Lidia Matter-Sadzinski

Neuronal specificity of the alpha 7 nicotinic acetylcholine receptor promoter develops during morphogenesis of the central nervous system.

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.

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.

Expression of neuronal nicotinic acetylcholine receptor genes in the developing chick visual system.

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.

Characterization of the Nicotinic Acetylcholine Receptor 3 Gene ITS REGULATION WITHIN THE AVIAN NERVOUS SYSTEM IS EFFECTED BY A PROMOTER 143 BASE PAIRS IN LENGTH

Genomic and cDNA clones encoding the chicken neuronal nicotinic acetylcholine receptor β3 subunit were isolated and sequenced. The β3 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, β3 mRNAs are relatively abundant in the developing retina and in the trigeminal ganglion. In situ hybridization and immunohistochemical analysis demonstrated that in retina, β3 transcripts and protein are confined to subpopulations of cells in the inner nuclear and ganglion cell layers. β3 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 β3 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.

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.

The Basic Domain of ATH5 Mediates Neuron-Specific Promoter Activity during Retina Development

ABSTRACT In the developing retina, the gene encoding the β3 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 β3 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 β3 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 β3 promoter in most neurons. The mechanisms used by bHLH proteins to activate β3 thus highlight how ATH5 is selected by the β3 promoter and coordinates the derepression and transcriptional activation of the β3 gene during the specification of retinal ganglion cells.

Functional Properties of the Neuronal Nicotinic Acetylcholine Receptor β3 Promoter in the Developing Central Nervous System

Within the chick central nervous system, expression of the β3 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 β3 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 β3. This fragment encompasses an E-box and a CAAT box, both of which are shown to be key positive regulatory elements of the β3 promoter. Co-transfection experiments into retinal, telencephalic, and tectal neurons with plasmid reporters of β3 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 β3 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.

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.

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.

Nicotinic acetylcholine receptor gene expression in developing chick autonomic ganglia

The developmental expression patterns of ten genes encoding nicotinic acetylcholine receptor subunits were analyzed using Northern blots and in situ hybridization in chick peripheral ganglia of neural crest, placodal and dual embryonic origin. The superior cervical and ciliary ganglia were investigated in detail because they accumulated relatively abundant transcripts of the alpha3, beta4, alpha5 and alpha7 genes. In the superior cervical ganglion, these four mRNA species had similar developmental time-courses. They appeared at embryonic day 8 (E8), increased steadily until E16 and maintained a rather high plateau level until E18. In the ciliary ganglion, alpha7 transcripts were already abundant at E6, increased until E10, and considerably decreased thereafter. High-resolution in situ hybridization showed that alpha7 transcripts were present in all cell types of the E6 ciliary ganglion, whereas they were restricted to large neuronal somas at E16. Transfections with a reporter gene under the control of the alpha7 promoter demonstrated that a sharp developmental divide occurred at E11-12, after which stage the promoter was activatable in neurons exclusively.