Ipou/abnormal chemosensory jump 6

DEVELOPMENTAL BIOLOGY

Embryonic

Overall expression of Ipou and tIpou transcripts is minimal in the early embryo, but increases strongly by mid-embryogenesis, peaking in late embryonic development. IPOU and tIpou expression decline with respect to the total RNA pool present at later developmental stages, but this may in part represent growth and development of non-expressing tissues. Ipou is the preferred splice variant in the embryo, but tIpou expression is also detected (Turner, 1996).

Ipou transcripts are first detectable after germ-band shortening (Stage 13), and are localized to a subset of neurons in the supraoesophageal ganglia (brain) and the ventral cord. The subset of neurons expressing Ipou correspond in position and arrangement to the RP1, aCC/pCC and CQ neuron clusters (median stained cells). The EL cluster and two serotonergic neurons also strongly express Ipou (Treacy, 1991).

Larval and Pupal

Axonal selection of synaptic partners is generally believed to determine wiring specificity in the nervous system. However, evidence has been found for specific dendritic targeting in the olfactory system of Drosophila: second order olfactory neurons (Projection Neurons) from the anterodorsal (adPN) and lateral (lPN) lineages send their dendrites to stereotypical, intercalating but non-overlapping glomeruli. POU domain transcription factors, Acj6 and Drifter, are expressed in adPNs and lPNs respectively, and are required for their dendritic targeting. Moreover, misexpression of Acj6 in lPNs, or Drifter in adPNs, results in dendritic targeting to glomeruli normally reserved for the other PN lineage. Thus, Acj6 and Drifter translate PN lineage information into distinct dendritic targeting specificity. Acj6 also controls stereotypical axon terminal arborization of PNs in a central target, suggesting that the connectivity of PN axons and dendrites in different brain centers is coordinately regulated (Komiyama, 2003).

Prior to this study of PNs, it was generally believed that synaptic connection specificity is conferred by selection of synaptic partners by presynaptic axons. Systematic lineage analysis strongly suggests that PN dendrites play an active role in establishing connection specificity. Specifically, a given PN's lineage and birth order predicts its glomerular target. However, the position of a given PN's target glomerulus is correlated neither with its neuroblast lineage nor with birth order. Thus, it is unclear how a PN's lineage contributes to its dendritic targeting specificity (Komiyama, 2003).

Molecular genetic evidence is provided that this active dendritic targeting is controlled by transcriptional programs within PNs. The data suggest that the observed dendritic targeting specificity is achieved in two steps: specification of a particular lineage and further intra-lineage specification. The POU domain transcription factors Acj6 and Dfr play critical roles in the first step (Komiyama, 2003).

Several lines of evidence support the idea that Acj6 and Drifter play analogous roles in translating lineage information into dendritic targeting specificity of adPNs and lPNs. (1) Acj6 and Dfr are mutually exclusively expressed in adPNs and lPNs; this lineage-specific expression could be used to regulate the distinct wiring specificity of these two PN lineages. (2) Loss-of-function phenotypes in neuroblast clones demonstrate that Acj6 and Dfr are required for proper dendritic targeting of at least a subset of PNs in their respective lineages. The neuroblast clone phenotypes likely underestimate the requirement of Acj6 or Dfr in PN dendritic targeting. Since each glomerulus is innervated by an average of 3 PNs, it might not be possible to detect inappropriate targeting if 1 or 2 PNs in the same class still innervate the glomerulus properly. This possibility is supported by the study of DL1 PNs. In neuroblast clone analysis, 11 out of 19 acj6^-/- clones exhibited no detectable defects in DL1 glomerular innervation; in single-cell clone analysis with a higher resolution, each of the 11 clones showed significant phenotypes. Results from single-cell clone analysis of other PN classes support the generality of the DL1 phenotype -- failure to innervate one specific glomerulus (Komiyama, 2003).

(3) Misexpression of Acj6 in lPNs, or Dfr in adPNs, leads to dendritic targeting defects. In the case of Acj6 misexpression in lPNs, where the phenotypes are stronger (possibly due to a higher ratio of transgene to endogenous Acj6 expression than could be observed for Dfr transgene/endogenous Dfr), there are two qualitatively different mistargeting phenotypes. The first is non-specific accumulation of dendrites in the lateral part of the antennal lobe with associated glomerular organization defects. This phenotype is analogous to the non-specific accumulation of adPN dendrites in the dorsal part of the antennal lobe in acj6^-/- adPN clones and may reflect a default response of dendrites deprived of targeting information. The second class of phenotypes is more revealing. In this case, lPN dendrites are mistargeted to well-defined dorsal landmark glomeruli distant from lPN cell bodies and areas of non-specific accumulation. Certain inappropriate glomeruli are specifically targeted, while their neighbors remain uninnervated; this observation argues against the alternative interpretation that misexpression simply causes non-specific dendritic spillover. The specificity of the mistargeting phenotypes caused by misexpression is further supported by the following two observations: (1) overexpression of Acj6 in adPNs, or Dfr in lPNs, never results in any phenotypes; and (2) specific mistargeting is not observed in loss-of-function mutants (Komiyama, 2003).

Taken together, these results strongly suggest that Acj6 and Dfr participate in instructing adPNs and lPNs to innervate a set of glomeruli appropriate to each lineage. At present, it remains probable that other transcription factors act in concert with Acj6 and Dfr to completely specify these lineage-dependent wiring programs. The existence of these other factors -- in addition to the likely underestimation of phenotypes in neuroblast clone analysis, or perdurance in the case of Dfr -- may explain why both loss-of-function and gain-of-function experiments affect only specific subsets of glomeruli (Komiyama, 2003).

It is important to note that Acj6 and Dfr alone cannot specify a particular PN to target its dendrites to a particular glomerulus. All adPNs express Acj6, yet they project their dendrites to a series of different glomeruli according to their birth order. There must be timing factors, probably also transcription factors, which further distinguish PNs within the same lineage based on their birth order. An elegant mechanism to specify different progeny from a common neuroblast has recently been described in the Drosophila embryonic CNS, where neuroblasts exhibit asymmetric cell division patterns similar to those giving rise to PNs. In the embryonic CNS, the neuroblast changes its transcription factor profile as a function of time, thereby specifying the fate of neurons born at different stages. It is suspected that analogous timing factors might exist in PN lineages. These timing factors, in collaboration with lineage-specific factors, will ultimately specify the expression of a repertoire of cell surface molecules that allow PNs to target their dendrites precisely to specific glomeruli (Komiyama, 2003).

Could the same hypothetical timing factors be used in both lineages? This was tested by attempting to switch the DL1 class of adPN to its lPN equivalent by simultaneously removing Acj6 and misexpressing Dfr. If the only differences between the DL1 adPN and its lPN equivalent are the POU domain lineage factors, it might be expected that the DL1 PNs lacking Acj6 but expressing Dfr now would target to a novel glomerulus. These PNs indeed acquire novel features compared to simple loss of Acj6. They no longer even partially innervate DL1. In a subset of clones, their axons also acquired novel branching patterns and terminal fields. However, a clear switch is not observed based both on these dendritic or axonal phenotypes. This could be due to inappropriate level and/or timing of transgene expression; it could also be because: (1) the hypothetical timing factors are not exactly the same in adPNs and lPNs; (2) Acj6 and Dfr are not the only factors distinguishing these two lineages, or (3) cell-cell interaction among PNs from the same lineage may play a role in determining targeting specificity (Komiyama, 2003).

Acj6 is necessary not only for PN dendritic targeting, but also for establishing highly stereotyped PN axon branching patterns and terminal fields in a higher olfactory center. This is best exemplified by the analysis of DL1 single-cell clones. acj6^-/- DL1 PNs are defective specifically in the dorsal branch without affecting general axon growth and guidance. This specific phenotype suggests that Acj6 plays a role in selecting synaptic connections with specific third order neurons. Axon terminal arborizations of other classes of PNs are also likely to be regulated by Acj6, as revealed by phenotypes from neuroblast clones containing ~13 classes of adPNs. As for Dfr, there is no evidence from loss-of-function studies that it plays a role in PN axon terminal arborization because there is no equivalent in the lateral lineage to the DL1 PN, which can be unambiguously identified independent of its dendritic innervation. However, the fact that simultaneous loss of Acj6 and gain of Dfr in DL1 clones result in qualitatively different axonal phenotypes compared with simple loss of Acj6 suggests that Dfr also plays a role in regulating axon terminal arborization in the lateral horn (Komiyama, 2003).

These observations bring back the question of why PNs are prespecified to project their dendrites to specific glomeruli and thereby receive specific olfactory input, and to have axons exhibiting specific branching patterns and terminal fields, presumably allowing stereotyped connections with third order neurons. By making PNs genetically distinct at the outset, it is possible to coordinate the dendritic choices of different glomeruli and the specific connections made by axons in higher centers. This coordination may contribute to innate behavioral responses to odorant stimuli by allowing a highly stereotyped relaying of olfactory information from the periphery to higher olfactory centers. Mechanistically, it is possible that PNs use similar cell surface molecules, whose expression depends on specific transcription factors such as Acj6 and Dfr, to guide both dendrites and axons to appropriate targets. The dual Acj6 phenotypes (both axonal and dendritic) provide support for this hypothesis. In ongoing forward genetic screens and candidate tests to identify genes necessary for PN dendritic and axonal connectivity, additional mutants have been found with simultaneous defects in dendritic targeting and axonal arborization (Komiyama, 2003).

In theory, the dual phenotypes in dendrites and axons could be caused by primary defects in dendritic targeting, with axon arborization defects as a secondary consequence, or vice versa. However, two lines of evidence argue against such possibilities: (1) developmental studies indicate that there is not a sequential development of dendritic and axonal arborization; (2) different mutants exhibit different ranges and specificity in their axonal and dendritic phenotypes -- even for individual PNs with the same mutant genotype, there was no clear correlation between the severity of dendritic and axonal phenotypes. The possibility is thus favored that the correct targeting of PN axons and dendrites are both directly regulated events rather than a sequential process in which, for example, the correct targeting of dendrites then instructs the corresponding axonal arborization (Komiyama, 2003).

POU domain transcription factors are used widely in C. elegans, Drosophila, and mammalian development. In particular, classes III and IV POU domain proteins play a variety of important roles in neural development. C. elegans UNC-86, the founding member of the POU IV class, is expressed shortly after asymmetric division in one of the two daughter cells. In unc-86 mutants, the daughter neuroblast that usually expresses UNC-86 now acquires the fate of its parental neuroblast, resulting in reiterations of cell lineage. UNC-86 also regulates differentiation of a number of neuronal classes such as touch sensory neurons or HSN motor neurons. In mammals, 3 class IV and 4 class III POU domain proteins are widely expressed in the nervous system during development. Knockout experiments demonstrate their important functions in different developmental processes. Because there is genetic redundancy between members of the same class, however, phenotypes resulting from single gene knockouts tend to reflect defects in cells that uniquely express that particular POU domain protein (Komiyama, 2003).

Acj6 and Dfr are respectively the single existing members of the class IV and class III POU domain proteins in Drosophila. Both genes have been shown to play a variety of roles in development. In particular, photoreceptor axon targeting is disrupted in acj6 mutants, however this phenotype is not cell autonomous (Acj6 is not expressed in photoreceptors) and is probably due to a requirement for Acj6 in the target lamina neurons. By restricting genetic manipulations to a small subset of neurons with well-defined connection specificity, the requirement of Acj6 and Dfr in other developmental events is bypassed and focus was placed on their function in olfactory projection neurons. This study assigns a new function for POU domain proteins: regulating lineage-dependent wiring specificity down to specific synapse formation. Interestingly, PNs from two lineages utilize two POU domain proteins of different classes for analogous functions. It remains to be seen whether the large number of mammalian POU domain proteins could be used in this way to regulate the specificity of numerous connections necessary to assemble the mammalian nervous system (Komiyama, 2003).

Lastly, Acj6 functions in a subset of ORs to regulate the expression of olfactory receptors; it is possible that it also regulates other molecules including putative ORN axon targeting molecules (which are likely to be distinct from the ORs themselves). The demonstration that Acj6 is necessary for dendritic targeting specificity of a subset of PNs raises an intriguing possibility that Acj6 may regulate matching ORNs and PNs destined to form synaptic connections. In fact, Acj6 is also expressed in a subset of neurons whose cell bodies are located near the lateral horn, one of the two central targets of PN axons. Thus, it is even feasible that Acj6 also regulates matching of synaptic partners in the next olfactory center. Molecular markers and other genetic tools are currently being developed to test these intriguing possibilities (Komiyama, 2003).

Intrinsic control of precise dendritic targeting by an ensemble of transcription factors

Proper information processing in neural circuits requires establishment of specific connections between pre- and postsynaptic neurons. Targeting specificity of neurons is instructed by cell-surface receptors on the growth cones of axons and dendrites, which confer responses to external guidance cues. Expression of cell-surface receptors is in turn regulated by neuron-intrinsic transcriptional programs. In the Drosophila olfactory system, each projection neuron (PN) achieves precise dendritic targeting to one of 50 glomeruli in the antennal lobe. PN dendritic targeting is specified by lineage and birth order, and their initial targeting occurs prior to contact with axons of their presynaptic partners, olfactory receptor neurons. A search was performed for transcription factors (TFs) that control PN-intrinsic mechanisms of dendritic targeting. Two POU-domain TFs, acj6 and drifter have been identified as essential players. After testing 13 additional candidates, four TFs were identified, (LIM-homeodomain TFs islet and lim1, the homeodomain TF cut, and the zinc-finger TF squeeze) and the LIM cofactor Chip, that are required for PN dendritic targeting. These results begin to provide insights into the global strategy of how an ensemble of TFs regulates wiring specificity of a large number of neurons constituting a neural circuit (Komiyama, 2007).

For technical simplicity, larval born GH146-Gal4-positive PNs, originating from three neuroblast lineages, anterodorsal (adPNs), lateral (lPNs), and ventral (vPNs), were studied. Out of ~25 classes defined by their glomerular targets, focus was placed on 17 classes whose target glomeruli are reliably recognized across different animals. The MARCM technique allows visualization and genetic manipulation of PNs in neuroblast and single-cell clones in otherwise heterozygous animals, so PN-intrinsic programs can be studied for dendritic targeting. GH146 is expressed only in postmitotic PNs (Komiyama, 2007).

acj6 and drifter have been identified as lineage-specific regulators of PN dendritic targeting. To identify additional transcription factors (TFs) that regulate dendritic targeting of different PN classes, candidates were tested that have been shown to regulate neuronal subtype specification and targeting specificity and have available loss-of-function mutants. The following was tested; (1) the expression of candidate genes in PNs at 18 hr after puparium formation (APF) when PN dendrites are in the process of completing their initial targeting, and/or (2) their requirement in PNs by examining dendritic targeting in homozygous mutant MARCM clones (Komiyama, 2007).

DL1 adPN expresses Acj6, an adPN lineage factor, but not Drifter or Cut. acj6^−/− DL1 PNs typically have diffuse dendrites that always innervate, but are not limited to, DL1. drifter misexpression alone did not affect their dendritic targeting. However, when loss of acj6 and gain of drifter were combined, the dendrites completely missed DL1 and targeted anterior glomeruli (Komiyama, 2007).

Misexpression of Cut alone caused DL1 PNs to target part of DL1 and the vicinity, similar to acj6^−/−. Notably, this diffuse phenotype was directional, because most mistargeted dendrites targeted medially to DL1 (Komiyama, 2007).

cut misexpression combined with loss of acj6 caused severe mistargeting of DL1 adPNs. The dendrites completely missed DL1 and occupied the medial to dorsomedial AL, typically VM2, DM6, and DC1. Interestingly, these glomeruli are all adPN targets near DM1 and DM2, the two glomeruli that most frequently fail to be innervated by cut^−/− lPNs. One interpretation is that loss of acj6 made the DL1 adPN more sensitive to the instructive information of cut to target the medial AL, but the remaining lineage information kept the dendrites within the adPN glomeruli in the area. If this were true, adding a lPN lineage factor drifter may bring the dendrites to DM1 or DM2, since this might recreate, based on partial knowledge of the TF code, a code for targeting these glomeruli. Loss of acj6 and misexpression of cut and drifter were combined simultaneously in DL1 adPNs. Under this condition, the dendrites again mostly targeted the medial to dorsomedial AL. However, glomerular preferences were strikingly different: they frequently innervated 1, DM2, and DA2. Notably, DA2 and DM2 are lPN targets (Komiyama, 2007).

These results suggest that cut and drifter have qualitatively different instructive information, with cut controlling global targeting and drifter controlling local glomerular choice according to their lineage (Komiyama, 2007).

Manipulation of an innate escape response in Drosophila: photoexcitation of acj6 neurons induces the escape response

The genetic analysis of behavior in Drosophila has linked genes controlling neuronal connectivity and physiology to specific neuronal circuits underlying a variety of innate behaviors. This study investigated the circuitry underlying the adult startle response, using photoexcitation of neurons that produce the abnormal chemosensory jump 6 (acj6) transcription factor. This transcription factor has previously been shown to play a role in neuronal pathfinding and neurotransmitter modality, but the role of acj6 neurons in the adult startle response was largely unknown. This study shows that the activity of these neurons is necessary for a wild-type startle response and that excitation is sufficient to generate a synthetic escape response. Further, it was shown that this synthetic response is still sensitive to the dose of acj6 suggesting that that acj6 mutation alters neuronal activity as well as connectivity and neurotransmitter production. These results extend the understanding of the role of acj6 and of the adult startle response in general. They also demonstrate the usefulness of activity-dependent characterization of neuronal circuits underlying innate behaviors in Drosophila, and the utility of integrating genetic analysis into modern circuit analysis techniques (Zimmermann, 2009).

Photostimulation of genetically specified neural circuits using the directed expression of the ChR2 channel rhodopsin can be effectively used to regulate neuronal output. The stimulation of aminergic neurons in Drosophila larvae, for example, was previously shown to induce aversive and appetitive reinforcement. More recently, ChR2 was used to manipulate odorant neurons in adult flies. This study demonstrates that ChR2 stimulation offers temporal control of neural activity with millisecond precision over a wide range of frequencies, and can be used to manipulate both larval and adult behaviors. Such tight control of neural activity is a prerequisite if such photostimulation is to be used to mimic environmental stimuli, or for activation-based screens to identify the neural circuitry underlying innate behaviors (Zimmermann, 2009).

To apply ChR2 to behaviorally significant circuitry, neurons necessary for the adult startle response were manipulated. The startle response is disrupted by mutations in the acj6 transcription factor. acj6 expression is found in neurons at multiple points along sensory pathways, and is required for their proper specification and wiring. Because acj6 appears to regulate the physiology of a subset of these neurons, it was hypothesized that activity of neurons expressing acj6 would be necessary and sufficient for the jump behavior that does not occur in acj6 mutants (Zimmermann, 2009).

This study has shown that the activity of acj6 neurons is necessary for a physical startle response response, and that silencing these neurons recapitulates the mutant phenotype. Stimulation of these neurons through ChR2 photostimulation induces a synthetic jump response that mimics the wild-type startle response. This suggests that the escape response associated with the acj6 phenotype is in fact mediated by acj6-expressing neurons, whose activity is necessary and sufficient for this behavior (Zimmermann, 2009).

Photostimulation is capable of inducing a wild-type response in males hemizygous for the acj6-GAL4 insertion, and induces an attenuated response of females carrying acj6-GAL4 in trans to an acj6 deficiency. The more robust effect in males may be attributable to dosage compensation, which could increase transcription of acj6, to a level sufficient for photoexcitation but not sufficient for wild-type behavior, or of gal4, to a level sufficient to overcome the acj6-GAL4 mutation. Several roles for acj6 are compatible with this effect: the acj6-GAL4 mutation may reduce or alter activity in some neurons, such that photoexcitation can induce a jump response but normal stimuli cannot. Alternatively, the acj6-GAL4 mutation may alter connectivity, as reported for acj6 mutation in photoreceptors and projection neurons (Zimmermann, 2009).

Preventing ChR2 expression in cholinergic neurons with cha-Gal80 partially eliminates the synthetic jump response. The remaining activity may be due to residual photoexcitation of some cholinergic acj6 neurons, or ChR2 function in non-cholinergic acj6 neurons - largely limited to CNS interneurons - may be sufficient to drive the synthetic jump response. The latter possibility would mean that a subset of acj6 neurons comprises a circuit whose activity is necessary and sufficient for the escape response (Zimmermann, 2009).

Several drawbacks to photostimulation remain. In most experiments, illumination levels are quite high, and reliable photostimulation of the CNS is limited by the ability to provide enough light to penetrate the cuticle without overheating the animal. More detailed analyses, such as eliciting a behavior through the excitation of single neurons, remains elusive but may be resolved by MARCM techniques or development of a library of GAL80 repressors (Zimmermann, 2009).

Photostimulation of genetically identified neural populations is a powerful tool for inhibiting or inducing behaviors in an awake animal. Directed stimulation of neuronal populations can be used to identify novel circuitries underlying other innate behaviors. Because these experiments rely upon genetic targeting, they will also reveal genes and enhancers whose expression is limited to specific neurons underlying a behavioral circuit and may therefore play a role in their formation or physiology (Zimmermann, 2009).

Photostimulation may also be used to manipulate neuronal activity in the behaving animal to understand how information is processed during behavior, and reveal the modulation of sensation, integration, and motor output during behavior. When integrated with genetic and physiological analyses, these approaches will contribute powerfully to revealing the genetic and neural basis of behavior (Zimmermann, 2009).

Effects of Mutation

Although the Drosophila visual system has been described extensively, little is known about the Drosophila olfactory system. A major reason for this discrepancy has been the lack of simple, reliable means of measuring response to airborne chemicals. This paper describes a jump response elicited by exposing Drosophila to chemical vapors. This behavior provides the basis for a single-fly chemosensory assay. The behavior exhibits dose dependence and chemical specificity: it is stimulated by exposure to ethyl acetate, benzaldehyde, and propionic acid but not ethanol. Animals can respond repeatedly at short intervals to ethyl acetate and propionic acid. The response relies on the third antennal segments. To illustrate the use of this behavior in genetic analysis of chemosensory response, nine acj (abnormal chemosensory jump) mutants defective in chemosensory response were isolated, and their responses to two chemicals were characterized. All of the acj mutants are normal in giant fiber system physiology, and two exhibit defects in visual system physiology (McKenna, 1989).

Mutations affecting olfactory behavior provide material for use in molecular studies of olfaction in Drosophila. Using the electroantennogram (EAG), a measure of antennal physiology, an adult antennal defect has been found in the olfactory behavioral mutant abnormal chemosensory jump 6 (acj6). The acj6 EAG defect was mapped to a single locus. The same mutation is responsible for both reduction in EAG amplitude and diminished behavioral response, as if reduced antennal responsiveness to odorant is responsible for abnormal chemosensory behavior in the mutant. acj6 larval olfactory behavior is also abnormal; the mutation seems to alter cellular processes necessary for olfaction at both developmental stages. The acj6 mutation exhibits specificity in that visual system function appears normal in larvae and adults. These experiments provide evidence that the acj6 gene encodes a product required for olfactory signal transduction (Ayer, 1991).

This article provides characterization of the electrical response to odorants in the Drosophila antenna and provides physiological evidence that a second organ, the maxillary palp, also has olfactory function in Drosophila. The acj6 mutation, previously isolated by virtue of defective olfactory behavior, affects olfactory physiology in the maxillary palp as well as in the antenna. Interestingly, abnormal chemosensory jump 6 (acj6) reduces response in the maxillary palp to all odorants tested except benzaldehyde (odor of almond), as though response to benzaldehyde is mediated through a different type of odorant pathway from the other odorants. In other experiments, different parts of the antenna are shown to differ with respect to odorant sensitivity. Evidence is also provided that antennal response to odorants varies with age, and that odorants differ in their age dependence (Ayer, 1992).

Mutations in the Drosophila class IV POU domain gene abnormal chemosensory jump 6 (acj6) cause physiological deficits in odor sensitivity. However, loss of Acj6 function also has a severe detrimental effect on coordinated larval and adult movement that cannot be explained by the simple loss in odorant detection. In addition to olfactory sensory neurons, Acj6 is expressed in a distinct subset of postmitotic interneurons in the central nervous system from late embryonic to adult stages. In the larval and adult brain, Acj6 is highly expressed in central brain, optic and antennal lobe neurons. Loss of Acj6 function in larval optic lobe neurons results in disorganized retinal axon targeting and synapse selection. Furthermore, the lamina neurons themselves exhibit disorganized synaptic arbors in the medulla of acj6 mutant pupal brains, suggesting that Acj6 may play a role in regulating synaptic connections or structure. To further test this hypothesis, two Acj6 isoforms were misexpressed in motor neurons where they are not normally found. The two Acj6 isoforms are produced from alternatively spliced acj6 transcripts, resulting in significant structural differences in the amino-terminal POU IV box. Acj6 misexpression causes marked alterations at the neuromuscular junction, with contrasting effects on nerve terminal branching and synapse formation associated with specific Acj6 isoforms. These results suggest that the class IV POU domain factor, Acj6, may play an important role in regulating synaptic target selection by central neurons and that the amino-terminal POU IV box is important for regulation of Acj6 activity (Certel, 2000).

The central brain neuropils include the antennal lobes (the first central region for processing olfactory information), the mushroom bodies (higher order structures involved in complex behaviors) and the central complex (a region necessary for the coordination and modulation of motor activities). A large subset of central brain interneurons express Acj6 including central complex neurons, which function to receive, process and convey information from one site within the nervous system to another. A loss of Acj6 function could therefore affect the ability to carry commands necessary to direct motor activity and thus serve to provide a reasonable hypothesis for the quantifiable reduction in coordinated movement exhibited by acj6 mutant flies. Acj6 is also expressed in the optic lobes in regions functioning as hierarchical processing sites for visual information. No Acj6 expression is detected in photoreceptor cells but instead Acj6 optic lobe expression is initiated in differentiated neurons that receive R cell synaptic input. Differentiated lamina and medulla neurons as well as neurons in the lobula express Acj6 through adult stages (Certel, 2000).

Although motor activity defects are probably due to Acj6 function in the central complex, as an initial step in determining whether Acj6 is required for events following lineage determination and initial axon guidance, focus was placed on the easily visible axonal projections and organized synapses found in the optic lobe. In the wild-type larval eye-brain complex, R cell axons project from the eye disc, through the optic stalk and into the optic ganglia. R1-R6 photoreceptors send their axons to the lamina ganglion layer of the brain where their growth cones form an array of postsynaptic 'cartridge' units. Each cartridge unit contains the set of R1-R6 axons, five lamina neurons (L1-5) and several glial cells. Acj6 expression is observed in the synaptic partners of the R cells, the lamina and medulla neurons, but not in the R cells themselves. To first analyze any effects that loss of Acj6 function might have on synapses as a post-synaptic target, the organization of R cell projections was assessed in acj6 mutants using the R cell-specific antibody mAb 24B10. The array of expanded R1-R6 growth cones appears as a continuous line of immunoreactivity in wild-type larvae. In acj6 mutants, R cell axons project into the brain in a wild-type manner; however, these fibers do not uniformly form the lamina neuropil. In a acj6 heteroallelic combination, occasional gaps are observed in the lamina plexus. In the acj6 null larvae, the entire lamina plexus is of variable thickness generating irregular small breaks. Therefore, the loss of Acj6 function in the R cell synaptic partners affects the ability of these R cell growth cones to establish connections with the appropriate lamina neuron column (Certel, 2000).

Defects in R cell connectivity could be due to a loss of neurons or abnormal lamina neuron specification. In acj6 mutants, lamina precursor cell (LPC) proliferation and initial lamina neuron differentiation are wild type, as assessed using anti-Dachshund and anti-Elav staining. At the level of antibody labeling, the organization of lamina neurons into columns also appears largely normal. Possible explanations for the connectivity defects may be the inability of the R cell growth cones to recognize their synaptic partners or to adhere correctly and form a stable synaptic cartridge (Certel, 2000).

To investigate possible pre-synaptic changes in Acj6- expressing lamina monopolar neurons, mAb 1D4, which is directed against the cell adhesion molecule Fasciclin II, was used. The axons and synapses of a subset of lamina neurons, L1 and L3, express Fasciclin II during selected stages of pupal development as they project in a highly structured pattern into the distal medulla. In acj6 mutants, the L1 and L3 arborizations and terminals are disorganized and unevenly spaced. In addition, the structure of the synaptic terminals is diffuse and overlaps are observed. These results provide further evidence that Acj6 is not required for initial differentiation steps such as axon pathfinding, but instead may be necessary for target cell selection and the establishment of synaptic connections (Certel, 2000).

To further test the hypothesis that Acj6 may regulate the formation of synaptic connections, Acj6 was misexpressed in motor neurons in regions where it is not normally found in order to observe any distinct morphological effects upon the well-characterized neuromuscular junction (NMJ). However, multiple acj6 transcripts have been identified and it is not clear whether different Acj6 isoforms are capable of unique functions. The Acj6 protein contains two domains with extensive homology to the vertebrate class IV members: Brn-3a, Brn-3b and Brn-3c. In addition to the DNA-binding POU domain, members of this group contain a class IV-specific 40-amino-acid POU IV box at the N-terminal end. Four of the five acj6 transcripts differ only in the use of the four small exons encoding the N-terminal POU IV box (amino acids 88-119). Neither the POU IV box nor the POU domain are affected in the fifth alternatively spliced transcript, which utilizes an alternative consensus acceptor site to generate a short form of exon 5 (Certel, 2000).

It was therefore imperative to determine whether the predicted Acj6 isoforms are expressed in vivo so that the significance of any differences in functional capability could be evaluated. Alternatively spliced Acj6 transcripts should produce proteins with predicted molecular masses of 40.0, 41.2, 42.2 and 43.5 kDa. A cluster of bands corresponding to proteins of the predicted size was detected using the Acj6 antibody on Western blots. The cluster of anti-Acj6 immunoreactive bands is absent in extracts from acj6 null flies demonstrating that multiple Acj6 isoforms are expressed (Certel, 2000).

Based upon previous experiments with other members of the POU domain family, it was not expected that alterations in the Acj6 POU IV box would have significant effects on DNA-binding activity. To verify that amino-terminal changes do not affect the ability of Acj6 isoforms to bind DNA, fusion proteins were generated and used in gel mobility-shift assays. All of the Acj6 isoforms differing in the highly conserved POU IV box are capable of binding octamer and neuronal DNA recognition elements with affinities comparable to wild type. Although the POU IV box does not appear to be important for DNA-binding, in vitro studies indicate that this region is necessary for both the transforming activity of Brn-3a and activation of specific promoters. The Gal4/UAS system was used to analyze the functional capabilities of two Acj6 isoforms, Acj6(1,4) and Acj6(1,3,4), through misexpression studies (the numbers in parentheses represent the protein primary structure in terms of the contributions of the first four exons) (Certel, 2000).

Transgenic strains carrying a UAS-acj6(1,4) or UAS-acj6(1,3,4) transposon were mated with either scabrous-GAL4 (sca-GAL4) or elav-GAL4 flies to express the Acj6 isoforms at high levels in all neurons. Acj6(1,4) and Acj6(1,3,4) differ only in the absence or presence of exon 3 encoding a portion of the conserved POU IV box. Multiple transgenic lines were tested to eliminate the possibility that differences in phenotypes might be due to the position of insertion. In addition, expression of Acj6 proteins at comparable levels, from each of the UAS-acj6 transposons, was confirmed by labeling with Acj6 antibody. In each abdominal hemisegment of the Drosophila embryo and larva, the axons of approximately 40 motor neurons exit the ventral nerve cord and specifically synapse with 30 identified muscle fibers. The analysis focused on the well described motor axons of the ISNb fascicle visualized using mAb 1D4. The ISNb fascicle contains motor axons from at least four motor neurons innervating the ventral muscles 6, 7, 12 and 13. The ISNb fascicles of sca-GAL4/UAS-acj6(1,3,4) embryos correctly leave the nerve cord, defasciculate from the ISN and project to target muscle clefts in nearly all late stage 16/stage 17 hemisegments examined. However, in many of the hemisegments with normal initial axon pathfinding, a striking increase in the number of nerve terminal branches and processes arising from each motor axon was observed. In addition, some of the ectopic terminal branches are able to extend and form connections onto inappropriate muscle fibers. To quantitate the observed increase in ectopic boutons, transgenic sca-GAL4/UAS-acj6(1,3,4) embryos were allowed to develop to crawling third instar larvae. Analysis of the larger NMJs in these larvae indicates that a subset of the ectopic terminal branches generated in the embryo were maintained, increasing the number of boutons by 27% at muscles 6 and 7 and 22% at muscles 12 and 13. The ectopic boutons also expressed the synaptic markers Synaptotagmin and Cysteine string protein, consistent with functional synapses. In sharp contrast, misexpression of the Acj6(1,4) isoform causes a failure of motor axons of the ISNb fascicle to defasciculate from the ISN fascicle, resulting in defective innervation of the ventrolateral muscle field in approximately 65% of embryonic hemisegments examined. In approximately 15% of the hemisegments, the ISNb motor axons stop at the correct location of their target muscles but do not branch out to innervate the ventral muscle groups. The remaining hemisegments (20%) show a wild-type pattern of muscle innervation. These studies suggest that distinct Acj6 isoforms can influence specific aspects of nerve terminal branching and synapse formation. In addition, this activity appears to be mediated by differential activities of the N-terminal POU IV box (Certel, 2000).

A new Drosophila POU gene, pdm3, acts in odor receptor expression and axon targeting of olfactory neurons; pdm3 interacts genetically with acj6

Olfaction depends on the differential activation of olfactory receptor neurons (ORNs) and on the proper transmission of their activities to the brain. ORNs select individual receptors to express, and they send axons to particular targets in the brain. Little is known about the molecular mechanisms underlying either process. This study has identified a new Drosophila POU gene, pdm3, that is expressed in ORNs. Genetic analysis shows that pdm3 is required for odor response in one class of ORNs. This study shows that pdm3 acts in odor receptor expression in this class and that the odor response can be rescued by the receptor. Another POU gene, acj6, is required for receptor expression in the same class, and a genetic interaction was found between the two POU genes. The results support a role for a POU gene code in receptor gene choice. pdm3 is also expressed in other ORN classes in which it is not required for receptor expression. For two of these classes, pdm3 is required for normal axon targeting. Thus, this mutational analysis, the first for a POU class VI gene, demonstrates a role for pdm3 in both of the processes that define the functional organization of ORNs in the olfactory system (Tichy, 2008).

Each olfactory receptor neuron (ORN) makes two remarkable choices that underlie the sense of smell. ORNs select individual odor receptor genes to express, a process that determines which odors they detect, and they send axons to particular targets in the brain, which determines what behaviors are elicited by the odors. The molecular mechanisms underlying both choices are largely unknown (Tichy, 2008).

ORNs in Drosophila are contained within two organs, the antenna and the maxillary palp. The odor response spectrum of an ORN class is conferred by the expression of one or a small number of odor receptor (Or) genes. The organization of ORN classes is stereotyped, and depends on the proper selection of individual Or genes from among a large repertoire. ORNs send axons to individual glomeruli, which are spheroidal modules in the antennal lobe of the brain. ORNs that express the same receptor converge on the same glomerulus (Tichy, 2008).

In Drosophila, the expression of particular Or genes, and hence the odor specificity of the ORN, depends on a regulatory code of cis-acting elements (Ray, 2007; Ray, 2008). Transcription factors that act in the process of receptor gene expression include abnormal chemosensory jump 6 (Acj6), a POU transcription factor. The acj6 gene was identified by a defect in olfactory behavior. In a null mutant of acj6, some maxillary palp ORNs respond normally to odors, some are present but have lost response to all odors, and some undergo changes in odor specificity. Correspondingly, acj6 is required for the expression of a subset of Or genes (Tichy, 2008).

The present study identified a new POU gene, pdm3, a POU gene of class VI. pdm3 is expressed in ORNs. Loss of pdm3 results in loss of odor response in a class of ORNs in the maxillary palp. Correspondingly, the defective ORNs lose Or gene expression. The phenotypes can be rescued with a cDNA representing either pdm3 or the affected Or gene, indicating a surprising degree of specificity to the odor-sensitivity phenotype. A genetic interaction between pdm3 and acj6, and ORNs can be divided into three categories, those that depend on both of these POU genes, those that depend on acj6 alone, and those that depend on neither for the acquisition of odor specificity. These results are consistent with a combinatorial code of POU genes. pdm3 is expressed in ORN classes in which it is not required for odor response, and in at least two of these classes pdm3 is required for normal axon targeting. pdm3 thus functions in both of the processes that dictate the precise organization of the Drosophila olfactory system: the expression of individual odor receptors and the targeting of individual glomeruli (Tichy, 2008).

To identify a new POU gene, the Drosophila genome sequence was searched with POU domain sequences from all six POU gene classes. The new gene belongs to class VI, and was named pdm3 (POU domain motif 3) by analogy to the previously named pdm1 and pdm2 genes. This is the first POU class VI gene in Drosophila, and no other members of this class were found in the fly genome (Tichy, 2008).

The POU domain of pdm3 consists of a POU-specific domain, a POU-homeodomain, and a linker region that separates the two. It is located in the C-terminal part of the gene, as in other POU genes of class VI. pdm3 spans ~8 kb and contains 10 exons. The predicted POU domain is the most highly conserved region of the gene, showing 70%-90% identity with other class VI POU proteins and 40%-50% identity with POU proteins of other classes (Tichy, 2008).

In initial RT-PCR experiments, pdm3 was amplified from adult head RNA. Two splice forms were amplified, henceforth called the 'long' and 'short' forms. They differ in that the long form includes exon 7, which is missing from the short form. This exon encodes 18 aa that interrupt the POU domain. An optional exon has likewise been found at the same location in Rpf-1, a human ortholog of pdm3 (Zhou, 1996).

The fruit fly detects odors with two types of olfactory organ, the antenna and the maxillary palp. In situ hybridization experiments showed that pdm3 is expressed in a subset of cells in the antenna. Expression was also detected in the maxillary palp, where a double-label experiment was performed to determine whether expression was neuronal. It was found that cells that express pdm3 RNA are labeled by an antibody against Elav, which labels the nuclei of differentiated neurons (Tichy, 2008).

To characterize the expression of pdm3 in more detail, an antibody was generated, using as antigen a portion of the protein that did not contain POU domain sequences. The antibody labeled cells in the maxillary palp. All or almost all of these Pdm3⁺ cells are Elav⁺. Many, but not all, of the Elav⁺ cells are Pdm3⁺. In the Pdm3⁺ Elav⁺ cells, the labeling appeared coincident. Together, these results show that pdm3 is expressed in the nuclei of a subset of maxillary palp neurons (Tichy, 2008).

A mutant line was obteind that contains a transposon insertion in pdm3. The insertion lies within an intron between two exons that encode the POU domain. This insertion eliminated Pdm3 expression, indicating specificity of the antibody. The insertion did not markedly reduce the number of Elav⁺ cells, suggesting that the loss of pdm3 does not cause a loss of neurons (Tichy, 2008).

To determine whether pdm3 is required for the function of ORNs, ORN responses was measured to odors using single-unit electrophysiology. The antenna and maxillary palp contain a number of functional types of sensilla, as defined by electrophysiological and molecular analysis. Each sensillum type contains up to four ORNs, usually two, which are combined in a stereotyped configuration. The maxillary palp is the simpler organ, in that it contains only three functional types of sensilla, pb1, pb2, and pb3, each of which contains a pair of ORNs: pb1A and pb1B; pb2A and pb2B; pb3A and pb3B. Odorants pass through pores in the sensillum walls, traverse the internal lymph, and activate the ORNs. Seven Or genes are expressed in the maxillary palp: of the six ORN classes, each expresses one odor receptor, except that one class, pb2A, expresses two (Tichy, 2008).

Of the six ORN classes of the maxillary palp, one is severely defective in pdm3. The pb1A ORN, which in wild type responds to a pulse of E2-hexenal with a train of action potentials, does not respond in pdm3. pb1A is present and yields spontaneous action potentials, but does not respond to the odorant (Tichy, 2008).

Systematic measurement of neuronal responses, measured in action potentials/s, after stimulation with a diverse panel of odorants, revealed that pb1A failed to respond to any tested odorant. In contrast, in the same sensillum, the neighboring pb1B ORN responded normally to 4-methyl phenol and 4-propyl phenol, its most effective odorants. Neurons of the pb2 and pb3 sensilla gave very similar responses in pdm3 and control animals (Tichy, 2008).

As a more stringent test of the role of pdm3 in ORN response, the pdm3 mutation was tested in heterozygous combination with a deletion for the region. In this pdm3/Df heterozygote, all responses were very similar to those of the pdm3 homozygote, indicating that the pdm3 insertion is a null allele (Tichy, 2008).

The olfactory phenotype is not limited to the maxillary palp. The large basiconic sensilla of the antenna were examined, and one of the four ORNs in the ab1 sensillum, ab1A, is present but has lost odor response. Limited analysis of the ORNs in ab2 and ab3 sensilla revealed no abnormalities. The detection of an olfactory phenotype in some antennal ORNs is consistent with detection of pdm3 expression in a subset of antennal cells (Tichy, 2008).

Because pb1A is present in pdm3 and yields spontaneous action potentials, but does not respond to odors, the hypothesis was tested that pdm3 affects expression of its odor receptor. Or42a is the odor receptor that is expressed in pb1A (Tichy, 2008).

As a first test of this hypothesis, it was asked whether Pdm3 is expressed in pb1A in wild type. A double-label experiment showed that Pdm3 is in fact expressed in Or42a-expressing cells of wild type. It was then found that Or42a expression is undetectable in pdm3. In contrast, the neighboring pb1B cell shows normal expression of its receptor gene, Or71a, and all other maxillary palp odor receptor genes are expressed in pdm3. Thus these expression studies coincide precisely with the functional studies: the one class of ORN that has lost function is the one class of ORN that has lost expression of a receptor gene (Tichy, 2008).

To determine whether the loss of pb1A odor response is attributable solely to loss of Or42a expression or whether other essential components are lost as well, expression of Or42a was drived in pb1A neurons of pdm3 using the GAL4-UAS system. Response was tested to the two odorants that elicit the strongest response from pb1A and it was found that responses to both were completely or largely restored when Or42a was driven by Elav-GAL4. These results suggest a surprising degree of specificity to the pdm3 phenotype at the molecular level (Tichy, 2008).

Next, the ability of pdm3 splice forms to rescue the phenotype was tested. Of the two splice forms, only the long form was amplified from adult maxillary palps in RT-PCR experiments. When expression of a long form cDNA was driven using the GAL4-UAS system, the phenotype of pb1A was rescued completely or largely in 7 of 29 pb1 sensilla, tested with three odorants. Expression of Or42a was also restored in a number of cells. In this experiment, an acj6-GAL4 driver was used, which drives expression in all or almost all maxillary palp ORNs. A faithful driver could not be constructed for pdm3, whose 5' end is separated from the next annotated upstream gene by a long region; it is possible that a pdm3 driver might restore the pb1A phenotype in a greater fraction of pb1 sensilla. Expression of a short form cDNA rescued in 1 of 19 pb1 sensilla tested (Tichy, 2008).

It is noted that in addition to the rescued pb1A cells, a number of pb1A cells were found that acquired an odor response profile different from that of wild type. Further testing revealed that this odor response profile is very similar to that conferred by a larval odor receptor, Or85c. Moreover, Or85c expression is detected in several cells of the 'rescued' maxillary palp. The proportion of cells with this response profile is greater when pdm3 is driven with a promoter that is expected to drive later expression in ORNs. Further testing beyond the scope of this study would be required to determine whether these results reflect the presence of different pdm3 interaction partners at different times at the promoters of Or42a and Or85c (Tichy, 2008).

Because pb1A is affected by both pdm3 and another POU gene, acj6, the relationship between them was investigated. Although the concept of a combinatorial code of transcription factors has been previously invoked in discussion of receptor gene choice in Drosophila (Ray, 2007; Ray, 2008), the interaction of two transcription factors within the same ORN has not yet been examined in Drosophila (Tichy, 2008).

It was found first that neither POU gene appears to regulate the other in the maxillary palp: pdm3 is expressed in a null mutant of acj6, and acj6 is expressed in a null mutant of pdm3. Although both pdm3 and acj6 are fully recessive, the transheterozygote shows a reduced response of pb1A. The response of pb1B is normal in this genotype. These results indicate a genetic interaction between the two POU genes in pb1A (Tichy, 2008).

Interestingly, two putative Pdm3 binding sites, i.e., sequences corresponding to binding sites for rat and zebrafish orthologs [(Brn-5) and (Pou[c]), respectively], lie near an Acj6 site, ~320 bp upstream of Or42a. One putative Pdm3 site overlaps with the Acj6 site, and the other is immediately adjacent to it. POU genes have been shown to heterodimerize on DNA, and this cluster of POU sites may bring Pdm3 and Acj6 in a position to do so upstream of a gene whose expression depends on both (Tichy, 2008).

From the initial analysis of pdm3 expression in the maxillary palp, it is clear that the number of cells expressing pdm3 is greater than the number of pb1A cells. To determine in which ORN classes pdm3 is expressed, a series of double-label experiments was performed using ORN class-specific probes and an anti-Pdm3 antibody. It was found that Pdm3 is expressed in four of the six maxillary palp ORN classes: pb1A, pb1B, pb3A, and pb3B. Expression was not detected in pb2A or pb2B. The expression of pdm3 in three ORN classes that do not require its expression for odor response suggested the possibility that pdm3 might have a different function in these cells (Tichy, 2008).

ORNs send axons to the antennal lobe of the brain, and axons of an individual ORN class converge precisely on an individual unit of the antennal lobe called a glomerulus. Thus, a spatial map of olfactory information is created in the antennal lobe by the stereotyped pattern of connectivity. There are 43 glomeruli in the antennal lobe, and both receptor-to-ORN and ORN-to-glomerulus maps have been constructed. A number of genes required for normal ORN axon targeting have been identified (Tichy, 2008).

The axon targeting was examined of two ORN classes that express pdm3 but that do not require pdm3 for odor response: pb1B, which expresses the Or71a receptor, and pb3A, which expresses Or59c. It was found that in a pdm3 mutant, the axonal projections of both ORN classes are abnormal. In the wild type, in both cases the projections converge on a single, discrete glomerulus in each antennal lobe. In pdm3, the convergence is less precise and the boundaries of the targeted areas are less discrete. In some cases, the axon tracts appear to bifurcate, with labeled axons occupying more than one region. In most cases, the axons appear to terminate in the general vicinity of the wild-type target, but not in the precise positions of the wild-type glomerular targets. In summary, pdm3 has effects on ORN axon targeting, in addition to its role in specifying odor response and receptor gene expression (Tichy, 2008).

This study has identified a new Drosophila POU gene, pdm3, that acts in both receptor gene expression and axonal targeting. Pdm3 is a POU transcription factor of class VI. Vertebrate members of class VI are expressed in brain and spinal cord. Notably, a mouse ortholog of pdm3, Emb, is expressed in the olfactory bulb (Tichy, 2008 and references therein).

The mechanism of ORN fate determination has been elegantly examined in Caenorhabditis elegans. The AWA, AWB, and AWC cells express multiple olfactory receptors and sense distinct but overlapping sets of odors. The specification of AWA fate requires lin-11, a LIM homeodomain gene. lin-11 regulates ODR-7, a nuclear hormone receptor that promotes expression of AWA-specific genes and represses AWC-specific genes. The Aristaless-related homeodomain gene alr-1 is also required for specification of AWA. ceh-37, an Otx homeodomain gene, regulates lim-4, another LIM homeobox gene, to promote AWB neuron fate. Another Otx gene, ceh-36, is required for AWC neuron fate (Tichy, 2008 and references therein).

The first olfactory phenotype detected for pdm3 was its defective odor response in an electrophysiological assay. The phenotype is striking in its specificity, at both the cellular and molecular levels. At the cellular level, only one ORN class in the maxillary palp, pb1A, was defective in a null mutant of pdm3. The pb1B neuron, which neighbors pb1A in the same sensillum, and the other four ORN classes all appeared to yield normal odor responses. At the molecular level, the pb1A physiological phenotype appeared to be attributable largely if not completely to the loss of a single gene: Or42a, the odor receptor gene normally expressed in this neuron. Moreover, Or42a is the only maxillary palp Or gene affected by pdm3 (Tichy, 2008).

Although the specificity of the molecular and cellular phenotypes was surprising, the specificity of one phenotype is in good agreement with the specificity of the other. The cascade of molecular steps between the presentation of an odor molecule and the production of an action potential is poorly understood but presumably requires the agency of a number of different genes. It seems likely, however, that the signaling pathway used by one maxillary palp ORN class is the same as that used by the others, with the exception of the odor receptor. Thus the ability to rescue the pb1A odor response with its receptor gene alone supports a model in which the rest of the signaling pathway does not depend on pdm3. In this case, pdm3 would affect odor response only in those maxillary palp ORNs whose receptor genes it affects, i.e., only in pb1A (Tichy, 2008).

The specificity of pdm3 is in contrast to that of acj6, which affects the odor response of four ORN classes in the maxillary palp (Clyne, 1999b). Moreover, in acj6 some of the unresponsive ORNs were not detected electrophysiologically, either because of a defect that made them physiologically silent or because they were absent. Thus acj6 appears to affect more ORNs and to affect some of them more severely than pdm3. Noted is the formal possibility that pdm3 acts in some ORNs other than pb1A but is functionally redundant in them (Tichy, 2008).

The requirement of both pdm3 and acj6 in pb1A led to an investigation of whether they act together or independently. A genetic interaction was found between these two POU genes in the analysis of pb1A odor response. The location of overlapping putative binding sites for both transcription factors upstream of Or42a suggests the possibility of a biochemical interaction between them. Heterodimerization, one possible means of interaction, has been shown previously for other POU proteins. The genetic interaction and coexpression of acj6 and pdm3 in ORNs is in contrast to the relationship between acj6 and another POU gene, drifter, in projection neurons (PNs), the postsynaptic partners of ORNs. Although both acj6 and drifter act in PNs, they are expressed in mutually exclusive subsets of PNs (Tichy, 2008).

A major problem in olfactory system biology is how individual ORNs select which of a large family of odor receptor genes to express. There are 60 Or genes in the genome of Drosophila melanogaster. The expression of particular Or genes, and hence the odor specificity of the ORN, has recently been found to depend on a regulatory code of cis-acting elements. Positive and negative regulatory elements named Dyad-1 and Oligo-1 are required for the selection of Or genes in the correct olfactory organ (Ray, 2007). Within the maxillary palp, additional elements act positively to promote expression of individual Or genes in a subset of ORN classes, whereas other elements act negatively to restrict expression of individual Or genes to a single ORN class (Ray, 2007, 2008). Evidence was found that a combinatorial code of transcription factors underlies the problem of receptor gene choice (Ray, 2007). Transcription factors that act in this process include Lozenge, a Runx domain-containing protein that is required for the expression of two Or genes (Ray, 2007), and Scalloped, which mediates repression (Ray, 2008). Another subset of maxillary palp Or genes depends on the POU gene acj6 (Tichy, 2008).

The current finding that two POU genes, acj6 and pdm3, are required in the same ORN for receptor gene expression suggests that the combinatorial action of POU genes may be an important part of such a code. The Or genes of the maxillary palp can be divided into three classes: those that require both POU genes (Or42a), those that require only acj6 (Or85e, Or33c, Or46a, and Or59c), and those that require neither (Or71a and Or85d). Heterodimer formation and alternative splicing could expand the number of components that act in selecting individual receptors from the entire family of 60 Or genes in the entire olfactory system, including the ORNs of the antenna (Tichy, 2008).

It was found that pdm3 is also required for axon targeting of pb1B and pb3A cells. Interestingly, acj6 is also required for axon targeting of these ORNs, but it is not known whether the two POU proteins act together on common transcriptional target genes or independently on different genes required for axonal wiring (Tichy, 2008).

The relationship between receptor expression and ORN axon targeting has been a topic of great interest. In vertebrates, there is evidence that the odor receptor acts in both processes. In Drosophila, the receptor does not appear to be required for normal axon targeting. Likewise, the effects of pdm3 on receptor expression and axon targeting appear to be separable, in that pb1B and pb3A show apparently normal receptor gene expression but abnormal targeting. A related issue is the relationship between ORN activity and axon targeting. In vertebrates, there is evidence that ORN activity is necessary for the establishment or maintenance of correct ORN axon targeting. It was not possible to ask whether in pdm3 the lack of odor response in pb1A correlates with a failure in axon targeting, for lack of a pb1A GAL4-driver that functions normally in the absence of pdm3. However, it was found that the normal odor responses of pb1B and pb3A ORNs are not sufficient for normal axon targeting (Tichy, 2008).

This is the first mutational analysis of a class VI POU gene and demonstrates the essential role that pdm3 plays in the development of a highly complex and precisely organized sensory system. Further study of pdm3 may uncover critical roles in other systems as well (Tichy, 2008).

Aguilar, J. I., Dunn, M., Mingote, S., Karam, C. S., Farino, Z. J., Sonders, M. S., Choi, S. J., Grygoruk, A., Zhang, Y., Cela, C., Choi, B. J., Flores, J., Freyberg, R. J., McCabe, B. D., Mosharov, E. V., Krantz, D. E., Javitch, J. A., Sulzer, D., Sames, D., Rayport, S. and Freyberg, Z. (2017). Neuronal depolarization drives increased dopamine synaptic vesicle loading via VGLUT. Neuron 95(5): 1074-1088 e1077. PubMed ID: 28823729

Ayer, R. K. and Carlson, J,. (1991). acj6: a gene affecting olfactory physiology and behavior in Drosophila. Proc. Natl. Acad. Sci. 88(12): 5467-71. PubMed Citation: 1905022

Ayer, R. K. and Carlson, J. (1992). Olfactory physiology in the Drosophila antenna and maxillary palp: acj6 distinguishes two classes of odorant pathways. J. Neurobiol. 23(8): 965-82. PubMed Citation: 1460467

Badea, T. C., Cahill, H., Ecker, J., Hattar, S. and Nathans, J. (2009). Distinct roles of transcription factors Brn3a and Brn3b in controlling the development, morphology, and function of retinal ganglion cells. Neuron 61(6): 852-64. PubMed Citation: 19323995

Bai, L., Goldman, A. L. and Carlson, J. R. (2009). Positive and negative regulation of odor receptor gene choice in Drosophila by acj6. J. Neurosci. 29(41): 12940-7. PubMed Citation: 19828808

Bai, L. and Carlson, J. R. (2010). Distinct functions of acj6 splice forms in odor receptor gene choice. J. Neurosci. 30(14): 5028-36. PubMed Citation: 20371823

Baumeister, R., Liu, Y. and Ruvkun, G. (1996). Lineage-specific regulators couple cell lineage asymmetry to the transcription of the Caenorhabditis elegans POU gene unc-86 during neurogenesis. Genes Dev. 10: 1395-1410. PubMed Citation: 8647436

Bouchard, M., et al. (2005). Identification of Pax2-regulated genes by expression profiling of the mid-hindbrain organizer region. Development 132: 2633-2643. 15872005

Budhram-Mahadeo, V., Parker, M. and Latchman, D. S. (1998). POU transcription factors Brn-3a and Brn-3b interact with the estrogen receptor and differentially regulate transcriptional activity via an estrogen response element. Mol. Cell. Biol. 18(2): 1029-1041. PubMed Citation: 9448000

Certel, S. J., et al. (2000). Regulation of central neuron synaptic targeting by the Drosophila POU protein, Acj6. Development 127: 2395-2405. PubMed Citation: 10804181

Clyne, P. J., et al. (1999). The odor specificities of a subset of olfactory receptor neurons are governed by Acj6, a POU-domain transcription factor. Neuron 22(2): 339-47. PubMed Citation: 10069339

Davie, K., Janssens, J., Koldere, D., De Waegeneer, M., Pech, U., Kreft, L., Aibar, S., Makhzami, S., Christiaens, V., Bravo Gonzalez-Blas, C., Poovathingal, S., Hulselmans, G., Spanier, K. I., Moerman, T., Vanspauwen, B., Geurs, S., Voet, T., Lammertyn, J., Thienpont, B., Liu, S., Konstantinides, N., Fiers, M., Verstreken, P. and Aerts, S. (2018). A single-cell transcriptome atlas of the aging Drosophila brain. Cell 174(4): 982-998 e920. PubMed ID: 29909982

Eng, S. R., et al. (2001). Defects in sensory axon growth precede neuronal death in Brn3a-deficient mice. J. Neurosci. 21(2): 541-549. 11160433

Eng, S. R., et al. (2004). Coordinated regulation of gene expression by Brn3a in developing sensory ganglia. Development 131: 3859-3870. 15253936

Erkman, L., et al. (1996). Role of transcription factors Brn-3.1 and Brn-3.2 in auditory and visual system development. Nature 381: 603-606. PubMed Citation: 8637595

Erkman, L., et al. (2000). A POU domain transcription factor-dependent program regulates axon pathfinding in the vertebrate visual system, Neuron 28: 779-792. 11163266

Estacio-Gómez, A., Hassan, A., Walmsley, E., Le, L. W. and Southall, T. D. (2020). Dynamic neurotransmitter specific transcription factor expression profiles during Drosophila development. Biol Open 9(5). PubMed ID: 32493733

Fedtsova, N. G. and Turner, E. E. (1995). Brn-3.0 expression identifies early post-mitotic CNS neurons and sensory neural precursors. Mech. Dev. 53: 291-304. PubMed Citation: 8645597

Fedtsova, N. and Turner, E. E. (1997). Inhibitory effects of ventral signals on the development of Brn-3.0-expressing neurons in the dorsal spinal cord. Dev. Biol. 190(1): 18-31. PubMed Citation: 9331328

Fedtsova, N. and Turner, E. E. (2001). Signals from the ventral midline and isthmus regulate the development of Brn3.0-expressing neurons in the midbrain. Mech. of Dev. 105: 129-144. 11429288

Fedtsova, N., Quina, L. A., Wang, S. and Turner, E. E. (2008). Regulation of the development of tectal neurons and their projections by transcription factors Brn3a and Pax7. Dev. Biol. 316(1): 6-20. PubMed Citation: 18280463

Gan, L., et al. (1996). POU domain factor Brn-3b is required for the development of a large set of retinal ganglion cells. Proc. Natl. Acad. Sci. 93: 3920-25. 8632990

Gan, L., et al. (1999). POU domain factor Brn-3b is essential for retinal ganglion cell differentiation and survival but not for initial cell fate specification. Dev. Biol. 210(2): 469-80. PubMed Citation: 10357904

Gruber, C. A., et al. (1997). POU domain factors of the Brn-3 class recognize functional DNA elements which are distinctive, symmetrical, and highly conserved in evolution. Mol. Cell. Biol. 17: 2391-2400. PubMed Citation: 9111308

Herr, W. and Cleary, M. A. (1995). The POU domain: versatility in transcriptional regulation by a flexible two-in-one DNA-binding domain. Genes Dev. 9: 1679-93. 7622033

Huang, E. J., et al. (1999). POU domain factor Brn-3a controls the differentiation and survival of trigeminal neurons by regulating Trk receptor expression. Development 126: 2869-2882. PubMed Citation: 10357931

Huang, E. J., et al. (2001). Brn3a is a transcriptional regulator of soma size, target field innervation and axon pathfinding of inner ear sensory neurons. Development 128: 2421-2432. 11493560

Hutcheson, D. A. and Vetter, M. L. (2001). The bHLH factors Xath5 and XNeuroD can upregulate the expression of XBrn3d, a POU-homeodomain transcription factor. Dev. Bio. 232: 327-338. PubMed Citation: 11401395

Jafari, S., et al. (2012). Combinatorial activation and repression by seven transcription factors specify Drosophila odorant receptor expression. PLoS Biol. 10(3): e1001280. PubMed Citation: 22427741

Kambadur, R., Koizumi, K., Stivers, C., Nagle, J., Poole, S. J. and Odenwald, W. F. (1998). Regulation of POU genes by castor and hunchback establishes layered compartments in the Drosophila CNS. Genes Dev. 12(2): 246-60. PubMed Citation: 9436984

Komiyama, T., Johnson, W. A., Luo, L. and Jefferis, G. S. X. E. (2003). From lineage to wiring specificity: POU Domain transcription factors control precise connections of Drosophila olfactory projection neurons. Cell 112: 157-167. 12553905

Komiyama, T. and Luo, L. (2007). Intrinsic control of precise dendritic targeting by an ensemble of transcription factors. Curr. Biol. 17(3): 278-85. Medline abstract: 17276922

Lanier, J., et al. (2004). Brn3a target gene recognition in embryonic sensory neurons. Dev. Biol. 302: 703-716. PubMed Citation: 17196582

Lee, M.-H. and Salvaterra, P. M. (2002). Abnormal chemosensory jump 6 is a positive transcriptional regulator of the cholinergic gene locus in Drosophila olfactory neurons. J. Neurosci. 22(13): 5291-5299. 12097480

Li, H., Li, T., Horns, F., Li, J., Xie, Q., Xu, C., Wu, B., Kebschull, J. M., McLaughlin, C. N., Kolluru, S. S., Jones, R. C., Vacek, D., Xie, A., Luginbuhl, D. J., Quake, S. R. and Luo, L. (2020). Single-cell transcriptomes reveal diverse regulatory strategies for olfactory receptor expression and axon targeting. Curr Biol. PubMed ID: 32059767

Lichtsteiner, S. and Tjian, R. (1995). Synergistic activation of transcription by UNC-86 and MEC-3 in Caenorhabditis elegans embryo extracts. EMBO J. 14: 3937-3945. PubMed Citation: 7664734

Liu, W., et al. (2000). All Brn3 genes can promote retinal ganglion cell differentiation in the chick. Development 127: 3237-3247. PubMed Citation: 10887080

Liu, W., Mo, Z. and Xiang, M. (2001). The Ath5 proneural genes function upstream of Brn3 POU domain transcription factor genes to promote retinal ganglion cell development. Proc. Natl. Acad. Sci. 98: 1649-1654. 11172005

Ma, L., et al. (2003). Brn3a regulation of TrkA/NGF receptor expression in developing sensory neurons. Development 130: 3525-3534. 12810599

McKenna, M., et al. (1989). A simple chemosensory response in Drosophila and the isolation of acj mutants in which it is affected. Proc. Natl. Acad. Sci. 86(20): 8118-22. PubMed Citation: 2510161

Mu, X., et al. (2004). Discrete gene sets depend on POU domain transcription factor Brn3b/Brn-3.2/POU4f2 for their expression in the mouse embryonic retina. Development 131: 1197-1210. 14973295

Ninkina, N. N., et al. (1993). A novel Brn3-like POU transcription factor expressed in subsets of rat sensory and spinal cord neurons. Nucleic Acids Res. 21: 3175-82. PubMed Citation: 8341591

Pan, L., et al. (2005). Functional equivalence of Brn3 POU-domain transcription factors in mouse retinal neurogenesis. Development 132: 703-712. 15647317

Phippard, D. et al. (1999). Targeted mutagenesis of the POU-Domain gene Brn4/Pou3f4 causes developmental defects in the inner ear. J. Neurosci. 19(14): 5980-5989. PubMed Citation: 10407036

Poggi, L., et al. (2004). The homeobox gene Xbh1 cooperates with proneural genes to specify ganglion cell fate within the Xenopus neural retina. Development 131: 2305-2315. 15102701

Ray, A., van Naters, W. G., Shiraiwa, T. and Carlson, J. R. (2007). Mechanisms of odor receptor gene choice in Drosophila. Neuron 53(3): 353-69. Medline abstract: 17270733

Ray, A., van der Goes van Naters, W. and Carlson, J. R. (2008). A regulatory code for neuron-specific odor receptor expression. PLoS Biol 6: 1069-1083. PubMed Citation: 18846726

Rohrig, S., et al. (2000). Protein interaction surface of the POU transcription factor UNC-86 selectively used in touch neurons. EMBO J. 19: 3694-3703. PubMed Citation: 10899123

Shaham, S. and Bargmann, C. I. (2002). Control of neuronal subtype identity by the C. elegans ARID protein CFI-1. Genes Dev. 16: 972-983. 11959845

Smith, M. D., Dawson, S. J. and Latchman, D. S. (1997a). The Brn-3a transcription factor induces neuronal process outgrowth and the coordinate expression of genes encoding synaptic proteins. Mol. Cell. Biol. 17: 345-354. 8972215

Smith, M. D., et al. (1997b). Coordinate induction of the three neurofilament genes by the Brn-3a transcription factor. J. Biol. Chem. 272(34): 21325-21333. PubMed Citation: 9261145

Sze, J. Y., Liu, Y, and Ruvkun, G (1997). VP16-activation of the C. elegans neural specification transcription factor UNC-86 suppresses mutations in downstream genes and causes defects in neural migration and axon outgrowth. Development 124: 1159-1168. 9102303

Sze, J. Y., et al. (2002). The C. elegans POU-domain transcription factor UNC-86 regulates the tph-1 tryptophan hydroxylase gene and neurite outgrowth in specific serotonergic neurons. Development 129: 3901-3911. 12135927

Tichy, A. L., Ray, A. and Carlson, J. R. (2008). A new Drosophila POU gene, pdm3, acts in odor receptor expression and axon targeting of olfactory neurons. J. Neurosci. 28(28): 7121-9. PubMed Citation: 18614681

Torres, M. and Giraldez, F. (1998). The development of the vertebrate inner ear. Mech. Dev. 71(1-2): 5-21. PubMed Citation: 9507049

Treacy, M. N., He, X. and Rosenfeld, M. G. (1991). I-POU: a POU domain protein that inhibits neuron-specific gene activation. Nature 350: 577-584. PubMed Citation: 1673230

Treacy, M. N., et al. (1992). Twin of I-POU: a two amino acid difference in the I_POU homeodomain distinguishes an activator from an inhibitor of transcription. Cell 68: 491-505. 1346754

Trieu, M., et al. (1999). Autoregulatory sequences are revealed by complex stability screening of the mouse brn-3.0 locus. J. Neurosci. 19(15): 6549-6558. PubMed Citation: 10414983

Trieu, M., et al. (2003). Direct autoregulation and gene dosage compensation by POU-domain transcription factor Brn3a. Development 130: 111-121. 12441296

Trudeau, L. E. and El Mestikawy, S. (2018). Glutamate cotransmission in cholinergic, GABAergic and monoamine systems: contrasts and commonalities. Front Neural Circuits 12: 113. PubMed ID: 30618649

Turner, E. E., Jenne, K. J. and Rosenfeld, M. G. (1994). Brn-3.2: a Brn-3-related transcription factor with distinctive central nervous system expression and regulation by retinoic acid. Neuron 12: 205-18. PubMed Citation: 7904822

Turner, E. E. (1996). Similar DNA recognition properties of alternatively spliced Drosophila POU factors. Proc. Natl. Acad. Sci. 93: 15097-15101. PubMed Citation: 8986770

Verrijzer, C. P. and Van der Vliet, P. C. (1993). POU domain transcription factors. Biochim. Biophys. Acta 1173: 1-21. PubMed Citation: 8485147

Wang, S. W., et al. (2001). Requirement for math5 in the development of retinal ganglion cells. Genes Dev. 15: 24-29. 11156601

Wang, S. W., et al. (2002). Brn3b/Brn3c double knockout mice reveal an unsuspected role for Brn3c in retinal ganglion cell axon outgrowth. Development 129: 467-477. 11807038

Wiggins, A. K., et al. (2004). Interaction of Brn3a and HIPK2 mediates transcriptional repression of sensory neuron survival. Jour. Cell Biol. 167: 257-267. 15492043

Xiang, M., et al (1997). Essential role of POU-domain factor Brn-3c in auditory and vestibular hair cell development. Proc. Natl. Acad. Sci. 94(17): 9445-9450. PubMed Citation: 9256502

Xiang, M. (1998a). Requirement for Brn-3b in early differentiation of postmitotic retinal ganglion cell precursors. Dev. Biol. 197(2): 155-169. PubMed Citation: 9630743

Xiang, M., et al. (1998b). Requirement for Brn-3c in maturation and survival, but not in fate determination of inner ear hair cells. Development 125(20): 3935-3946. PubMed Citation: 9735355

Xue, D., Tu, Y. and Chalfie, M. (1993). Cooperative interactions between the Caenorhabditis elegans homeoproteins UNC-86 and MEC-3. Science 261: 1324-8. PubMed Citation: 8103239

Zhang, F., Bhattacharya, A., Nelson, J. C., Abe, N., Gordon, P., Lloret-Fernandez, C., Maicas, M., Flames, N., Mann, R. S., Colon-Ramos, D. A. and Hobert, O. (2014). The LIM and POU homeobox genes ttx-3 and unc-86 act as terminal selectors in distinct cholinergic and serotonergic neuron types. Development 141: 422-435. PubMed ID: 24353061

Zhang, Q., Zagozewski, J., Cheng, S., Dixit, R., Zhang, S., de Melo, J., Mu, X., Klein, W. H., Brown, N. L., Wigle, J. T., Schuurmans, C. and Eisenstat, D. D. (2017). Regulation of Brn3b by Dlx1 and Dlx2 is required for retinal ganglion cell differentiation in the vertebrate retina. Development. PubMed ID: 28356311

Zimmermann, G., et al. (2009). Manipulation of an innate escape response in Drosophila: photoexcitation of acj6 neurons induces the escape response. PLoS One. 4(4): e5100. PubMed Citation: 19340304

date revised: 17 August 2020