InteractiveFly: GeneBrief

charlatan: Biological Overview | Regulation | Developmental Biology | Effects of Mutation and Overexpression | References

BIOLOGICAL OVERVIEW

The proneural genes achaete (ac) and scute (sc) are necessary for the formation of the external sensory organs (SOs) of Drosophila. ac and sc are expressed in proneural clusters and impart their cells with neural potential. For this potential to be realized, and the SO precursor cell (SOP) to arise within a cluster, sufficient proneural protein must accumulate in the cluster. charlatan (chn) encodes a zinc finger transcription factor that facilitates this accumulation by forming a stimulatory loop with ac/sc. Loss of function of chn decreases the accumulation of Sc in proneural clusters and partially removes notum macrochaetae, while overexpression of chn enhances ac/sc expression and the formation of extra SOs. Moreover, chn is activated by ac/sc in proneural clusters. Chn apparently stimulates ac/sc by physically interacting with the proneural cluster-specific enhancers and increasing enhancer efficiency, thus acting as a stimulator of ac/sc expression in proneural clusters. chn is also required for the proper development of the embryonic peripheral nervous system; its absence leads to loss of neurons and causes aberrant development of chordotonal organs (Escudero, 2005).

A classical example of two-dimensional pattern is that formed by the bristles and other types of sensory organs (SOs) in the epidermis of the adult Drosophila fly. On the head and the dorsal mesothorax (notum), conspicuous large bristles (macrochaetae) arise in stereotyped positions, while smaller bristles (microchaetae) appear in density patterns. During the third instar larval and early pupal stages, the location of each macrochaeta is specified by the emergence of a precursor cell (SO precursor cell, SOP) at a stereotyped position of the imaginal discs, the larval epithelia that give rise to a large part of the adult epidermis. This accurate positioning of SOPs in the imaginal discs is thought to be the culmination of a multistep process in which positional information is gradually refined (Escudero, 2005).

A key step of this process is the expression of the proneural genes achaete (ac) and scute (sc) in groups of cells, the proneural clusters, that prefigure the sites of the future macrochaetae. These genes, members of the achaete-scute complex (ASC), encode transcriptional factors of the basic helix-loop-helix (bHLH) family. These factors confer to cells the potential to become SOPs, presumably by implementing neural differentiation programs. From each proneural cluster, a fixed number of SOPs are born, usually one or two. The proneural clusters of the wing imaginal discs (the precursors of each heminotum, wing and mesothoracic pleura) not only appear in constant positions, but each of them has a characteristic size, shape and time of appearance and disappearance. Moreover, a typical cluster that gives rise to one bristle may consist of 20 to 30 cells, but the SOP is selected from a smaller subgroup of cells that accumulate higher levels of Ac-Sc proteins than their neighbors, which constitute the proneural field. This subgroup and the SOP, which accumulates the highest levels of Ac-Sc, always occupy the same position within the cluster. Hence, the expression of ac/sc in proneural clusters is exquisitely regulated (Escudero, 2005).

The regulation of ac/sc is effected by means of two classes of cis-regulatory sequences, namely, cluster-specific and SOP-specific enhancers. The first type normally directs expression of both ac and sc in one specific proneural cluster and defines many of its characteristics, such as position, size and shape. These cluster-specific enhancers appear to be controlled by local combinations of transcription factors that together form a prepattern. Expression occurs only at sites with the appropriate combinations of factors. Although in a few cases some of the prepattern factors have been identified, most of them remain unknown. Moreover, a clear understanding of how the inputs of the prepatterning factors are integrated into the patterns of proneural gene expression characteristic of each cluster is still lacking (Escudero, 2005).

The second type of enhancer mediates the strong expression of proneural genes in SOPs by allowing self-stimulatory loops of expression of ac, sc and asense (ase), another bHLH member of the ASC. The activation of these loops in one of the cells of the proneural field is an early and essential step of SOP commitment. This loop is also dependent on the presence of the Senseless (Sens) protein. The SOP-specific enhancers are also the targets of the inhibitory interactions that occur within the cells of the proneural cluster mediated by the Notch signaling pathway via E(spl) proteins. By antagonizing these enhancers, N signaling (activated by Ac/Sc in the cells of the cluster) maintains cells in the cluster in a non-SOP state (mutual inhibition). However, in a little-understood process, one cell of the proneural field escapes this inhibition, starts the proneural self-stimulatory loop and becomes an SOP. The developing SOP then signals via Notch in order to impede the remaining cells of the field from becoming SOPs (lateral inhibition). These SOP-specific enhancers are also the targets of positive interactions between the cells of proneural clusters mediated by the EGFR, which is necessary for the emergence of the SOPs of the notum macrochaetae (lateral cooperation. To prevent the determination of excess SOPs from a proneural cluster, the levels of EGFR signaling must be regulated. This event seems to be accomplished in part by a negative effect on EGFR signaling of the N-mediated interactions that occur among cells of the proneural cluster (Escudero, 2005).

The ac, sc and ase genes are also necessary for the formation of the external SOs of embryos and larvae. The process is similar to that in the imaginal discs. Other proneural genes are responsible for the development of the internal chordotonal organs (atonal) and other neurons of the larval peripheral nervous system (PNS) (amos) (Escudero, 2005).

A novel gene, charlatan (chn), has been identified that is involved in the development of the adult pattern of macrochaetae. chn defines a new level of control of ac/sc that is intermediate between the prepattern genes and the ac/sc self-stimulation mediated by the SOP-specific enhancers. Thus, chn, which encodes a zinc finger transcription factor, is activated by ac/sc in the proneural clusters of the wing disc. In turn, chn stimulates the expression of ac/sc in these clusters. This enhanced expression facilitates the formation of SOPs. The data indicate that the Chn protein reinforces the expression of ac/sc by acting, probably directly, on the proneural cluster-specific enhancers of the ASC. chn is also required for correct development of the embryonic/larval PNS; its absence removes neurons and causes malformations of chordotonal organs (Escudero, 2005).

Thus chn is involved in the development of the PNS of the Drosophila embryo and the adult fly. The function of chn in the formation of the stereotyped pattern of notum macrochaetae was examined in detail. Complete removal of chn expression leads to a relatively mild phenotype; namely, the failure of each notum macrochaetae to develop in 25% to 45% of the flies. Any macrochaeta was subject to loss. This loss was strongly enhanced when, concomitant to the removal of chn, the proneural function of ac/sc was reduced by either halving the doses of the ASC or by introducing alleles that decreased accumulation of Ac/Sc in proneural clusters. This result suggests a positive interaction between proneural and chn functions in macrochaetae development, an inference that was verified by overexpression experiments. Thus, overexpression of chn gives rise to a large number of extra macrochaetae, an effect that is strongly dependent on the number of doses of the ASC. Reciprocally, the number of extra macrochaetae that arise when overexpressing sc is sharply decreased by compromising chn function. In all cases, the extra macrochaetae that are formed upon chn overexpression are not contiguous to one another and epidermal cells are present between them. This indicates that N-mediated lateral inhibition is still operating and that chn is unlikely to antagonize this process (Escudero, 2005).

The presence of chn mRNA in the proneural clusters of the wing disc is dependent on ac/sc. Moreover, ectopic accumulation of Sc results in ectopic expression of chn. These observations place chn downstream of ac/sc, and suggest a positive, possibly direct, regulation of chn by ac/sc. Consistent with this, two clusters of four and eight E-boxes, putative binding sites for bHLH proteins of the proneural type, are found approximately 15 kb upstream of the chn structural sequences and within the first intron of the gene, respectively (Escudero, 2005).

In turn chn stimulates the accumulation of Sc in proneural clusters; loss of function of chn results in decreased accumulation of Sc. However, some Sc still accumulates in the complete absence of Chn, which probably explains why many SOPs and their corresponding macrochaetae develop in its absence. The upregulation of sc by chn is even more manifest by the overexpression of UAS-chn, which causes a strong accumulation of Sc and leads to the formation of large numbers of SOPs and extra macrochaetae. ac is also upregulated by overexpression of chn. Although it cannot be ruled out that Chn may slow the turnover of Sc/Ac and thereby promote Sc/Ac accumulation, the data clearly show that Chn stimulates the transcription of ac/sc. Indeed, the overexpression of chn greatly increases in vivo the expression of the reporter gene lacZ driven by proneural group-specific enhancers of the ASC and its removal decreases the expression of these constructs. The stimulation is also observed with enhancer constructs that do not have the endogenous sc promoter (rather, they carry an hsp70 minimal promoter). These data suggest that chn acts mainly on the ASC enhancers, but it cannot be ruled out at present that the endogenous promoter might additionally favor this effect. However, the results argue against a stimulatory action of Chn directly on the sc and/or ac promoters, since generalized expression of UAS-chn does not lead to widespread expression of the constructs carrying the sc promoter. Moreover, the stimulation is equally observed in the presence or absence of the endogenous ac/sc genes, which indicates that it is not mediated by positive feedback loops of ac/sc on the ASC enhancers. Considering that the ASC enhancers act in vivo on both the sc and the ac promoters, it is to be expected that Chn also stimulates ac expression (Escudero, 2005).

Interestingly, Chn not only increases the levels of lacZ expression within the proneural cluster for which the enhancer is specific, but in general it also expands the expression into a larger area surrounding the proneural cluster, so that more cells express the reporter gene. Perdurance of ß-galactosidase should not be responsible for this effect, because when chn is not overexpressed, DC-lacZ directs ß-galactosidase accumulation only in the cells that also express sc at the DC cluster. Moreover, the stimulation by chn seems to require the presence of at least some of the prepattern factors that normally act on the enhancers and drive the expression of ac and sc in proneural clusters, as is the case for Pnr, the prepattern activating factor of the DC cluster. It is proposed that excess Chn makes the proneural cluster enhancers responsive to suboptimal concentrations of the prepattern activators that are normally too low to permit activity. Hence, the domains of expression of lacZ are expanded. The dependence of Chn stimulation on different prepattern factors suggests that Chn acts as a coactivator, increasing the effective interaction of prepattern activators with the ac and sc promoters. Moreover, the finding that a fragment of Chn that contains the five Zn-finger motifs of the protein can bind in vitro to a 316 bp fragment of the DC enhancer DNA further suggests that Chn stimulates ac/sc expression by directly binding to ASC proneural cluster-specific enhancers. The possible functional relevance of this binding is reinforced by the fact that the 316 bp fragment is found within a 508 bp segment that possesses residual DC enhancer activity and that Chn is capable of strongly stimulating this activity in vivo (Escudero, 2005).

Chn does not appear to act in vivo as a general stimulator of the enhancer action of proneural genes. The ASC enhancer(s) responsible for expression of ac/sc during microchaetae formation does not require Chn, as judged by the independence of microchaetae density from the activity of chn. Note that downregulation of ac and/or sc normally leads to a strong loss of microchaetae. By contrast, overexpression of UAS-chn does increase microchaetae density, suggesting that the microchaetae enhancer(s) can potentially respond to Chn. chn^ECJ1 clones and UAS-chni do not alter the anterior wing margin bristles. However, overexpression of UAS-chn impairs the expression of sc at the anterior wing margin, although the idea is favored that this inhibition results from an interference of Chn with the general patterning of the wing, as suggested by the inhibition of sens expression even in the posterior wing margin. The lack of an identified ASC wing margin enhancer has prevented a more direct test of these possibilities. It was also found that the ASC SOP-specific enhancer can not be activated in the absence of ac/sc and that the stimulation that is observed occurs in isolated cells, rather than in the majority of cells of the domain of UAS-chn expression. Probably, the stimulation results from extra SOPs arising from the overexpression of the endogenous sc gene. Finally, the proneural gene atonal, which is not a member of the ASC, is not affected in the wing or in the eye discs by UAS-chn. It is concluded that in the wing disc, Chn is mostly specific for the ASC enhancers that direct ac/sc expression in the proneural clusters of the macrochaetae and other landmark sensilla, such as the twin sensilla of the anterior wing margin (TSM) and the L3 wing vein sensilla campaniformia (Escudero, 2005).

Taken together, the data indicate that chn and ac/sc form a mutually stimulatory loop that enhances accumulation of Ac/Sc in the proneural clusters of the notum macrochaetae. These and other findings suggest the following consecutive levels of genetic control during SOP selection. The process starts by the deployment of combinations of prepattern factors that trigger the expression of ac/sc in proneural clusters. Then, ac/sc activate chn and their stimulatory loop reinforces the expression of ac/sc. This allows increasing levels of Ac/Sc to accumulate in the cells of the proneural cluster and the formation of the proneural field, which includes the few cells of the cluster with the highest levels of Ac/Sc. The SOP will be selected from one of these cells by the Ac/Sc-mediated activation of sens, which in turn allows the autostimulatory loops of the proneural genes mediated by the SOP-specific enhancers. These enhancers are the targets of two antagonistic signaling systems, both triggered by the accumulation of Ac/Sc. The positive one is mediated by the EGF receptor and Sens. The EGFR pathway allows the cells of the proneural cluster to signal positively to each other (lateral cooperation) and helps activate the SOP-specific enhancers, whereas Sens directly activates proneural gene expression in a positive feedback loop when the proneurals reach a certain threshhold in the SOP. Sens and EGFR are in turn antagonized by the negative loop, which is mediated by the Dl/N pathway and the E(spl) proteins and prevents more than one cell from turning on the proneural gene self-stimulation and becoming an SOP (lateral inhibition). Thus, three loops of self-stimulation of ac/sc exist: the first is mediated by chn and targets the proneural cluster enhancers; the second is mediated by the EGFR pathway and targets the SOP-specific enhancers; the third is mediated by Sens and also directly targets the SOP-specific enhancers. It is interesting to note that the first and third stimulatory loops are mediated by Zn-finger transcription factors of the C2H2 type with homologs in mammals and other species. The negative loop, mediated by Dl/N and the E(spl) proteins, maintains most cells of the proneural cluster in a non-SOP state, allowing them to differentiate as epidermal cells. It is tempting to speculate that these consecutive layers of control facilitate the refinement of the position where SOPs arise within proneural clusters (Escudero, 2005).

In the embryo, chn is expressed in regions where the neurons of the PNS will arise and later in the developing neural cells. Its removal causes loss of PNS neurons and defects in the morphology of the chordotonal organs, suggesting that chn is required for the proper formation of many or most elements of the PNS. So far, the reported effects of insufficiency of proneural gene function in the embryonic PNS have mostly been the removal of neurons and chordotonal organs, rather than defective morphologies. Hence, it is suggested that in the embryonic PNS chn acts more as a neuronal differentiation gene than a proneural gene activator. In agreement with this suggestion it was observed that overexpression of UAS-chn does not modify the embryonic PNS, as detected with the 22c10 antibody. By contrast, overexpression of proneural genes promotes development of extra neurons and chordotonal organs. Moreover, loss of function of cousin of atonal (cato) and ase, two genes that can act as neuronal differentiation genes, also causes malformations of the lateral clusters of chordotonal organs. It is not known whether the removal of chn may also affect the differentiation of the adult bristles, but the observation that, with low frequency, a shaft can be missing, but not the basal cell, also suggests a role of chn in the differentiation of these SOs. Moreover, the fact that UAS-chni partially suppressed the extra macrochaetae induced by UAS-sc, a transgene not subjected to chn modulation, may additionally indicate that chn favors macrochaetae formation. However, it should be kept in mind that UAS-sc may promote accumulation of Sc not only through its own expression, but also by the activation of chn, which would in turn stimulate the endogenous ac/sc genes. This latter stimulation should be sensitive to UAS-chni and its inhibition might partially suppress the formation of extra macrochaetae. At present, it is not possible to decide between these alternatives (Escudero, 2005).

REGULATION Transcriptional Regulation

An NRSF/REST-like repressor downstream of Ebi/SMRTER/Su(H) regulates eye development in Drosophila; charlatan is repressed by Ebi/SMRTER and promotes Dl expression

The corepressor complex that includes Ebi and SMRTER is a target of epidermal growth factor (EGF) and Notch signaling pathways and regulates Delta (Dl)-mediated induction of support cells adjacent to photoreceptor neurons of the Drosophila eye. A mechanism is described by which the Ebi/SMRTER corepressor complex maintains Dl expression. charlatan (chn) is repressed by Ebi/SMRTER corepressor complex by competing with the activation complex that includes the Notch intracellular domain (NICD). Chn represses Dl expression and is critical for the initiation of eye development. Thus, under EGF signaling, double negative regulation mediated by the Ebi/SMRTER corepressor complex and an NRSF/REST-like factor, Chn, maintains inductive activity in developing photoreceptor cells by promoting Dl expression (Tsuda, 2006).

The corepressor complex that includes Ebi, SMRTER and Su(H) is required for expression of Dl in Drosophila photoreceptor cells. To identify genetic loci that are transcriptionally repressed by the Ebi corepressor, a screen was set up using an ectopic gene expression system (Gene Search System). Insertion of a Gene Search (GS) vector, a modified P-element carrying the Gal4 upstream activating sequence (UAS_G) near its 3' end, causes overexpression of a nearby gene under the control of the Gal4-UAS_G system. GS insertions into the chn locus were identified, whose overexpression phenotype in the eye using an eye-specific Gal4 driver (GMR-Gal4) was modified by reducing ebi activity. Thus the regulation of chn by Ebi-dependent transcriptional repression was studied (Tsuda, 2006).

In third instar larval-stage eye discs, the chn transcript is highly expressed in the morphogenetic furrow (MF), where photoreceptor differentiation initiates, but is downregulated in cells in the later stage photoreceptor development. In ebi mutant eye discs, however, chn expression becomes detectable in differentiating photoreceptor cells, and its expression in the MF is increased, suggesting that Su(H) in association with Ebi and SMRTER represses chn transcription in the eye disc (Tsuda, 2006).

To reveal the role of Su(H) as an activator, chn expression was examined when the level of Su(H) expression was reduced. Removing one copy of Su(H) suppresses the loss-of-Dl expression phenotype in ebi mutants. It was found that reducing one copy of Su(H) suppresses ectopic chn expression in ebi mutants, suggesting that ectopic expression of chn in ebi mutants is Su(H)-dependent. RT-PCR analysis of chn expression in ebi^- eye discs differing in the dosage of Su(H) gene also supported these results. Strong reduction of Su(H) expression alone reduced expression of chn in the MF; this expression became weaker and was slightly broader. The phenotype of ebi, Su(H) double mutants is almost the same as Su(H) single mutants , suggesting that Su(H) acts as an activator in the absence of Ebi. This might be due to dual functions of Su(H) as an activator or repressor. Hence, reducing the amount of Ebi in the corepressor complex involving Su(H) might convert Su(H) to an activator by permitting the replacement of the corepressor complex with NICD (Tsuda, 2006).

To reveal the molecular nature of transcriptional regulation of chn by Su(H), Su(H) target sites were sought in the genomic region of chn. Since Su(H) binds slightly degenerate sequences, it was not easy to identify the functional Su(H) binding region from a simple genomic search. An alternative approach was taken to map the chn genomic region, which is regulated by Su(H) in the normal chromosomal context. Ebi-mediated repression involves SMRTER, a corepressor that recruits histone deacetylases and induces the formation of inactive chromatin, which spreads from the site where Su(H) recruits the corepressor complex. Promoters near the Su(H)-binding site are thus expected to be downregulated in an Ebi-dependent manner. Four insertion lines of the GS vector were identified in the chn promoter region. All these GS lines caused ectopic expression of chn with consequent abnormal eye morphology when they were crossed with GMR-Gal4. If the effect of the Ebi/SMRTER corepressor complex reaches the UAS_G in those insertions, reduction of Ebi activity will derepress UAS_G and further enhance activation by GMR-Gal4. One copy of a dominant-negative construct of ebi (GMR-ebi^DN) caused only a mild defect in eye morphology and weak, if any, ectopic expression of chn. GMR-ebi^DN strongly enhanced the overexpression phenotype of chn^GS17605 and chn^GS11450, which contained GS vector insertions (-474 and -734, respectively) upstream of the transcriptional start site. However, GMR-ebi^DN failed to enhance the overexpression phenotype of other GS lines (chn^GS2112 and chn^GS17892) that were inserted downstream (+773 and +1040, respectively) of the first exon. From these results, it is concluded that Ebi-dependent transcriptional repression is targeted to the proximity of the transcriptional initiation site of the chn promoter (Tsuda, 2006).

Chn is a 1108-amino-acid protein with multiple C2H2-type zinc-finger motifs. Although no highly homologous gene within the mammalian genome could not be detected using BLAST, a small sequence of similarity between the N-terminal zinc-finger motif of Chn and the fifth zinc-finger of human NRSF/REST was found. Chn has several structural and functional similarities to human NRSF/REST, as follows. First, Chn and NRSF/REST each contain an N-terminal region with multiple zinc-finger motifs (five motifs in 264 residues in Chn and eight motifs in 251 residues in NRSF/REST), followed by a cluster of S/T-P motifs (serine or threonine followed by a proline) and a single zinc-finger motif at the C terminus. Second, the C-terminal region of NRSF/REST binds a corepressor, CoREST, which serves as an adaptor molecule to recruit a complex that imposes silencing activities. The Drosophila homolog of CoREST (dCoREST) (Andres, 1999; Dallman, 2004) can associate with the C-terminal half of Chn in cultured S2 cells. Finally, NRSF/REST binds to NRSE/RE1, a 21-bp sequence located in the promoter region of many types of neuron-restricted genes, via the N-terminal zinc-finger motifs. It was found that a recombinant protein containing the N-terminal zinc-fingers of Chn bound specifically to the NRSE/RE1 sequence in vitro. Thus, the structural similarity to NRSF/REST, binding to dCoREST and the DNA-binding specificity of Chn suggest that it is a candidate for a functional Drosophila homolog of NRSF/REST (Tsuda, 2006).

If Chn acts as a regulator of neural-related functions, as suggested for NRSF/REST, then Chn would be expected to bind to a regulatory region common to many types of neural-related genes in Drosophila. Numerous sequences similar to NRSE/RE1 were identified in the Drosophila genome, and their binding to Chn was assessed by EMSA. Using these sequences, a consensus binding sequence for Chn (Chn-binding element (CBE), 5'-BBHASMVMMVCNGACVKNNCC-3') was derived. 26 CBEs were identified within 10 kb of annotated genes from the Drosophila genome. Binding to Chn was confirmed for 18 CBEs using EMSA competition assay. Genes containing the CBE include dopamine receptor 2 (DopR2) and the potassium channel, ether-a-go-go, for which the mammalian homologs are target genes of NRSF/REST. These results suggest that the CBE is a good indicator of Chn binding sites and that Chn regulates many types of neural-related genes, as is implicated for NRSF/REST. However, it was found that divergent forms of CBE adjacent to hairy and extramacrochaetae were bound specifically by Chn. Likewise, some of the CBE sites failed to bind to Chn. Thus, a further refinement will be necessary to predict a definitive set of Chn binding sites in the Drosophila genome (Tsuda, 2006).

Although it has been established that mammalian NRSF/REST is a key regulator of neuron-specific genes, attempts to isolate invertebrate homolog of NRSF/REST have so far failed to identify a true homologous factor in invertebrates. The properties of Chn, including the similarity in DNA-binding specificity, association with CoREST and transcriptional repressor activity, suggest that Chn is a strong candidate for a functional Drosophila homolog of NRSF/REST. chn was originally identified by its requirement in the development of the PNS. This study identified a number of candidate target genes of Chn, a large fraction of which is implicated in neural function and/or gene expression. It is expected that further analysis of these candidates will provide valuable information about chn function in vivo, which may be extended to the understanding of NRSF/REST (Tsuda, 2006).

The Chn mutation blocks eye development by preventing the initiation of MF, a process requiring Notch signaling. This phenotype is likely owing to a loss of Notch function, because elevated Dl expression is known to block Notch signaling. The function of Chn during the early stage of eye development might be to regulate Notch signaling at an appropriate level by downregulating Dl. It is possible that Chn-mediated modulation places a variety of Notch functions in eye under the influence of EGFR signaling and provides flexibility in its regulation (Tsuda, 2006).

Although chn is expressed in the MF, genetic analyses show that small clones of chn mutant cells permit progression of the MF and photoreceptor differentiation. It is speculated that the repressive effect of Chn is overcome by other signals in the MF, such as hedgehog signaling, which strongly induces Dl (Tsuda, 2006).

Developing photoreceptor cells are exposed to the EGFR ligand, Spitz, and the Notch ligand, Dl, and each cell must assess the level of the two signals and respond appropriately to perform each task of photoreceptor cell specification and induction of non-neural cone cells. This question was investigated by studying the expression of Dl in photoreceptor cells. chn was identified as a direct target of Ebi/SMRTER-dependent transcriptional repression and as a repressor of Dl expression. The abrogated expression of Dl in ebi mutants was recovered by reducing one copy of chn, suggesting that the negative regulation of chn by ebi is indeed prerequisite for photoreceptor cell development (Tsuda, 2006).

Genetic data suggest that Su(H) may activate or repress chn expression. This idea is supported by data showing that Ebi/SMRTER and NICD are recruited to the promoter region of chn. The Ebi/SMRTER complex formed in this region did not contain any detectable level of the intracellular domain of Notch (NICD), suggesting that the binding of Ebi/SMRTER and NICD to this region may be mutually exclusive, and therefore it is expected that a regulatory system controls the balance between the active and repressive states of Su(H). Taken together, these results suggest that chn is a key factor in the crosstalk between two major signal transduction pathways: the EGFR-dependent pathway and the Notch/Delta-dependent pathway (Tsuda, 2006).

In the mammalian system, competition between SMRT and NICD for interaction with RBPJkappa determines the state of RBPJkappa-dependent transcriptional activity. Extracellular signaling may modulate this competition; diverse signaling pathways modulate the functions of N-CoR/SMRT. The current findings would prompt investigations of potential interaction of two repression systems of NRSF/REST and N-CoR/SMRT, and their regulation by Notch and EGF signaling in mammalian neuronal differentiation (Tsuda, 2006).

Targets of Activity

The strong genetic interaction between the LOF conditions for chn and ac/sc, together with the presumed activation of chn by ac/sc, led to an examination of whether chn might in turn stimulate ac/sc expression. Initially examined was whether the overexpression of chn affects Sc accumulation in third instar wing discs. In these discs, ac and sc are coexpressed in a stereotyped pattern of well-resolved proneural clusters from which SOPs emerge. With the MS248-Gal4 driver, UAS-chn promotes strong and generalized expression of sc in most of the domain of expression of the driver, namely, the medial and part of the lateral prospective notum. Many SOPs arose from this enlarged region of Sc accumulation, as detected by the Sens marker, consistent with the additional macrochaetae that develop on the notum of these flies. With the MS1096-Gal4 driver, which is expressed most strongly in the dorsal part of the wing anlage, there is also ectopic expression of sc and emergence of extra SOPs in the wing territory. Interestingly, expression of UAS-chn disrupts the characteristic double row expression of sc and sens at the wing margin, suggesting interference with its formation. This is also consistent with the presence of small, crumpled adult wings that carry many bristles and other types of sensilla. Overexpression of UAS-chn with the ubiquitous wing disc driver C765-Gal4 activates sc but fails to stimulate atonal, a proneural gene which is not a member of the ASC and is normally expressed in a few cells at the presumptive tegula and ventral radius. Conversely, overexpression of atonal does not stimulate chn in the wing disc (Escudero, 2005).

The expression of ac/sc in proneural clusters is controlled by a series of separable enhancer elements in the ASC. Each enhancer is responsible for expression in one or in a few proneural clusters. Thus, whether the ectopic activation of sc could be mediated by the overexpression of UAS-chn acting upon these enhancers was examined. UAS-chn strongly stimulates the activity of a construct in which the lacZ gene is under the control of the ASC L3/TSM enhancer [construct 2.3-lacZ; MS1096-Gal4 driver], which directs expression at the wing vein L3 and the twin sensilla of the wing margin proneural clusters. Similar observations were made with the dorsocentral (DC) enhancer [construct AS1.4DC=DC-lacZ; C765-Gal4 ubiquitous driver], which promotes expression in the central part of the notum. It also activates expression directed by the ANP enhancer. Since the DC-lacZ construct bears the heterologous hsp70 promoter, these data indicate that the sc endogenous promoter is dispensable for the stimulation by UAS-chn. The Ac and Sc proneural proteins are also not essential for the increased activity of the enhancers; DC-lacZ expression is strongly increased by Chn in an In(1)sc^10.1 background. By contrast, the sc SOP-dedicated enhancer (SRV-lacZ construct), which is responsible for the strong accumulation of Sc in SOPs, is only clearly activated by UAS-chn (C734-Gal4 driver) in the presence of ac/sc, and this stimulation occurred in individual cells. This observation suggests that the upregulation of this enhancer results from the formation of ectopic SOPs by the UAS-chn-induced overexpression of sc, rather than from a direct effect of Chn on the enhancer. Still, the possibility remains that Chn and Sc cooperate in the activation of this enhancer (Escudero, 2005).

UAS-chn upregulates the activity of these enhancers, but it does not lead to a generalized expression of lacZ in all the domains of UAS-chn expression. These data indicate that despite the elevated activation, the enhancers are still dependent on the prepattern factors that define their spatial domains of activity. This fact was verified by the observation that the overstimulation of DC-lacZ is strongly dependent on its prepattern activator, the transcription factor Pnr. Moreover, 2.3-lacZ, which is active only in the wing pouch, is not stimulated by the overexpression of UAS-chn in the prospective notum (MS248-Gal4); this indicates that the sc promoter present in this construct was not responsive to UAS-chn (Escudero, 2005).

Next examined were mosaic wing discs to determine whether removal of chn function affects expression of sc or enhancer-lacZ constructs in proneural clusters. Homozygous chn^ECJ1 cells generally display reduced expression of sc or ß-galactosidase under the control of proneural enhancers, when compared with neighboring heterozygous chn^ECJ1/+ cells. Note however, that the expression is not completely abolished. Similar decreased expression of sc is observed by misexpressing UAS-chni^S in cell clones. These effects appear to be cell-autonomous. While SOPs can still emerge from homozygous chn^ECJ1 cells with reduced levels of Sc, SOPs were often missing, in agreement with the partial suppression of macrochaetae observed within the chn^ECJ1 clones. When both homozygous and heterozygous cells were near a position where an SOP emerged, a heterozygous cell appeared to be preferentially selected. These findings clearly indicate that chn⁺ is required for proneural proteins to accumulate in proneural clusters at levels sufficient to ensure SOP selection. Moreover, the observation that expression of enhancer-lacZ constructs is reduced in chn^- cells and increased in chn overexpressing cells indicates that the effect of chn⁺ is not due to an enhanced perdurance of the Sc protein, but to the increased transcription of the sc gene (Escudero, 2005).

To analyze whether Chn is a direct regulator of the ASC enhancers, the ability of the Chn protein to bind to the DC enhancer was assayed in vitro. A fragment of the Chn protein containing the five zinc fingers was produced in and purified from E. coli. The enhancer DNA was divided into six partially overlapping fragments of approximately 300 bp each, and each of them was assayed in gel retardation experiments. Only the fragment that comprised the proximal-most region of the enhancer (fragment DC6) shows binding of the Chn polypeptide. Interestingly, the DC6 fragment is included within the PB0.5DC sequence, the smallest subfragment of AS1.4DC, which still retains enhancer activity. For unclear reasons, the PB0.5DC enhancer drives expression only in the PDC SOP. Still, misexpression of UAS-chn expands this expression to many cells of the posterior notum. This suggests that the binding of Chn protein to the DC6 region of the DC enhancer may prompt its response to Chn in vivo (Escudero, 2005).

DEVELOPMENTAL BIOLOGY

The patterns of expression of chn in embryos and imaginal discs were examined using in-situ hybridization. In early blastoderm stages, the expression of chn is ubiquitous, but before stage 5, chn mRNA disappears from the poles of the embryo and faint stripes become visible. At stage 5, chn mRNA also accumulates in the dorsal region, cephalic furrow and in the presumptive mesoderm. At stage 11, chn mRNA is found mostly in the mesoderm, and in ectodermal patches between the tracheal pits, where neurons of the PNS appear. Older embryos (stage 15) show strong expression, which is mostly restricted to the central nervous system (CNS) and PNS. In the latter case, the pattern suggests that expression occurs in many of the neurons of the ventral, lateral and dorsal clusters of neurons (Escudero, 2005).

In third instar wing discs , expression of chn is observed in rows of cells on either side of the prospective anterior wing margin and in groups of cells that coincide with proneural clusters of ac/sc expression. This pattern suggests that chn may be positively regulated by ac/sc. Indeed, expression of chn in proneural clusters is abolished in discs null for ac/sc (In(1)sc^10.1). Moreover, overexpression of a UAS-sc transgene in the posterior compartment of the disc (en-Gal4 driver) induces ectopic expression of chn. Note that chn is also expressed independently of ac/sc in certain areas of the disc, such as the postnotum and posterior dorsal proximal wing. chn is also expressed in proneural clusters of the leg discs and in the eye/antenna disc. In the latter case, the pattern seems very similar to that of the proneural gene atonal (ato), and includes the region of the morphogenetic furrow, and the presumptive cephalic capsule and second antennal segment (Escudero, 2005).

EFFECTS OF MUTATION

When driven by en-Gal4, the EPIL6 insertion induces the overexpression of both chn and bda transcripts in wing imaginal discs. Hence, flies were created carrying either a UAS-chn or a UAS-bda transgene. Overexpression of UAS-bda using several drivers (MS1096-Gal4, C765-Gal4, ap-Gal4 and MS248-Gal4) does not cause noticeable phenotypic effects. By contrast, overexpression of UAS-chn with these and other drivers gives rise to extra bristles. Ubiquitous expression with the C765-Gal4 driver causes the appearance of many macrochaetae near wild-type bristles, and it increases the density of microchaetae [141±10 microchaetae per female hemi-notum versus 103±7 in Oregon R controls (averages of 10 hemi-nota). However, the bristles were always separated by epidermal cells, suggesting that Notch-mediated lateral inhibition is still active. With earlier-expressing drivers such as MS248-Gal4, overexpression gives rise to many bristles. In addition, this early expression reduces the size of the hemi-nota and interfers with their dorsal fusion. With the appropriate drivers many extra bristles appear on other regions of the fly body, including wings (C765-Gal4, MS1096-Gal4 and nub-Gal4), head (MS248-Gal4) and the metathorax (MS1096-Gal4) (Escudero, 2005).

Formation of additional bristles by overexpression of chn depends on the presence of the proneural genes ac/sc. Halving the dose of these genes sharply reduces the number of extra macrochaetae induced by the overexpression of chn. Removal of both ac and sc completely suppresses bristle formation. The genetic interaction between sc and chn is also manifested by the synergism of their overexpression in bristle formation, which gives rise to many macro- and mesochaetae in ectopic positions, such as the anterior notum (Escudero, 2005).

To examine the effects of the removal of chn, new LOF alleles were obtained by generating imprecise excisions of the Pl(2)42/18 insertion. One of these, chn^ECJ1, is probably a null, since the excision removed at least part of the promoter region of chn, and homozygous embryos lack the chn mRNA (as detected by in-situ hybridization) and die as embryos. In keeping with the expression of chn in the cells of the PNS, chn^ECJ1 embryos display conspicuous anomalies in PNS cells. These include the absence of many neurons, especially in the dorsal and ventral clusters, and an abnormal morphology of chordotonal lateral neurons, which appear bunched and lack the typical apical dendrites. Individually identifiable neurons such as the v'chn1 and the dbp are generally absent. Some of these defects are similar to the phenotype described for the original Pl(2)42/18 insertion but are more severe. Ubiquitous expression of UAS-chn in the epidermis (69B-Gal4 driver) largely rescues many of the missing neurons and the morphological defects of the lateral chordotonal neurons. However, overexpression of UAS-chn in a wild-type background (using da-Gal4, 69B-Gal4 and 1407-Gal4 drivers) does not appreciably affect the larval PNS (Escudero, 2005).

In the adult, the effects of chn^ECJ1 were examined through clonal analysis. Clones of cells that lack chn often fail to generate macrochaetae. These were observed to be missing in all notum positions, but scutellar bristles were the most sensitive. However, the penetrance was far from complete: clones including the dorsocentral or the posterior postalar positions lost approximately 25% of the bristles, while in scutellar clones about 45% of the bristles were removed. It was also observed, with low frequency, that the bristle shaft was missing, but not the tormogen cell, suggesting a role for chn in formation of the sensory organ. No defects were seen in the pattern of notum microchaetae (Escudero, 2005).

The effects of the removal of chn in macrochaetae formation were examined with the help of an RNA-interference construct -- UAS-chni. A strong (UAS-chni^S) and a weak (UAS-chni^W) expressing line were used. UAS-chni^S clearly antagonized chn function, because it largely rescued the extra bristle and heminota fusion phenotypes of MS248-Gal4; UAS-chn. No rescue was observed by replacing UAS-chni^S by UAS-lacZ or UAS-GFP, which indicated that the effect was not caused by reduced expression of UAS-chn in the presence of an additional UAS transgene. Moreover, overexpression of UAS-chni^S with MS1096-Gal4 sharply decreased accumulation of the endogenous chn mRNA in the wing margin These experiments indicate that the UAS-chni^S acts as a LOF allele of chn. With the drivers ap-Gal4 or sca-Gal4 (the latter promotes expression in proneural clusters), UAS-chni^S moderately removes notum macrochaetae while microchaetae are not affected. With the MS248-Gal4 driver, the macrochaetae in the medial part of the notum, dorsocentrals and scutellars, were often missing, but, similar to the homozygous chn^ECJ1 clones, in no case was the phenotype fully penetrant (Escudero, 2005).

The effectiveness of both UAS-chni^S and UAS-chni^W in removing macrochaetae was increased when the accumulation of ac/sc in proneural clusters was compromised. Thus, flies overexpressing UAS-chni^W with C765-Gal4 or pnr-Gal4 had, respectively, wild-type or almost wild-type phenotypes, but a sizable number of macrochaetae were lost in the heterozygous In(1)sc^10.1/+ genetic background. The N allele Ax^M1 has been shown to reduce ac/sc expression in proneural clusters. Hence, in Ax^M1/+ individuals, several notum macrochaetae are missing. UAS-chni^S almost completely eliminates the remaining bristles in Ax^M1/+ with the exception of the ANP and APA that are always present. The pnr prepattern gene is necessary for the formation of the dorsocentral and scutellar macrochaetae. In the hypomorphic pnr^V1/pnr-Gal4 background, expression of ac/sc is diminished and these bristles are partially removed. In this genetic background, UAS-chni^W almost completely eliminates all these bristles. Interestingly, UAS-chni^W was able to partially suppress the extra macrochaetae that are generated by the expression of UAS-sc driven by C765-Gal4 -- the number of macrochaetae per notum decreased in the dorsocentral area from 12±1.2 to 7.5±1.2 and, in the scutellum, from 18±1.3 to 10.2±1.3. Moreover, macrochaetae that appeared away from the normal macrochaeate-bearing regions, such as the anterior notum, were almost completely eliminated (from 5.4±1.3 to 1±1.2). This suggested that these extra macrochaetae have a stronger requirement for chn than the extant bristles. Taken together, these and the data obtained from the chn^ECJ1 clones indicate that chn is not essential for macrochaetae formation, but that it facilitates the process (Escudero, 2005).

A genome-wide RNAi screen reveals multiple regulators of caspase activation

A genome-wide RNA interference screen was performed to systematically identify regulators of apoptosis induced by DNA damage in Drosophila cells. Forty-seven double- stranded RNAs were identified that target a functionally diverse set of genes, including several with a known function in promoting cell death. Further characterization uncovers 10 genes that influence caspase activation upon the removal of Drosophila inhibitor of apoptosis 1. This set includes the Drosophila initiator caspase Dronc and, surprisingly, several metabolic regulators, a candidate tumor suppressor, Charlatan, and an N-acetyltransferase, ARD1. Importantly, several of these genes show functional conservation in regulating apoptosis in mammalian cells. These data suggest a previously unappreciated fundamental connection between various cellular processes and caspase-dependent cell death (Yi, 2007).

The genes that are specifically involved in caspase-dependent cell death were classified. Substantial induction of caspase activity was observed 8 h after treatment with a topoisomerase II inhibitor, doxorubicin (dox), to induce dose-dependent cell death. Any RNAi suppressing this activity implicates the target gene in early regulation of caspase activation. In addition to dcp-1 RNAi, knockdown of dronc and jra (the Drosophila homolog of c-Jun) significantly suppressed caspase-3/7-like activity in the presence of dox, whereas the negative control, RNAi against calpain A, a calcium-dependent cysteine protease, did not affect this pathway (Yi, 2007).

This analysis was expanded to all of the genes identified in the initial RNAi screen and 20 dsRNAs were discovered that suppressed caspase activation induced by DNA damage. Interestingly, 12 of these genes were found to be epistatic to diap1 (Yi, 2007).

diap1 epistatic analysis was performed to further categorize the genes. DIAP1, the fly orthologue of the mammalian inhibitors of apoptosis proteins, is a direct inhibitor of caspases, and deficiency in DIAP1 leads to rapid caspase activation and apoptosis in vivo. Thus, apoptosis induced by the loss of DIAP1 presents an alternative apoptotic assay independent of DNA damage. Silencing of genes that regulate activation of the core apoptotic machinery may provide protection against apoptosis induced by both DNA damage and the loss of DIAP1. RNAi against dcp-1 partially suppressed cell death induced by the depletion of DIAP1 in Kc cells. Also, dronc RNAi potently protected cells against apoptosis induced by deficiency in DIAP1. Altogether, 32 of the genes confirmed from the primary screen provided significant protection against cell death induced by the silencing of DIAP1 (Yi, 2007).

Interestingly, 12 dsRNAs suppressed caspase-3/7-like activity after dox treatment and protected against cell death induced by diap1 RNAi, suggesting that these genes are required for apoptosis induced by multiple stimuli. To confirm that these genes are necessary for the full activation of caspases, it was determined whether these dsRNAs could suppress spontaneous caspase activity induced by diap1 RNAi. Maximal induction of caspase activity by diap1 RNAi was observed after 24 h, and this effect was completely suppressed by dsRNA against dcp-1. Importantly, ablating 10/12 dsRNAs resulted in the significant suppression of caspase activity compared with diap1 RNAi only (Yi, 2007).

In addition to dronc RNAi, dsRNAs targeting chn and dARD1 provided the strongest suppression of spontaneous caspase activity. Consistent with the observation that RNAi against chn protects against DNA damage-induced cell death, the mammalian orthologue neuron-restrictive silencer factor (NRSF)/RE1-silencing transcription factor (REST) was recently identified as a candidate tumor suppressor in epithelial cells (Westbrook, 2005). Previous work indicates that Chn and NRSF/REST function as a transcriptional repressor of neuronal-specific genes (Chong, 1995; Schoenherr, 1995; Tsuda, 2006), suggesting that cellular differentiation may render cells refractory to caspase activation and apoptosis. Also, several metabolic genes, CG31674, CG14740, and CG12170, were identified that may be involved in the general regulation of caspase activation. It has been demonstrated that NADPH produced by the pentose phosphate pathway regulates the activation of caspase-2 in nutrient-deprived Xenopus laevis oocytes. Together with these results, these observations provide further evidence for an intimate link between the regulation of metabolism and induction of apoptosis (Yi, 2007).

To further explore the significance of these findings, whether silencing the mammalian orthologues of the fly genes identified from the RNAi screen confers protection against dox-induced cell death was investigated in mammalian cells. A set of mammalian orthologues was selected that are believed to be nonredundant. The list includes the orthologues of dMiro, which functions as a Rho-like GTPase; dARD1, which functions as an N-acetyltransferase; CG12170, which functions as a fatty acid synthase; and Chn, which functions as a transcriptional repressor (RHOT1, hARD1, OXSM, and REST, respectively; FlyBase). In addition, Plk3, a mammalian orthologue of Polo, was tested since dsRNA targeting polo potently protected against dox treatment (Yi, 2007).

The ability of siRNAs targeting a gene of interest to protect against DNA damage was tested in HeLa cells. As a positive control, cells were transfected with siRNAs targeting Bax or Bak, two central regulators of mammalian cell death. Indeed, silencing of Bax or Bak resulted in significant protection against dox- induced cell death. It was observed that plk3 RNAi provided partial protection against dox treatment, which is consistent with previous studies implicating Plk3 in stress-induced apoptosis. Interestingly, the knockdown of hARD1 dramatically enhanced cell survival in the presence of dox to levels similar to that of Bak. This protective effect was also evident at the morphological level. In cells transfected with a nontargeting control siRNA, dox treatment resulted in typical apoptotic morphology, including cell rounding and membrane blebbing. In direct contrast, cells transfected with siRNAs against hARD1 maintained a normal and healthy morphology and continued to proliferate in the presence of dox (Yi, 2007).

To examine whether the protection provided by siRNAs targeting hARD1 and plk3 is associated with the suppression of caspase activation, caspase activity was measured in these cells treated with dox. RNAi against plk3 provided partial suppression of caspase activity, again supporting the observed protection phenotype. Interestingly, the depletion of REST resulted in some suppression of caspase activity in the presence of dox even though the protection against cell death was not statistically significant. Consistent with the viability assay, complete suppression of caspase-3/7 activity was observed in cells transfected with hARD1 siRNA. These results indicate that hARD1 is required for caspase-dependent cell death induced by DNA damage. Furthermore, all four siRNAs targeting hARD1 were individually capable of providing robust protection against cell death, strongly suggesting that these siRNAs target hARD1 specifically (Yi, 2007).

Because the silencing of hARD1 dramatically suppressed activation of the downstream caspases, whether activation of the upstream caspases in response to dox treatment is also perturbed was also examined. Remarkably, hARD1 RNAi inhibited the cleavage of caspase-2 and -9 in cells treated with dox, whereas caspase cleavage was readily detected in control cells. Thus, it is proposed that hARD1 regulates the signal transduction pathway apical to the apoptotic machinery in the DNA damage response itself or the activation of upstream caspases (Yi, 2007).

Consistent with the results of the caspase-3/7 assay, silencing of hARD1 completely inhibited the appearance of activated caspase-3 induced by dox. This assay was used for a hARD1 complementation experiment to demonstrate the proapoptotic role of hARD1 in response to DNA damage. A new siRNA pool was used, targeting the 5' untranslated region of hARD1 (5'si); this treatment inhibited caspase-3 cleavage induced by dox treatment. Furthermore, caspase-3 cleavage was observed in reconstituted hARD1 knockdown cells. Because six out of six siRNAs against hARD1 provided strong protection against DNA damage-induced apoptosis and complementation of hARD1-sensitized cells to caspase activation, it is concluded that the functional role of ARD1 for dox-induced apoptosis is evolutionally conserved from Drosophila to mammals (Yi, 2007).

In summary, this study used an unbiased RNAi screening platform in Drosophila cells to identify genes involved in promoting DNA damage-induced apoptosis. Forty-seven dsRNAs were isolated that suppress cell death induced by dox. These genes encode for known apoptotic regulators such as Dronc, the Drosophila orthologue of the known proapoptotic transcriptional factor c-Jun, and an ecdysone-regulated protein, Eip63F-1, thereby validating the primary screen. Furthermore, this study implicates a large class of metabolic genes that were previously not suspected to have a role in modulating caspase activation and apoptosis, such as genes involved in fatty acid biosynthesis (CG11798), amino acid/carbohydrate metabolism (CG31674), citrate metabolism (CG14740), complex carbohydrate metabolism (CG10725), and ribosome biosynthesis (CG6712). These results support the proposal that the cellular metabolic status regulates the threshold for activation of apoptosis and thus plays a critical role in the decision of a cell to live or die (Yi, 2007).

Of particular interest is the identification of ARD1. Evidence is presented that RNAi against ARD1 provides protection against cell death and leads to the suppression of caspase activation induced by DNA damage in fly cells and HeLa cells. Furthermore, deficiency in dARD1 renders fly cells resistant to the spontaneous caspase activity and cell death associated with loss of Diap1. Importantly, substantial evidence is provided that hARD1 is required for caspase activation in the presence of DNA damage in mammalian cells. Cleavage of initiator and executioner caspases are suppressed in hARD1 RNAi cells treated with dox, suggesting that hARD1 functions further upstream of caspase activation, and the complementation of hARD1 knockdown cells restores caspase-3 cleavage. These data indicate that ARD1 is necessary for DNA damage-induced apoptosis in flies and mammals (Yi, 2007).

ARD1 functions in a complex with N-acetyltransferase to catalyze the acetylation of the N^α-terminal residue of newly synthesized polypeptides and has been implicated in the regulation of heterochromatin, DNA repair, and the maintenance of genomic stability in yeast. These studies suggest that ARD1 may be involved in regulating an early step in response to DNA damage. It is anticipated that future studies will focus on determining whether ARD1 functions in similar processes in mammals. The diversity of genes identified in the screen illustrates the complex cellular integration of survival and death signals through multiple pathways (Yi, 2007).

Lineage tracing of lamellocytes demonstrates Drosophila macrophage plasticity

Leukocyte-like cells called hemocytes have key functions in Drosophila innate immunity. Three hemocyte types occur: plasmatocytes, crystal cells, and lamellocytes. In the absence of immune challenge, plasmatocytes are the predominant hemocyte type detected, while crystal cells and lamellocytes are rare. However, upon infestation by parasitic wasps, or in melanotic mutant strains, large numbers of lamellocytes differentiate and encapsulate material recognized as 'non-self'. Current models speculate that lamellocytes, plasmatocytes and crystal cells are distinct lineages that arise from a common prohemocyte progenitor. This study shows that over-expression of the CoREST-interacting transcription factor Charlatan (Chn) in plasmatocytes induces lamellocyte differentiation, both in circulation and in lymph glands. Lamellocyte increases are accompanied by the extinction of plasmatocyte markers suggesting that plasmatocytes are transformed into lamellocytes. Consistent with this, timed induction of Chn over-expression induces rapid lamellocyte differentiation within 18 hours. Double-positive intermediates between plasmatocytes and lamellocytes were observed, and it was shown that isolated plasmatocytes can be triggered to differentiate into lamellocytes in vitro, either in response to Chn over-expression, or following activation of the JAK/STAT pathway. Finally, plasmatocytes were marked, and lineage tracing showed that these differentiate into lamellocytes in response to the Drosophila parasite model Leptopilina boulardi. Taken together, these data suggest that lamellocytes arise from plasmatocytes and that plasmatocytes may be inherently plastic, possessing the ability to differentiate further into lamellocytes upon appropriate challenge (Stofanko, 2010).

Drosophila provide a genetically tractable model system to investigate cellular innate immune function. This report examined the origins of lamellocytes, which are Drosophila hemocytes that differentiate in response to parasite infestation. Over-expression of Chn in plasmatocytes induces lamellocyte differentiation, both in circulation and in lymph glands. The data indicate that Chn over-expression transforms plasmatocytes into lamellocytes. Consistent with this, double-positive intermediates between plasmatocytes and lamellocytes were detected, and it was shown that isolated plasmatocytes in vitro can be triggered to differentiate into lamellocytes following Chn over-expression. This property is not limited to Chn since it was observed that other stimuli, including activation of the JAK/STAT pathway and the natural response to parasitic wasp infestation, also induced lamellocyte formation from plasmatocytes (Stofanko, 2010).

The data suggest that Chn may control lamellocyte development. Previously defined regulators of lamellocyte development include the transcription factor STAT92E, the FOG-1 homologue Ush, and the NURF chromatin remodelling complex. STAT92E functions as an inducer of lamellocyte development, as gain-of-function hopTum-l mutants that activate the JAK/STAT pathway cause lamellocyte over-production. In contrast, both loss-of-function ush and Nurf mutants exhibit increased lamellocyte numbers. Like the homologous FOG-1-GATA-1 pairing, Ush modulates activity of the Drosophila GATA factor Srp to favour plasmatocyte differentiation. Recent data in mammalian systems indicates that FOG-1 mediates its effect on GATA-1 in part via recruitment of the transcriptional co-repressor NURD, suggesting that Ush functions similarly to repress expression of gene targets required for lamellocyte differentiation in plasmatocytes. Likewise, NURF also inhibits lamellocyte differentiation, in this case by preventing activation of targets of the JAK/STAT pathway (Stofanko, 2010).

The current biochemical data suggest that Chn is a transcription repressor since Chn recruits the co-repressor complex CoREST. Indeed it has been shown that Chn over-expression represses Delta expression in the eye imaginal disk, while this study has shown that Chn over-expression is accompanied by repression of some plasmatocyte markers. However, it was also shown that Chn over-expression leads to elevated expression of lamellocyte markers, and it has been demonstrated that Chn over-expression increases expression of the proneural genes Achaete and Scute. These data do not allow discrimination of whether Chn functions entirely as a transcriptional repressor or whether it may also activate transcription. However, the temporally-controlled Chn induction system (Pxn-Gal4 TARGET) that was utilized in this study will allow the primary gene targets of Chn to be determined. By analyzing transcriptional profiles of hemocytes at defined time points after Chn over-expression the primary responders to Chn over-expression will be able to be identified. It will be possible to discriminate whether these targets are preferentially activated or repressed, and also subsequently determine recruitment of transcription co-activator or co-repressor complexes such as CoREST at these targets using chromatin immunoprecipitation (Stofanko, 2010).

The data demonstrating that lamellocytes can originate from plasmatocytes sheds new light on hemocyte lineages. Current models of hemocyte lineages speculate that plasmatocytes, crystal cells and lamellocytes are distinct lineages that arise separately from a common stem cell or prohemocyte. This study proposes, however, that prohemocytes generate either crystal cells or plasmatocytes. It is suggested that plasmatocytes are a plastic population that can generate other frequently observed hemocyte types including lamellocytes. This model is strikingly reminiscent of the initial hemocyte lineages first proposed more than 50 years ago. According to that analysis prohemocytes were predicted to generate either crystal cells or plasmatocytes, with plasmatocytes differentiating further into activated cells (podocytes) and then lamellocytes. This model has support from a number of experimental studies including this study. Foremost among these are recent studies of hemocyte functions of Ush. Dominant-negative Ush variants are able to induce lamellocyte differentiation and it has been suggested that Ush regulates lamellocyte differentiation from a potential plasmatocyte. Secondly, lamellocyte differentiation in response to Salmonella infection is blocked in decapentaplegic mutants with a corresponding increase in plasmatocyte number, suggesting that lamellocytes arise from plasmatocytes or a common precursor (Stofanko, 2010).

Two recent studies also suggest that plasmatocytes are a plastic population that may be able to differentiate into lamellocytes. Marking of embryonic plasmatocytes using the gcm-GAL4 or sn-GAL4 drivers and an act5C>stop>GAL4 flip-out transgene shows that lamellocytes that arise in larvae after wasp infestation may originate from cells that had expressed gcm-GAL4 or sn-GAL4 in embryos. Similar results have also been observed using the act5C>stop>GAL4 flip-out transgene and Pxn-GAL4 and eater-GAL4. In both these cases the elicitor of the FLP/FRT activation event and the subsequent sustained marker are the same, namely GAL4 expression. However, in the current lineage tracing experiments, GAL4 expression initiates the FLP/FRT activation of a distinct marker, lacZ protein. These data, taken together with lineage tracing experiments and in vitro differentiation studies suggest that the plasmatocyte is an inherently plastic cell type that is capable of being reprogrammed to tailor immune responses to suit the infectious threats faced by the host. In humans, lymphocyte and leukocyte plasticity has a significant impact on immune responses. An important future challenge is to establish the full spectrum of Drosophila plasmatocyte heterogeneity and exploit the utility of the Drosophila genetic system to dissect the mechanisms that regulate such leukocyte plasticity (Stofanko, 2010).

REFERENCES

Search PubMed for articles about Drosophila Charlatan

Andres, M. E, et al. (1999). CoREST: a functional corepressor required for regulation of neural-specific gene expression. Proc. Natl. Acad. Sci. 96: 9873-9878. 10449787

Chong, J. A., et al. (1995). REST: a mammalian silencer protein that restricts sodium channel gene expression to neurons. Cell 80: 949-957. PubMed Citation: 7697725

Dallman, J. E., et al. (2004). A conserved role but different partners for the transcriptional corepressor CoREST in fly and mammalian nervous system formation. J. Neurosci. 24: 7186-7193. 15306652

Escudero, L. M, Caminero, E.. Schulze, K. L., Bellen, H. J. and Modole, J. (2005). Charlatan, a Zn-finger transcription factor, establishes a novel level of regulation of the proneural achaete/scute genes of Drosophila. Development 132: 1211-1222. 15703278

Schoenherr, C. J. and Anderson, D. J. (1995). The neuron-restrictive silencer factor (NRSF): a coordinate repressor of multiple neuron-specific genes. Science 267: 1360-1363. PubMed Citation: 7871435

Stofanko, M., Kwon, S. Y. and Badenhorst, P. (2010). Lineage tracing of lamellocytes demonstrates Drosophila macrophage plasticity. PLoS One 5(11): e14051. PubMed Citation: 21124962

Tsuda, L., et al. (2006). An NRSF/REST-like repressor downstream of Ebi/SMRTER/Su(H) regulates eye development in Drosophila. EMBO J. 25(13): 3191-202. 16763555

Westbrook, T. F., et al. (2005). A genetic screen for candidate tumor suppressors identifies REST. Cell 121: 837-848. PubMed Citation: 15960972

Yi, C. H., et al. (2007). A genome-wide RNAi screen reveals multiple regulators of caspase activation. J. Cell Biol. 179: 619-626. PubMed Citation: 17998402

date revised: 28 December 2011

Home page: The Interactive Fly © 2017 Thomas Brody, Ph.D.