hedgehog

DEVELOPMENTAL BIOLOGY

Embryonic

hh expression is first detectable in the maxillary segment of the head and the hindgut. By gastrulation, 14 parasegments express hh. Later non-metameric expression is seen in the following sites: clypeolabrum, mandibular segment, intercalary segment, antennal segment, hindgut, posterior spiracles, proctodeum, anal plate, posterior pharynx and esophagus, and antennal maxillary sense organ [Images] (Lee, 1992).

During neurogenesis, the transmembrane protein Patched promotes a wingless-mediated specification of a neuronal precursor cell, NB4-2. Wg, secreted by row 5 cells promotes wingless expression in adjacent row 4 cells; Wg in turn represses gooseberry. Novel interactions of these genes with engrailed and invected during neurogenesis have been uncovered. While in row 4 cells Ptc represses gsb and wg, in row 5 cells en/inv relieve Ptc repression of gsb by a non-autonomous mechanism that does not involve hedgehog. The non-autonomous mechanism originates in Row 6/7 cells where en/inv engender hedgehog and another unknown secreted signal which acts in turn on adjacent row 5 cells to heighten wingless, and consequently, the expression of gooseberry. This differential regulation of gsb leads to the specification of NB5-3 and NB4-2 identities to two distinct neuroblasts. The row 5, NB5-3, neuroblasts are specified by high levels of gsb, expressed autonomously in row 5. The fate of row 4, NB4.2, requires an absence of gooseberry, assured by Patched repression and Wingless signaling from adjacent row 5 cells. The uncoupling of the ptc-gsb regulatory circuit by hedgehog and the unknown secreted signal from row 6/7 cells enables gsb to promote Wg expression in row 5 cells. These results suggest that the en/inv->ptc->gsb->wg pathway uncovered here and the hh->wg are distinct pathways that function to maintain the wild-type level of Wg. These results also indicate that Hh is not the only ligand for Ptc and similarly, that Ptc is not the only receptor for Hh (Bhat, 1997).

Hedgehog, compartments and boundary formation

The adult abdomen of Drosophila is a chain of anterior (A) and posterior (P) compartments. The engrailed gene is active in all P compartments and selects the P state. Hedgehog enters each A compartment across both its anterior and posterior edges; within A its concentration confers positional information. The A compartments are subdivided into an anterior and a posterior domain that each make different cell types in response to Hedgehog. The relationship between Hedgehog, engrailed and cell affinity was studied. Twin clones can be made in which one sister clone lacks smoothened (smo) a gene essential for a response to Hedgehog protein and the other is normal, apart from a marker. If these twins are generated in the posterior region of the A compartment, the smo minus clone frequently moves back and into territory normally occupied by P compartment cells, leaving its twin in A territory. This 'sorting back' may imply that the cells of the smo minus clone, which no longer see Hh, have more affinity with P than with the nearby A cells (Lawrence, 1999b and references).

Twin clones were made and the shape, size and displacement of the experimental clone, relative to its control twin, were tested. The perceived level of Hedgehog was varied in the experimental clone and it was found that, if this level is different from the surround, the clone fails to grow normally, rounds up and sometimes sorts out completely, becoming separated from the epithelium. Also, clones are displaced towards cells that are more like themselves: for example groups of cells in the middle of the A compartment that are persuaded to differentiate as if they were at the posterior limit of A, move posteriorly. Similarly, clones in the anterior domain of the A compartment that are forced to differentiate as if they were at the anterior limit of A, move anteriorly. Quantitation of these measures and the direction of displacement indicate that there is a U-shaped gradient of affinity in the A compartment that correlates with the U-shaped landscape of Hedgehog concentration. Since affinity changes are autonomous to the clone it is believed that, normally, each cellís affinity is a direct response to Hedgehog. By removing engrailed in clones it is shown that A and P cells also differ in affinity from each other, in a manner that appears independent of Hedgehog. Within the P compartment, some evidence was found for a U-shaped gradient of affinity, but this cannot be due to Hedgehog which does not act in the P compartment (Lawrence, 1999b).

For experimental purposes, the abdomen has an advantage over the wing: even in small clones, the types of cuticle being made can be assessed. Thus smo minus clones of A provenance can make the type of cuticle (a3) found in the middle of the A compartment. Such clones made near the back end of the A compartment come to lie between two sorts of alien cells. Behind them are P cells, and in front of them are posterior A cells (a6, a5). In the abdomen the results are unequivocal -- the smo minus clones fail to mix with either type of alien cell, forming straight boundaries with both. P clones lacking both smo and en can also form epidermal cells of the a3 type, and at the front of the P compartment, these behave the same way as a3 cells of A provenance. By contrast, en clones of P provenance, those that form a5 cells, cross over the boundary into A and mix there with a5 cells. It is concluded that the A/P boundary in the abdomen (and presumably in the wing) depends on two independent factors: the difference between A and P due to en and the differences within A due to the Hh signal (Lawrence, 1999b).

The Hh signal enters each A compartment from two directions: the results suggest that it acts to set up two opposing gradients of cell affinity. The behaviors of twin clones were examined: one having a different identity from its neighbors and the other acting as a control. The most detailed results concern the posterior domain within the A compartment. (1) A spatial gradient of clone survival is found; clones of different positional identity sort out most readily when there is a large disparity between their positional value and that of the surrounding cells. For example, ptc;en minus clones sort out rapidly when they are induced anteriorly, while they survive well in the posterior part. The same type of clones when induced later survive further to the anterior, which suggests there is a continuous gradient of affinity. (2) The wiggliness of the boundary made between the clone and its surrounding is a measure of the degree of affinity between the two types of cells. It is noted that with ptc;en minus clones induced at a certain stage in the pupa, the clones are more circular the more anterior their location. This also suggests that affinities change continuously. (3) Further evidence is provided for polarity in the epidermis, because relative to its twin, the clone moves toward the level appropriate to its own differentiation: if the clone differentiates as a5 cuticle, then it moves towards the a5 region. This implies that a vector is present in the epithelium, for if the clone were simply uncomfortable being surrounded by a uniform field of a3 cells, it might round up or sort out, but it would not migrate in a specific direction. This vector is imagined to be defined by a gradient of cell affinity; one would expect cells to take whatever opportunity they have to move in the direction that maximizes their affinity with their neighbors (Lawrence, 1999b).

The adult abdominal epidermis develops as a fairly loose sheet and cells might be somewhat free to exchange neighbors, perhaps during mitosis. The results also indicate that the anterior domain of the A compartment correlates with an affinity gradient of the opposite polarity -- accordingly, while the a5 or a6 clones in the a3 region move back, the a1 clones in the a2 region move forward. Both these findings suggest that the prime agent responsible for affinity in the A compartment is Hh itself. The response to Hh is cell autonomous and it is imagined the affinity depends on how much Hh is perceived: it is a scalar output from the Hh gradients (Lawrence, 1999b).

Why would gradients of cell affinity be biologically efficacious? The general model is that morphogen gradients define basic aspects of pattern: positional information is encoded in the scalar of the primary gradient, information relating to size and growth in the steepness and polarity encoded in the vector of a secondary gradient. To this hypothesis is now added the notion that affinity is also encoded in the scalar, giving a graded readout, perhaps in the amount of a homophilic adhesion molecule such as a cadherin. It is thought that gradients of cell affinity will prove to be basic properties of all cell sheets in vivo, where they act to ensure the integrity and stability of the sheet by keeping the cells adherent to their neighbors and reducing any tendency to roam. Without this gradient, even if all cells tended to cohere to one another, their intrinsic motility could allow them to move around by exchanging equally adhesive neighbours. Mobility like this could compromise pattern formation; it might be problematic if cells were to receive information of position from, for example, the ambient level of Hh, begin to respond to it, and then migrate to a different position too late to readjust their response. The stripes of different types of cuticle in the A compartment are a consequence of threshold responses to a continuously varying Hh concentration. In general, once differentiation has begun in any group of cells (such as one of these stripes) they might acquire additional affinity label(s) that would reduce mixing with neighbors, thus sharpening the border line. Maybe this explains the straightness of the line between a5 and a6 cuticle, which seems straighter than one would expect if the a5 and a6 cells were mixing as much as cells do elsewhere. In the wing, the Hh gradient is responsible for pattern only close to the A/P boundary, with the differentiation of cells further away in thrall to a gradient of Decapentaplegic (Dpp). There is some evidence that the gradient of affinity extends into parts of the wing outside the Hh territory: for example, clones of activated receptor for Dpp take up a circular shape, showing that their cell affinities are different from the surround. Thus affinity changes may accompany positional information even when this information depends on more than one morphogen (Lawrence, 1999b).

In Drosophila embryos, segment boundaries form at the posterior edge of each stripe of engrailed expression. An HRP-CD2 transgene has been used to follow by transmission electron microscopy the cell shape changes that accompany boundary formation. The first change is a loosening of cell contact at the apical side of cells on either side of the incipient boundary. Then, the engrailed-expressing cells flanking the boundary undergo apical constriction, move inwards and adopt a bottle morphology. Eventually, grooves regress, first on the ventral side, then laterally. Groove formation and regression are contemporaneous with germ band retraction and shortening, respectively, suggesting that these rearrangements could also contribute to groove morphology. The cellular changes accompanying groove formation require that Hedgehog signalling be activated, and, as a result, a target of Ci is expressed at the posterior of each boundary (obvious targets like stripe and rhomboid appear not to be involved). In addition, Engrailed must be expressed at the anterior side of each boundary, even if Hedgehog signalling is artificially maintained. Thus, there are distinct genetic requirements on either side of the boundary. In addition, Wingless signalling at the anterior of the domains of engrailed (and hedgehog) expression represses groove formation and thus ensures that segment boundaries form only at the posterior (Larsen, 2003).

Segmental boundary formation is initiated shortly after germ-band retraction has begun. They are recognizable as periodic indentations in the epidermis that separate cells expressing engrailed at the anterior from those expressing rhomboid at the posterior. To allow identification of cells in electron micrographs, a transgenic membrane marker was devised based on horseradish peroxidase (HRP), which catalyses the production of an electron-dense product from diaminobenzidine (DAB). HRP was fused to the transmembrane protein CD2 so that the marker would outline cells and thus reveal cell shapes. This inert fusion protein was expressed under the control of engrailed-Gal4, so that the membrane of engrailed-expressing cells appears dark under the electron microscope (Larsen, 2003).

Engrailed has both a cell autonomous and a non-cell autonomous function in the establishment of the compartment boundary in wing imaginal discs. Although the compartment boundary does not trace its embryonic origin to segment boundaries, there is a striking parallel between the two. For segmental grooves to form, Hedgehog signaling is required in cells at the posterior of the boundary, even if engrailed expression is artificially maintained at the anterior side. Conversely, Hedgehog signaling is not sufficient as exogenous expression of hedgehog in the absence of engrailed does not lead to groove formation (Larsen, 2003).

It is the cells that line the anterior side of segment boundaries (the most posterior engrailed-expressing cells) that undergo the most distinctive behavior during groove formation. This behavior requires Hedgehog signalling, and yet engrailed-expressing cells are not responsive to this signal. Therefore, their morphological changes must be in response to a signal originating from neighboring non-engrailed expressing cells. This could be achieved through standard paracrine signaling or by contact-dependent signal mediated by cell surface proteins. Whatever the mechanism, Hedgehog-responsive cells influence the behavior of adjoining engrailed-expressing cells across the boundary, and crosstalk between the two cells takes place. This is reminiscent of the situation found during eye morphogenesis; at rhombomere boundaries cross communication between neighboring rhombomere cells are required for rhombomere formation (Larsen, 2003).

Because boundaries form in the complete absence of Ci (in ci⁹⁴), it is concluded that the activator form of Ci is not required for segment boundary formation. However, no boundary forms in ci^Cell mutant embryos, indicating that the presence of Ci[75] (the repressor) prevents boundary formation. It is suggest therefore that boundary formation requires the expression of a gene (x) that is repressed by Ci[75] but does not require Ci[155] to be activated. Presumably, an activator of x is constitutively present but, in the absence of Hedgehog, it is prevented from activating x expression by Ci[75]. Hedgehog signaling would remove Ci[75] and thus allow activation to occur. Two characterized target genes of Hedgehog (wingless and rhomboid) follow the same mode of regulation. For example, expression of wingless in the embryonic epidermis decays in ci^Cell but is still present in the complete absence of Ci, in ci⁹⁴ embryos (Larsen, 2003).

Although Hedgehog signaling is activated both at the anterior and the posterior of its source, segment boundaries only form at the posterior. One reason for this asymmetry is that Wingless signaling represses boundary formation at the anterior. Indeed, in the absence of Wingless, boundaries are duplicated, as long as expression of Engrailed and Hedgehog is artificially maintained. It is concluded that expression of x is repressed by Wingless signalling. Two obvious candidates for x are Rhomboid and Stripe. Genes encoding both these proteins are activated by Hedgehog signaling and repressed by Wingless signaling and, indeed, both are expressed in cells that line the segment boundary. To determine if either gene could mediate the role of Hedgehog in boundary formation, the respective mutants were examined. No effect on grooves could be seen. It is concluded that neither rhomboid nor stripe is required for boundary formation although the possibility that these genes could contribute in a redundant fashion cannot be excluded. Overall the genetic analysis suggests that additional targets of Hedgehog must be involved in boundary formation. It will be interesting to find out whether any of these targets will turn out to be implicated in compartment boundary maintenance as well (Larsen, 2003).

Although the role of a Hedgehog target gene in boundary formation has been emphasized, it is clear from this analysis that engrailed also has a cell-autonomous role. Even though Engrailed represses ci expression, its role in boundary formation is likely to involve the transcriptional regulation of another target gene. One possibility is that Engrailed could be a repressor of x and that boundaries would form at the interface between x-expressing and non-expressing cells. However, it is thought that instead, or in addition, Engrailed has a Hedgehog-independent effect on cell affinity and that this could contribute to boundary formation. Of note is the observation that engrailed-expressing cells remain together in small groups even when boundaries are lost for lack of hedgehog. This suggests that engrailed-expressing cells have increased affinity for one another. Thus, Engrailed could specify P specific cell adhesion independently of Hedgehog. Clearly, future progress will require the identification of Engrailed target genes that control such preferential affinity and/or contribute to boundary formation (Larsen, 2003).

Drosophila T box proteins break the symmetry of hedgehog-dependent activation of wingless: Slp is a negative regulator of midline expression

Segmentation of the Drosophila embryo is a classic paradigm for pattern formation during development. The Wnt-1 homolog Wingless (Wg) is a key player in the establishment of a segmentally reiterated pattern of cell type specification. The intrasegmental polarity of this pattern depends on the precise positioning of the Wg signaling source anterior to the Engrailed (En)/Hedgehog (Hh) domain. Proper polarity of epidermal segments requires an asymmetric response to the bidirectional Hh signal: wg is activated in cells anterior to the Hh signaling source and is restricted from cells posterior to this signaling source. This study reports that Midline (Mid) and H15, two highly related T box proteins representing the orthologs of zebrafish hrT and mouse Tbx20, are novel negative regulators of wg transcription and act to break the symmetry of Hh signaling. Loss of mid and H15 results in the symmetric outcome of Hh signaling: the establishment of wg domains anterior and posterior to the signaling source predominantly, but not exclusively, in odd-numbered segments. Accordingly, loss of mid and H15 produces defects that mimic a wg gain-of-function phenotype. Misexpression of mid represses wg and produces a weak/moderate wg loss-of-function phenocopy. Furthermore, it has been shown that loss of mid and H15 results in an anterior expansion of the expression of serrate (ser) in every segment, representing a second instance of target gene repression downstream of Hh signaling in the establishment of segment polarity. The data presented indicate that mid and H15 are important components in pattern formation in the ventral epidermis. In odd-numbered abdominal segments, Mid/H15 activity plays an important role in restricting the expression of Wg to a single domain (Buescher, 2004).

Previous work has suggested that Slp permits the Hh-dependent activation of Wg anterior to the En/Hh stripe by antagonizing a repressor of Wg. Based on the data presented above, Mid/H15 appear to be such repressors. To determine if Slp is a negative regulator of mid expression, the effect of slp loss-of-function and slp misexpression was studied on the distribution of mid RNA. In slp mutant embryos the early mid expression is normal. However from early stage 9 onward the mid stripes broaden to approximately twice their normal width. Using mid-positive neuroblasts as a landmark (these remain unchanged in slp mutant embryos), it was possible to characterize the increase in mid expression as an anterior expansion. This aberrant mid expression pattern is unstable; from stage 11 onward mid decays in odd-numbered segments. Conversely, misexpression of slp in the ventral ectoderm from early stage 9 onward led to a complete loss of ectodermal mid expression. These data show that Slp functions as a repressor of mid expression. Taken together with the observation that misexpression of mid in otherwise wild-type embryos results in the loss of Wg expression, these results lead to the conclusion that the Slp-mediated repression of mid anterior to the En/Hh stripe is an important component of wg competence (Buescher, 2004).

As a further test of the relationship between slp and mid, the effect was compared of expressing mid and slp, alone or in combination, on Wg expression. Ectopic expression of mid results in a rapid and almost complete loss of Wg expression, whereas ectopic expression of slp results in weak ectopic expression of Wg posterior to the En/Hh stripe. This slp-induced phenotype resembles that of the loss of mid, except that ectopic Wg expression is weaker and appears randomly in even- and odd-numbered segments. The ectopic Wg expression is blocked when mid and slp are expressed together, suggesting that in this context mid acts downstream of slp. The Wg expression anterior to the En/Hh stripe still decays in UAS-mid/UAS-slp embryos, albeit more slowly and variably than in UAS-mid alone. This result may reflect that Wg expression is sensitive to the amounts of available Mid and Slp. It may also indicate that anterior to the En/Hh stripe, Slp function is required for more than just repression of mid and may possibly have independent activating functions. An analysis of slp1,slp2;mid/H15 quadruple mutants would be highly helpful in clarifying the relationship between slp genes and mid/H15. Unfortunately, the generation of such a quadruple mutant by genetic recombination is impossible because the slp deletion that removes these genes (Δ34B) is on a balancer chromosome that precludes recombination (Buescher, 2004).

Hedgehog and Decapentaplegic instruct polarized growth of cell extensions in the Drosophila trachea

The trachea is a respiratory organ consisting of a network of tubular epithelia that delivers outside air directly to target organs. The tracheal primordium forms six primary branches that migrate towards specific target tissues expressing Branchless (Bnl), a Drosophila homolog of FGF. Bnl activates Breathless (Btl), an FGF receptor, at the tip of primary branches and cell process formation. The dorsal branch (DB) migrates toward the dorsal midline, where it fuses with another DB from the contralateral side. Two specialized cells are present at each DB tip. Fusion cells lead the migration and form anastomoses of tracheal tubules, whereas terminal cells extend a long cell process called a terminal branch. The terminal branch is also present in other branches such as visceral and ganglionic branches, and in all cases spreads over the surface of target tissues and serves as an interface for gas exchange by extending unicellular processes containing a dead-ended lumen, a structure known as the tracheole. Terminal branching in postembryonic stages is regulated by Bnl, which is induced as a hypoxic response. The regulation of terminal branch migration in the CNS uses the same molecules involved in axon guidance. Current knowledge is lacking, however, on the regulation of directed terminal branch growth over the epidermis, as well as the mechanism by which it is positioned over the epidermis to maximize oxygen transfer. The guidance roles of the morphogens Hedgehog and Decapentaplegic during directed outgrowth of cytoplasmic extensions in the Drosophila embryonic trachea were investigated. A subset of tracheal terminal cells adheres to the internal surface of the epidermis and elongates cytoplasmic processes called terminal branches. Hedgehog promotes terminal branch spreading and its extension over the posterior compartment of the epidermis. Decapentaplegic, which is expressed at the onset of terminal branching, restricts dorsal extension of the terminal branch and ensures its monopolar growth. Orthogonal expression of Hedgehog and Decapentaplegic in the epidermis instructs monopolar extension of the terminal branch along the posterior compartment, thereby matching the pattern of airway growth with that of the epidermis (Kato, 2004).

Terminal branches extend numerous cell processes that rapidly and repeatedly extend and retract in many directions. Although cell processes that extend anteriorly and dorsally are unstable, a subset of cell processes that extend ventrally along the posterior (P) segmental compartment becomes selectively stabilized. The behavior of terminal cells in the anterior compartment is strikingly different from that in the P compartment. In the anterior compartment, the number and size of the cell processes are much smaller, suggesting that the P compartment constitutes the preferred substrate for terminal cell spreading (Kato, 2004).

Hh is important for promoting terminal cell spreading. Hh is secreted from the P compartment and forms symmetrical gradients of cellular responses in both the anterior and posterior directions within the epidermis. It is proposed that Hh stimulates the adhesion of terminal cells to the epidermis by activating Ci. Because Hh signaling is submaximal in terminal cells, terminal branch filopodia that extend randomly would be preferentially stabilized near the source of Hh. Thus, terminal cell bodies are placed at the point of highest Hh concentration and terminal branches are stabilized at the apex of the Hh concentration gradient. Because terminal cell growth continues while the level of Hh signaling remains below maximum within terminal cells, terminal branches would be expected to extend along the P compartment (Kato, 2004).

The mechanism(s) of terminal branch guidance by Hh through regulation of Ci-dependent transcription differs from those that guide the behavior of the growth cone, which is primarily regulated at the level of cytoskeletal motility. The latter mechanism has the advantage of maintaining a small cell-surface area receiving guidance cues to minimize the chances of making aberrant connections during synapse formation. Terminal cells, however, use the entire basal cell surface to receive a guidance signal and to stabilize their association with the epidermis. This mechanism of terminal branching by cell spreading meets the physiological requirement that the terminal branch serves as an interface for gas exchange (Kato, 2004).

Hh signaling has been implicated in another cell adhesion-related process, namely cell sorting behavior at the AP compartmental boundary in the wing imaginal disc. Because this behavior is regulated transcriptionally by Ci, there may be a common downstream target of Ci that acts in both wing disc cells and terminal cells (Kato, 2004).

Terminal branch extension is limited to the AP compartmental border, suggesting that there is an additional mechanism that shifts the terminal branch to the anterior side of the P compartment. Bnl was expressed as short stripes in the dorsal epidermis (DE) at the time of terminal branching. It has been reported that mesodermal cells also contribute to correct patterning of dorsal branch (DB). It will be interesting to address guidance functions of those components on terminal branch outgrowth (Kato, 2004).

Dpp is expressed at the dorsal edge of the DE during dorsal closure. This corresponds to the time when terminal branch outgrowth in the DB starts, suggesting that Dpp affects the initial stage of terminal branch outgrowth. Prolonged activation of Dpp signaling in terminal cells by expression of Dpp prevents its elongation. These observations suggest that in normal development Dpp prevents dorsally directed terminal branch extension at the onset of terminal branching, thereby shunting terminal branch extension towards the ventral direction. It is proposed that Dpp converts the initial bipolar shape of a terminal branch into one that is monopolar. Once terminal branch extension is initiated, its direction may be maintained by localized Bnl expression at the AP compartment border. Whether this inhibitory effect of Dpp is mediated by direct signaling to cytoskeletons at the cell periphery or mediated by a nuclear transduction of the signal, remains to be determined (Kato, 2004).

Terminal cells undergo an enormous increase in cell volume and surface area during terminal branching, which continues throughout embryonic and post-embryonic stages. FGF signaling promotes this process in two ways, first by activating the target gene SRF, which is required for terminal branch growth, and second by stimulating rapid filopodial movement. These two FGF signaling effects seem to be independent of Hh and Dpp. The FGF ligand Bnl is expressed in epidermal cells beneath terminal cells during terminal branching. Because this expression is limited to a relatively small region, it is considered unlikely that FGF signaling is sufficient to provide vectorial information for terminal branching. It is suggested that FGF-driven growth and the motility of terminal branches are restricted to the P compartment by Hh signaling, wherein they are further limited by Dpp to establish a monopolar growth pattern (Kato, 2004).

Hh functions as both a cell fate determinant and a guidance molecule for terminal branch extension. But how are these two distinct Hh functions coordinated? Inactivation of Hh after initiation of terminal branching via a temperature shift of hh^ts2 mutant embryos causes a loss of terminal cells, suggesting that maintenance of tracheal cell fate also depends on a late Hh function. Thus, the striped expression of Hh in the epidermis is used simultaneously for epidermal patterning, tracheal cell fate determination and terminal branch guidance, exemplifying a simple strategy to coordinate the patterning of complex organs having multiple tissue types (Kato, 2004).

Hox-controlled reorganisation of intrasegmental patterning cues underlies Drosophila posterior spiracle organogenesis: Hh, Wg and Egfr pathways provide specific inputs for posterior spiracle morphogenesis

Hox proteins provide axial positional information and control segment morphology in development and evolution. Yet how they specify morphological traits that confer segment identity and how axial positional information interferes with intrasegmental patterning cues during organogenesis remains poorly understood. This study investigates the control of Drosophila posterior spiracle morphogenesis, a segment-specific structure that forms under Abdominal-B (AbdB) Hox control in the eighth abdominal segment (A8). The Hedgehog (Hh), Wingless (Wg) and Epidermal growth factor receptor (Egfr) pathways provide specific inputs for posterior spiracle morphogenesis and act in a genetic network made of multiple and rapidly evolving Hox/signalling interplays. A major function of AbdB during posterior spiracle organogenesis is to reset A8 intrasegmental patterning cues, first by reshaping wg and rhomboid expression patterns, then by reallocating the Hh signal and later by initiating de novo expression of the posterior compartment gene engrailed in anterior compartment cells. These changes in expression patterns confer axial specificity to otherwise reiteratively used segmental patterning cues, linking intrasegmental polarity and acquisition of segment identity (Merabet, 2005).

In the dorsal ectoderm of stage 10 embryos, hh and wg follow the same striped expression patterns in A8 as in other abdominal segments. rho expression, which marks cells secreting an active form of the Egf ligand, occurs in all primordia of tracheal pits, in A8 as in more anterior segments (Merabet, 2005).

Specification of posterior spiracle primordia occurs at early stage 11. The primordia can then be recognised by Cut expression in spiracular chamber cells and by Sal, the homogenous expression of which in A8 becomes restricted dorsally to stigmatophore cells (forming the external structure of the posterior spiracle) that form a crescent surrounding Cut-positive cells. From mid-stage 11, wg and rho adopt in the dorsal ectoderm expression patterns specific to A8, with wg transcribed in two cells only and rho in a second cell cluster, dorsal and posterior to the tracheal placode. To localise wg- and rho-expressing cells with regard to stigmatophore and spiracular chamber cells, co-labelling experiments for wg or rho transcripts and for Cut or Sal proteins were performed: the two wg cells lie between Cut- and Sal-positive cells; the second cell cluster expressing rho in A8 also expresses Cut but not Sal. This cluster is likely to produce the Egf ligand required for posterior spiracle development, since mutations that alleviate rho expression in the tracheal placodes do not abolish spiracles formation. At mid-stage 11, the hh pattern in A8, along a stripe lying posterior and adjacent to the spiracular chamber and overlapping stigmatophore presumptive cells, resembles expression in other abdominal segments. Analyses at later stages indicate that the relationships between posterior spiracle cells and hh, wg and rho patterns are maintained (Merabet, 2005).

Null mutations of wg, hh or Egfr result in the absence of posterior spiracles. The strong cuticular defects observed raise the possibility that the phenotypes result indirectly from early loss of segment polarity. Removing the Wg, Hh or Egfr signals from 5-8 hours of development using thermosensitive alleles causes strong segment polarity defects but allows filzkörpers, stigmatophores or even complete posterior spiracles to form. Thus, spiracular chamber and stigmatophore can develop in embryos that have pronounced segment polarity defects (Merabet, 2005).

It was next asked whether defects in primordia specification could account for posterior spiracle loss, and Cut and Sal expression was examined in the dorsal A8 ectoderm of hh, wg and Egfr mutant embryos. Expression of Cut and Sal is initiated at stage 11 in all of these mutants, although the somewhat disorganised patterns, especially from late stage 11, may reveal roles for these genes in signalling in sizing or shaping the posterior spiracle primordia. Alternatively, these defects may result from altered morphology of mutant embryos. In any case, the induction of the early markers Sal and Cut in A8 dorsal ectoderm of mutant embryos indicates that posterior spiracle primordia specification does occur in the absence of signalling by Wg, Hh or Egfr. Transcription of ems, another AbdB target that is activated slightly later than Cut, although not affected in hh mutants, is lost in wg or Egfr mutants. Thus, proper regulation of AbdB downstream targets activated following primordia specification appears dependent on signalling activities (Merabet, 2005).

The role was examined of Wg, Hh and Egfr signalling pathways in posterior spiracle organogenesis (i.e., after the specification of presumptive territories). Co-labelling experiments performed on embryos expressing GFP driven by ems-Gal4 or by sal-Gal4 indicate that whereas Cut and Sal are already expressed at early stage 11, GFP is detected from late stage 11 only. These two drivers, which promote expression approximately 1 hour after primordia specification, were used to express DN molecules for each pathway, counteracting Wg (DN-TCF), Egfr (DN-Egfr) or Hh [DN-Cubitus interuptus (Ci)] signalling from that time on. Blocking either pathway in spiracular chamber cells does not perturb stigmatophore morphogenesis, but specifically leads to the loss of differentiated filzkörpers. Conversely, blockade in stigmatophore cells provokes in each case its flattening, while differentiated filzkörpers do form (Merabet, 2005).

To ask how signalling inhibition interferes with the genetic modules initiated downstream of AbdB, expression of Sal and Cut was examined from stages 11 to 13. No major defects are seen until late stage 12. Strong deviation from the wild-type patterns is, however, observed slightly later, from stage 13 onwards: Sal expression in basal cells of the stigmatophore is lost and Cut expression remains in only a few scattered cells. The 2-hour delay seen between the onset of DN molecules expression and the detection of Sal and Cut could reflect the time required for shutting down the pathways. Alternatively, Sal and Cut expression may not require signalling activities before stage 13. To discriminate between these possibilities, an earlier expression of the DN molecules was forced, using the 69B-Gal4, known to promote protein accumulation by the onset of stage 11 (i.e., slightly before posterior spiracle primordia specification). Strong defects in Sal and Cut expression were again seen only in stage 13 embryos, supporting the notion that signalling activities are dispensable before the end of stage 12, but are required from stage 13 onwards to maintain Sal in basal stigmatophore cells and Cut in the spiracle chamber (Merabet, 2005).

A8-specific modulation of rho and wg patterns at mid-stage 11 suggests a regulation by AbdB. In AbdB mutants, rho expression in the spiracle-specific cell cluster is lost, and wg transcription does not evolve towards an A8-specific pattern. In embryos expressing AbdB ubiquitously, ectopic posterior spiracle formation in the trunk can be identified as ectopic sites of Cut accumulation. In such embryos, rho and wg are induced in trunk segments following patterns that resemble their expression in A8: rho in a cluster that overlaps the Cut domain, and wg in few cells abutting ectopic Cut-positive cells. These transcriptional responses to loss and gain of function of AbdB indicate that the Hox protein controls the A8-specific expression patterns of wg and rho. The lines gene (lin), which is known to be required for Cut and Sal activation by AbdB, also controls wg and rho patterns respecification (Merabet, 2005).

In contrast to wg and rho, hh does not adopt an A8-specific expression pattern at mid-stage 11. At that stage, hh expression pattern is not affected upon AbdB mutation. The hh stripe in A8 lies posterior and adjacent to spiracular chamber cells and overlaps stigmatophore cells, suggesting that Hh signalling may participate in the regulation of rho and wg transcription by AbdB. In support of this, it was found that the AbdB-dependent aspects of rho and wg transcription patterns are missing in hh mutant embryos. Thus, inputs from both Hh and AbdB are required to remodel Wg and Egfr signalling in A8 (Merabet, 2005).

The dependence of wg and rho A8 expression patterns on Hh, and the loss of ems expression in wg and rho but not in hh mutants, suggest that transcription of ems requires Wg and Egfr signalling prior to wg and rho pattern respecification by AbdB and Hh. To explore this point further, the time course of ems, wg and rho expression was comparatively analyzed. Embryos bearing an ems-lacZ construct stained for ß-Gal and for wg or rho transcripts show that ems expression precedes wg pattern respecification, and occurs at the same time as rho acquires an A8-specific pattern. Importantly, A8-specific rho clusters were never observed before the onset of ems expression. Thus, ems transcription starts before wg and at the same time as rho pattern respecification, supporting that signalling by Wg and Egfr is required prior to mid-stage 11. These observations also indicate that respecification of the wg pattern occurs slightly later than that of rho, which could not been concluded from changes in embryo morphology (Merabet, 2005).

To determine whether signalling by Wg and Egfr from local sources is important for posterior spiracle organogenesis, the production of Wg and Spi^S (the mature form of Spi) ligands was forced from domains broader than normal in A8 dorsal ectoderm. This was performed after posterior spiracle specification, using the ems-Gal4 and sal-Gal4 drivers. Ectopic signalling results in abnormally shaped posterior spiracles: stigmatophores are reduced in size and filzkörpers do not elongate properly. Ectopic signalling from all presumptive stigmatophore cells results in stronger defects than those produced when ectopic signals emanate from all spiracular chamber cells. This can be correlated to the fact that sal-Gal4 drives expression in a pattern that more strongly diverges from the wild-type situation than ems-Gal4 does. Thus, restricted delivery of Wg and Spi^S signals is required for accurate posterior spiracle organogenesis (Merabet, 2005).

It was next asked whether, downstream of Hh, the Wg and Egfr pathways provide separate inputs for posterior spiracle organogenesis. Two sets of experiments were conducted and it was found that: (1) in embryos respectively mutant for Egfr or wg, wg and rho acquire A8-specific patterns; (2) epistasis experiments performed by forcing in spiracular or stigmatophores cells the activity of one pathway while inhibiting the other indicate that loss of one pathway could not be rescued by the other. Thus, Egfr and Wg pathways do not act as hierarchically organised modules, but provide independent inputs for posterior spiracle organogenesis (Merabet, 2005).

The expression of the posterior compartment selector gene engrailed (en) until stage 12 follows a striped pattern identical in all trunk segments. Later on, En adopts a pattern that is specific to A8: it is no longer detected in the ventral part of the segment; dorsally, the En stripe has turned to a circle of cells that surround the future posterior spiracle opening and express the stigmatophore marker Sal. The transition from a striped to a circular pattern depends on AbdB. This transition could result either from a migration of en posterior cells towards the anterior, or from transcriptional initiation in cells that were not expressing en before stage 12, and that can therefore be defined as anterior compartment cells (Merabet, 2005).

To distinguish between the two possibilities, en-Gal4/UAS-lacZ embryos were simultaneously stained with anti ß-Gal and anti-En antibodies. If circle formation results from cell migration, one would expect ß-Gal and En to be simultaneously detected in all cells of the circle since the two proteins are already co-expressed in the posterior compartment stripe earlier on. Conversely, if the circle results from de novo expression, one would expect anterior cells in the circle to express En before ß-Gal, since ß-Gal production requires two rounds of transcription/translation compared with one for En. It was found that cells from the anterior part of the circle express En but not ß-Gal in stage 13 embryos, which demonstrates that de novo expression of En occurs in anterior compartment cells. Further supporting En expression in anterior compartment cells, it was found that precursors of anterior spiracle hairs that do not express En at stage 12 do so at stage 13. Engrailed function in A8 is essential for posterior spiracle development, since stigmatophores do not form in en mutants, and are restored if En is provided in stigmatophore cells (Merabet, 2005).

It was also found that although identical in all abdominal segments at stage 11, hh transcription adopts an A8-specific pattern from stage 12 onwards: transcripts are then localised only at the anterior border of the En stripe. This expression of hh is lost in AbdB mutants and still occurs in en mutant. The uncoupling of hh transcription from En activity in the dorsal A8 ectoderm correlates with the distinct phenotypes seen for en mutants, which do differentiate filzkörper like structures, and for hh mutants, which do not (Merabet, 2005).

Data in this paper allow the distinguishing of four phases in functional interactions between AbdB and signalling by Wg, Hh and Egfr during posterior spiracle formation. The first phase corresponds to the specification of presumptive territories of the organ. The signalling activities are not involved in this AbdB-dependent process, since they are not required for the induction of the earliest markers of spiracular chamber and stigmatophore cells, Cut and Sal, in the dorsal ectoderm of A8. The second phase, which immediately follows primordia specification, concerns the regulation of AbdB target genes activated slightly later. Inputs from the Hox protein and the Wg and Egfr pathways are then simultaneously needed, as seen for transcriptional initiation of the ems downstream target. This function of Wg and Egfr signalling precedes and does not require the reallocation of signalling sources in A8-specific patterns; impairing A8-specific expression of wg and rho by loss of hh signalling does not affect ems expression. Within the third phase, AbdB and Hh activities converge to reset wg and rho expression patterns. The three phases take place in a narrow time window, less than 1 hour during stage 11, and could only be distinguished by studying the functional requirements of Wg, Hh and Egfr for transcriptional regulation in the posterior spiracle (Merabet, 2005).

The fourth phase is referred to as an organogenetic phase. Data obtained using DN variants to inhibit the pathways in cells already committed to stigmatophore or filzkörper fates, indicate that Wg, Egfr and Hh pathways are required for organ formation after specification and early patterning of the primordia. Their roles are then to maintain the AbdB downstream targets' expression in posterior spiracle cells as development proceeds, as shown for Cut and Sal at stage 13 (Merabet, 2005).

A salient feature of AbdB function during posterior spiracle development is to relocate Wg and Egfr signalling sources in the dorsal ectoderm at mid-stage 11. wg and rho then adopt expression patterns that differ from expressions in other abdominal segments, conferring axial properties unique to A8 to otherwise segmentally reiterated patterning cues. Resetting Wg and Egfr signalling sources into restricted territories is of functional importance for organogenesis, as revealed by the morphological defects that result from the delivery of Wg or Spi^S signals in all spiracular chamber or stigmatophore cells after the specification phase. During stage 12, AbdB also relocates the Hh signalling source by inducing En-independent expression of hh in the dorsal ectoderm. Thus, later than Wg and Egfr signalling, the Hh signal also acquires properties unique to A8. In generating this pattern, AbdB plays a fundamental role in uncoupling hh transcription from En activity, providing a context that prevents anterior compartment En-positive cells to turn on hh transcription, and that allows hh expression in the absence of En in other cells. Slightly later, at stage 13, AbdB modifies the expression of the posterior selector gene en, initiating de novo transcription in anterior compartment cells. In these cells, En fulfils different regulatory functions than in posterior cells, as discussed above for hh regulation. Changes in En expression and function can be interpreted as a requisite to loosen AP polarity in A8 and gain circular coordinates required for stigmatophore formation (Merabet, 2005).

Cytoskeletal dynamics and cell signaling during planar polarity establishment in the Drosophila embryonic denticle

Many epithelial cells are polarized along the plane of the epithelium, a property termed planar cell polarity. The Drosophila wing and eye imaginal discs are the premier models of this process. Many proteins required for polarity establishment and its translation into cytoskeletal polarity were identified from studies of those tissues. More recently, several vertebrate tissues have been shown to exhibit planar cell polarity. Striking similarities and differences have been observed when different tissues exhibiting planar cell polarity are compared. This study describe a new tissue exhibiting planar cell polarity -- the denticles, hair-like projections of the Drosophila embryonic epidermis. the changes in the actin cytoskeleton that underlie denticle development are described in real time, and this is compared with the localization of microtubules, revealing new aspects of cytoskeletal dynamics that may have more general applicability. An initial characterization is presented of the localization of several actin regulators during denticle development. Several core planar cell polarity proteins are asymmetrically localized during the process. Finally, roles for the canonical Wingless and Hedgehog pathways and for core planar cell polarity proteins in denticle polarity are described (Price, 2006).

Among the hallmarks of PCP in structures as diverse as Drosophila wing hairs to stereocilia in the mammalian ear is polarization of the actin cytoskeleton. The polarized actin cytoskeleton underlying wing hair polarity has been described and defects in polarization in fz and dsh mutants have been documented. Microtubules (MTs) are also polarized in developing wing hairs, and disruption of either actin or MTs disrupts wing hair formation. The data suggest that basic features of cytoskeletal polarity in pupal wing hairs are also seen in denticles. Denticles, like wing hairs, arise from polarized actin accumulations – in denticles this occurs along the posterior cell margin. Further, like wing hairs, denticles all elongate in the same direction. The less detailed analysis of dorsal hairs suggests that they also arise from polarized actin accumulations, but these are more complex; different cell rows accumulate actin either along the anterior or posterior cell margin (Price, 2006).

The effect of Wg and Hh on denticle development is mediated in part by their regional activation of the Shaven-baby transcription factor (Ovo), which is necessary and sufficient for cells to generate actin-based denticles. Therefore genes that are targets of Shaven-baby are likely to be triggers for actin accumulation and cytoskeletal rearrangements. Wg and Hh signaling may also trigger polarization of cellular machinery that is not typically thought to be involved in PCP – e.g. the polarity of Arm that was observed. It will be useful in the future to examine whether proteins polarized during germband extension, such as Bazooka, are also polarized during denticle formation. Mutations in both hh and wg also affected the normal changes in cell shape accompanying denticle formation – rather than elongating along the dorsal-ventral axis, cells remain columnar. A similar failure of cells to polarize during dorsal closure is observed in wg mutants. These effects may reflect alterations in cell polarization or cytoskeletal regulation. It will be of interest to determine whether changes in cell shape are coupled to the establishment of cytoskeletal polarity (Price, 2006).

Thus far the analysis of actin in wild-type and mutant pupal wings has been restricted to snapshots in fixed tissue. This was extended by examining F-actin in developing denticles in real time, revealing features of polarization that have not been noted previously; these features may be shared with wing hairs or other polarized structures. The initial cytoskeletal change observed was actin accumulation all across the apical surface of the cell. This actin gradually 'condenses', becoming more restricted to the posterior cell margin and forming distinct condensations, which then brighten and sometimes merge. They then elongate, all in the posterior direction. It will be interesting to learn whether the dynamic aspects of condensation involve de novo actin polymerization and/or collection of preexisting actin filaments (Price, 2006).

It is only in late condensations that enrichment was seen of any of the actin regulators that were examined. Arp3 and Dia are weakly enriched in late condensations, with enrichment increasing as denticles elongate, and Ena is enriched even later. Of course, the localization of these actin regulators to developing denticles does not by itself demonstrate that they play an important role there, but it is consistent with the possibility that they have a role in actin remodeling associated with denticle elongation. To test this hypothesis, genetic analyses will be necessary. This presents significant obstacles, since Arp2/3 and Dia are required for much earlier events (syncytial stages and cellularization), while maternal Ena plays a role in oogenesis, complicating analysis of loss-of-function mutants. Surprisingly, none of these actin regulators localizes in an informative fashion during the initial formation of actin condensations (though APC2 localizes there during this time). Thus additional regulators functioning during early denticle development need to be identified. Studies of cytoskeletal regulation in the larger adult sensory bristles may guide this. EM studies, the use of cytoskeletal inhibitors, and FRAP, which has proved informative in studies of wing hairs and bristles, may reveal how actin in denticles is assembled. Finally, it will be important to study in denticles additional actin regulators that regulate bristle development (Price, 2006).

What signals regulate denticle polarity? As examples of PCP have proliferated, understanding of the signals that instruct cells about their orientation in epithelial sheets has evolved. Certain features are shared in many, if not all, tissues. Fz receptors play a key role. Other core polarity proteins including Dsh, Fmi, Van Gogh/Strabismus and Prickle act in many if not all places. The current data extend this analysis to the denticles. Intriguing differences were found between the phenotypes of loss of Wg or Hh signaling, in which polarity was severely altered or abolished and loss of proteins that play dedicated roles in PCP, such as embryos null for either fz or stbm, which exhibit more subtle defects. A strong polarity bias was retained in these latter mutants, with cells in the posterior denticle rows correctly polarized and only cells in the anterior two rows making frequent mistakes. Interestingly, occasional mistakes are also observed in wild-type embryos (albeit at much lower frequency) and these are also restricted to the anterior most rows. This is in strong contrast to the effects of these mutants in the wing disc, where they globally disrupt polarity (Price, 2006).

One possible reason for this difference is the different scales of the tissues. The embryonic segment is only 12 cells across, while the wing disc encompasses hundreds of cells. Many core polarity proteins help mediate a feedback loop that amplifies an initially small difference in signal strength between the two sides of a wing cell. Perhaps the small scale of the embryonic segment makes this reinforcement less essential. It is also intriguing that the polarity is most sensitive to disruption in the anterior two denticle rows. If signal emanated from the posterior, signal strength might be lower in the anteriormost cells, rendering the reinforcement process more important. The lower frequency of defects in pk¹ mutants may also reflect the reduced role of the feedback loop, but this is subject to the caveat that pk is a complex locus with different mutations having different consequences. Future work will be needed to test these possibilities (Price, 2006).

Significant questions also remain about the signal(s) activating Fz receptors during PCP. Wnts were initial candidates, since Fz proteins are Wnt receptors. In vertebrates, this may be the case – Wnt11 regulates convergent extension and Wnt proteins can regulate PCP in the inner ear. By contrast, Drosophila Wnt proteins may not play a direct role. The Wg expression pattern in the eye and wing discs is not consistent with a role as the PCP ligand. Detailed studies of PCP in the eye and abdomen are most consistent with the idea that neither Wg nor other Wnt proteins are polarizing signals, but suggest that Wg regulates production of a secondary signal [dubbed `X'). Recent work suggests that Fj, Ds and Fat may be this elusive signal, with Drosophila Wg acting as an indirect cue of polarity. In fact, one cannot rule out the possibility Wnt11's role in vertebrate convergent extension is also indirect (Price, 2006).

Roles were found for Wg, Dsh and Arm in establishing denticle polarity. At face value, Arm's role is surprising, since the current view is that the Wg pathway diverges at Dsh, with a non-canonical branch mediating PCP and the canonical pathway playing no role in this. However, the data do not imply that Arm is required in denticle PCP per se. Wg acts in a paracrine feedback loop to maintain its own expression. In embryos maternally and zygotically mutant for arm alleles that cannot transduce Wg, Wg expression is lost by late stage 9. Thus, even though Arm is not in the non-canonical pathway, loss of Arm could still disrupt PCP indirectly due to the loss of Wg expression (Price, 2006).

While the data demonstrate that Wg is required for denticle PCP, two things suggest its role is indirect. wg mutants retain segmental periodicity in denticle orientation, suggesting that polarity is not totally disrupted, while in hh mutants there is no segmental periodicity. Second, when Wg signaling was reduced but did not eliminated, many cells retained normal polarity and there was segmental periodicity to which cells lost polarity or exhibited polarity reversals. This is consistent with the idea that Wg regulates production of another ligand. In fact, Wg's role may be even more indirect – given the more dramatic effect of hh, Wg's primary role in polarity may be to maintain Hh expression (this is also consistent with a requirement for canonical pathway components like Arm). Global activation of Hh signaling in the ptc mutant also disrupts polarity. Hh thus remains a possible directional cue. In the abdomen, Hh also plays an important role in polarity, but it does not seem to be the directional cue either but rather regulates its production; this may also be the case in the embryo. Thus the precise roles for canonical Wg and Hh signaling in denticle polarization must be addressed by future experiments. If neither Wnts nor Hh are directional signals, what is? Data from the eye, wing and abdomen suggest roles for Ds, Fj, Fat and Fmi but details differ in different tissues. It thus will also be useful to examine Ds, Fj and Fat's roles in embryonic PCP (Price, 2006).

Wnt, Hedgehog and junctional Armadillo/beta-catenin establish planar polarity in the Drosophila embryo

To generate specialized structures, cells must obtain positional and directional information. In multi-cellular organisms, cells use the non-canonical Wnt or planar cell polarity (PCP) signaling pathway to establish directionality within a cell. In vertebrates, several Wnt molecules have been proposed as permissible polarity signals, but none has been shown to provide a directional cue. While PCP signaling components are conserved from human to fly, no PCP ligands have been reported in Drosophila. This paper reports that in the epidermis of the Drosophila embryo two signaling molecules, Hedgehog (Hh) and Wingless, provide directional cues that induce the proper orientation of Actin-rich structures in the larval cuticle. Proper polarity in the late embryo also involves the asymmetric distribution and phosphorylation of Armadillo (Arm or β-catenin) at the membrane and that interference with this Arm phosphorylation leads to polarity defects. These results suggest new roles for Hh and Wg as instructive polarizing cues that help establish directionality within a cell sheet, and a new polarity-signaling role for the membrane fraction of the oncoprotein Arm (Colosimo, 2006).

These results indicate that Wg and Hh act as instructive cues in the Drosophila embryonic epidermis to establish planar cell polarity. Though the complete molecular mechanisms that control the complex system of PCP in the ventral epidermis remain to be determined, this process appears to occur in part through the asymmetric localization of Arm at the membrane. Further, proper polarity signaling is abolished if specific phosphorylation sites within the alpha-catenin binding domain of Arm are mutated. These sites were originally found to increase the affinity of β-catenin for alpha-catenin when phosphorylated by Casein Kinase II in vitro, suggesting one mechanism for stabilizing junctions. These findings provide in vivo support for this hypothesis, since low levels of Arm^AA Arm in which two threonines were mutated to alanines) rescues cellular junction defects to a similar extent as expression of an alpha-catenin/E-cadherin fusion protein, a protein that makes overly stable junctions. Higher levels of Arm^AA expression lead to apparent polarity defects. Since Arm^AA does not localize asymmetrically the way that wild-type Arm does, it is inferred that CKII phosphorylation may be required for the accumulation of junctions in specific regions of cells implying that stable junctions at specific sites in a cell are required for proper planar cell polarity. Further, these findings revealed that when all signaling activity is abolished through null mutations in the Wg or Hh signaling pathways, both cell identity and polarity determination was disrupted. It remains to be determined how Wg and Arm proteins function in polarity signaling, specifically whether they work through known PCP components, function similarly to their role in dorsal closure, or perhaps through novel signaling mechanisms like the interaction with Notch or Axin (Colosimo, 2006).

The wg and hh genes are required for the proper establishment of cell identities within segments. Uniform expression of Wg in the embryo leads to a completely naked cuticle, but short early bursts of expression establish what appears to be relatively normal patterning. Upon closer inspection, however, the denticle orientations of these early expression rescue experiments do not entirely resemble the wild-type patterning. This suggests that early expression of Wg can rescue several aspects of cell identity, including development of naked cuticle, but Wg is also required in the later stages when denticles form to specify proper orientations. Expression of ectopic Wg has been observed to correlate with denticles pointing toward the source of Wg, and expression of ectopic Hh also leads to denticles pointing away from its source. These previous studies, however could not distinguish between cell fate transformation and changes in cell polarity since the sources of both ligands were in the normal orientation. The current observations argue that Hh and Wg can have direct effects on cell polarity since denticles and their precursors (the Actin foci) are rotated 90ƒ away from the anterior-posterior axis corresponding to the direction of ligand expression (Colosimo, 2006).

In the early embryo, expression of Wg and Hh is determined by pair-rule genes, but this effect is transient and requires mutually reinforcing positive activation loops to form between cells expressing Wg and En/Hh. This is the early signaling event that establishes an organizer region in each parasegment. Therefore, if either Hh or Wg is missing, expression of both is lost. The early effects of Hh and Wg expression are important for the establishment of segment boundaries, and these boundaries function in limiting Wg function, giving this morphogen an asymmetric range. The current findings agree with these observations, because it was observed that the Wg effect is best observed when hh is absent, suggesting that when the hh gene is present a boundary may be formed, thus preventing Wg from orienting the denticles to the same extent. It also appears that the distance over which Wg can act is longer in the absence of hh as expected from previous observations. According to the proposed boundary model, the extent of Wg influence is to the first denticle-secreting cell, but not beyond. This finding, along with the discovery that denticles orient toward the source of Wg, may explain why the first row of denticles in wild-type larvae points toward the anterior of the embryo. Only this row of cells receives Wg signal as the segment boundary blocks further action by Wg to the next row of cells. In contrast, Hh can and does affect the next two rows of cells. It was found that expression of Hh causes a rotation away from the source, and could explain why the next two rows of denticles point toward the posterior of the embryo. These results do not explain the final orientation of all rows of denticles, and one likely complication is that in late embryonic stages the Notch and EGFR signaling pathways affect the identities of cells within the denticle belt. It will be interesting to test what effects these signals have on the final orientation of the orientation of denticles, and whether the Notch pathway functions in polarity as well (Colosimo, 2006).

The PCP signaling pathway determines planar polarity in a variety of tissues. In vertebrate and C. elegans studies, Wnts have been implicated in the establishment of polarity, but only one study in Drosophila suggested a role for Wg in PCP (Price, 2006). In fact, the present model excludes the known morphogens, and suggests that PCP is established through cell-cell interactions involving atypical cadherins like Flamingo or through an as yet unidentified factor X. Though this study does not address the function of the known components of PCP signaling in the embryo, it is interesting that mutants in PCP signaling pathway components affect the polarity of the first two rows of denticles. The current findings support the possibility that Wg and Hh lead to the expression of an unknown factor affecting the polarization of denticles, because blocking the transcriptional readout of either Wg or Hh with tcf or ci mutations respectively prevents the polarizing activity of both pathways. This is similar to the PCP disruptions found in the Drosophila eye model for Wg signaling components. The current observations do, however, offer a further possibility, namely that by blocking all Wg signaling with null mutations the underlying polarity organizing function of Wg may be obscured. In the weak arm^F1A mutant the orientation of denticles can be affected by the expression of Wg without affecting the cell-fates, suggesting that perhaps Wg can affect polarity directly. This effect of Wg was not observed in stronger arm mutant embryos suggesting that Arm protein is required for the Wg effect on denticle orientation. Interestingly, cell culture work has recently implicated Wg in controlling adherens junction strength (Colosimo, 2006).

The use of the embryonic epidermis led to the the interesting possibility that Arm functions in cell polarity. Since some of the molecules involved in the PCP signaling pathway are similar to Cadherins, it seems logical that adhesion is involved in the establishment of polarity. However, adherens junctions have not been implicated so far. This is likely due to the difficulty of working with adherens junction component mutations that are often cell-lethal in the systems that have been used to study PCP. Use of the embryo allows relatively simple perturbation of arm function, and efficient ubiquitous or directional ectopic expression. Unfortunately, the major limitation of the ventral midline expression assay is that it only works for secreted, diffusible ligands. Thus, cell-autonomous activation of Hh or Wg pathway components (such as with activated Arm or Smo) along the ventral midline cannot be observed, since these cells invaginate and do not become a part of the external epidermis (Colosimo, 2006).

The fact that β-catenin is both an oncogene and a component of adherens junctions has led to many studies attempting to link the phosphorylation state of β-catenin in adherens junctions to the epithelial to mesenchymal transition (EMT) in cancer cells and during development. Phosphorylation of tyrosine residues in β-catenin is thought to lead to disassembly of adherens junctions, but recent studies both in vivo and in vitro have challenged this. Certainly these discrepancies will have to be resolved, but this study provides evidence for a different mechanism for regulating junctions, and perhaps EMT, through threonine phosphorylation-based stabilization or dephosphorylation-based destabilization of junctions. It will be crucial to establish which is the regulated step, and whether there are any phosphatases involved in this process in addition to the known kinase CKII (Colosimo, 2006).

Interestingly, the recent findings that alpha-catenin and ţ-catenin do not form a stable complex in junctions, suggests a possible explanation for these findings. It is speculated that expression of Arm^AA can rescue the basic activity of junctions lost in strong arm mutant embryos, which is to hold a tissue together. However, its reduced affinity for alpha-catenin does not cause a local increase in alpha-catenin levels and therefore Actin levels do not become asymmetric. This leads to a skewing of the normal polarization of the Actin cytoskeleton. It will be crucial to determine how junctions are localized asymmetrically in the first place, and whether this is dependent on extracellular signaling. These findings, and the effects of alpha-catenin mutations on inflammation and tumor progression in the mouse epidermis make analysis of the interaction between alpha- and β-catenin particularly important (Colosimo, 2006).

These experiments provide some of the first evidence that the Hh signaling pathway is involved in polarity. It is particularly interesting that Hh expression leads to the reorganization of Actin structures within epithelial cells, since this suggests that Hh can affect the polarity of the Actin cytoskeleton. This finding is also relevant to cancer biology, because during metastasis, cancer cells lose polarity and essentially ignore their environment. Our results show that Wnts and Hh can affect cell polarity, in addition to their well-known effects on cell proliferation. Along with the recent report that TGFβ signaling affects polarity and EMT, these findings imply that this dual role may be a general feature of oncogenic signaling pathways (Colosimo, 2006).

Lipoprotein particles are required for Hedgehog and Wingless signalling

Wnt and Hedgehog family proteins are secreted signalling molecules (morphogens) that act at both long and short range to control growth and patterning during development. Both proteins are covalently modified by lipid, and the mechanism by which such hydrophobic molecules might spread over long distances is unknown. The Drosophila lipoprotein particle, Lipophorin (Retinoid- and fatty acid-binding glycoprotein), bears lipid-linked morphogens on its surface and is required for long-range signaling activity of Wingless and Hedgehog. Wingless, Hedgehog and glycophosphatidylinositol-linked proteins copurify with lipoprotein particles marked by lipophorin, and co-localize with these particles in the developing wing epithelium of Drosophila. In larvae with reduced lipoprotein levels, Hedgehog accumulates near its site of production, and fails to signal over its normal range. Similarly, the range of Wingless signalling is narrowed. A novel function is proposed for lipoprotein particles, in which they act as vehicles for the movement of lipid-linked morphogens and glycophosphatidylinositol-linked proteins (Panakova, 2005).

In the developing wing of Drosophila, Hedgehog activates short-range target gene expression up to five cells away from its source of production, and longer-range targets over more than twelve cell diameters. Wingless can signal through a range of over 30 cell diameters. These morphogens are anchored to the membrane via covalent lipid modification. The mechanisms that allow long-range movement of molecules with such strong membrane affinity are unclear (Panakova, 2005).

Like Wingless and Hedgehog, glycophosphatidylinositol (gpi)-linked proteins transfer between cells with their lipid anchor intact. Gpi-linked green fluorescent protein (GFP) expressed in Wingless-producing cells spreads into receiving tissue at the same rate as Wingless, where it co-localizes with Wingless in endosomes. Thus, it is proposed that these proteins travel together on a membranous particle, which has been called an argosome. How might argosomes form? One possibility is that argosomes are membranous exovesicles. Such particles could be generated by plasma membrane vesiculation, or by an exosome-related mechanism. Alternatively, argosomes might resemble lipoprotein particles like low-density lipoprotein (LDL). Vertebrate lipoprotein particles are scaffolded by apolipoproteins and comprise a phospholipid monolayer surrounding a core of esterified cholesterol and triglyceride. Insects construct similar particles called lipophorins. Lipid-modified proteins of the exoplasmic face of the membrane (such as GFPgpi, Wingless or Hedgehog) might insert into the outer phospholipid monolayer of such a particle via their attached lipid moieties. This study use biochemical fractionation to determine the sort of particle with which lipid-linked proteins associate, and genetic means to address its function (Panakova, 2005).

Lipid-linked proteins copurify with lipophorin: Sedimentation of Wingless, Hedgehog and gpi-linked proteins were compared to that of transmembrane proteins, exosomes and lipophorin particles. To mark exosomes, flies were used expressing a vertebrate CD63:GFP fusion construct. CD63 is a tetraspanin that localizes to internal vesicles of multivesicular endosomes, and is released on exosomes. In Drosophila imaginal discs, CD63:GFP localizes to late endosomes in producing cells, consistent with vertebrate studies. It is released and endocytosed by neighbouring cells between one and three cell diameters away, indicating that it is present on exosomes (Panakova, 2005).

To mark lipoprotein particles, antibodies were made to Drosophila apolipophorins I and II (ApoLI and ApoLII); these proteins are generated by cleavage of the precursor pro-Apolipophorin (Sundermeyer, 1996; Kutty, 1996). Lipophorin is produced in the fat body (Kutty, 1996); consistent with this, apolipophorin transcripts cannot be detected in imaginal discs. Nevertheless, the ApoLI and ApoLII proteins are as abundant in discs as in the fat body (Panakova, 2005).

Plasma membrane and exosomal markers are completely pelleted after centrifugation for 3 h at 120,000g, whereas most ApoLII remains in the supernatant. Most Wingless:GFP and Hedgehog is present in the pellet, as are the gpi-linked proteins Fasciclin, Connectin, Klingon and Acetylcholineasterase; this is not unexpected, because these proteins localize to the plasma membrane and internal membrane compartments. Surprisingly, however, some Wingless:GFP (6%), Hedgehog (2%) and gpi-linked proteins (14%-22%) remain in the supernatant (Panakova, 2005).

The 120,000g supernatant (S120) contains both free soluble proteins and lipoprotein particles. To separate them, isopycnic density centrifugation was performed. In these gradients, lipophorin moves to the top low-density fraction whereas soluble proteins are present in higher-density fractions. Gpi-linked proteins are found almost entirely in the top fraction with lipophorin. Treating the S120 with Phosphatidylinositol-specific phospholipase C (PI-PLC) before density centrifugation shifts their migration to higher-density fractions. This suggests that gpi-linked proteins associate with low-density particles via their gpi anchor (Panakova, 2005).

Similarly, when S120s from larvae that express Wingless:GFP or Hedgehog:HA in imaginal discs are subjected to isopycnic density centrifugation, these proteins are found in the lowest-density fraction with ApoLII, as is endogenous Hedgehog. Antibodies to endogenous Wingless detect a doublet in the top fraction and a band of somewhat higher mobility in high-density fractions. These data indicate that non-membrane-bound Wingless and Hedgehog associate with low-density particles in imaginal discs in vivo; other larval tissues may secrete Wingless in a non-lipophorin-associated form (Panakova, 2005).

To ask whether lipid-linked proteins associate with lipophorin, or with some other low-density particle, ApoLII was immunoprecipitated from larval S120s and precipitates were probed for Wingless, Hedgehog or GFPgpi. These proteins are immunoprecipitated by anti-ApoLII. Hedgehog and Fas-1 also immunoprecipitate with ApoLII from the more purified top fraction of KBr gradients. Thus, lipid-linked morphogens and gpi-linked proteins associate directly with lipophorin particles (Panakova, 2005).

Lipophorin-RNAi perturbs lipid transport: To assess the role of lipophorin in larval growth and development, the levels of ApoLI and II were reduced by RNA interference directed against two different regions of the apolipophorin messenger RNA. Similar phenotypes were produced by each construct. To express double-stranded (ds)RNA, a modified GAL4:UAS system was used in which expression of inverted repeats can be temporally controlled by heat-shock-dependent excision of an intervening HcRed cassette by the flippase (FLP) recombinase. Extracts were tested from wild-type larvae or larvae harbouring hs-flp, GAL4 driver and UAS dsRNA constructs at various times after heat shock to see how fast lipophorin levels were reduced. Larvae of the latter genotype made only 50% of the wild-type level of ApoLII, even in the absence of heat shock; basal activity of the heat-shock promoter in the fat body causes HcRed excision in approximately 50% of fat-body cells, although excision strictly depends on heat shock in other larval tissues. Although they survive less frequently, these flies have no obvious phenotype (Panakova, 2005).

After heat shock, all fat-body cells excise the HcRed cassette and ApoLII levels decrease further. After four days, ApoLII is reduced to 5% of wild-type levels. ApoLI levels are reduced with similar kinetics. These animals prolong the third larval instar and rarely pupariate. All the experiments described below were performed on third-instar larvae 4-6 days after heat shock (Panakova, 2005).

To investigate the requirement for lipophorin in lipid transport, the accumulation of neutral lipids in larval tissues was assessed by staining them with Nile Red. Cells of the posterior midgut normally contain many small lipid droplets. Lipophorin reduction causes a dramatic expansion of these droplets, suggesting that lipophorin is required for the efficient extraction of lipid from the midgut (Panakova, 2005).

The wild-type fat body contains both small and large lipid droplets. Fat bodies of lipophorin-RNAi larvae are reduced in size and have fewer small lipid droplets, although larger droplets appeared normal. These data suggest that lipophorin delivers lipid to the fat body (Panakova, 2005).

Lipid droplets in discs from lipophorin-RNAi larvae are fewer and smaller than in the wild type. Their discs are also reduced in size, particularly in the wing pouch. Thus, discs require lipophorin for accumulation of lipid droplets and for growth. Neither Caspase3 activation nor membrane phosphatidylinositol 3,4,5-phosphate (PIP₃) accumulation is altered in lipophorin- RNAi discs , suggesting that their small size is not due to cell death or reduced insulin signalling (Panakova, 2005).

Hedgehog function requires lipophorin: To test whether lipophorin association is required for Hedgehog function, Hedgeghog distribution and signalling was examined in lipophorin-RNAi larval discs. In wild-type discs, Hedgehog expressed in the posterior compartment moves across the anterior-posterior (AP) compartment boundary and activates transcription of short and long-range target genes. Cells closest to the source respond by activating the transcription of collier and patched. Further away, Hedgehog activates transcription of decapentaplegic. Levels of Collier and a decapentaplegic reporter construct (dpplacZ) were monitored in wild-type and lipophorin-RNAi discs stained in parallel and imaged under identical conditions. Discs from lipophorin-RNAi larvae activate collier at least as efficiently as those of the wild type. In contrast, the range of activation of dppLacZ is significantly narrowed in lipophorin RNAi discs. dppLacZ is expressed up to 11 cells away from the AP boundary in wild-type discs, but only up to six cells away in lipophorin-RNAi larvae. These data suggest that lipophorin knockdown decreases the range of Hedgehog signalling (Panakova, 2005).

To discover whether Hedgehog trafficking was altered, discs were stained for Hedgehog and Patched. In wild-type discs, Hedgehog moves into the anterior compartment, where it is found in endosomes, often with Patched. Patched-mediated endocytosis is thought to sequester Hedgehog and limit its spread. Hedgehog is most abundant up to five cell rows away from the AP boundary; although Hedgehog signals over a wider range, specific staining there cannot be distinguished from background. In lipophorin-RNAi discs, Hedgehog accumulates to abnormally high levels in the first five rows of anterior cells. 380 Hedgehog spots were found in the most apical 10 µm of the wild-type disc. The lipophorin-RNAi disc contained 1,208 Hedgehog spots in the same region. Most accumulated Hedgehog colocalizes with Patched in endosomes. Furthermore, Patched co-accumulates more extensively with Hedgehog in endosomes than it does in wild-type. These data indicate that lipophorin RNAi either increases the susceptibility of Hedgehog to Patched-mediated endocytosis, or prevents subsequent degradation of the protein (Panakova, 2005).

Drosophila cannot synthesize sterols and relies on dietary sources. To assess whether reduced uptake of sterols or other lipids might cause the changes seen, the effects of lipid deprivation on larval development were explored. Larvae were allowed to hatch and feed on sucrose/agarose plates supplemented with yeast for 2-3 days, then transferred to plates containing chloroform-extracted yeast autolysate, rather than yeast. These larvae are developmentally delayed; after 7 days of lipid deprivation, their discs are much smaller than those of younger late-third-instar larvae. In contrast, yeast-fed siblings pupariate and begin to eclose by this time. Those flies that infrequently eclose after larval lipid depletion are small (35%-60% of normal body weight) but normally patterned. Thus, lipid depletion stalls imaginal growth (Panakova, 2005).

To discover whether lipid starvation affected Hedgehog trafficking or signalling, larvae were deprived of lipid 2 days after hatching and their discs were stained 6 days later. No changes in Hedgehog or Patched distribution are apparent in these discs compared with younger yeast-fed discs of similar size. Furthermore, the range of dpp and collier expression does not differ in lipid-starved and yeast-fed discs. Thus, lipid starvation does not mimic the effects of lipophorin knockdown. It is speculated that lipid-starvation-induced growth arrest prevents membrane sterol from dropping to levels that would interfere with the Hedgehog pathway. Thus, lipophorin does not indirectly affect the Hedgehog pathway via lipid deprivation (Panakova, 2005).

Wingless function requires lipophorin: To discover whether lipophorin RNAi perturbed Wingless trafficking, Wingless distribution was examined. In lipophorin- RNAi discs, extracellular Wingless is less abundant on both the apical and basolateral epithelial surfaces and spreads over shorter distances. However, no consistent alterations were detected in intracellular Wingless. Thus, lipophorin promotes accumulation of extracellular Wingless (Panakova, 2005).

To investigate whether Wingless signalling requires lipophorin, the activation of two target genes was examined. Senseless is produced only in cells near the Wingless source and its expression is unaffected by lipophorin RNAi. Distalless is normally produced in a gradient throughout most of the wing pouch. In lipophorin-RNAi discs, the Distalless gradient is abnormally narrow. This suggests that lipophorin knockdown specifically perturbs long-range Wingless signalling (Panakova, 2005).

Conclusions: This study establishes the principle that lipid-linked proteins of the exoplasmic face of the membrane associate with lipoproteins. These include many gpi-linked proteins with diverse functions, as well as the lipid-linked morphogens Wingless and Hedgehog. The mechanism allowing long-range dispersal of lipid-linked proteins is not yet understood. The finding that these proteins exist in both membrane-associated and lipoprotein-associated forms suggests reversible binding to lipoprotein particles as a plausible mechanism for intercellular transfer, and the consequences of lowering lipoprotein levels in Drosophila larvae supports this idea (Panakova, 2005).

Lipophorin knockdown narrows the range of both Wingless and Hedgehog signalling. Hedgehog accumulates to an abnormally high level in cells near the source of production and long-range signalling is inhibited; short-range target genes, however, are expressed normally. These data suggest that Hedgehog does not move as far when lipophorin levels are low. The range over which Hedgehog moves is normally restricted by Patched-mediated endocytosis. In discs from lipophorin RNAi larvae, accumulated Hedgehog co-localizes with Patched in endosomes, suggesting that it is more efficiently sequestered by Patched. How might lipophorin antagonize Patched-mediated sequestration and promote long-range movement (Panakova, 2005)?

The data are consistent with the idea that lipophorin is continuously needed for movement, rather than required only for the release of morphogens. If lipophorin were important only for Hedgehog secretion, lipophorin RNAi would be expected to decrease the amount of Hedgehog found in receiving tissue; this seems not to be the case. Furthermore, altered Hedgehog trafficking in receiving tissue is consistent with a model in which lipophorin is required at each step of intercellular transfer. The idea is favored that reversible association of Hedgehog with lipophorin particles facilitates its transfer from the plasma membrane of one cell to that of the next. This model predicts that lowering lipophorin levels should increase the length of time that Hedgehog spends in the plasma membrane before becoming associated with lipophorin. This would slow its rate of transfer and increase the probability of Patched endocytosing Hedgehog before it moved to the next cell. Hedgehog would then signal efficiently in the short range, but be so efficiently sequestered by Patched that very little protein would travel far enough to activate long-range target genes. These predictions are completely consistent with the current observations (Panakova, 2005).

This model differs significantly from the original concept of argosome function. It was initially speculated that argosomes were exosome-like particles with an intact membrane bilayer, and that lipid-linked morphogens needed to be assembled on these particles to be secreted by producing cells. Instead, it was found that argosomes are exogenously derived lipoproteins that facilitate the movement of morphogens through the epithelium. Many questions remain as to how morphogens become associated with argosomes, and how the spread and cell-interactions of these particles are regulated. Clearly, heparan sulphate proteoglycans are essential for the movement of Hedgehog and Wingless into receiving tissue. Because heparan sulphate binds to vertebrate lipoprotein particles, one might speculate that heparan sulphate proteoglygans (HSPGs) facilitate morphogen movement through lipoprotein binding. Conversely, many gpi-linked proteins, including the HSPG's Dally and Dally-like, are found on lipoprotein particles themselves. These associated proteins have the potential to modulate the cellular affinities or trafficking properties of lipoproteins and the morphogens they carry (Panakova, 2005).

The data suggest that lipophorin particles not only mediate intercellular transfer of Hedgehog, but may also be endocytosed together with the morphogen. Interestingly, LDL-receptor-related proteins Arrow and Megalin have demonstrated roles in Wingless signalling and Hedgehog endocytosis, respectively. It is intriguing to speculate that these receptors might be important for interaction with the lipoprotein-associated form of the morphogen (Panakova, 2005).

Cholesterol has the potential to modulate the activity of the Hedgehog pathway at many different points. Whether changes in the level of cellular cholesterol normally play a role in regulating the activity of the pathway is unclear. This study shows that Hedgehog interacts with the particle that delivers sterol to cells. This observation raises the possibility that internalization of Hedgehog is linked to sterol uptake, and suggests new mechanisms to link nutrition, growth and signalling during development (Panakova, 2005).

A Hedgehog- and Antennapedia-dependent niche maintains Drosophila haematopoietic precursors

The Drosophila lymph gland is a haematopoietic organ in which pluripotent blood cell progenitors proliferate and mature into differentiated haemocytes. Previous work (Jung, 2005) has defined three domains, the medullary zone, the cortical zone and the posterior signalling centre (PSC), within the developing third-instar lymph gland. The medullary zone is populated by a core of undifferentiated, slowly cycling progenitor cells, whereas mature haemocytes comprising plasmatocytes, crystal cells and lamellocytes are peripherally located in the cortical zone. The PSC comprises a third region that was first defined as a small group of cells expressing the Notch ligand Serrate. This study shows that the PSC is specified early in the embryo by the homeotic gene Antennapedia (Antp) and expresses the signalling molecule Hedgehog. In the absence of the PSC or the Hedgehog signal, the precursor population of the medullary zone is lost because cells differentiate prematurely. It is concluded that the PSC functions as a haematopoietic niche that is essential for the maintenance of blood cell precursors in Drosophila. Identification of this system allows the opportunity for genetic manipulation and direct in vivo imaging of a haematopoietic niche interacting with blood precursors (Mandal, 2007).

The Drosophila lymph gland primordium is formed by the coalescence of three paired clusters of cells that express Odd-skipped (Odd) and arise within segments T1-T3 of the embryonic cardiogenic mesoderm. At developmental stages 11-12, mesodermal expression of Antp is restricted to the T3 segment. A fraction of these Antp-expressing cells will contribute to the formation of the dorsal vessel, whereas the remainder, which also express Odd, give rise to the PSC. By stages 13-16, the clusters coalesce and Antp is observed in 5-6 cells at the posterior boundary of the lymph gland. The expression of Antp is subsequently maintained in the PSC through the third larval instar. The embryonic stage 16 PSC can also be distinguished by Fasciclin III expression and at stage 17 these are the only cells in the lymph gland that incorporate BrdU (Mandal, 2007).

Previous studies have identified the transcription factor Collier (Col) as an essential component regulating PSC function. The gene for this protein is initially expressed in the entire embryonic lymph gland anlagen and by stage 16 is refined to the PSC. In col mutants, the PSC is initially specified, but is entirely lost by the third larval instar. To address further the role of Antp and Col in embryonic lymph gland development, the expression of each gene was investigated in the loss-of-function mutant background of the other. It was found that loss of col does not affect embryonic Antp expression. In contrast, col expression is absent in the PSC of Antp mutant embryos, establishing that Antp functions genetically upstream of Col in the PSC (Mandal, 2007).

In imaginal discs, the expression of Antp is related to that of the homeodomain cofactor Homothorax (Hth). In the embryonic lymph gland, Hth is initially expressed ubiquitously but is subsequently downregulated in PSC cells, which become Antp-positive. In hth loss-of-function mutants, the lymph gland is largely missing, whereas misexpression of hth causes loss of PSC and the size of the embryonic lymph gland remains relatively normal. It is concluded that a mutually exclusive functional relationship exists between Antp and Hth in the lymph gland such that Antp specifies the PSC, whereas Hth specifies the rest of the lymph gland tissue. Interestingly, knocking out the mouse homologue of Hth, Meis1, eliminates definitive haematopoiesis (Hisa, 2004; Azcoitia, 2005). Meis1 is also required for the leukaemic transformation of myeloid precursors overexpressing HoxB9 (Mandal, 2007).

Although lymph gland development is initiated in the embryo, the establishment of zones and the majority of haemocyte maturation takes place in the third larval instar. At this stage, Antp continues to be expressed in the wild-type PSC. To investigate how the loss of PSC cells affects haematopoiesis, Antp expression was examined in third instar col mutant lymph glands. In this background, all Antp-positive PSC cells are missing, consistent with the previously described role for col in PSC maintenance. Overexpression of Antp within the PSC increases the size of PSC from the usual 30-45 cells to 100-200 cells. These PSC cells are scattered over a larger volume, often forming two or three large cell clusters rather than the single, dense population seen in wild type (Mandal, 2007).

To determine the role of PSC in haematopoiesis, the expression pattern of various markers was investigated in lymph glands of larvae of the above genotypes, which either lack a PSC or have an enlarged PSC. The status of blood cell progenitors was directly assessed using the medullary-zone-specific markers ZCL2897, DE-cadherin (Shotgun) and domeless-gal4. In col mutant lymph glands, expression of these markers is absent or severely reduced and when the PSC is expanded, the medullary zone is greatly enlarged. Previous work demonstrated that medullary zone precursors are relatively quiescent, a characteristic similar to the slowly cycling stem cell or progenitor populations in other systems. BrdU incorporation in the wild-type lymph gland is largely restricted to the cortical zone, but in third-instar col mutants incorporation of BrdU is increased relative to wild type and becomes distributed throughout the lymph gland, suggesting that the quiescence of the medullary zone haematopoietic precursors is no longer maintained in the absence of the PSC. Similarly, when the PSC domain is expanded, BrdU incorporation is significantly suppressed throughout the lymph gland (Mandal, 2007).

P1 and ProPO were used as markers for plasmatocytes and crystal cells, respectively, to assess the extent of haemocyte differentiation within lymph glands of the above genotypes. Loss of the PSC does not compromise haemocyte differentiation; rather, mature plasmatocytes and crystal cells are found abundantly within the lymph gland. Furthermore, the distribution of these differentiating cells is not restricted to the peripheral region that normally constitutes the cortical zone and many cells expressing ProPO and P1 can be observed medially throughout the region normally occupied by the medullary zone. Increasing the PSC domain causes a concomitant reduction in the differentiation of haemocytes (Mandal, 2007).

In summary, loss of the PSC causes a loss of medullary zone markers, a loss of the quiescence normally observed in the wild-type precursor population and an increase in cellular differentiation throughout the lymph gland. Similarly, increased PSC size leads to an increase in the medullary zone, a decrease in BrdU incorporation and a decrease in the expression of maturation markers. It is concluded that the PSC functions as a haematopoietic niche that maintains the population of multipotent blood cell progenitors within the lymph gland. The observed abundance of mature cells in the absence of the PSC suggests that the early blood cell precursors generated during the normal course of development will differentiate in the absence of a PSC-dependent mechanism that normally maintains progenitors as a population. This situation is reminiscent of the Drosophila and C. elegans germ lines in which disruption of the niche does not block differentiation per se, but lesser numbers of differentiated cells are generated as a result of the failure to maintain stem cells. It is also interesting to note that col mutant larvae are unable to mount a lamellocyte response to immune challenge. It is speculated that this could be because of the loss of precursor cells that are necessary as a reserve to differentiate during infestation (Mandal, 2007).

Recent work on several vertebrate and invertebrate developmental systems has highlighted the importance of niches as unique microenvironments in the maintenance of precursor cell populations. Examples include haematopoietic, germline and epidermal stem cell niches that provide, through complex signalling interactions, stem cells with the ability to self-renew and persist in a non-differentiated state. The work presented in this report demonstrates that the PSC is required for the maintenance of medullary zone haematopoietic progenitors. The medullary zone represents a group of cells within the lymph gland that are compactly arranged and express the homotypic cell-adhesion molecule, DE-cadherin. These cells are pluripotent, slowly cycling and undifferentiated and are capable of self-renewal. It is presently uncertain whether Drosophila has blood stem cells capable of long-term repopulation as haematopoietic stem cells are in vertebrates. Nevertheless, it is clear that the maintenance of medullary zone cells as precursors is niche dependent (Mandal, 2007).

In order for the PSC to function as a haematopoietic niche there should exist a means by which the PSC can communicate with precursors. As such, a signal emanating from the PSC and sensed by the medullary zone represents an attractive model of how this might occur. Although it has been reported that Ser and Upd3 are expressed in the PSC, preliminary analysis suggests that elimination of either of these ligands alone will not cause the phenotype seen for Antp and col mutants. Therefore the haematopoietic role of several signalling pathways was investigated and the hedgehog (hh) signalling pathway was identified as a putative regulator in the maintenance of blood cell progenitors. The hh^ts2 lymph gland is remarkably similar in its phenotype to that seen for Antp hypomorphic or col loss-of-function mutants. Blocking Hh signalling in the lymph gland through the expression of a dominant-negative form of the downstream activator Cubitus interruptus (Ci, the Drosophila homologue of Gli) also causes a phenotype similar to that observed in Antp and col loss-of-function backgrounds. This is true when expressed either specifically in the medullary zone or throughout the lymph gland (Mandal, 2007).

Consistent with the above functional results, Hh protein is expressed in the second instar PSC and continues to be expressed in third instar PSC cells. In the hh^ts2 mutant background, the PSC cells continue to express Antp at the restrictive temperature indicating that, unlike col and Antp, Hh is not essential for the specification of the PSC. Rather, Hh constitutes a component of the signalling network that allows the PSC to maintain the precursor population of the medullary zone. Consistent with this notion, downstream components of the Hh pathway, the receptor Patched (Ptc) and activated Ci, are found in the medullary zone. On the basis of both functional and expression data, it is proposed that Hh in the PSC signals through activated Ci in medullary zone cells, thereby keeping them in a quiescent precursor state (Mandal, 2007).

The Hh pathway has been studied extensively in the context of animal development. Although the Hh signal does not disperse widely on secretion, many studies have shown that this signal can be transmitted over long distances. The mechanism by which this occurs is not fully clear and this is also true of how the PSC delivers Hh to medullary zone progenitors. However, when labelled with green fluorescent protein (GFP), it was found that PSC cells extend numerous thin processes over many cell diameters. The morphology of the PSC cells, taken together with the long-range function of Hh revealed by the mutant phenotype, indicates that the long cellular extensions may deliver Hh to receiving cells not immediately adjacent to the PSC. In this respect, the Drosophila haematopoietic system shows remarkable similarity to the C. elegans germline. In both cases, precursors are maintained as a population over some distance from the niche and in both instances, the niche cells extend long processes when interacting with the precursors (Mandal, 2007).

Several studies have highlighted the importance of homeodomain proteins in stem cell development and leukaemias. Likewise, the role of Hh in vertebrate and invertebrate stem cell maintenance has recently received much attention. This study describes direct roles for Antp in the specification and Hh in the functioning of a haematopoietic niche. The medullary zone cells are blood progenitors that are maintained in the lymph gland at later larval stages by Hh, a signal that originates in the PSC. The maintenance of these progenitors provides the ability to respond to additional developmental or immune-based haematopoietic signals. On the basis of these findings, understanding the specific roles of Hh signalling and Hox genes in the establishment and function of vertebrate haematopoietic niches warrants further investigation. The identification of a haematopoietic niche in Drosophila will allow future investigation of in vivo niche/precursor interactions in a haematopoietic system that allows direct observation, histological studies and extensive genetic analysis (Mandal, 2007).

Larval

Hedgehog is secreted by cells of posterior compartments in wing, leg and eye-antennal discs, and is involved in structuring the boundary between anterior and posterior compartments in disc development (Diaz-Benjumea, 1994).

In the Drosophila wing imaginal disc, the Hedgehog (Hh) signal molecule induces the expression of decapentaplegic in a band of cells abutting the anteroposterior (A/P) compartment border. It has been proposed that Dpp organizes the patterning of the entire wing disc. This proposal was tested by studying the response to distinct levels of ectopic expression of Hh and Dpp, using the sensory organ precursors (SOPs) of the wing and notum and the presumptive wing veins as positional markers. Mis-expression of Dpp does not mimic all the effects of mis-expression of Hh. Dpp specifies the position of most SOPs in the notum and of some of them in the wing. Close to the A/P compartment border, however, SOPs are specified by Hh rather than by Dpp alone. Late signaling by Hh, after setting up dpp expression, is responsible for the formation of vein 3 and the scutellar region, and also for the determination of the distance between veins 3 and 4. One of the genes that mediates the Hh signal is the zinc-finger protein Cubitus interruptus (Ci). Ectopic Ci has an effect on SOP distribution similar to that observed by the induction of Hh expression. These results indicate that Hh has a Dpp-independent morphogenetic effect in the region of the wing disc near the A/P border (Mullor, 1997).

Developmental analysis and squamous morphogenesis of the peripodial epithelium in Drosophila imaginal discs: the role of hedgehog in the wing disc

Imaginal discs of Drosophila provide an excellent system with which to study morphogenesis, pattern formation and cell proliferation in an epithelium. Discs are sac-like in structure and are composed of two epithelial layers: an upper peripodial epithelium and lower disc proper (DP). Although development of the disc proper has been studied extensively in terms of cell proliferation, cell signaling mechanisms and pattern formation, little is known about these same processes in the peripodial epithelium (PE), the cell layer opposing the disc proper. This topic was addressed by focusing on morphogenesis, compartmental organization, proliferation and cell lineage of the PE in wing, second thoracic leg (T2) and eye discs. A subset of peripodial cells in different imaginal discs undergo a cuboidal-to-squamous cell shape change at distinct larval stages. This shape change requires both Hedgehog and Decapentapelagic, but not Wingless, signaling. Additionally, squamous morphogenesis shifts the anteroposterior (AP) compartment boundary in the peripodial epithelium relative to the stationary AP boundary in the disc proper. Finally, by lineage tracing cells in the PE, it was surprisingly found that peripodial cells are displaced into the disc proper during larval development and this movement leads to Ubx repression (McClure, 2005).

Little is known about when and how disc cells acquire their diverse morphologies. Although Hh and Dpp are well-known for their roles in cell proliferation and patterning it is known that they are also active in epithelial morphogenesis. In eye discs, Hh is both necessary and sufficient to initiate cell shape changes that occur in the morphogenetic furrow. In wing discs, columnar cells require Dpp signaling for normal cytoskeletal organization, shape and pseudostratified organization. This study describes precisely and for the first time when cuboidal, columnar and squamous cell morphologies arise in the epithelia of different imaginal discs. This study examines how the genesis of different morphologies in imaginal discs are affected by loss of a non-autonomous signal (wg, hh and dpp). Hh-dependent Dpp signaling is shown to be required for squamous morphogenesis in the PE of wing and leg discs. Additionally, Dpp signaling is activated as PE cells transition to a squamous morphology. The results indicate that the establishment of columnar morphology in the DP of wing and leg discs is independent of Dpp signaling activity. Clearly, one question still remains: what is the mechanism which causes DP cells to become columnar? The information from these studies provides, at least, an initial framework of how epithelial morphogenesis occurs in imaginal discs (McClure, 2005).

Since both hh and dpp are expressed in wing and leg discs prior to the onset of squamous morphogenesis in the PE, it is clear that their ability to instruct these shape changes must be regulated by additional temporal signals. An obvious candidate for such a temporal signal is ecdysone, which initiates the onset of the larval molts and adult differentiation. The ecdysone signal is mediated by a heterodimer complex consisting of the ecdysone receptor (EcR) and RXR-homolog Ultraspriacle (Usp). To test whether squamous morphogenesis is triggered by ecdysone signaling, usp^–/– clones were induced and it was found that cells of such clones still exhibited normal cuboidal-to-squamous shape changes. Therefore, the temporal cue(s) that initiate disc morphogenesis is independent of ecdysone signaling and remains unknown (McClure, 2005).

Previous studies document that shape change of epithelial cells can activate certain signaling pathways. Thus, squamous morphogenesis of the PE may enhance planar and/or vertical epithelial signaling to promote growth and patterning of the disc. Two observations were made that support this statement: (1) where PE cells fail to undergo squamous morphogenesis, both the disc and adult wing show an obvious reduction in size; (2) in discs that lack PE-specific Dpp signaling, folded clefts in the presumptive wing blade primordia are consistently apposed to a region of squamous PE cells, suggesting communication between the disc epithelia where shape changes do occur. Alternatively, the aberrant apposition of AP compartment boundaries in the PE and DP, owing to a failure in squamous morphogenesis, may result in epithelial abnormalities such as folded clefts in the DP. Resolving the mechanisms by which cell shape can affect disc growth and pattern will integrate both morphogenetic and signaling processes that are crucial for disc development (McClure, 2005).

A lineage analysis of cells has been performed in the wing disc using Ubx-Gal4, UAS flp and act5C>stop>nuclacZ (Pallavi, 2003). Since Ubx-Gal4 is initially expressed in both disc epithelia prior to the second larval instar, cells of both the PE and DP were marked. This analysis concluded that cells of the PE and DP share a common origin in the disc primordium but later become separate lineages, although cells that make up the PE and DP lineages are never specified. The current results, based on lineage-tracing cells born in the PE, are in overall agreement with these conclusions; however, there are some differences. Although Pallavi (2003) identified cell clones spanning both disc epithelia, it could not be determined when or where these clones were born. Furthermore, clones that encompassed cells from both PE and DP were interpreted as either fusions between two independent clones or as clones that originated early in the embryo before separation of the two lineages (PE and DP) (McClure, 2005).

Using four different methods, it has now been found that cells that originate within the PE produce progeny that are a part of the DP. The MARCM and estrogen-inducible systems were used to perform a clonal analysis specific to cells within the PE. These two methods indicate that cells born within the PE produce daughter cells that contribute to the DP. Additionally, a twinspot clonal analysis leads to a similar conclusion and has the advantage of marking cells more directly than either the MARCM- or estrogen-inducible systems. Thus, this analysis indicates a lineage relationship between margin cells in both the PE and DP, and squamous cells in the wing disc, and provides evidence that together these cells comprise the peripodial lineage (McClure, 2005).

As cells are displaced from the PE and into the DP they lose Ubx expression. The loss of Ubx may cause cells to acquire a more distal fate, forging a possible link between displacement and cell fate changes. Similar dynamic cell movements, along with changes in gene expression, have been observed in the chick during somite segmentation. In addition, cell movements and changes in gene expression, similar to what is described here, have been reported by Weigmann (1999), who observed that leg disc cells born in the most proximal regions of the disc contribute to more distal leg segments. Finally, it is proposed that once PE cells are displaced into the DP they may change their cell fate by altered cell signaling. Displaced cuboidal cells at the margins of the disc receive not only planar signals from both epithelial layers, which they are a part of at different stages in larval development, but also vertical signals from overlying PE cells after displacement into the DP. These new planar and/or vertical signals may lead to Ubx repression. It is suggested that the mechanisms that play a role in the development of the imaginal discs may be functionally similar to mechanisms that regulate primary neurogenesis in vertebrates. Neural plate formation and patterning cues arise from two sources: a horizontal source within the plane of an epithelium and a vertical source that arises from the underlying mesodermal cells. The current study suggests that patterning of the imaginal discs is a much more dynamic process with cells exposed to not only signals within the plane of an epithelium but also vertical signals between disc epithelia (McClure, 2005).

Hedgehog signaling and eye development

The eye-antennal imaginal discs of Drosophila melanogaster form the head capsule, the eyes and the antenna of the adult fly. Unlike the limb primordia, each eye-antennal disc gives rise to morphologically and functionally distinct structures. As a result, these discs provide an excellent model system for determining how the fates of primordia are specified during development. An investigation has been carried out of how the adjacent primordia of the compound eye and dorsal head vertex are specified. Subdivision of the eye-antennal disc is not based on compartmentalization: this is in contrast to the basis for subdivision in the wing and leg discs. Therefore, selector gene-mediated division of the disc into compartments, mediated by engrailed and invected, as in the wing disc for example, is not likely to be the basis for regionalization within the antennal primordium. Instead, in this region, the genes wingless and orthodenticle are expressed throughout the entire second instar eye-antennal disc, conferring a default fate of dorsal vertex cuticle. Mutations that decrease dpp expression in the eye primordia lead to the formation of severely reduced eyes. Similarly, the loss of otd or wg function in the vertex primordia causes the elimination of dorsal head structures (Royet, 1997).

Transplantation experiments show that the eye primordium occupies most of the posterior half of the eye-antennal disc (the so-called 'eye disc'). The head vertex forms from the dorsomedial region of the disc, while the antenna develops from the anterior half of the disc (the so-called 'antennal disc'). During the early third instar stage (70-80 hours after egg laying), dpp is expressed in a horseshoe-shaped domain along the ventral, posterior and dorsal periphery of the eye disc. Dorsal dpp expression does not extend as far anteriorly as ventral expression, but instead ends at the vertex primordium. At this stage, otd expression covers the vertex primordium and extends along the edge of the antennal disc. The posterior boundary of otd expression in the vertex anlage coincides, approximately, with the anterior boundary of the dpp domain. At the same stage of disc development, wg is expressed in two regions of the eye disc. One region corresponds to the future gena (the lateral part of the head capsule bounded above by the eye) and the other to the head vertex (Royet, 1997).

dpp expression prevents dorsal head development in the eye primordium. Flies homozygous for the dppd-blk allele that reduces dpp activity in the eye primordium greatly reduces the compound eye giving rise to an eye with only a few residual ommatidia. In these mutants the eyes are largely replaced by frons cuticle, which normally appears only on the dorsal areas of the head. This ectopic frons lies between the orbital cuticle and the remaining ommatidia, and to the anterior, between the shingle cuticle and the ommatidia. In other eye loss mutants, such as sine oculis or eyes absent, the eyes are completely lost but are not replaced by ectopic frons. This suggests that dorsal head cuticle does not result simply from loss of the eyes, but is caused instead by loss of dpp function. Clones of Mothers against dpp, coding for a protein involved in transmission of the Dpp signal, likewise transform ommatidia into frons (Royet, 1997).

Activation of decapentaplegic expression in the posterior eye disc eliminates wg and otd expression, thereby permitting eye differentiation. In dppd-blk mutants, the otd domain expands toward the anlagen of the shingle cuticle and the compound eyes, consistent with the location of ectopic frons cuticle on dppd-blk mutant heads. wg expression also expands in these mutant discs. Ectopic activation of the wingless pathway (the result of the generation of clones mutant for shaggy/zeste-white 3) in the eye primordium induces otd expression and vertex formation. Loss of shaggy function results in constitutively activated wg signaling and ectopic otd expression. This suggests that otd expression in the vertex primordium is normally activated or maintained by wingless. Early activation of dpp depends on hedgehog expression in the eye anlage prior to morphogenetic furrow formation. Loss of hh activity during the second instar larval stage eliminates dpp expression along the posterior and lateral margins of the eye disc and in the antennal primordium. This loss of dpp expression is associated with a dramatic expansion of the otd expression domain. wg expression also expands into the eye primordium (Royet, 1997).

Unlike the limb discs, which derive from single trunk segments, each eye-antennal disc arises from multiple embryonic head segments. Divisions between segment primordia within the disc could contribute to certain aspects of regional specification. It is proposed that wg and otd expression in the eye-antennal discs are inherited from the embryo, where the two genes are expressed in segments from which these discs are derived. The almost ubiquitous expression of these two genes serves to program the early disc for a vertex fate. Later, hh expression in the posterior region of the future eye disc induces dpp expression along the margins of the eye primordium. dpp represses wg, permitting the formation of the eye primordium (Royet, 1997).

hedgehog and wingless have roles in specifying adult head structures. Reduction of hedgehog activity results in flies completely lacking medial head structures, while loss of wingless results in deletion of lateral (orbital) and mediolateral (frons) head structures. Ectopic expression of hh results in the induction of ectopic ocelli at more lateral locations, while ectopic wg results in an invasion of mediolateral frons cuticle into the ocellar region. In orthodenticle mutants, specifically ocelliless regulatory mutations, wg expression fails to disappear from the medial region and instead persists across the entire primordium of the head vertex. In addition hh expression in lost. In a complementary manner, hh also seems to have a positive role in otd expression; ectopic hh activates otd, suggesting that otd expression in the head vertex primordium may be activated by hh during normal eye-imaginal disc development. Thus otd is required for regional head development, and has a critical role in regulating wg and hh expression (Royet, 1996).

Hedgehog is required for progression of the morphogenetic furrow in the eye antennal disc. Differentiation of the Drosophila retina is asynchronous: it starts at the posterior margin of the eye imaginal disc and over two days moves progressively more toward the anterior. During this time the disc continues to grow, increasing approximately eightfold. An indentation in the epithelium, the morphogenetic furrow, marks the front edge of the differentiation wave. Anterior progression of the furrow is driven by Hedgehog signals emanating from differentiating photoreceptor cells in the posterior eye disc. Hedgehog is required for continued furrow movement. Ectopic expression of hedgehog sets in motion ectopic furrows in the anterior eye disc. In addition to changes in cell shape, these ectopic furrows are associated with a tightly orchestrated series of events that parallel normal furrow progression and include proliferation, cell cycle synchronization and pattern formation (Heberlein, 1995).

The progression of retinal morphogenesis in the Drosophila eye is controlled to a large extent by Hedgehog (HH), a signaling protein emanating from differentiating photoreceptor cells. Adjacent, more anterior cells in the morphogenetic furrow respond to HH by expressing dpp, suggesting that the relationship between HH and DPP might be similar to that in the limb imaginal discs where DPP mediates the organizing activity of HH. This study contradicts that suggestion. Analysis of somatic clones of cells lacking the DPP receptors Punt or Tkv reveals that DPP plays only a minor role in furrow progression and no critical role in subsequent ommatidial development. Within tkv and punt clones traversing the furrow at the time of dissection, neuronal differentiation, as shown by ELAV staining, is somewhat retarded, especially in the middle of large clones. The function of DPP in this context must be nonessential or redundant as the furrow is only slightly slowed, but not stopped. Normal ommatidial development occurs in the complete absence of DPP. In contrast, HH-independent dpp expression around the posterior and lateral margins of the first and second instar eye discs is important for the growth of the eye disc and for initiation of the morphogenetic furrow at these margins. Tkv and Punt are absolutely required for cell proliferation in the early developing eye imaginal disc. tkv clones are severly restricted in their ability to grow, implying a strong requirement for the DPP signal for cell proliferation in the early eye disc. There is a posterior requirement for punt function in eye development, which suggests a role for DPP signaling in the initiation of the furrow at the posterior margin Adult eyes containing predominantly punt mutant tissue are regularly observed, but such eyes always have some wild-type tissue at the posterior margin. Both punt and tkv clones cause local overproliferation and block neural differentiation. The tissue in these marginal clones must die, as loss of head cuticle and eye structures is observed in eyes containing mutant clones (Burke, 1996).

The movement of the morphogenetic furrow is dependent upon the secretion of the signaling protein Hedgehog by more posterior cells. It has been suggested that Hh acts as an inductive signal to induce cells to enter a furrow fate and begin differentiation. Nevertheless, hh loss-of-function clones have a negligible effect on furrow progression. To further define the role of Hh in the process of furrow progression, clones of cells were examined lacking the function of the smoothened gene. smo is required for transduction of the Hh signal and allows the investigation of the autonomous requirement for hh signaling. These experiments demonstrate that the function of hh in furrow progression is indirect. Cells that cannot receive/transduce the Hh signal, by virtue of being smo mutants, are still capable of entering a furrow fate and differentiating normally. This suggests that a second signal, received from adjacent cells, is required for entering a furrow fate and differentiating normally. However, hh is required to promote furrow progression and regulate its rate of movement across the disc, since the furrow is significantly delayed in smo clones. Activation of the hh pathway anywhere anterior to the furrow (as occurs in pka-C1 mutant clones) does not immediately trigger ectopic photoreceptor differentiation. The inability of pka-C1 loss-of-function clones to induce ectopic entry to furrow fate, except when close to the endogenous furrow, is not due to insufficient activation of the hh signaling pathway in the mutant cells. That is, double mutants for smo and pka-C1 have identical fates (ectopic photoreceptor differentiation) to clones mutant for pka-C1 alone. Entry into furrow fate only occurs when pka-C1 comes to lie close to the advancing furrow. This has lead to the proposal that a "zone of competence" lies immediately anterior to the furrow. The identity of the second, furrow-inducing signal is unknown, but it is possible that it is provided by a physical relay from cell to cell (Strutt, 1997).

In the course of a genetic mosaic screen for new mutations affecting early eye development, alleles of a conserved gene were isolated and the gene was called capulet or act up (acu) for its effect on actin. acu encodes a homolog of yeast cyclase-associ