Ipou/abnormal chemosensory jump 6


Transcriptional regulation

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).

Targets of Activity

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).

Transcriptional regulation of odorant receptors; Mechanisms of odor receptor gene choice in Drosophila

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).

Positive and negative regulation of odor receptor gene choice in Drosophila by acj6

Little is known about how individual olfactory receptor neurons (ORNs) select, from among many odor receptor genes, which genes to express. Abnormal chemosensory jump 6 (Acj6) is a POU domain transcription factor essential for the specification of ORN identity and odor receptor (Or) gene expression in the Drosophila maxillary palp, one of the two adult olfactory organs. However, the mechanism by which Acj6 functions in this process has not been investigated. This study systematically examine the role of Acj6 in the maxillary palp and in a major subset of antennal ORNs. An Acj6 binding site was defined by a reiterative in vitro selection process. The site is found upstream of Or genes regulated by Acj6, and Acj6 binds to the site in Or promoters. Mutational analysis shows that the site is essential for Or regulation in vivo. Surprisingly, a novel ORN class in acj6 adults is found to arise from ectopic expression of a larval Or gene, which is repressed in wild type via an Acj6 binding site. Thus, Acj6 acts directly in the process of receptor gene choice; it plays a dual role, positive and negative, in the logic of the process, and acts in partitioning the larval and adult receptor repertoires (Bai, 2009).

How individual ORNs select individual odor receptors to express is a remarkable problem in molecular neurobiology. Most ORN classes in the fly select one or a small number of receptor genes from a large family of 60 Or genes. The choice dictates the odor response spectrum of the ORN and, consequently, its contribution to the coding of olfactory information (Bai, 2009).

The simplest model for receptor gene choice in the fly is that it is governed by a combinatorial code of transcription factors. Although there is evidence to support this model, much remains to be learned about the elements of the code and the logic by which it operates. Distinct combinations of six transcription factors could in principle dictate the specification of 26=64 distinguishable ORN classes, a number comparable to the number of Or genes and of the same order as the number of ORN classes. Thus for factors A–F, one ORN class might require factors A, B, and D and be specified by the combination (A+B+CD+EF), while another class might be specified by the combination (ABC+D+EF+). In this simple model, each factor would be required for the specification of half of the 64 ORN classes. Alternatively, a much larger number of such factors could act, each in a small number of classes. In the 14 ORN classes for which data are now available, six in the maxillary palp and eight in the antenna, Acj6 is required for the specification of eight, approximately half. Thus evidence was found that at least one transcription factor acts broadly across the ORN repertoire (Bai, 2009).

One aspect of the problem of receptor gene choice is that each ORN must express one or a small number of receptor genes; another aspect is that each ORN must not express all the other receptor genes. In principle, each aspect of the problem could be governed by separate sets of factors. This study found that Acj6 plays a role in both positive and negative regulation of Or genes. This duality of Acj6 function imparts economy to the process, an economy that may be critical in a process that controls such a large number of Or genes in a large number of ORN classes. Allowing individual factors to act in two aspects of the problem reduces the number of required factors (Bai, 2009).

Another example of dual regulation by a single transcription factor was found in the differentiation of photoreceptor subtypes in the Drosophila visual system. Otd, a homeodomain transcription factor, activates the rhodopsin genes rh3 and rh5 but represses rh6 in other photoreceptors, through a common binding site. The modes of regulation are likely to depend on the availability of a coactivator and a corepressor of Otd. An example of dual regulation in a chemosensory system is provided in C. elegans by the UNC-3 Olf/EBF protein. This protein represses a number of genes, including the odr-10 olfactory receptor gene, in the ASI chemosensory neuron, and it activates other chemoreceptor genes. UNC-3 was shown to repress multiple genes by direct binding to sites in the promoters, but it may activate the expression of ASI-specific genes through an indirect mechanism (Bai, 2009).

Drosophila contains two distinct olfactory systems, one in the larva and one in the adult. The two systems differ in morphology and developmental origins, and they operate under different conditions: the larva burrows in semi-solid medium whereas the adult walks and flies through air. Both olfactory systems, however, depend on members of the Or gene family. Some members of the Or family are larval-specific, others are adult-specific, and some are expressed in both. By what mechanism are members of the same gene family partitioned into two different systems (Bai, 2009)?

Acj6 acts in the partitioning mechanism. It is required to restrict at least one member, Or45b, to the larval system: when acj6 is mutated, Or45b is ectopically expressed in the adult. Or45b contains in its upstream region two regulatory elements, Dyad-1 and Oligo-1, which are found adjacent to all or most maxillary palp Or genes and are required for their expression in the maxillary palp. It is not known if the Dyad-1 and Oligo-1 elements play a role in the expression of Or45b in larvae. These observations, taken together, suggest the possibility that Or45b may have shifted over evolutionary time from a maxillary palp gene to a larval gene. If so, the shift is likely to have occurred before the divergence of D. melanogaster and D. pseudoobscura tens of millions of years ago, since the odor response spectra of the maxillary palp ORNs in these two species are similar (Bai, 2009).

Using an in vitro DNA binding assay a positional weight matrix and a consensus Acj6 binding site was defined. Only one Acj6 site has been experimentally identified and tested previously, in a study of sequences upstream of the gene encoding choline acetyltransferase, Cha. These Cha sequences were scanned for motifs similar to those found for the mammalian POU-IV transcription factors, and of seven identified, one was found to bind Acj6 protein in an EMSA assay. It does not show close similarity to the site (matrix score=3.4) defined in this paper and its activity in vivo was not evaluated (Bai, 2009).

A strong correlation was found between the presence of the Acj6 site that this study has identified and Acj6-dependence in vivo. Moreover, it was shown directly, in vivo, that these sites are required for normal expression of flanking Or genes. These results are consistent with a direct role for Acj6 in mediating Or expression, as opposed to an indirect role via another transcription factor (Bai, 2009).

One of the Acj6 sites, the Or45b site, acts in negative regulation. Its score based on the positional weight matrix is intermediate among the sites that act in positive regulation, which is consistent with the finding from a binding-site-swap experiment that repression of Or45b depends not on the specific sequence of an Acj6 site but on its flanking sequences--the Or46a site also mediated repression when placed upstream of Or45b (Bai, 2009).

In several cases Acj6 sites lie within 8 bp of conserved sequence elements that are required for appropriate Or expression. An Acj6 site also lies very near a putative binding site for Pdm3, another POU domain transcription factor that regulates ORN development and that interacts genetically with Acj6. The proximity of these regulatory elements is likely to reflect the interaction of Acj6 and its cofactors. Identification of the cofactors of Acj6 will further contribute to understanding of the combinatorial regulation of specific Or expression (Bai, 2009).

Acj6 is likely to have a number of interesting transcriptional regulatory targets in addition to odor receptors and choline acetyltransferase. It may regulate other molecules that are essential for neuronal signaling: while some ORNs in acj66 exhibited spontaneous action potentials but did not respond to odorants, other ORNs showed no electrical activity, consistent with a loss of both odor receptors and other components (Bai, 2009).

Acj6 is also likely to regulate molecules required for synaptic connectivity. Acj6 is required for axon targeting specificity of a subset of ORN classes, some of which require Acj6 autonomously and some non-autonomously. The second-order neurons of the Drosophila olfactory system, the projection neurons, also require Acj6 for both normal dendritic targeting and axon terminal arborization. Moreover, the role of Acj6 extends beyond the olfactory system: acj6 mutants have defects in retinal axon targeting and synapse selection as well (Bai, 2009).

The definition of an Acj6 binding site may now facilitate the identification of some of its transcriptional targets. There are many sites in the genome with matrix scores >7, and analysis of the adjacent coding regions may be useful in identifying novel components required for synaptic connectivity and other processes (Bai, 2009).

Distinct functions of acj6 splice forms in odor receptor gene choice

Individual olfactory receptor neurons (ORNs) selectively express one or a small number of odor receptors from among a large receptor repertoire. The expression of an odor receptor dictates the odor response spectrum of the ORN. The process of receptor gene choice relies in part on a combinatorial code of transcription factors. In Drosophila, the POU domain transcription factor Acj6 is one element of the transcription factor code. In acj6 null mutants, many ORNs do not express an appropriate odor receptor gene and thus are not correctly specified. acj6 is alternatively spliced to yield many structurally distinct transcripts in the olfactory organs. Flies were generated that express single splice forms of acj6 in an acj6- background. Different splice forms are functionally distinct; they differ in their abilities to specify ORN identities. Some individual splice forms can fully rescue the specification of some ORNs. Individual splice forms can function both positively and negatively in receptor gene regulation. ORNs differ in their requirements for splice forms; some are not fully rescued by any single splice form tested, suggesting that some ORNs may require the combinatorial action of multiple splice forms. Late expression of some acj6 splice forms is sufficient to rescue some ORN classes, consistent with a direct role for Acj6 isoforms in receptor gene expression. The results indicate that alternative splicing may add another level of richness to the regulatory code that underlies the process of odor receptor gene choice (Bai, 2010).

This study provides evidence that acj6 is spliced in at least eight different ways in the maxillary palp, potentially producing eight structurally distinct proteins. Different splice forms are functionally distinct, and different ORNs differ in their requirements for acj6 splice forms. Individual splice forms are capable of acting both positively and negatively in regulating the expression of odor receptor genes. It has been shown previously that odor receptor gene choice depends on a combinatorial code of transcription factors; the results presented here provide evidence that that the process also depends on a multiplicity of splice forms. Thus alternative splicing may expand the combinatorial code that underlies the process of receptor gene choice and the generation of ORN diversity (Bai, 2010).

These results show that different splice forms that are expressed in the maxillary palp are not functionally equivalent in maxillary palp ORN specification. This finding is in agreement with a study showing that two acj6 splice forms produced different phenotypes when misexpressed in embryonic motor neurons, which do not normally express acj6. The distinct functions of Acj6 variants are also consistent with results demonstrating that the mammalian POU genes Brn3a and Brn3b, which are highly homologous to each other and are mammalian orthologs of acj6, have distinct roles in the programming of retinal ganglion cell diversity (Bai, 2010).

A single splice form of acj6 is capable of full rescue of the odor response profile of some ORNs, such as the full rescue of pb1A by acj6-F. This finding argues against a model in which Acj6 functions as an obligate heterodimer of splice forms. The ability of a single splice form to function is consistent with structural data indicating that the POU domain protein Oct-1 binds to DNA as a monomer and that Pit-1 can bind as a homodimer (Bai, 2010).

Individual splice forms are able to act both positively and negatively. pb2A, which expresses Or85e, is not observed in acj6 null mutants, whereas a novel pb2C cell appears due to the ectopic expression of Or45b. acj6-F, -M, and -J are able to activate the expression of Or85e and repress the expression of Or45b. These results argue against a model in which activation is mediated uniquely by one subset of splice forms and repression is mediated uniquely by a complementary subset (Bai, 2010).

None of the splice forms fully rescued either pb2 or pb3 sensilla, although two of these splice forms fully rescued pb1. The simplest interpretation of these results is that ORNs differ in their requirements for acj6 splice forms. One explanation for the lack of full rescue is that some ORNs may require more than a single acj6 splice form. The possibility that an untested splice form might fully rescue pb2 or pb3 cannot be excluded. However, it is noted that the number of splice forms identified in the maxillary palp (eight) exceeds the number of ORN classes (six), suggesting that some ORNs express more than one form. Single-cell RNA profiling may allow a determination of how many splice forms are expressed in each ORN class. It is noted with interest that there is ample precedent for heterodimerization of different POU proteins. Heterodimerization of Acj6 isoforms could provide yet another level of functional complexity: in principle, 8 Acj6 isoforms could form 28 distinguishable heterodimers (Bai, 2010).

Expression of a single splice form in some cases led to novel ORN fates, including pb1C, pb2D, and pb2E. Each of these fates is likely to arise from the ectopic expression of a larval Or gene. pb2D and pb2E have not been described previously; interestingly, a neuron with an odor response spectrum like that of pb1C was recently observed when a cDNA encoding pdm3 was expressed in the maxillary palp. Or85c was found to be ectopically expressed as well, supporting the interpretation that pb1C derives from the misexpression of this gene. pdm3 is a POU gene that genetically interacts with acj6 in the activation of Or42a, which confers the response of pb1A. Perhaps certain forms of the two POU proteins cooperate in misexpressing Or85c. The occurrence of pb1C in flies expressing acj6-J, but not other splice forms, could reflect differential interactions between Acj6 isoforms and other transcription factors, perhaps expanding the capacity of the combinatorial code to specify odor receptor gene choice (Bai, 2010).

There are several degrees of freedom in the splicing of acj6. Exons 2 and 3 can each be included or excluded, and exons 5 and 8 can each be included in either long or short forms. If all four of these splicing events occur independently, 24=16 splice forms could be produced. This study has identified 13 of these 16 forms in extensive but not exhaustive RT-PCR analysis (Bai, 2010).

Inclusion of either exon 2 or 3 disrupts the POU IV box, which is believed to mediate protein-protein interactions. acj6-M, which contains exon 3, conferred complete rescue of pb1A. These results suggest that the POU IV box is not required for all acj6 functions. Moreover, acj6-F and acj6-M, which differ only in the presence or absence of exon 3, conferred very similar or identical phenotypes in all tests in this study. Interestingly, Brn-3a and Brn-3b, mammalian orthologs of acj6, also undergo alternative splicing to generate transcripts that either include or lack the POU IV box (Bai, 2010).

acj6-I and acj6-J differ from each other only in the form of exon 5 that they contain. acj6-I and acj6-F differ from each other only in the form of exon 8 that they contain. The simplest interpretation of these results is that functional differences are conferred by the inclusion of extra amino acids in exons 5 and 8. The two amino acids that differ between exons 8s and 8l are conserved in C. elegans and mammalian orthologs of Acj6, and they are located in the POU homeodomain. One possibility is that the exclusion of these amino acids alters the DNA-binding properties of Acj6; alternative splicing of other transcription factors has previously been shown to affect DNA sequence discrimination (Bai, 2010).

acj6 is capable of acting late in development. When driven by a late-acting promoter, acj6 splice forms were able to rescue pb1A, pb2B, and pb3A cells. The ability of acj6 to act late is consistent with a direct role in Or gene regulation, which in turn is consistent with the finding that Acj6 binds directly to several Or promoters. It is noted that studies with a temperature-sensitive allele of lozenge, which encodes a Runx domain-containing transcription factor, showed that it could modulate expression of Or genes after eclosion. These results, taken together, are of special interest in light of recent findings that there is plasticity in the expression of some chemoreceptor genes. For example, Or59b was found to be expressed at higher levels in old males than in young males (Bai, 2010).

The process of odor receptor gene choice in Drosophila relies on a combinatorial code of transcription factors, which act in concert in the olfactory system to activate individual odor receptors while repressing all the others in individual ORNs. This study examined one of these transcription factors in detail and found that it is expressed in a variety of functionally distinct forms in the maxillary palp via alternative splicing. This variety expands the combinatorial code of receptor gene choice by adding another level of complexity. In this manner the number of transcription factors needed to govern the selection of odor receptor genes may be reduced. Moreover, the efficiency and flexibility of this regulatory system may be increased by the availability of an additional level at which natural selection can operate (Bai, 2010).

In addition to the problem of receptor gene choice, the olfactory system also faces the challenge of wiring specificity in the brain. Each ORN sends an axon to a particular destination in the antennal lobe of the brain, where it forms connections with projection neurons. acj6 plays a role in this axonal targeting, and it will be interesting to determine whether alternative splicing of acj6 adds a degree of freedom to this process as well. Moreover, acj6 is required for dendritic targeting of projection neurons, and a single splice form, acj6-F, when individually expressed in acj6 flies, rescued some of the acj6 dendritic targeting defects. Future studies may determine whether different acj6 splice forms play distinct roles in the regulation of cell adhesion molecules, for example, and thereby act in the differential selection of targets by different neurons (Bai, 2010).

Protein Interactions

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).

Ipou/abnormal chemosensory jump 6: Biological Overview | Evolutionary Homologs | Developmental Biology | Effects of Mutation | References

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