Gene name - abnormal chemosensory jump 6
Synonyms - Ipou, I-POU
Cytological map position - 13C1--13C3
Function - Transcription factor
Symbol - acj6
Genetic map position - 1-
Classification - Homeobox, POU domain
Cellular location - nuclear
The odor specificities of a subset of olfactory receptor neurons are governed by Acj6, the POU-domain transcription factor formerly known as Ipou. A jump response is elicited by exposing Drosophila to chemical vapors. acj6 was isolated in a search for flies that respond abnormally to this simple chemosensory stimulus. acj6 mutants were analyzed for the basis of their deficiency in response to chemical vapors. Olfactory coding depends on the existence of a functionally diverse population of olfactory receptor neurons (ORNs). Each class of ORN exhibits a distinct odor response profile, as revealed by physiological measurements of individual ORNs (see the Odorant Receptor site for more information). The odor specificity of these classes presumably reflects the expression of different odorant receptor genes. Little is known, however, about how the odor specificities of ORNs are generated during development. The olfactory system of Drosophila melanogaster allows convenient genetic, molecular, and physiological analysis of this problem. The fly contains two olfactory organs: the third segment of the antenna and the maxillary palp. Each organ is covered with sensory hairs that contain ORNs, with each hair on the maxillary palp housing two ORNs and hairs on the antenna containing up to four. Olfactory response can be measured in vivo, either behaviorally or physiologically. One means of physiological measurement is to record the receptor potentials of populations of ORNs in the vicinity of an extracellular electrode, employing recordings called electroantennograms (EAGs) or electropalpograms (EPGs). Another means is to record the action potentials of the neurons within a single sensory hair, in single-unit recordings (Clyne, 1999 and references).
Wild-type flies jump in response to a sudden pulse of odor, presumably as an escape response; acj6 was isolated by virtue of a reduced jump response to odor stimuli. Further analysis has revealed that acj6 flies have severe physiological deficits in response to some, but not all odors, as revealed by reductions in amplitudes of EAGs and EPGs (Ayer, 1991 and 1992). One kind of defect that might have different effects on responses to different odors is a defect affecting the specification of a subset of olfactory neurons (Clyne, 1999).
How are the identities of olfactory neurons specified? By analogy to other processes, such as early embryonic development in Drosophila, the generation of ORN phenotypes is likely to be governed largely by combinatorial interactions of transcription factors. Attractive candidates for key regulatory molecules in olfactory system development are POU-domain transcription factors. POU-domain proteins consist of a highly conserved 75 amino acid POU-specific domain, tethered by a linker of variable length and sequence to a 60-amino-acid homeodomain. A large number of POU-domain proteins from a broad range of organisms have now been identified, and members of the class IV subfamily are required for the development of specific phenotypes in subsets of sensory system neurons. In the mouse retina, three such class IV POU-domain proteins, Brn-3a, Brn-3b, and Brn-3c, are expressed in overlapping subsets of ganglion cells. Targeted disruption of Brn-3b leads to a selective loss of 70% of retinal ganglion cells; other neurons in the retina and brain are affected little if at all. Brn-3c is also expressed in hair cells in the auditory system, and a null mutation causes a failure of hair cells to differentiate. In C. elegans, the class IV POU-domain transcription factor encoded by the unc-86 gene is required for the proper differentiation of mechanosensory neurons (Clyne, 1999 and references).
Physiological analysis of individual olfactory neurons shows that in acj6 mutants, a subset of neurons acquires a different odorant response profile. To extend the functional and phenotypic analysis of the acj6 mutations to the level of the single cell, a single-unit recording technique was used to record from Drosophila olfactory hairs. Single-unit recording is an extracellular measure of the action potentials of the neurons housed in an individual sensillum, or sensory hair. Based on its amplitude and shape, each action potential from a recording can be assigned to a single neuron in the sensillum under study. This technique was used first to identify the various wild-type ORNs based on their odor-induced responses and then to determine whether these neurons are modified in the acj6 mutants. Single-unit studies were begun with the maxillary palp, one of the two olfactory organs in the adult fly, because of its numerical simplicity. On the surface of the maxillary palp, there are 60 olfactory sensilla, with each sensillum housing just two ORNs. The entire maxillary palp was mapped and it was found that the 120 ORNs can be grouped into six classes of about 20 neurons each, based upon their odor response spectra. There is also a second level of organization: these six classes of neurons are housed in characteristic pairs in three types of sensilla: types 1, 2, and 3. These six classes of neurons have been called 1A, 1B, 2A, 2B, 3A, and 3B. Thus, each 1A neuron, for example, has a particular odor response spectrum, and is always paired with a 1B neuron, which has a distinct spectrum (Clyne, 1999).
The odor specificities of single ORNs were examined in null mutants of acj6 to determine if they differed from wild type. The results clearly show that of the two neurons in the type 1 sensillum, only the 1A neuron is altered in acj6 flies. In wild type, the 1A neuron responds to several of the tested stimuli: it responds most strongly to ethyl acetate and moderately to several others. The wild-type 1B neuron, by contrast, is narrowly tuned: it responds strongly to 4-methylphenol, but shows no response to the other tested odors. In acj6, sensilla were found with neurons that show the same response spectrum as the 1B neuron, i.e., a strong response to 4-methylphenol and nothing else. However, a neuron with a response spectrum like 1A was never found in acj6 (Clyne, 1999). What happens to the 1A neuron in acj6 mutants? The recording data suggest that 1A can take on two distinct identities: (1) in some acj6 sensilla two 1B neurons are found, as if the 1A neuron is transformed into a second 1B neuron; (2) in other acj6 sensilla a neuron that does not respond to any of the tested odors is found alongside a single 1B neuron, as if the 1A neuron is transformed into this unresponsive neuron. Similarly, in the type 3 sensillum, the 3A neuron also appears to become an unresponsive neuron that is housed alongside a normal 3B neuron (Clyne, 1999).
acj6 alters the 2A and 2B neurons in a surprising manner. The wild-type 2A neuron responds weakly to most tested odors, and the wild-type 2B neuron is inhibited by some odors and excited by others. No sensilla on acj6 flies were found with neurons that have response spectra like either 2A or 2B. Instead, on acj6 flies, sensilla are found that house a neuron with a novel response spectrum unlike any of the wild-type neuronal classes: this neuron responds strongly to benzaldehyde, moderately to 4-methylphenol, and weakly to other tested odors. The response spectrum of this new neuron, which has been called 2C, cannot be explained in terms of any linear amplification or summation of the response spectra of other neuronal classes. The simplest interpretation is that the acj6 mutations transform either the 2A or the 2B neuron into the 2C neuron. In recordings from sensilla containing the novel 2C neuron, electrophysiological evidence is found for a second neuron. This neuron produces spontaneous action potentials but does not respond to any of the tested odors (Clyne, 1999).
Thus, in acj6 null mutants, four of the six classes of maxillary palp ORNs are altered: wild-type 1A and 3A are never observed, and 2A and 2B are transformed into an ORN with a novel response spectrum and a second, unresponsive ORN. 1B and 3B, by contrast, are unaffected by acj6 mutations; they retain their characteristic odor response profiles. It is noted that the unresponsive neurons in each sensillar type did not respond to any of 50 additional odors tested, nor to mechanical stimuli, nor to changes in temperature or humidity, and that the numbers of each sensillum type are normal in the acj66 maxillary palp. Single-unit recordings from acj61, a partial loss-of-function mutant, show a phenotype intermediate between that of acj66 and wild type, in the sense that in acj61 those neurons that are affected in acj66 are either like those of acj66, normal, or in an intermediate state (Clyne, 1999).
The single-unit data suggest that acj6 functions to specify the developmental fates of a subset of ORNs. Immunohistochemical techniques were used to examine Acj6 expression patterns in developing and adult olfactory tissue. A monoclonal antibody generated against a subregion of the Acj6 protein that is common to all isoforms was used. In the developing third antennal segment (the olfactory organ whose development has been best characterized) Acj6 is first detectable in a few cells at about 16 hr after puparium formation (APF). Interestingly, about 16 hr APF is also when the earliest differentiating ORNs are found in the third antennal segment. The number of Acj6-expressing neurons increases with developmental time and then stabilizes at about 36 hr APF, when Acj6 is expressed throughout the third antennal segment. Expression of Acj6 is absent in the third antennal segment of acj66 mutants. All identified isoforms of acj6 are expressed in the antenna throughout pupal development and in the adult (Clyne, 1999).
In the developing maxillary palp, Acj6 is first expressed in a few cells at about 31 hr APF. By 48 hr APF, there are 84 ± 1 (n = 8) Acj6-expressing cells. Expression of Acj6 is absent in developing maxillary palps of acj66 mutants. Only a subset of the acj6 isoforms are present in the adult maxillary palp. These studies reveal that Acj6 is expressed in all maxillary palp ORNs (Clyne, 1999).
If acj6 is not required for specification of all neuronal types in the maxillary palp, are there counterparts of acj6 that specify the other types? One attractive possibility is that other POU-domain genes are required for specifying other subsets of olfactory neurons. Of the four Drosophila POU-domain genes, at least two others, drifter and pdm-1, in addition to acj6, are also expressed in adult olfactory organs (unpublished data cited by Clyne, 1999). It seems plausible, then, that multiple POU-domain proteins function in concert, perhaps combinatorially, in the specification of all types of olfactory neurons. POU-domain proteins are able to function as heterodimers with other POU-domain proteins, further enriching the possibilities for combinatorial coding of neuronal type (Clyne, 1999).
Another degree of freedom is afforded by the alternative splicing of acj6. Evidence has been found for at least seven alternative splice forms of acj6, of which a subset is expressed in the adult maxillary palp. An intriguing possibility is that different isoforms of acj6 specify different ORN classes. It is also possible that different isoforms are required at different steps in the specification of ORNs. In acj6 null mutants, some neurons do not respond to any tested odors whatsoever (e.g., the mutant 3A cell), whereas others respond to an abnormal set of odors (e.g., the mutant 2C cell), as if different neuronal classes are disrupted at different steps in the ORN differentiation pathway. Perhaps acj6 functions at different steps in different ORNs, establishing a cellular context capable of odorant response in one ORN class, while selecting the particular type of odorant response in another class. If so, different functions of Acj6 might be carried out by different splice forms (Clyne, 1999).
How might Acj6 act in controlling the identity of receptor neurons? Most interesting, how might loss of acj6 function alter odor specificity, as in the transformation of a 2A or 2B neuron into the novel 2C neuron? It seems likely that Acj6 regulates the expression of certain downstream genes essential to establishing ORN identity, and a simple interpretation of how Acj6 determines odor specificity is that Acj6 regulates odorant receptor genes. Therefore, the results suggest that acj6 may regulate the expression of a subset of odorant receptor genes (Clyne, 1999).
The mechanism that specifies olfactory sensory neurons to express only one odorant receptor (OR) from a large repertoire is critical for odor discrimination but poorly understood. This study describes the first comprehensive analysis of OR expression regulation in Drosophila. A systematic, RNAi-mediated knock down of most of the predicted transcription factors identified an essential function of acj6, E93, Fer1, onecut, sim, xbp1, and zf30c in the regulation of more than 30 ORs. These regulatory factors are differentially expressed in antennal sensory neuron classes and specifically required for the adult expression of ORs. A systematic analysis reveals not only that combinations of these seven factors are necessary for receptor gene expression but also a prominent role for transcriptional repression in preventing ectopic receptor expression. Such regulation is supported by bioinformatics and OR promoter analyses, which uncovered a common promoter structure with distal repressive and proximal activating regions. Thus, these data provide insight into how combinatorial activation and repression can allow a small number of transcription factors to specify a large repertoire of neuron classes in the olfactory system (Jafari, 2012).
How many OR selector genes are required to uniquely express one OR in each OSN class? This study identified seven OR selector genes, but given the limitations of RNAi, it is likely that there are a total of at least ten critical TFs to specify all OSN classes. Even this probably low estimate generates a rather high number of TFs considering that Drosophila antennae have 34 OSN classes that express ORs. Theoretically the number of TFs needed for a binary combinatorial code to generate 34 unique outcomes is six (26 = 64). Seven TFs can in theory separate 27 = 128 combinations, and ten TFs designate more than 1,000 combinations, suggesting a large number of unused combinations. This surplus of combinations may be due to the inherent randomness of evolution and the impossibility of creating a streamlined code by chance. Another possibility for this large number is the need for a high degree of fidelity, with little or no ectopic OR expression tolerable for proper functioning of the olfactory system. Extrapolation of these observations to the regulatory requirements of the mammalian olfactory system indicates that at least 200-300 TFs would be required to provide a regulatory system that controls >1,000 mammalian ORs, a daunting number. Therefore, it is reasonable to suspect that the stochastic OR selection mechanism found in vertebrates was added during evolution to accommodate the heavy increase in regulatory costs resulting from an expanded number of OR genes (Jafari, 2012).
To date very few TFs have been found to be restricted to small neuronal populations in neuroepithelia or in the developing brain in general. This situation has motivated the suggestion that combinatorial TF regulation defines broad expression patterns of molecules such as neurotransmitters, but is insufficient to generate the large number of neuron classes in, for example, the olfactory system. Similarly, all seven selector genes in this study are expressed across the antenna but still are required for the expression of some few ORs. How can widely expressed TFs then produce restricted expression patterns? Two explanations were formulated. First, the promoter analysis suggests that the OSN class specificity is in part due to repression. Most ORs have a proximal regulatory region next to the gene that is sufficient for expression in OSNs but requires repression from more distal regions for the spatial restriction to each OSN class. In this model, the expression of the TFs that produce OR expression does not need to be particularly specific as long as they are counteracted by repressive factors. Second, the identified TFs can both activate and repress OR expression dependent on the location of the binding site or by the available cofactors. Dual use of the TFs might increase their regulatory power and as a likely consequence the number of TFs required for OR expression to be reduced. It is therefore suggested that specification of large numbers of neuron classes in the olfactory system and likely in the nervous system, require two layers of combinatorial coding, one layer of terminal selector genes that produce expression and a layer of repressors that restrict the expression to each class (Jafari, 2012).
tIpou and Ipou, alternatively spliced varients of the same gene both possess a POU domain and a homeodomain. Ipou is more closely related to the neuro-specific protein BRN-3 in mammals and UNC-86 in C. elegans and thus belongs to the POU-IV class. It shares (respectively) 82% and 77% amino-acid identity with BRN-3 and UNC-86, over the entire POU domain. Ipou differs from POU-IV class proteins in that it lacks two basic amino acids in the N-terminal cluster of basic residues found in the POU homeodomains. The alternatively spliced variant of Ipou, Twin of Ipou, contains the two basic amino acid residues absent in Ipou (Treacy, 1991 and 1992).
Among different POU-homeodomain proteins, the 75 amino acid POU-specific (POUs) domain and a 60 amino acid carboxy-terminal homeo (POUh) domain are joined by a hypervariable linker segment. In different POU domain proteins, this segment may vary in length from 15 to 56 amino acids. Thus the POU domain is not a single structural domain; indeed, the POUs and POUh segments form structurally independent, separate domains. The POUs and POUh domains are, however, always found together and have therefore coevolved. Both POUs and POUh domains contain helix-turn-helix motifs. The POUs-domain structure is very similar to that of lambda and 434 bacteriophage proteins, but there are significant differences in the length of the first alpha helix. The "turn" connecting the two HTH alpha helices is also longer. Both POUs and POUh bind DNA, and the length of the linker regulates the efficacy of binding various DNA sequence motifs, especially because POUs and POUh DNA binding sites have different spacings in different promoter elements (Herr, 1995).
Nine exons have been identified for acj6/ipou five new alternative splice forms have been found in addition to the two described previously (Treacy, 1992). There is a series of alternative splices at the 5' end of the transcript that either disrupt or leave intact the POU IV box protein motif. There exist alternative splice junctions in exon 5 and exon 8 but there is only one transcriptional start site. A splice form has been identified that lacks both exon 2 and exon 3 (Clyne, 1999).
date revised: 13 March 99
Home page: The Interactive Fly © 1995, 1996 Thomas B. Brody, Ph.D.
The Interactive Fly resides on the
Society for Developmental Biology's Web server.