Ipou/abnormal chemosensory jump 6
The role of Castor in regulating pdm genes raises the possibility that it may regulate expressions of other POU genes. To test this, the expression domains of Cas and Drifter/Ventral veins lacking were examined. Drf expression was examined in cas- embryos. In addition to its established role in midline glia and tracheal development, Drf is also expressed in a subset of NB progeny in both the developing brain and ventral cord. Many Cas-expressing NB sublineages also express Drf. Thus, it appears that Cas does not repress drf expression: to the contrary, a marked reduction in late-lineage Drf expression is observed in cas- embryos, suggesting Cas either directly or indirectly plays a role in activating and/or sustaining drf expression in these sublineages. Ectopically activated Cas has no effect on Drf expression. In the absence of castor function, I-POU expression is lost in a subset of ventral cord cells, but ectopic Cas has no effect on the I-POU wild-type expression pattern. It is not known if Cas is a direct activator of drf and/or I-POU. However, the data indicate that if Cas is playing a direct activator role, it most likely requires co-factors that are not expressed outside of its normal domain (Kambadur, 1998).
Cholinergic neurons acquire their neurotransmitter phenotype, in part, by expressing the cholinergic gene locus. Previous studies have indicated that the 5' flanking DNA of the locus contains both positive and negative regulatory elements important for expression in different subsets of cholinergic neurons in Drosophila and other animals. Approximately 300 bases of proximal 5' flanking DNA control expression in Drosophila CNS neurons essential for viability, whereas more distal regulatory elements are important for expression in PNS sensory neurons. In this study the POU domain transcription factor abnormal chemosensory jump 6 (Acj6) has been identified as a necessary positive transcriptional regulator for cholinergic locus expression in primary olfactory neurons. Choline acetyltransferase enzyme activity, protein levels, mRNA, and a fluorescent cholinergic reporter gene are all decreased in olfactory neurons of acj6 mutants. Decreased cholinergic expression was observed in both adults and larvae. The presence of a specific Acj6 binding site has been identified in the cholinergic locus 5' flanking DNA, suggesting that Acj6 may play a direct role in specifying the cholinergic neurotransmitter phenotype of most olfactory neurons. Transgenic expression of two different isoforms of Acj6 restricted to olfactory neurons indicates that additional trans factors may be required for cholinergic locus expression. Transgenic expression in all cholinergic neurons, however, results in lethality when a POU IV box element is absent but is essentially benign when present, indicating the importance of this motif in specifying different functional roles for Acj6 (Lee, 2002).
No direct DNA binding motifs in regulatory elements are known for Drosophila Acj6 protein. Acj6, however, is a homolog of the vertebrate class IV POU proteins of the Brn3 family and contains a POU specific domain and a POU homeodomain with extensive homology to Brn3a, b, and c. Because Brn3 DNA binding motifs have been studied extensively, the cholinergic locus 5' flanking DNA was scanned for similar motifs. Initially concentration was placed on a region of the cholinergic locus 5' flanking DNA known to be important for expression in peripheral sensory olfactory neurons. This region is in the distal part of a 3.3 kb DNA fragment because deletion of the 5' flanking DNA to 1.2 kb eliminates reporter gene expression in olfactory neurons. Therefore, it is likely that any direct interaction of Acj6 protein with cholinergic regulatory DNA important for olfactory neuron expression will be localized upstream of the 1.2 kb 5' flanking DNA. Seven candidate binding motifs (A-G) were identified on the basis of the fact that they contained either of two core elements (ATAATT or ATAAAT) identified in the two known Brn3 binding motifs, ATAATTAAT and GCATAAATAAT. Nineteen to twenty-two base oligonucleotides, representing these core sequences along with their flanking bases, were synthesized and tested for their ability to bind recombinant Acj6 protein. Only motif D showed strong specific binding with Acj6 recombinant protein. No Acj6 binding was detected to motif B, which is representative of the six negative sites tested. Specificity of Acj6 binding to motif D was established by competitive inhibition using unlabeled oligonucleotide and tested further by constructing clustered point mutations in the target oligonucleotide D. As expected, binding was abolished when the core sequence was mutated. Mutations introduced into the 5' or 3' flanking bases of motif D have little or no effect on Acj6 binding. These results show that Acj6 protein can interact directly with a specific site in the cholinergic locus regulatory DNA and suggest that Acj6 may function as a direct transcriptional regulator of the cholinergic locus in peripheral sensory olfactory neurons (Lee, 2002).
The principal conclusion of this study is that the Drosophila class IV POU domain transcription factor Acj6 is required for normal expression of the cholinergic gene locus in primary olfactory neurons. Evidence supporting this conclusion comes primarily from genetic experiments demonstrating a reduction in cholinergic locus expression in animals carrying mutations in the acj6 gene. A decrease in ChAT enzyme activity and protein, a substantial decrease in antennal ChAT-specific mRNA, and a loss of fluorescent cholinergic reporter gene expression in olfactory neurons of antennae and maxillary palps of acj6 mutants have been documented. The decrease in cholinergic locus expression is proportional to the level of Acj6 function because ChAT activity is decreased more in an acj66 null genetic background than in an acj61 hypomorphic background. The reduction of ChAT mRNA in antennae from acj66 mutants is consistent with a decrease in transcription. The decrease in locus expression is not complete because some residual fluorescence is observed in antennal, maxillary palp neurons as well as in larval and embryonic olfactory neurons. Perhaps there are redundant transcriptional regulators expressed in some types of olfactory neurons, or alternatively, some types of cholinergic neurons may be only partly dependent on Acj6 for locus expression (Lee, 2002).
Acj6 does not regulate the cholinergic locus in all types of cholinergic neurons. There are at least three classes of cholinergic neurons that can be distinguished relative to Acj6 function. First are those that are dependent on Acj6, such as most primary olfactory neurons. Eighty-four of the 120 total maxillary palp olfactory neurons express Acj6, whereas it is estimated ~97 are cholinergic and show reduced or absent expression in acj66 mutants. Second are neurons that are independent of Acj6, such as the second antennal segment mechanosensory neurons, which do not express Acj6 and show no change in cholinergic fluorescence in acj66 mutants. Third are neurons that express Acj6 but maintain cholinergic expression even in acj66 mutants, such as the larval SP interneurons. There is also likely to be some overlap in the cholinergic central complex, antennal lobe, and optic lobe interneurons, because all of these regions have numerous Acj6-positive and cholinergic neurons. In addition, acj66 null mutants are viable, whereas loss-of-function mutations in either product of the cholinergic locus (the Cha or Vacht genes) are late embryo or early larval lethal. Thus the reduction in ChAT enzyme activity, protein, mRNA, and fluorescent cholinergic reporter expression seen in acj6 mutants is likely attributed to the dependence of cholinergic locus expression on Acj6 function only in nonessential cholinergic neurons, such as the primary olfactory neurons (Lee, 2002).
A remarkable problem in neurobiology is how olfactory receptor neurons (ORNs) select, from among a large odor receptor repertoire, which receptors to express. Computational algorithms and mutational analysis were used to define positive and negative regulatory elements that are required for selection of odor receptor (Or) genes in the proper olfactory organ of Drosophila, and an element was identified that is essential for selection in one ORN class. Two odor receptors are coexpressed by virtue of the alternative splicing of a single gene, and dicistronic mRNAs were identified that each encode two receptors. Systematic analysis reveals no evidence for negative feedback regulation, but provides evidence that the choices made by neighboring ORNs of a sensillum are coordinated via the asymmetric segregation of regulatory factors from a common progenitor. Receptor gene choice in Drosophila also depends on a combinatorial code of transcription factors to generate the receptor-to-neuron map (Ray, 2007).
Transcription factors were investigated whose expression had been reported in at least one olfactory organ and whose mutations had been shown to cause olfactory defects. One such protein, the Runx domain-containing transcription factor Lozenge, was found had predicted binding sites (RACCRCA, R = purine) adjacent to four maxillary palp Or genes. Specifically, it was found that two maxillary palp Or genes, Or59c and Or85d, had two Lz binding sites, and two genes, Or71a and Or85e, had one Lz binding site, within 1 kb upstream or downstream of the coding region. Lz is required for the specification of cell fate in the eye and for normal numbers of olfactory sensilla in the antenna. In the maxillary palp the numbers of sensilla are normal, but electropalpogram recordings showed large reductions in odor responses (Ray, 2007).
To investigate the possibility that Lz is required for normal receptor gene expression, it was first asked whether it is expressed in ORNs of the maxillary palp. Lz is coexpressed with Elav, indicating that it is expressed in the nuclei of all maxillary palp ORNs. Then the expression of six maxillary palp Or genes was examined, one from each ORN class, in lz3, a strong hypomorphic mutant. The four genes that are flanked by predicted Lz binding sites all showed reduced levels of expression; the two genes that contain two Lz binding sites, Or59c and Or85d, showed particularly severe reductions (of 47% and 87%, respectively) in the number of labeled cells. The mildest reduction, 18%, was observed for Or85e; consistent with this result, a 14% reduction was observed when DNA including the predicted Lz binding site was removed from an Or85e-GAL4 driver (the construct containing 3 kb of upstream DNA labeled 13.4 ± 0.4 cells, whereas the construct containing 0.45 kb labeled 11.5 ± 0.3 cells; n = 12). The two genes that did not contain Lz binding sites did not show a reduction in labeling in lz3. These results demonstrate that lz is required for the expression of a subset of Or genes in the maxillary palp (Ray, 2007).
Next a weaker, temperature-sensitive allele, lzts1, was used to investigate the possibility that levels of Or gene expression are susceptible to modulation during the adult stage. It was found that Or85d is expressed in 18% fewer cells (p < 0.05) when lzts1 flies are raised at the restrictive temperature (29°) than when raised at the permissive temperature (18°). When flies were raised at the restrictive temperature and then shifted to the permissive temperature for 24 hr, 1 week after eclosion, the number of Or85d-expressing cells showed an increase of 19%, to a level indistinguishable from that of flies that had been cultured continuously at the permissive temperature. These results confirm the finding of a functional role for lz in Or expression, provide direct evidence that levels of Or expression can be altered after eclosion, and invite investigation of epigenetic modulation of odor receptor expression in Drosophila (Ray, 2007).
Only one other transcription factor, the POU domain protein Acj6, has previously been demonstrated to be required for odor receptor expression in Drosophila. Specifically, expression of Or33c, Or42a, Or46a, Or59c, and Or85e was severely reduced by the null allele acj66, whereas expression of Or71a and Or85d was unaffected. It has been shown in this study that expression of Or59c, Or71a, Or85e, and Or85d was reduced by lz3, but expression of Or42a and Or46a was not. Thus, the maxillary palp Or genes can be divided into three classes based on their sensitivity to these mutations: those sensitive to both acj66 and lz3 (Or59c and Or85e), to acj66 alone (Or42a and Or46a), or to lz3 alone (Or71a and Or85d). These results support a model in which Or gene expression depends not only on a combinatorial code of regulatory elements but also on a combinatorial code of transcription factors (Ray, 2007).
In summary, in mammals, it is thought that transcriptional regulatory mechanisms direct expression of OR genes in specific zones of the olfactory epithelium, but that within a zone, OR gene choice is based on a stochastic selection mechanism. A third mechanism, negative feedback, could then operate to limit the number of OR genes expressed in individual neurons (Ray, 2007).
In Drosophila, the process of receptor gene choice achieves a conceptually simple end: it produces a highly stereotyped receptor-to-neuron map. However, the large number of receptors and neurons presents a regulatory problem of great complexity. To achieve such a precise and highly ordered organization, Drosophila has evolved a sophisticated suite of regulatory mechanisms. This study has documented organ-specific and neuron-specific levels of transcriptional control, including both positive and negative mechanisms. A posttranscriptional mechanism, alternative splicing, was identified and the system has even evolved a relatively rare innovation, dicistronic mRNAs (Ray, 2007).
The worm Caenorhabditis elegans has a much larger repertoire of odor receptor genes than Drosophila, but the number of ORNs to which it allocates them is very limited. Thus the number of receptor genes per neuron is increased, but the complexity of the regulatory problem is decreased. In vertebrates, however, the repertoire is very large and the number of receptor genes expressed per neuron is very low. Perhaps as the receptor gene repertoire expanded in vertebrate evolution, the complexity of the regulatory problem eventually exceeded the ability of the system to execute a deterministic plan with sufficient fidelity, and deterministic mechanisms were replaced by a stochastic mechanism and a negative feedback mechanism. In any case, the ultimate result of receptor gene choice in Drosophila is the same as in vertebrates: a spectacular diversity of ORNs that underlie the detection and discrimination of odors (Ray, 2007).
There is no unique inhibitory mechanism to Ipou as reported previously (Treacy, 1991 and 1992). The DNA recognition profiles of Ipou, tIpou and Brn-3.0 are similar, with only minor relative differences in affinity for some oligonucleotide competitors. Ipou does not form a complex with Drifter as reported previously. The principal contacts of POU-IV class proteins with DNA are highly conserved. The deletion of the Arg-Lys residues at homeodoman positions 3 and 4 in Ipou, relative to tIpou, results in the occurrence of Gly-Glu residues in positions 1 and 2, and substitution of Lys for Arg in position 3. The amino acid residues at homeodomain positions 1 and 2 do not contact DNA. It is not surprising that a substitution at this position does not eliminate DNA binding. The substitution of Lys for Arg at position 3 in Ipou would also not be expected to alter DNA binding, as either residue may occur at this postion in various POU proteins (Turner, 1996).
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