A positive feedback loop between ATOH7 and a Notch effector regulates cell-cycle progression and neurogenesis in the retina.

The HES proteins are known Notch effectors and have long been recognized as important in inhibiting neuronal differentiation. However, the roles that they play in the specification of neuronal fate remain largely unknown. Here, we show that in the differentiating retinal epithelium, the proneural protein ATOH7 (ATH5) is required for the activation of the transcription of the Hes5.3 gene before the penultimate mitosis of progenitor cells. We further show that the HES5.3 protein slows down the cell-cycle progression of Atoh7-expressing cells, thereby establishing conditions for Atoh7 to reach a high level of expression in S phase and induce neuronal differentiation prior to the ultimate mitosis. Our study uncovers how a proneural protein recruits a protein known to be a component of the Notch signaling pathway in order to regulate the transition between an initial phase of selection among uncommitted progenitors and a later phase committing the selected progenitors to neuronal differentiation.

Conserved regulatory sequences in Atoh7 mediate non-conserved regulatory responses in retina ontogenesis.

The characterisation of interspecies differences in gene regulation is crucial to understanding the molecular basis of phenotypic diversity and evolution. The atonal homologue Atoh7 participates in the ontogenesis of the vertebrate retina. Our study reveals how evolutionarily conserved, non-coding DNA sequences mediate both the conserved and the species-specific transcriptional features of the Atoh7 gene. In the mouse and chick retina, species-related variations in the chromatin-binding profiles of bHLH transcription factors correlate with distinct features of the Atoh7 promoters and underlie variations in the transcriptional rates of the Atoh7 genes. The different expression kinetics of the Atoh7 genes generate differences in the expression patterns of a set of genes that are regulated by Atoh7 in a dose-dependent manner, including those involved in neurite outgrowth and growth cone migration. In summary, we show how highly conserved regulatory elements are put to use in mediating non-conserved functions and creating interspecies neuronal diversity.

Highly conserved sequences mediate the dynamic interplay of basic helix-loop-helix proteins regulating retinogenesis.

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.

The adhesion molecule Necl-3/SynCAM-2 localizes to myelinated axons, binds to oligodendrocytes and promotes cell adhesion.

BACKGROUND: Cell adhesion molecules are plasma membrane proteins specialized in cell-cell recognition and adhesion. Two related adhesion molecules, Necl-1 and Necl-2/SynCAM, were recently described and shown to fulfill important functions in the central nervous system. The purpose of the work was to investigate the distribution, and the properties of Necl-3/SynCAM-2, a previously uncharacterized member of the Necl family with which it shares a conserved modular organization and extensive sequence homology. RESULTS: We show that Necl-3/SynCAM-2 is a plasma membrane protein that accumulates in several tissues, including those of the central and peripheral nervous system. There, Necl-3/SynCAM-2 is expressed in ependymal cells and in myelinated axons, and sits at the interface between the axon shaft and the myelin sheath. Several independent assays demonstrate that Necl-3/SynCAM-2 functionally and selectively interacts with oligodendrocytes. We finally prove that Necl-3/SynCAM-2 is a bona fide adhesion molecule that engages in homo- and heterophilic interactions with the other Necl family members, leading to cell aggregation. CONCLUSION: Collectively, our manuscripts and the works on Necl-1 and SynCAM/Necl-2 reveal a complex set of interactions engaged in by the Necl proteins in the nervous system. Our work also support the notion that the family of Necl proteins fulfils key adhesion and recognition functions in the nervous system, in particular between different cell types.

The basic domain of ATH5 mediates neuron-specific promoter activity during retina development.

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.

A bHLH transcriptional network regulating the specification of retinal ganglion cells.

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.

Highly specific interactions between bHLH transcription factors and chromatin during retina development.

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.

Gene expression analysis of the critical period in the visual cortex.

The development of the primary visual cortex in animals possessing binocular vision is a classical paradigm for the study of activity-dependent neuronal plasticity. To elucidate the genetic determinants of this period of substantial plasticity, we conducted an unbiased and comprehensive transcript profiling analysis with differential display and DNA array techniques. We characterized the transcripts that change significantly between the critical and postcritical periods in the rat binocular visual cortex. We determined if these changes are specific for the visual cortex by simultaneously profiling the hippocampus and examined the impact of sensory experience on the accumulation of the identified transcripts. Our results uncover visual cortex-specific and unspecific transcription programs. Transcripts for protein kinases and phosphatases are particularly regulated. The identified transcripts support the notion that the critical period provides a permissive state for plasticity.

Amyloid beta(1-42) peptide alters the gating of human and mouse alpha-bungarotoxin-sensitive nicotinic receptors.

The beta-amyloid(1-42) peptide (Abeta(1-42)), a major constituent of the Alzheimer's disease amyloid plaque, specifically binds to the neuronal alpha-bungarotoxin (alpha-BuTx)-sensitive alpha7 nicotinic acetylcholine receptor (alpha7 nAChR). Accordingly, Abeta1-42 interferes with the function of alpha7 nAChRs in chick and rodent neurons. To gain insights into the human disease, we studied the action of Abeta(1-42) on human alpha7 nAChRs expressed in Xenopus oocytes. In voltage-clamped oocytes expressing the wild-type receptor, Abeta(1-42) blocked ACh-evoked currents. The block was non-competitive, required over 100 s to develop and was partially reversible. In oocytes expressing the mutant L248T receptor, Abeta(1-42) activated methyllycaconitine-sensitive currents in a dose-dependent manner. Peptide-evoked unitary events, recorded in outside-out patches, showed single-channel conductances and open duration comparable to ACh-evoked events. Abeta(1-42) had no effect on the currents evoked by glutamate, GABA or glycine in oocytes expressing human or mouse receptors for these transmitters. Muscle nAChRs are also alpha-BuTx-sensitive and we therefore investigated whether they respond to Abeta(1-42). In human kidney BOSC 23 cells expressing the fetal or adult mouse muscle nAChRs, Abeta(1-42) blocked ACh-evoked whole-cell currents, accelerating their decay. Outside-out single-channel recordings showed that the block was due to a reduced channel open probability and enhanced block upon ACh application. We also report that the inverse peptide Abeta(42-1), but not Abeta(40-1), partially mimicked the effects of the physiological Abeta(1-42) peptide. Possible implications for degenerative neuronal and muscular diseases are discussed.

Early steps in the production of sensory neurons by the neurogenic placodes.

Neurogenic placodes are specialized regions of the embryonic ectoderm that generate the majority of the neurons of the cranial sensory ganglia. Here we have accurately determined the onset of neurogenesis in each of the placodes in the chick, and we have also analyzed the expression profiles of genes that are believed to be involved in determining the types of sensory neurons produced by each placode. Interestingly, we find that there is a major difference in the expression domains of neurogenin-1 and neurogenin-2 in the chick, when compared with those reported for the mouse. We do find, however, that Brn-3a and Phox-2a and Phox-2b which are also associated with the specification of neuronal type are expressed in the same domains in the chick as they are in the mouse. These results suggest that neurogenin-1 and neurogenin-2 are functionally interchangeable in neurogenic placodes. We have also found major differences between the ophthalmic and maxillomandibular trigeminal placodes, and while all of the other placodes generate mitotically active cells the ophthalmic trigeminal placode seems to throw off postmitotic neuronal cells.