Interactive Fly, Drosophila

Recent advances have shed new light on how the Q50 homeoproteins act in Drosophila. Q50 homeoproteins all contain a glutamine residue at position 50 of the homeodomain. These transcription factors, encoded by the segmentation genes even-skipped, fushi-tarazu and engrailed, have remarkably similar and promiscuous DNA-binding specificities in vitro, yet they each specify distinct developmental fates in vivo. One current model suggests that because the Q50 homeoproteins have distinct biological functions, they must each regulate different target genes. According to this 'co-selective binding' model, significant binding of Q50 homeoproteins to functional DNA elements in vivo would be dependent upon cooperative interactions with other transcription factors (cofactors). If the Q50 homeoproteins each interact differently with cofactors, they could be selectively targeted to unique, limited subsets of their in vitro recognition sites and thus control different genes. Thus cofactors would selectively target different Q50 homeoproteins to bind to different DNA sites. However, a variety of experiments question this model. Molecular and genetic experiments suggest that the Q50 homeoproteins do not regulate very distinct sets of genes. Instead, they mostly control the expression of a large number of shared targets. The distinct morphogenic properties of the various Q50 homeoproteins may result principally from the different manners in which they either activate or repress these common targets. Further, in vivo binding studies indicate that at least two Q50 homeoproteins, Eve and Ftz, have very broad and similar DNA-binding specificities in embryos, a result that is inconsistent with the 'co-selective binding' model. Based on these and other data, it is suggested that Q50 homeoproteins bind many of their recognition sites without the aid of cofactors. In this 'widespread binding' model, cofactors act mainly by helping to distinguish the way in which homeoproteins regulate targets to which they are already bound (Biggin, 1997).

Engrailed regulation of segment polarity genes

The Drosophila wing is formed by two cell populations, the anterior and posterior compartments, distinguished by the activity of the selector gene en in posterior cells. en governs growth and patterning in both compartments by controlling the expression of the secreted proteins Hedgehog and Decapentaplegic. en activity programs wing cells to express hh , whereas the absence of en activity programs wing cells to respond to HH by expressing dpp. As a consequence, posterior cells secrete HH and induce a stripe of neighboring anterior cells across the compartment boundary to secrete DPP (Zecca, 1995).

hedgehog expression is a target of engrailed regulation. hh expression in epidermal cells is confined to the anterior parasegmental compartments and coincides precisely with that of engrailed . Despite similar patterns of expression in the cellular blastoderm, early hh expression seems to be independent of en, becomes sensitive to and dependent on en later during the extended germ band stage (Tabata, 1992).

en has a dual role: a general one for patterning of the wing, achieved through the activation of secreted proteins like HH, and indirectly DPP, and a more specific role, determining posterior identity, in which the inv gene is implicated. Engrailed, along with Hedgehog, regulates invected. engrailed expression has been targeted to different regions of the wing disc. In the anterior compartment, ectopic en expression gives rise to the substitution of anterior structures by posterior ones, thus demonstrating its role in specification of posterior patterns. The en-expressing cells in the anterior compartment also induce high levels of Hedgehog andDecapentaplegic gene products, which results in local duplication of anterior patterns. When ectopically expressed in the anterior compartment, hh is also able to activate en and invected. In the posterior compartment, elevated EN result in partial inactivation of endogenous en and inv, indicating the existence of a negative autoregulatory mechanism. (Guillen, 1995).

In posterior compartments, Engrailed and Invected repress anterior segment specific proteins Decapentaplegic and Patched. This serves to maintain the integrity of the posterior compartment. Mutant clones completely lacking both en and invected activity ectopically express dpp in the posterior compartment, where dpp activity ordinarily is repressed. Similarly, patched (ptc) is also ectopically expressed in such posterior compartment en-inv- null clones. These en-inv- clones also exhibit loss of hedgehog expression. Absence of dpp expression in the posterior compartment is due to direct repression by EN. Ubiquitious expression of en in imaginal disks eliminates the expression of dpp- in its normal A/P boundary stripe. There are three Engrailed binding sites in a dpp-lacZ reporter gene. Mutagenesis of these Engrailed binding sites results in ectopic expression of this reporter gene, but does not alter the normal stripe of expression at the A/P boundary. Thus an en-hh-ptc regulatory loop used in the embryo is reutilized in imaginal disks to create a stripe of dpp expression along the A/P compartment boundary (Sanicola, 1995).

Engrailed and Cubitus interruptus regulate patched. Early ubiquitous expression of patched is followed by its repression in the anterior portion of each parasegment; subsequently each broad band of expression splits into two narrow stripes. The first step in patched regulation is under the control of en whereas the second requires the activity of both cubitus interruptus and patched itself. Furthermore, the products of en, wingless and hedgehog are essential for maintaining the normal pattern of patched expression (Hidalgo, 1990).

deformed, cubitus interruptus, (ci) and engrailed itself are all targets of Engrailed. Engrailed is involved in an auto-regulatory loop in posterior compartments, and both deformed and cubutis interruptus are limited to anterior compartments by engrailed function in adjacent posterior compartments. Engrailed binding sites have been found in the promoters of both engrailed and ci (Saenz-Robles, 1995).

cubitus interruptus is expressed in all anterior segmental compartment cells in embryos and imaginal discs. Separate elements regulate its expression in embryos and imaginal discs. Mutants that delete a portion of this regulatory region express ci ectopically in the posterior compartments of wing imaginal disks and have wings with malformed posterior compartments. Evidence that the Engrailed protein normally represses ci in posterior compartments includes the expansion of ci expression into posterior compartment cells that lack engrailed function, diminution of ci expression upon overexpression of Engrailed protein in anterior compartment cells, and the ability of Engrailed protein to bind to the ci regulatory region in vivo and in vitro (Schwartz, 1995).

A dominant interaction between combgap and engrailed/invected mutations that gives rise to a gap in vein L4 strongly suggests that Cg and En/Inv act together to repress posterior cubitus interruptus transcription. Posterior expression of En represses the transcription of ci resulting in anterior specific expression. En has been shown to interact directly with the ci regulatory elements. In cg mutant wing imaginal discs, weak ectopic expression of ci-lacZ reporter constructs are found in posterior cells, thus Cg may act in concert with En to repress posterior ci. Hypomorphic mutants in either cg or en/inv can give rise to the reduction in vein L4 that is characteristic of ectopic ci expression (Svendsen, 2000).

Many proteins with multiple C2H2 zinc finger motifs like those found in cg have been shown to be transcription factors, DNA-binding proteins or chromatin proteins. The widespread localization of Cg on salivary gland chromosomes is consistent with all of these activities. While the data have not yet established direct action of Cg on the ci regulatory elements, binding of Cg to the ci region of polytene chromosomes suggests that Cg could be a direct regulator of ci transcription. Direct binding of Cg (produced in E. coli) to DNA from the ci regulatory region has not been detected. However, given that the transcriptional regulation of ci is likely to be complex, Cg may not act at the level of direct DNA binding. The involvement of the Pc-group genes in the repression of ci suggests that intricate regulatory modes are necessary to maintain the correct levels and spatial patterns of ci transcription during imaginal disc development. Furthermore, the ci-regulatory regions have been shown to be subject to transvection effects, indicating that interchromosomal interactions also govern ci regulation. Thus Cg may act at any level, from generally influencing the chromosome pairing through to direct binding of ci enhancer elements. Finally, the positive and negative effects of cg mutants on ci transcription and the genetic interaction with en/inv suggest that Cg may be required in conjunction with other transcription factors for the function of ci enhancers and that Cg may not specify activation or repression itself (Svendsen, 2000).

The regulation and function of the Hedgehog pathway activity has been compared in eye and wing discs, and there are significant differences. Whereas in the wing disc, engrailed function is required for hedgehog expression, in the eye disc activation and maintenance of hedgehog expression is achieved independently of engrailed. Nevertheless, engrailed functions in the eye disc, as elevated engrailed expression represses dpp, patched and cubitus interruptus in the eye disc, but does not disrupt morphogenesis. Regulation of decapentaplegic expression also differs: in the wing disc it is repressed in the anterior compartment by patched and in the posterior compartment by engrailed. In the eye disc, however, it is repressed posterior to the morphogenetic furrow in the absence of either patched or engrailed activity (Strutt, 1996).

Analysis of the expression of 18 wheeler in different mutant backgrounds shows that it is under control of segment polarity and homeotic genes. Initial accumulation of 18w is normal in wingless mutants. However, by full germband extension, the ventrolateral expression of 18wis narrower than in wild type. These changes appear well before cell death is seen in wg mutants. In patched mutants, the domains of wg and of 18w expand to include the expression domains of wingless and engrailed. These results suggest that wg and en positively regulate 18w expression within the ventromedial stripes (Eldon, 1994).

Genetic analysis shows that Engrailed has both negative and positive targets. Negative regulation is expected from a factor that has a well-defined repressor domain but activation is harder to comprehend. VP16En, a form of En that has its repressor domain replaced by the activation domain of VP16, has been used to show that En activates targets using two parallel routes, by repressing a repressor and by being a bona fide activator. The intermediate repressor activity has been identified as being encoded by sloppy paired 1 and 2 and bona fide activation is dramatically enhanced by Wingless signaling. Thus, En is a bifunctional transcription factor and the recruitment of additional cofactors presumably specifies which function prevails on an individual promoter. Extradenticle (Exd) is a cofactor thought to be required for activation by Hox proteins. However, in thoracic segments, Exd is required for repression (as well as activation) by En. This is consistent with in vitro results showing that Exd is involved in recognition of positive and negative targets. Moreover, genetic evidence is provided that, in abdominal segments, Ubx and Abd-A, two homeotic proteins not previously thought to participate in the segmentation cascade, are also involved in the repression of target genes by En. It is suggested that, like Exd, Ubx and Abd-A could help En recognize target genes or activate the expression of factors that do so (Alexandre, 2003).

slp1 and slp2 are repressed by En and their products repress en expression. Importantly, Slp1 and Slp2 are the only dominant repressors that stand between En and its positive targets, hh and en -- at least in the paired-Gal4 domain. If another such repressor existed, it would prevent VP16En from activating the expression of hh (or en) in a slp mutant. Expression of slp at the anterior, and of en at the posterior, of prospective parasegment boundaries is initiated by the activity of pair-rule genes. Mutual transcriptional repression ensures that neither factor can subsequently 'invade' the other's domain of expression after pair-rule genes have ceased to function and when cell communication starts to dominate segmental patterning and thus contributes to the stability of parasegment boundaries. Note that slp is expressed only at the anterior of each stripe of en expression (not at the posterior). It may be that no analogous repressive function is needed at the posterior because the Wg pathway, which contributes to activation by En, is not active there. Indeed, in otherwise wild-type embryos, ectopic activation of Wg signaling is sufficient to cause posterior expansion of en stripes (Alexandre, 2003).

The key evidence for En being a bona fide activator is that, in the absence of slp, both En and VP16En activate hh transcription. Either En activates hh directly or it activates an intermediate activator of hh transcription. Either way, it is suggested that En must be capable of transcriptional activation (in addition to repression). Note that in otherwise wild-type embryos, VP16En formally represses the expression of hh and en. This led initially to the belief that wild-type En acts solely via an intermediate repressor since no positive effect of VP16En on the expression of en or hh could be observed. As is know now, however, this was masked by the presence of Slp. It was therefore essential to identify the intermediate repressor and assess the effect of removing its activity in order to infer the true activation function of En (Alexandre, 2003).

Wg signaling contributes to the activation of En's positive targets. The temporal aspect of this requirement has not been investigated, but earlier results suggest that it is probably transient. Note that Wg signaling is irrelevant to repression by En and that, even in cells that are within the range of Wg, repression and activation (of distinct targets) coexist. For example, in the normal domain of en expression, ci is repressed and hh is activated. Therefore, Wg signaling does not convert En from an activator to a repressor. Perhaps Wg signaling helps the recruitment, on specific targets, of a cofactor needed to mask the repressor domain of En, while at the same time providing an activation domain. One candidate cofactor that could be regulated by Wg is the homeodomain protein encoded by exd, a known cofactor of Hox gene activity in vivo. However, Exd is not an activation-specific cofactor and more work is therefore needed to understand how Wg signaling contributes to the activating function of En (Alexandre, 2003).

Two types of activities have been ascribed to Exd. According to the selective binding model, Exd could help En recognize positive targets and assemble a transcription complex. Alternatively, or in addition, Exd could mask the repressor domain of En and, at the same time, recruit an activator (the so-called activity regulation model). Adding a functional activation domain to En (as in VP16En) does not override the need for Exd. This gives in vivo support to the selective binding model and is consistent with in vitro studies, which have shown that Exd and En can dimerize and bind DNA cooperatively. Cooperativity requires the eh2 domain of En, a domain that is left intact in VP16En. Because VP16En requires Exd for in vivo activity, it is concluded that the N-terminal half of En, which is absent in VP16En, is not required for the interaction with Exd (Alexandre, 2003).

In thoracic segments, VP16En requires exd to act on all En targets, positive and negative. This is the first indication that Exd could be involved in negative (as well as positive) target recognition by En. Indeed in thoracic segments wild-type En requires Exd for repression of its natural targets. This had presumably not been noticed previously because endogenous expression of En is lost in the absence of Exd. That Exd could be involved in repression is consistent with in vitro studies with PBX proteins and earlier suggestions from in vivo work with Drosophila. Because Exd is required for both repression and activation, the issue of what distinguishes activated targets from repressed ones remains unresolved. Throughout the present study, it has been found that the two En-positive targets, en and hh, are expressed identically in a variety of experimental conditions. It may therefore be that the regulatory regions of these two genes might contain unique features that make them positive targets (Alexandre, 2003).

En must be capable of activating transcription in the appropriate context. Because En harbors a robust repressor domain, it is likely that one or several cofactor(s) mask this domain and recruit an activation function and, it is unlikely that Exd alone provides such an activity. Nevertheless, the possible role of Hth is worth discussing. In vitro, Hth binds DNA as a part of a ternary complex with Exd and a Hox protein. Intriguingly, overexpression of an activator form of Hth (VP16Hth) phenocopies the overexpression of wild-type Hth (VP16Hth mimics overactive Hth). This suggests that the normal role of Hth is to bring an activation domain to a complex -- a conclusion that contradicts the observation that Hth is required for both repression and activation by En. One way to resolve this paradox would be to suggest that Hth has two distinct roles: to help target recognition on negative and positive targets and, in addition, to bring an activation domain onto positive targets. Of course activation by En could also involve as yet unidentified activating cofactors. Further progress will require the identification, within natural targets, of enhancers that confer either activation or repression. Comparing these sites and subsequent mutational and biochemical analysis could lead to a molecular understanding of what distinguishes negative from positive targets (Alexandre, 2003).

The most unexpected aspect of these results is that, in abdominal segments, the Hox proteins Ubx and Abd-A are involved in repression by En. In formal genetic assays, Ubx and Abd-A can substitute for Exd in helping En act on negative targets. In the absence of Ubx, Abd-A and Exd, En can no longer repress target genes. By contrast, two other Hox proteins (Antp and Abd-B) appear not to be involved in En function. Antp does not help En repress targets in vivo even though its homeodomain differs from that of Abd-A at only five positions. Likewise, Abd-B, a more distantly related Hox protein, is also unlikely to participate in En function. It is concluded that the role of Ubx and Abd-A in repression by En is specific (Alexandre, 2003).

How could ectopic Ubx or Abd-A allow En to repress targets in the absence of Exd? It could be that this is mediated by wholesale transformation of segmental identity [although such transformation would have to be exd/hth-independent. Alternatively, Ubx and Abd-A could have a more immediate involvement in En function. One can envisage that they could regulate an as yet unidentified corepressor of En (although such regulation would not require Exd). Alternatively, and more speculatively, Ubx and Abd-A could serve as cofactors themselves in regions of the embryo where Exd levels are low. Again, molecular analysis of negative targets will be needed to discriminate these possibilities (Alexandre, 2003).

Homeotic genes have not been previously implicated in En function despite many years of genetic analysis of the Bithorax complex. It is suggested that the role of Ubx and Abd-A in En function has been overlooked previously because, in the absence of these two genes, Exd is upregulated in the presumptive abdomen and thus takes over as a repression cofactor. However, the present results establish that homeotic genes do participate in the segmentation cascade and link two regulatory networks previously thought to be independent (Alexandre, 2003).

Engrailed regulation of homeotic genes

Ubx is shown to be down-regulated by Engrailed in the posterior compartment of parasegment 6. In the posterior compartment, Dll is normally expressed in a small cluster of cells. If Ubx is expressed uniformly, Dll is inappropriately repressed in these posterior compartment cells. In the anterior compartment of parasegment 6, Dll is normally repressed by high levels of Ubx expression. However, if en is expressed uniformly, Ubx is repressed and Dll is derepressed. Because Dll is required for the development of larval sensory structures, these results demonstrate that EN-mediated repression of Ubx in the posterior compartment is necessary for the morphology of parasegment 6 (Mann, 1994).

fushi tarazu en in particular, appear to act as transcriptional activating factors of abdominal-A. abd-A is normally expressed in parasegments 7 to 13. The initial distribution of the product is approximately uniform within this domain, but the subsequent elaboration of the expression pattern results in differences between, as well as within, parasegments. A recent study investigated the possible role of several pair-rule genes ( fushi tarazu, even-skipped, runt, hairy, paired,) and segment polarity genes (en, wingless, naked, patched and cubitus interruptus) on the patterning of abd-A expression (Macias, 1994). It concluded that the establishment of the original abd-A expression domain was independent of any of these genes, but most of them are required for the subsequent elaboration of abd-A expression within the domain (Macias, 1994).

Engrailed and neuroblast specification

How is neuroblast-specific gene expression established? This paper's focus was on the huckebein gene, because it is expressed in a subset of neuroblasts and is required for aspects of neuronal and glial determination. hkb is required within the neuroblast 1-1, 2-2 and 4-2 lineages for proper axon pathfinding of interneurons and motoneurons and for proper muscle target recognition by motoneurons. The secreted Wingless and Hedgehog proteins activate huckebein expression in distinct but overlapping clusters of neuroectodermal cells and neuroblasts, whereas the nuclear Engrailed and Gooseberry proteins repress huckebein expression in specific regions of neuroectoderm or neuroblasts. Hedgehog activates hkb in cells that give rise to the 5HT expressing lineage), while Wingless activates hkb in cells that give rise to an eve expressing motorneuron lineage). Wingless and Hedgehog activate hkb in the neuroectoderm of hemisegment row 5 neuroblast precursors. Early-forming neuroblasts of rows 5 and 6 never express hkb even though they develop from Hkb+ neuroectoderm (row 5). Gooseberry functions to repress hkb expression in row 5 neuroblasts while Engrailed represses hkb expression in rows 6/7 neuroectoderm. Integration of these activation and repression inputs is required to establish the precise neuroectodermal pattern of huckebein, which is subsequently required for the development of specific neuroblast cell lineages (McDonald, 1997).

The mechanisms leading to the specification and differentiation of ventral nerve cord neuroblast lineages in Drosophila are largely unknown. Mechanisms that lead to cell differentiation within the NB 7-3 lineage have been analyzed. Analogous to the grasshopper, NB 7-3 is the progenitor of the Drosophila serotonergic neurons. The zinc finger protein Eagle (Eg) is expressed in NB 7-3 just after delamination and is present in all NB 7-3 progeny until late stage 17. eagle is required for normal pathfinding of interneuronal projections and for restricting the cell number in the thoracic NB 7-3 lineage. eg is required for serotonin expression. Ectopic expression of Eg protein forces specific additional CNS cells to enter the serotonergic differentiation pathway. Like NB 7-3, the progenitor(s) of these ectopic cells express Huckebein (Hkb), another zinc finger protein. However, and in contrast to the NB 7-3 lineage, where en acts upstream of eg, the ectopic progeny do not express engrailed. It is concluded that eg and hkb act in concert to determine serotonergic cell fate, while en is more distantly involved in this process by activating eg expression. This is the first functional evidence for a combinatorial code of transcription factors acting early but downstream of segment polarity genes to specify a unique neuronal cell fate (Dittrich, 1997).

A variety of factors could influence how far developmental signals spread. For example, the Patched receptor limits the range of its ligand Hedgehog. Somehow, the Frizzled2 receptor has the opposite effect on its ligand. Increasing the level of Frizzled2 stabilizes Wingless and thus extends the Wingless gradient in Drosophila wing imaginal disks. Here it is asked whether Frizzled or Frizzled2 affects the spread of Wingless in Drosophila embryos. In the embryonic epidermis, the combined expression of both receptors is lowest in the engrailed domain. This is because expression of Frizzled is repressed by the Engrailed transcription factor, whereas that of Frizzled2 is repressed by Wingless signaling. Receptor downregulation correlates with an early asymmetry in Wingless distribution, characterized by the loss of Wingless staining in the engrailed domain. Raising the expression of either Frizzled or Frizzled2 in this domain prevents the early disappearance of Wingless-containing vesicles. Apparently, Wingless is captured, stabilized, and quickly internalized by either receptor. As far as is possible to tell, captured Wingless is not passed on to further cells and does not contribute to the spread of Wingless. Receptor downregulation in the posterior compartment may contribute to dampening the signal at the time when cuticular fates are specified (Lecourtois, 2001).

Both Frizzled and Frizzled2 proteins are expressed in a dynamic fashion during the first 12 h of development. In particular, the level of Frizzled is down in the engrailed domain and Frizzled2 is relatively less abundant in the apparent domain of Wingless action. The patterns of transcription around Stages 8 and 11 (3.5-7 h AEL) were studied. Although frizzled expression is initially uniform during gastrulation, it begins to resolve into a periodic pattern by Stage 9 (4 h AEL). Double staining shows that, at Stage 10 (4.5-5 h AEL), frizzled transcripts are abundant in all cells except those that express engrailed. Expression of frizzled2 also becomes segmental around Stage 9, a pattern that is clearly marked at Stage 10: broad stripes of frizzled2 expression are detected at the posterior of each engrailed stripe. Thus, at Stage 10 (4.5-5 h AEL), combined expression of frizzled and frizzled2 is lowest in engrailed-expressing cells, especially those nearest to the source of Wingless. Note, however, that residual mRNA remains, possibly as a result of maternal contribution or low-level zygotic transcription. In fact, intensive studies support the view that Engrailed directly represses frizzled (Lecourtois, 2001).

At Stage 10 of Drosophila embryogenesis, the amount of detectable Wingless decreases within the engrailed domain. This corresponds to the time when both frizzled and frizzled2 are transcriptionally downregulated there. Artificially increasing the expression of frizzled or frizzled2 prevents the early loss of Wingless staining; binding of Wingless to its receptors may render it inaccessible to extracellular proteases. This suggests that, in the wild type, transcriptional downregulation of the receptors causes the early loss of Wingless immunostaining. Two distinct mechanisms repress the transcription of frizzled and frizzled2: Engrailed itself appears to repress frizzled, whereas Wingless signaling represses frizzled2. Repression of frizzled expression by Engrailed is not seen in imaginal disks where, presumably, a cofactor is missing. In contrast, repression of frizzled2 by Wingless signaling appears to be a general feature. As a result of two distinct repression mechanisms, the combined expression of frizzled and frizzled2 is lowest in the engrailed cells, especially those nearest to the source of Wingless. Nevertheless, residual activity must remain because engrailed-expressing cells respond to Wingless as late as 8.5 h AEL, whereas the complete absence of frizzled and frizzled2 activity phenocopies a wingless null mutation (Lecourtois, 2001).

The results suggest that downregulation of the Frizzled receptors reduce the spread of Wingless into the posterior compartment, not by affecting its transport but rather by reducing its stability. This would lead to a reduced number of effective receptor-ligand complexes and hence dampened signaling. This is thought to commence during Stage 10. Transcriptional repression of receptor expression has been shown to contribute to dampening of signaling in other instances. Additional strategies such as desensitization are also at work. Likewise, additional mechanisms for dampening Wingless signaling are likely to exist. Indeed, after Stage 11, residual Wingless/receptor complexes are rapidly degraded (and hence rendered ineffective) in prospective denticle-secreting cells. This targeted degradation of Wingless can account for the fact that row 1 denticles still form in embryos that massively express frizzled or frizzled2. Both mechanisms of signal downregulation (repression of receptor transcription and degradation of receptor/ligand complexes) dampen the action of Wingless toward the posterior, although more work is needed to assess their relative importance. Another outstanding issue is whether Frizzled and Frizzled2 are equivalent with respect to signal downregulation. Clearly, these receptors differ in terms of affinity for the ligand. It may also be that differences in intracellular trafficking lead to distinct effects on Wingless signal downregulation (Lecourtois, 2001).

Engrailed regulation of polyhomeotic

Continued: Engrailed Targets of activity part 2/2

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