of an upstream genomic fragment of Nr2f6. Taken together, we propose that when putative direct targets of Math1 are being selected for detailed studies on DNA microarray hybridization, the enrichment and clustering of binding E-boxes in multiple species may be helpful criteria. Our findings may be useful to the study of other bHLH transcription factors, many of which control the development of the nervous system.
Journal of Molecular Neuroscience 02/2006; 28(2):211-29. · 2.50 Impact Factor
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ABSTRACT: Cerebellar granule cells, the most abundant neurons in the mammalian brain, arise in the rhombic lip located at the roof of the brain's fourth ventricle. Bordering the rhombic lip is the choroid plexus, a non-neuronal structure, composed of blood vessels enveloped by epithelial cells. Here, we show a striking decrease in neural differentiation of rhombic lip-derived cells, which failed to extend neuritic processes and attenuate Math1 promoter activity, when co-cultured with choroid plexus cells. Moreover, a blocking antibody against BMP7, a morphogenetic protein expressed in the choroid plexus, blocked the inhibitory effect of the choroid plexus, whereas purified BMP7 mimicked this effect, demonstrating causal involvement of BMP. On the other hand, the BMP antagonist NBL1 promoted neurogenesis in rhombic lip cultures from Math1 null mice displaying arrested differentiation. Our data indicate that besides its secretory and barrier functions, the choroid plexus has a novel role in attenuating the differentiation of adjacent neural progenitors.
Mechanisms of Development 02/2006; 123(1):67-75. · 2.83 Impact Factor
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BioTechniques 12/2005; 39(5):650, 652, 654 passim. · 2.67 Impact Factor
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ABSTRACT: Long-chain neurotoxins derived from the venom of the Buthidae scorpions, which affect voltage-gated sodium channels (VGSCs) can be subdivided according to their toxicity to insects into insect-selective excitatory and depressant toxins (beta-toxins) and the alpha-like toxins which affect both mammals and insects. In the present study by the aid of reverse-phase HPLC column chromatography, RT-PCR, cloning and various toxicity assays, a new insect selective toxin designated as BjalphaIT was isolated from the venom of the Judean Black Scorpion (Buthotus judaicus), and its full primary sequence was determined: MNYLVVICFALLLMTVVESGRDAYIADNLNCAYTCGSNSYCNTECTKNGAVSGYCQWLGKYGNACWCINLPDKVPIRIPGACR (leader sequence is underlined). Despite its lack of toxicity to mammals and potent toxicity to insects, BjalphaIT reveals an amino acid sequence and an inferred spatial arrangement that is characteristic of the well-known scorpion alpha-toxins highly toxic to mammals. BjalphaITs sharp distinction between insects and mammals was also revealed by its effect on sodium conductance of two cloned neuronal VGSCs heterloguously expressed in Xenopus laevis oocytes and assayed with the two-electrode voltage-clamp technique. BjalphaIT completely inhibits the inactivation process of the insect para/tipE VGSC at a concentration of 100 nM, in contrast to the rat brain Na(v)1.2/beta1 which is resistant to the toxin. The above categorical distinction between mammal and insect VGSCs exhibited by BjalphaIT enables its employment in the clarification of the molecular basis of the animal group specificity of scorpion venom derived neurotoxic polypeptides and voltage-gated sodium channels.
Insect Biochemistry and Molecular Biology 04/2005; 35(3):187-95. · 3.25 Impact Factor
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ABSTRACT: Preparations of β-adrenergic receptor and Gs from turkey erythrocytes were delipidated by previously developed procedures. Three synthetic phospholipids, dioleoylglycerophosphoethanolamine, dioleoylglycerophosphocholine and dioleoylglycerophosphoserine plus an unphosphorylated lipid, were all required to restore receptor-mediated activation of Gs by GTP[γS]. The same lipids were necessary for the reconstitution of the isoproterenol-enhanced GTPase. The requirement for the unphosphorylated lipid could be fulfilled by 1-monooleoyl glycerol, α-tocopherol or oleic acid.
European Journal of Biochemistry. 03/2005; 176(3):649 - 654.
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ABSTRACT: Knockout mice are widely used in all fields of biomedical research. Determining the genotype of every newborn mouse is a tedious task, usually performed by Southern blot hybridization or Polymerase Chain Reaction (PCR). We describe here a quick and simple genotype identification assay based on real-time PCR and SYBR Green I dye, without using fluorescent primers. The discrimination between the wild type and targeted alleles is based on a PCR design that leads to a different melting temperature for each product. The identification of the genotype is obvious immediately after amplification, and no post-PCR manipulations are needed, reducing cost and time. Therefore, while the real-time PCR amplification increases the sensitivity, the fact that the reactions tubes are never opened after amplification, reduces the risk of contamination and eliminates errors, which are common during the repeated handling of dozens of samples from the same mouse line. The protocol we provide was tested on Math1 knockout mice, but is general, and may be utilized for any knockout line and real-time thermocycler, without any further modification, accessories or special reagents.
Journal of Neuroscience Methods 08/2004; 136(2):187-92. · 1.98 Impact Factor
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Daniela Canzoniere,
Stefano Farioli-Vecchioli,
Filippo Conti,
Maria Teresa Ciotti,
Ada Maria Tata,
Gabriella Augusti-Tocco,
Elisabetta Mattei,
Madepalli K Lakshmana,
Valery Krizhanovsky,
Steven A Reeves,
Roberto Giovannoni,
Francesca Castano,
Antonio Servadio, Nissim Ben-Arie,
Felice Tirone
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ABSTRACT: Growing evidence indicates that cell cycle arrest and neurogenesis are highly coordinated and interactive processes, governed by cell cycle genes and neural transcription factors. The gene PC3 (Tis21/BTG2) is expressed in the neuroblast throughout the neural tube and inhibits cell cycle progression at the G1 checkpoint by repressing cyclin D1 transcription. We generated inducible mouse models in which the expression of PC3 was upregulated in neuronal precursors of the neural tube and of the cerebellum. These mice exhibited a marked increase in the production of postmitotic neurons and impairment of cerebellar development. Cerebellar granule precursors of PC3 transgenic mice displayed inhibition of cyclin D1 expression and a strong increase in the expression of Math1, a transcription factor required for their differentiation. Furthermore, PC3, encoded by a recombinant adenovirus, also induced Math1 in postmitotic granule cells in vitro and stimulated the Math1 promoter activity. In contrast, PC3 expression was unaffected in the cerebellar primordium of Math1 null mice, suggesting that PC3 acts upstream to Math1. As a whole, our data suggest that cell cycle exit of cerebellar granule cell precursors and the onset of cerebellar neurogenesis are coordinated by PC3 through transcriptional control of cyclin D1 and Math1, respectively.
Journal of Neuroscience 04/2004; 24(13):3355-69. · 7.11 Impact Factor
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ABSTRACT: Cerebellar granule cells (CGC) are the most abundant neurons in the mammalian brain, and an important tool for unraveling molecular mechanisms underlying neurogenesis. Math1 is a bHLH transcription activator that is essential for the genesis of CGC. To delineate the effects of Math1 on CGC differentiation, we generated and studied primary cultures of CGC progenitors from Math1/lacZ knockout mice. Rhombic lip precursors appeared properly positioned, expressed CGC-specific markers, and maintained Math1 promoter activity in vivo and in vitro, suggesting that Math1 is not essential for the initial stages of specification or survival of CGC. Moreover, the continuous activity of Math1 promoter in the absence of MATH1, indicated that MATH1 was not necessary for the activation of its own expression. After 6, but not 3, days in culture, Math1 promoter activity was downregulated in control cultures, but not in cells from Math1 null mice, thus implying that Math1 participates in a negative regulatory feedback loop that is dependent on increased levels of MATH1 generated through the positive autoregulatory feedback loop. In addition, Math1 null CGC did not differentiate properly in culture, and were unable to extend processes. All Notch signaling pathway receptors and ligands tested were expressed in the rhombic lip at embryonic date 14, with highest levels of Notch2 and Jag1. However, Math1-null rhombic lip cells presented conspicuous downregulation of Notch4 and Dll1. Moreover, of the two transcriptional repressors known to antagonize Math1, Hes5 (but not Hes1) was downregulated in Math1-null rhombic lip tissue and primary cultures, and was shown to bind MATH1, thus revealing a negative regulatory feedback loop. Taken together, our data demonstrate that CGC differentiation, but not specification, depends on Math1, which acts by regulating the level of multiple components of the Notch signaling pathway.
Development 03/2004; 131(4):903-13. · 6.60 Impact Factor
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ABSTRACT: To explore neuronal mechanisms underlying long-term consequences of stress, we studied stress-induced changes in the neuritic translocation of acetylcholinesterase (AChE) splice variants. Under normal conditions, we found the synaptic AChE-S mRNA and protein in neurites. Corticosterone, anticholinesterases, and forced swim, each facilitated a rapid (minutes), yet long-lasting (weeks), shift from AChE-S to the normally rare AChE-R mRNA, promoted AChE-R mRNA translocation into neurites, and induced enzyme secretion. Weeks after stress, electrophysiological measurements in hippocampus slices displayed apparently normal evoked synaptic responses but extreme hypersensitivity to both anticholinesterases and atropine. Our findings suggest that neuronal hypersensitivity under stress involves neuritic replacement of AChE-S with AChE-R.
Science 02/2002; 295(5554):508-12. · 31.20 Impact Factor
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ABSTRACT: Long-chain neurotoxins derived from the venom of the Buthidae scorpions, which affect voltage-gated sodium channels (VGSCs) can be subdivided according to their toxicity to insects into insect-selective excitatory and depressant toxins (β-toxins) and the α-like toxins which affect both mammals and insects.In the present study by the aid of reverse-phase HPLC column chromatography, RT-PCR, cloning and various toxicity assays, a new insect selective toxin designated as BjαIT was isolated from the venom of the Judean Black Scorpion (Buthotus judaicus), and its full primary sequence was determined: MNYLVVICFALLLMTVVESGRDAYIADNLNCAYTCGSNSYCNTECTKNGAVSGYCQWLGKYGNACWCINLPDKVPIRIPGACR (leader sequence is underlined).Despite its lack of toxicity to mammals and potent toxicity to insects, BjαIT reveals an amino acid sequence and an inferred spatial arrangement that is characteristic of the well-known scorpion α-toxins highly toxic to mammals. BjαITs sharp distinction between insects and mammals was also revealed by its effect on sodium conductance of two cloned neuronal VGSCs heterloguously expressed in Xenopus laevis oocytes and assayed with the two-electrode voltage-clamp technique. BjαIT completely inhibits the inactivation process of the insect para/tipE VGSC at a concentration of 100 nM, in contrast to the rat brain Nav1.2/β1 which is resistant to the toxin.The above categorical distinction between mammal and insect VGSCs exhibited by BjαIT enables its employment in the clarification of the molecular basis of the animal group specificity of scorpion venom derived neurotoxic polypeptides and voltage-gated sodium channels.
Insect Biochemistry and Molecular Biology.
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