The diverse physiological effects of dopamine are mediated by multiple receptor systems. The dDA1 represents one of the Drosophila dopamine receptors that activate the cAMP cascade. To gain insight into the role of dDA1, a polyclonal antibody was generated against the unique sequence in dDA1 and dDA1 distribution in the central nervous system (CNS) was investigaed. In both larval and adult CNS pronounced dDA1 immunoreactivity is present in the neuropil of the mushroom bodies, a brain structure crucial for learning and memory in insects, and four unpaired neurons in each thoracic segment. In addition, the larval abdominal ganglion contained two dDA1 cells in each segment. This expression pattern appears to be maintained in the condensed adult abdominal ganglion although the precise number and the intensity of staining were somewhat variable. The adult CNS also exhibits intense dDA1 immunoreactivity in the central complex, a structure controlling higher-order motor function, moderate expression in several neurosecretory cells, and weak staining in two unpaired neurons in the mesothoracic neuromere. The dDA1 expression in these areas was detected only in the adult, but not in third instar larval CNS (Kim, 2003).
A set of peptidergic neurons is conserved throughout all developmental stages in the Drosophila central nervous system. A small complement of 28 apterous-expressing cells (Ap-let neurons) in the ventral nerve cord (VNC) of Drosophila larvae co-express numerous gene products. The products include the neuroendocrine-specific bHLH regulator called Dimmed (Dimm), four neuropeptide biosynthetic enzymes (PC2, Fur1, PAL2, and PHM), and a specific dopamine receptor subtype (dDA1). For the PC2, Fur1, and PAL2 enzymes, and for the dDA1 receptor, this neuronal pattern represents the vast majority of their total expression in the VNC. In addition, while Dimm and PHM are present in the peritracheal Inka cells in larvae, pupae, and adults, Ap, PC2, Fur1, PAL2, and dDA1 are not. PC2, PAL2, and DA1 receptor expression are all controlled by both dimm and ap. Previous genetic analysis of animals deficient in PC2 revealed an abnormal larval ecdysis phenotype. Together, these data support the hypothesis that the small cohort of Ap-let interneurons regulates larval ecdysis behavior by secretion of an unidentified amidated peptide(s). This hypothesis further predicts that the production of the Ap-let neuropeptide(s) is dependent on each of four specific enzymes, and that a certain aspect(s) of its production and/or release is regulated by dopamine input (Park, 2004).
One of the Drosophila D1-like dopamine receptors, dDA1 (CG9562), is expressed in a subset of the larval and adult CNS neurons. In the VNC, dDA1 immunoreactivity is evident in a single Dorsal neuron in each thoracic and abdominal hemi-neuromere, and a single lateral neuron in each thoracic hemi-neuromere. This expression pattern is similar to that of the Ap-let group. To test whether dDA1 is expressed in Ap-let neurons, the larval CNSs of apGAL4/UAS-GFP or of c929/UAS-GFP were stained with the dDA1 antibody. All of the dDA1 cells were positive with apGAL4, and with c929, and they included the Dorsal chain and one of the two Tv neurons. This indicates the dDA1-positive T neuron is either Tv or Tvb. When the CNS was double-stained with anti-FMRFa antibody that stains the larval Tv neuron, the dDA1 and dFMRFa immunosignals were in distinct cells. Together, these data indicate that the dDA1-positive cell in the T cluster os the Tvb neuron. Similar to PHM, PC2, Fur1, and PAL2, none of the ap-positive ventral neuron pairs expressed dDA1 immunosignals (Park, 2004).
Neuronal specification is often seen as a multistep process: earlier regulators confer broad neuronal identity and are followed by combinatorial codes specifying neuronal properties unique to specific subtypes. However, it is still unclear whether early regulators are re-deployed in subtype-specific combinatorial codes, and whether early patterning events act to restrict the developmental potential of postmitotic cells. This study used the differential peptidergic fate of two lineage-related peptidergic neurons in the Drosophila ventral nerve cord to show how, in a feedforward mechanism, earlier determinants become critical players in later combinatorial codes. Among the progeny of neuroblast 5-6 are two peptidergic neurons: one expresses FMRFamide and the other one expresses Nplp1 and the dopamine receptor DopR. The HLH gene collier functions at three different levels to progressively restrict neuronal identity in the 5-6 lineage. At the final step, collier is the critical combinatorial factor that differentiates two partially overlapping combinatorial codes that define FMRFamide versus Nplp1/DopR identity. Misexpression experiments reveal that both codes can activate neuropeptide gene expression in vast numbers of neurons. Despite their partially overlapping composition, the codes are remarkably specific, with each code activating only the proper neuropeptide gene. These results indicate that a limited number of regulators may constitute a potent combinatorial code that dictates unique neuronal cell fate, and that such codes show a surprising disregard for many global instructive cues (Baumgardt, 2007).
In the developing Drosophila VNC, approximately 90 neurons express the LIM-homeodomain regulator Apterous (Ap), and these represent at least six different cell types. This study focuses on three of the Ap neurons: two cells of the Ap cluster, the Tv cells, which express FMRFa, and the Tvb cells, which together with the dAp cells express DopR. A number of regulators involved in Tv neuron specification have been identified, but to better understand specification of the related Tvb/dAp neurons, it was important to identify the putative neuropeptide gene expressed by Tvb/dAp neurons. The completion of the Drosophila genome led to the prediction of several additional neuropeptide genes, including the Neuropeptide like precursor protein 1-4 genes (Nplp1-4). The validity of these predictions has been confirmed by the identification of expressed sequence tags (ESTs) matching these genes, and by the detection of amidated and secreted peptides in circulation, and/or in brain extracts. Expression of gene products from one of these genes, Nplp1, was found in a set of cells in the VNC reminiscent of the Tvb/dAp neurons. In situ hybridization for Nplp1 verified that these cells indeed correspond to the dAp neurons and to one Ap cluster neuron. To further identify this Ap cluster neuron, antibodies were raised against pro-Nplp1 and against one of the processed and amidated peptides, IPNamide, and a similar pattern was detected. Markers were used for specific subsets of Ap neurons, and the Nplp1-expressing cell in the Ap cluster was identified as the Tvb neuron. In Tvb/dAp neurons, Nplp1 and DopR expression commences in the late embryo (18 h after egg laying [AEL]) and persists at least into the third larval stage. Nplp1 and DopR are thus specifically expressed by the 28 embryonic and larval Tvb/dAp neurons (Baumgardt, 2007).
Recent studies have revealed that ap and dimm are important for DopR expression in Tvb/dAp. It was asked whether these Ap neuron determinants, as well as eya, also affected Nplp1 expression. Nplp1 expression was found to depended on eya, ap, and dimm, and eya also regulates DopR. How is Tv versus Tvb/dAp cell fate then determined? Although both cell types express ap, dimm, and eya, only Tv neurons express dac and have activated the BMP pathway. Could the mere absence of dac and/or BMP activation be sufficient to specify the Tvb/dAp fate? To test this, expression of Nplp1 was analyzed in dac and BMP mutants, but no evidence was found of ectopic Nplp1 expression in Tv neurons. Thus, specification of Tvb/dAp neurons likely requires additional factors restricted to this cell type (Baumgardt, 2007).
The COE family of HLH regulators is highly evolutionary conserved, and is represented in Drosophila by a single member, col (Flybase, knot). COE genes play important roles during nervous system development in Caenorhabditis elegans and vertebrates, and col is expressed in the developing Drosophila central nervous system (CNS), although no function has yet been assigned to it there. The involvement of members of this gene family in nervous system development in other species, and the embryonic CNS expression of col, prompted an investigation of the possible role of col during Ap neuron specification. col has a dynamic expression pattern in the VNC, and focus was initially placed on its expression in mature Ap neurons, at 18 h AEL, and larval stages. At these stages, col is expressed specifically in Tvb/dAp neurons, and expression is maintained in these neurons at least into the third larval stage (see below). This raised the possibility that col plays a role in Tvb/dAp cell fate specification. This notion was supported by the complete loss of Nplp1 and DopR expression in col mutants (Baumgardt, 2007).
Previous studies have addressed the regulatory interactions between several of the Ap neuron determinants. However, these had not been addressed in the case of dac, eya, and dimm. As expected from the late onset of dimm expression, no evidence was found of dimm regulation of Dac or Eya. Similarly, as expected from the mild effect of dac upon FMRFa, dac does not regulate Dimm. In contrast, in eya mutants, a nearly complete loss of Dimm expression was found in the Tv, Tvb, and dAp neurons (Baumgardt, 2007).
With a more complete picture of how previously identified Ap neuron determinants interact genetically, whether col acts upstream, downstream, or in parallel to other Ap neuron determinants was determined. The expression of Col was examined in embryos mutant for these other regulators. In general, no severe effects on Col expression were found. The one exception was in sqz, in which a reproducible increase in the number of Col cells was found. This was, however, expected, since sqz affects the composition of Ap cluster cells, with an increase both in the number of Ap cluster cells (specifically in T1) and an increase in Tvb cells at the expense of Tv cells (in T1-T3). In line with this, an increase was also found in Nplp1 cells in sqz mutants (Baumgardt, 2007).
Does col act upstream of other Ap neuron determinants instead? To address this, the expression of these other regulators was examined in col mutant embryos. This analysis was facilitated by the fact that col2 and col3 mutants, which are both genetically strong alleles, are not protein null, thus allowing for detection of Col in col mutants. In addition, Col is expressed by all four Ap cluster neurons at stage 15. In col mutants, it was found that although sqz and Dac are largely unaffected, ap, Eya, and Dimm are all completely absent from Ap neurons. Because ap, eya, and dimm all regulate FMRFa, the loss of these regulators prompted a look at the expression of FMRFa as well, and as expected, in col mutants, a complete loss was found of FMRFa in the Tv neurons. However, FMRFa is still expressed in the SE2 neurons, a feature common to all identified FMRFa regulators except dimm. These results suggest that Ap cluster neurons are generated in col mutants, but are incompletely specified because they express part of their normal specification code such as dac and sqz, but not other elements of the code such as ap, Eya, Dimm, FMRFa, Nplp1, and DopR (Baumgardt, 2007).
The severe effect of col upon ap, eya, and dimm within all Ap cluster neurons, and upon FMRFa within the Tv neuron, is at odds with the restricted expression of Col in Tvb/dAp neurons at late embryonic stages. Therefore whether Col is more widely expressed at earlier embryonic stages was analyzed, focusing on the Ap cluster neurons. This revealed that although col is restricted to Tvb neurons at stages 17 and 16, expression was observed in all four Ap cluster neurons at stage 15, the stage at which these neurons are first identifiable using ap, Eya, Dac, and sqz as specific markers (Baumgardt, 2007).
Is Col expressed even in the progenitor cells generating Ap cluster neurons? Because ap, Eya, Dac, and sqz are not expressed in Ap cluster neurons prior to stage 15, resolving this issue required the identification of the neuroblast lineage generating the Ap cluster. Extensive work during the last two decades has provided a detailed lineage map of most, if not all, of the 30 neuroblasts found in each hemisegment, and has identified regulatory genes expressed by different neuroblasts. These studies, together with the extreme lateral positioning of early Ap cluster neurons and their appearance only in thoracic hemisegments, allowed use a series of specific markers and determine that the Ap cluster is generated by NB 5-6 -- a lateral-most neuroblast that has been shown to generate larger lineages in the thoracic segments. These results, combined with previous detailed studies of NB 5-6 development, suggested a tentative model for the thoracic NB 5-6 lineage, and lead to placement Col expression within this lineage (Baumgardt, 2007).
Col is expressed by all four newly born Ap cluster neurons and is essential for Ap cluster specification, as evident from the complete loss of ap and Eya expression in col mutants. Col is rapidly down-regulated from three Ap cluster cells and maintained only in Tvb. Is the down-regulation of col important for proper Ap cluster differentiation? To test this, col was misexpressed using the apGAL4 driver, which is not expressed until stage 16, thus maintaining col expression in all four Ap cluster neurons at the time when Col is normally down-regulated. This experiment led to frequent activation of Nplp1 in one additional Ap cluster neuron, and staining for Dimm reveals that this cell is indeed the Tv neuron. FMRFa expression is frequently down-regulated in Tv, but no Tv cells were observed that co-express FMRFa and Nplp1. The finding that col misexpression in the Ap cluster only leads to one ectopic Nplp1 cell and no ectopic Dimm cells indicates that col cannot induce a peptidergic cell fate, at least not with this late driver. However, because the two unaffected cells already are expressing ap and Eya, it was predicted that co-misexpression of dimm, together with col, should trigger Nplp1 expression in all four Ap cluster neurons. This is indeed what was found. In summary, down-regulation of col in three Ap cluster neurons is essential for proper Ap cluster specification (Baumgardt, 2007).
Col is expressed prior to ap and Eya in the Ap cluster neurons, and it is essential for ap and Eya expression within these cells. To address whether col is also sufficient to activate ap and Eya, col was misexpressed in all neurons, using the elav-GAL4 driver. This led to ectopic activation of both ap and Eya. In addition, some activation of Dimm, Nplp1, and DopR expression was found. Although ectopic activation of ap or Eya alone was found in several regions, co-activation was largely confined to neuroblast row 5 -- the anterior region of Gsbn expression. Typically six to ten ap/Eya co-expressing cells were observed in the lateral-most part of row 5. Ectopic activation of ap/Eya, together with Dimm, Nplp1, and DopR, was also confined to lateral-most row 5, i.e., the posterior-most part of gsblacZ cells, and further confined to thoracic segments. Ectopic Nplp1/DopR expression is not overlapping with FMRFa, and there is clear evidence of ectopic Dimm expression, indicating that additional peptidergic neurons are being generated. Ectopic generation of ap/Eya double-expressing cells, i.e., ectopic 'Ap cluster' neurons, was observed already at stage 13, i.e., prior to when Ap cluster neurons are normally born (Baumgardt, 2007).
These results show that col can activate ap and Eya in a number of neurons, but can act to generate bona fide Ap cluster neurons only in a highly context-dependent manner: in lateral, thoracic, row 5 neurons. The appearance of six to ten Eya-expressing cells, but only three to five Nplp1/DopR-expressing cells, and no evidence of ectopic FMRFa expression, suggests that the generation of ectopic Ap cluster neurons is biased toward Tvb (Nplp1/DopR expressing) as opposed to Tv (FMRFa expressing) cell fate. In contrast, although col function depends upon these three positional cues, these results indicate that col is able to override the temporal coding within lateral row 5, and activate Nplp1 and DopR in earlier-born neurons (Baumgardt, 2007).
The loss- and gain-of-function studies place col clearly upstream of ap and eya. Does col act merely to regulate ap and eya in early postmitotic Ap cluster neurons, or does it play additional roles during Ap cluster formation? To further address this issue, attempts were made to 'cross-rescue' col with ap and eya, by expressing ap and eya in a col mutant background. First, as a positive control, attempts were made to rescue col by providing col activity using elav-GAL4/UAS-col. This led to a robust rescue, both of Ap cluster determinants (Eya, ap, and Dimm) and of terminal differentiation genes (Nplp1, DopR, and FMRFa). Similar to the col misexpression experiments, a clear increase was found in 'Ap cluster' neurons, primarily of the Tvb type, as evident from the finding of six to ten ap/Eya- and three to five Nplp1/DopR-expressing neurons per hemisegment. Next, attempts were made to 'cross-rescue' col mutants with ap and eya, and indeed a significant degree of rescue of Ap cluster formation was found, as evident both from Dimm and FMRFa expression. In contrast, no evidence of rescue of Nplp1 or DopR was found in these embryos. Because Col can be detected in the col2 and col3 mutant backgrounds, a Dimm/Col-expressing cell was identified adjacent to the Tv/FMRFa neuron. This indicates that ap/eya can partially rescue Tvb cell fate, but in the absence of col activity, these 'Tvb' neurons do not activate Nplp1. In summary, the finding that in col mutants, ap and eya can partially rescue the Tv cell fate, but not Tvb cell fate, suggests additional roles for col in Tvb specification (Baumgardt, 2007).
The results indicate that Tvb cell fate is not specified by a linear col-->ap/eya-->dimm-->Nplp1/DopR genetic cascade. To further address this issue, the sufficiency was examined of col, ap, and eya to activate Dimm when misexpressed both alone and in combination. These experiments reveal that although col can trigger some ectopic activation of Dimm, there is little effect upon Dimm when misexpressing ap, eya, or ap/eya. In contrast, co-misexpression of col with either ap or eya, and in particular, co-misexpression of all three genes, leads to striking ectopic Dimm expression (Baumgardt, 2007).
Does col play a role even at the final step of Tvb differentiation, i.e., in the activation of Nplp1? Attempts were made to address the possible late role of col by misexpressing it alone and together with other Ap neuron determinants, and then assay its potency in activating Nplp1. Importantly, if there is a simple linear col-->ap/eya-->dimmNplp1/DopR genetic cascade at work, the effect of triple co-misexpression of ap/eya/dimm should not be enhanced by addition of col to this code. However, a striking enhancement was found of ectopic Nplp1 expression when adding col to this code. One particular double co-misexpression combination, col/ap, was more potent than others in activating both Dimm and Nplp1. A likely explanation for this effect is that co-misexpression of col/ap activates significant ectopic eya expression (Baumgardt, 2007).
To further address the late role of col, a transgenic RNA-interference (RNAi) line, (UAS-col-dsRNA), was generated, and attempts were made to suppress col gene activity by crossing this line to apGAL4. Because apGAL4 also drives expression in the developing wing disc, the efficiency was examined of this novel tool in suppressing col gene activity in this tissue. This phenocopied the effects of col mutants on wing development, with a clear L3-L4 wing vein fusion, indicating that this RNAi transgene specifically blocks col gene activity. However, upon analyzing late larval (third instar) CNSs, no effect upon Col expression in Tvb neurons was found, and as expected, no effect was found upon Nplp1 expression. Recent studies reveal that RNAi can be efficiently enhanced by overexpression of components of the RNAi pathway, in particular of the Dicer-2 (Dcr-2) gene. Therefore Dcr-2 was co-expressed with col dsRNA (UAS-Dcr-2/+; apGAL4/UAS-col-dsRNA), and a clear effect was found not only upon Col, but importantly, also upon Nplp1 expression. No obvious effect was found in the first instar larvae, but in third instar larvae, Col expression is specifically and completely lost from all Tvb/dAp cells. This leads to a complete loss of Nplp1 in 44% of Tvb/dAp cells, and strongly reduced expression in the remaining expressing cells. Strikingly, this strong effect upon Nplp1 is not an indirect effect of down-regulation of ap, Eya, or Dimm. As anticipated, col RNAi has no effect upon FMRFa expression in the third instar larvae (Baumgardt, 2007).
Previous studies have identified several regulators acting to specify Tv fate and to control FMRFa expression. Although co-misexpression of parts of this code had been previously tested, all possible combinations had not. Similar to the combinatorial activation of Nplp1 and DopR, it was found that whereas co-misexpression of ap/sqz, ap/dac, or ap/dimm has limited effect upon FMRFa expression, triple co-misexpression of these regulators, and in particular of ap/dimm/dac, leads to a dramatic ectopic activation of FMRFa (Baumgardt, 2007).
The identification of two partly overlapping and highly potent combinatorial codes allowed lead to asking of an important question: Does combinatorial misexpression of these regulators merely lead to a general confusion with a mixed neuronal identity, or are these codes truly instructive and specific? To address this issue, the expression of Nplp1 and FMRFa was examined in the various misexpression backgrounds. Not surprisingly, when common and partial components of these codes are misexpressed, such as ap/dimm (common to both Tv and Tvb/dAp neurons), ectopic activation of both Nplp1 and FMRFa was found in different subsets of cells. However, as a third, and cell-type specific, regulator is added, not only does the amount of ectopic, terminal differentiation gene expression increase dramatically, but less evidence of cross-activation of the inappropriate downstream gene was found. This surprising finding reveals that combinatorial misexpression may act in a highly specific and instructive manner, and that these combinatorial codes may be viewed as potent binary switches for cell fate specification. In addition, for both codes, ectopic activation of FMRFa and Nplp1 is observed in neurons throughout the VNC and brain, and traverses many developmental boundaries, such as anteroposterior, dorsoventral, and mediolateral boundaries (Baumgardt, 2007).
This study has identified a sequential regulatory cascade of combinatorial coding that acts to specify two unique neuronal cell fates during Drosophila CNS development. Combined with previous studies, the findings provide the following model for Ap cluster generation and specification. Neuroblast 5-6 forms in the first wave of neuroblast delamination, at late stage 8, and generates a mixed lineage of glia and neurons. At stage 13, Col expression is turned on specifically in thoracic NB 5-6, in two subsequent ganglion mother cells (GMCs) at stages 13/14, and in the four Ap cluster neurons generated from these GMCs, during stages 14/15. The birth order of the four Ap cluster neurons has not been resolved, and the sibling relationship of Tv and Tvb is thus unclear. When Ap neurons are born, col activates ap and eya, whereas sqz and dac are activated by unknown regulator(s). sqz appears to play an early postmitotic role, apparently acting in the Notch pathway, to ensure proper Ap cluster composition, and sqz mutants display both additional Ap cluster cells (in T1) and additional Tvb cells (in T1-T3). col is rapidly down-regulated from three Ap neurons, but remains expressed in the Tvb, where it acts with ap and eya to activate dimm at stage 16. At late embryogenesis, col acts with ap, eya, and dimm to activate Nplp1 and DopR in Tvb. In the Tv neuron, ap and eya act, apparently independently of col, to activate dimm expression. In the Tv neuron, eya furthermore plays a role in setting up competence to respond to the BMP signal. At stage 17, the Tv axon reaches the dorsal neurohemal organ (DNH) and receives the BMP ligand Gbb that activates the Wit receptor, and then triggers activation of the BMP pathway in the Tv neuron. At 18 h AEL, ap, eya, sqz, dac, dimm, and BMP signaling cooperate to activate FMRFa in the Tv neuron. In addition to their roles in neuropeptide regulation and BMP signaling, both ap and eya act to ensure proper axon pathfinding of Tv neurons (eya), as well as Tvb and dAp neurons (ap and eya). The role that col may play more directly in axon pathfinding has not been resolved due to the fact that the expression of the appropriate axonal markers (apGAL4, Nplp1, DopR, and FMRFa) is completely absent in col mutants. Given the complexity of axon pathfinding, it is anticipated that several other regulators are yet to be identified before a combinatorial code for 'Tv-type' or 'Tvb-type' axonal pathfinding is deciphered. Indeed, no evidence was found that combinatorial misexpression of the abovementioned regulators can dictate axonal projections, because ectopic Nplp1 or FMRFa axons are following many different routes in the VNC. Finally, dimm also plays additional roles to those described above and is necessary for the expression of neuropeptide-processing enzymes in peptidergic neurons. Importantly, dimm acts independently to control expression of the neuroamidase gene PHM in the Tv and Tvb neurons, and dimm is sufficient to activate PHM in most, if not all, VNC neurons. Thus, during the specification and differentiation of the Ap neurons, there exists a remarkable diversity in the division of labor between the identified regulators, with most of them participating in more than one, but never all, of the identified events (Baumgardt, 2007).
This study has identified a multistep process for specifying the Tv and Tvb cell fates. What would be the purpose of this type of sequential combinatorial coding? In other model systems with higher genetic resolution, such as Escherichia coli and yeast, extensive genetic analysis has revealed that this type of sequential gene regulation is quite common, and a recent study in C. elegans suggests it may also function during neuronal specification. These regulatory nodes, also known as feedforward loops (FFL), have been shown to ensure fidelity in gene regulation. For instance, in a simple FFL in which gene A regulates gene B, and A/B then co-operate to regulate C, activation of C depends upon prolonged A expression such that A/B will have time to activate C. If A is only active in a short burst, B may be activated, but C is not, because A/B never co-express for a sufficiently prolonged period of time. The role of col during Ap cluster specification provides an excellent example of a FFL used during neuronal cell fate specification. col is expressed in all four Ap cluster cells and plays an early role in activating ap and Eya, but is only maintained in Tvb, where it plays a later role in activating first dimm, then Nplp1. Maintained expression of col in all four Ap cluster neurons, by driving col expression from apGAL4, leads to activation of the Tvb program also in the Tv neuron. Thus, a burst of col expression has a different informational value than persistent col expression -- general Ap cluster specification versus Tvb specification (Baumgardt, 2007).
Misexpression of each of the two identified combinatorial codes leads to striking ectopic activation of the Nplp1 and FMRFa genes, and two particular aspects of these findings were surprising. First, the global potency of these codes: co-misexpression triggers ectopic FMRFa of Nplp1 in a number of neurons, located in many different anteroposterior, dorsoventral, and mediolateral positions. Thus, it would appear that early regulators mainly act to ensure proper combinatorial coding in each neuron, and play a minor role in restricting cell fate by limiting the cell's competence. Once the proper code is in place, the cell fate specification program will be carried out irrespective of the history of the cell. Second, the striking binary effect of these codes is noteworthy: the change of one single player in a code completely alters target gene choice. For instance, misexpression of ap/dimm/dac leads almost exclusively to strong FMRFa activation, but simply replacing dac with col leads to almost exclusively Nplp1 activation. Thus, it appears that more complete codes not only have great potency, but also have great specificity (Baumgardt, 2007).
Col shows a very dynamic expression pattern in the VNC, exemplified in NB 5-6 by the expression in the neuroblast, in two GMCs, in all four Ap cluster neurons, and finally only in Tvb. This poses three obvious questions: what activates col in the neuroblast, what shuts it down in three of the Ap cluster neurons, and finally, what maintains col in Tvb? As for the activation of col in the late 5-6 neuroblast, row 5 neuroblast determinants, thoracic determinants, and late temporal determinants are obvious candidates. Indeed, current work has identified input from a number of such upstream regulators. It is less clear why col expression is lost from three Ap cluster cells and maintained in Tvb. It is possible that the initial expression in all four Ap cluster cells merely reflects residual expression, as an effect of the activation by earlier determinants acting in the neuroblast. But why is col then maintained in Tvb, and similarly, what maintains eya and ap in all four Ap cluster cells? One simple solution would be autoregulation of each gene. But surprisingly, there is no evidence of autoregulation of col. In addition, no clear evidence was found of cross-regulation between col, ap, or eya, at least not during embryonic stages. Thus, it seems likely that other mechanisms, either unidentified regulators or, perhaps, epigenetic mechanisms, act to ensure the continual expression of these regulators during larval (and perhaps adult) life (Baumgardt, 2007).
To identify dDA1 mutants, multiple fly lines with lesions that are known to map at the chromosomal location 88A where the dDA1 gene resides were surveyed. Two lines showed abnormal dDA1 immunoreactivities in the brain. One of them is the inversion line In(3LR)234, which has the break points at 67D and 88A-88B. The other is f02676 containing the transposable element piggyBac inserted at the fimmunoreactivitiest intron in the dDA1 locus. dDA1 is highly enriched in the MB lobes, the central complex, a few scattered cells in the brain, and the Apterous-positive cells in the thoracico-abdominal ganglion (Kim, 2003; Park, 2004). Both In(3LR)234 and f02676 have negligible dDA1 immunoreactivities in the MB and the central complex but intact immunoreactivities in the scattered and Apterous-positive cells. Consistently, full-length dDA1 transcripts were detected in both lines by RT-PCR. Thus, In(3LR)234 and f02676 appear to have lesions in the regulatory sequence for tissue-specific dDA1 expression, representing hypomorphic dDA1 alleles, and are designated as dumb1 [D1(uno) in mushroom bodies] and dumb2, respectively (Kim, 2007b).
The observations that dDA1 is concentrated in the MB neuropil and can activate the cAMP pathway (Sugamori, 1995; Kim, 2003) prompted an investigation of the role of dDA1 in olfactory conditioning. When subjected to aversive conditioning using odorants octanol (OCT) and benzaldehyde (BA) as conditioned stimuli and electric shock as a US, dumb1 homozygous mutants showed severely impaired performance immediately after training. Performance of dumb1 did not decline at 1 h after training, suggesting that dumb1 is defective in learning rather than memory. dumb1 has two break points caused by inversion. Thus, to investigate the lesion accountable for poor performance of dumb1, two deficiency lines were used, Df(3L)AC1 and Df(3R)su(Hw)7, having deletion between chromosomes 67A2 and 67D13 and chromosomes 88A9 and 88B2, respectively, which include each break point. Similar to dumb1, dumb1/Df(3R)su(Hw)7 trans-heterozygous mutants exhibited poor performance immediately or 1 h after training. In contrast, performance of dumb1/Df(3L)AC1 was comparable to that of Canton-S and dumb1/+ or Df(3R)su(Hw)7/+ heterozygous flies immediately after training. These data indicate that the lesion in chromosome 88A is responsible for poor learning of dumb1 mutants. The flies heterozygous for both deficiency chromosomes had slightly lower performance scores compared with those of Canton-S and dumb1/+ at 1 h after training. This could be attributable to putative memory genes in the deleted chromosomes (Kim, 2007b).
It was next asked whether the dumb1 phenotype is linked to the lesion in dDA1 by examining the independent dumb allele dumb2 and dumb1/dumb2 trans-heterozygous mutants in aversive conditioning. Like dumb1, both genotypes had negligible performance scores immediately after or 1 h after training, supporting the potential role of dDA1 in punishment-mediated olfactory learning. dumb1 and dumb2 heterozygous flies exhibited normal performance; thus, a single copy of dDA1 may be sufficient for mediating this process (Kim, 2007b).
To test whether dumb mutants could learn better with different conditioned stimuli, other odorants were used in electric shock-mediated olfactory conditioning. When trained with ethyl acetate (EA) and isoamyl acetate (IAA) as conditioned stimuli, dumb1 mutants also displayed severely impaired learning. This suggests that dDA1 is involved in aversive learning induced by diverse odor inputs. The impaired performance of dumb mutants is not attributable to anomalous sensory modalities because all dumb alleles and the control Canton-S and w1118 flies showed comparable avoidance of the CS odors and electric shock presented at two different concentrations or intensities, respectively. Thus, poor learning of dumb mutants is likely attributable to their inability to associate CS+ with US (Kim, 2007b).
Synaptic output of dopamine neurons has been shown to be required during training for aversive learning (Schwaerzel, 2003), implicating the similar requirement of dDA1 at the time of learning. To test this, the pan-neuronal driver Elav-GAL4 and GAL80ts, which allows the temporal control of GAL4 activities, was used. GAL80 binds to GAL4 to sequester it from activating upstream activating sequence (UAS). The temperature-sensitive GAL80ts can no longer bind to GAL4 at 30°C, allowing it to act on UAS to induce downstream gene expression. The piggyBac inserted at the first intron of the dDA1 gene in dumb2 has UAS. Although the piggyBac insertion itself interferes with endogenous dDA1 expression in dumb2, UAS in piggyBac, after binding to GAL4, may induce dDA1 transcription from the second exon containing the 5' untranslated sequence and the start codon. Thus, dumb2 was crossed with dumb1 carrying Elav-GAL4 and GAL80ts to generate Elav-GAL4,GAL80ts/+;dumb1/dumb2 flies. The Elav-GAL4,GAL80ts/+;dumb1/dumb2 kept at room temperature did not have any detectable dDA1 induction; however, when the flies were reared at 30°C for 3 d, conspicuous dDA1 IR was visible in the MB lobes and pedunculi, the central complex, and other brain areas including antennal lobes. Whereas Elav-GAL4 is expressed in all neurons, membrane-bound GFP reporters driven by Elav-GAL4 are enriched in certain brain areas including the aforementioned structures. Therefore, the temporal manipulation of GAL80ts and Elav-GAL4 activities was effective in restricting dDA1 expression at the adult stage in dumb mutants (Kim, 2007b).
When Elav-GAL4,GAL80ts/+;dumb1/dumb2 flies reared at room temperature were subjected to electric shock-mediated conditioning, they showed poor learning; however, their performance was dramatically improved after temperature shift to 30°C. The performance score of Elav-GAL4,GAL80ts/+;dumb1/dumb2 with the restored dDA1 expression was slightly lower than that of Canton-S; nonetheless, it was not significantly different from that of Canton-S treated with the same temperature shift but was different from that of uninduced Elav-GAL4,GAL80ts/+;dumb1/dumb2 (p = 0.0009). Therefore, dDA1 is required in the adult neurons, presumably at the time of training, for aversive memory formation. Notably, the same manipulation in the dumb2 heterozygous background (Elav-GAL4,GAL80ts/+;dumb2/+) did not alter the performance scores after brief training (2 pulses of electric shock) or regular training (12 pulses of electric shock). This indicates that the ectopically expressed dDA1 has a negligible effect on normal learning of the heterozygous flies and thus unlikely contribute to the reinstated performance of dumb1/dumb2 mutants (Kim, 2007b).
It was next asked whether the learning phenotype of dumb mutants is attributable to deficient dDA1 function in the MB rather than in the central complex or other neurons. MB247-GAL4 contains 247 bp of dMEF2 regulatory sequence that allows GAL4 expression rather specifically in a subset of the MB neurons projecting to the α/ß lobes and the gamma lobes, but not the α'/ß' lobes. When MB247-GAL4/UAS-GFP in the wild-type background was stained with the dDA1 antibody, the GFP-labeled (thus MB247-GAL4-expressing) MB neurons were positive for dDA1 immunoreactivities although the relative intensities of GFP and dDA1 signals varied in the different MB lobes. Thus, MB247-GAL4 was used to reinstate dDA1 expression in the MB of dumb mutants. After staining with anti-dDA1 antibody, dDA1 expression was apparent in the MB lobes and pedunculi but not in other neural structures of MB247-GAL4/+;dumb1/dumb2. When subjected to electric shock-mediated conditioning, MB247-GAL4/+;dumb1/dumb2 or MB247-GAL4/+;dumb2/dumb2 had the learning scores comparable to those of Canton-S. Moreover, fully reinstated performance was observed in MB247-GAL4/+;GAL80ts,dumb2/dumb1 reared at 30°C for 3 d before training but not in MB247-GAL4/+;GAL80ts,dumb2/dumb1 reared at room temperature. Therefore, dDA1 expressed only in the subset of the adult MB neurons is necessary and sufficient to rescue the dumb mutant's impaired learning, indicating the indispensable role of the MB dDA1 in aversive memory formation (Kim, 2007b).
Dopamine is crucial in appetitive learning in mammals; however, the previous study (Schwaerzel, 2003) of TH-GAL4/UAS-Shits flies suggests that this is not the case in Drosophila. To investigate this further, dumb mutants were tested in sugar-mediated olfactory conditioning. Surprisingly, both dumb1 and dumb2 mutants exhibited poor performance immediately after training. Although dumb mutants' performance in appetitive learning was not as severely impaired as in aversive conditioning, it was significantly different from that of Canton-S. As in electric shock-mediated conditioning, dumb mutants' performance did not decline at 1 h after training, indicating a crucial role of dDA1 in acquisition, as opposed to short-term memory, of appetitive conditioning. Moreover, dumb2 homozygous or dumb1/dumb2 trans-heterozygous mutants carrying MB247-GAL4 displayed fully reinstated learning in sugar-mediated conditioning. These data indicate that dDA1 is required in the same subset of the MB neurons for aversive and appetitive learning (Kim, 2007b).
Reference names in red indicate recommended papers.
Search PubMed for articles about Drosophila Dopamine receptor
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Cannon, C. M., Scannell, C. A. and Palmiter, R. D (2005). Mice lacking dopamine D1 receptors express normal lithium chloride-induced conditioned taste aversion for salt but not sucrose. Eur. J. Neurosci. 21: 2600-2604. Medline abstract: 15932618
Cheer, J. F., et al. (2007). Coordinated accumbal dopamine release and neural activity drive goal-directed behavior. Neuron 54(2): 237-44. Medline abstract: 17442245
Dalley, J. W., et al. (2005). Time-limited modulation of appetitive Pavlovian memory by D1 and NMDA receptors in the nucleus accumbens. Proc. Natl. Acad. Sci. 102: 6189-6194. Medline abstract: 15833811
El-Ghundi, M., O'Dowd, B. F. and George, S. R. (2001). Prolonged fear responses in mice lacking dopamine D1 receptor. Brain Res. 892: 86-93. Medline abstract: 11172752
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Han, K. A., Millar, N. S., Grotewiel, M. S. and Davis, R. L. (1996). DAMB, a novel dopamine receptor expressed specifically in Drosophila mushroom bodies. Neuron 16: 1127-1135. Medline abstract: 8663989
Han, K.-A., Millar, N. S., Davis, R. L. (1998) A novel octopamine receptor with preferential expression in Drosophila mushroom bodies. J Neurosci 18: 3650-3658. Medline abstract: 9570796
Holmes, A., et al. (2001). Behavioral characterization of dopamine D5 receptor null mutant mice. Behav Neurosci 115: 1129-1144. Medline abstract: 11584926
Hsouna, A., Lawal, H. O., Izevbaye, I., Hsu, T. and O'Donnell, J. M. (2007). Drosophila dopamine synthesis pathway genes regulate tracheal morphogenesis. Dev. Biol. 308(1): 30-43. Medline abstract: 17585895
Kehren, V. and Baumann, A. (2005). Characterization of the 5' regulatory region of the Drosophila Dmdop1 dopamine receptor-gene. Arch. Insect Biochem. Physiol. 59(3): 118-31 . Medline abstract: 15986377
Kim, O. J., et al. (2004). The role of phosphorylation in D1 dopamine receptor desensitization: evidence for a novel mechanism of arrestin association. J. Biol. Chem. 279(9): 7999-8010. Medline abstract: 14660631
Kim, Y. C., Lee, H. G., Seong, C. S. and Han, K. A. (2003). Expression of a D1 dopamine receptor dDA1/DmDOP1 in the central nervous system of Drosophila melanogaster. Gene Expr. Patterns 3: 237-245. Medline abstract: 12711555
Kim, Y. C., Lee, H. G. and Han, K. A. (2007a). Classical reward conditioning in Drosophila melanogaster. Genes Brain Behav. 6: 201-207. Medline abstract: 16740144
Kim, Y. C., Lee, H. G. and Han, K. A. (2007b). D1 dopamine receptor dDA1 is required in the mushroom body neurons for aversive and appetitive learning in Drosophila. J. Neurosci. 27(29): 7640-7647. Medline abstract: 17634358
Kindt, K. S., et al. (2007). Dopamine mediates context-dependent modulation of sensory plasticity in C. elegans. Neuron 55(4): 662-76. Medline abstract: 17698017
Kipnis, J., et al. (2004). Dopamine, through the extracellular signal-regulated kinase pathway, downregulates CD4+CD25+ regulatory T-cell activity: implications for neurodegeneration. J. Neurosci. 24(27): 6133-43. Medline abstract: 15240805
Kong, M., et al. (2007). Regulation of D1 dopamine receptor trafficking and signaling by caveolin-1. Mol Pharmacol. [Epub ahead of print]. Medline abstract: 17699686
Kume, K., Kume, S., Park, S. K., Hirsh, J. and Jackson, F. R. (2005). Dopamine is a regulator of arousal in the fruit fly. J. Neurosci. 25(32): 7377-84. Medline abstract: 16093388
Lemon, N. and Manahan-Vaughan, D. (2006). Dopamine D1/D5 receptors gate the acquisition of novel information through hippocampal long-term potentiation and long-term depression. J. Neurosci. 26(29): 7723-9. Medline abstract: 16855100
Mao, Z, Roman, G., Zong, L. and Davis, R. L (2004). Pharmacogenetic rescue in time and space of the rutabaga memory impairment by using Gene-Switch. Proc. Natl. Acad. Sci. 101: 198-203. Medline abstract: 14684832
McGuire, S. E., Le, P. T., Osborn, A. J., Matsumoto, K. and Davis, R. L. (2003). Spatiotemporal rescue of memory dysfunction in Drosophila. Science 302: 1765-1768. Medline abstract: 14657498
Park, D., Han, M., Kim, Y.-C., Han, K.-A. and Taghert, P. H. (2004). Ap-let neurons-a peptidergic circuit potentially controlling ecdysial behavior in Drosophila. Dev. Biol. 269: 95-108. Medline abstract: 15081360
Park, S. K., et al. (2006). Cell-type-specific limitation on in vivo serotonin storage following ectopic expression of the Drosophila serotonin transporter, dSERT. J. Neurobiol. 66(5): 452-62. Medline abstract: 16470720
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Schwaerzel, M., Monastirioti, M., Scholz, H., Friggi-Grelin, F., Birman, S. and Heisenberg, M. (2003). Dopamine and octopamine differentiate between aversive and appetitive olfactory memories in Drosophila. J. Neurosci. 23: 10495-10502. Medline abstract: 14627633
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date revised: 22 September 2007
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