InteractiveFly: GeneBrief

pou domain motif 3: Biological Overview | References

BIOLOGICAL OVERVIEW

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, 1999). 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).

Diversity of internal sensory neuron axon projection patterns is controlled by the POU-domain protein Pdm3 in Drosophila larvae

Internal sensory neurons innervate body organs and provide information about internal state to the CNS to maintain physiological homeostasis. Despite their conservation across species, the anatomy, circuitry, and development of internal sensory systems are still relatively poorly understood. A largely unstudied population of larval Drosophila sensory neurons, termed tracheal dendrite (td) neurons, innervate internal respiratory organs and may serve as a model for understanding the sensing of internal states. This study characterized the peripheral anatomy, central axon projection, and diversity of td sensory neurons. Evidence for prominent expression of specific gustatory receptor genes in distinct populations of td neurons, suggesting novel chemosensory functions. This study identified two anatomically distinct classes of td neurons. The axons of one class project to the subesophageal zone (SEZ) in the brain, whereas the other terminates in the ventral nerve cord (VNC). This study identified expression and a developmental role of the POU-homeodomain transcription factor Pdm3 in regulating the axon extension and terminal targeting of SEZ-projecting td neurons. Remarkably, ectopic Pdm3 expression is alone sufficient to switch VNC-targeting axons to SEZ targets, and to induce the formation of putative synapses in these ectopic target zones. These data thus define distinct classes of td neurons, and identify a molecular factor that contributes to diversification of axon targeting. These results introduce a tractable model to elucidate molecular and circuit mechanisms underlying sensory processing of internal body status and physiological homeostasis (Qian, 2018).

High-resolution studies of sensory axon morphology in embryos identified unusual axon projections of td neurons beyond their segment of origin to a common target in thoracic neuromeres. Whether this neuromere represented an intermediate or terminal axon target was unknown because mature td axon projections in the third instar larva were not described. This study shows that all td neurons make long-range projections but have dichotomous terminal zones anteriorly in the SEZ and in the VNC. The SEZ receives chemosensory inputs and contains numerous peptidergic fibers. Based on their location along trachea, td neurons were proposed to function as proprioceptors or gas sensors, although the function of td neurons is as yet unknown. Anatomical data from this study are more consistent with roles for td neurons as internal chemosensors. It is noted that axons that project to the SEZ form en passant synapses throughout the VNC, suggesting distributed input to central circuits. SEZ- and VNC-targeting axons could conceivably share postsynaptic partners in the VNC, with SEZ-targeting axons connecting with an additional population of targets in the SEZ, although precise connectivity remains to be determined. A recent electron microscopic study of the SEZ identified ascending sensory projections that form synapses with a subset of peptidergic Hugin neurons (Schlegel, 2016). These sensory projections likely correspond to a subset of td neurons. Functional interrogation of this Hugin circuit and reconstruction of additional downstream targets of SEZ- and VNC-projecting td neurons will provide insights into possible roles for the td system in behavior and physiology (Qian, 2018).

This study identified expression of multiple gustatory and ionotropic receptor (GR and IR) reporters in td neurons. These findings, together with anatomical data, suggests that td neurons may function to sense internal chemical stimuli. In Drosophila, the combinatorial coexpression of specific GRs determines the tuning of gustatory neurons to specific ligands. The patterns of coexpressed GRs that were observed in td neurons have not been observed in other gustatory neurons, suggesting possible tuning to novel ligands. Two GRs that appear to be expressed in td neurons, Gr33a and Gr89a, are expressed in all adult bitter neurons, and Gr33a is broadly required for responses to aversive cues in the context of feeding. These GRs have been proposed to function as 'core bitter coreceptors'. It is possible that at least a subset of td neurons may detect aversive chemical stimuli. Given that td dendrites appear to be bathed in hemolymph and associated with the trachea, td neurons may detect both dissolved circulating stimuli (e.g., ingested toxins, metabolites, electrolytes) and gaseous stimuli (e.g., CO2, O2). The expression of a reporter for Ir76b, a detector of low salt, and oxygen-sensitive guanylyl cyclase in different subsets of td neurons is consistent with this idea. It is speculated that td neurons may detect chemical imbalances and relay signals to the SEZ and VNC to elicit behavioral or physiological responses to restore homeostasis. Neurons in the SEZ could regulate feeding, and neurons in the VNC could regulate locomotion or fluid balance. In mammals, lung-innervating sensory neurons comprise molecularly distinct subtypes with different anatomical projections and functions. This study shows that larval Drosophila trachea-innervating sensory neurons similarly comprise molecularly distinct subtypes with distinct axon projections. Future studies to image and manipulate td activity, and disrupt chemosensory receptor gene function, should clarify the sensory functions of td neurons and the underlying molecular mechanisms (Qian, 2018).

This study uncovered multiple levels of specificity of td neuron dendrite-substrate relationships, including strict association with a tracheal substrate, arborization across specific tracheal branches, and dendritic specializations at tracheal fusion cells. The factors that specify sensory dendrite organization of td neurons are unknown and do not appear to include Pdm3. Whether dendrite specializations are important for detection of chemicals in the tracheal lumen or whether trachea merely serve as an attachment site to allow sensing of abdominal hemolymph status is not clear. The positioning of td dendrites may place them out of direct contact with the tracheal interior; however, association across tracheal cells could still permit sensing of tracheal physiology. Future studies to monitor tracheal system and td dendrite development will help to sort out mechanisms of dendrite-substrate interactions and the importance of this association for td neuron function (Qian, 2018).

Many of the guidance decisions made by sensory axons involve decisions to terminate at specific mediolateral and dorsoventral positions or in specific neuropil layers. For td axons, the guidance decisions are complex. Single td axons switch between medial and lateral positions, and dorsal and ventral positions and do so at specific locations along their length. Moreover, the terminal position of td axons varies according to cell identity and segment of origin. It is predicted that studies of td neurons may be especially useful for understanding sequential and regionally restricted guidance switches in axons, a model more akin to long-range projections, such as vertebrate corticospinal tract axons that navigate multiple choice points, than other locally projecting Drosophila sensory axons (Qian, 2018).

This study provides initial insight into one major choice of td axons: the choice to project, or not, to far anterior regions of the CNS (SEZ). The Pdm3 transcription factor is expressed in most, but not all, td neurons that project to the SEZ and is expressed in none of the td neurons that terminate in the VNC. Ectopic Pdm3 expression promoted anterior axon growth along the canonical td axon path, indicating that Pdm3 expression is sufficient for SEZ projections. This effect depends on sensory context because misexpression of Pdm3 in cIV dendritic arborization neurons did not convert axons to a td-like projection, but rather led to axon defasciculation, overgrowth, and axon straying, occasionally into the SEZ. Loss of Pdm3 led to modest disruptions of terminal targeting in SEZ-projecting tds, suggesting sufficiency, but redundancy with other factors, in SEZ targeting. This study noted specific patterns of axon-axon segregation among axons that project to the SEZ and those that project to the VNC. Thus, in addition to the possibility that Pdm3 functions as a growth-promoting factor, other explanations could account for Pdm3 misexpression phenotypes, such as promoting specific patterns of axon-axon interactions that underlie pathfinding to anterior CNS (Qian, 2018).

These results extend the roles for Pdm3 in axon targeting and chemosensory receptor expression. Prior studies identified roles for Pdm3 in targeting of olfactory sensory neurons, in olfactory receptor expression and in ellipsoid ring (R) neuron axon targeting (Chen, 2012). In R neurons, Pdm3 controls axon terminal targeting, without impacting dendritic arborization, cell fate determination, or initial axon outgrowth. The results for td neurons support a role in axon terminal growth and targeting, or maintenance, and in regulation of GR expression. Thus, this study demonstrates that Pdm3 regulates multiple aspects of td cellular identity, consistent with prior findings in the olfactory system. With respect to fine terminal targeting, one potential role for Pdm3 may be to inhibit midline contact of sensory axon terminals, which could account for the Pdm3 loss-of-function phenotype in td neurons and part of the Pdm3 misexpression phenotype in cIV neurons. The normal functions of Pdm3 in different cell types suggest context-dependent roles to promote terminal targeting. Identifying whether conserved transcriptional targets are shared between these different systems will be an important future step. Studies of Pdm3 might reveal how axon initial growth, pathfinding, terminal targeting, and maintenance are regulated in a modular fashion across different neurons, which could be important not only for axon wiring during development but also for regeneration (Qian, 2018).

The POU-domain protein Pdm3 regulates axonal targeting of R neurons in the Drosophila ellipsoid body

The ability of axons to project correctly to the target is essential for the formation of complex neural networks. The intrinsic regulation of this process is still unclear. This study shows that POU domain motif 3 (Pdm3) is required in ring (R) neurons to control precise axon targeting to the Drosophila ellipsoid body (EB). Pdm3 is expressed in neurons of the central nervous system in larvae and adults and required for the normal development of the EB of the central complex in the adult brain. The normal EB structure is abolished in pdm3 mutants, and this phenotype is rescued by pdm3 expression in R neurons, suggesting that the defect in axonal targeting of R neurons is the major cause in EB malformation in pdm3 mutants. Cell fate determination, dendritic arborization, and initial axon projection of R neurons are normal while the axonal targeting to the EB is defective in pdm3 mutants (Chen, 2012).

This study has generated pdm3 mutant alleles to study pdm3 function in neural development. Pdm3 is expressed in differentiated neurons in Drosophila central brain and is required for its normal development. In pdm3 mutants, the central complex is disarrayed, which can be explained by the disorganized axonal terminals of R neurons in the EB (Chen, 2012).

In previous studies, it has been shown that pdm3 is expressed in olfactory neurons and required for axonal targeting of olfactory neurons. In pdm3 mutants, a low percentage of axonal tracts of olfactory neurons are bifurcated. In addition, most axonal tracts fail to terminate precisely in the glomeruli. Instead, the fasciculation of R neuron axonal tracks is normal without bifurcation in pdm3 mutants. However, the precise controlling for axon targeting to the EB is severely disturbed. Interestingly, the POU-domain protein Acj6 has a role in axonal targeting as well. In acj6 mutants, projection neurons still send their axons to the lateral horn but fail to present the same axon arborization pattern as wild type. Moreover, overexpression of the POU-domain protein Drifter can modify this phenotype in acj6 projection neurons. These results indicate that the POU-domain proteins do not regulate the general axon outgrowth and guidance but are relatively specific for properly targeting of axonal terminals (Chen, 2012).

Through transcription activity, Pdm3 may regulate expression of receptors or downstream signaling components for precise targeting of axonal tracts. Several studies suggest some candidates could be regulated by Pdm3 to mediate normal EB development. Robo2 and Robo3, the cell-surface proteins mediating axon guidance, are required for EB development. In robo2 and robo3 mutants, the doughnut-like structure of EB is abolished as in pdm3 mutants. Neuralized, an ubiquitin ligase involved in the processing of the Notch ligand Delta, is required for normal EB morphology. The EB surface is reduced in neuralized mutant animals. Thus, Pdm3 may regulate expression of these molecules, which can be tested in the future (Chen, 2012).

Drosophila mutants containing abnormal central complex are capable of performing locomotion, but show defects in the control of locomotion. These mutants show slower walking, abnormal orientation behavior and losing activity quickly. Although pdm3 mutants have the defective central complex, behavior assay for pdm3 mutant files could not be performed, as adult flies display severe locomotion defects and lethality. Pdm3 is also expressed in the ventral nerve cord and abnormal ventral nerve cord morphology has been found in pdm3 mutants, which might explain the severe locomotion defect. The expression and rescue experiment by pdm3-GAL4 implied that the critical timing for normal EB development is within the 0-48 hours APF, consistent with previous study presenting EB development with the same timing. The development of EB in the central complex is formed laterally by two compartments during early pupal stage. The R neurons innervating the EB are derived from the same DALv2 lineage (Pereanu, 2011). This study observed Pdm3 expression in the same region of anterior cortex. pdm3-GAL4 is expressed specifically in the larger field R neurons of the EB rather than the other type of neurons innervating the EB, suggesting that the R neurons play the dominant roles in the formation of the doughnut-like structure of the EB. Obviously, pdm3 is required cell-autonomously in this process. It is important to address downstream components regulated by pdm3 in this coordinated developmental process of the EB (Chen, 2012).

A transcriptional network controlling glial development in the Drosophila visual system

In the nervous system, glial cells need to be specified from a set of progenitor cells. In the developing Drosophila eye, perineurial glia proliferate and differentiate as wrapping glia in response to a neuronal signal conveyed by the FGF receptor pathway. To unravel the underlying transcriptional network, this study silenced all genes encoding predicted DNA-binding proteins in glial cells using RNAi. Dref and other factors of the TATA box-binding protein-related factor 2 (TRF2) complex were previously predicted to be involved in cellular metabolism and cell growth. Silencing of these genes impaired early glia proliferation and subsequent differentiation. Dref was found to control proliferation via activation of the Pdm3 transcription factor, whereas glial differentiation was regulated via Dref and the homeodomain protein Cut. Cut expression was controlled independently of Dref by FGF receptor activity. Loss- and gain-of-function studies showed that Cut was required for glial differentiation and was sufficient to instruct the formation of membrane protrusions, a hallmark of wrapping glial morphology. This work discloses a network of transcriptional regulators controlling the progression of a naïve perineurial glia towards the fully differentiated wrapping glia (Bauke, 2015).

Using a genome-wide RNAi-based screen this study has unravelled the transcriptional machinery responsible for such a switch during gliogenesis in the Drosophila eye. During embryonic development the anlage of the eye imaginal disc is formed. It is attached to the forming brain through the so-called Bolwig's nerve. A few glial cells reside along this nerve, presumably generated in the segmental nerves, as are most of the glia. These glial cells proliferate extensively during larval stages to form ~300 glial cells within each eye imaginal disc. During the third larval stage ~50 of these cells differentiate into wrapping glia in an FGFR-dependent manner. This study shows that the proliferation of the glial progenitor pool requires the activity of Pdm3 and the DNA replication-related element-binding factor (Dref), which are both strongly expressed by proliferating perineurial glia. Dref was first identified as an important factor required for efficient transcription of the proliferating cell nuclear antigen (PCNA), a key regulator of replication. Dref protein associates with the TATA box-binding protein related factor 2 (TRF2), which functions as a core promoter selectivity factor that governs a restricted subset of genes co-ordinately regulated. Interestingly, pan-glial knockdown of TRF2 also results in lethality, suggesting that the Dref/TRF2 complex is active in glia. Knockdown of CG30020, encoding a member of the Dref/TRF2 complex, or osa and moira, which had been shown previously to interact with Dref, caused similar glial phenotypes in the visual system (Bauke, 2015).

TRF2 targets several classes of TATA-less promoters present in more than 1000 genes, including a cluster of ribosomal protein genes. Likewise, Dref was found to associate with many genes involved in protein synthesis and cell growth, and loss of Dref results in reduced organismal growth rates. Most likely, dividing glial cells as well as differentiating wrapping glia have an increased protein synthesis demand, which might explain the observed defects in proliferation and differentiation. This study shows that expression of the transcription factor Pdm3 depends on Dref. Previously, Pdm3 has been associated with axonal pathfinding. The current results indicate that Pdm3 also regulates cell number. In contrast to Dref, Pdm3 expression is repressed by FGFR signalling, ensuring that perineurial glia routed to differentiation do not express Pdm3 anymore (Bauke, 2015).

Previous work suggested that in the Drosophila eye imaginal disc perineurial glial cells at the anterior margin of the eye field are competent to react to a neuronal signal inducing their glial differentiation. During this phase glial cells have reduced Dref expression but increased FGFR activity. Whereas in the absence of FGFR signalling no glial differentiation can be observed, high levels of FGF signalling trigger the expression of Cut specifically in wrapping glia. In addition to Cut, Dref is essential for proper glial differentiation. Dref is required for normal Cut expression levels but gain of Dref function is unable to activate Cut ectopically in perineurial glial cells. This requires additional FGFR signalling, indicating that two parallel molecular pathways converge on the activation of the transcription factor Cut to orchestrate wrapping glial differentiation (Bauke, 2015).

In the Drosophila PNS Cut controls the ES/ChO lineage decision. By contrast, during glial cell development this work defined Cut as a master regulator organizing elaborated membrane growth, which is required during the wrapping of axons. Similarly, Cut instructs the morphogenesis of multi-dendritic neurons. In mammals, the Cut homologues Cux1/2 also control dendritic branching, the number of dendritic spines and synapses. The number of filopodial extensions correlates to the level of Cut expression, corresponding to these findings. It was recently shown that Cut-dependent filopodia formation depends on the function of CrebA, which activates components of the secretory pathway. Cut might not only orchestrate membrane organization through the modulation of the secretory pathway, it also directly controls cytoskeletal dynamics. In larval sensory da neurons, the actin bundling protein Fascin is necessary for a Cut-dependent induction of spiked cell protrusions. However, eye disc glial cells still form long cell processes when fascin expression is suppressed by RNAi. Further understanding of wrapping glial cell differentiation will require the identification of the transcriptional targets of Cut (Bauke, 2015).

In conclusion, this study demonstrates that the specification of wrapping glial cells in the developing visual system does not require a single lineage switch gene but rather appears as a gradual process. The specification of wrapping glia is orchestrated by a transcriptional network comprising Pdm3, Dref and Cut that is modulated by the activity of the FGFR (Bauke, 2015).

POU domain motif3 (Pdm3) induces wingless (wg) transcription and is essential for development of larval neuromuscular junctions in Drosophila

Wnt is a conserved family of secreted proteins that play diverse roles in tissue growth and differentiation. Identification of transcription factors that regulate wnt expression is pivotal for understanding tissue-specific signaling pathways regulated by Wnt. This study identified pdm3^m7, a new allele of the pdm3 gene encoding a POU family transcription factor, in a lethality-based genetic screen for modifiers of Wingless (Wg) signaling in Drosophila. Interestingly, pdm3^m7 larvae showed slow locomotion, implying neuromuscular defects. Analysis of larval neuromuscular junctions (NMJs) revealed decreased bouton number with enlarged bouton in pdm3 mutants. pdm3 NMJs also had fewer branches at axon terminals than wild-type NMJs. Consistent with pdm3^m7 being a candidate wg modifier, NMJ phenotypes in pdm3 mutants were similar to those of wg mutants, implying a functional link between these two genes. Indeed, lethality caused by pdm3 overexpression in motor neurons was completely rescued by knockdown of wg, indicating that pdm3 acts upstream to wg. Furthermore, transient expression of pdm3 induced ectopic expression of wg-LacZ reporter and wg effector proteins in wing discs. It is proposed that pdm3 expressed in presynaptic NMJ neurons regulates wg transcription for growth and development of both presynaptic neurons and postsynaptic muscles (Kim, 2020).

Transcription factors play essential roles by inducing genes during the formation of body plans, organ development, tissue specificity, and generation of diverse cell types. Numerous transcription factors are grouped based on similarity in their sequences and domain structures. Pituitary-specific positive transcription factor 1, Octamer transcription factor-1, Uncoordinated-86 domain (POU) transcription factors belong to a subfamily of homeodomain transcription factors, and are highly conserved in all metazoans. POU domain consists of two DNA binding domains, POU homeodomain and POU specific domain, and these two domains are linked by a flexible linker. Based on sequence homology of the POU domain and the linker, POU proteins are grouped into six classes. POU proteins are often expressed in spatiotemporally restricted patterns during development, implying that they may be specialized for differentiation of specific cells or tissues by activating required signal transduction pathways (Kim, 2020).

The class VI Drosophila POU domain motif 3 (Pdm3) protein is reported to function in olfactory receptor neurons (ORNs) by regulating olfactory receptor gene expression and axon targeting, and in ring (R) neurons by regulating the development of ellipsoid body (EB) and axon targeting to EB in the central brain. pdm3 is also important for the axon targeting of a type of tracheal dendrite (td) neurons. In particular, td neurons that normally form synapse in the nerve cord change their target to the central brain by ectopic expression of Pdm3. Besides the neuronal functions of Pdm3, pdm3 also acts as a repressor of abdominal pigmentation in D. melanogaster, and plays a role in female-limited color dimorphism in abdomen of D. montium. Despite these studies, it is still unknown how pdm3 performs these neuronal and non-neuronal functions (Kim, 2020).

pdm3^f00828 and pdm3¹ homozygotes exhibit defects in axon targeting, odor perception, and locomotion. pdm3^f00828 allele has insertion of a piggyback element in an intron near the 3' end of the pdm3 gene, and pdm3¹ has a premature stop codon in the middle of the coding region that results in the deletion of the POU domain. This study identified a new pdm3 allele, pdm3^m7, as a suppressor of lethality induced by Sol narae (Sona) overexpression in a genetic screen. Sona is a fly ADAMTS (A disintegrin and metalloprotease with thrombospondin motif) whose family members are secreted metalloproteases important for cell proliferation, cell survival and development. This study has shown that Sona positively regulates Wingless (Wg) signaling and is essential for fly development, cell survival, and wg processing. wg is a prototype of Wnt family that initiates signal transduction cascade as extracellular signaling proteins, and activation of Wnt signaling leads to transcriptional induction of multiple genes for regulation of cell proliferation, cell survival, cell fate decision, and cell migration. wg is important for the development of all appendages, and the wing imaginal disc has been a great tool to study wg signaling because wg secreted from its dorsal-ventral midline is crucial for growth and development of wings (Kim, 2020).

Wg also plays an essential role in the development of NMJ. During larval development, NMJs continue to form synaptic boutons that are specialized structures with axon terminals of motor neurons surrounded by reticular subsynaptical reticulum (SSR) formed by the plasma membrane of postsynaptic muscle19. Among multiple types of boutons such as type Ib, Is, II, and III, wg is secreted at a high level from the glutamatergic type Ib bouton known as the main localization site of wg protein and wg signaling components, and is absent or at very low levels in other types of boutons (Packard, 2002; Kim, 2020).

Type Ib boutons also have more extensive SSR compared to other bouton types, so are easily detected by the high level of Discs-Large (Dlg) as a postsynaptic marker. Type Ib boutons in NMJs of wg mutants show reduction in bouton number but increase in bouton size. Components in wg signaling such as Arrow (Arr) that positively regulates wg signaling as a coreceptor of wg also shows its mutant phenotype similar to wg, but Shaggy (Sgg)/GSK3β that negatively regulates wg signaling as a kinase shows opposite phenotype to wg. Thus, dynamic regulation of wg signaling is essential for the development of NMJ (Kim, 2020).

Secreted wg also signals to the presynaptic motor neuron to regulate Futsch, one of the microtubule-associated proteins (MAPs). Futsch is a homolog of mammalian MAP1B, and both Futsch and MAP1B are phosphorylated at a conserved site by Sgg/GSK3β. The phosphorylated MAP1B does not bind microtubules, which results in reduced stability of microtubules. Therefore, localization of Futsch at NMJ faithfully reflects the stability of microtubules that is dynamically regulated by wg signaling. Loss of futsch phenotype is similar to the loss of wg phenotype in NMJ (Kim, 2020).

This study reporta that pdm3 is identified as a suppressor of Sona-induced lethality. Based on the involvement of Sona in wg signaling and the neuronal role of Pdm3, the roles of pdm3 in NMJ were specifically studied. Similar to loss of wg, loss of pdm3 in NMJ caused decrease in number but increase in size of boutons. Lethality induced by overexpressed pdm3 was completely rescued by the knockdown of wg in motor neurons but not vice versa. This indicated that pdm3 functions upstream to wg, and prompted a test whether pdm3 can induce wg transcription. Indeed, transient expression of pdm3 in wing discs induced wg transcription and wg effector proteins. Based on these data, it is propose that one of the main functions of pdm3 in NMJ is to induce wg transcription (Kim, 2020).

This study reports that pdm3 regulates growth and development of NMJs. pdm3 mutants showed increase in bouton size and decrease in bouton number, which are similar to the phenotype of wg mutants. Lethality induced by the overexpression of pdm3 was rescued by knockdown of wg in NMJ, indicating that pdm3 functions upstream to wg. Furthermore, overexpression of pdm3 induced wg transcription in wing discs. It is proposed that a major function of pdm3 in motor neurons is to induce wg transcription, and secreted wg from motor neurons regulates growth, development, and maturation of both pre- and post-synaptic regions of NMJ (Kim, 2020).

The mammalian homolog of pdm3 is Brain-5 (Brn-5)/POU class 6 homeobox 1 (POU6F1) mainly expressed in brain and spinal cord. Brn-5 is heavily expressed in embryonic brain but also expressed in adult brain and multiple adult organs such as kidney, lung, testis, and anterior pituitary. In developing brain, Brn-5 is expressed in postmitotic neurons after neuronal progenitor cells exit cell cycle in the early process of terminal neuronal differentiation. Therefore, both pdm3 and Brn-5 function in differentiation of neurons. Interestingly, ectopic expression of Brn-5 inhibits DNA synthesis, which is similar to cell cycle arrest phenotype by wg overexpression. Given the homology between pdm3 and Brn-5 as well as functional similarities, Brn-5 may also induce wnt transcription (Kim, 2020).

Most of pdm3 functions identified so far are related to the maturation of neurons such as olfactory neurons, R neurons and td neurons as well as their postsynaptic partners. Ectopic expression of pdm3 induced lethality without exception, indicating that expression of pdm3 in fly tissues is generally repressed in vivo in order to express wg under the strict spatiotemporal control. An important question is whether pdm3 directly transcribe wg. This study found that wg transcription is induced only after 36 hours of transient overexpression of Pdm3. It is possible that the level of pdm3 needs to be over a threshold to induce wg transcription. Alternatively, pdm3 may need to turn on other components to indirectly induce wg transcription. DNA sequence of Brn-5 binding site has been reported, so analysis on wg and wnt regulatory regions will help understand the mechanism of wnt induction by pdm3 and Brn-5 (Kim, 2020).

This study consistently found more significant NMJ phenotypes in A2 than A3 in both pdm3 and wg mutants. Therefore, pdm3 and wg may play more prominent roles in the A2 than the A3 segment. In fact, the level of pdm3 was higher in the anterior region than the posterior region of ventral ganglion, which suggests that more wg may be present in the NMJs of anterior abdominal segments. Consistent with this idea, the number of type Ib boutons in the A2 segment was 1.8 times more than A3 segment. One difference between pdm3 and wg mutants is the lack of certain phenotypes in the A3 segment of pdm3 NMJs: the size of boutons and the number of axon terminals in A3 were not affected in pdm3 mutant. It is possible that pdm3 turns on both common and segment-specific genes besides wg, and A3 segment-specific components may alleviate the loss of wg phenotype in the A3 segment. Similarly, other proteins induced by pdm3 may also play important roles in NMJ growth, differentiation and maintenance. In fact, multiple signaling pathways including Glass-bottom-boat (Gbb) pathway also play roles in NMJ development. Gbb is secreted from muscles and induces development of both pre- and post-synaptic structures, similar to wg signaling (Kim, 2020).

This study identified a defective hobo element in the pdm3^m7 allele. The hobo element belongs to Ac family found in maize and has short inverted terminal repeats. Laboratory and wild strains of D. melanogaster have average 28 and 22 copies of hobo elements in the genome that are either full-length or defective, respectively. Because other suppressors identified in the genetic screen using Sona overexpression did not have hobo element in the pdm3 gene, the transposition of the hobo element to the pdm3 gene may have occurred subsequent to the generation of a point mutation in the arr gene by EMS. Since both arr and pdm3 are positively involved in wg signaling, this hobo insertion may have helped the original arr^m7 mutation to further decrease the activity of wg signaling under the condition of Sona overexpression (Kim, 2020).

Besides the neuronal roles of Pdm3, all pdm3 mutants show minor but consistent defects in planar cell polarity in a restricted region of the wing as well as adhesion between the dorsal and ventral wing blades. Other phenotypes such as wing drooping and premature death were also observed in all pdm3 mutants, but these may be due to malformation of synaptic structures. pdm3 also plays a role in female-limited color dimorphism in abdomen of D. montium. The authors found in sexually dimorphic females that the first intron of the pdm3 gene has four tandem sets with predicted binding sites for the HOX gene Abdominal-B (Abd-B) and the sex determination gene doublesex (dsx). Interestingly, it has been shown that wg expression is repressed by the combinatory work of Abd-B and Dsx proteins. Taken together, it is possible that transcription of wg and pdm3 is co-repressed by Abd-B and Dsx. Such co-repression of wg and pdm3 transcription may be also required for synaptic growth and differentiation in neurons. Further studies on pdm3 will help understand how this understudied transcription factor is involved in the final differentiation of various cell types (Kim, 2020).

Search PubMed for articles about Drosophila Pdm-3

Bauke, A.C., Sasse, S., Matzat, T. and Klämbt, C. (2015). A transcriptional network controlling glial development in the Drosophila visual system. Development 142(12):2184-93. PubMed ID: 26015542

Chen, C. K., Chen, W. Y. and Chien, C. T. (2012). The POU-domain protein Pdm3 regulates axonal targeting of R neurons in the Drosophila ellipsoid body. Dev Neurobiol 72(11): 1422-1432. PubMed ID: 22190420

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 ID: 10069339

Kim, Y. and Cho, K. O. (2020). POU domain motif3 (Pdm3) induces wingless (wg) transcription and is essential for development of larval neuromuscular junctions in Drosophila. Sci Rep 10(1): 517. PubMed ID: 31949274

Packard, M., et al. (2002). The Drosophila Wnt, Wingless, provides an essential signal for pre- and postsynaptic differentiation. Cell 111: 319-330. 12419243

Pereanu, W., et al. (2011). Lineage-based analysis of the development of the central complex of the Drosophila brain. J. Comp. Neurol. 519(4): 661-89. PubMed ID: 21246549

Qian, C. S., Kaplow, M., Lee, J. K. and Grueber, W. B. (2018). Diversity of internal sensory neuron axon projection patterns is controlled by the POU-domain protein Pdm3 in Drosophila larvae. J Neurosci 38(8): 2081-2093. PubMed ID: 29367405

Ray, A., van der Goes van Naters, W., Shiraiwa, T. and Carlson, J. R. (2007). Mechanisms of odor receptor gene choice in Drosophila. Neuron 53: 353-369. PubMed ID: 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 ID: 18846726

Schlegel, P., Texada, M. J., Miroschnikow, A., Schoofs, A., Huckesfeld, S., Peters, M., Schneider-Mizell, C. M., Lacin, H., Li, F., Fetter, R. D., Truman, J. W., Cardona, A. and Pankratz, M. J. (2016). Synaptic transmission parallels neuromodulation in a central food-intake circuit. Elife 5. PubMed ID: 27845623

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 ID: 18614681

date revised: 5 April 2021

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