and their precursor cells, binds to this sequence motif (Wang and Reed 1993). In transgenic mice, a 300 bp fragment of the OMP 5’ flanking region containing the Olf-1 binding motif is sufficient for olfactory-specific expression of a reporter gene (Kudrycki et al. 1993). Surprisingly, a mutation that prevents the interaction of Olf-1 with its binding site does not alter the expression pattern of a reporter construct in the olfactory epithelium of transgenic mice (Kudrycki et al. 1998). As the mutated DNA sequence is still able to bind nuclear proteins from nervous tissue, the results are difficult to interpret.
The finding of related genes establishes Olf-1 as the founding member of the Olf-1/EBF-like HLH transcription factor family (Garel et al. 1997; Malgaretti et al.
1997; Wang et al. 1997). The expression of the Olf-1-relatives O/E2 and O/E3 in the olfactory epithelium indicates that there may be functional redundancy in their regulation of transcription (Wang et al. 1997). Their expression in other parts of the nervous system suggests that they may have additional functions in neuronal development.
Olf proteins represent transcription factors which may regulate an entire aspect of function in sensory neurons related to their stimulus transduction cascade. Still, the demonstration of their necessity in vivo is lacking due to technical difficulties. The expression of several family members in the olfactory epithelium and their possible redundant action compromises simple gene inactivation strategies. The mutation of promoter binding sites in transgene approaches is complicated by the creation of new, even more powerful binding sites due to the mutation. Thus, it still remains to be proven that a number of olfactory neuron-specific proteins involved in transduction of the sensory stimulus are regulated as a synexpression group in vivo.
3.7
Specifying neuronal functions at the transcriptional
3.7.1 Specifying transmitter phenotypes may include transcriptional repression
In general neurons use either one of the transmitters glutamate, GABA, glycine, acetyl-choline, noradrenaline, and some others, even though they may coexpress a range of neuropeptides and other mediators. In sympathetic neurons of birds and mammals, two populations of neurons exist which differ in their transmitter phenotype (Ernsberger and Rohrer 1999). A majority of neurons uses noradrenaline and is called
‘noradrenergic’, whereas a smaller population of neurons uses acetylcholine and is called ‘cholinergic’. During development cholinergic neurons may be derived from noradrenergic ones (Landis 1990), but mature neurons are characterized by the usage of one of the transmitters and by a specific expression of the respective transmitter-synthesizing enzymes.
The analysis of the 5′flanking regions of the genes for the transmitter-synthesizing enzymes choline acetyltransferase (ChAT) and dopamine β-hydroxylase (DBH) has provided some evidence that, in addition to inducing the ‘right’ enzyme in a neuron population, the expression of the ‘wrong’ enzyme is suppressed. A luciferase reporter driven by several kb of 5′flanking sequences of the human ChAT gene expresses differ-ently in cholinergic and non-cholinergic cell lines (Li et al. 1993). Elements with silencer activity were characterized which repress promoter activity in an adrenergic cell line to a much higher degree than in cholinergic cell lines. One of the silencer elements containes E-box motifs required for silencing activity (Li et al. 1995). Nuclear proteins from adrenergic cells specifically bind to these E-boxes. Using 2.3 kb of 5′
flanking region of the rat ChAT gene to drive a CAT reporter demonstrates enhancer activity in cholinergic and repressor activity in non-cholinergic cell lines and targets transgene expression to cholinergic sites (Lönnerberg et al. 1995). The activity of a RE1/NRSE motif does not discriminate between cholinergic and non-cholinergic neu-ronal cells but silences activity in non-neuneu-ronal cells (Lönnerberg et al. 1996). A differ-ent part of the flanking sequences demonstrates cholinergic-specific enhancer activity and is inactive in non-cholinergic neuronal and in non-neuronal cells. These data demonstrate that there is an interaction of positive and negative regulating elements which enhance ChAT expression in cholinergic cells and represses its expression in non-cholinergic and specifically adrenergic cells. They also suggest that the RE1/ NRSE may not be involved in this aspect of neuron subpopulation-specific silencing.
Analysis of 5′flanking sequences of the DBH gene in transgenic mice complements this picture (Hoyle et al. 1994). Comparing reporter gene expression driven by 5′ flanking fragments of different length shows expression in noradrenergic and to differ-ent degrees in non-noradrenergic neuron populations. The comparison of a 1.1 and 1.5 kb fragment indicates the presence of sequences that may be responsible for the repression of expression in three sites which normally do not express DBH.
These studies illustrate the possible interaction of population-specific activation and silencing of gene expression. The proteins involved in this antagonistic regulation still
have to be determined. The Phox2 transcription factors are implicated in noradrenergic induction (Ernsberger 2000). One has to bear in mind, however, that they are also expressed in other autonomic neurons (Ernsberger et al. 2000) and may induce the expression of both noradrenergic and cholinergic properties (Stanke et al. 1999).
3.7.2 Specifying receptive properties in sensory cells — locus control regions
Red–green color vision is based on different visual pigments encoded by an array of genes on the human X chromosome (Neitz and Neitz 1995). Individual photoreceptor cells express typically one gene. Analysis of the DNA sequences upstream of the visual pigment genes provides evidence that a locus control region is involved in the decision as to which of the genes from the cluster to express.
5′flanking regions of visual pigment genes fused to a lacZ reporter direct expression to cone cells in transgenic mouse retina (Wang et al. 1992). A 600 bp fragment 5′to the human red pigment gene is essential for expression and contains sequences highly conserved between vertebrate species. In an exciting set of experiments, a minimal human X chromosome visual pigment gene array was constructed, where the red and green pigment gene transcription units were replaced, respectively, by alkaline phosphatase and β-galactosidase reporters (Wang et al. 1999). In transgenic mice, the two enzymes were in the large majority of cases expressed in different cones. More than 70% of the expressing cones showed either one or the other enzyme activity.
30% expressed both activities. A fascinating aspect of this result is that the mutually exclusive expression of the two transgenes in different cones is achieved in a dichromat mammal.
An even more demanding task is the control of odorant receptor gene expression, where some thousand receptor encoding genes are expressed in unique patterns in the sensory neurons of the olfactory epithelium (Buck and Axel 1991; Buck 1992). The receptor genes are clustered across the genome (Ben-Arie et al. 1994; Sullivan et al.
1996) and expressed in a specific mode that has been hypothesized to occur via sto-chastic selection (Ressler et al. 1993). Comparing sequences surrounding the β-globin locus in mice and humans shows a high degree of conservation (Bulger et al. 1999).
Surprisingly, the β-globin loci turned out to be embedded within an array of odorant receptor genes, suggesting a role of the β-globin locus control region in control of these odorant receptors. Deletion of the endogenous β-globin locus control region in mice did, however, not result in altered expression of neighbouring olfactory receptor genes (Bulger et al. 2000).
3.7.3 Conclusions
The hierarchical action of transcription factors in cascades during the rapid progression from neuronal precursors to immature neurons leads to the expression of a number of neuron-specific genes which in a large number of cases may be restricted to distinct neuronal subpopulations. A least in part, the expression of these genes may occur in a
coordinate manner as synexpression groups. To sharpen the functional profile of a developing neuron, the induction of a certain set of genes may go along with the repression of others resulting in a mature phenotype or gene expression profile that distinguishes a certain neuronal subpopulation from others. Growth factors are involved in these induction processes. Neuronal activity may also shape gene expres-sion profiles and contribute to plasticity in the mature nervous system.
3.8