Targets of Activity
(part 1/2)

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).

Role of en and novel interactions between msh, ind, and vnd in dorsoventral patterning of the Drosophila brain and ventral nerve cord

Subdivision of the neuroectoderm into discrete gene expression domains is essential for the correct specification of neural stem cells (neuroblasts) during central nervous system development. This study extends knowledge on dorsoventral (DV) patterning of the Drosophila embryonic brain and uncovers novel genetic interactions that control expression of the evolutionary conserved homeobox genes ventral nervous system defective (vnd), intermediate neuroblasts defective (ind), and muscle segment homeobox (msh). Cross-repression between Ind and Msh was shown to stabilize the border between intermediate and dorsal tritocerebrum and deutocerebrum, and both transcription factors are competent to inhibit vnd expression. Conversely, Vnd segment-specifically affects ind expression; it represses ind in the tritocerebrum but positively regulates ind in the deutocerebrum by suppressing Msh. These data provide further evidence that in the brain, in contrast to the trunk, the precise boundaries between DV gene expression domains are largely established through mutual inhibition. Moreover, it was found that the segment-polarity gene engrailed (en) regulates the expression of vnd, ind, and msh in a segment-specific manner. En represses msh and ind but maintains vnd expression in the deutocerebrum, is required for down-regulation of Msh in the tritocerebrum to allow activation of ind, and is necessary for maintenance of Ind in truncal segments. These results indicate that input from the anteroposterior patterning system is needed for the spatially restricted expression of DV genes in the brain and ventral nerve cord (Seibert, 2010)

The spatial and temporal order in which the DV genes (vnd, ind, and msh) are activated in neuromeres of the brain differs from their appearance in the trunk neuroectoderm, and those differences seem to be basic for the segment-specific regulation of vnd, ind, and msh expression. In the early trito- and deutocerebrum, Vnd is expressed not only in the ventral but also in the intermediate neuroectoderm, where cross-repression between Vnd and dorsally expressed Msh establishes the border between intermediate and dorsal neuroectoderm. Since Msh was found to be an ind repressor, the repression of msh via Vnd is a prerequisite for ind to become activated in the intermediate tritocerebrum (anterior) and deutocerebrum. In the trunk, ind expression in the intermediate neuroectoderm starts before that of msh in the dorsal neuroectoderm, and msh and vnd domains do not abut; accordingly, repressive interaction between Msh and Vnd is not required (Seibert, 2010)

In the tritocerebrum, Vnd not only acts as repressor of msh but also of ind, in contrast to the deutocerebrum. When the level of Vnd protein in the intermediate tritocerebrum declines with time (down-regulated through the activity of Ems), ind becomes subsequently activated. In the trito- and deutocerebrum, instead of Vnd, increasing levels of Ind, together with the recently uncovered msh-repressor Nkx6, still keep msh expression limited to the dorsal neuroectoderm. Since it was found that Nkx6 expression starts earlier and persists longer than that of ind in both brain neuromeres, and additionally, that msh is expanded into the intermediate neuroectoderm in Nkx6 but not in ind mutants, it is proposes that Nkx6 represses msh more efficiently (Seibert, 2010)

The most striking difference in DV gene regulation leads to the question how vnd and ind can be co-expressed in the anterior deutocerebrum (during stages 6–9), if Vnd is a repressor of ind and, vice versa, Ind is also capable of preventing vnd expression in the neuroectoderm. It has been reported recently that the repressor activity of Ind on vnd seems to be stage-specific, not taking place before stage 9. By contrast, Vnd repression of ind seems independent of the developmental period. In this context, it is interesting that activity of Vnd can be modified by EGFR signalling, which is supposed to affect the selective interaction of Vnd with co-factors necessary to mediate repression or activation of target genes. Availability of co-factors might also account for the specific situation of vnd and ind co-expression in the anterior deutocerebrum that was observed specifically during early stages of development (Seibert, 2010)

Involvement of en, which can act as transcriptional repressor as well as activator, has been implicated in diverse developmental processes in Drosophila such as compartmentalization in the early embryo, modulation of Hox gene expression, or regulation of molecules that directly govern axon growth (e.g. frazzled). This study demonstrates a novel function for En in the early embryo, that is to control the spatially restricted expression of the DV genes in the neuromeres of the posterior brain (trito- and deutocerebrum) and ventral nerve cord. In the posterior compartment of the deutocerebrum, En represses expression of msh and ind, but maintains expression of vnd. Since it was found that Ind (later) becomes a vnd repressor, this indicates that En maintains expression of vnd by repressing ind. In the posterior compartment of the tritocerebrum, En is also required for down-regulation of Msh, but opposite to the deutocerebrum, En is necessary for activation of ind. This study shows that Msh is an ind repressor, its repression by En seems to allow for activation of ind; yet, it cannot be excluded that En in addition directly activates ind expression. Similar to the situation in the tritocerebrum, En seems to negatively regulate expression of msh and to positively regulate expression of ind (as a maintenance factor) in the neuroectoderm of the ventral nerve cord. Together, these data suggest that the AP patterning gene engrailed is crucially involved in fine-tuning the regionalized expression of distinct DV genes in the posterior compartment of neuromeres in the brain and ventral nerve cord. En may act as a positive or negative transcriptional regulator depending on the gene that is regulated and on the segmental context. For DV genes it is known that they control formation and specification of brain neuroblasts. Since all the genetic interactions between En and DV genes take place during the period when neuroblasts develop, it is likely that En, via regulation of DV genes, controls formation and fate specification of neuroblasts in the brain (Seibert, 2010)

It was observed that cross-repressive interaction between pairs of DV gene factors in the brain (i.e. in trito- and deutocerebrum) is essential for the establishment and maintenance of discrete DV gene expression domains. Early, cross-repression between Ems/Vnd pre-patterns the ventral and intermediate neuroectoderm in both neuromeres. Mutual repression between Msh/Nkx6 and Msh/Ind maintains the dorsal/intermediate neuroectodermal border in trito- and deutocerebrum, and between Ind/Vnd the intermediate/ventral border in the tritocerebrum. All these genetic interactions, and the observation that Msh and Vnd act as mutual repressors, are not in compliance with the concept of ventral dominance (as proposed in the neuroectoderm of the ventral nerve cord where the more ventral gene represses the gene expressed more dorsally) but rather support the model that in the brain cross-repression between DV factors is crucial for stabilizing these borders (Seibert, 2010)

However, despite the ability of Msh and Ind to repress vnd, neither factor seems to be sufficient to define the dorsal border of vnd expression in trito- and deutocerebrum, as has been shown for Ind in the ventral nerve cord (from stage 9 onwards). Instead of reinforcing this border through repressive interaction, vnd expression in the brain could also be limited by a (too) low concentration or absence of an activator, like Dorsal (as has been speculated for the trunk), or be regulated by BMP signalling in a dosage-dependent fashion. Neuromere-specific differences are also observed regarding limitation of ind and msh expression domains along the DV axis. Vnd establishes the ventral border of ind expression in the trunk and tritocerebrum, but not in the deutocerebrum or protocerebrum (where the expression domains of ind and vnd do not abut). ind expression was found to be limited dorsally by repression through Msh in the trito- and deutocerebrum, but not in the protocerebrum (where msh is not expressed before stage 11) or trunk, although evidence is available that Msh might act in rendering the dorsal border of ind expression more precisely in the ventral nerve cord. Taking into account that ind expression does not expand into the complete dorsal neuroectoderm of trito- and deutocerebrum in msh mutants, this may also indicate an involvement of the nuclear Dorsal gradient, possibly in concert with graded activity of EGFR (as was shown for the trunk neuroectoderm), or BMP (which can repress ind in the trunk neuroectoderm), in establishing a rough dorsal border of ind expression that is further defined and stabilized via repression by Msh. Whereas Vnd is initially responsible for keeping msh expression confined to the dorsal neuroectoderm in trito- and deutocerebrum, it is only indirectly involved in defining the ventral border of msh expression in the trunk neuroectoderm. Later in development Ind helps to maintain repression of msh in trito- and deutocerebrum (together with Nkx6), which is in contrast to the trunk where Ind directly establishes the ventral limit of msh expression from the beginning (Seibert, 2010)

DV neuroectodermal and corresponding stem cell domains in the Drosophila brain become established and maintained through cross-repressive regulation, and it has been speculated that such genetic interactions are more common in the fly brain. This study has presented further examples supporting this hypothesis. Notably, this is a feature that bears similarity to DV patterning in the neural tube of vertebrates where cross-repressive interactions of homeodomain proteins are common and indeed crucial for the establishment of discrete DV progenitor domains (Seibert, 2010)

All interactions between DV genes in the brain identified so far are based on the interplay of transcriptional repressors. Likewise, this study shows that Vnd does not act as a direct activator to positively regulate ind, but according to a double-negative mechanism, it suppresses the ind-repressor Msh. It has been shown previously, that interactions of the AP patterning gene ems with the DV genes (vnd, ind, msh, and Nkx6) are indispensable for proper development of the trito- and deutocerebrum. This study demonstrates that the segmentation gene en is significantly involved in regionalization of DV gene expression domains, thus representing a further example of an AP patterning gene integrating into the DV gene regulatory network that patterns the brain. This study has shown that En acts differently on the respective DV genes, but no evidence is available that, vice versa, DV genes control en, as has been observed for expression of ems. DV genes, as well as En and Ems, all contain an Eh1 repressor domain and are able to interact with the co-repressor Groucho (Gro), and thus are capable of mediating repression on target genes (including each other). But how could it be possible that all DV genes interact with the same co-factor to stabilize expression domains by conferring repression onto genes expressed in neighboring domains? In the first place, the DV genes display spatio-temporal differences in their respective expression. In addition, conformational changes of the protein seem to be necessary to enable binding of Gro which has been at least shown for Nkx6. It has been observed that Vnd can be phosphorylated by activated MAPK and is present in different isoforms in the developing embryo, which most likely leads to a change in its binding partners. Another critical point could be inactivation of the co-repressor, in case of Gro also through phosphorylation by activated MAPK, or modification of target genes so that binding of the repressor complex is impaired. Still, that the DV genes are able to interact with Groucho, does not exclude that their repressor activity is Gro-independent, since also other repressor domains have been reported for these genes, as well as activator domains, at least for Vnd, Ind, and Nkx6. Whether the DV gene products function as repressors or activators seems to depend on co-factor availability as well as on the respective target gene, since not only the presence of a transcriptional binding site, but also its accessibility is limiting in this context (Seibert, 2010)

Engrailed cooperates directly with Extradenticle and Homothorax on a distinct class of homeodomain binding sites to repress sloppy paired

Even skipped (Eve) and Engrailed (En) are homeodomain-containing transcriptional repressors with similar DNA binding specificities that are sequentially expressed in Drosophila embryos. The sloppy-paired (slp) locus is a target of repression by both Eve and En. At blastoderm, Eve is expressed in 7 stripes that restrict the posterior border of slp stripes, allowing engrailed (en) gene expression to be initiated in odd-numbered parasegments. En, in turn, prevents expansion of slp stripes after Eve is turned off. Prior studies showed that the two tandem slp transcription units are regulated by cis-regulatory modules (CRMs) with activities that overlap in space and time. An array of CRMs that generate 7 stripes at blastoderm, and later 14 stripes, surround slp1. Surprisingly given their similarity in DNA binding specificity and function, responsiveness to ectopic Eve and En indicates that most of their direct target sites are either in distinct CRMs, or in different parts of coregulated CRMs. Cooperative binding sites for En, with the homeodomain-containing Hox cofactors Extradenticle (Exd) and Homothorax (Hth), were located within two CRMs that drive similar expression patterns. Functional analysis revealed two distinct, redundant sites within one CRM. The other CRM contains a single cooperative site that is both necessary and sufficient for repression in the en domain. Correlating in vivo and in vitro analysis suggests that cooperativity with Exd and Hth is a key ingredient in the mechanism of En-dependent repression, and that apparent affinity in vitro is an unreliable predictor of in vivo function (Fujioka, 2012).

Consistent with the fact that Eve is expressed earlier than En, with some overlap at embryonic stages 8–9, slp CRMs tended to respond to ectopically expressed Eve at earlier stages than to En. Transgenic dissections further showed that they have distinct responsive regions within CRMs, suggesting that many of their binding sites are distinct. This is somewhat surprising because they are both homeodomain-containing repressors that set the posterior borders of slp stripes, and they have been seen to have similar in vitro binding specificities. A possible explanation is that they cooperate in DNA binding with different cofactors, making their functional sites distinct. Despite detailed analyses of Eve function in segmentation, no candidate co-factors for specifying target genes have emerged (Fujioka, 2012).

A recent study showed that CRM u8172 drives ectopic expression within odd-numbered parasegments in cells that normally do not express detectable levels of slp RNA. However, when combined with the promoter-proximal CRM u3125, which drives properly restricted expression within even-numbered parasegments, ectopic expression is repressed, suggesting that an Eve-responsive element resides within this region. Consistent with these findings, transgenes containing this region responded to ectopically expressed Eve, and rescue-type transgenes carrying u8172 without this region drove ectopic Slp, causing embryonic defects (Fujioka, 2012).

Recently, a striking number of distinct CRMs surrounding the slp1 transcription unit were found to drive expression that overlaps in both space and time. Extensive dissection of this regulatory region and rescue of slp mutants with various transgenes suggested that apparent redundancy may be necessary to provide fully functional levels of expression across the various stages of slp expression. This study shows that there are functionally redundant En/Exd/Hth binding sites within CRM u1523. In vitro binding analysis identified a strongly cooperative binding site and a weaker, but still highly cooperative site. Despite the apparent difference in in vitro binding affinity, either site is sufficient to confer repression in the En domain, and both sites must be mutated to cause significant derepression. Thus, apparent redundancy exists at multiple levels in slp regulation. Whether apparent redundancy at this level has a function in increasing the robustness of functional gene expression within the organism, as does apparent redundancy among multiple enhancers regulating the same gene, remains to be determined. Furthermore, cooperativity with cofactors in vitro seems to be a significant indicator of function in vivo, in addition to affinity. It was found that while the B1b site has the same apparent affinity as A2a, A2a confers considerably stronger repression activity, and shows greater cooperativity in binding by En with Exd/Hth. The discrepancy between relative affinity and functionality may be attributed to the challenge of reproducing functional binding conditions in vitro, where protein–protein interactions leading to cooperativity may be less sensitive to the differences in conditions than are protein–DNA interactions. Relatedly, competition with a variety of DNA binding proteins in vivo for sites on the DNA may lead to a greater reliance on cooperativity in vivo for occupancy of functional sites (Fujioka, 2012).

Previous studies indicated that En requires the Hox co-factors Exd and Hth to efficiently repressslp, especially in the anterior half of the embryo, and En was found to act cooperatively on target sites in the distalless gene with both Exd/Hth and posteriorly-expressed Hox gene products. Although it remains possible that the relatively weak, yet functional binding site (A2a) within i1523 might bind En with other cofactors in addition to Exd/Hth, dissection and construction experiments with this and other sites have not revealed any clear anterior–posterior differences in their activity that might suggest a functional interaction with cofactors such as Hox proteins that are restricted in expression along the anterior–posterior axis. Nonetheless, previous studies suggested that regulation of slp by En might utilize posterior-specific factors. Further analysis will be required to more fully explore this possibility (Fujioka, 2012).

The relative arrangement of consensus En and Exd sites that facilitate cooperative binding appears to be quite flexible. For example, the A2a site contains no canonical consensus core for En binding (ATTA), while for the other two functional sites, the distance between the centers of the En and Exd sites is 10–12 bp for A1a and only 2 bp for B1a. The latter is reminiscent of En–Exd/Hth binding in distalless, where simultaneous Hox binding occurs, although the position of the En site is on the opposite side of the Exd core consensus ATCA. This relative arrangement of En and Exd sites (En binding 5′ of the Exd core ATCA) is seen for all of the functional sites analyzed in this study. This arrangement is similar to the relative positions of Hox and Exd binding to sites where there is no En involvement. The flexibility overall is consistent with that seen for Exd/Hth binding in conjunction with the Hox gene products, and suggests that while homeodomain family transcription factors are able to function combinatorially in vivo on a wide variety of binding sites, there are significant constraints on the positions of contact by the individual homeodomains. A full understanding of the similarities and differences between En binding in conjunction with Exd and Hth, and Hox binding with these cofactors, will require further investigation (Fujioka, 2012).

The highly cooperative, strong En/Exd/Hth binding site B1a was both necessary and sufficient for repression of u4734 in the En domain. However, it did not fully substitute for the entire repression element that contains it, located between −3.9 and −3.4 kb from the slp1 TSS. This finding suggests that there may be other functional En binding sites in this region. Consistent with this, in vitro binding suggested that other subregions (B2 and/or B3) harbor some binding activity. Thus, like i1523, there may be partial redundancy in En complex binding within u4734, despite the existence of a single essential binding site (Fujioka, 2012).

This study has established the functional significance of three cooperative En/Exd/Hth binding sites within slp. Interestingly, two of them are well conserved among the 12 species of Drosophila whose genomes have been sequenced, and the other site is conserved within the more closely related species. The duplication that generated the twin slp transcription units apparently took place before the divergence of these 12 species, as all drosophilids (but not mosquitoes) contain two tandem slp-related protein coding regions. This might suggest that the two conserved En/Exd/Hth sites were duplicated along with the locus as a whole. It has been shown that Drosophila enhancers contain clusters of conserved sequences blocks, and the two CRMs analyzed in this study contain such conserved sequence clusters. However, the patterns of conservation in the regions surrounding the conserved En/Exd/Hth sites do not suggest that they are directly related to each other. Furthermore, both CRMs are more closely linked to slp1 than to slp2. Clearly, there have been other chromosomal rearrangements in the history of the slp locus, precluding a simple description of its evolution (Fujioka, 2012).

A recent study investigating the genome-wide distribution of En binding showed a peak on i1523, but not on u4734. The data were derived from 7–24 h-old embryos, which were mostly at later stages than those at which these CRMs are active. In addition, the data show peaks where our analysis has not identified functional CRMs. Such sites may function to assist those within the core enhancer regions, or they might be functional during larval or adult stages to keep slp in the off state. Alternatively, they might not be functionally important. Further study will be required to address these issues (Fujioka, 2012).

JAK/STAT controls organ size and fate specification by regulating morphogen production and signalling

A stable pool of morphogen-producing cells is critical for the development of any organ or tissue. This study presents evidence that JAK/STAT signalling in the Drosophila wing promotes the cycling and survival of Hedgehog-producing cells, thereby allowing the stable localization of the nearby BMP/Dpp-organizing centre in the developing wing appendage. The inhibitor of apoptosis dIAP1 and Cyclin A were identified as two critical genes regulated by JAK/STAT and contributing to the growth of the Hedgehog-expressing cell population. JAK/STAT was found to have an early role in guaranteeing Wingless-mediated appendage specification, and a later one in restricting the Dpp-organizing activity to the appendage itself. These results unveil a fundamental role of the conserved JAK/STAT pathway in limb specification and growth by regulating morphogen production and signalling, and a function of pro-survival cues and mitogenic signals in the regulation of the pool of morphogen-producing cells in a developing organ (Recasens-Alvarez, 2017).

Morphogens of the Wnt/Wg, Shh/Hh and BMP/Dpp families regulate tissue growth and pattern formation in vertebrate and invertebrate limbs. This study has unraveled a fundamental role of the secreted Upd ligand and the JAK/STAT pathway in facilitating the activities of these three morphogens in exerting their fate- and growth-promoting activities in the Drosophila wing primordium. Early in wing development, two distinct mechanisms ensure the spatial segregation of two alternative cell fates. First, the proximal-distal subdivision of the wing primordium into the wing and the body wall relies on the antagonistic activities of the Wg and Vn signalling molecules. While Wg inhibits the expression of Vn and induces the expression of the wing-determining genes, Vn, through the EGFR pathway, inhibits the cellular response to Wg and instructs cells to acquire body wall fate. Second, growth promoted by Notch pulls the sources of expression of these two morphogens apart, alleviates the repression of wing fate by Vn/EGFR, and contributes to Wg-mediated appendage specification. Expression of Vn is reinforced by a positive amplification feedback loop through the activation of the EGFR pathway. This existing loop predicts that, in the absence of additional repressors, the distal expansion of Vn/EGFR and its targets would potentially impair wing development. The current results indicate that Upd and JAK/STAT restrict the expression of EGFR target genes and Vn to the most proximal part of the wing primordium, thereby interfering with the loop and allowing Wg to correctly trigger wing development. Evidence is presented that JAK/STAT restricts the expression pattern and levels of its own ligand Upd and that ectopic expression of Upd is able to bypass EGFR-mediated repression and trigger wing development de novo. This negative feedback loop between JAK/STAT and its ligand is of biological relevance, since it prevents high levels of JAK/STAT signalling in proximal territories that would otherwise impair the development of the notum or cause the induction of supernumerary wings, as shown by the effects of ectopic activation of the JAK/STAT pathway in the proximal territories. Thus, while Wg plays an instructive role in wing fate specification, the Notch and JAK/STAT pathways play a permissive role in this process by restricting the activity range of the antagonizing signalling molecule Vn to the body wall region (Recasens-Alvarez, 2017).

Later in development, once the wing field is specified, restricted expression of Dpp at the AP compartment boundary organizes the growth and patterning of the whole developing appendage. Dpp expression is induced in A cells by the activity of Hh coming from P cells, which express the En transcriptional repressor. This study shows that JAK/STAT controls overall organ size by maintaining the pool of Hh-producing cells to ensure the stable and localized expression of the Dpp organizer. JAK/STAT does so by promoting the cycling and survival of P cells through the regulation of dIAP1 and CycA, counteracting the negative effects of En on these two genes. Since the initial demonstration of the role of the AP compartment boundary in organizing, through Hh and Dpp, tissue growth and patterning, it was noted that high levels of En interfered with wing development by inducing the loss of the P compartment. The capacity of En to negatively regulate its own expression was subsequently shown to be mediated by the Polycomb-group genes and proposed to be used to finely modulate physiological En expression levels. Consistent with this proposal, an increase was observed in the expression levels of the en-gal4 driver, which is inserted in the en locus and behaves as a transcriptional reporter, in enRNAi-expressing wing discs. The negative effects of En on cell cycling and survival reported in this work might also contribute to the observed loss of the P compartment caused by high levels of En. As is it often the case in development, a discrete number of genes is recurrently used to specify cell fate and regulate gene expression in a context-dependent manner. It is proposed that the capacity of En to block cell cycle and promote cell death might be required in another developmental context and that this capacity is specifically suppressed in the developing Drosophila limbs by JAK/STAT, and is modulated by the negative autoregulation of En, thus allowing En-dependent induction of Hh expression and promoting Dpp-mediated appendage growth. It is interesting to note in this context that En-expressing territories in the embryonic ectoderm are highly enriched in apoptotic cells. Whether this apoptosis plays a biological role and relies on En activity requires further study (Recasens-Alvarez, 2017).

Specific cell cycle checkpoints appear to be recurrently regulated by morphogens and signalling pathways, and this regulation has been unveiled to play a major role in development. Whereas Notch-mediated regulation of CycE in the Drosophila eye and wing primordia is critical to coordinate tissue growth and fate specification by pulling the sources of two antagonistic morphogens apart, the current results indicate that JAK/STAT-mediated regulation of CycA is critical to maintain the pool of Hh-producing cells in the developing wing and to induce stable Dpp expression. The development of the wing hinge region, which connects the developing appendage to the surrounding body wall and depends on JAK/STAT activity, has been previously shown to restrict the Wg organizer and thus delimit the size and position of the developing appendage. The current results support the notion that JAK/STAT and the hinge region are also essential to restrict the organizing activity of the Dpp morphogen to the developing appendage. Taken together, these results reveal a fundamental role of JAK/STAT in promoting appendage specification and growth through the regulation of morphogen production and activity, and a role of pro-survival cues and mitotic cyclins in regulating the pool of morphogen-producing cells in a developing organ. The striking parallelisms in the molecules and mechanisms underlying limb development in vertebrates and invertebrates have contributed to the proposal that an ancient patterning system is being recurrently used to generate body wall outgrowths. Whether the conserved JAK/STAT pathway plays a developmental role also in the specification or growth of vertebrate limbs by regulating morphogen production or activity is a tempting question that remains to be elucidated (Recasens-Alvarez, 2017).

Engrailed regulation of polyhomeotic

Continued: Engrailed Targets of activity part 2/2

engrailed: Biological Overview | Evolutionary Homologs | Transcriptional regulation | Protein Interactions | Developmental Biology | Effects of Mutation | References

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