Gene name - charlatan
Cytological map position - 51E1--2
Function - transcription factor
Keywords - PNS, neurogenesis, macrochaeta
Symbol - chn
FlyBase ID: FBgn0015371
Genetic map position - 2R
Classification - C2H2 zinc finger
Cellular location - nuclear
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. chnECJ1 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).
date revised: 30 April 2005
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