Brn3b mutation (Erkman et al. 2000) and may contribute to the altered expression of target genes and axonal outgrowth disruption.
3.5.4 Conclusions
The results from different parts of vertebrate and invertebrate nervous systems provide a first impression of transcription factor cascades involved in neuronal differentiation and specification. A general theme is the early role of bHLH proteins which may excert proneural functions and are involved in the specification of developing neurons.
Cascades of bHLH proteins found in vertebrate neurogenesis indicate the diversifica-tion of their funcdiversifica-tion along different stages during neurogenesis. A crucial role for the differentiation of the diverse neuronal lineages can be attributed to transcription factors of distinct homeobox protein families. There is currently no unifying concept for the relative roles played by the different homeobox factor families during differenti-ation of the diverse neuronal lineages.
A classical example for the combinatorial action of different homeobox transcrip-tion factors is the specificatranscrip-tion of neuron lineages in the nematode C. elegans. The POU domain protein unc-86 and the LIM domain protein mec-3, both homeobox-containing transcription factors, are necessary for development of distinct neurons (Finney et al. 1988; Way and Chalfie 1989). The sequential expression of unc-86 before mec-3 suggest hierarchical actions in the touch neuron differentiation cascade (Duggan et al. 1998). Their action must not be strictly sequential, however. unc-86/
mec-3 heterodimers can form and bind to the mec-3 promoter (Xue et al. 1993;
Rockelein et al. 2000) as well as presumed target gene promoters (Duggan et al. 1998).
This illustrates how sequential activation of transcription factors may propel cell fate determination not only by successive production of distinct transcription factors but also by synergistic action of these transcription factors recruited into hetero-oligomere complexes.
Direct target genes of the respective transcription factors are known for only a limited number of examples such as Pit-1, Phox2, Ath-5, or unc-86. Moreover, the proof that the transcription factors bind to the presumed promoter target sequences in vivo is, in the majority of cases, not existent. Nevertheless, the methods to undertake this analysis are available. By the combination of these approaches with the increasing knowledge of model organism genomes, we can within the foreseeable future expect to have a profound look into the similarities and differences between transcription factor cascades specifying different classes of neurons.
3.6
Organizing neuronal function at the transcriptional
of a multitude of proteins which belong to different protein families. Consequently, the coordinate expression of a range of proteins is necessary to enable a neuron to perform specific tasks. To achieve this goal, mechanisms exist to coordinate the expression of neuron-specific genes. Synexpression groups are groups of genes whose protein products are involved in a certain functional context and whose expression is regulated by common mechanisms (Niehrs and Pollet 1999).
3.6.1 Specifying neurotransmitter phenotypes as synexpression groups
Substantial evidence exists to indicate that neurotransmitter phenotypes are regulated as synexpression groups. The neurotransmitter phenotype is a term that describes the ability of a neuron to synthesize, store, and release a certain neurotransmitter. Neurons using glutamate as transmitter are called glutamatergic to indicate their phenotype;
those which use GABA are referred to as GABA-ergic; cholinergic neurons use acetyl-choline, dopaminergic neurons dopamine, noradrenergic neurons noradrenaline, and so forth. For transmitter synthesis they employ specific enzymes, and for the loading of the transmitter or its precursor into vesicles they use specific vesicular transport proteins. In addition, specific plasma membrane transporters may be expressed for reuptake of transmitter or breakdown products. Evidence has been obtained that the synthesizing enzymes and transport proteins for a certain transmitter can be expressed in coordinate manner, thus qualifying them as synexpression groups.
Coordinated transcriptional regulation has been observed for glutamic acid decar-boxylase and the vesicular GABA transporter in C. elegans GABA-ergic neurons (Eastman et al. 1999). The homeodomain protein unc-30, which upon ectopic expres-sion can induce a GABA-ergic phenotype (Jin et al. 1994), sequence-specifically binds to the promoters of both genes. Mutation in the unc-30 binding sites abolishes expression of reporter genes with promoters of glutamic acid decarboxylase and of the vesicular GABA transporter.
The ETS domain transcription factor Pet-1 colocalizes with serotonergic neurons in the mouse brain (Hendricks et al. 1999). Conserved Pet-binding sites are present in genes coding for tryptophan hydroxylase, aromatic L-amino acid decarboxylase, serotonin transporter, and 5HT-1a receptor, and are capable of supporting transcrip-tional activation through interaction with the Pet-1 ETS domain.
A particular interesting example is the cholinergic neurotransmitter phenotype encoded by the cholinergic gene locus (Mallet et al. 1998; Eiden 1998). The locus con-tains the genes coding for the enzyme for acetylcholine biosynthesis, choline acetyl-transferase (ChAT), as well as the vesicular acetylcholine transporter (VAChT) gene (Fig. 3.4). The VAChT gene is located in the first intron of the ChAT gene, indicating that the expression of both genes may be driven by the same regulatory mechanism.
Indeed, both genes can be coregulated by growth factor-treatment (Misawa et al. 1995, Lopez-Coviella et al. 2000) and by the common 5′flanking region including the NRSE/RE1 motif (De Gois et al. 2000). Regulatory regions in the locus directing
expression in cholinergic cells have been identified (Lönnerberg et al. 1995, 1996;
Naciff et al. 1999) but the transcription factors interacting with these sites remain to be determined.
The close packing of genes into loci such as the cholinergic one constitutes an obvi-ous way to coordinate gene expression in a synexpression group. In the case of the noradrenergic transmitter phenotype, genes encoding two different transmitter syn-thesizing enzymes may be coordinately regulated (Ernsberger 2000) and, at least in humans, are located on different chromosomes (www.ncbi.nlm.nih.gov:80/LocusLink).
Tyrosine hydroxylase, the rate-limiting enzyme in the noradrenaline biosynthesis
Fig. 3.4 Regulation of neuronal genes as synexpression groups. Genes regulated as synexpres-sion groups can be located on different chromosomes (A). Gene structures are given schemat-ically with the 5’ upstream region as line and the coding region as hatched box. Different symbols in the 5’ upstream region indicate distinct transcription factor binding sites. Common transcription factor binding sites in the regulatory regions of different genes are considered to mediate the coordinate regulation in certain classes of neurons as observed for tyrosine hydroxylase and dopamine β-hydroxylase in noradrenergic neurons. Different elements in the regulatory region may contribute to different regulation in other classes of neurons as observed for tyrosine hydroxylase and dopamine β-hydroxylase in noradrenergic versus dopaminergic neurons. Genes regulated as synexpression group can also be located within one locus (B) as shown for the cholinergic locus encoding choline acetyltrasferase (ChAT) and the vesicular acetylcholine transporter (VAChT). Exons are indicated by hatched boxes and transcription start sites by arrows. (B) Modified from Eiden (1998) and Mallet et al. (1998).
Chromosome A
Chromosome B
Cholinergic locus
ChAT Exon R
VAChT ORF
ChAT exon N
ChAT exon M
first ChAT coding exon Tyrosine hydroxylase coding region
Dopamine β-hydroxylase coding region
A) Synexpression group of genes on different chromosomes
B) Synexpression from gene locus
cascade, and dopamine β-hydroxylase, the final enzyme converting dopamine into noradrenaline, become detectable at the same time during sympathetic neuron development (Ernsberger et al. 2000). Coordinate induction by overexpression of the transcription factors Phox2a and 2b (Stanke et al. 1999) and lack of expression after functional inactivation of the Phox2b gene (Pattyn et al. 1999) strongly support the hypothesis that the two enzymes are regulated as a synexpression group in nor-adrenergic neuron populations. Dopaminergic neurons require TH but not DBH for transmitter synthesis. This indicates that coregulation of TH and DBH expression is not obligatory but depends on the neuron population and the respective transcription factor equipment. This may be mediated by transcription factor binding sites which in part are common and in part distinct between the genes (Fig. 3.4).
3.6.2 Coexpression of ion channel subunits — a question to be addressed
Ligand-gated ion channels such as the acetylcholine receptors or the glutamate recep-tors are composed of several subunits that contibute to the channel pore. In the case of the voltage-dependent ion channels, the same situation can be found for potassium-specific channels. In the case of voltage-dependent sodium and calcium channels, there is one subunit sufficient for pore formation and additional subunits may affect chan-nel properties such as kinetic behavior. For this reason, the coordinate expression of channel subunits in individual neurons has important impact on the electrical properties of the neuronal plasmamembrane.
In the case of the neuronal AChR subunits alpha 3, alpha 5, and beta 4, the location of the genes in a cluster of 60 kb has been observed in the rat genome (Boulter et al.
1990). As there is extensive overlap of expression in the peripheral nervous system (Yang et al. 1997), this has sparked speculations on a common regulation of these clus-tered genes. Due to only partial overlap of the expression in the central nervous system, the issue may not have a simple solution. A reporter construct driven by a fragment spanning the region from the alpha 3 promoter to the beta 4 untranslated exon in transgenic mice results in CNS but not PNS expression (Yang et al. 1997). CNS expres-sion is localized to a subset of CNS nuclei that expresses endogenous alpha 3, shows some overlap with beta 4 and none with alpha 5. The precise mechanism by which sub-unit expression is established in some neuron populations and prevented in others and the role of the clustering of genes therein still needs to be worked out.
3.6.3 Olfactory neurons — establishing neuronal identity by coordinately regulated gene expression
Analysing the regulatory region of a number of proteins specifically expressed in olfac-tory neurons, such as the olfacolfac-tory marker protein (OMP) and components of olfacolfac-tory signal transduction, identified a novel sequence motif binding specifically nuclear proteins present in olfactory neuroepithelium (Kudrycki et al. 1993; Wang et al. 1993).
Olf-1, a helix-loop-helix transcription factor expressed exclusively in olfactory neurons
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