robo2 and robo3 interact with eagle to regulate serotonergic neuron differentiation

The function of the central nervous system (CNS) depends crucially upon the correct differentiation of neurons and formation of axonal connections. Some aspects of neuronal differentiation are known to occur as axonal connections are forming. Although serotonin is a highly conserved neurotransmitter that is important for many CNS functions, little is known about the process of serotonergic neuron differentiation. In Drosophila, expression of the serotonin transporter (SerT) is both temporally and physically related to midline crossing. Additionally, the axon guidance molecules roundabout2 and roundabout3 (robo2/3) are necessary for serotonergic neuron differentiation and function independently of their ligand, slit. Loss of robo2 or robo3 causes a loss of SerT expression in about half of neurons, and resembles the phenotype seen in mutants for the transcription factor eagle (eg). A direct relationship is shown between robo2/3 and eg: robo2/3 mutants lose Eg expression in serotonergic neurons, and robo2 and eg interact genetically to regulate SerT expression. It is proposed that post-midline expression of Robo2/3 is part of a signal that regulates serotonergic neuron differentiation and is transduced by the transcription factor Eg (Couch, 2004).

Thus SerT expression in the fly is temporally and physically tied to axon guidance across the midline. The data further indicate that the axon guidance molecules robo2 and robo3 (robo2/3) positively regulate serotonergic neuron differentiation; a loss of robo2/3 function causes a loss of SerT expression in ~50% of neurons. A robo2/3 loss of function closely resembles an eg mutant phenotype. Finally, the data show a dose-sensitive relationship between Robo2/3, Eg and SerT expression, suggesting that they function in the same genetic pathway to control serotonergic neuron differentiation. This interpretation is supported by the fact that loss of robo2 or robo3 causes a loss of Eg expression, and by genetic rescue experiments (Couch, 2004).

By visualizing serotonergic axonal projections with tau-lacZ, it was determined that SerT expression begins at the end of stage 15, just after growth cones complete midline crossing and reach the contralateral side. This temporal correlation between midline crossing and SerT induction suggests that the midline is important for serotonergic neuron differentiation in the fly, as it is in the grasshopper (Condron, 1999). Further evidence for the importance of the midline comes from data showing that in wild-type cords, axons physically separated from the midline fail to express SerT. These results recapitulate similar experiments in the grasshopper. Additionally, when the repulsive axon guidance receptor Unc5 is expressed in serotonergic neurons, a partial loss of SerT expression is observed. Although these results suggest a role for the midline in serotonergic neuron differentiation, it remains unclear whether this role is temporally restricted as it is in the grasshopper, and, additionally, what factors act as the presumptive midline signal. FGF signaling in the grasshopper is crucial for SerT induction (Condron, 1999), and plays a role in the differentiation of vertebrate serotonergic neurons. In the fly, experiments indicate that FGF signaling also appears to be important for SerT regulation (Couch, 2004).

One problem with interpreting the role for the midline is the lack of an abnormal serotonergic phenotype in mutants for the master regulatory gene sim, where midline cells fail to properly differentiate. It is difficult to speculate about what factors may allow normal differentiation in the absence of normal midline cells, since there are many changes in gene regulation throughout sim mutants. Although the results suggest a role for the midline in serotonergic neuron differentiation, it is likely to be more complicated than a simple switch acting to induce differentiation (Couch, 2004).

The data show that a loss of robo2 or robo3 causes a loss of SerT expression, suggesting that Robo2/3 function positively to regulate serotonergic neuron differentiation. A positive role for Robo2 is further supported by results showing that overexpression of Robo2 prevents a loss of SerT in neurons physically separated from the midline. Possibly, Robo2 functions downstream of the midline signal required for SerT induction and thus allows differentiation to proceed in the absence of such a signal. An alternative hypothesis suggests that Robo2/3 function indirectly to induce SerT, by guiding serotonergic axons to an unknown signal in the contralateral neuropil. Such indirect signaling occurs in the developing vertebrate CNS where trophic support is required by commissural axons at the floorplate, an intermediate axonal target. Although the possibility that Robo2/3 act indirectly to regulate serotonergic neuron differentiation cannot be ruled out at this time, several lines of evidence suggest a more direct role. These data shows that overexpression of Robo2 not only spares SerT loss following a midline cut but also rescues an eg hypomorph, and furthermore, that an Eg gain of function rescues a robo2 loss of function; these results strongly suggests that Robo2 functions autonomously in the serotonergic neurons. Additionally, SerT loss is not seen in other guidance mutants that disrupt midline crossing or cause general disorganization of the CNS. However, it is difficult to clearly resolve the presence of Robo2/3 protein specifically in the serotonergic neurons because of the broad distribution of neuronal processes and the fact that serotonergic neuron branching does not correspond simply to any Fas2 pathway where axons are known to express Robo2/3 (Couch, 2004).

Robo2 and Robo3 appear to act as overall regulators of differentiation rather than specific regulators of SerT, since robo2/3 mutants not only lose SerT expression (mRNA and reuptake activity) but also have defects in serotonin synthesis later in development. Thus, the role of Robo2/3 in serotonergic neuron differentiation parallels that of other genes, including eg and the LIM-homeodomain transcription factor islet, that cause both a loss of SerT as well as serotonin synthesis when disrupted. The data further indicate that robo2/3 are not required in the formation of the serotonergic neurons from their progenitor neuroblast 7-3. All serotonergic neurons in a robo2/3 mutant express eg-lacZ, even those with a loss of SerT expression. This may at first appear to contradict the result showing a loss of Eg at stage 16 in these mutants, since eg expression must have occurred in order to produce lacZ. It is hypothesized that lacZ staining in stage 16 robo2 mutants is likely to be due to a lengthy persistence of lacZ rather than continued expression of eg, since eg mRNA is not detectable by in situ hybridization after stage 14. Most probably, eg-lacZ expression in robo2/3 mutants occurs in the progenitors of serotonergic neurons when other factors, such as engrailed (en), are known to control eg expression. Even in eg mutants, all serotonergic neurons continue to express eg-lacZ, despite a disruption in SerT, serotonin synthesis and expression of Ddc. Thus, a robo2/3 mutant, like an eg mutant, does not affect the early specification of serotonergic neurons, including early eg expression, but instead affects later maturation (Couch, 2004).

Interestingly, an effect is observed of robo2/3, but not robo, on serotonergic neuron differentiation. Disparities between Robo and Robo2/3 function have been previously observed in the lateral positioning of axons where only Robo2/3 appear to play a role, and in dendritic guidance, synapse formation and midline crossing, where all three Robo receptors have separable functions. Furthermore, Robo2 and Robo3 show greater homology to each other than to Robo. Robo2/3 have cytoplasmic domains that diverge from Robo, and lack two motifs considered important for Robo signaling. Possibly, Robo2 and Robo3 regulate a Robo-independent signaling cascade that is critical for serotonergic neuron differentiation. Additionally, a loss of slit, the ligand for all three Robo receptors, does not perturb SerT expression, indicating that either another ligand exists or the function of Robo2/3 in serotonergic neuron differentiation is ligand independent. In C. elegans, some activities of the Robo homolog SAX-3 are thought to be Slit independent (Couch, 2004).

The transmembrane protein Comm has been shown to negatively regulate the levels of all three Robo receptors. After midline crossing, Comm expression decreases and Robo levels increase in order to prevent inappropriate midline crossing. In serotonergic neuron differentiation, Comm may play a role in regulating Robo2/3, such that levels of both Robo2/3 increase following midline crossing and thereby permit differentiation to proceed. To test this possibility, Comm was expressed using egGal4 to specifically induce a loss of Robo2/3 in the serotonergic neurons. In these experiments, expression of Comm causes a loss of SerT activity in only a few cells and with low penetrance. This is believed to be due to expression of Comm at levels insufficient for total loss of Robo2/3. Alternatively, other regulators of Robo2/3 may exist. However, neither a loss of Comm nor overexpression of Robo2/3 results in precocious serotonergic neuron differentiation, indicating a requirement for other signals (Couch, 2004).

In both a robo2 and a robo3 loss-of-function mutant, expression of the zinc-finger transcription factor eg is lost in the same cells that lose SerT expression. Additionally, overexpression of Robo2 rescues the loss of SerT observed in an eg hypomorph in a dose-sensitive manner. Finally, Eg gain of function rescues the SerT loss seen in robo2 loss-of-function mutants. These results indicate that Robo2/3 function in the same genetic pathway as Eg to control serotonergic neuron differentiation. Although these results suggest that Robo2/3 regulate Eg in stage 16 embryos, other genes such as en and hunchback (hb) also have an established role in regulating Eg during serotonergic neuron differentiation. At present, it remains unclear if Robo2/3 cooperate with these genes to regulate Eg expression (Couch, 2004).

Additionally, in both robo2 and robo3 loss-of-function mutants only a percentage of neurons lose SerT expression (and serotonin synthesis), indicating the presence of a redundant mechanism for serotonergic neuron differentiation. The pattern of SerT and serotonin loss in robo2/3 mutants appears random and differs between nerve cords. At this point, it remains unclear why differentiation is affected in only some cells and not others, or what factors allow remaining cells to maintain normal SerT expression. One possibility is that cells must maintain a threshold level of Eg expression to differentiate properly. This is supported by differences in the degree of SerT loss according to the severity of the mutation in eg, since a hypomorphic allele displays a loss of SerT in ~30% of hemisegments while a null allele displays closer to 80% loss of SerT. Many studies have also suggested that a combinatorial code of transcription factors act to specify serotonergic properties: (1) loss-of-function mutations in several genes required for differentiation, including eg, en and hb show an incomplete loss of SerT phenotype; (2) if Eg is inappropriately expressed throughout the nervous system, only a few ectopic serotonin positive cells appear. These ectopic serotonergic cells always express the transcription factor hkb. Robo2 and Robo3 may also function redundantly. Further studies should indicate the relationship of Robo2/3 to other genes involved in serotonergic neuron differentiation, and the mechanism by which Robo2/3 regulate Eg expression (Couch, 2004).

One question that readily follows from these observations: how does Robo2 influence Eg expression? Robo2 and Robo3 are cell-surface axon guidance receptors, while Eg is a transcription factor. It is likely that other factors interact with both Robo2/3 and Eg to mediate their roles in serotonergic neuron differentiation. Although their relationship remains obscure, data indicate that Robo2 may regulate Eg post-transcriptionally. In a series of real-time RT-PCR experiments, no difference in eg mRNA levels was detected when EP2582 (UAS-robo2) was expressed using egGal4, scabrousGal4 or elavGal4, suggesting that Robo2 is insufficient to induce ectopic Eg expression. However, when Robo2 is overexpressed, a rescue of Eg protein expression is seen in egMz360 hypomorphs. Through the same series of PCR experiments, it was discovered that the egMz360 allele produces mRNA, although no Eg staining is observed. This suggests that the Gal4 insertion responsible for the egMz360 allele affects Eg protein expression, which in turn causes a disruption in SerT expression. Thus, expression of Robo2 appears to somehow rescue Eg protein expression in an egMz360 hypomorph sufficiently to rescue SerT activity. At this point, the mechanism of such a post-transcriptional rescue is unclear. Identifying the genetic and intracellular links between Robo2, Robo3 and Eg with more molecular approaches such as RNAi studies will probably reveal how Robo2/3 regulate not only Eg but eventually serotonergic neuron differentiation as well (Couch, 2004).

Functions of the segment polarity genes midline and H15 in Drosophila melanogaster neurogenesis: negative regulation of eagle

The Drosophila ventral nerve cord derives from neural progenitor cells called neuroblasts. Individual neuroblasts have unique gene expression profiles and give rise to distinct clones of neurons and glia. The specification of neuroblast identity provides a cell intrinsic mechanism which ultimately results in the generation of progeny which are different from one another. Segment polarity genes have a dual function in early neurogenesis: within distinct regions of the neuroectoderm, they are required both for neuroblast formation and for the specification of neuroblast identity. Previous studies of segment polarity gene function largely focused on neuroblasts that arise within the posterior part of the segment. This study shows that the segment polarity gene midline is required for neuroblast formation in the anterior-most part of the segment. Moreover, midline contributes to the specification of anterior neuroblast identity by negatively regulating the expression of Wingless and positively regulating the expression of Mirror. In the posterior-most part of the segment, midline and its paralog, H15, have partially redundant functions in the regulation of the NB marker Eagle. Hence, the segment polarity genes midline and H15 play an important role in the development of the ventral nerve cord in the anterior- and posterior-most part of the segment (Buescher, 2006).

To investigate whether loss of mid/H15 results in additional misspecifications of NBs, midGA174 mutant embryos were stained with the anti-Eg antibody. In wt, mid/H15 and Eg expressing NBs are close neighbors but expression of these genes appears mutually exclusive. In mid mutant embryos, ectopic Eg expression was found in NB7-4 (12%), in both odd- and even-numbered abdominal segments. By contrast, removal of the H15 gene alone did not result in ectopic Eg expression. However, removal of both copies of mid and H15 caused an enhancement of the phenotype (37.8%) indicating that in the absence of mid, H15 contributes to the regulation of Eg expression. These results indicate that both mid and, to a lesser extent, H15, act as negative regulators of Eg expression (Buescher, 2006).

A role for mid in the negative regulation of Eg expression was surprising since in wt embryos, two of the Eg-positive NBs (NB6-4 and NB7-3) derive from mid/H15-positive NE. However, in contrast to NB7-4, NB6-4 and NB7-3 rapidly lose mid/H15 expression during NB formation while mid/H15 expression in NB7-4 is not only maintained but also upregulated. These results suggest that, in NB7-3 and NB6-4, mid and Eg expression occurs consecutively while prolonged expression of mid in NB7-4 prevents Eg expression. To test this hypothesis, the expression of mid was extended in NB6-4 and NB7-3 using enGal4 to drive the expression of mid. Staining of stage 11 embryos with anti-Eg revealed a complete loss of Eg expression in the NB6-4 and NB7-3 positions. In addition, ectopic expression of mid with the pan-neural driver scabrousGal4 (scaGal4;UAS-mid) resulted in a nearly complete absence of Eg expression. These results confirm that high levels of mid negatively regulate the expression of Eg in NBs. The ectopic expression of Eg in mid mutant embryos is restricted to NB7-4, indicating that the absence of mid per se is not sufficient to permit Eg expression. The regulation of Eg expression involves positive acting factors such as Huckebein and En which have locally restricted expression patterns. The finding that mid negatively regulates Eg in NB7-4 indicates that Eg expression in individual NBs is achieved by a combinatorial code of positively and negatively acting factors (Buescher, 2006).

Targets of Activity

eagle (eg) is expressed in neuroblasts and is involved in the fate determination of serotonergic neurons. Serotonin is an evolutionarily conserved neurotransmitter, found in both invertebrates and vertebrates, and involved in locomotor and behavioral roles. Serotonin is produced in descendents of neuroblast NB 7-3. NB7-3 expresses several genes including engrailed, huckebein, seven-up, pdm1 and eagle. Although eg is expressed in both lateral and medial NB 7-3 derived serotonin cells, the eg loss-of-function mutants often affect the development of only one serotonin cell from each pair. The two cells can be distinguished from one another by differential expression of Zn finger homeodomain 2 (zfh-2). This dual domain transcription factor has been shown to bind to and activate the DDC gene (Lundell, 1992 and 1994). The differential expression of zfh-2 and of another gene, pdm-1, can be used to determine that the remaining serotonin positive single cell in eg mutants expresses markers characteristic of the more lateral serotonin cell. In a wild-type CNS, both zfh-2 and pdm1 are selectively expressed in the more lateral serotonin cell but not in the more medial cell. engrailed and eagle are expressed in both these serotonin cells. In eg mutants only the medial cell consistently fails to become a serotonin cell. Therefore, even though eg is normally expressed in both serotonin cells, the absence of Eg protein has a more dramatic effect on the fate of the more medial neuron. This important observation suggests that the lateral serotonin cell can maintain its fate in the absence of Eg (Lundell, 1998).

Analysis of gene expression in eg mutants shows that expression of zfh-2 and en is dependent on eg function but expression of pdm1 is independent of eg function. Loss of eg function appears to have no affect on the expression of pdm1. Clearly, the serotonin cell phenotype in eg mutants is not directly related to the expression of pdm1. Loss of eg function affects the expression of zfh-2 in the lateral serotonin neuron. Loss of eg function affects the expression of en in both serotonin neurons. Thus, eagle is necessary for the maintenance of both engrailed and zfh-2 expression in the serotonin neurons (Lundell, 1998).

The simplest explanation for the difference between the medial and lateral serotonin neurons is that the lateral cell contains a redundant mechanism that allows continued synthesis of serotonin in the absence of Eg protein. This redundant mechanism is not 100% efficient, since not all segments in an eg mutant CNS contain serotonin cells. Since zfh-2 but not pdm1, expression is affected in eg mutants, it is suggested that zfh-1 is a potential factor for this redundant pathway, which establishes eg-independent serotonin synthesis. In an eg-loss-of-function mutant, the loss of Ddc expression is always accompanied by the loss of en expression, but can occur independently in the two serotonin cells. In a hemisegment where both cells fail to express Ddc, neither cell shows en expression. In a hemisegment where only the lateral serotonin cells continues to express Ddc, this lateral cell shows en expression but the medial cell does not. It is concluded that the two serotonin cells have distinctive regulatory networks. In the medial cell, eagle is required for the serotonin fate, while in the lateral cell, engrailed and zhf-1 are required but eagle is not. It is shown that hypomorphic alleles of eagle can produce viable adults that have a dramatic reduction in the number of serotonin-producing neurons (Lundell, 1998).



The spatial distribution of eagle transcripts has been examined, using in situ hybridization. Transcripts first become discernible in a subset of lateral CNS cells at stages 10 or 11. The fact that eg transcripts are detected only in large cells indicates that the eg-positive cells are neuroblasts (NBs). eg expression in the CNS is still detected at stage 12, but virtually disappears by stage 13, except for some thoracic cells. At stage 14, eg expression starts at or near the dorsal sac (Higashijima, 1996).

eg expression was examined in more detail in abdominal NBs (NBs in abdominal segments A1-A7). eg-RNA-positive cells first appear as a pair of lateral NBs in every hemisegment. At the beginning of eg expression, these NBs are situated not in the NB layer but near the ventral ectodermal surface, indicating that they are newly formed NBs. The inner NB array at this point conforms to S3 (the third wave of neuroblast determination) of the NB map. The relative positions of these eg NBs indicate that they are NB2-4 and NB3-3. The eg expression in both NB2-4 and NB3-3 reaches a maximum at the very beginning of expression (S3/S4 transition period) and thereafter becomes weaker. The amount of eg RNA in NB2-4 is extensively reduced at S4, while EG mRNA in S4 NB3-3 is much more abundant. Weak eg expression in NB2-4 and NB3-3 persists until the beginning of stage 12. A few smaller stained cells are present in the dorsal vicinity of NB2-4 and NB3-3 during S4 and S5. These stained cells might be GMCs of NB2-4 and NB3-3. However, eg RNA is hardly detectable, suggesting that eg is not transcribed in the GMCs of NB2-4 and NB3-3 (Higashijima, 1996).

The second class of eg NBs is NB6-4, in which EG RNA expression becomes discernible at late S4. This EG RNA expression appears quite transient, because as a whole, only 1-4 NB6-4s per 10 hemisegments were scored EG-RNA-positive. Although NB6-4 is a component of the NB layer at S3, EG RNA expression occurs at late S4. Thus, unlike NB2-4 and NB3-3, newly formed NB6-4 appears unable to express EG RNA. NB6-4 appears to divide quasi-symmetrically and to generate a pair of glial cells, even where no EG RNA expression is observed. During the S4/S5 transition period, another class of eg NBs is detected at or near the ventral ectodermal surface at a frequency of one cell per hemisegment. The cell corresponds to NB7-3. The maximum eg expression in NB7-3 is observed at the very beginning of its expression but strong expression persists from early to middle S5. By late S5, NB7-3 has been replaced by a pair of smaller EG-RNA-positive cells, which are most likely the daughter cells of NB7-3 (Higashijima, 1996).

The lineage of NB 7-3 is described in Bossing (1996). At stage 17, most NB 7-3 clones consist of 4 mediodorsal neurons: three interneurons, called 7-3I neurons, which project contralaterally across the posterior commissure and one motoneuron, called 7-3M, projecting ipsilaterally. Differentiation of 7-3M starts rather late (stage 15/16). At stage 17, the projection of 7-3M reaches the posterior root of the intersegmental nerve. The same projection pattern is described for the EW and GW neurons (Higashijima, 1996). Based on cell position, cell number and projection pattern, it is concluded that EW neurons are the same as 7-3 lateral neurons and the GW neuron is the same as the 7-3 medial cell. Therefore, Eg is expressed in NB 7-3 just after delamination and is present in all NB 7-3 progeny (both 7-3 lateral and medial) until late stage 17. In the grasshopper, the serotonergic neurons represent contralaterally projecting interneurons, which are the progeny of NB 7-3, resulting from the division of the first ganglion mother cell (GMC). In Drosophila, the serotonergic neurons of the ventral nerve cord also project contralaterally across the posterior commissure, and hkb and en genes are coexpressed uniquely in the serotonin cells and in NB 7-3. This suggests that, as in the grasshopper, NB 7-3 is the progenitor of the serotonergic neurons (Lundell, 1996).

For more information on Drosophila neuroblast lineages, see Linking neuroblasts to their corresponding lineage, a site carried by Flybrain, an online atlas and database of the Drosophila nervous system.

Effects of mutation or deletion

Close inspection of the morphologies of cells at later stages in mutant embryos indicates that the fates of some cells are altered. At stage 13, wild-type EG neurons begin sending axons toward the anterior commissure, and by the end of the stage, some of them meet at the midline. In the mutant, there is no sign of axonogenesis throughout this stage. At stage 14, stained axon bundles are observed in some anterior commissures, but they are much thinner or more weakly stained than those of wild type. In contrast to wild type, cell bodies of mutant EG neurons are heavily stained. These results suggest that only a small fraction of mutant EG cells are capable of sending axons at this stage. During stages 14-16, stained axon bundles become progressively thicker; in accordance with this, the level of staining in the cell bodies becomes weaker. However, even at stage 16, many cells remain heavily stained in the mutant, suggesting that they correspond to EG cells whose differentiation into neurons is either extremely delayed or has totally failed. The absence of eagle causes either failure of, or extreme delay in, neuron differentiation (Higashijima, 1996).

In order to investigate the function of Eg within the NB 7-3 lineage, fly strains carrying mutations at the eg locus were analyzed. The P-element insertions of eg Mz360 and eg P289 are homozygously viable and cause, as shown by P-element remobilization, a `wings held out' phenotype, which is allelic to classical eg mutations. In eagle deletion mutants, the abdominal projections no longer cross the midline, but remain ipsilateral, although the orientation of these projections is quite variable. In one case, the mutant cells project anteriorly and posteriorly and, in the remaining cases, they project out of the CNS. Thus, NB 7-3 in eg P289 embryos produces interneurons (7-3I) with significant axon pathfinding defects, whereas 7-3M seems to have a normal axonal projection. In addition to the interneuronal pathfinding defects, eg P289 embryos have a thorax-specific abnormality in the NB 7-3 clones. The mutant clones consist of 6 to 8 cells, as compared to 4 to 5 cells in wild type. This suggests that eg is necessary to restrict the cell number in the thoracic NB 7-3 lineage (Dittrich, 1997).

Since the serotonergic neurons of the ventral nerve cord are among the progeny of NB 7-3, serotonin expression was investigated within the eagle mutants. In abdominal segments of a wild-type first instar larval CNS, there are two serotonergic neurons per hemineuromere, except for A8, where there is only one. In eg P289 and eg 18B mutants of the same stage, truncal serotonergic cells are almost entirely lacking. Only 2 to 4 escaper serotonergic cells are generally present and occur mostly as single cells in the thoracic NB 7-3 lineage. eg is therefore an important factor for serotonin expression in the NB 7-3 lineage, yet other genes must exist that are able to promote serotonin expression at a low frequency in the absence of eg function (Dittrich, 1997).

Loss-of-function eagle mutations produce an unusual differential phenotype with respect to the sister serotonin cells. eagle is necessary for the maintenance of engrailed and zfh-2 (see zhf-1) expression in the serotonin neurons. A model is presented that uniquely identifies all progeny neurons in the neuroblast 7-3 lineage, based on three criteria: the expression of specific molecular markers, the position within the nerve cord and the effect of eagle loss-of-function mutations. Although serotonin is an important neurotransmitter conserved throughout the animal kingdom, it is shown that hypomorphic alleles of eagle can produce viable adults that have a dramatic reduction in the number of serotonin-producing neurons (Lundell, 1998).

Insights into the function of a gene can be gained in multiple ways, including loss-of-function phenotype, sequence similarity, expression pattern, and by the consequences of its misexpression. Analysis of the phenotypes produced by expression of a gene at an abnormal time, place, or level may provide clues to a gene's function when other approaches are not illuminating. An eye-specific, enhancer-promoter present in the P element expression vector pGMR is able to drive high level expression in the eye of genes near the site of P element insertion. Cell fate determination, differentiation, proliferation, and death are essential for normal eye development. Thus the ability to carry out eye-specific misexpression of a significant fraction of genes in the genome, given the dispensability of the eye for viability and fertility of the adult, should provide a powerful approach for identifying regulators of these processes. To test this idea two overexpression screens were carried out for genes that function to regulate cell death. A screen was carried out for insertion-dependent dominant phenotypes in a wild-type background, and for dominant modifiers of a reaper overexpression-induced small eye phenotype. Multiple chromosomal loci were identified, including an insertion 5' to hid, a potent inducer of apoptosis, and insertions 5' to DIAP1, a cell death suppressor. To facilitate the cloning of genes near the P element insertion, new misexpression vectors were created. A screen with one of these vectors identified eagle as a suppressor of a rough eye phenotype associated with overexpression of an activated Ras1 gene. This suggests that eg may be involved in the Ras1 signal transduction pathway (Hay, 1997).

Mammalian cell culture studies have shown that several members of the nuclear receptor super family such as glucocorticoid receptor, retinoic acid receptor and thyroid hormone receptor can repress the activity of AP-1 proteins (referring to Drosophila Kayak and Jun) by a mechanism that does not require the nuclear receptor to bind to DNA directly, but that is otherwise poorly understood. Several aspects of nuclear receptor function are believed to rely on this inhibitory mechanism, which is referred to as transrepression. This study presents evidence that nuclear receptor-mediated transrepression of AP-1 occurs in Drosophila melanogaster. In two different developmental situations, embryonic dorsal closure and wing development, several nuclear receptors, including Seven up, Tailless, and Eagle antagonize AP-1. The inhibitory interactions with nuclear receptors are integrated with other modes of AP-1 regulation, such as mitogen-activated protein kinase signaling. A potential role of nuclear receptors in setting a threshold of AP-1 activity required for the manifestation of a cellular response is discussed (Gritzan, 2002).

The best understood AP-1-dependent process in Drosophila development is a coordinated cell sheet movement known as dorsal closure. During DC, lateral epidermal cells migrate dorsally and close the epidermis on the dorsal side of the embryo. Failure to undergo DC results in a characteristic dorsal open phenotype, the cuticle of affected embryos displays a dorsal hole. Mutations in genes encoding the Drosophila homologs of JNKK, (JNK, Jun and Fos) all give rise to similar dorsal open phenotypes. Thus, it is thought that DC requires activation of Jun/Fos heterodimers by a JNK-type MAPK cascade. Embryos homozygous for kay1, a fos null allele are devoid of zygotic Fos activity and DC fails. A large dorsal hole forms and the cuticle collapses. In an embryo homozygous kay2, a hypomorphic fos-allele, AP-1 activity is reduced but not eliminated. Correspondingly, the DC phenotype is weaker. The embryo displays a small dorso-anterior hole (Gritzan, 2002).

To test whether Drosophila NRs can antagonize AP-1, a variety of AP-1 constructs were in the embryonic epidermis. Interestingly, expression of some, but not all, NRs tested result in DC phenotypes of different strengths. Expression of Svp in the dorsal epidermis under the control of pnrGal4 results in a DC phenotype reminiscent of that of kay2 homozygotes. This finding is consistent with a suppression of AP-1 activity by Svp. Similarly, expression of Tll under the control of a heat shock promoter causes a weak dorsal open phenotype. The differentiation of ventral cells does not seem to be disturbed by Tll expression since the pattern of denticles in this part of the epidermis appears grossly normal. Thus, Tll expression specifically affects the dorsal epidermis where AP-1 activity is required. The expression of Knrl under the control of pnrGal4 elicits stronger DC phenotypes with the dorsal hole frequently extending over several segments (Gritzan, 2002).

If the DC defects caused by NR expression reflect a negative effect on AP-1, the defects should be sensitive to changes in Fos or Jun activity. In genetic interaction experiments, the dorsal open phenotypes caused by NR expression were compared in a wild-type background and in embryos with altered levels of AP-1 activity. Embryos heterozygous for kay1 carry only one copy of the fos gene. While these embryos are phenotypically normal, their levels of AP-1 are reduced and they might therefore be more susceptible to a further decrease of this activity. If expression of NRs causes DC defects by antagonizing AP-1, it should have stronger phenotypic consequences in embryos heterozygous for kay1 than in wild type embryos. Indeed, while expression of Eagle (Eg) in a wild type background mostly results in DC phenotypes of intermediate strength, NR expression in embryos heterozygous for kay1 typically elicits complete failure of DC, indicative of a severe reduction of AP-1 activity. Since embryos of both genotypes display somewhat variable phenotypes, the effect of kay1 heterozygosity is best appreciated by quantitative analysis. A clear reduction in size, a collapsed folded cuticle and a dorsal hole extending over at least half of the body length are described as characteristics of a strong DC phenotype. Embryos with a smaller dorso-anterior hole and normal body size were scored as showing weak DC phenotypes. This analysis confirms that Eg expression has more severe phenotypic consequences in kay1 heterozygotes than in wild type embryos and supports the suggestion that NRs cause defective DC by suppressing AP-1 activity (Gritzan, 2002).


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Lundell, M. J. and Hirsh, J. (1992). The zfh-2 gene product is a potential regulator of neuron-specific dopa decarboxylase gene expression in Drosophila. Dev. Biol. 154: 84-94. 1426635

Lundell, M. J., Chu-LaGraff, Q., Doe, C. Q. and Hirsh, J. (1996). The engrailed and huckebein genes are essential for development of serotonin neurons in the Drosophila CNS. Mol. Cell. Neurosci. 7(1): 46-61. 8812058

Lundell, M. J. and Hirsh, J. (1998). eagle is required for the specification of serotonin neurons and other neuroblast 7-3 progeny in the Drosophila CNS. Development 125(3): 463-472. 9425141

Matsuzaki, M. and Saigo, K. (1996). hedgehog signaling independent of engrailed and wingless required for post-S1 neuroblast formation in Drosophila CNS. Development 122: 3567-3575. 8951072

Rothe, M., Nauber, U. and Jackle, H. (1989). Three hormone receptor-like Drosophila genes encode an identical DNA-binding finger. EMBO J. 8(10): 3087-3094. 2555153

eagle: Biological Overview | Regulation | Developmental Biology | Effects of Mutation

date revised: 10 August 2006  

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