Targets of Activity (part 2/2)

Engrailed regulation of polyhomeotic

Engrailed regulates polyhomeotic proximal and distal by two classes of high affinity Engrailed-binding sites upstream of each of the two polyhomeotic transcription units. These proteins are part of the Polycomb Group (PC-G) that repress homeotic gene expression, and thus help maintain segment identity in the developing fly (McKeon 1991 and Fauvarque, 1995). Immunochemical methods, the precipitation of chromatin using anti-Engrailed antibody, detects the presence of Engrailed in DNA fragements containing these sites, suggesting that polyhomeotic activation in germ-band-elongated embryos could be mediated by Engrailed-binding (Serrano, 1995).

Engrailed is a nuclear regulatory protein with essential roles in embryonic segmentation and wing morphogenesis. One of its regulatory targets in embryos has been shown to be the Polycomb group gene, polyhomeotic. Transheterozygous adult flies, mutant for both engrailed and polyhomeotic, show a gap in the fourth vein. In the corresponding larval imaginal discs, a polyhomeotic-lacZ enhancer trap is not normally activated in anterior cells adjacent to the anterior-posterior boundary. This intermediary region corresponds to the domain of low engrailed expression that appears in the anterior compartment, during L3. This en expression depends on the putative serine-threonine kinase protein fused and on the level of hh expression in the posterior cells abutting the A/P boundary, and so depends indirectly on en expression in the posterior compartment. The exact role of this late L3 anterior compartment en expression is still not understood (Maschat, 1998).

Several arguments show that engrailed is responsible for the induction of polyhomeotic in these cells. The role of polyhomeotic in this intermediary region is apparently to maintain the repression of hedgehog in the anterior cells abutting the anterior-posterior boundary, since these cells ectopically express hedgehog when polyhomeotic is not activated. Analysis of the expression patterns of different genes of the Hh signaling pathway that are normally expressed in this intermediary region showed that the segmentation gene patched is highly affected in ph/en mutant discs. The gap in the fourth vein can therefore be correlated with a misregulation of ptc in the posterior compartment. Interestingly, this ectopic ptc expression appears not only in the cells where ph is affected, but also in neighboring posterior cells. This ectopic expression of ptc progressively invades the posterior compartment during the third instar to fill the whole compartment in mature larvae. Genetic data indicate that the level of hh expression is involved in this phenomenon, suggesting that the progressive invasion of the posterior compartment by Ptc is due to an increased secretion of Hh by the cells of the anterior intermediary region, towards cells localized more posteriorly. As a consequence of this ptc misregulation, cubitus-interruptus (ci) and decapentaplegic (dpp) are activated in the posterior compartment, suggesting that the intermediary region, where dpp expression is normally confined, expands posteriorly. As a result of the absence of ph activation by En in cells abutting the A/P boundary, this boundary is not maintained at its normal position, but is progressively shifted posteriorly, while cells lose their posterior identity. Thus posterior cells express a new set of genes that are normally characteristic of anterior cells, suggesting a change in the cell identity. Altogether, these data indicate that engrailed and polyhomeotic interactions are required to maintain the anterior-posterior boundary and the posterior cell fate, just prior to the evagination of the wing (Maschat, 1998).

Considering that hh is responsible for the changes appearing in the posterior compartment of ph/en flies implies that posterior cells might become competent to respond to the Hh signal. Such competence could be attributed to the presence of a low level of posterior compartment ci, which is present ectopically in a [ph-; en-/+] background. Indeed, ph has been shown to be a repressor of ci in the posterior compartment and now it seems both en and ph are likely to be responsible for ci repression in the posterior compartment. Transcriptional repression of ci in the posterior compartment could be initiated by en and maintained by ph, the ph expression depending itself on en expression. Indeed, posterior heterozygous en/+ cells do not show any phenotype unless they are also mutant for ph. One could hypothesize a feedback loop involving en and ph to maintain the level of en expression and ci repression in the posterior compartment. If the basic level of en expression in the posterior compartment depends on both en and ph, en could be maintained at a lower level in a [ph-; en-/+] background. These cells might now produce enough Ci and Ptc to become competent to receive the Hh signal. If posterior cells are not competent to receive an Hh signal, higher amounts of Hh would not affect the posterior cells. Such a feedback loop mechanism between en and ph, maintaining the level of en expression, could also explain the lack of hypomorphic en mutants, since such mutants would be detectable only when ph is affected (Maschat, 1998).

The Drosophila Engrailed homeoprotein has been shown to directly activate a Polycomb-group gene, polyhomeotic, during embryogenesis. A study of the molecular mechanism involved in this activation detected two different types of Engrailed-binding fragments within the polyhomeotic locus. The P1 and D1 fragments contain several 'TTAATTGCAT' motifs, whereas the D2 fragment contains a long 'TAAT' stretch to which multiple copies of Engrailed bind cooperatively. Another homeodomain-containing protein, Extradenticle, establishes protein-protein interactions with Engrailed on the D2 fragment. Both types of Engrailed-binding sites (P1 or D1 and D2), as well as Extradenticle, are necessary to obtain activation by Engrailed. In vivo, normal polyhomeotic expression depends on extradenticle expression. Moreover, in the absence of Extradenticle, overexpression of Engrailed protein represses polyhomeotic expression (Serrano, 1998).

On the basis of these results, a model is proposed to explain ph regulation by En. In the absence of Exd, En protein is probably bound to P1 and D1, while D2 might be bound either by En or by other homeodomain-containing proteins. Under such conditions, no activation of ph is observed. This situation might represent what happens in the embryo prior to full germband extension. When Exd is present, activation of ph occurs. Although Exd is expressed prior to the time at which En activates ph, Exd might not be active at those earlier stages: Exd activity has been shown to be regulated by nuclear import and was described as exclusively cytoplasmic in the embryo before germband extension. Because the En-binding sites are so far apart within the ph locus and because D2 is required for activation, acting as an enhancer on both ph transcription units, it is further suggested that DNA bending might be involved in activation. In particular, En binding to P1 and D1 could be involved in bringing the proximal and distal ph promoters close to D2 in the presence of Exd, allowing ph activation from both transcription units. En binding to P1 and D1 prior to germband extension might thus prepare the chromatin for rapid activation. At least two lines of evidence support the idea that D2 interacts with P1 as well as D1, to mediate activation of both the proximal and the distal transcription units: (1) the P1-D2 and D1-D2 fusions behave similarly in CAT assays and (2) the in vivo activation was observed with the phlac enhancer trap, which corresponds to an insertion in the proximal transcription unit of ph (Serrano, 1998).

Engrailed targets hedgehog in wing discs

In Drosophila, the Trithorax-group (trxG) and Polycomb-group (PcG) proteins interact with chromosomal elements, termed Cellular Memory Modules (CMMs). By modifying chromatin, this ensures a stable heritable maintenance of the transcriptional state of developmental regulators, like the homeotic genes, that is defined embryonically. It was asked whether such CMMs could also control expression of genes involved in patterning imaginal discs during larval development. The results demonstrate that expression of the hedgehog gene, once activated, is maintained by a CMM. In addition, the experiments indicate that the switching of such CMMs to an active state during larval stages, in contrast to embryonic stages, may require specific trans-activators. Thes results suggest that the patterning of cells in particular developmental fields in the imaginal discs does not only rely on external cues from morphogens, but also depends on the previous history of the cells, as the control by CMMs ensures a preformatted gene expression pattern (Maurange, 2002).

Immunoprecipitation using cross-linked chromatin (XChIP) allows the mapping of in vivo DNA target sites of chromatin proteins. Because one Polycomb (PC, a member of the PcG) binding site on polytene chromosomes coincides with the cytological position of hh at 94E, this method was applied to ask whether there are PC and GAGA factor (GAF/Trl, a member of the trxG) binding sites in the hh genomic region. These two factors had previously been found to be hallmarks of CMMs, and the GAF has been shown to be associated with some PcG complexes and necessary for the silencing function of PREs. Initially the immunoprecipitated material was hybridized to a genomic stretch of 45 kb encompassing the hh gene. This led to the identification of PC/GAF-binding sites in regions close to the transcription unit. To further fine-map the location of the PC/GAF-binding sites, the region around the hh gene was subdivided into 1-kb-sized PCR fragments (from 4 kb upstream of the hh transcription start site according to the transcript CG4637 from Flybase, to 13.4 kb downstream to the end of the gene). Slot-blot hybridizations of immunoprecipitated material revealed two main sites where PC and GAF are strongly enriched. The first site (A) is located in a region between 0.07 and 1.06 kb upstream of the transcription start site, whereas the second binding site (B) is found in a region spanning the second exon of the hh gene and spreading about 0.4 kb on both sides of the exon. On both sites a substantial overlap was observed between PC- and GAF-binding sites. The presence of this particular arrangement of PC- and GAF-binding sites in the hh genomic region suggests that these PcG and trxG proteins directly control hh expression (Maurange, 2002).

During embryonic and larval development, En induces transcription of hh in the posterior compartment of leg and wing imaginal discs, where the two factors substantially colocalize. Even though it is not presently clear whether En directly activates hh expression, this regulatory feature provides a tool to test for CMM activity at the hh gene. UAS-en was expressed at the D-V boundary using a vestigial-GAL4 driver (vg-GAL4). This transgene combination allows expression of GAL4 in a thin stripe (1 or 2 cells thick) along the D-V boundary during wing disc development. Double stainings of such late third-instar wing discs reveal that, surprisingly, En not only induces a thin stripe of hh-lacZ expression (reflecting the hh expression pattern in the P30 enhancer trap line) in cells along the D-V boundary as expected, but also in all the posterior and anterior wing pouch cells (except in a stripe along the A-P boundary). Strong UAS-en expression is detected in cells at the D-V boundary and lower levels of En in some regions of the anterior wing pouch. The repression of the endogenous en observed in some parts of the posterior compartment is explained by the fact that high levels of En could cause repression of the endogenous en in the P compartment. Strikingly, the overlay of Hh-LacZ and En stainings clearly reveals large domains, in both anterior and posterior wing pouch, with strong hh expression in the absence of En, suggesting that the transcription of hh in these cells becomes independent of En. Furthermore, it is known that En represses cubitus interruptus (ci) expression, and it has been shown that clones of A cells lacking Ci express low levels of Hh protein. These observations suggest that hh expression is activated by En at the D-V boundary in early larval development, and is inherited, even in the absence of the initial trans-activator (En), through mitosis in the cells forming, in later stages, the wing pouch (Maurange, 2002).

Alternatively, hh inheritance of transcription to daughter cells could be explained by the existence of a positive feedback loop allowing continuous maintenance of hh expression. This positive feedback loop would be activated once hh is expressed, either by autoactivation or cross-activation with another factor, like En, for instance. To investigate this possibility, hh was misexpressed along the D-V boundary, using the vg-GAL4 driver and a UAS-hh transgene. Although UAS-hh is continuously strongly expressed at the D-V boundary from the second instar larval stage, in situ stainings do not reveal any inheritance of hh transcription to daughter cells, because the presence of hh mRNA is always restricted to a thin row of cells at the D-V boundary, even in late third-instar wing discs. This result demonstrates that the previously observed inheritance of hh expression in wing pouch cells of vg-GAL4; UAS-en flies is not caused by autoactivation by Hh itself nor by any positive feedback loop (Maurange, 2002).

Furthermore, antibody stainings in such discs display a progressive activation of en expression along the D-V boundary during development. In late third-instar larvae, a strong En signal is observed, testifying to the functional activity of the protein produced by UAS-hh. Higher magnification shows that in these discs, Hh is able to induce en expression non-cell-autonomously in a stripe of ~7 rows of cells. However, the fact that at this stage, hh expression is only limited to a stripe of 2 rows of cells indicates that En is no longer able to induce transcription of the endogenous hh gene, in contrast with early larval stages. It implies that the low levels of En protein observed in some of the anterior wing pouch cells of vg-GAL4; UAS-en third-instar larvae is most probably caused by a late activation of en transcription by Hh. In addition, hh expression in these cells cannot be due to activation by low or undetectable levels of En protein, because even strong doses of En do not activate hh transcription in this region at this stage of development (Maurange, 2002).

When UAS-en is misexpressed at the D-V boundary in a wild-type genetic background using vg-GAL4, it induces hh expression in most of the cells of the wing pouch except in a stripe along the A-P boundary where hh seems to be repressed. Whereas UAS-en is strongly misexpressed at the D-V boundary, the endogenous en gene is weakly misactivated in some cells of the anterior wing pouch (Maurange, 2002).

Repeating the same experiment in a genetic background hemizygous mutant for an hypomorphic allele of polyhomeotic (ph409) leads to a broader domain of expression of hh. Remarkably, the region along the A-P boundary seems to be less refractory to activation of hh transcription, given that the territory of the repressed domain is reduced. Endogenous en is itself overexpressed in the anterior compartment. This is consistent with the findings demonstrating that en expression can be derepressed in a PcG gene mutant background. In this case in the anterior wing pouch cells, the activation of en transcription by Hh is probably more efficient than in a wild-type background because en cannot be correctly silenced by PH (Maurange, 2002).

The same experiment repeated in a genetic background now doubly heterozygote for the trxG genes trithorax (trxE2) and brahma (brm2) consistently shows that hh expression is activated at the D-V boundary, but can hardly be maintained through cell divisions in the anterior compartment, because with in situ staining, the Hh signal progressively fades away from the D-V boundary. As expected, in such a case, en expression in the anterior compartment is restricted to the D-V boundary, because Hh might not be present in a sufficient amount to activate transcription of the endogenous en gene in the subsequent wing pouch cells (Maurange, 2002).

Furthermore, it is known that PcG-mediated silencing is enhanced at higher temperature, and this hyperrepressed state can be inherited through cell divisions. Based on these observations, it was reasoned that raising embryos at 28°C instead of 18°C would make the Pc-mediated silencing more difficult to derepress, and influence the activation of hh transcription by En. vg-GAL4; UAS-en embryos were allowed to develop at 28°C until the beginning of second instar larvae, when the D-V boundary is established in wing discs and UAS-en is expressed there. As expected, stainings on third instar imaginal discs reveal ectopic clones of wing pouch cells expressing hh. However, the frequency of cells expressing hh is lower than in discs of larvae grown at 18°C, indicating that the Pc-mediated silencing was harder to erase at 28°C. Nevertheless, in contrast with trxG mutant flies, once the transcription has initially been activated in this case, it is maintained in the subsequent daughter cells as suggested by the presence of clones spreading from the D-V midline to the limits of the wing pouch (Maurange, 2002).

These experiments demonstrate that once initiated by En, the maintenance of the transcriptional state of hh to the daughter cells can be attributed to the action of the PcG and trxG proteins. It is concluded that the CMM activity of the hh upstream region described in the transgenic assay is also efficient when considered in its natural chromatin environment and is responsible for the inheritance of the initial transcriptional state of hh from the initiation to the completion of the wing pouch development (Maurange, 2002).

In the GAL4/UAS system, a GAL4 pulse, when provided in larval stages, is only able to transiently activate transcription of the reporter gene, but no heritable switching of the Fab7-CMM is observed because transcription is lost as soon as the trans-activator (GAL4) is down-regulated. These observations led to the hypothesis that Pc-mediated silencing might be more stable in larval stages than in embryonic stages, and CMMs cannot be switched to mitotically heritable activity at these later stages. Consistent with these data, the upstream 3.4-kb fragment showing a CMM activity cannot not be switched to an active state through a GAL4 pulse produced during larval stages as demonstrated by the lack of miniwhite derepression in the eyes of the adult flies (Maurange, 2002).

However, in contrast to these experiments, the endogenous hh CMM can be switched to an active state in larval wing pouch cells upon an En pulse. The switch occurs in second instar larval stages, when the D-V boundary is established through the action of the Notch pathway and GAL4 expressed by the vg driver. At this moment, en misexpression induces a switch of the endogenous hh CMM at the D-V boundary to an active state, leading to maintenance of hh transcription in all wing pouch cells. It was of interest to test whether GAL4 is also able to directly switch the endogenous hh CMM, in its natural chromatin environment, in larval stages or whether this feature is restricted to specific trans-activators like En. To perform this experiment, the previously described line containing an EP-element inserted into the hh promoter region (EP-hh) was used. By inducing GAL4 in the cells it is possible to activate expression of the endogenous hh gene. It was postulated that, by promoting transcription of the endogenous hh gene, the hh CMM may be switched to an active state in wing pouch cells. As observed on in situ preparations of late third-instar discs, endogenous hh transcription is activated by GAL4 at the D-V boundary, but is not maintained through cell division in wing pouch cells. In comparison, also the well-characterized Fab7-CMM is itself not switched to the active state after GAL4 induction at the D-V boundary because expression of the reporter gene is not maintained in daughter wing pouch cells. It is concluded that the GAL4 trans-activator is not able to switch a CMM in larval stages, although this can be carried out by the action of a gene-specific trans-activator, alone or more likely in association with other factors (Maurange, 2002).

Large clones lacking en/inv expression in the posterior compartment of wing discs show reduced or no Hh protein, although this was not a universal feature of small clones. Apparently, in this situation the loss of en/inv in the cells, especially when induced early in development, might cause a substantial reprogramming of the gene expression pattern leading to repression of hh, perhaps owing to the appearance of new repressors. In this case, the initially activated CMM would not be able to overcome the repression (Maurange, 2002).

From these results, it is likely that CMMs have major direct roles in the inheritance of the expression of hh in the development of wing imaginal discs (it could also be imagined that the well-defined en-PRE could also act as a CMM). Furthermore, hh and its vertebrate homologs are expressed in many other tissues during development, in which its activation and/or maintenance are independent of En and not yet elucidated (i.e., eye, gut, lung). Further studies will help develop an understanding of how the hh CMM may be involved in regulating the gene in different tissues (Maurange, 2002).

It is important to note that the state of activation of a CMM does not have to be established, once and for all, during embryogenesis, but can be modified or stably switched later in development. This may be especially true for genes patterning imaginal discs for which the expression pattern is established during larval development in contrast to homeotic genes defining the A-P axis during embryogenesis. However, it seems that general trans-activating factors like GAL4, which are able to establish the active state of a CMM during embryogenesis, are not able to modify or switch the CMM state later in development, suggesting that the chromatin state of a CMM is more difficult to reprogram at late developmental stages. During larval stages, many cell divisions have been accomplished and cells are getting more and more restricted in their determination state. The chromatin could then be in a 'mature' conformation stable enough to transmit a previously established transcriptional state despite the potentially contradictory actions of other transcription factors found simultaneously in the nucleus. Nevertheless, other transcription factors such as En (in the case where En directly activates hh) seem to be able, alone or by recruiting cofactors, to stably switch a CMM from a repressed to an active state during larval stages. At these stages, the switching of CMMs could require specific factors to set epigenetic marks. It could be envisaged that the En complex is able to attract some kind of chromatin-remodeling machinery that would have the potency to erase the memory and leave the chromatin competent to be reprogrammed (Maurange, 2002).

In this way, it seems that the cell memory system is a complex and dynamic process during development, in which the role of CMMs is to heritably maintain a previously established transcriptional state until new specific patterning cues are able to redirect the epigenetic marks of the CMMs. However, this also makes it quite clear that during the establishment of a morphogenetic field, besides the local specifying signaling events, the previous history of a determining gene should be taken into account (Maurange, 2002).

Additional Engrailed targets

araucan-caupolican expression is restricted to two symmetrical patches located one at each side of the dorsoventral compartment border of the wing imaginal disc. ara-caup expression in these patches is necessary for the specification of the prospective vein L3 and associated sensory organs. Here, ara-caup expression is mediated by the Hedgehog signal through its induction of high levels of Cubitus interruptus in anterior cells near the the AP compartment border. The high levels of CI activate decapentaplegic expression, and together, CI and DPP positively control ara-caup. patched overexpression is equivalent to a reduced hh function in that accumulation of CI and DPP at the AP border are strongly depressed. The wing pouch of patched mutants have much reduced or absent ara-caup L3 patches. dpp by itself is insufficient to account for ara-caup expression. wingless is expressed in a narrow strip of cells straddling the DV compartment boundary of the wing disc, corresponding to the prospective wing margin. The dorsal and ventral ara-caup L3 patches are separated by a gap that corresponds to the cells that accumulate detectable amounts of WG. Clones of mutant wg expressing cells spanning the gap between the L3 patches extend these patches toward the DV border and a narrow gap of only one or two cell diameters remains. Thus WG represses ara-caup expression at the prospective wing margin domain. Likewise repression by Engrailed is most likely to be responsible for the posterior border of ara-caup expression in the L3 patches (Gómez-Skarmeta, 1996)

The beta3-tubulin gene is a direct target of Engrailed. The cytological location of beta3-tubulin, 60C, is a strong site for Engrailed binding on polytene chromosomes. Immunostaining analysis of a transgenic line containing a P[beta3-tubulin-lacZ] construct shows an additional site for Engrailed binding at the location of the transgene. Molecular analysis allows identification of several Engrailed binding sites, both in vitro and in vivo, within the first intron of the beta3-tubulin locus. Sequence analysis of beta3-tubulin fragments that bind En reveal the presence of ten sites related to the En consensus binding sequence TTAATTGCAT. Engrailed binding sites identified in vitro are active in larvae. Expression of beta3-tubulin has been shown to be derepressed in the ectoderm of engrailed mutant embryos (Serrano, 1997).

During embryogenesis, expression in the visceral and late somatic musculatures is regulated by sequences contained within the first intron of beta3-tubulin, while upstream sequences are necessary for early expression in somatic musculature, suggesting that beta3-tubulin regulation is achieved by an early transient program until stage 12 and a more stable late program in later stages. Two different sets of Engrailed binding sites are shown to be involved in the early and late regulation of beta3-tubulin by Engrailed during embryogenesis. The intronic beta3-tubulin region can be subdivided into 5 subregions (A-E), each containing one to four En binding sites. Evidence is provided that fragments A to D are involved in late en expression, while sequences in the E fragment are implicated in the early regilation of beta3-tubulin expression by En. Sequences in the E fragment are able to respond in vivo to differences in En protein concentration and might be part of the regulation of beta3-tubulin expression in one half of the larval hindgut. It is suggested that En might be responsible for the observed repression of beta3-tubulin, at least in the posterior compartment, through its binding to the D region. Repression of beta3-tubulin by Engrailed is obtained when Engrailed is ectopically expressed in embryonic mesoderm. A 500 base pair F fragment (which does not contain En binding sites and is located between region D and E), is shown to be sufficient to confer a pattern similar to the endogenous beta3-tubulin expression in the visceral mesoderm and in the late somatic mesoderm. The addition of the D fragment to this 500 base pair F fragment shows a particularly high level of expression in the chordotonal organs, a place for the normal expression of beta3-tubulin. The absence of En binding sites in the F fragment points to a complicated network of regulation of beta3-tubulin in different types of cells in early development (Serrano, 1997).

Engrailed is expressed in subsets of interneurons that do not express Connectin or appreciable Neuroglian, whereas other neurons that are Engrailed negative strongly express these adhesion molecules. Connectin and Neuroglian expression are virtually eliminated in interneurons when engrailed expression is driven ubiquitously in neurons, and greatly increased when engrailed genes are lacking in mutant embryos. The data suggest that Engrailed is normally a negative regulator of Connectin and neuroglian. These are the first two effector genes identified in the nervous system of Drosophila as regulatory targets for Engrailed. It is argued that differential Engrailed expression is crucial in determining the pattern of expression of cell adhesion molecules and thus constitutes an important determinant of neuronal shape and perhaps connectivity. In wild-type embryos, all neurons that express engrailed also express invected. The converse is not true, however. Neurons, which lie anterior to the predominant Engrailed/Invected stripe in the CNS, express invected but not engrailed. This is the best example to date of differing expression of engrailed and invected in an identified cell type. Connectin is also expressed in SNa and SNc motor neurons, which are Engrailed negative. When Engrailed is expressed in all neurons, Connectin is not downregulated but slightly upregulated in these motor neurons, in contrast to the effect on interneurons. Since Engrailed can act either as a repressor or as an activator, it is possible that ectopic Engrailed directly activates Connectin in the motor neurons (Siegler, 1999).

mtv shapes the activity gradient of the Dpp morphogen through regulation of thickveins

One of the pleiotropic functions of scribbler is an effect of wing morphogenesis. This function has been addressed by Funakoshi (2001), who shows that sbb shapes the activity gradient of the Dpp morphogen through regulation of thickveins. Drosophila wings are patterned by a morphogen, Decapentaplegic, a member of the TGFbeta superfamily, that is expressed along the anterior and posterior compartment boundary. The distribution and activity of Dpp signaling is controlled in part by the level of expression of its major type I receptor, thickveins (tkv). The level of tkv is dynamically regulated by Engrailed and Hedgehog. sbb, termed master of thickveins (mtv) by Funakoshi, downregulates expression of tkv in response to Hh and En. mtv expression is controlled by En and Hh, and is complementary to tkv expression. mtv integrates the activities of En and Hh that shape tkv expression pattern. Thus, mtv plays a key part of regulatory mechanism that makes the activity gradient of the Dpp morphogen (Funakoshi, 2001).

The fact that tkv expression is repressed by hh at the A/P border and mtv is highly expressed in the same cells has prompted an examination of whether mtv mediates hh dependent tkv repression. tkv-lacZ levels were examined in clones of cells mutant for patched (ptc), which encodes the Hh receptor. Hh signal transduction is constitutively active in the absence of ptc activity. Anterior compartment ptc clones cause cell-autonomous repression of tkv. In the clones of cells mutant both for mtv and ptc, however, tkv levels are elevated as in the mtv singly mutant clones. This indicates that mtv mediates hh-dependent tkv regulation along the A/P border. mtv-lacZ expression was monitored within clones of cells mutant for smoothened (smo), which encodes a component of the Hh receptor complex and is required for Hh signaling. Within the clones located at the A/P border, mtv-lacZ expression is repressed, indicating that Hh signaling induces the high level of mtv expression along the A/P border. This was also confirmed by the fact that mtv-lacZ levels are elevated within pka mutant clones, in which Hh signaling is constitutively active. These results show that Hh represses tkv levels by upregulating its negative regulator, mtv (Funakoshi, 2001).

As described earlier, the basal tkv level in the P compartment is higher than it is in the A compartment and it is responsible for the asymmetric structure of the wing. The mtv expression pattern is complementary to the tkv expression pattern. The possibility that mtv is also responsible for regulating the basal level of tkv expression was also examined. Initially, it was asked whether the level of tkv is regulated by en in the P compartment. Within clones of cells mutant for en in the P compartment, the tkv-lacZ level is lower than that seen in the A compartment, suggesting that tkv expression is regulated by en. This is confirmed by the observation that, within clones of cells ectopically expressing en in the A compartment, the tkv level is elevated in comparison to that seen in the P compartment in an autonomous way. Within en and mtv double mutant clones, tkv transcription levels are derepressed as in mtv single mutant clones, indicating that mtv mediates en dependent tkv regulation. Ectopically expressed en downregulates mtv-lacZ in the A compartment, implying that low levels of mtv in the P compartment are under the control of en regulation. These results altogether indicate that en regulates the high basal level of tkv expression in the P compartment by downregulating mtv expression (Funakoshi, 2001).

It is concluded that the central region of the wing is patterned by Hh but not by Dpp although Dpp expression is induced by Hh in this region. This is because Dpp signaling is downregulated by Hh by upregulating mtv, which causes repression of tkv expression. The patterning by Hh appears to be ensured by lowering the Dpp signaling, which would otherwise interfere with the Hh morphogen activity, because upregulation of Dpp signaling by overexpressing tkv or by eliminating mtv activity can alter the vein patterns there. Therefore, the mtv-dependent tkv regulation is required both for Hh and Dpp morphogen activities. The patterning along the A/P border between veins 3 and 4 may be more complicated. The dorsal mtv mutant clone at the A/P border disrupts the vein pattern; vein 3 is displaced posteriorly, which is similar to the phenotype associated with sal mutant clones. The fact that mtv is downregulated in sal mutant clones might explain the phenotype if Mtv has a role in mediating Sal activity, which positions vein 3 through regulating target genes such as the iroquois gene complex. Further analysis is required to elucidate whether tkv function is linear or parallel to this regulatory cascade (Funakoshi, 2001).

The mechanism that shapes the tkv pattern and hence the Dpp morphogen gradient is unique in that it makes the mtv expression pattern that is made by integrating En and Hh signals complementary to the tkv pattern. Thus, the mtv expression pattern acts as a 'negative' for generating the tkv pattern. en positive cells initiate the cascade of the patterning along the A/P axis by expressing Hh, which both acts as short-range morphogen and induces the long-range morphogen, Dpp. Here it is proposed that not only does En induce expression of the morphogen, but it also shapes the morphogen activity gradient by regulating its receptor level via Mtv (Funakoshi, 2001).

Notch signaling controls cell fate specification along the dorsoventral axis of the Drosophila gut

The genetic programs that control patterning along the gut dorsoventral (DV) axis have remained largely elusive. The activation of the Notch receptor occurs in a single row of boundary cells that separates dorsal from ventral cells in the Drosophila hindgut. rhomboid, which encodes a transmembrane protein, and knirps/knirps-related, which encode nuclear steroid receptors, are Notch target genes required for the expression of crumbs, which encodes a transmembrane protein involved in organizing apical-basal polarity. Notch receptor activation depends on the expression of its ligand Delta in ventral cells, and localizing the Notch receptor to the apical domain of the boundary cells may be required for proper signaling. The analysis of gene expression mediated by a Notch response element suggests that boundary cell-specific expression can be obtained by cooperation of Suppressor of Hairless and the transcription factor Grainyhead or a related factor. These results demonstrate that Notch signaling plays a pivotal role in determining cell fates along the DV axis of the Drosophila hindgut. The finding that Notch signaling results in the expression of an apical polarity organizer, one which, in turn, may be required for apical Notch receptor localization, suggests a simple mechanism by which the specification of a single cell row might be controlled (Fusse, 2002).

To study whether En, which is expressed in the adjacent dorsal cells, contributes to the boundary cell fate, the expression of kni/knrl, rho, and crb was examined in en mutants and in en; invected double mutants (enE), since en and invected are known to act redundantly. Whereas the expression of the Notch target genes remains unchanged in en mutants, it is absent in the large intestine of en; invected double mutants. Morphological studies indicate that the dorsal and the boundary cell fates are not established in these mutants, and the large intestine seems to consist entirely of the ventral cell fates. To investigate the cause for this effect, the expression of Delta was studied in these mutants and it was found to be expressed ubiquitously in the large intestine. These data indicate that a boundary between Delta expressing and nonexpressing cells is required for Notch receptor activation. Ectopic expression of En in the large intestine using the 14-3 fkh driver and UAS-En effector lines results in a repression of kni/knrl and rho gene expression. This indicates that En bears the potential to act as a negative regulator of Notch target genes. Upon ectopic activation of Notch signaling in the entire hindgut by expressing Nicd, En is repressed on the dorsal side of the large intestine, thus allowing ectopic activation of Notch target genes (Fusse, 2002).

Dally regulates Dpp morphogen gradient formation in the Drosophila wing

The expression pattern of dally, monitored by dally::lacZ enhancer-trap expression, in the developing wing along the AP axis shows a peak of expression at the A/P border cells, and dally levels are lowest in cells adjacent to this region. Furthermore, dally levels gradually increase toward the anterior and posterior distal cells. This pattern correlates with the expression patterns of several genes involved in pattern formation along the AP axis, such as tkv and master of thick veins (mtv; also known as scribbler/breakless); this suggests that dally also participates in this process. Expression of the tkv gene is controlled by two distinct pathways. (1) Hh represses tkv expression at the A/P border cells, and En regulates a high basal level of tkv in the P compartment. The activities of both hh and en genes are mediated by a putative transcription factor, Mtv. (2) tkv levels are downregulated by Dpp signaling. By this mechanism, tkv expression is maintained at low levels in the center of the disc and at higher levels toward the anterior and posterior edges. The correlation between expression patterns of dally and tkv prompted an analysis of the dally function in Dpp signaling in this tissue. Regulatory pathways controlling dally expression were analyzed and compared with those controlling tkv expression (Fujise, 2003).

Hh signaling induces dally expression at the A/P border cells. dally expression is absent in smoothened (smo) mutant clones generated in the anterior compartment, where the Hh signaling is blocked, indicating that Hh signaling is required for activation of dally at the A/P border cells. To further determine whether Hh signaling is sufficient for the induction of dally, clones were examined that ectopically express hhCD2, which encodes a membrane-tethered form of Hh, using the FLP-OUT system. In the A compartment, dally expression levels are increased in hhCD2-expressing cells and in cells immediately adjacent to them. This result shows that Hh expression is sufficient to induce dally expression in the A compartment. To determine if dally expression is controlled by en, which upregulates tkv expression, clones of en-mutant cells were induced using the FLP-FRT mosaic analysis system. Within en-mutant clones in the P compartment, dally levels are dramatically increased; this indicates that dally expression is negatively regulated by en (Fujise, 2003).

To determine whether modulation of Dpp signaling affects dally expression, the dally::lacZ expression was compared between wild-type and tkv heterozygous cells. Clones mutant for tkv were generated in a heterozygous background (tkva12/+) using the FLP-FRT system, which should, as a consequence, produce both mutant (tkva12/tkva12) and wild-type sister clones (+/+). However, tkv cells do not survive in the wing pouch since Tkv activity is indispensable for growth and, thus, only wild-type sister clones survive. In resultant mosaic discs with wild-type and tkv-heterozygous cells, dally expression is decreased cell autonomously in wild-type (+/+) clones at the AP border and peripheral to the border. To further confirm this result, the effects were examined of tkv-hypomorphic clones on dally expression. In such clones, where tkv activity is partially compromised, the levels of dally expression are elevated. In the notum region of the wing disc, tkv-null clones can be generated in which a substantial increase of dally expression is observed. Finally, the effect of increased Dpp signaling on dally expression was tested by using the FLP-OUT method to induce clones of cells that express tkvQ253D, a constitutively active form of tkv, in the wing pouch. The level of dally::lacZ expression was found to be autonomously reduced in the tkvQ253D-expressing clones. All of these results consistently indicate that dally expression in the wing disc is negatively regulated by Dpp signaling, as has been shown for tkv. Thus, dally and tkv are regulated by the same set of molecular pathways: Hh, En and Dpp signaling (Fujise, 2003).

Although dally expression is regulated by the same set of signaling pathways that control expression of tkv the effects of Hh and En on dally are the opposite of those on tkv. In addition, dally expression is negatively controlled by Dpp signaling. Through this mechanism, relative levels of dally expression are higher at the anterior and posterior distal edges. Therefore, dally and tkv show similar patterns of expression with one exception: the level of dally expression is high in A/P border cells, where Dpp is synthesized and secreted, but by contrast, tkv expression levels are low in this region. The high levels of dally in the peripheral regions could sensitize cells to low levels of Dpp, as has been shown for tkv. These regulatory pathways appear to form negative feedback loops, which may stabilize the shape of the Dpp morphogen gradient. Thus, the regulated expression and function of Dally are crucial factors in the generation and maintenance of the Dpp morphogen gradient (Fujise, 2003).

Genome-wide identification of in vivo Drosophila Engrailed-binding DNA fragments and related target genes

Chromatin immunoprecipitation after UV crosslinking of DNA/protein interactions was used to construct a library enriched in genomic sequences that bind to the Engrailed transcription factor in Drosophila embryos. Sequencing of the clones led to the identification of 203 Engrailed-binding fragments localized in intergenic or intronic regions. Genes lying near these fragments, which are considered as potential Engrailed target genes, are involved in different developmental pathways, such as anteroposterior patterning, muscle development, tracheal pathfinding or axon guidance. This approach was validated by in vitro and in vivo tests performed on a subset of Engrailed potential targets involved in these various pathways. Strong evidence is presented showing that an immunoprecipitated genomic DNA fragment corresponds to a promoter region involved in the direct regulation of frizzled2 expression by engrailed in vivo (Solano, 2003).

Forty-seven percent of the fragments were localized within gene introns. In this case, it was assumed that the corresponding intron is a part of the engrailed regulated target gene. Fifty-three percent were present in intergenic regions. In this case, analysis was restricted to the nearest transcription unit, whatever its orientation and its distance with respect to the Engrailed-binding fragment. Half of the intergenic fragments are localized at less than 5 kb upstream of the genes, suggesting that the Engrailed-binding fragment may be a part of their promoter region. In rare cases (5%), when the Engrailed-binding fragment lies between two transcription units among which only one encodes a known function, the latter was considered as the putative target (Solano, 2003).

In 55% of the cases, the Engrailed-binding fragments could be associated with a gene whose function is known or that contains a recognizable protein domain. In all the other cases (45%), the binding fragments were associated to genes with an unknown function, which is approximately the ratio of this category in the Drosophila genome. According to GO annotation, the majority of the known genes are involved in cell communication and developmental processes. As expected from previous work, potential Engrailed targets identified using this approach include genes that are involved in the establishment and the maintenance of the AP axis body. Several genes involved in wing development, tracheal development, muscle development and axon guidance were identified. Furthermore, different categories of genes encoding proteins involved in signal transduction were found (signal proteins, receptors, protein kinases, protein phosphatases, transcription factors and cell adhesion protein). Interestingly, cell adhesion proteins and receptors were particularly well represented (see http://www.igh.cnrs.fr/equip/WebFM/). This suggests that engrailed could act at different molecular levels in several developmental processes (Solano, 2003).

A motif research analysis was performed on a subset of 107 sequences from the 203 clones selected in the UV-X-ChIP library. It revealed that the most frequent motifs were a group of 49 related octanucleotides, compiled in a position weight matrix and resolved as a 'YAATYANB' consensus. This consensus sequence largely overlaps those already described for Engrailed (Solano, 2003).

Gel shift assays were then performed on 14 Engrailed-binding fragments isolated from the library, and the results are shown in four cases where the associated target genes are involved in different signaling pathways. 1A4 clone corresponds to a genomic fragment lying 5 kb downstream of frizzled 2 (fz2), which encodes one of the wingless (wg) receptors. 2H10 clone corresponds to a genomic fragment lying within hibris (hbs), which encodes a member of the immunoglobulin superfamily involved in muscle guidance. 1B12 clone corresponds to a genomic DNA fragment lying within the first intron of branchless (bnl), encoding the Drosophila homolog of the Fibroblast Growth Factor (FGF) involved in tracheal morphogenesis. 2C5 clone corresponds to a genomic fragment lying in the first intron of frazzled (fra), which encodes a netrin receptor involved in motor axon guidance (Solano, 2003).

In each case, two sets of experiments were performed, either with the entire immunoprecipitated fragment (150 bp to 350 bp), or with a shorter 100 bp fragment, surrounding the YAATYANB motifs. All these DNA fragments form retarded complexes in the presence of HS-EN protein. Addition of a known specific Engrailed target DNA (D2) was able to compete the formation of the complexes. Moreover, addition of Engrailed specific 4F11 antibody super-shifted the complexes. These data show the specificity of Engrailed binding, which was also confirmed using purified Engrailed protein. The addition of the cold DNA fragment itself allowed a comparison of the affinity of Engrailed on this fragment to the affinity of the strong Engrailed-binding fragment D2. The affinities are at least 10-9 M. In conclusion, Engrailed is shown to be able to bind specifically to these four in vivo immunoprecipitated DNA fragments, which lie close to genes involved in different developmental processes, most probably via the 'YAATYANB' consensus sequence identified (Solano, 2003).

In order to discriminate among the list of putative targets, a simple screen was used. The expression of several potential target genes was monitored, after ectopic expression of Engrailed using the UAS-GAL4 system. Because Engrailed can act as a repressor or an activator, either the wild-type Engrailed protein (UAS-En) or a chimeric activator form (UAS-VP16-En) was overexpressed, under the control of MS1096-Gal4, in third instar wing imaginal disc. This approach was tested on ß3-tubulin, which is known to be directly repressed by engrailed. As expected, overexpression of wild-type Engrailed protein led to a repression of endogenous ß3-tubulin in the wing disc, whereas overexpression of the activator form of Engrailed had no detectable effect, probably because of the strong expression of endogenous ß3 tubulin in the discs (Solano, 2003).

Using this assay, the expression of 14 genes was studied that are localized close to the genomic DNA fragments isolated in the library and tested previously for their Engrailed-specific binding ability. The results are shown for four genes (frizzled2, hibris, branchless, frazzled) that are representative of the different pathways where engrailed seems to be involved. frizzled 2 expression is activated in the presence of (VP16-En) and repressed in the presence of En. This suggests that engrailed might act as a repressor on fz2 expression. hibris is expressed along the wing margin and in the presumptive region of wing vein L3 and L4 in wild type. This expression is slightly activated in the presence of (VP16-En), but strongly repressed when En is overexpressed, suggesting that hbs expression is regulated by engrailed in vivo. branchless is essentially expressed in a dorsal/posterior territory surrounding the wing pouch in wild type. In the presence of (VP16-En), several additional patches of bnl expression are detected within the wing pouch, whereas no activation of bnl is observed after wild type En overexpression. As expected, because MS1096 drives Gal4 expression only in the wing pouch, endogenous bnl expression outside the wing pouch is not affected, showing the specificity of the experiment. Finally, frazzled is slightly expressed in wild-type wing disc. This expression is activated when (VP16-En) is overexpressed, and repressed upon En overexpression (Solano, 2003).

In conclusion, these data demonstrate that the expression of several potential target genes identified via UV-X-ChIP is modulated when engrailed is misexpressed. This test has been successfully performed on 12 genes of the 14 that were tested (Solano, 2003).

Interactions between engrailed and the wingless signaling pathway have been extensively described. A direct regulation of frizzled receptor expression by engrailed has been documented. The other wingless receptor gene, frizzled2 (fz2), might also be directly regulated by engrailed. A high-affinity Engrailed-binding fragment (1A4) was detected in the close vicinity of the fz2 transcription unit. In wild-type embryos, fz2 expression becomes segmentally repeated around stage 9, in two or three rows of cells just anterior to engrailed. In stage 9 engrailed mutant embryos, fz2 expression is extended posteriorly, being detected in 4 rows of cells. This shows that Engrailed acts as a repressor of fz2 expression in embryos, as has been suggested with the previous test in the wing disc. Whether the 1A4 Engrailed-binding fragment was able to drive the expression of a reporter gene was verified in vivo and whether it responds to engrailed regulation was examined. For this purpose, this 170 bp fragment, either as a monomer or a trimer, was cloned upstream of a GFP reporter gene and hsp70 minimal promoter and introduced into the Drosophila genome by P element-mediated transposition. In these transgenic lines, GFP expression was essentially detected in the embryonic hindgut and in half of the larval hindgut. GFP is expressed in the ventral cells of the larval hindgut that do not express engrailed, which mimics endogenous fz2 expression. This demonstrates that the 1A4 DNA fragment might be a part of endogenous fz2 regulatory sequences. Overexpression of (VP16-En) fusion protein driven by hs-Gal4 leads to ectopic GFP expression in the entire hindgut, but also in tissues that do not express the transgene in wild type, such as the midgut, the salivary glands, and the wing disc. Overexpression of (VP16-En) fusion protein driven by en-Gal4 in embryos leads to ectopic GFP expression in a striped pattern. Such activation does not occur with overexpression of wild-type Engrailed, confirming a repressor role of Engrailed on fz2 expression through this 1A4 fragment. These results show that 1A4 is able to respond to engrailed regulation in vivo. Altogether, these data show that the 1A4 fragment that was isolated by UV-X-ChIP is a part of the fz2 regulatory regions and is able to directly respond to engrailed regulation in vivo (Solano, 2003).

Direct integration of Hox and segmentation gene inputs during Drosophila development

During Drosophila embryogenesis, segments, each with an anterior and posterior compartment, are generated by the segmentation genes while the Hox genes provide each segment with a unique identity. These two processes have been thought to occur independently. This study shows tha abdominal Hox proteins work directly with two different segmentation proteins, Sloppy paired and Engrailed, to repress the Hox target gene Distalless in anterior and posterior compartments, respectively. These results suggest that segmentation proteins can function as Hox cofactors and reveal a previously unanticipated use of compartments for gene regulation by Hox proteins. The results suggest that these two classes of proteins may collaborate to directly control gene expression at many downstream target genes (Gebelein, 2004).

The segregation of groups of cells into compartments is fundamental to animal development. Originally defined in Drosophila, compartments are critical for providing cells with their unique positional address. The first compartments to form during Drosophila development are the anterior and posterior compartments and the key step to defining them is the activation of the gene engrailed (en). Expression of en, which encodes a homeodomain transcription factor, results in a posterior compartment fate, and the absence of en expression results in an anterior compartment fate. Once activated by gap and pair-rule genes, en expression and, consequently, the anterior-posterior compartment boundary later become dependent upon the protein Wingless (Wg), which is secreted from adjacent anterior compartment cells. Concurrently with anterior-posterior compartmentalization and segmentation, the expression of the eight Drosophila Hox genes is also initially established by the gap and pair-rule genes. The Hox genes, however, which also encode homeodomain transcription factors, do not contribute to the formation or number of segments but instead specify their unique identities along the anterior-posterior axis (Gebelein, 2004).

This flow of genetic information during Drosophila embryogenesis has led to the idea that anterior-posterior compartmentalization and segment identity specification are independent processes. In contrast to this view, this study shows that these two pathways are interconnected in previously unrecognized ways. Evidence is provided that Hox factors directly interact with segmentation proteins such as En to control gene expression. Moreover, Hox proteins collaborate with two different segmentation proteins in anterior and posterior cell types to regulate the same Hox target gene, revealing a previously unknown use of compartments to control gene expression by Hox proteins (Gebelein, 2004).

Distalless (Dll) is a Hox target gene that is required for leg development in Drosophila. In each thoracic hemisegment, wg, expressed by anterior cells adjacent to the anterior-posterior compartment boundary, activates Dll in a group of cells that straddle this boundary. A cis-regulatory element derived from Dll, called DMX, drives accurate Dll-like expression in the thorax. The abdominal Hox genes Ultrabithorax (Ubx) and abdominalA (abdA) directly repress Dll and DMX-lacZ in both compartments, thereby blocking leg development in the abdomen. DMX is composed of a large activator element (DMXact) and a 57-base-pair (bp) repressor element referred to here as DMX-R. Previous work demonstrated that Ubx and AbdA cooperatively bind to DMX-R with two homeodomain cofactors, Extradenticle (Exd) and Homothorax (Hth). In contrast, the thoracic Hox protein Antennapedia (Antp) does not repress Dll and does not bind DMX-R with high affinity in the presence or absence of Exd and Hth. Thus, repression of Dll in the abdomen depends in part on the ability of these cofactors to selectively enhance the binding of the abdominal Hox proteins to DMX-R (Gebelein, 2004).

Exd and Hth, as well as their vertebrate counterparts, are used as Hox cofactors at many target genes. Moreover, Hox/Exd/Hth complexes are used for both gene activation and repression, raising the question of how the decision to activate or repress is determined. One view posits that these complexes do not directly recruit co-activators or co-repressors, but instead are required for target gene selection. Accordingly, other DNA sequences present at Hox/Exd/Hth-targeted elements would determine whether a target gene is activated or repressed. Consistent with this notion, DMX-R sequences isolated from six Drosophila species show extensive conservation outside the previously identified Hox (referred to here as Hox1) Exd and Hth binding sites, suggesting that they also play a role in Dll regulation (Gebelein, 2004).

To test a role for these conserved sequences, a thorough mutagenesis of DMX-R was performed. Each mutant DMX-R was cloned into an otherwise wild-type, full-length DMX and tested for activity in a standard reporter gene assay in transgenic embryos. Thoracic expression was normal in all cases. However, surprisingly, many of the DMX-R mutations, such as X5, resulted in abdominal de-repression only in En-positive posterior compartment cells, whereas other mutations, such as X2, resulted in abdominal de-repression only in En-negative anterior compartment cells. Single mutations in the Hox1, Exd, or Hth sites also resulted in de-repression predominantly in posterior cells. In contrast, deletion of the entire DMX-R (DMXact-lacZ), or mutations in both the X2 and X5 sites (DMX[X2 + X5]-lacZ), resulted in de-repression in both compartments. These results suggest that distinct repression complexes bind to the DMX-R in the anterior and posterior compartments and that segmentation genes play a role in Dll repression (Gebelein, 2004).

One clue to the identity of the proteins in these repression complexes is that the sequence around the Hth site is nearly identical to a Hth/Hox binding site that had been identified previously by a systematic evolution of ligands by exponential enrichment (SELEX) approach using vertebrate Hox and Meis proteins. This similarity suggested the presence of a second, potentially redundant Hox binding site, Hox2. In agreement with this idea, mutations in both the Hox1 and Hox2 binding sites resulted in de-repression in both the anterior and posterior compartments of the abdominal segments. Similarly, although individual mutations in the Exd and Hth binding sites lead predominantly to de-repression in the posterior compartment, mutation of both sites resulted in de-repression in both compartments. These results suggest that a Hox/Exd/Hth/Hox complex may be used for repression in both compartments. Furthermore, they suggest that although single mutations in these binding sites are sufficient to disrupt the activity of this complex in the posterior compartment, double mutations are required to disrupt its activity in the anterior compartment (Gebelein, 2004).

To provide biochemical evidence for a Hox/Exd/Hth/Hox tetramer, DNA binding experiments were performed using DMX-R probes and proteins expressed and purified from E. coli. Previous experiments demonstrated that a Hox/Exd/Hth trimer cooperatively binds to the Hox1, Exd and Hth sites. The function of the Hox2 site was tested in two ways. First, binding was measured to a probe, DMX-R2, that includes the Exd, Hth and Hox2 sites, but not the Hox1 site. It was found that Exd/Hth/AbdA and Exd/Hth/Ubx trimers cooperatively bind to this probe and that mutations in the Hth, Exd or Hox2 binding sites reduced or eliminated complex formation (Gebelein, 2004).

Second, if both the Hox1 and Hox2 sites are functional, the full-length DMX-R may promote the assembly of Hox/Exd/Hth/Hox tetramers. Using a probe containing all four binding sites (DMX-R1 + 2), the formation of such complexes was observed. Mutation of any of the four binding sites reduced the amount of tetramer binding whereas mutation of both Hox sites or both the Exd and Hth sites eliminated tetramer binding. Furthermore, Antp, which does not repress Dll, formed tetramers with Exd and Hth that were approximately tenfold weaker than with Ubx or AbdA, but bound well to a consensus Hox/Exd/Hth trimer binding site. Because mutation of both Hox sites or both the Exd and Hth sites resulted in de-repression in both compartments, these experiments correlate the binding of a Hox/Exd/Hth/Hox complex on the DMX-R with the ability of this element to mediate repression in both compartments (Gebelein, 2004).

Although binding of a Hox/Exd/Hth/Hox tetramer is sufficient to account for the necessary abdominal Hox-input into Dll repression, it does not explain the compartment-specific de-repression exhibited by some DMX-R mutations. The X2 and X5 mutations, for example, result in abdominal de-repression but do not prevent the formation of the Hox/Exd/Hth/Hox tetramer. Sequence inspection of the DMX-R revealed that the X2 mutation, which resulted in de-repression specifically in the anterior compartment, disrupts two partially overlapping matches to a consensus binding site for Forkhead (Fkh) domain proteins. With this in mind, the expression pattern of Sloppy paired 1 (Slp1), a Fkh domain factor encoded by one of two partially redundant segmentation genes, slp1 and slp2, was examined. The two slp genes are expressed in anterior compartment cells adjacent and anterior to En-expressing posterior compartment cells. In the thorax, cells expressing Dll and DMX-lacZ co-express either Slp or En at the time Dll is initially expressed. In the abdomen, the homologous group of cells, which express DMXact-lacZ (a reporter lacking the DMX-R), co-express either Slp in the anterior compartment or En in the posterior compartment. The expression patterns of Slp and En were compared with Ubx and AbdA. Ubx levels are highest in anterior, Slp-expressing cells whereas AbdA levels are elevated in posterior, En-expressing cells. In contrast, both Exd and Hth are present at similar levels in both compartments throughout the abdomen (Gebelein, 2004).

On the basis of these data, a model is presented for Hox-mediated repression of Dll in both the anterior and posterior compartments of the abdominal segments. In the anterior compartment it is proposed that Slp binds to DMX-R directly with a Ubx/Exd/Hth/Ubx tetramer. In the posterior compartment it is suggested that En binds to DMX-R directly with an AbdA/Exd/Hth/AbdA tetramer. One important feature of this model is that Antp/Exd/Hth/Antp complexes fail to form on this DNA, thereby accounting for the lack of repression in the thorax. Furthermore, the model proposes that Slp and En should, on their own, have only weak affinity for DMX-R sequences because repression does not occur in the thorax, despite the presence of these factors. The Hox/Exd/Hth/Hox complex, perhaps in conjunction with additional factors, is required to recruit or stabilize Slp and En binding to the DMX-R. Both Slp and En are known repressor proteins that directly bind the co-repressor Groucho. Thus, the proposed complexes in both compartments provide a direct link to this co-repressor and, therefore, a mechanism for repression. DNA binding and genetic experiments are presented that test and support this model (Gebelein, 2004).

To test the idea that En is playing a direct role in Dll repression, the ability of En and Hox proteins to bind to DMX-R probes was examined. On its own, En binds to DMX-R very poorly. Surprisingly, it was found that En binds DMX-R with the abdominal Hox proteins Ubx or AbdA in a highly cooperative manner. The thoracic Hox protein Antp does not bind cooperatively with En to this probe. Mutations in the Hox1 or X5 binding sites block AbdA/En binding in vitro, consistent with these mutations showing posterior compartment de-repression in vivo. In contrast, the X6, X7 and Hth mutations do not affect AbdA/En complex formation (Gebelein, 2004).

On the basis of DMX-R's ability to assemble a Hox/Exd/Hth/Hox tetramer, whether En could bind together with an AbdA/Exd/Hth/AbdA complex was tested. Addition of En to reactions containing AbdA, Exd and Hth resulted in the formation of a putative En/AbdA/Exd/Hth/AbdA complex. This complex contains En because its formation is inhibited by an anti-En antibody. A weak antibody-induced supershift is also observed. Moreover, this complex fails to form on the X5 mutant, which causes posterior compartment-specific de-repression. It is noted that En/Exd/Hth complexes also bind to the DMX-R and that it cannot be excluded that an En/Exd/Hth/AbdA complex may be important for Dll repression. The model emphasizes a role for an En/AbdA/Exd/Hth/AbdA complex because it better accommodates the cooperative binding observed between En and AbdA on the DMX-R (Gebelein, 2004).

Repression in the anterior compartments of the abdominal segments requires the sequence defined by the X2 mutation, which is similar to a Fkh domain consensus binding site. The model predicts that this sequence is bound by Slp. Consistent with this view, Slp1 binds weakly to wild type, but not to X2 mutant DMX-R probes. However, in contrast to En, no cooperative binding was observed between Slp and Hox or Hox/Exd/Hth/Hox complexes, suggesting that additional factors may be required to mediate interactions between Slp and the abdominal Hox factors (Gebelein, 2004).

Together, these results suggest that En and Slp play a direct role in DMX-lacZ and Dll repression. However, these experiments do not unambiguously determine the stoichiometry of binding by these factors. Furthermore, in vivo, additional factors may enhance the interaction between these segmentation proteins and Hox complexes, thereby increasing the stability and/or activity of the repression complexes (Gebelein, 2004).

The model for Dll repression is supported by previous genetic experiments that examined the effect of Ubx and abdA mutants on Dll expression in the abdomen. Ubx abdA double mutants de-repress Dll in both compartments of all abdominal segments. In contrast, Ubx mutants de-repress Dll in the anterior compartment of only the first abdominal segment, which lacks AbdA. abdA mutant embryos de-repress Dll in the posterior compartments of all abdominal segments, where Ubx levels are low (Gebelein, 2004).

Several genetic experiments were performed to provide in vivo support for the idea that Slp and En work directly with Ubx and AbdA to repress Dll. The design of these experiments had to take into consideration that the activation of Dll in the thorax depends on wg, and that wg expression depends on both slp and en. Consequently, Dll expression is mostly absent in en or slp mutants, making it impossible to characterize the role that these genes play in Dll repression from examining en or slp loss-of-function mutants. However, some of the mutant DMX-Rs described here provide the opportunity to test the model in alternative ways (Gebelein, 2004).

According to the model, DMX[X5]-lacZ is de-repressed in the posterior compartments of the abdominal segments because it fails to assemble the posterior, En-containing complex. Repression of DMX[X5]-lacZ in the anterior compartments still occurs because it is able to assemble the anterior, Slp-containing complex. According to this model, DMX[X5]-lacZ should be fully repressed if Slp is provided in posterior cells. A negative control for this experiment is that ectopic Slp should be unable to repress DMX[X2]-lacZ because this reporter gene does not have a functional Slp binding site. To mis-express Slp, paired-Gal4 (prd-Gal4), which overlaps both the Slp and En stripes in the odd-numbered abdominal segments, was used. As predicted, ectopic Slp repressed DMX[X5]-lacZ but not DMX[X2]-lacZ, providing strong in vivo support for Slp's direct role in Dll repression in the anterior compartments (Gebelein, 2004).

Conversely, the model posits that DMX[X2]-lacZ is de-repressed in the anterior compartment because it cannot bind Slp, but remains repressed in the posterior compartment because it is able to assemble the En-containing posterior complex. Thus, providing En in the anterior compartment should repress DMX[X2]-lacZ. A complication with this experiment is that En is a repressor of Ubx, which is the predominant abdominal Hox protein in the anterior compartment. It was confirmed that prd-Gal4-driven expression of En represses Ubx and that AbdA levels remain low at the time Dll is activated in the thorax. Consequently, ectopic En expression is not sufficient to repress DMX[X2]-lacZ, consistent with the observation that low levels of abdominal Hox proteins are present. Therefore, to promote the assembly of the posterior complex in anterior cells, En was co-expressed with AbdA using prd-Gal4. As predicted, this combination of factors repressed DMX[X2]-lacZ but not DMX[X5]-lacZ, providing strong in vivo evidence for En playing an essential role in Dll repression in the posterior compartments (Gebelein, 2004).

Several observations provide additional support for the model. First, ectopic expression of AbdA or Ubx in the second thoracic segment (T2) represses DMX[X5]-lacZ in the anterior compartment, but not in the posterior compartment. Conversely, expression of AbdA or Ubx in T2 represses DMX[X2]-lacZ only in posterior compartment cells. Second, co-expression of Slp with Ubx completely represses DMX[X5]-lacZ in T2 but does not repress DMX[X2]-lacZ in T2. Third, in those cases where repression is incomplete (for example, En + AbdA repression of DMX[X2]-lacZ in the abdomen), cells that escape repression have low levels of either an abdominal Hox protein or Slp/En. Together, these data provide additional evidence that the abdominal Hox proteins work together with Slp and En to repress Dll (Gebelein, 2004).

The segregation of cells into anterior and posterior compartments during Drosophila embryogenesis is essential for many aspects of fly development. The results presented in this study reveal an unanticipated intersection between anterior-posterior compartmentalization by segmentation genes and segment identity specification by Hox genes. Specifically, it is suggested that the abdominal Hox proteins collaborate with two different segmentation proteins, Slp and En, to mediate repression of a Hox target gene (Dll) in the anterior and posterior compartments of the abdomen, respectively. This mechanism of transcriptional repression suggests a previously unknown use of compartments in Drosophila development. The mechanism proposed here contrasts with the alternative and simpler hypothesis in which the abdominal Hox proteins would have used the same set of cofactors to repress Dll in all abdominal cells, regardless of their compartmental origin (Gebelein, 2004).

These results provide further support for the view that Hox/Exd/Hth complexes do not directly bind co-activators or co-repressors but instead indirectly recruit them to regulatory elements. Consistent with previous analyses, it is suggested that Hox/Exd/Hth complexes are important for the Hox specificity of target gene selection. Additional factors, such as Slp or En in the case of Dll repression, are required to determine whether the target gene will be repressed or activated. In the future, it will be important to dissect in similar detail other Hox-regulated elements, to assess the generality of this mechanism (Gebelein, 2004).

These results also broaden the spectrum of cofactors used by Hox proteins to regulate gene expression. Although the analysis of Exd/Hth in Drosophila and Pbx/Meis in vertebrates has provided some insights into how Hox specificity is achieved, there are examples of tissues in which these proteins are not available to be Hox cofactors and of Hox targets in which Exd and Hth seem not to play a direct role. This study shows that En, a homeodomain segmentation protein, is used as a Hox cofactor to repress Dll in the abdomen. Although the complex defined at the DMX-R includes Exd and Hth, the DNA binding studies demonstrate that Hox and En proteins can bind cooperatively to DNA in the absence of Exd and Hth. These findings suggest that En may function with Ubx and/or AbdA to regulate target genes other than Dll, and perhaps independently of Exd and Hth. Consistent with this idea are genetic experiments showing that, in the absence of Exd, En can repress slp and this repression requires abdominal Hox activity. Although these experiments were unable to distinguish whether the Hox input was direct or indirect, the results suggest that En may bind directly with Ubx and AbdA to repress slp, and perhaps other target genes (Gebelein, 2004).

Finally, these results raise the question of why a compartment-specific mechanism is used by Hox factors to repress Dll. The activation of Dll at the compartment boundary by wg may be important for accurately positioning the leg primordia within each thoracic hemisegment, but this mode of activation requires that Dll is repressed in both compartments in each abdominal segment. The utilization of segmentation proteins such as En and Slp may be the simplest solution to this problem. Compartment-specific mechanisms may also provide additional flexibility in the regulation of target genes by Hox proteins by allowing them to turn genes on or off specifically in anterior or posterior cell types. For these reasons, compartment-dependent mechanisms of gene regulation may turn out to be the general rule instead of the exception (Gebelein, 2004).

Mechanosensilla in the adult abdomen of Drosophila: engrailed and slit help to corral the peripheral sensory axons into segmental bundles

The abdomen of adult Drosophila bears mechanosensory bristles with axons that connect directly to the CNS, each hemisegment contributing a separate nerve bundle. In this study the amount of Engrailed protein was altered and the Hedgehog signalling pathway was manipulated in clones of cells to study their effects on nerve pathfinding within the peripheral nervous system. It was found that high levels of Engrailed make the epidermal cells inhospitable to bristle neurons; sensory axons that are too near these cells are either deflected or fail to extend properly or at all. Attempts were made to find the engrailed-dependent agent responsible for these repellent properties. slit was found to be expressed in the P compartment and, using genetic mosaics, evidence is presented that Slit is the responsible molecule. Blocking the activity of the three Robo genes (putative receptors for Slit) with RNAi supported this hypothesis. It is concluded that, during normal development, gradients of Slit protein repel axons away from compartment boundaries - in consequence, the bristles from each segment send their nerves to the CNS in separated sets (Fabre, 2010).

The peripheral sensory system of arthropods is segmented: neurons originate in sensilla in segmental groups in the epidermis and axons project from each of them to the corresponding segmental ganglion in the CNS. To achieve this, the nerves coming from each epidermal segment or compartment must not mix with nerves from neighbouring compartments. The epidermis of the fly and other arthropods is subdivided into a chain of anterior (A) and posterior (P) compartments, the P/A compartment boundary being the true segmental boundary. This segment boundary is recognised by neurons as they build the embryonic nervous system and is not crossed by peripheral sensory neurons in later stages. However, little is known of the molecular mechanisms responsible for this process (Fabre, 2010).

In the adult abdomen of Drosophila, the mechanoreceptive bristles are confined to a region of each A compartment; they develop de novo as sensory organ precursor cells that derive from the epidermal cells or `histoblasts' that proliferate during the pupal stage. Sensory organ precursor cells divide asymmetrically to generate a bristle and their associated neurons and supporting cells; the neurons then extend axons towards the CNS in an orderly manner (Fabre, 2008). These axons remain within their compartments of origin because they are oriented with respect to the body axes: within each A compartment, the more anteriorly situated bristle axons grow backwards, while the posteriorly situated bristle axons grow forwards, and thus both sets of axons meet to form a segmental nerve bundle in the middle of the A compartment (Fabre, 2010).

A and P compartments differ fundamentally: all the P cells but not the A cells, except for a6, express engrailed (en). The en gene encodes a homeodomain-containing transcription factor that induces hedgehog (hh) expression in P cells. Hh is a secreted morphogen that spreads into the A compartment, forming a U-shaped gradient that patterns cell fate and determines cell affinity. Only the epidermal cells of the A compartment produce Patched (Ptc) and Smoothened (Smo), proteins that act as receptors for Hh. Although the mechanosensory neurons are related to epidermal cells by lineage, it is not clear whether they retain all the compartmental properties of their origin (Fabre, 2010).

This study asked how En- and Hh-dependent information positions the neuronal cell bodies, affects the dendrites and influences the pathways followed by axons. To investigate this, cell identities wer altered by manipulating the relevant genes (en, hh, ptc and smo) within clones of cells, and effects on the neurons were examined. Strikingly, cells with P identity, but located within an A compartment, repel nearby neurons. This neuronal repulsion is not directly mediated by En or Hh, but indirectly by activating the expression of slit, a molecule previously implicated in neuronal pathfinding. Also, the response to Slit appears to be mediated by one or more of the Robo proteins. It is proposed that, during normal development, the secretion of Slit from P cells creates a Slit gradient in each A compartment that helps position neurons and orient axon outgrowth and thereby ensures segmental bundling of axons (Fabre, 2010).

In the wild-type fly (and perhaps therefore also in many other invertebrates), it was found that Sli is normally made in the P compartments, spreading forwards and backwards to repel neurons at the back and the front of the A compartments. As a consequence, the axons meet in the middle of the A compartments. Thus, En regulates sli expression to form a Sli gradient, the axons growing away from the source of Sli and down that gradient. Sli may also drive oriented nucleokinesis of the mechanosensory cell bodies away from the compartmental boundaries (Fabre, 2010).

Gradients of morphogens, such as Wingless (Wg), Hh and Decapentaplegic, can act at short or long range to specify cell identity and have also been implicated in axon pathfinding. Numerous studies have concluded that Hh can act as an axonal repellent or attractant, and that axons can respond directly to the gradient of Hh. Surprisingly, in the abdomen evidence is presented that Hh does not guide the mechanosensory neurons. No dependence on the Hh receptors Ptc or Smo is seen. This raises the possibility that some of the previously described effects of Hh might also be indirect. Indeed, in the zebrafish forebrain (Barresi, 2005), Hh acts to guide commissural and retinal axons indirectly by regulating sli expression (Fabre, 2010).

In vertebrates, En affects axon routing. In invertebrates, En modifies axon morphology via the expression of cell adhesion molecules such as Connectin and Neuroglian or the cell adhesion receptor Frazzled. In the cockroach cercus, En is essential for axonal pathfinding, perhaps acting directly on genes needed for guidance and synaptic recognition. There is a hypothesis that En acts directly: En protein has structural domains that could regulate nuclear export, secretion and cell-internalisation, processes also needed for axon pathfinding and target recognition. However, the current experiments in the fly abdomen point to a different conclusion. When smo- en- and ptc- en- clones were produced in the P compartment, mechanosensory axons traversed anterior cells of the P compartment and a6 cells in which en is expressed. Thus, it is unlikely that En itself repels axons in the abdomen of Drosophila. Evidence suggests instead that En drives the expression of sli autonomously, the effects of En on pathfinding being due to local gradients of Sli concentration. The behaviour of axons emanating from smo- en- and ptc- en- clones in the P compartment can be understood in this context: the clones are small and even though they do not themselves secrete Sli (because they are transformed into A cells), they nevertheless find themselves in a Sli gradient, high behind and lower in front. Axons leaving such clones behave as expected and grow down that gradient (Fabre, 2010).

The mode of action of Sli in neuronal and axonal repulsion has been studied in numerous systems. In vertebrates, a gradient of one or more of the three Slit genes can induce the arrest of growth cones, similar to that observed here with ptc- and en-expressing clones, which are ectopic sources of Sli (Fabre, 2010).

In other systems, Sli is received by one or more Robo receptors acting with Dock. In Drosophila, the three Robo genes are typically expressed in distinct but overlapping regions, but only Robo binds to Dock. This study presents evidence that Robo and the co-receptor Dock are expressed in the mechanosensory neurons and also that robo3 is expressed in the multidendritic neurons. Results with RNAi suggest that all three Robo genes are required for the normal fasciculation of the mechanosensory axons; the strongest effect was found with RNAi for robo2. Note that knockdown of any one of the Robo genes is unlikely to produce a very clear phenotype as they can partially substitute for each other (Fabre, 2010).

Flies carrying sli.lacZ suggest that sli is normally strongly expressed only at the back of the P compartments, raising the question of how its expression is controlled in the wild type. In the adult tergites, wg is expressed at the rear of each A compartment and Wg protein is thought to cross over the A/P border to form a gradient that patterns the P compartment. If so, and if a high concentration of Wg were to inhibit sli expression, then sli expression might be blocked in the anterior part of P (p3), but allowed in the posterior part of P. There are two other arguments supporting this hypothesis. First, wg is not (or is weakly) expressed in the most lateral tergite, which could explain why the band of sli expression is broader laterally and fills, or almost fills, the P compartment there. Second, wg is not expressed in the pleura, where sli.lacZ expression is ubiquitous. By contrast, in the sternites, wg is expressed and there sli.lacZ is confined to the P compartments. It could therefore be that ptc- as well as the en-expressing clones that are transformed towards P identity would not express wg themselves. Thus, when located far from the endogenous source of Wg they should escape repression and transform into p1, which is of extreme posterior P identity, and become sources of Sli, as observed (Fabre, 2010).

Sli might work with other guidance cues in the fly abdomen. In the Drosophila eye, disruption of the Sli/Robo mechanism disturbs the boundary between the lamina and the distal cell neurons. It has been suggested that the Fasciclin adhesion molecules also support the boundary: Fas3 is expressed in the region where distal cell neurons are found, and Fas2 is expressed by the photoreceptor axons that carry Hh to the lamina. It is suspected that Fas2 and Fas3 might contribute to corralling neurons inside of the A compartment by promoting axonal bundling to the APN (Fabre, 2010).

An individual axon might be pushed from behind by a chemorepellent, pulled from afar by a chemoattractant, and hemmed in by attractive and repulsive local cues. These signals constitute what Ramón y Cajal proposed to be an 'intelligent force' guiding axons. It is not easy to dissect out these various signals, this paper has documented one repulsive signal, Sli, that hems in neurons and helps bundle segmental sets of sensory neurons in an arthropod (Fabre, 2010).

back to Engrailed Targets of activity part 1/2

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

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