Regulation of engrailed by segment polarity genes

Shaggy/zeste-white 3 acts as a repressor of en autoregulation. Genetic epistasis experiments indicate that wg signaling operates by inactivating the zw3 repression of en autoactivation (Siegfried, 1992).

hopscotch (hop), the Drosophila JAK kinase homolog, is required maternally for the establishment of the normal array of embryonic segments. In hop mutant embryos, although expression of the gap genes appears normal, there are defects in the expression patterns of the pair-rule genes even-skipped, runt, and fushi tarazu, as well as the segment-polarity genes engrailed and wingless. The effect of hop on the expression of these genes is stripe-specific (Binari, 1994).

In wingless mutants, engrailed expression comes on normally but fails to be maintained. In a null pangolin mutant, engrailed expression is initiated normally, but the stripes of engrailed expression begin to decay by late stage 9, particularly in midlateral regions and along the ventral midline. This effect resembles that of a zygotic armadillo mutation or of removal of functional Wingless at the end of stage 9 (van de Wetering, 1997).

The normal growth of the wing disc requires that posterior segmental-specific genes, such as hedgehog and engrailed not be expressed in cells of the anterior compartment. Hedgehog has the capacity to activate engrailed in the anterior compartment but both hedgehog and engrailed are specifically repressed in anterior cells by the activity of the neurogenic gene groucho. In groucho mutant discs, hedgehog and engrailed are expressed at the dorsoventral boundary of the anterior compartment, leading to the ectopic activation of decapentaplegic and patched and to a localised increase in cell growth associated with pattern duplications. The presence of engrailed in the anterior compartment (brought on by a failure to regulate en expression) causes the transformation of anterior into posterior structures and structural duplication (de Celis, 1995).

It is not known whether the secretion of Wingless protein acts only on cells near its site of synthesis or whether it has a longer range. Mosaic Drosophila embryos have been used to estimate the range of Wingless as it acts to maintain expression of engrailed. Expression of engrailed is often sustained in those wingless- cells that are located near a wildtype patch of tissue, but this is not invariably so. Also, the numbers of cells maintaining engrailed expression are small. It seems that wingless-expressing cells sustain engrailed expression only in adjoining cells. The wingless gene is also needed for maintenance of its own expression; using mosaics, it has been shown that the range of this action is similarly short (Vincent, 1994).

Wg and Wnt molecules tightly associate with membrane and extracellular matrix and appear not to be readily soluble. Thus, it is unlikely that these proteins freely diffuse through extracellular spaces. Rather, Wg appears to be transported via active cellular processes. This phenomenon was first demonstrated using the shibirets (shits) mutation to block endocytosis. shi encodes the fly dynamin homolog, a GTPase required for clathrin-coated vesicle formation. Rather than the broad, punctate Wg protein distribution normally found over several cell diameters on either side of the wg-expressing cells, shi mutant embryos show high level accumulation of Wg around the wg-expressing cells. Structure/function analysis of the Wg molecule further supports the idea that active transport of the ligand is essential. Four mutations within wg have been isolated that specifically disrupt Wg transport without abolishing signaling activity. These mutant molecules generate a restricted response within the segment, as assayed by both cuticular pattern elements and molecular events. Homozygous mutant embryos produce naked cuticle but little denticle diversity, and show narrowed domains of Wg protein distribution and Arm stabilization. Three of these four mutations are single amino acid substitutions; each affects a residue that is highly conserved throughout the Wnt family, suggesting that ligand transport may be an important general aspect of Wnt function (Moline, 1999 and references).

Since wild-type Wg signaling activity is required for stabilization of en expression, En stripes of normal width indicate that sufficient functional Wg contacts both rows of en-expressing cells to produce normal target gene regulation. This result demonstrates that expression of shiD does not interfere with Wg signal transduction and supports the idea that moderate level shiD expression reduces, but does not eliminate, transport of Wg across the affected domain. In contrast, embryos expressing high level shiD in the en domain show a narrowed stripe of En antibody staining, suggesting that Wg can no longer traverse the first row of en-expressing cells to stabilize en in the second row. However, because of the severe effects of a more complete endocytotic block, these embryos do not secrete cuticle properly and so the effects on cuticle pattern are not interpretable (Moline, 1999).

During early stages of wild-type embryogenesis, Wg protein can be detected at high levels in cells both anterior and posterior to the wg-expressing row of cells. Diversity of denticle types, as well as stabilization of en expression in the adjacent cells, are specified by Wg activity during these early stages of embryonic development. By mid-stage 10, when Wg is no longer required for denticle specification or en stabilization, the Wg protein distribution shifts and Wg appears to be excluded from the en-expressing cells. This exclusion is not observed in naked cuticle (nkd) mutants at the same stage. Rather, Wg protein continues to be detected in cells on either side of the wg-expressing row of cells and the levels become substantially higher due to the ectopic stripe of wg expression. These results suggest that nkd gene function may play a role in the posterior restriction of Wg protein that occurs during stage 10. Hence the mutant phenotype is rescued dramatically when this restriction is produced artificially, by expressing shiD in the en-expressing row of cells. All stage 11 and 12 embryos derived from this cross show posterior restriction of Wg protein, indicating that the nkd homozygotes do not exhibit excess posterior movement of Wg under these conditions. It is suspected that, in wild-type embryos, this restrictive function is not limited to the en-expressing cells. If this were the case, then one would expect to observe excess naked cuticle replacing denticle belts when wg+ is expressed in the en domain. Instead, en-Gal4-driven wg+, either alone or when co-expressed with shiD, does not produce substantial amounts of ectopic naked cuticle. Thus, it seems likely that some ability to restrict posterior Wg movement during later stages is shared by the rows of cells at the anterior of each segment (Moline, 1999).

Specialized groups of cells known as organizers govern the establishment of cell type diversity across cellular fields. Segmental patterning within the Drosophila embryonic epidermis is one paradigm for organizer function. Here cells differentiate into smooth cuticle or distinct denticle types. At parasegment boundaries, cells expressing Wingless confront cells co-expressing Engrailed and Hedgehog. While Wingless is essential for smooth cell fates, the signals that establish denticle diversity are unknown. wg mutants are shown to have residual mirror-symmetric patterns that are due to an Engrailed-dependent signal specifying anterior denticle fates. The Engrailed-dependent signal acts unidirectionally and Wg activity imposes this asymmetry. Reciprocally, the Engrailed/Hedgehog interface imposes asymmetry on Wg signaling. Thus, a bipartite organizer, with each signal acting essentially unidirectionally, specifies segmental pattern (Gritzan, 1999).

Reciprocal signaling between Wg- and En/Hh-expressing cells stabilizes one another’s expression, consolidating the parasegmental body plan. At this time, signaling is effective only locally, thereby restricting the expression of the other signal to a narrow strip of cells. This ensures that the bipartite organizer remains a line source rather than broadening during patterning. Ventrally, the organizer specifies half the fates as smooth cell types and compelling evidence demonstrates that Wg specifies these. This step occurs after Wg stabilizes En/Hh expression. In contrast, the identity of the signals responsible for specifying the diverse denticle cell types has been less clear. By bypassing the need for Wg input to En cells, row 2- to 4-type denticles are specified in the absence of Wg. This conclusion was confirmed by analyzing the control of Rhomboid expression in cells flanking the En domain, since Rhomboid is essential for the proper differentiation of row 1- 4 fates. Previous studies have indicated that denticle diversity arises early, around the time Wg stabilizes En/Hh expression. The data suggest that the stabilization of En/Hh expression establishes the conditions to generate denticle diversity, but that diversity is not specified until later as reflected in the induction of Serrate and Rhomboid (~7-8 hours AEL). Indeed, excess Hh delivered at late times can broaden Rho stripes and this still affects denticle diversity. En/Hh influence can only extend up to row 4. Consistent with this, Hh signaling sets the anterior Serrate expression boundary. Since Wg signaling sets Serrate’s posterior boundary, the Serrate domain defines a region of positional values within the segment where Hh and Wg cooperate in patterning. In fact, Serrate expression is perhaps a molecular marker for a default state, as its expression is almost global in cases where only row 5 cell types are specified, such as in wg;en doubly mutant embryos. Serrate would indeed be globally expressed in a wg;en;hh triple mutant (Gritzan, 1999).

The data suggest that both Wg- and the En/Hh-expressing cells establish a block so that each signal operates largely unidirectionally. Rho is repressed by Wg signaling and it is important to block activation of the Wg pathway from cells posterior to the En/Hh domain. This block seems to be Hh-dependent because Rho expression is greatly reduced in hh mutants but maintained in wg;smo double mutants. Also, Wg imparts asymmetry to signals from En cells. Without Wg function, Hh can signal more strongly to the anterior, as compared to wild type. Importantly, the cuticle pattern generated is also now symmetric relative to the En/Hh cells, strongly suggesting that a normally biased signal is now sent or received bidirectionally (Gritzan, 1999).

The signals expressed from an organizer are developmentally potent, because they confer pattern over a large cellular field. Thus, once the appropriate expression of these signals is established, an important facet to organizer function is the temporal and spatial restriction of signaling. In some cases, activation of a signaling pathway induces an inhibitor of that same pathway. In examining fate across the En domain, Rho-dependent activation of the Egfr pathway posterior to the En cells leads to the induction of the diffusible inhibitor, Argos. Argos attenuates Egfr activation in anterior En cells, allowing Wg signaling to win out, leading to proper fate specification of anterior En cells (Gritzan, 1999).

In discs, signals emanate from compartment boundaries, which are inherited from the embryonic parasegment boundaries. For the compartment organizer, Hh locally induces a line source for a long-range morphogen, either Wg or Dpp, which each act symmetrically. Cells exposed to the same ligand concentration on opposite sides of the source, adopt the same positional value. However, the anterior compartment cell will select a different fate from the posterior compartment cell at the same positional value. This is because the posterior compartment expresses a unique transcription factor, En, and therefore is programmed intrinsically with a different response repertoire to the morphogen. In parasegments, 10 of 12 rows of cells are intrinsically equivalent anterior compartment cells, while the posterior En-expressing compartment only accounts for 2 cells. Thus, compartmental organization, with each compartment sporting unique transcription factors, can only make a small contribution toward distinguishing cell fate selection. Perhaps for this reason patterning cannot rely on induction of one longer-range morphogen. Instead, a bipartite organizer is used with each signal acting essentially unidirectionally. Equivalent cells to each side of the parasegment boundary develop differently because they are exposed to different signals (Gritzan, 1999).

The secreted protein Hedgehog (Hh) transmits a signal from posterior to anterior cells that is essential for limb development in insects and vertebrates. In Drosophila, Hh has been thought to act primarily to induce localized expression of Decapentaplegic and Wingless, which in turn relay patterning cues at long range. Hh plays an additional role in patterning the wing. Engrailed is expressed in the posterior compartment and in anterior cells close to the AP boundary. Anterior En levels decrease rapidly with distance from the posterior comparment. Expression of En in anterior cells is thought to be regulated by Hh. Consistent with this, anterior clones of smoothened fail to express En, whereas posterior smo clones express En normally. Likewise, anterior expression of Ptc is regulated by Hh. As Hh acts directly to induce Dpp, to upregulate Ptc in a broad domain of 8-10 cell diameters, and to induce En in a narrow band of cells close to the AP boundary, it is possible that the broad spatial domains of Ptc and Dpp and the narrow domain of En reflect requirements for different levels of Hh activity. Use of a temperature sensitive allele of hh shows that dpp can be activated by levels of Hh activity that are below the minimal levels required to activate En (Strigini, 1997).

By replacing endogenous Hh activity with that of a membrane-tethered form of Hh, it has been shown that Hh acts directly to pattern the central region of the wing, in addition to its role as an inducer of Dpp. Comparing the biological activities of secreted and membrane-tethered Hh provides evidence that Hh forms a local concentration gradient and functions as a concentration-dependent morphogen in the fly wing. Such tethered Hh can only induce En in immediately adjacent cells. Tethered Hh expressing cells also act on adjacent cells to induce dpp. It is noted that dpp expression is reduced in cells that express En at high levels, suggesting that En represses dpp. Membrane-tethered Hh flies develop to pharate adults that completely lack wings. Restoring Hh during development allows flies to form wings that show substantial rescue of anterior and posterior structures while missing structures from the cental region. These results suggest that Hh plays an important role in directly patterning the central region of the wing, while Dpp is primarily responsible for patterning at long range (Strigini, 1997).

The Suppressor of fused [Su(fu)] encodes a protein with a PEST sequence involved in rapid protein turn-over. Fused is phosphorylated in response to the Hh signal. A large protein complex that includes Cubitus interruptus, Costal-2 and Fused binds to microtubules and has been implicated in the regulation of Ci cleavage and accumulation, and may be involved in mediating the Hh signal. Although Su(fu) activity is apparently dispensable in a wild-type background, its absence fully suppresses all the fused mutant phenotypes. These data suggest that the activation of Fused in cells receiving the Hh signal relieves the negative effect of Su(fu) on the pathway (Alves, 1998 and references).

The roles of Fused and Su(fu) proteins were examined in the regulation of Hh target gene expression in wing imaginal discs, by using different classes of fu alleles and an amorphic Su(fu) mutation. The fused phenotype consists of a vein 3 thickening and vein 4 disappearance with reduction of the intervein region. At the wing margin, the anterior double row bristles reach the fourth vein. Fused protein is present throughout the entire wing level, but its level is much higher in the anteior compartment. In contrast, fused transcripts are uniformly distributed, suggesting that fused is regulated post-transcriptionally. Observations using fused clones indicate that only fused minus clones located in the region extending between veins 3 and 4 generate a mutant phenotype, consisting of extra-veins, which often bear campaniform sensillae characteristic of vein 3. Thus Fused kinase activity is required at the anterior/posterior (AP) boundary in the anterior compartment. At the AP boundary, Fu kinase activity is involved in the maintenance of high ptc expression and in the induction of late anterior engrailed expression. These combined effects can account for the modulation of Ci accumulation and for the precise localization of the Dpp morphogen stripe. Here, at the AP boundary, Hh signal activates the Fu kinase, leading to a modified active form of Ci required for anterior en expression and high ptc expression. Su(fu) suppresses all fused phenotypes associated with the AP boundary, suggesting that Su(fu) normally functions to antagonize the effects of Fused (Alves, 1998).

Surgically fragmented Drosophila appendage primordia (imaginal discs) engage in wound healing and pattern regulation during short periods of in vivo culture. Prothoracic leg disc fragments possess exceptional regulative capacity, highlighted by the ability of anterior (A) cells to convert to posterior (P) identity and establish a novel posterior compartment. This AP conversion violates developmental lineage restrictions essential for normal growth and patterning of the disc, and thus provides an ideal model for understanding how cells change fate during epimorphic pattern regulation. Evidence is presented that the secreted signal encoded by hedgehog directs AP conversion by activating the posterior-specific transcription factor engrailed in regulating anterior cells. In the absence of hedgehog activity, prothoracic leg disc fragments fail to undergo AP conversion, but can still regenerate missing anterior pattern elements. It is suggested that hedgehog-independent regeneration within the anterior compartment (termed integration) is mediated by the positional cues encoded by wingless and decapentaplegic. Taken together, these results provide a novel mechanistic interpretation of imaginal disc pattern regulation (Gibson, 1999).

The observation that A cells switch to P identity during pattern regulation in L1 first thoracic leg disc fragments is a curious exception to the rule of compartmental lineage restriction during Drosophila appendage development. This prompted an inquiry into how AP conversion is achieved on the molecular level. Since En is normally expressed in all P cells, a test was performed to see if AP conversion correlates with activation of En in cultured disc fragments. Third instar L1 discs were cut into fragments and cultured in vivo. The anterior 1/4 (A1/4), consists of the anterior/dorsal 1/4 segment of the disc, and posterior 3/4 (P3/4) consists of the posterior 1/2 of the disc, in addition to the anterior/ventral 1/4 of the disc. Approximately 50% of A1/4 and P3/4 fragments produced novel En expression domains within 96 hours in vivo. The size and shape of nascent posterior compartments vary from small clusters of En-expressing cells to large domains almost indistinguishable from the endogenous P compartment. This suggests that stochastic variations (subtle differences in cut sites, disc morphology and precise age of the disc donor) might influence the timing and extent of pattern regulation. In a significant number of cases, however, A1/4 disc fragments regenerate a new P compartment, which restore the proportions of a whole disc, and P3/4 fragments duplicate a new En-expression domain in mirror-symmetric opposition to the endogenous P compartment (Gibson, 1999).

To directly observe the cellular origin of nascent En domains in regulating disc fragments, random GFP-labeled clones were generated in larvae 1 day prior to fragmentation and injection into host animals. Half of clone-bearing P3/4 fragments produce a duplicated En domain; many with a well-defined novel compartment boundary suitable for detailed analysis. Anterior cells are shown to be able to directly convert to P identity during pattern regulation in L1 disc fragments. The size and distribution of GFP + clones in duplicated discs provide additional insights into the dynamics of proliferation in the blastema. Nascent P compartments are never entirely composed of GFP + cells (0/12), indicating that AP conversion is a polyclonal event in duplicating disc fragments. Single GFP + clones often occupy up to 10%-20% of the whole duplicated area, suggesting that as few as 5-10 founder cells participate in duplicative growth. This agrees with a previous clonal analysis, which suggests that similar cell numbers generate disc duplications during cell-lethality-mediated pattern regulation (Gibson, 1999).

Endogenous en expression in the L1 disc is activated during embryogenesis by the transcription factor Fushi tarazu. However, ftz-lacZ expression is not detected in cultured disc fragments, suggesting an alternate mechanism for activation of en during pattern regulation. Ectopic hh activity is known to induces en in A cells of the abdominal tergites, and wing and leg imaginal discs, indicating that hh might be required to activate en in cultured disc fragments. To test this hypothesis, L1 discs from flies with temperature sensitive hh larvae raised at permissive temperature were fragmented and cultured under restrictive conditions. In the absence of Hh, cultured A1/4 fragments are small and extremely difficult to recover from hosts. Of those recovered, only a few regenerate new En domains, compared with 37% at the permissive temperature. The effect of hh loss is more pronounced in P3/4 fragments; none duplicate at restrictive temperature while 39% duplicate under permissive conditions. Loss of hh clearly blocks en-activation in both fragments. At non-permissive temperature, temperature sensitive hh disc fragments also fail to produce regenerated/duplicated posterior leg structures upon forced differentiation in host larvae. Taken together, these experiments demonstrate that hh is necessary to activate en and establish a new P compartment in cultured L1 disc fragments. Because loss of hh does affect endogenous En expression in cultured P3/4 fragments, the possibility that Hh is simply required to maintain En during culture can be ruled out (Gibson, 1999).

P3/4 fragments normally duplicate existing pattern elements, but only rarely regenerate missing structures. In the absence of hh, however, P3/4 fragments gain the ability to regenerate all missing anterior leg structures. This observation suggests a paradox: if hh is required for normal development, how do P3/4 fragments regenerate in its absence? During normal leg disc development, Hh secreted from P cells acts primarily through induction of wg and dpp in A cells along the compartment boundary. Secreted Wg and Dpp subsequently pattern both compartments through a variety of mechanisms, including activation of Distalless (Dll) at the center of the disc. Once established, late-third instar wg and dpp expression domains may be sufficient to direct anterior pattern regulation even if Hh levels are severely reduced. In intact discs, ectopic Wg interacts with Dpp to cause overgrowth and anterior pattern duplications suggesting that wg/dpp interactions might be sufficient to direct anterior compartment regeneration without direct input from hh. This assertion is supported by the fact that hh is not required for maintenance of dpp in third instar wing discs, and predicts that Wg and Dll domains are maintained in the absence of Hh in cultured disc fragments (Gibson, 1999).

To test this hypothesis, P3/4 fragments cultured in the absence of hh were immunostained with antibodies directed against Wg and Dll. Temperature sensitive hh P3/4 fragments cultured at restrictive temperature maintain Wg and Dll at reduced levels (relative to wild-type) in their endogenous domains. These fragments do not produce duplicated Wg or Dll domains. At permissive temperature Wg and Dll levels are also significantly reduced (possibly due to weak hypomorphic effects of temperature sensitive hh), but both domains are duplicated or expanded in most fragments analyzed. These data show that wild-type levels of Hh are required for activation, but not maintenance, of Wg and Dll domains in cultured disc fragments. It is concluded that P3/4 fragments regenerate missing anterior pattern elements in an hh-independent fashion and it is suggested that this occurs by a process of integration between established wg/dpp domains juxtaposed through wound healing (Gibson, 1999).

Peripodial cells contribute to a squamous epithelium that covers the columnar epithelium of the disc proper and participates in disc eversion and metamorphosis. In wing disc fragments, peripodial cells form a transient heterotypic contact with regulating columnar cells at the site of wound healing. Substantial evidence is found that a specific population of dorsolateral peripodial membrane cells act as the source of Hh in cultured L1 disc fragments. In both A1/4 and P3/4 disc fragments, peripodial cells express hh-lacZ and En prior to and during in vivo culture, and appear to fuse with anterior cells in the regenerating disc epithelium during wound healing. In A1/4 disc fragments, hh-lacZ is not expressed in nascent P cells until after a new En-domain is clearly visible. This rules out the possibility that loss of AP conversion in temperature sensitive hh mutants results from hh-dependent growth effects within the nascent P compartment and makes peripodial cells the sole potential source of Hh in regenerating A1/4 fragments. These observations indicate that Hh from peripodial cells activates en during a transient fusion between peripodial and columnar cells at the wound site. As confirmation, En-expressing peripodial cells still fuse to the wounded epithelium in the absence of Hh, but do not induce En in surrounding columnar cells. It is shown that L2 discs lack En/Hh-expressing peripodial cells and cannot regenerate posterior leg structures (Gibson, 1999).

During Drosophila wing development, Hedgehog (Hh) signaling is required to pattern the imaginal disc epithelium along the anterior-posterior (AP) axis. The Notch (N) and Wingless (Wg) signaling pathways organise the dorsal-ventral (DV) axis, including patterning along the presumptive wing margin. A functional hierarchy of these signaling pathways is described that highlights the importance of the competing influences of Hh, N, and Wg in establishing gene expression domains. Investigation of the modulation of Hh target gene expression along the DV axis of the wing disc has revealed that collier/knot (col/kn), patched, and decapentaplegic are repressed at the DV boundary by N signaling. Attenuation of Hh signaling activity caused by loss of fused function results in a striking down-regulation of col, ptc, and engrailed (en) symmetrically about the DV boundary. This down-regulation depends on activity of the canonical Wg signaling pathway. It is proposed that modulation of the response of cells to Hh along the future proximodistal (PD) axis is necessary for generation of the correctly patterned three-dimensional adult wing. These findings suggest a paradigm of repression of the Hh response by N and/or Wnt signaling that may be applicable to signal integration in vertebrate appendages (Glise, 2002).

The Hh target genes col/kn and ptc, in contrast to en, are repressed in a wild type wing in cells corresponding to the presumptive wing margin. It has been demonstrated, using both gain- and loss-of-function experiments, that this repression is mediated by N signaling and that its inhibition results in aberrant morphogenesis of the wing. Hh signaling, achieved either by overexpression of Hh or loss of Ptc activity, is not sufficient to give maximum activation of Hh targets in cells of the prospective wing margin, suggesting that a finely tuned balance of activation and repression is required to achieve the appropriate biological output. However, overexpression of a stabilized form of Ci under the ptc-Gal4 driver results in the activation of Col in the prospective wing margin and defects in wing margin differentiation, indicating that N repression can be overcome by hyperactivity of the Hh signaling pathway. N signaling may lead to the repression of col, ptc, and dpp directly or it may act indirectly by affecting the ability of Ci to act as a transcriptional activator. Since expression of en, which requires the highest level of Hh signaling and Ci activity, appears immune to N repression, the former possibility is favored (Glise, 2002).

Differential regulation of Hedgehog target gene transcription by Costal2 and Suppressor of Fused

The mechanism by which the secreted signaling molecule Hedgehog (Hh) elicits concentration-dependent transcriptional responses from cells is not well understood. In the Drosophila wing imaginal disc, Hh signaling differentially regulates the transcription of target genes decapentaplegic (dpp), patched (ptc) and engrailed (en) in a dose-responsive manner. Two key components of the Hh signal transduction machinery are the kinesin-related protein Costal2 (Cos2) and the nuclear protein trafficking regulator Suppressor of Fused [Su(fu)]. Both proteins regulate the activity of the transcription factor Cubitus interruptus (Ci) in response to the Hh signal. This study analyzed the activities of mutant forms of Cos2 in vivo and found effects on differential target gene transcription. A point mutation in the motor domain of Cos2 results in a dominant-negative form of the protein that derepresses dpp but not ptc. Repression of ptc in the presence of the dominant-negative form of Cos2 requires Su(fu), which is phosphorylated in response to Hh in vivo. Overexpression of wild-type or dominant-negative cos2 represses en. These results indicate that differential Hh target gene regulation can be accomplished by differential sensitivity of Cos2 and Su(Fu) to Hh (Ho, 2005).

The data suggest that the activities of Cos2 and Su(fu) are independently regulated by different concentrations of Hh along the gradient that forms from posterior to anterior. In the anterior cells distant from the AP boundary, little or no Hh is received and target genes are silent. In these cells, Cos2 is required for proteolytic processing of Ci into its repressor form and possibly for the delivery of CiFL for lysosomal degradation. The data suggest that Cos2 requires an intact P-loop for its role in these events. Cos2 ATPase activity may be inhibited in cells receiving very low levels of Hh, preventing Ci proteolysis and stabilizing CiFL. The stabilization of CiFL results in the activation of dpp. Nearer the AP border, where higher levels of Hh are received, Su(fu) becomes phosphorylated, inactivating its negative regulatory hold on Ci, while inhibition of the ATPase activity of Cos2 continues to allow stabilization of Ci. In this situation, ptc and dpp are transcribed. Finally, at the highest levels of Hh signaling adjacent to the AP border, Cos2 is required for activation of the pathway and the expression of en. S182N expression, or cos2 over-expression, inhibits the induction of en by endogenous Hh in these cells. The elements of this model are addressed below (Ho, 2005).

Ci plays a central role in determining which genes are repressed or activated in response to different concentrations of Hh. In order to activate target genes such as dpp or ptc, Ci must be stabilized in its full-length form. In wild-type discs, Hh stabilizes Ci by antagonizing molecular events that reduce the concentration of nuclear CiFL. In addition to the constitutive nuclear export of Ci, there are two ways CiFL concentration is reduced: full-length Ci is proteolytically processed into a repressor form; and CiFL is degraded by a lysosome-mediated process involving a novel protein called Debra. In these experiments, the stabilization of CiFL was accomplished by expressing S182N in responsive cells, which antagonizes Cos2 repressor activity and results in the accumulation of high levels of CiFL, with minimal effects on the levels of CiR. This same type of differential effect on CiR and CiFL is accomplished by Debra, which causes the lysosomal degradation of CiFL without affecting the production of CiR. Cos2 and Debra may act in concert to destabilize CiFL, while Cos2 may also aid in the production of CiR via a Debra-independent mechanism. This would involve presenting Ci to the kinases, PKA, CKI and GSKß (Shaggy) for phosphorylation and processing by the proteasome. Since Debra regulates Ci stability in limited areas of the wing disc but S182N can stabilize Ci throughout the anterior compartment, it is likely that S182N interferes with both Debra-dependent and Debra-independent mechanisms of Ci stability to achieve the observed effect: cell-autonomous stabilization of CiFL leading to derepression of dpp (Ho, 2005).

These results suggest that Cos2 may use its ATPase activity to transport Ci to a location where it becomes phosphorylated in preparation for processing, or to the site of processing itself. Alternatively, the ATPase activity may be important for regulating the conformation of Cos2 and its binding to partners such as Smo, Su(fu), Fu and Ci, which would be a novel role for the P-loop in a kinesin-related protein. The S182N mutation may lock Cos2 in a conformation that changes association with binding partners. For example, S182N may decrease the ability of Cos2 to bind Ci, releasing Ci from the cytoplasm, resulting in an increased level of CiFL in the nucleus and the activation of dpp (Ho, 2005).

The human ortholog of Suppressor of fused is a tumor suppressor gene. Su(fu) can associate with Ci, and with the mammalian homologs of Ci, the Gli proteins, through specific protein-protein interactions. Through these interactions, Su(fu) controls the nuclear shuttling of Ci and Gli, as well as the protein stability of CiFL and CiR. Flies homozygous for Su(fu) loss-of-function mutations are normal, so the importance of Su(fu) becomes evident only when other gene functions are thrown out of balance, as in a fu mutant background, with extra or diminished Hh signaling caused by ptc, slimb and protein kinase A mutations or when altered Cos2 is produced (Ho, 2005).

To activate ptc transcription in the wing disc, two conditions have to be met simultaneously: CiFL must be stabilized, and the activity of Su(fu) must be reduced. Removal of Su(fu) changes S182N from a ptc repressor into a ptc activator. Removal of Su(fu) may result in the modification, activation or relocalization of CiFL, or in further sensitizing the system to stabilized CiFL. In Su(fu) homozygous animals, the quantity of CiFL and CiR proteins is greatly diminished, and Su(fu) mutant cells are more sensitized to the Hh signal. The lower levels of both CiFL and CiR in mutant Su(fu) cells may contribute to the sensitivity of these cells to Hh, since a small Hh-driven change in the absolute concentration of either form of Ci would result in a significant change in the ratio between the two proteins. Both CiFL and CiR bind the same enhancer sites, so their relative ratio is likely to be important in determining target gene expression. S182N expression tips the ratio of CiFL to CiR toward CiFL, and reducing the absolute quantities of both Ci isoforms by removing Su(fu) will enhance this effect. Furthermore, Su(fu) binds Ci and sequesters it in the cytoplasm in a stoichiometric manner Reducing the amount of Su(fu) should release more CiFL to the nucleus to activate ptc (Ho, 2005).

The activity of Su(fu) must be regulated or overcome so that target genes can be activated at the right times and places in response to Hh. The regulation of Su(fu) activity may occur by Hh-dependent phosphorylation. A phosphoisoform of Su(fu), Su(fu)-P, was detected in discs where GAL4 was used to drive extra Hh expression. At high concentrations of Hh, the phosphorylation of Su(fu) is not antagonized by overexpression of cos2 or either of the cos2 mutants, suggesting that phosphorylation of Su(fu) occurs independently of Cos2 function. One kinase involved in the phosphorylation of Su(fu) is the Ser/Thr kinase Fused, a well-established component of Hh signal transduction. It is not known whether the phosphorylation of Su(fu) by Fu is direct or indirect (Ho, 2005).

The phosphorylation state of Su(fu) may be an important factor in determining Hh target gene activity. Phosphorylation of an increasing number of Su(fu) molecules with increasing Hh signal may gradually release Ci from all of the known modes of Su(fu)-dependent inhibition, such as nuclear export and recruitment of repressors to nuclear Ci, leading to higher levels of CiFL in the nucleus and the activation of Hh target genes such as ptc (Ho, 2005).

Anterior en expression was used as an in vivo reporter of high levels of Hh signaling. cos2 mutant cells at the AP boundary fail to activate en, suggesting that Cos2 plays a positive regulatory role in en regulation. S182N, S182T and Cos2 overexpression mimics the cos2 loss-of-function condition with respect to en: en remains off in these cells. One interpretation of these data is that all the Cos2 proteins are able to associate with another pathway component, such as Smo, and overproduction of any of them inactivates some of the Smo in non-productive complexes not capable of activating en (Ho, 2005).

In contrast to the activity of all the other mutations generated, deletion of the C terminal domain creates a protein (Cos2DeltaC) that represses normal dpp, ptc and en expression in the wing disc. In this in vivo assay, Cos2DeltaC acts just like wild-type Cos2. A similar deletion has been shown to retain function in cell culture assays. This mutant, expressed under the control of its endogenous promoter, rescues the lethality and wing duplication phenotypes of a cos2 loss-of-function allele over a cos2 deficiency. The results of the rescue experiment bring up a new possibility: that the C-terminal domain of Cos2, and the Cos2-Smo interaction via the C terminus of Cos2, is not necessary for repressor activities of Cos2. Alternatively, Cos2DeltaC could complement or boost the activity of the hypomorphic allele cos211, which was used for the rescue experiment (Ho, 2005).

Determination of cell fate along the anteroposterior axis of the Drosophila ventral midline

The Drosophila ventral midline has proven to be a useful model for understanding the function of central organizers during neurogenesis. The midline is similar to the vertebrate floor plate, in that it plays an essential role in cell fate determination in the lateral CNS and also, later, in axon pathfinding. Despite the importance of the midline, the specification of midline cell fates is still not well understood. This study shows that most midline cells are determined not at the precursor cell stage, but as daughter cells. After the precursors divide, a combination of repression by Wingless and activation by Hedgehog induces expression of the proneural gene lethal of scute in the most anterior midline daughter cells of the neighbouring posterior segment. Hedgehog and Lethal of scute activate Engrailed in these anterior cells. Engrailed-positive midline cells develop into ventral unpaired median (VUM) neurons and the median neuroblast (MNB). Engrailed-negative midline cells develop into unpaired median interneurons (UMI), MP1 interneurons and midline glia (Bossing, 2006).

The determination of midline cells appears to take place during germband elongation, since by germband retraction most midline subsets can be identified by the expression of unique molecules. The anteroposterior position of midline siblings was determined during germband elongation. Midline precursors were labelled with the lipophilic dye DiD or DiI in embryos expressing GFP in the Engrailed domain (en-GAL4/UAS-tauGFP). After division of the precursors, the daughter cells were followed throughout development, recording their segmental position at stage 10 and stage 11. MP1 interneurons, UMI and MNB neurons each arise from one precursor, and their daughter cells occupy fixed anteroposterior positions during germband elongation. The four daughter cells of the two glial precursors can be located either in the middle of the segment or just anterior to the Engrailed domain. VUM neurons arise from three midline precursors, and the six daughter cells of these precursors are located inside the Engrailed domain and immediately posterior to the domain, in the anterior of the next segment (Bossing, 2006).

In summary, the midline glia and MP1 interneurons are the most anterior midline subsets, followed by a second pair of midline glia and a pair of UMIs, and, finally, the VUM and MNB neurons. DiI labelling cannot resolve whether the MP1 interneurons or the midline glia are the most anterior cells. Since determination of the MP1 interneurons depends on Notch/Delta signalling, it is possible that the anteroposterior position of the most anterior midline cells, the midline glia and MP1 interneurons, is random. Interestingly, four VUM neurons and the MNB neurons seem to arise from the anterior compartment of the next posterior segment. These cells initiate Engrailed expression half-way through germband elongation, and, during germband retraction, they join the adjacent anterior segment to become the most posterior midline subsets (Bossing, 2006).

The separation of midline cells into two compartments is an early and crucial step in midline cell determination. During germband elongation, a second phase of Engrailed expression is initiated at the midline in the anterior cells of the next posterior segment. During germband retraction, these cells join the anterior segment where they develop into posterior midline cells. Expression of late Engrailed depends on Hedgehog signalling and the proneural gene lethal of scute. Lethal of scute precedes Engrailed expression and is also activated by Hedgehog. Hedgehog and Wingless signalling counteract each other to define the position of the Lethal of scute cluster, and to divide the 16 midline daughter cells into eight non-Engrailed- and eight Engrailed-expressing cells (Bossing, 2006).

It has generally been believed that the determination of the different subsets of midline cells occurs before the precursors undergo their simultaneous division at stage 8. This view is challenged by the observation that expression of the proneural gene lethal of scute, and the subsequent expression of Engrailed, is initiated in midline daughter cells at stage 10, about one hour after the precursors divide. In the neuroectoderm, proneural genes confer neural competence to a cluster of ectodermal cells. Lateral inhibition by Notch/Delta signalling then limits the expression of proneural genes to a single cell, which delaminates from the ectoderm and becomes a neural precursor (neuroblast). Because the only neuroblast at the ventral midline (median neuroblast, MNB) originates from the proneural Lethal of scute cluster, it seems likely that the MNB is selected by lateral inhibition from a cluster of midline daughter cells. However, the process of lateral inhibition in the midline differs from that in the adjacent neuroectoderm. In the neuroectoderm, a single cell delaminates and the remaining cells of the cluster cease proneural expression and give rise to the epidermis. The proneural cluster in the midline consists of three pairs of siblings generated by the division of three separate precursors. Labelling of single precursors shows that, during the selection of the MNB, only one of the two labelled siblings enlarges, but both delaminate from the embryo. In contrast to the neuroectoderm, the remaining cells of the midline cluster continue to express Lethal of scute after delamination of the MNB. This extended proneural expression might be necessary to maintain neural competence in the non-delaminating cells that develop into VUM neurons (Bossing, 2006).

The results cannot exclude the possibility that some of the midline subsets are determined as precursors, but at least two of the five midline subsets, the VUM neurons and the MNB, are determined after precursor cell division. There are striking similarities between the development of the ventral midline of Drosophila and grasshopper embryos. In grasshopper, Engrailed expression can be detected in the MNB, its progeny and the midline precursors MP4 to MP6, which each give rise to two neurons with projection patterns comparable to the Drosophila VUM neurons. Hence, the same types of midline cells express Engrailed in grasshopper and Drosophila, but in grasshopper Engrailed expression is initiated in all midline precursors prior to division (Bossing, 2006).

In the ectoderm from stage 10 onwards, Wingless, Engrailed and Hedgehog maintain the expression of one another by a feedback loop: Wingless maintains Engrailed expression, Engrailed is needed for the expression of Hedgehog and Hedgehog maintains Wingless expression. In the developing CNS, Wingless and Hedgehog expression seem to be independent of each other. At the ventral midline there are two separate stages of Engrailed expression: the early phase is maintained by Wingless; the late phase does not require Wingless and is instead activated at stage 10 by Hedgehog signalling and Lethal of scute. In the ectoderm, Wingless and Hedgehog act in concert to maintain Engrailed expression, but at the midline Wingless and Hedgehog act in opposition: Wingless represses and Hedgehog activates Lethal of scute expression (Bossing, 2006).

Wingless may repress Lethal of scute expression indirectly, via its maintenance of early Engrailed. As in the ectoderm, midline Engrailed represses expression of the Hedgehog receptor Patched and the Hedgehog signal transducer Cubitus interruptus. It is possible that early Engrailed-expressing midline cells are not able to receive the Hedgehog signal. However, ectopic expression of Hedgehog is able to induce Lethal of scute in all midline cells, suggesting that Wingless may repress Lethal of scute by a yet unknown mechanism. Recently it has been reported that a vertebrate wingless orthologue, Wnt2b, can maintain the naïve state of retinal progenitors by attenuating the expression of proneural and neurogenic genes (Bossing, 2006).

The differentiation of midline cells was studyed in wingless and hedgehog mutants. Consistent with earlier reports, many midline cells become apoptotic in both mutants. The surviving midline cells are not integrated into the CNS and show no morphological differentiation. The reduction in the number of Engrailed-positive midline cells in hedgehog mutant embryos may be mainly due to the loss of midline cell identity. In hedgehog mutants, midline cells lose the expression of Sim, the master regulator of midline development. As described for sim mutants, the loss of midline identity results in increased cell death and misspecification of the surviving midline cells as ectoderm (Bossing, 2006).

Ectopic expression of Hedgehog in the neuroectoderm and the developing CNS induces the expression of Lethal of scute and, approximately 40 minutes later, the expression of late Engrailed in all midline cells. It seems likely that Lethal of scute is an early target of Hedgehog signalling, and its activation may only require release from repression by the short form of Cubitus interruptus. By contrast, the delay in induction of late Engrailed in the same midline cells indicates that Engrailed activation may not only require release from repression, but also activation by the long form of Cubitus interruptus (Bossing, 2006).

Uniformly high levels of ectopic Hedgehog prevent the differentiation of most midline subsets and cause increased cell death. A single source of ectopic Hedgehog, achieved by cell transplantation, does not result in midline cell death, but reveals that the differentiation of the MP1 interneurons is more sensitive to Hedgehog levels than is the differentiation of midline glia. No other midline subsets are affected. It seems likely that Hedgehog not only activates Lethal of scute and late Engrailed, but also acts as a morphogen to control the differentiation of the MP1 neurons and midline glia (Bossing, 2006).

The phenotypes caused by ectopic Hedgehog are due to the induction of Engrailed in all midline cells. Expression of ectopic Hedgehog and ectopic Engrailed blocks the differentiation of midline glia and MP1 interneurons, and also prevents the formation of the anterior commissure. Labelling single midline precursors enabled examination of cell fates in embryos expressing ectopic Engrailed in the midline. The frequency of clones obtained indicates that ectopic Engrailed expression does not transform non-Engrailed-expressing midline subsets (MP1 interneurons, midline glia and UMI) into Engrailed-expressing subsets (VUM and MNB). Instead, embryos expressing midline Engrailed show increased cell death. In particular, the MP1 interneurons seem to be affected and were never obtained during this analysis. The low frequency of midline glia also points to apoptosis caused by expression of Engrailed. Surviving midline glia are not able to differentiate properly and cannot enwrap the remaining, posterior, commissure. All other midline subsets, including the UMIs, are able to differentiate, but they show a variety of axonal pathfinding defects that may result from the loss of anterior midline subsets and the absence of the anterior commissure (Bossing, 2006).

It is likely that genes other than hedgehog and wingless are crucial for midline cell determination. In these experiments, non-Engrailed-expressing midline subsets are never transformed into Engrailed-expressing subsets, or vice versa. gooseberry-distal may be one of these genes. From the blastoderm stage, Gooseberry-distal is expressed by two midline precursors and their four daughter cells. During early embryogenesis Gooseberry-distal expression at the midline does not depend on Wingless and Hedgehog. The anterior Gooseberry-distal cells also express Wingless and most likely give rise to the UMIs. The posterior Gooseberry-distal pair also express early Engrailed and Hedgehog, and develop into the most anterior VUM neurons. At stage 10, Hedgehog activates the expression of Lethal of scute and Engrailed in midline cells posterior to the Gooseberry-distal domain. Lateral inhibition by Notch/Delta signalling selects one cell from the Lethal of scute cluster to become the MNB. The remaining cells become VUM neurons. At stage 10, the absence of Engrailed in the six midline cells anterior to the Gooseberry-distal domain defines a cell cluster that will give rise to midline glia and MP1 interneurons. Based on the expression of Odd, Delta mutants have an increased number of MP1 interneurons, up to six per segment. In Notch mutants, midline glial-specific markers are absent and the number of cells expressing a neuronal marker increases. Therefore, Notch/Delta signalling appears to determine midline glial versus MP1 interneuron cell fates in the anterior cluster. In the current model, midline cell determination takes place mainly after the division of the precursors. Although the initial determination of midline cells appears to be directed by a small number of genes, a far larger number is needed to control the differentiation of the various midline subsets. This work, and the recent identification of more than 200 genes expressed in midline cells, is the beginning of a comprehensive understanding of the differentiation of the ventral midline (Bossing, 2006).

Continued: see engrailed Transcription regulation part 3/3 | back topart 1/3

engrailed: Biological Overview | Evolutionary Homologs | Targets of activity | Protein Interactions | Developmental Biology | Effects of mutation | References

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