Christine Alliod

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.

Localization of the Main Immunogenic Region and Toxin Binding Site of the Nicotinic Acetylcholine Receptora

We have used two approaches in order to map the main immunogenic region (MIR) and the a-bungarotoxin (aBTX) binding site of the nicotinic acetylcholine receptor (nAChR). Digestion of Torpedo nAChR by papain, followed by SDS-PAGE and immunoblot analysis of the products, allows us to localize the MIR to the NH,terminal 169 amino acids of the a chain and the aBTX binding site COOH-terminal to residue 15 1.’ Analysis of fusion proteins generated using restriction fragments from cDNA coding for mouse a-chain allows us to map the MIR and the aBTX binding site to residues 6-85 and 160-215, respectively.

Monoclonal anti-torpedo receptor antibodies used to study antibody heterogeneity in myasthenic sera

Monoclonal antibodies against Torpedo acetylcholine receptor were used to define the binding regions of cross-reacting, anti-receptor antibodies from sera of myasthenic patients. Cross-reacting antibodies were directed mainly against the toxin-binding region of the receptor and a region remote from the acetylcholine-binding site. Few patients had antibodies against the acetylcholine-binding site region. The monoclonal antibodies provide probes for defining the heterogeneity of anti-receptor antibodies in myasthenic sera.

Neuronal Nicotinic Acetylcholine Receptor Genes in the Avian Genome

In vertebrates, the nicotinic acetylcholine receptor (nAChR) present in the post-synaptic membrane of electrocytes or squeletal muscle cells is very well characterised biochemically (Changeux et al., 1984) and physiologically (Sakmann et al., 1983). In contrast, the related cholinergic receptors found in the central or peripheral nervous system are poorly understood. In the past few years, however, significant progress has been made in the identification and functional characterisation of neuronal nAChRs. Progress has resulted mainly from two different experimental approaches: the use of monoclonal antibodies has allowed the localisation, functional identification and purification of several distinct neuronal cholinoceptor species (Jacob et al., 1984; Whiting and Lindstrom 1986, 1987; Whiting et al., 1987) while molecular biological techniques have resulted in the isolation of numerous genes and cDNA clones encoding neuronal proteins that are related in sequence to the muscle endplate nAChR subunits (in rodents: Boulter et al., 1986; Goldman et al., 1987; Wada et al., 1988; Deneris et al., 1988; in birds: Nef, 1986; Nef et al., 1988).