Abstract: Motility is a universal property of newly generated neurons. How cell migration is coordinately regulated with other aspects of neuron production is not well understood. Here we show that the proneural protein neurogenin 2 (Neurog2), which controls neurogenesis in the embryonic cerebral cortex, directly induces the expression of the small GTP-binding protein Rnd2 (ref. 3) in newly generated mouse cortical neurons before they initiate migration. Rnd2 silencing leads to a defect in radial migration of cortical neurons similar to that observed when the Neurog2 gene is deleted. Remarkably, restoring Rnd2 expression in Neurog2-mutant neurons is sufficient to rescue their ability to migrate. Our results identify Rnd2 as a novel essential regulator of neuronal migration in the cerebral cortex and demonstrate that Rnd2 is a major effector of Neurog2 function in the promotion of migration. Thus, a proneural protein controls the complex cellular behaviour of cell migration through a remarkably direct pathway involving the transcriptional activation of a small GTP-binding protein.
Abstract: So far, the computational identification of transcription factor binding sites is hampered by the complexity of vertebrate genomes. Here we present an in silico procedure to predict target sites of a transcription factor in complex genomes using its binding site. In a first step sequence, comparison of closely related genomes identifies the binding sites in conserved cis-regulatory regions (phylogenetic footprinting). Subsequently, more remote genomes are introduced into the comparison to identify highly conserved and therefore putatively functional binding sites (phylogenetic filtering). When applied to the binding site of atonal homolog 5 (Ath5 or ATOH7), this procedure efficiently filters evolutionarily conserved binding sites out of more than 300,000 instances in a vertebrate genome. We validate a selection of the linked target genes by showing coexpression with and transcriptional regulation by Ath5. Finally, chromatin immunoprecipitation demonstrates the occupancy of the target gene promoters by Ath5. Thus, our procedure, applied to whole genomes, is a fast and predictive tool to in silico filter the target genes of a given transcription factor with defined binding site.
Abstract: The atonal homolog 5 (ATH5) protein is central to the transcriptional network regulating the specification of retinal ganglion cells, and its expression comes under the spatiotemporal control of several basic helix-loop-helix (bHLH) proteins in the course of retina development. Monitoring the in vivo occupancy of the ATH5 promoter by the ATH5, Ngn2, and NeuroM proteins and analyzing the DNA motifs they bind, we show that three evolutionarily conserved E-boxes are required for the bHLH proteins to control the different phases of ATH5 expression. E-box 4 mediates the activity of Ngn2, ATH5, and NeuroM along the pathway leading to the conversion of progenitors into newborn neurons. E-box 1, by mediating the antagonistic effects of Ngn2 and HES1 in proliferating progenitors, controls the expansion of the ATH5 expression domain in early retina. E-box 2 is required for the positive feedback by ATH5 that underlies the up-regulation of ATH5 expression when progenitors are going through their last cell cycle. The combinatorial nature of the regulation of the ATH5 promoter suggests that the bHLH proteins involved have no assigned E-boxes but use a common set at which they either cooperate or compete to finely tune ATH5 expression as development proceeds.
Abstract: Proneural proteins play a central role in vertebrate neurogenesis, but little is known of the genes that they regulate and of the factors that interact with proneural proteins to activate a neurogenic program. Here, we demonstrate that the proneural protein Mash1 and the POU proteins Brn1 and Brn2 interact on the promoter of the Notch ligand Delta1 and synergistically activate Delta1 transcription, a key step in neurogenesis. Overexpression experiments in vivo indicate that Brn2, like Mash1, regulates additional aspects of neurogenesis, including the division of progenitors and the differentiation and migration of neurons. We identify by in silico screening a number of additional candidate target genes, which are recognized by Mash1 and Brn proteins through a DNA-binding motif similar to that found in the Delta1 gene and present a broad range of activities. We thus propose that Mash1 synergizes with Brn factors to regulate multiple steps of neurogenesis.
Abstract: In the developing retina, the production of ganglion cells is dependent on the proneural proteins NGN2 and ATH5, whose activities define stages along the pathway converting progenitors into newborn neurons. Crossregulatory interactions between NGN2, ATH5 and HES1 maintain the uncommitted status of ATH5-expressing cells during progenitor patterning, and later on regulate the transition from competence to cell fate commitment. Prior to exiting the cell cycle, a subset of progenitors is selected from the pool of ATH5-expressing cells to go through a crucial step in the acquisition of a definitive retinal ganglion cell fate. The selected cells are those in which the upregulation of NGN2, the downregulation of HES1 and the autostimulation of ATH5 are coordinated with the progression of progenitors through the last cell cycle. This coordinated pattern initiates the transcription of ganglion cell-specific traits and determines the size of the ganglion cell population.
Abstract: In the developing retina, the gene encoding the beta3 subunit of the neuronal nicotinic receptor, a specific marker of retinal ganglion cells, is under the direct control of the atonal homolog 5 (ATH5) basic helix-loop-helix (bHLH) transcription factor. Although quite short (143 bp in length), the beta3 promoter has the remarkable capacity to discriminate between ATH5 and the other neuronal bHLH proteins expressed in the developing nervous system. We have identified three amino acids within the basic domain that confer specificity to the ATH5 protein. These residues do not mediate direct DNA binding but are required for interaction between ATH5 and chromatin-associated proteins during retina development. When misexpressed in neurons, the myogenic bHLH factor MyoD is also able to activate the beta3 gene. This, however, is achieved not by binding of the protein to the promoter but by dimerization of MyoD with a partner, a process that depends not on the basic domain but on the HLH domain. By sequestering an E-box-binding protein, MyoD relieves the active repression that blocks the beta3 promoter in most neurons. The mechanisms used by bHLH proteins to activate beta3 thus highlight how ATH5 is selected by the beta3 promoter and coordinates the derepression and transcriptional activation of the beta3 gene during the specification of retinal ganglion cells.
Abstract: Basic helix-loop-helix (bHLH) transcription factors such as atonal homolog 5 (ATH5) and neurogenin 2 (NGN2) determine crucial events in retinogenesis. Using chromatin immunoprecipitation, we demonstrate that their interactions with target promoters undergo dynamic changes as development proceeds in the chick embryo. Chick ATH5 associates with its own promoter and with the promoter of the beta3 nicotinic receptor specifically in retinal ganglion cells and their precursors. NGN2 binds to the ATH5 promoter in retina but not in optic tectum, suggesting that interactions between bHLH factors and chromatin are highly tissue specific. The transcriptional activations of both promoters correlate with dimethylation of lysine 4 on histone H3. Inactivation of the ATH5 promoter in differentiated neurons is accompanied by replication-independent chromatin de-methylation. This report is one of the first demonstrations of correlation between gene expression, binding of transcription factors and chromatin modification in a developing neural tissue.
Abstract: Genetic studies in Drosophila and in vertebrates have implicated basic helix-loop-helix (bHLH) transcription factors in neural determination and differentiation. In this report, we analyze the role that several bHLH proteins play in the transcriptional control of differentiation in chick retina. Our experimental system exploits the properties of the promoter for the beta 3 subunit of the neuronal acetylcholine receptors, important components of various phenotypes in the CNS of vertebrates. The beta 3 subunit contributes to define ganglion cell identity in retina and its promoter, whose activation is an early marker of ganglion cell differentiation, is under the specific control of the chick atonal homolog ATH5. Functional analysis of the ATH5 promoter indicates that interactions between ATH5 and several other bHLH transcription factors underlie the patterning of the early retinal neuroepithelium and form a regulatory cascade leading to transcription of the gene for beta 3. ATH5 appears to coordinate the transcriptional pathways that control pan-neuronal properties with those that regulate the subtype-specific features of retinal neurons.
Abstract: The promoters driving transcription of the neuronal nicotinic genes alpha 7 and beta 3 have been characterized in the chicken. Although their regulatory modalities are thoroughly different, they nevertheless lead to co-expression in the same neurons.
Abstract: Within the chick central nervous system, expression of the beta3 nicotinic acetylcholine receptor gene is restricted to a subset of retinal neurons, the majority of which are ganglion cells. Transient transfection in retinal neurons and in neural and non-neural cells from other regions of the chick embryo allowed the identification of the cis-regulatory domain of the beta3 gene. Within this domain, a 75-base pair fragment located immediately upstream of the transcription start site suffices to reproduce the neuron-specific expression pattern of beta3. This fragment encompasses an E-box and a CAAT box, both of which are shown to be key positive regulatory elements of the beta3 promoter. Co-transfection experiments into retinal, telencephalic, and tectal neurons with plasmid reporters of beta3 promoter activity and a number of vectors expressing different neuronal (ASH-1, NeuroM, NeuroD, CTF-4) and non-neuronal (MyoD) basic helix-loop-helix transcription factors indicate that the cis-regulatory domain of beta3 has the remarkable property of discriminating accurately between related members of the basic helix-loop-helix protein family. The sequence located immediately 3' of the E-box participates in this selection, and the E-box acts in concert with the nearby CAAT box.
Abstract: We examine some of the biological and physiological properties of the avian alpha6 neuronal nicotinic acetylcholine receptor (nAChR) subunit. We show here that, beginning at embryonic day 5, alpha6 mRNA is abundantly expressed in the developing chick neuroretina, where it coexists with other nicotinic receptor subunit mRNAs such as alpha3, beta2 and beta4. In contrast, alpha6 mRNA is absent from the optic tectum and from the peripheral ganglia. Despite numerous efforts, the alpha6 subunit has long failed the critical test of functional reconstitution. Here we use patch-clamp techniques and confocal laser microscopy to measure ACh-activated currents and nicotine-elicited Ca2+ transients in human BOSC 23 cells transfected with chick alpha6 in combination with other chick nAChR neuronal subunits. Heterologously expressed alpha6 and beta4 subunits form functional heteromeric nAChRs, which are permeable to Ca2+ ions and blocked by the nicotinic antagonist methyllycaconitine (10 microM). Likewise, ACh elicits measurable currents in cells transfected with alpha6 and beta2. Hill analysis of the dose-response curves in cells transfected with alpha3, beta4 and alpha6 cDNAs, suggests the assembly of functional alpha3beta4alpha6 receptor, with an apparent affinity for ACh threefold lower than alpha3beta4. Our results indicate that alpha6-containing nAChRs assemble in heterologous expression systems and are probably present in retinal cells.
Abstract: Genes encoding transcription factors of the helix-loop-helix family are essential for the development of the nervous system in Drosophila and vertebrates. Screens of an embryonic chick neural cDNA library have yielded NeuroM, a novel neural-specific helix-loop-helix transcription factor related to the Drosophila proneural gene atonal. The NeuroM protein most closely resembles the vertebrate NeuroD and Nex1/MATH2 factors, and is capable of transactivating an E-box promoter in vivo. In situ hybridization studies have been conducted, in conjunction with pulse-labeling of S-phase nuclei, to compare NeuroM to NeuroD expression in the developing nervous system. In spinal cord and optic tectum, NeuroM expression precedes that of NeuroD. It is transient and restricted to cells lining the ventricular zone that have ceased proliferating but have not yet begun to migrate into the outer layers. In retina, NeuroM is also transiently expressed in cells as they withdraw from the mitotic cycle, but persists in horizontal and bipolar neurons until full differentiation, assuming an expression pattern exactly complementary to NeuroD. In the peripheral nervous system, NeuroM expression closely follows cell proliferation, suggesting that it intervenes at a similar developmental juncture in all parts of the nervous system. We propose that availability of the NeuroM helix-loop-helix factor defines a new stage in neurogenesis, at the transition between undifferentiated, premigratory and differentiating, migratory neural precursors.
Abstract: Genomic and cDNA clones encoding the chicken neuronal nicotinic acetylcholine receptor beta 3 subunit were isolated and sequenced. The beta 3 gene consists of six protein-encoding exons and the deduced protein has the structural features found in all other members of the neuronal nicotinic acetylcholine receptor subunit family. Although they are undetectable in most brain compartments, beta 3 mRNAs are relatively abundant in the developing retina and in the trigeminal ganglion. In situ hybridization and immunohistochemical analysis demonstrated that in retina, beta 3 transcripts and protein are confined to subpopulations of cells in the inner nuclear and ganglion cell layers. Beta 3 is expressed in the proximal and distal regions of the developing trigeminal ganglion, i.e. in both placode- and neural crest-derived neurons. Transient transfection assays in cells freshly dissociated from selected regions of the central nervous system at different developmental stages allowed the identification of genetic elements involved in the neuronal-selective expression of the beta 3 gene. A promoter fragment 143 base pairs in length and containing TATA, CAAT, and other consensus sequences is sufficient to restrict reporter gene expression to a subpopulation of retinal neurons. This promoter is totally inactive upon transfection into neuronal and non-neuronal cells from other regions of the central nervous system.
Abstract: We have previously shown that transcription of the beta 3 nicotinic receptor gene within the chick CNS is regulated by a promoter 143 base pairs (bp) in length. Here, we demonstrate that in the developing visual system this promoter is active in a subset of retinal cells, the majority of which are ganglion cells. Because the beta 3 promoter is activated very early during retina development, it can provide a marker of ganglion cell induction and differentiation. Transfection of neuroretina explants enabled us to detect activity of the beta 3 promoter in premigratory cells localized on the ventricular side of the retina. Double-labeling experiments showed that activation of the beta 3 promoter takes place before the last S-phase, suggesting that this particular phenotypic trait is determined when precursor cells are still proliferating. The beta 3 phenotype is induced in about one-tenth of the total pool of retinal progenitor cells and is stable upon changing the cellular environment. Our study suggests that at the very early stages of retina neurogenesis, some lineage restrictions have already occurred in the population of retinal progenitor cells.
Abstract: A transient transfection assay has been developed to analyse promoter activity in neuronal cells freshly dissociated from the chick central nervous system. The assay enabled us to identify cis-acting regulatory elements within the 5'-flanking region of the alpha 7 nicotinic acetylcholine receptor gene. In differentiated retina, regulatory elements direct reporter gene expression to a small subset of neurons which has been identified as ganglion cells, i.e. to the population of neurons in which alpha 7 transcripts were localized by in situ hybridization. However, these promoter elements exhibit ubiquitous activity in undifferentiated neural cells and in mesodermal stem cells. Our study supports the idea that alpha 7 regulatory elements acquire their neuronal specificity in the course of embryogenesis.
Abstract: Expression of the neuronal non-alpha nicotinic acetylcholine receptor (n alpha nAChR) gene is transiently stimulated in the chick optic tectum between embryonic days 7 and 16 with a peak value reached around embryonic day 12. This stimulation takes place at the time when optic nerve axons are invading this region of the brain and proceeds along a rostral to caudal gradient. Transcripts of the n alpha nAChR gene are localized in the superficial layers of the tectum at the time when cells in these layers are forming synapses with retina axons. The transient expression of n alpha nAChR gene does not take place in the optic tectum of 'eyeless' embryos. The results of our study suggest that the neuronal n alpha nAChR gene may play a role in neurogenesis of retino-tectal connections.
Abstract: cDNA and genomic clones encoding alpha 7, a novel neuronal nicotinic acetylcholine receptor (nAChR) alpha subunit, were isolated and sequenced. The mature alpha 7 protein (479 residues) has moderate homology with all other alpha and non-alpha nAChR subunits and probably assumes the same transmembrane topology. alpha 7 transcripts transiently accumulate in the developing optic tectum between E5 and E16. They are present in both the deep and the superficial layers of E12 tectum. In Xenopus oocytes, the alpha 7 protein assembles into a homo-oligomeric channel responding to acetylcholine and nicotine. The alpha 7 channel desensitizes very rapidly, rectifies strongly above -20 mV, and is blocked by alpha-bungarotoxin. A bacterial fusion protein encompassing residues 124-239 of alpha 7 binds labeled alpha-bungarotoxin. We conclude that alpha-bungarotoxin binding proteins in the vertebrate nervous system can function as nAChRs.
