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 cell’s 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).

Segment boundary formation in Drosophila embryos

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 90° 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 ß-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. These 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).

Hedgehog signaling plays a conserved role in inhibiting fat formation

Hedgehog (Hh) signals regulate invertebrate and vertebrate development, yet the role of the cascade in adipose development was undefined. To analyze a potential function, Drosophila and mammalian models were examined. Fat-body-specific transgenic activation of Hh signaling inhibits fly fat formation. Conversely, fat-body-specific Hh blockade stimulated fly fat formation. In mammalian models, sufficiency and necessity tests showed that Hh signaling also inhibits mammalian adipogenesis. Hh signals elicit this function early in adipogenesis, upstream of PPARgamma, potentially diverting preadipocytes as well as multipotent mesenchymal prescursors away from adipogenesis and toward osteogenesis. Hh may elicit these effects by inducing the expression of antiadipogenic transcription factors such as Gata2. These data support the notion that Hh signaling plays a conserved role, from invertebrates to vertebrates, in inhibiting fat formation and highlighting the potential of the Hh pathway as a therapeutic target for osteoporosis, lipodystrophy, diabetes, and obesity (Suh, 2006).

Adipose tissues play crucial roles in many biological processes and the ability to store fat is found over a wide evolutionary distance. Homologous molecules control aspects of worm and mammalian fat formation, so mechanisms underlying fat biology, such as those that might be important in human diseases, may well be conserved. Signaling pathways, such as BMP, Wnt and Hh, are conserved, not only in terms of the components that constitute the pathways, but also in their biological roles. Wnt and BMP signals regulate mammalian adipogenesis, however the role of the Hh pathway was undefined. Since a variety of small molecule activators and inhibitors of the Hh cascade have been developed and because diseases of fat tissues, such as lipodystrophy, obesity, and diabetes, are major causes of morbidity and mortality, identifying a potential role for the Hh pathway in adipose biology could have therapeutic implications (Suh, 2006).

This study provides evidence that the Hh pathway might play a conserved role in fat biology. It was found that Hh pathway components were expressed in the fly fat body. Hh pathway components were also expressed in developing and adult mouse fat and their levels were regulated by adipogenesis and obesity. The function of the Hh pathway also appeared to be conserved as sufficiency and necessity tests support the notion that the Hh pathway blocks fly and mammalian fat formation (Suh, 2006).

Mechanistic studies showed that Hh signals blocked the early steps of adipogenesis, presumably after mitotic clonal expansion. Consistent with that temporal placement, epistasis studies support the idea that Hh acts upstream of PPARγ. It was also found that Hh signals increased the expression levels of three antiadipogenic genes: Gata2, Gata3, and Gilz. This expression appeared to be functionally significant as Hh signals required Gata to elicit antiadipogenic actions. So it is possible that Hh signals inhibit adipogenesis at least in part by regulating Gata expression (Suh, 2006).

Based upon the timing and epistasis results, it seemed plausible that Hh signals keep 3T3-L1s as preadipocytes or divert the cells to alternative fates. Molecular analyses support both notions; an increase was observed in the expression of Pref-1, a preadipocyte marker, and also of bone markers. Hh was found to be antiadipogenic and pro-osteogenic in C3H10T1/2 pluripotent mesenchymal cells. Gata appeared to have similar functions to Hh, but this does not exclude the possibility that other mechanisms downstream of Hh exist. It appears that the Hh cascade induces an antiadipogenic program leading to down-regulation of the key adipogenic transcription factor PPARγ as well as a pro-osteogenic program inducing expression of osteogenic transcription factors such as Runx2. So Hh may act upon an unidentified population of mesenchymal stem cells to promote osteogenesis at the expense of adipogenesis. If so, the increase in fat and decrease in bone mass observed with aging might be accounted for by a reduction in Hh signals or may be reversed or prevented by activating the cascade (Suh, 2006).

Taken together, the results support the notion that the Hh pathway inhibits fat formation in a conserved manner from invertebrates to mammalian cellular models. Signaling pathways and receptors, especially seven transmembrane receptors such as Smo, are often amenable to therapeutics as demonstrated by the identification of small molecules that modulate the Hh pathway. So these data support the notion that the Hh pathway might be an appropriate target for drugs to treat lipodystrophy, obesity, and diabetes (Suh, 2006)

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).

Increased cell bond tension governs cell sorting at the Drosophila anteroposterior compartment boundary

Subdividing proliferating tissues into compartments is an evolutionarily conserved strategy of animal development. Signals across boundaries between compartments can result in local expression of secreted proteins organizing growth and patterning of tissues. Sharp and straight interfaces between compartments are crucial for stabilizing the position of such organizers and therefore for precise implementation of body plans. Maintaining boundaries in proliferating tissues requires mechanisms to counteract cell rearrangements caused by cell division; however, the nature of such mechanisms remains unclear. This study quantitatively analyzed cell morphology and the response to the laser ablation of cell bonds in the vicinity of the anteroposterior compartment boundary in developing Drosophila wings. Mechanical tension was found to be approximately 2.5-fold increased on cell bonds along this compartment boundary as compared to the remaining tissue. Cell bond tension is decreased in the presence of Y-27632, an inhibitor of Rho-kinase whose main effector is Myosin II. Simulations using a vertex model demonstrate that a 2.5-fold increase in local cell bond tension suffices to guide the rearrangement of cells after cell division to maintain compartment boundaries. These results provide a physical mechanism in which the local increase in Myosin II-dependent cell bond tension directs cell sorting at compartment boundaries (Landsberg, 2009).

A long-standing hypothesis to explain the maintenance of compartment boundaries is based on differential cell adhesion (or cell affinity). Cell adhesion molecules required for the maintenance of compartment boundaries, however, have not been identified. More recently, it has been proposed that actin-myosin-based tension is important for keeping the dorsoventral compartment boundary of the developing Drosophila wing smooth and straight. However, whether a similar mechanism operates at the anteroposterior compartment boundary (A/P boundary) is unclear. Moreover, a physical measurement of differential mechanical tension at compartment boundaries has not been reported. Furthermore, whether and how differential mechanical tension governs cell sorting at compartment boundaries is not well understood (Landsberg, 2009).

To test whether actin-myosin-based tension is increased at the A/P boundary, the levels of Filamentous (F)-actin and nonmuscle Myosin II (Myosin II) were quantified. The A/P boundary in the wing disc epithelium was particularly well defined by the cell bonds located at the level of adherens junctions, indicating that mechanisms maintaining the boundary operate at this cellular level. F-actin and the regulatory light chain of Myosin II (encoded by spaghetti squash, sqh) were increased at these cell bonds along the A/P boundary. Cell bonds displaying elevated levels of Myosin II correlate with decreased levels of Par3 (Bazooka in Drosophila), a protein organizing cortical domains, at the dorsoventral compartment boundary and during germ-band extension in Drosophila embryos. Likewise, Bazooka was decreased at cell bonds along the A/P boundary, indicating a common mechanism of complementary protein distribution of Myosin II and Bazooka. The level of E-cadherin, a component of adherens junctions, was not altered along the A/P boundary (Landsberg, 2009).

To identify signatures of increased tension in the vicinity of the A/P boundary, the morphology of cells were quantitatively analyzed at the level of adherens junctions. Line tension and mechanical properties of cells have been proposed to contribute to cell shape and to influence angles between cell bonds. Line tension associated with adherens junctions, here termed cell bond tension, can be defined as the work, per unit length, performed as a cell bond changes its length. Cell bond tension results from actin-myosin bundles and other structural components at junctional contacts that generate tensile stresses. Wing discs from late-third-instar larvae were stained for E-cadherin and engrailed-lacZ, a marker for the posterior compartment. Cell bonds were identified, and morphological parameters were analyzed. Adjacent anterior and posterior cells (A1 and P1, respectively) displayed a significantly enlarged apical cross-section area compared to cells farther away from the compartment boundary, indicating that apposition of anterior and posterior cells alters specifically the properties of A1 and P1 cells. Angles between adjacent cell bonds along the A/P boundary were larger compared to angles between bonds of the remaining cells and were significantly smaller in mutants for Myosin II heavy chain (encoded by zipper; zip2/zipEbr). Thus, the unique morphology of A1 and P1 cells depends on Myosin II. These data are consistent with an increased Myosin II-based tension of cell bonds located along the A/P boundary (Landsberg, 2009).

Cells on opposite sides of the A/P boundary differ in gene expression. The homeodomain-containing proteins Engrailed and Invected as well as the Hedgehog ligand are only expressed on the posterior side. The Hedgehog signal is transduced exclusively on the anterior side. Hedgehog signal transduction and the presence of Engrailed and Invected are required to maintain this compartment boundary. Whether the altered cell morphology at the A/P boundary could be reproduced by ectopically juxtaposing Hedgehog signaling and non-Hedgehog signaling cells was tested. Clones of cells that expressed Hedgehog from a transgene and that were also mutant for the gene smoothened (encoding an essential transducer of the Hedgehog pathway) were generated. In the P compartment, which is refractory to Hedgehog signal transduction, clones displayed a normal morphology. In the A compartment, a response to Hedgehog that is secreted by the clones is elicited in the surrounding wild-type cells. These clones had a rounder appearance, and at the clone border, but not away from it, apical cross-section area and bond angles were increased. Similarly, juxtaposing cells expressing engrailed and invected with cells that are mutant for these genes resulted in increased apical cross-section area and increased bond angles at the clone border. It is concluded that the morphology that is characteristic of cells at the A/P boundary can be imposed on cells within a compartment by juxtapositioning cells with different activities of Hedgehog signal transduction or Engrailed and Invected (Landsberg, 2009).

Ablating cell bonds generates cell vertex displacements, providing direct evidence for tension on cell bonds. Individual cell bonds were ablated by using a UV laser beam focused in the plane of the adherens junctions. Single-cell bonds were cut, and the displacement of vertices of neighboring cells, visualized by E-cadherin-GFP, was recorded. The P compartment was visualized by expression of GFP-gpi under control of the engrailed gene via the GAL4/UAS system. The increase in distance between the two vertices of the ablated cell bond and the initial velocity of this vertex separation were analyzed. The ratio of initial velocities in response to cell bond ablation is a measure of the tension ratio on these cell bonds. Initial velocity and extent of vertex separation were indistinguishable between anterior (A/A) and posterior (P/P) cell bonds located away from the A/P boundary. This was also the case when specifically cell bonds between the first and second row of anterior cells were ablated. By contrast, ablation of bonds between adjacent anterior and posterior cells (A/P cell bonds) gave rise to a larger vertex separation. This result was not due to the fact that A/P cell bonds have a preferred orientation. Moreover, the initial velocity of ablated A/P bonds was 2.37-fold higher compared to the mean of initial velocities of A/A and P/P bonds. This value provides an estimate of the ratio λ of cell bond tension along the A/P boundary relative to the average tension of cell bonds. In the presence of the Rho-kinase inhibitor Y-27632, the ratio of initial velocity of vertex separation of A/P cell bonds relative to A/A cell bonds was reduced to 1.46. Given that Myosin II is the main effector of Rho-kinase, these results strongly suggest that Myosin II-based tension acting on cell bonds is locally increased along the A/P boundary (Landsberg, 2009).

To quantify λ by an independent method, the displacement field was calculated after laser ablation. Using a vertex model, two populations of adjacent cells were introduced and cell bond ablations were simulated, varying λ between 1 and 4. When λ = 2.5, the vertex displacement, and in particular the anisotropy of displacements, in the simulations closely matched the vertex displacements in the experiment. In the vertex model, λ = 2.5 also resulted in increased bond angles at the interface of the two cell groups, similar to the A/P boundary in the wing disc. Thus, on the basis of two different methods, the data demonstrate that cell bond tension is increased approximately 2.5-fold along the A/P boundary compared to the remaining tissue (Landsberg, 2009).

To test whether a 2.5-fold increase in cell bond tension is sufficient to maintain a compartment boundary, the vertex model was used to simulate the growth of two adjacent cell populations for λ = 1, 2.5, and 4. For λ = 1, the interface between two growing cell populations became increasingly irregular. By contrast, for λ = 2.5 and 4, a well-defined interface was maintained. Moreover, corresponding changes in cell bond tension at borders of simulated clones resulted in the morphology and sorting behavior of cell patches that resembled those of experimental cell clones compromised for Hedgehog signal transduction or Engrailed and Invected activity. The roughness of the interface in the simulations decreased with increasing λ, showing that cell bond tension is sufficient to maintain straight interfaces between growing cell populations. For λ = 2.5, the roughness of the interface was still larger than the roughness of the A/P boundary in wing discs. This suggests that additional mechanisms might contribute to further reduce the roughness of the A/P boundary. Also, because of the uncertainty of the mechanical properties of A1 and P1 cells, which differ from those of the remaining cells, the value of λ, inferred from laser ablation of cell bonds, might be underestimated. Remarkably, the roughness of the A/P boundary could be altered in mutant conditions. In zip2/zipEbr mutant wing discs, the roughness of the compartment boundary was significantly larger than in controls, demonstrating a role for Myosin II in maintaining a sharp and straight A/P boundary (Landsberg, 2009).

In summary, by applying physical approaches and quantitative imaging, this work for the first time demonstrates and quantifies an increase in tension confined to the cell bonds along the A/P boundary. Moreover, simulations show that this increase in tension suffices to maintain a stable interface between two proliferating cell populations. Genetic studies demonstrated that cells of the two compartments differ in their expression profiles and signaling activities. It has therefore been proposed that biophysical properties of cells within the P compartment differ from those within the A compartment, and that such differences could drive cell sorting. When quantifying cell morphology and vertex displacements after laser ablation, no differences were detected in the biophysical properties of cells between the two compartments. However, the two rows of abutting A and P cells show clear differences in biophysical properties from other cells. Most importantly, the cell bond tension along the A/P boundary is increased. Cell divisions in the vicinity of the A/P boundary were randomly oriented in the epithelial plane. Thus, taken together with the simulations, these results suggest a sorting mechanism by which an increased cell bond tension guides the rearrangement of cells after cell division to maintain a straight interface. Increased cell bond tension and the roughness of the A/P boundary depend on Rho kinase activity and Myosin II, indicating a role for actin-myosin-based tension in this process. Because cell bond tension also depends on cell-cell adhesion, differences in the adhesion between A1 and P1 cells as compared to the remaining cells might also contribute to sorting. The heterotypic, but not homotypic, interaction of molecules presented on the surface of A and P cells might trigger the local increase in cell bond tension. Hedgehog signal transduction and the presence of Engrailed and Invected might control the expression of these heterotypically interacting molecules. These data indicate an important role for cell bond tension directing cell sorting during animal development (Landsberg, 2009).

Drosophila genome-wide obesity screen reveals hedgehog as a determinant of brown versus white adipose cell fate

Over 1 billion people are estimated to be overweight, placing them at risk for diabetes, cardiovascular disease, and cancer. A systems-level genetic dissection of adiposity regulation was performed using genome-wide RNAi screening in adult Drosophila. As a follow-up, the resulting approximately 500 candidate obesity genes were functionally classified using muscle-, oenocyte-, fat-body-, and neuronal-specific knockdown in vivo; hedgehog signaling was the top-scoring fat-body-specific pathway. To extrapolate these findings into mammals, fat-specific hedgehog-activation mutant mice were generated. Intriguingly, these mice displayed near total loss of white, but not brown, fat compartments. Mechanistically, activation of hedgehog signaling irreversibly blocked differentiation of white adipocytes through direct, coordinate modulation of early adipogenic factors. These findings identify a role for hedgehog signaling in white/brown adipocyte determination and link in vivo RNAi-based scanning of the Drosophila genome to regulation of adipocyte cell fate in mammals (Pospisilik, 2010).

To assess the in vivo relevance of hedgehog signaling in mammalian adipogenesis, fat-specific Sufu knockout animals (aP2-SufuKO) were generated. Sufu is a potent endogenous inhibitor of hedgehog signaling in mammals. Sufu^flox/flox mice were crossed to the adipose tissue deleting aP2-Cre transgenic line, and the resulting aP2- SufuKO animals were born healthy and at Mendelian ratios. PCR amplification revealed target deletion in both white adipose tissue (WAT) and brown adipose tissue (BAT). aP2-SufuKO mice displayed an immediate and obvious lean phenotype. MRI analysis revealed a significant and global reduction in white adipose tissue mass, including subcutaneous, perigonadal, and mesenteric depots. Intriguingly, though, in contrast to the gross loss of WAT, cross-sectional examination of the interscapular region revealed fully developed BAT depots of both normal size and lipid content. Direct measurement of WAT and BAT depot weights corroborated the divergent WAT/BAT phenotype, with an ~85% reduction in perigonadal fat pad mass in aP2-SufuKO mice concomitant with unaltered BAT mass. Tissue weight and histological analyses confirmed lack of any remarkable phenotype in multiple other tissues including pancreas and liver (no indication of steatosis), and muscle mass was unaffected. Cutaneous adipose was also markedly diminished. Whereas the morphology of Sufu-deficient BAT depots was largely indistinguishable from that of control animals, examination of multiple WAT pads revealed marked and significant reductions in both adipocyte size and total numbers in mutant animals. Of note, qPCR showed elevated Gli1, Gli2, and Ptch2 expression in both WAT and BAT verifying the intended pathway activation in both tissues. Thus, deletion of Sufu in fat tissue results in a markedly decreased white fat cell number and, remarkably, in normal brown adipose tissue (Pospisilik, 2010).

When the literature was cross-referenced focusing on adipogenesis, an impressive 18 of 65 key regulators of adipogenesis were found to be described as Gli targets in other systems. Intriguingly, when examined in 3T3-L1 preadipocytes, hedgehog activation induced a coordinated downregulation of the proadipogenic targets Bmp2, Bmp4, Egr2/Krox20, Sfrp1, and Sfrp2 by an average of ~50% after only 24 hr. In contrast, quantification of the antiadipogenic target set showed upregulation of the multiple critical repressors (Nr2f2, Gilz, Hes1, and Ncor2); the negative regulators Jag1 and Pref1 remained unchanged at this time point. Analysis of the master regulatory machinery downstream of these effectors revealed critical reductions in Pparg, Cebpb, and Cebpd and increases in the antiadipogenic factors Cebpg and Ddit3. Outside of this dramatic antiadipogenic profile, elevated levels of Cebpa were observed. Importantly, a similar coordinate downregulation of Pparg, Cebpb, Cebpd, as well as Cebpa was observed in WAT-derived primary murine adipocyte progenitors (stromal vascular cell, SVC, preparations) following genetic activation of hedgehog signaling (Pospisilik, 2010).

To establish a direct link between hedgehog activation and adipogenic block in white adipose, in silico predictions were used to identify clusters of probable Gli-binding sites in the highly SAG-responsive genes Ncor2, Nr2f2, Sfrp2, and Hes1. To assess the functionality of these putative binding sites, the relevant promoter fragments were cloned and luciferase reporter assays were performed. Gli1 and Gli2 induced activation of all Ncor2 and Nr2f2 reporter constructs, with the binding site clusters Ncor2_B, Nr2f2_A, and Nr2f2_B showing responses comparable to the hallmark target Ptch. Further, chromatin immunoprecipitations on 3T3-L1 preadipocytes using Gli2- and Gli3-specifc antibodies revealed increases in Gli2 and Gli3 binding within the endogenous Ncor2, Hes1, Nr2f2, and Sfrp2 regulatory regions following SAG treatment. Together, these findings demonstrate endogenous Gli2/Gli3 binding to multiple adipogenic loci and implicate direct modulation of Ncor2 and Nr2f2 in the dysregulation of adipogenesis (Pospisilik, 2010).

The power of D. melanogaster RNAi transgenics to probe gene function on a genome-wide scale has allowed screening of ~78% coverage of the Drosophila genome. One significant advantage of this inducible approach is the ability to interrogate the fat regulatory potential of the ~30% of the Drosophila genome that is developmentally lethal under classic mutation conditions. Indeed, the result that cell differentiation genes scored as the most enriched ontology subcategory substantiates the inducible strategy employed and identifies a large number of developmentally lethal genes with strong lipid storage regulatory potential. Consistent with a previous feeding-induced RNAi C. elegans screen, the fraction of candidate genes resulting in decreased fat content upon knockdown (360 of 516; 70%) exceeded that of obesity-causing candidates (216 of 516; 30%), which is consistent with the hypothesis that the major evolutionary pressures for animals have been to favor nutrient storage. The screen identified a large number of genes already known to play a key role in mammalian fat or lipid metabolism, including enzymes of membrane lipid biosynthesis, fatty acid and glucose metabolism, and sterol metabolism. Further, the whole-genome screen has uncovered a plethora of additional candidate genes of adiposity regulation, a large proportion of which had no previous annotated biological function. Moreover, multiple genes were identified that either positively or negatively regulate whole fly triglyceride levels when targeted specifically in neurons, the fly liver (oenocyte), the fat body, or muscle cells. Analyses of the hits allowed definition of either gene sets that function globally in all these tissues or others that display coordinate regulation of adiposity when targeted in metabolically linked organs such as the fat and the liver. Since >60% of the candidate genes are conserved across phyla to humans, this data set is a unique starting point for the elucidation of novel regulatory modalities in mammals (Pospisilik, 2010).

The top-scoring signal transduction pathway in the GO-based enrichment analysis was the hedgehog pathway. Tissue-specificity assessment revealed further that this enrichment was primarily derived from a pronounced fat-body restriction in function. Hedgehog signaling has been previously implicated in adipose tissue biology. In Drosophila larvae, hedgehog activation reduces lipid content consistent with what was found in adult flies and the fat-specific fly knockdown lines (Suh, 2006). Similarly, knockdown of the C. elegans equivalent of the inhibitory hedgehog receptor Ptch results in a prominent adiposity reducing phenotype in a feeding-based RNAi screen. Therefore this study homed into the hedgehog pathway to provide proof of principle for the fly screen and to translate Drosophila results directly into the mammalian context (Pospisilik, 2010).

Several reports exist describing systemic manipulation of hedgehog signaling, either by injection of ligand-depleting antibody or through examination of a systemic hypomorphic mutant, the Ptchmes/mes mouse. Indeed Ptchmes/mes mice display largely normal white adipose tissue depots albeit reduced in size (Li, 2008). Hedgehog signaling plays a crucial role in multiple organs systems including at least one intimately involved in nutrient storage and the etiologies of obesity and insulin resistance, namely, the pancreatic islet. In vitro and in vivo data using the adipose-specific Sufu mutant mice clearly show that hedgehog activation results in a complete and cell-autonomous inhibition of white adipocyte differentiation. The residual white adipose tissue observed in aP2-SufuKO mice is most likely due to late inefficient deletion and/or is due to developmental timing effects. Indeed, aP2 (and thus aP2-Cre) are expressed relatively late during adipocyte differentiation. The remarkable finding was that genetic activation of hedgehog signaling in vivo and in vitro blocks only white but not brown adipocyte differentiation (Pospisilik, 2010).

Fat is mainly stored in two cell types: WAT, which is the major storage site for triglycerides, and BAT, which, through the burning of lipids to heat (through uncoupling of mitochondrial oxidative phosphorylation), serves to regulate body temperature. Recent PET-CT data have revealed that adult humans contain functional BAT and that the amount of BAT is inversely correlated with body mass index. These new data in humans rekindle the notion that a functional BAT depot in humans could represent a potent therapeutic target in the context of obesity control. Lineage tracking and genetic studies have shown that WAT and interscapular BAT cells derive from two different but related progenitor pools. The current genetic data now demonstrate both in vitro and in vivo that hedgehog activation results in a virtually complete block of WAT development but leaves the differentiation process of brown adipocytes wholly intact. These data further support the concept that white and brown adipocytes are derived from distinct precursor cells (Pospisilik, 2010).

aP2-SufuKO mice are the first white adipose-specific lipoatrophic mice with a fully functional BAT depot over the long-term and normal glucose tolerance and insulin sensitivity. The capacity of an intact BAT depot to burn energy in aP2-SufuKO mice likely underlies, at least in part, their lack of ectopic lipid accumulation and insulin resistance. This largely normal metabolic picture highlights the potent regulatory capacity of brown adipose tissue and should prove invaluable in understanding the distinct roles of brown and white adipose tissues (Pospisilik, 2010).

The adult Drosophila gastric and stomach organs are maintained by a multipotent stem cell pool at the foregut/midgut junction in the cardia (proventriculus)

Stomach cancer is the second most frequent cause of cancer-related death worldwide. Thus, it is important to elucidate the properties of gastric stem cells, including their regulation and transformation. To date, such stem cells have not been identified in Drosophila. Using clonal analysis and molecular marker labeling, this study has identified a multipotent stem-cell pool at the foregut/midgut junction in the cardia (proventriculus). Daughter cells migrate upward either to anterior midgut or downward to esophagus and crop. The cardia functions as a gastric valve and the anterior midgut and crop together function as a stomach in Drosophila; therefore, the foregut/midgut stem cells have been named gastric stem cells (GaSC). JAK-STAT signaling regulates GaSC proliferation, Wingless signaling regulates GaSC self-renewal, and hedgehog signaling regulates GaSC differentiation. The differentiation pattern and genetic control of the Drosophila GaSCs suggest the possible similarity to mouse gastric stem cells. The identification of the multipotent stem cell pool in the gastric gland in Drosophila will facilitate studies of gastric stem cell regulation and transformation in mammals (Singh, 2011).

This study has identified multipotent gastric stem cells at the junction of the adult Drosophila foregut and midgut. The GaSCs express the Stat92E-GFP reporter, wg-Gal4 UAS-GFP, and Ptc, and are slowly proliferating. The GaSCs first give rise to the fast proliferative progenitors in both foregut and anterior midgut. The foregut progenitors migrate downward and differentiate into crop cells. The anterior midgut progenitors migrate upward and differentiate into midgut cells. However, at this stage because of limited markers availability and complex tissues systems at cardia location, it is uncertain how many types of cells are produced and how many progenitor cells are in the cardia. Clonal and molecular markers analysis suggest that cardia cells are populated from gastric stem cells at the foregut/midgut (F/M) junction; however, it cannot be ruled out that there may be other progenitor cells with locally or limited differential potential that may also take part in cell replacement of cardia cells. Nevertheless, the observed differentiation pattern of GaSCs in Drosophila may be similar to that of the mouse gastric stem cells. Gastric stem cells in the mouse are located at the neck-isthmus region of the tubular unit. They produce several terminally differentiated cells with bidirectional migration, in which upward migration towards lumen become pit cells and downward migration results in fundic gland cells (Singh, 2011).

Three signal transduction pathways differentially regulate the GaSC self-renewal or differentiation. The loss of JAK-STAT signaling resulted in quiescent GaSCs; that is, the stem cells remained but did not incorporate BrdU or rarely incorporated BrdU. In contrast, the amplification of JAK-STAT signaling resulted in GaSC expansion (Singh, 2011).

These observations indicate that JAK-STAT signaling regulates GaSC proliferation. In contrast, the loss of Wg signaling resulted in GaSC loss, while the amplification of Wg resulted in GaSC expansion, indicating that Wg signaling regulates GaSC self-renewal and maintenance. Finally, the loss of Hh signaling resulted in GaSC expansion at the expense of differentiated cells, indicating that Hh signaling regulates GaSC differentiation. The JAK-STAT signaling has not been directly connected to gastric stem cell regulation in mammal. However, the quiescent gastric stem cells/progenitors are activated by interferon γ (an activator of the JAK-STAT signal transduction pathway), indicating that JAK-STAT pathways may also regulate gastric stem cell activity in mammals. Amplification of JAK-STAT signaling resulted in expansion of stem cells in germline, posterior midgut and malpighian tubules of adult Drosophila. In the mammalian system, it has been reported that activated STAT contributes to gastric hyperplasia and that STAT signaling regulates gastric cancer development and progression. Wnt signaling has an important function in the maintenance of intestinal stem cells and progenitor cells in mice and hindgut stem cells in Drosophila, and its activation results in gastrointestinal tumor development. Tcf plays a critical role in the maintenance of the epithelial stem cell. Mice lacking Tcf resulted in depletion of epithelial stem-cell compartments in the small intestine as well as being unable to maintain long-term homeostasis of skin epithelia. A recent study even demonstrates that the Wnt target gene Lgr5 is a stem cell marker in the pyloric region and at the esophagus border of the mouse stomach. Further, it has been found that overactivation of the Wnt signaling can transform the adult Lgr5^+ve stem cells in the distal stomach, indicating that Wnt signaling may also regulate gastric stem cell self-renewal and maintenance in the mammal. Sonic Hedgehog (Shh) and its target genes are expressed in the human and rodent stomach. Blocking Shh signaling with cyclopamine in mice results in an increase in the cell proliferation of gastric gland, suggesting that Shh may also regulate the gastric stem cell differentiation in mice. These data together suggest that the genetic control of the Drosophila GaSC may be similar to that of the mammalian gastric stem cells (Singh, 2011).

The potential GaSCs niche. In most stem cell systems that have been well characterized to date, the stem cells reside in a specialized microenvironment, called a niche.66 A niche is a subset of neighboring stromal cells and has a fixed anatomical location. The niche stromal cells often secrete growth factors to regulate stem cell behavior, and the stem cell niche plays an essential role in maintaining the stem cells, which lose their stem-cell status once they are detached from the niche (Singh, 2011).

Loss of the JAK-STAT signaling results in the GaSCs being quiescent; the stem cells remain but do not proliferate or rarely proliferate. The Dome receptor is expressed in GaSCs, while the ligand Upd is expressed in adjacent cells. Upd-positive hub cells function as a germline stem cell niche in the Drosophila testis. Further, thia study demonstrated that overexpression of upd results in GaSC expansion, suggesting that the Upd-positive cells may function as a GaSC niche. Furthermore, while Stat92E-GFP expression is regulated by the JAK-STAT signaling in other systems, its expression at the F/M junction seems independent of the JAK-STAT signaling because Stat92E-GFP expression is not significantly disrupted in the Stat92E^ts mutant flies, suggesting that the GaSCs may have unique properties (Singh, 2011).

The stomach epithelium undergoes continuous renewal by gastric stem cells throughout adulthood. Disruption of the renewal process may be a major cause of gastric cancer, the second leading cause of cancer-related death worldwide, yet the gastric stem cells and their regulations have not been fully characterized. A more detailed characterization of markers and understanding of the molecular mechanisms control gastric stem cell behavior will have a major impact on future strategies for gastric cancer prevention and therapy. The information gained from this report may facilitate studies of gastric stem cell regulation and transformation in mammals (Singh, 2011).

A local difference in Hedgehog signal transduction increases mechanical cell bond tension and biases cell intercalations along the Drosophila anteroposterior compartment boundary

In the developing Drosophila wing disc, maintenance of the straight anteroposterior (AP) compartment boundary involves a local increase in mechanical tension at cell bonds along the boundary. This study shows that a local difference in Hedgehog signal transduction activity between anterior and posterior cells is necessary and sufficient to increase mechanical tension along the AP boundary. This difference in Hedgehog signal transduction is also required to bias cell rearrangements during cell intercalations to keep the characteristic straight shape of the AP boundary. Moreover, severing cell bonds along the AP boundary does not reduce tension at neighboring bonds, implying that active mechanical tension is upregulated, cell bond by cell bond. Finally, differences in the expression of the homeodomain-containing protein Engrailed also contribute to the straight shape of the AP boundary, independently of Hedgehog signal transduction and without modulating cell bond tension. These data reveal a novel link between local differences in Hedgehog signal transduction and a local increase in active mechanical tension of cell bonds that biases junctional rearrangements. The large-scale shape of the AP boundary thus emerges from biochemical signals inducing patterns of active tension on cell bonds (Rudolf, 2015).

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).

A novel interaction between hedgehog and Notch promotes proliferation at the anterior-posterior organizer of the Drosophila wing

Notch has multiple roles in the development of the Drosophila melanogaster wing imaginal disc. It helps specify the dorsal-ventral compartment border, and it is needed for the wing margin, veins, and sensory organs. Evidence is presented for a new role: stimulating growth in response to Hedgehog. This study shows that Notch signaling is activated in the cells of the anterior-posterior organizer that produce the region between wing veins 3 and 4, and strong genetic interactions are described between the gene that encodes the Hedgehog pathway activator Smoothened and the Notch pathway genes Notch, presenilin, and Suppressor of Hairless and the Enhancer of split complex. This work thus reveals a novel collaboration by the Hedgehog and Notch pathways that regulates proliferation in the 3-4 intervein region independently of Decapentaplegic (Casso, 2011).

This article shows activation of N signaling at the wing AP organizer by defining with cellular resolution the expression patterns of N protein and N pathway reporters in relation to the AP organizer, and dependence on Hh signaling is shown. Strong interactions are also shown between hh- and N-signaling pathways, and it is confirmed that the activation of N signaling is necessary for the normal growth of the AP organizer. This work uncovers a previously unknown activity of the Hh pathway in mitogenesis at the AP organizer: the activation of N signaling. These results are surprising in that they show that the roles of N signaling in the growth of the wing are not limited to the function of the DV organizer and a general growth-promoting function in the wing: N signaling also induces growth downstream of hh at the AP organizer (Casso, 2011).

N is essential for the cells that give rise to the DV margin, veins, and sensory organs of the wing, and its expression is elevated in the progenitors that produce these structures. The DV margin progenitors, which transect the wing disc in a band that is orthogonal to the Hh-dependent AP organizer, express wg in response to N. These wg-expressing cells function as a DV organizer, and several lines of evidence suggest that the AP and DV organizers function independently: Hh signaling along the AP axis is not N-dependent, N signaling along the DV axis is not hh-dependent, and targets regulated by the AP and DV organizers are not the same. The findings reported in this study show that, separately from its roles elsewhere in the wing disc, N signaling has an essential mitogenic role in the cells of the AP organizer region (Casso, 2011).

While N can stimulate growth by inducing expression of wg (as it does in the DV organizer), hyper-activation of N signaling near the AP border of the wing pouch causes overgrowth that is independent of wg. wg is not normally expressed along the AP axis, but this study found that N signaling is activated at the AP compartment border in late third instar discs, pupal discs, and pupal wings. Through vn expression, Hh signaling at the AP compartment border increases expression of Dl flanking the organizer, and Hh signaling activates N in the 3-4 intervein region. While a role for Ser at the AP organizer has not been directly investigated, Ser expression in the wing disc is very similar to that of Dl, with high levels of Ser in the vein 3 and 4 primordia as well as along the DV border. The results show that growth of the 3-4 intervein region, long known to be dependent on Hh, is also dependent on Hh-induced activation of N (Casso, 2011).

Expression of N pathway reporters and components and genetic interactions support this model of regulation of the intervein region. The reporters Su(H)lacZ and E(spl)m-α-GFP express at the AP border in a Hh-dependent manner. Elevated levels of N protein expression on the anterior side of the AP border require Vn signaling. This N region is flanked by Dl expression in the vein 3 and vein 4 primordia; Dl expression is known to be dependent upon expression of the Hh target vn. Genetic interactions between smo RNAi and N and between smo RNAi and N pathway components [e.g., the Psn intramembrane protease, which activates N; the Su(H) transcriptional co-activator; the Su(dx) E3 ubiquitin ligase, which monitors levels of N protein; and the E(spl) complex of N transcriptional targets] also indicate a functional link between the Hh and N systems (Casso, 2011).

The model for the role of N in the 3–4 intervein region is consistent with previous reports of expression patterns of the E(spl) genes E(spl)m8, M-β, and M-α. Ectopic expression of HLH-mδ and m8 rescues smo RNAi. Although HLH-mδ does not appear to be expressed in the AP organizer in a wild-type wing because the E(spl) genes are thought to have partially overlapping functions, the fact that mδ phenocopies the rescue by m8 reinforces the conclusion that the function of the E(spl) genes is critical to inducing growth at the AP organizer. Importantly, these findings show that the cells that activate N are the anterior cells of the AP organizer and are not associated with development of veins in pupal wings. Vein 4 develops within the posterior compartment and in many cases has posterior cells between it and the AP border. Since activation of these reporters was never observed extending into posterior territory, their expression correlates better with the position of the AP organizer than with vein/intervein territories at the stages that were examined. It should be noted that no single readout currently available marks all tissues in which N is activated. The E(spl) genes, for example, express in a variety of spatial and temporal patterns in response to N, and these patterns are only partially overlapping. The possibility cannot be excluded that N signaling is also activated along the stripe of Dl expression in the vein 3 primordium or that signaling could be occurring in the entire broad stripe of elevated N expression in the AP organizer. No changes were seen in proliferation using a direct readout such as phosphohistone staining of mitotic cells to visualize increases or decreases in growth at the AP organizer. These proliferation assays mark cell cycle progression at a single time point in fixed tissues, and the changes that were seen in the adult wing could be due to one or two fewer cell division cycles occurring over the course of days of development (Casso, 2011).

The findings indicate a link between the Hh and N pathways and suggest a model in which the domain of N activation at the AP border [manifested by Su(H)lacZ expression] is a consequence both of flanking cells that express high levels of Dl and of Hh signaling. The proposed role for Hh signaling is multifaceted: Hh is required for vn expression, which is itself required for high levels of Dl expression in the vein 3 stripe and the vein 4 stripe and for N expression at the AP organizer. Although whether Dl expression in veins 3 and 4 activates N signaling has not been directly tested, vn function is necessary for N activation, and the reciprocal relationship between cells expressing high levels of Dl and neighboring cells expressing high levels of N is well established (Casso, 2011).

Interactions between the Sonic hedgehog (SHH) and N signaling pathways have been identified previously in vertebrates. Particularly noteworthy for their relevance to the interactions that were found in the Drosophila wing disc are the increased expression of the Serrate-related N ligand, Jagged 1, in the mouse Gli3^Xt mutant; reduced expression of Jagged1 and Notch2 in the cerebella of mice with reduced SHH signaling; regulation of the Delta-related ligand, DNER, by SHH in Purkinje neurons and fetal prostate; activation of N signaling in neuroblastomas in Ptch^+/– mice with elevated SHH signaling; and Notch2 overexpression in mice carrying an activated allele of smo. These studies establish a positive effect of SHH signaling on the N pathway, consistent with the current data (Casso, 2011).

In Drosophila, there have been several reports of interactions between the N and Hh pathways. In the wing pouch, for example, expression levels of the Hh targets ptc, ci, col, and en are markedly lower at the intersection of the AP and DV borders than elsewhere in the AP organizer. This repression is mediated by wg. In addition, N and col function together to determine the position of wing veins 3 and 4. However, loss of function of either col or vn did not show interactions with smo RNAi (Casso, 2011).

N functions in two types of settings. One is associated with binary fate choices; it involves adjacent cells that adopt either of two fates on the basis of the activation of N signaling in one cell and inactivation in the other. In these settings, activation of N not only induces differentiation in a designated cell, but also blocks activation of N in the neighbors. The second type of setting does not induce a binary fate choice, but instead activates the pathway at the junction of two distinct cell types. N pathway activation at the DV border in the wing is one example; in this setting, N is activated in a band that straddles the DV border and the N ligands Dl and Ser signal from adjacent domains from either the dorsal (i.e., Dl) or the ventral (i.e., Ser) side. Activation of N in the 3-4 intervein region at the AP border appears to be of this second type: it occurs adjacent to regions of elevated Dl expression at the apposition of anterior and posterior cell types. There is no apparent binary fate choice in this region of the wing (Casso, 2011).

In ways that are not understood well, development of the 3-4 intervein region is controlled differently from other regions of the wing pouch. Whereas Hh induces expression of Dpp, and Dpp orchestrates proliferation and patterning of wing pouch cells generally, Dpp does not have the same role in the 3-4 intervein cells. For these cells, Hh appears to control proliferation and patterning directly. For example, the lateral regions of wings that develop from discs with compromised Dpp function are reduced, but their central regions, between veins 3 and 4, are essentially normal. Downregulation of Dpp activity and repression of expression of the Dpp receptor appears to be the basis for this insensitivity. In contrast, partial impairment of Hh signal transduction that is insufficient to reduce Dpp function (such as in fu mutants or in the smo RNAi genotypes that were characterized) results in wings that are normal in size and pattern except for a small or absent 3-4 intervein region. Since the 3-4 intervein cells divide one to two times in the early pupa during disc eversion and wing formation, the direct role of Hh in regulating these cells may be specific to this post-larval period. N signaling has a well-described mitogenic function in the wing. Ectopic signaling causes hyper-proliferation, while clones that impair the activation of the pathway reduce growth. The current findings indicate that Hh regulates proliferation of cells in the 3-4 intervein region at least in part by activating N signal transduction (Casso, 2011).

The idea that this model promotes is that Hh-dependent activation of N at the AP organizer is stage- and position-specific. This model is consistent with the complex pattern of N expression and activation in the wing, since different pathways may regulate N in different locations. It is also consistent with the proposed role of N regulating the width and position of veins 3 and 4, since the processes that establish the veins and control proliferation of the intervein cells need not be the same, even if they are interdependent. The temporal specificity that this study describes represents an example of how complex patterns are generated with a limited number of signaling pathways -- in this case by using N signaling for different outcomes at different times and in different places. Throughout larval development, Dpp regulates proliferation and patterning in the wing disc. In the pupal wing, Dpp takes on a new instructive vein-positioning function. There is no evidence that Hh regulates Dpp in the pupal wing, and moreover, the cells that had produced Dpp at the AP organizer no longer do so and no longer function as a AP organizers. These data show that N also takes on a new role during late larval and pupal stages: functioning at the AP organizer to regulate growth in response to Hh signaling (Casso, 2011).

Specificity of Drosophila cytonemes for distinct signaling pathways

Cytonemes are types of filopodia in the Drosophila wing imaginal disc that are proposed to serve as conduits in which morphogen signaling proteins move between producing and target cells. The specificity was investigated of cytonemes that are made by target cells. Cells in wing discs made cytonemes that responded specifically to Decapentaplegic (Dpp) and cells in eye discs made cytonemes that responded specifically to Spitz (the Drosophila epidermal growth factor protein). Tracheal cells had at least two types: one made in response to Branchless (a Drosophila fibroblast growth factor protein, Bnl), to which they segregate the Bnl receptor, and another to which they segregate the Dpp receptor. It is concluded that cells can make several types of cytonemes, each of which responds specifically to a signaling pathway by means of the selective presence of a particular signaling protein receptor that has been localized to that cytoneme (Roy, 2011).

Cells in developing tissues are influenced by multiple signals that they process and integrate to control cell fate, proliferation, and patterning. An example is in the Drosophila wing imaginal disc, where cells depend on several signaling systems that are intrinsic to the disc. Dpp, Wingless (Wg), Hedgehog (Hh), and epidermal growth factor (EGF) are produced and released by different sets of disc cells, and receipt of these signaling proteins programs their neighbors to develop and grow. The mechanisms by which morphogen signaling proteins influence target cells must ensure both specificity and accuracy, and one possibility is that these proteins transfer at points of direct contact. Imaginal discs are flattened sacs that have a monolayer of columnar cells on one side and squamous peripodial cells on the other. Many cells in wing discs make filopodial extensions that lie along the surfaces of the monolayers, oriented toward morphogen-producing cells. These extensions have been termed cytonemes to denote their appearance as cytoplasmic threads and to distinguish them as specialized structures that polarize toward morphogen-producing regions (Roy, 2011).

In wing discs dissected from third instar larvae, cytonemes can be seen as filaments extending from randomly generated somatic clones engineered to express a fluorescent protein such as soluble, cytoplasmic green fluorescent protein (GFP) or a membrane-bound form such as mCD8:GFP (the extracellular and transmembrane domains of the mouse lymphocyte protein CD8 fused to GFP). To image disc cytonemes, unfixed discs were placed peripodial side down on a coverslip, covered with a 1-mm-square glass, and mounted over a depression slide with the disc hanging from the coverslip. Because fluorescence levels in cytonemes were low relative to background, recorded images were processed to increase intensity and were subjected to de-convolution. Expression of CD8:GFP in wing disc clones revealed cytonemes emanating from both the apical and basal surfaces of columnar cells, as well as from peripodial cells (whose apical and basal surfaces could not be distinguished). Most cytonemes were perpendicular to the anterior/posterior (A/P) axis of the disc and oriented toward the cells that produce Dpp at the A/P compartment border; others were oriented toward the cells that produce Wingless at the dorsal/ventral (D/V) compartment border. Disc-associated myoblasts also had filopodia (Roy, 2011).

In the eye disc, cells in the columnar layer organize into ommatidial clusters as a wave of differentiation [the morphogenetic furrow (MF)] passes from posterior to anterior. A second axis, centered at the equator, is orthogonal to the MF and defines a line of mirror-image symmetry where dorsal and ventral ommatidia are juxtaposed. The columnar cells divide during the third instar period but stop or divide only once after the MF passes. CD8:GFP expression was induced in somatic clones and the columnar cells were examined. Whereas clones of six to eight cells were present on both sides of the MF, only cells anterior to the MF had visible cytonemes. Cytonemes emanating from these clones oriented either toward the axis defined by the MF or toward the axis defined by the equator. Single clones with cytonemes oriented both toward the MF and toward the equator were not observed, and there was no apparent correlation between clone position and cytoneme orientation or cytoneme length. Cells in the peripodial layer of the eye disc also had cytonemes (Roy, 2011).

The EGF pathway is a key signaling system for eye development, and cells in the MF express the EGF protein Spitz (Spi). Because one of the two types of anterior cell cytonemes extended toward the MF and to explore the distribution of membrane-bound receptor proteins, clones were induced that expressed an epidermal growth factor receptor:GFP (EGFR:GFP) fusion protein. Anterior cells expressing EGFR:GFP had cytonemes that oriented toward the MF, and most of these cytonemes had fluorescent puncta; no cytonemes that were marked by EGFR:GFP oriented toward the equator. Other than their 'furrow-only' orientation, the cytonemes marked by EGFR:GFP were similar to those marked by CD8:GFP. In contrast, co-expression of CD8:GFP with (nonfluorescent) EGFR marked both furrow-directed and equator-directed cytonemes. Thus, expression of EGFR:GFP does not eliminate the equator-directed cytonemes, suggesting that the specific localization of EGFR:GFP to furrow-directed cytonemes is not a consequence of ectopic (over)expression of this fusion protein (Roy, 2011).

Evidence that the furrow-directed cytonemes depend on Spi/EGF signaling was obtained by expressing a dominant negative form of EGFR. Although EGFR is required for cell proliferation in the disc, small clones expressing EGFR^DN were recovered that co-expressed EGFR^DN and CD8:GFP; in these clones, only cytonemes that appeared to be randomly oriented were present, indicating that the long, furrow-directed cytonemes may require EGFR signal transduction in the cytoneme-producing cells (Roy, 2011).

Wing disc-associated tracheal cells also make cytonemes. The transverse connective (TC) is a tracheal tube that nestles against the basal surface of the wing disc columnar epithelium and that sprouts a new branch [the air sac primordium (ASP)] during the third instar period in response to Branchless (Bnl) expressed by the wing disc. Tracheal tubes are composed of a monolayer of polarized cells whose apical surfaces line a lumen. Expression of CD8:GFP throughout the trachea (btl-Gal4 UAS-CD8:GFP) made it possible to detect GFP fluorescence in several types of cytonemes emanating from the basal surfaces of the TC and ASP. Cytonemes at the tip of the ASP (length range, 12 to 50 μm; average length of 23 μm) contained the Breathless (Btl); the Drosophila fibrobast growth factor receptor (FGFR) and appeared to contact disc cells that express Bnl. Short cytonemes (length range, 2 to 15 μm; average length of 8.5 μm) extended from the TC cells in the vicinity of the ASP (Roy, 2011).

Tests were carried out to se whether Dpp, Spi, Bnl, and Hh affected wing disc, eye disc, and tracheal cytonemes differentially. Ubiquitous expression of Spi, Bnl, or Hh (induced by heat shock) did not alter the A/P-oriented apical cytonemes in the wing disc, and, in the eye disc, the long cytonemes of the columnar layer were unaltered after ubiquitous expression of Dpp, Bnl, or Hh. In contrast, long oriented cytonemes were absent in wing discs after ubiquitous expression of Dpp, and only short cytonemes that appeared to be randomly oriented were observed. Similarly, 0.5 to 3 hours after cSpi, a constitutively active form of EGF, was expressed ectopically by heat shock induction, clones expressing CD8:GFP in the eye disc had many short cytonemes that lacked apparent directional bias; in contrast to controls, no long cytonemes oriented toward the MF were observed. Cytonemes with normal orientation and length (including MF-directed cytonemes) were present in eye discs that were examined later, 8 hours after a pulse of cSpi expression. To monitor EGFR-containing cytonemes for sensitivity and responsiveness to Spi, cSpi was expressed by heat shock induction, and cells in clones expressing EGFR:GFP were examined. After a pulse of cSpi expression, the extensions oriented outward without apparent directional bias, and the EGFR:GFP puncta were present in all cytonemes (Roy, 2011).

To examine responses of the ASP tip cytonemes, Hh, Spi, Dpp, and Bnl were overexpressed by heat shock and GFP-marked cytonemes at the ASP tip were examined. No differences in number of cytonemes were detected until about 3 hours after heat shock. Four to 5 hours after heat shock, expression of Bnl increased the number of tip cytonemes by ~2.6 times, and although most of the cytonemes were <30 μm, the cytonemes >30 μm also increased (~3.2 times). Most of the long cytonemes in these preparations were oriented in directions other than toward the cells that normally express Bnl. The number of long cytonemes >30 μm did not change after overexpression of Hh, Spi, and Dpp (0.6 to 0.8 times); the number of short cytonemes increased after Dpp overexpression (~1.7 times) but not after overexpression of Hh or Spi (Roy, 2011).

Thus, the responses of apical wing disc cytonemes to overexpressed Dpp, of eye disc cytonemes to ubiquitous Spi, and of ASP tip cytonemes to exogenous Bnl (Drosophila FGF) are similar. These results suggest that the cytonemes detected in the wing discs and eye discs may have orientations and lengths that are dependent specifically on the respective sources of Dpp and Spi, whereas the ASP may extend cytonemes in response to more than one signaling protein. These results are, however, complicated by the heat shock mode of induction because both the cells that expressed GFP (and extended marked cytonemes) as well as the surrounding cells expressed the signaling proteins. To overcome this problem, a method was developed to induce two types of somatic clones in the same tissue, one that expressed GFP and another that expressed Dpp (Roy, 2011).

The GAL4 system was used to label cytonemes with CD8:GFP. Clones of GAL4-expressing cells were generated with heat shock-induced flippase (FLP recombinase). The second type of clone expressed a Dpp:Cherry fusion and was generated with a variant Cre-progesterone receptor recombinase that could be activated with a regime of heat shock and RU486. By adjusting the timing and strength of induction, wing discs were produced with small, independent, and relatively infrequent clones. In discs with clones that expressed ectopic Dpp as well as clones that expressed CD8:GFP, apical cytonemes tagged with GFP were detected that oriented toward nearby Dpp:Cherry-expressing cells and not toward either the A/P or D/V signaling centers. Such 'abnormally directed' cytonemes were never observed in control discs. The abnormally oriented cytonemes suggest that apical cytonemes in the wing blade respond directly to sources of Dpp and that their orientation reflects extant sources of signaling protein (Roy, 2011).

To characterize the relationship between tracheal ASP tip cytonemes and FGF signaling from the wing disc, the distribution of Btl (FGFR) was examined in ASP cells and in ASP cytonemes. In preparations from larvae with tracheal expression of both CD8:GFP and Btl:Cherry (btl-GAL4 UAS-CD8:GFP;UAS-Btl:Cherry), cytonemes were marked by CD8:GFP, some of which had fluorescent Btl:Cherry puncta. Each ASP had only a few long (>30 μm) cytonemes, most of which contained Btl:Cherry puncta. Few of the more numerous short cytonemes (<30 μm) contained Btl:Cherry puncta. To characterize Btl:Cherry after overexpression of Bnl, focus was placed on preparations obtained 1 to 2 hours post-induction (genotype btl-GAL4 UAS-CD8:GFP/HS-Bnl;UAS-Btl:Cherry/Gal80^ts), because during this time interval the ASP morphology was close to normal but cytonemes had changed. ASPs were ignored after longer postinduction intervals because of major malformations to ASP morphology after 3 to 4 hours. Long cytonemes with Btl:Cherry puncta were present 1 hour after a pulse of Bnl expression; but 2 hours after the pulse, most ASPs had no long cytonemes, and the number of short puncta-containing cytonemes increased at the tip and along the shaft of the ASPs. After control heat shock or heat shock-induced expression of Dpp, the distribution of Btl:Cherry puncta in the ASP tip cytonemes was similar to normal controls: Long cytonemes had Btl:Cherry puncta, but most short cytonemes did not (Roy, 2011).

Because the number of small cytonemes at the ASP tip may have increased after ectopic Dpp expression, whether the thickveins (tkv) gene, which encodes a subunit of the Dpp receptor, is expressed in the ASP was investigated. Expression of the tkv reporter, tkv-lacZ (P{lacW}tkv¹⁶⁷¹³), was detected in the ASP. When Tkv:GFP and Btl:Cherry were expressed together, Tkv:GFP and Btl:Cherry segregated to separate tip cytonemes at the ASP tip. Whereas Tkv-containing cytonemes were short (<30 μm), most of the Btl-containing cytonemes were longer (three of four of the Btl:Cherry-containing cytonemes were longer than 30 μm), and they lay in focal planes closer to the disc. These properties were consistent in all preparations examined in which both green Tkv and red Btl cytonemes were intact. Imaging these marked ASPs revealed that overexpressed Tkv:GFP and Btl:Cherry were present not only in the plasma membranes (as expected) but also in separate puncta in the cell bodies. This shows that Tkv and Btl receptors also segregated to separate locations in the ASP cell bodies (Roy, 2011).

These findings suggest that the ASP has long cytonemes that are specific to Bnl and specifically harbor Btl-containing puncta and that the ASP also has cytonemes that are specific to Dpp and specifically harbor Tkv. Similarly in the eye disc, the presence of EGFR:GFP in furrow-oriented cytonemes and not in equator-oriented cytonemes suggests that cytonemes in the eye disc also selectively localize receptors. And as was previously shown, apical cytonemes in the wing disc selectively localize Tkv. The apparent ligand specificities and contrasting makeup of these cytonemes suggest a diversity of functionally distinct subtypes: Cells appear to make cytonemes that respond specifically to the Dpp, EGF, or Bnl signaling proteins. The basal filopodia implicated in Delta-Notch signaling in the wing disc may represent yet another type (Roy, 2011).

The mechanism that endows cytonemes with specificity for a particular signaling protein cannot be based solely on tissue-specific expression of a receptor. Spi, Dpp, and Hh are active in eye discs, but only changes in Spi signaling affected the furrow-directed cytonemes. And in the wing disc, both the Hh and EGF signal transduction pathways are active in cells at the A/P compartment border, but the apical cytonemes only responded to overexpressed Dpp. The findings that tracheal cells in the ASP respond to both Dpp and Bnl and that the Tkv and Btl receptors are present in different cytonemes that the ASP cells extend suggest that specificity may be a consequence of the constitution of the cytoneme, not on which receptors the cells make. The mechanism that localizes receptors to different cytonemes is not known, but because the marked receptors that were expressed also segregated to different intracellular puncta, the processes that concentrate these receptors in separate locations may not be exclusive to cytonemes. There is a precedent for segregation of proteins to different cellular extensions, neurons segregate proteins to dendrites or axons, so extending projections with specific and distinct attributes may be a general property of cells (Roy, 2011).

Essential basal cytonemes take up Hedgehog in the Drosophila wing imaginal disc

Morphogen concentration gradients that extend across developmental fields form by dispersion from source cells. In the Drosophila wing disc, Hedgehog (Hh) produced by posterior compartment cells distributes in a concentration gradient to adjacent cells of the anterior compartment. This study monitored Hh:GFP after pulsed expression and analyzed movements and co-localization of Hh, Patched (Ptc) and Smoothened (Smo) proteins tagged with GFP or mCherry and expressed at physiological levels from bacterial artificial chromosome transgenes. Hh:GFP moved to basal sub-cellular locations prior to release from posterior compartment cells that express it, and was taken up by basal cytonemes that extend to the source cells. Hh and Ptc were present in puncta that moved along the basal cytonemes and formed characteristic apical-basal distributions in the anterior compartment cells. The basal cytonemes required diaphanous, Scar, neuroglian, and synaptobrevin, and both the Hh gradient and Hh signaling declined under conditions in which the cytonemes were compromised. These findings show that in the wing disc, Hh distributions and signaling are dependent upon basal release and uptake, and on cytoneme-mediated movement. No evidence for apical dispersion was obtained (Chen, 2017).

Cytoneme-mediated delivery of hedgehog regulates the expression of bone morphogenetic proteins to maintain germline stem cells in Drosophila

Stem cells reside in specialised microenvironments, or niches, which often contain support cells that control stem cell maintenance and proliferation. Hedgehog (Hh) proteins mediate homeostasis in several adult niches, but a detailed understanding of Hh signalling in stem cell regulation is lacking. Studying the Drosophila female germline stem cell (GSC) niche, this study shows that Hh acts as a critical juxtacrine signal to maintain the normal GSC population of the ovary. Hh production in cap cells, a type of niche support cells, is regulated by the Engrailed transcription factor. Hh is then secreted to a second, adjacent population of niche cells, the escort cells, where it activates transcription of the GSC essential factors Decapentaplegic (Dpp) and Glass bottom boat (Gbb). In wild-type niches, Hh protein decorates short filopodia that originate in the support cap cells and that are functionally relevant, as they are required to transduce the Hh pathway in the escort cells and to maintain a normal population of GSCs. These filopodia, reminiscent of wing disc cytonemes, grow several fold in length if Hh signalling is impaired within the niche. Because these long cytonemes project directionally towards the signalling-deficient region, cap cells sense and react to the strength of Hh pathway transduction in the niche. Thus, the GSC niche responds to insufficient Hh signalling by increasing the range of Hh spreading. Although the signal(s) perceived by the cap cells and the receptor(s) involved are still unknown, these results emphasize the integration of signals necessary to maintain a functional niche and the plasticity of cellular niches to respond to challenging physiological conditions (Rojas-Rios, 2012).

The study of the mechanisms behind Hh signalling in the Drosophila ovary has allowed the identification of Hh-coated cytonemes in a cellular stem cell niche, emphasizing the idea that cytonemes mediate spreading of the activating signal from the producing cells. Recently, it has been reported that the Hh protein localises to long, basal cellular extensions in the wing disk. In addition, filopodial extensions in the wing, eye, and tracheal system of Drosophila have been shown to segregate signalling receptors on their surface, thus restricting the activation of signalling pathways in receiving cells. Hence, cytonemes, as conduits for signalling proteins, may be extended by receiving cells (and so are involved in uptake) or may be extended by producing cells (and so are involved in delivery and release) (Rojas-Rios, 2012).

Interfering with actin polymerisation in adult niches leads to a significant reduction in the number of cap cells (CpCs) growing Hh cytonemes, concomitant with precocious stem cell differentiation, demonstrating that these actin-rich structures are required to prevent stem cell loss and thus are functionally relevant. Importantly, because this study disturbed actin dynamics in post-mitotic CpCs that still produce wild-type levels of Hh protein and express CpC markers (but fail to activate the Hh pathway in ECs), the observed effects on stem cell maintenance are most likely specific to Hh delivery from CpCs to their target ECs via short cytonemes. This interpretation is further reinforced by the observation that CpCs can sense decreased Hh levels and/or a dysfunction in the transduction of the Hh pathway in the niche and respond to it by growing Hh-rich membrane bridges up to 6-fold longer than in controls. In this regard, it is interesting to note that the two lipid modifications found in mature Hh protein act as membrane anchors and give secreted Hh a high affinity for membranes and signalling capacities. In fact, it has been recently described that a lipid-unmodified form of Hh unable to signal does not decorate filopodia-like structures in the wing imaginal disc epithelium, confirming the link between Hh transport along cytonemes and Hh signalling. Thus, cytonemes may ensure specific targeting of the Hh ligand to the receiving germline cells in a context of intense signalling between niche cells and the GSCs. Interestingly, in both en^- and smo^- mosaic niches, the long processes projected towards the signalling-deficient area of the niche, which showed that competent CpCs sense the strength of Hh signalling activity in the microenvironment. While the nature of the signal perceived by the CpCs or the receptor(s) involved in the process are unknown, it is postulated that Hh-decorated filopodial extensions represent the cellular synapsis required for signal transmission that is established between the Hh-producing cells (the CpCs) and the Hh-receiving cells (the ECs). In this scenario (and because Ptc, the Hh receptor, is a target of the pathway) the membranes of mutant ECs, in which the transduction of the pathway is compromised, contain lower Ptc levels. Thus, longer and perhaps more stable projections ought to be produced to allow proper signalling. In addition, the larger the number of en mutant cells (and hence the stronger the deficit in Hh ligand concentration or target gene regulation), the longer the cellular projections decorated with Hh, which indicates that the niche response is graded depending on the degree of signalling shortage (Rojas-Rios, 2012).

Do the longer cytonemes found in mosaic germaria represent structures created de novo, or do they simply reflect a pre-existing meshwork of thin intercellular bridges that can regulate the amount of Hh protein in transit across them? Because an anti-Hh antibody was utilised to detect the cytonemes and all attempts to identify other markers for these structures have failed, it is not possible to discriminate between these two possibilities. In any case, since no increased was detected in Hh levels in wild-type CpCs that contained cytonemes relative to those that did not, it is clear that long filopodia do not arise solely by augmenting Hh production in the CpCs. Rather, if long cytonemes are not synthesised in response to a Hh signalling shortage and if they already existed in the niche, they ought to restrict Hh spreading independently of significant Hh production. Furthermore, because the strength of Hh signalling in the niche determines the distance of Hh spreading, either cytoneme growth or Hh transport (or both) are regulated by the ability of the CpCs to sense the Hh signalling output (Rojas-Rios, 2012).

The demonstration that a challenged GSC niche can respond to insufficient signalling by the cytoneme-mediated delivery of the stem cell survival factor Hh over long distances has wider implications. Niche cells have been shown to send cellular processes to their supporting stem cells in several other scenarios: the Drosophila ECs of the ovary and the lymph gland, the ovarian niche of earwigs, and the germline mitotic region in the hermaphrodite Caenorhabditis elegans. Similarly, wing and eye disc cells project cytonemes to the signalling centre of the disc. However, definitive proof that the thin filopodia described in the lymph gland, the earwig ovary, or imaginal discs deliver signals from the producing to the effector cells is lacking. The current findings strongly suggest that cytonemes have a role in transmitting niche signals over distance, a feature that may underlie the characteristic response of more complex stem cell niches to challenging physiological conditions. Careful analysis of the architecture of sophisticated niches, such as the bone marrow trabecular zone for mouse haematopoietic stem cells, will be needed to further test this hypothesis and to determine whether it represents a conserved mechanism for stem cell niche signalling (Rojas-Rios, 2012).

Cytonemes are required for the establishment of a normal Hedgehog morphogen gradient in Drosophila epithelia

Hedgehog (Hh) signalling is important in development, stem cell biology and disease. In a variety of tissues, Hh acts as a morphogen to regulate growth and cell fate specification. Several hypotheses have been proposed to explain morphogen movement, one of which is transport along filopodia-like protrusions called cytonemes. This study analysed the mechanism underlying Hh movement in the wing disc and the abdominal epidermis of Drosophila melanogaster. In both epithelia, cells were shown to generate cytonemes in regions of Hh signalling. These protrusions are actin-based and span several cell diameters. Various Hh signalling components localize to cytonemes, as well as to punctate structures that move along cytonemes and are probably exovesicles. In vivo imaging was used show that cytonemes are dynamic structures and that Hh gradient establishment correlates with cytoneme formation in space and time. Indeed, mutant conditions that affect cytoneme formation reduce both cytoneme length and Hh gradient length. The results suggest that cytoneme-mediated Hh transport is the mechanistic basis for Hh gradient formation (Bischoff, 2013).

The localization of several Hh signalling components at cytonemes has suggested a role for these structures in Hh signalling. This study characterize cytonemes in two Drosophila paradigms, the wing disc and the abdominal epidermis, and investigate their role in Hh gradient formation. Evidence is presented that cytonemes play an active role in gradient formation: the establishment of the Hh signalling gradient correlates dynamically in space and time with cytoneme formation in vivo; experimental shortening and lengthening of cytonemes affects the gradient accordingly; the analysis of ttv^−/−,botv^−/− mutant clones implicates cytonemes in Hh transport. Overall, the results support a model in which cytonemes of signal-producing cells are involved in long-range Hh transport (Bischoff, 2013).

In wing discs, however, both sending and receiving cells generate cytonemes raising the question of which role the cytonemes of receiving cells play. Expression of Ihog in A compartment cells leads to a depletion of Hh from the P compartment cells close to the A/P border, which suggests that A compartment cytonemes might actively engage in Hh reception. Hence, cytonemes of both sending and receiving cells might contribute to Hh transport. Interestingly, A compartment cytonemes are rare in histoblasts, suggesting that cytonemes of receiving cells play a minor role in the abdomen (Bischoff, 2013).

Ihog–RFP puncta were observed that associated with and moved along cytonemes. Frequently, such puncta were released from cytonemes. Puncta were also observed when labelling cytonemes with CD4–Tomato. This suggests that cytonemes might transport exovesicles that act as a vehicle for Hh or are the structure where exovesicles are being released. Accordingly, the knockdown of genes involved in exovesicle production/release has a significant effect on Hh gradient length, and Ihog can be detected in baso-lateral exovesicles at the ultrastructural level. However, the characterization of these exovesicles as well as their implication in Hh gradient formation requires further analysis. A role of exosomes in morphogen gradient formation has recently been suggested. Active Wnt proteins are secreted in exosomes in cultured cells and in the wing disc. In addition, vesicular release of SonicHh has been implicated in the determination of left–right asymmetry in vertebrates. Very recently, particles containing SonicHh and CDO (the vertebrate homologue of Ihog) that travel along filopodia-like extensions have been described in the chicken limb bud (Bischoff, 2013).

The mechanisms by which cytonemes could transport morphogens to their targets must ensure specificity and accuracy. One possibility is that cytonemes established contact between sending and receiving cells. Alternatively, cytonemes could act as a structure of morphogen release and uptake without cell–cell contacts involved. In vivo imaging showed that cytonemes are dynamic structures. Cytonemes might grow towards a receiving cell and then retract after a signalling event has taken place, or their dynamics could be determined intrinsically by the stability of their cytoskeleton. Moreover, not just cytoneme length but also their number could shape the gradient, as its brightest section coincides with the dense array of shorter cytonemes. This cytoneme-based model challenges the previous diffusion-based models (Bischoff, 2013).

Cytonemes have been described in a variety of signalling pathways. The Dpp receptor Thickveins is present in punctate structures moving along cytonemes. Air sac precursors extend cytonemes towards FGF-expressing cells. Tracheal cells were reported to have at least two types of cytoneme; one type that carries an FGF receptor, and another type that carries the Dpp receptor. This suggests that cytonemes are ligand specific. In the context of Notch signalling, filopodia mediate lateral inhibition between non-neighbouring cells of the pupal notum. Interestingly, the dynamic behaviour of these processes is crucial for signalling. Spitz/EGF is delivered through polarized actin protrusions to spatially bias the specification of a particular cell of the Drosophila leg. In another example, short cytonemes mediate the delivery of a juxtacrine Hh signal to maintain germline stem cells in the Drosophila ovary. This study has shown that cytonemes also play a pivotal role in long-range Hh signalling in wing disc cells, histoblasts and LECs. Therefore, it is believed that cytonemes are a general feature of signalling events of all epithelial cells (Bischoff, 2013).

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).

act up controls actin polymerization to alter cell shape and restrict Hedgehog signaling in the Drosophila eye disc

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-associated protein (CAP) that sequesters monomeric actin; acu is required to prevent actin filament polymerization in the eye disc. acu is required for cells to change shape in the morphogenetic furrow, and it also prevents premature neuronal differentiation in the eye disc, probably by restricting the movement of Hedgehog (Benlali, 2000).

The Drosophila CAP homolog, acu, is required for the organized progression of photoreceptor differentiation. In the eye disc, clones of acu mutant cells overlapping the morphogenetic furrow express markers of photoreceptor differentiation, such as Neuroglian, Atonal (Ato), and the decapentaplegic marker dpp-lacZ more anteriorly than surrounding wild-type cells. As differentiation proceeds from posterior to anterior, this represents an acceleration of differentiation in acu mutant cells. However, many cells in more posterior acu clones fail to differentiate as neurons and later appear to die, leaving scars in the adult eye. Removal of acu function from the entire eye disc by inducing clones in a Minute background with FLP recombinase expressed from the eyeless promoter results in a severe disorganization of the pattern of differentiation in the eye disc. dpp-lacZ is normally expressed in the morphogenetic furrow, preceding expression of the neuronal protein Elav. In the absence of acu, dpp-lacZ is expressed in very abnormal patterns and Elav-expressing photoreceptors appear to be randomly scattered over the eye disc (Benlali, 2000).

Since premature photoreceptor differentiation was observed both in acu mutant clones, in which actin filament levels are increased, and in chic clones, in which actin filament levels are decreased, an effect on cell shape might be common to both mutations. Using an antibody to the membrane-associated protein Armadillo (Arm), it was observed that acu mutant cells in the region of the morphogenetic furrow do not undergo the normal shape changes, and instead retain large apical profiles. The same phenotype was observed in chic mutant clones. This suggests both that coordinated alterations in actin filaments are required for apical constriction of cells in the morphogenetic furrow, and that this cell shape change is important in controlling the pattern and timing of differentiation (Benlali, 2000).

One hypothesis that might explain these results is suggested by the observation that Hh is a concentration-dependent regulator of ato. The extent of Hh diffusion is thus critical to determine the position, and therefore the developmental stage, at which photoreceptors differentiate. The Hh protein is modified by cholesterol addition and N-terminal acylation, and its movement through tissues appears to be a highly regulated process requiring the sterol-sensing-domain protein Dispatched and heparan sulfate proteoglycan synthesis by the product of the tout velu (ttv) gene. The rate-limiting step for Hh movement might therefore be transfer between cells rather than diffusion through the extracellular space. Constriction of the apical profiles of cells increases the density of their packing at the apical surface, where signaling is thought to occur; it would thus increase the number of such transfers required to travel a given distance (Benlali, 2000 and references therein).

Using an antibody to Hh, a tight band of Hh protein was indeed observed at the morphogenetic furrow in wild-type cells, and more anterior Hh staining in both large and small clones of acu mutant cells. To test whether this ectopic anterior Hh is functional, the accumulation of full-length Cubitus interruptus (Ci) protein was examined. Such accumulation is normally associated with a shift of Ci from its repressor to its activator form, and is the earliest response to Hh reception that can be visualized. In clones of cells mutant for acu, accumulation of Ci is observed more anteriorly than in surrounding wild-type cells. chic mutant cells do not themselves accumulate Ci, suggesting that actin filaments may be required for the response to Hh, but wild-type cells anterior to chic clones show precocious Ci accumulation. Ectopic Hh signaling has been shown to be sufficient to induce apical constriction of surrounding cells. Consistent with the presence of ectopic Hh in the clones, apical constriction of cells has been observed at the lateral edges of acu and chic mutant clones and anterior to smaller clones. It thus appears that apical constriction of cells in the morphogenetic furrow prevents long-range Hh movement, perhaps by increasing the number of rate-limiting active transport steps required (Benlali, 2000).

Eyeless collaborates with Hedgehog and Decapentaplegic signaling in Drosophila eye induction

eyeless (ey) is a key regulator of the eye development pathway in Drosophila. Ectopic expression of ey can induce the expression of several eye-specification genes (eya, so, and dac) and induce eye formation in multiple locations on the body. However, ey does not induce eye formation everywhere where it is ectopically expressed, suggesting that Ey needs to collaborate with additional factors for eye induction. Ectopic eye induction by Ey has been examined in the wing disc; eye induction was spatially restricted to the posterior compartment and the anterior-posterior (A/P) compartmental border, suggesting a requirement for both Hh and Dpp signaling. Although Ey in the anterior compartment induces dpp and dac, these are not sufficient for eye induction. Coexpression experiments show that Ey needs to collaborate with high level of Hh and Dpp to induce ectopic eye formation. Ectopic eye formation also requires the activation of an eye-specific enhancer of the endogenous hh gene (Kango-Singh, 2003).

These results indicated that Ey needs to collaborate with high levels of Hh and Dpp for eye induction. Since Hh and Dpp are secreted molecules and can act over long range, the requirement for their high levels restricts the site of eye development. At the time of MF initiation, hh and dpp are expressed at the posterior margin of the eye disc, and ey is expressed throughout the eye disc. So the coexistence of Ey, high Hh, and high Dpp occurs only at the posterior margin to induce MF initiation. After MF initiation, ey is downregulated in the developing photoreceptor cells posterior to the MF, where hh is expressed (Kango-Singh, 2003).

dpp is expressed only at the MF. Thus, the only location where Ey, high Hh, and high Dpp levels coexist is just anterior to the MF, allowing the MF to progress anteriorly. These results clearly show that even when ey, hh, and dpp are all provided, eye induction still does not occur everywhere, suggesting that additional factors are required. It is proposed that the coexpression of the eye-specification genes, eya, so, dac, ey, toy, and eyg, occurring first in the second instar eye disc, specifies the eye fate (Kango-Singh, 2003).

Mechanism of hedgehog signaling during Drosophila eye development

Although Hedgehog (Hh) signaling is essential for morphogenesis of the Drosophila eye, its exact link to the network of tissue-specific genes that regulate retinal determination has remained elusive. In this report, it is demonstrated that the retinal determination gene eyes absent (eya) is the crucial link between the Hedgehog signaling pathway and photoreceptor differentiation. Specifically, it has been shown that the mechanism by which Hh signaling controls initiation of photoreceptor differentiation is the alleviation of eya repression and decapentaplegic (dpp) expression by the zinc-finger transcription factor Cubitus interruptus (Ci^rep). Furthermore, the results suggest that stabilized, full length Ci (Ci^act) plays little or no role in Drosophila eye development. Moreover, while the effects of Hh are primarily concentration dependent in other tissues, hh signaling in the eye acts as a binary switch to initiate retinal morphogenesis by inducing expression of the tissue-specific factor Eya (Pappu, 2003).

Misexpression of eyeless (ey) in the wing disc causes ectopic photoreceptor differentiation only in regions where both dpp and hh signaling are normally active. The simplest explanation for this effect invokes a linear regulatory hierarchy where hh induces dpp, which in turn cooperates with ey to initiate retinal morphogenesis. While misexpression of ey and dpp together does indeed lead to synergistic photoreceptor differentiation, this occurs only in the posterior compartment of the wing disc. Notably, Hh signaling is not transduced in the posterior compartment of the wing disc due to the repression of ci by En. Furthermore, dpp and ey expression does not induce Ci expression in the posterior compartment of the wing disc. Thus, it is concluded that dpp and ey can induce Eya expression and photoreceptor differentiation in the posterior compartment of the wing disc in the absence of Hh signaling and Ci^rep. Misexpression of hh and ey induces robust eya expression and photoreceptor differentiation in the wing disc, but only in the anterior compartment. This result is consistent with a model in which Hh signaling normally blocks the production of Ci^rep and converts it into an activated form, Ci^act, in the anterior compartment of the wing disc. Ci^act can induce dpp expression in the anterior compartment and dpp can in turn cooperate with ey to induce robust Eya expression and photoreceptor differentiation. Consistent with this model, co-expression of hh, dpp and ey leads to Eya expression and photoreceptor differentiation in both compartments of the wing disc. Taken together, these results suggest that, in the wing disc, ey and dpp can activate eya expression only in the absence of Ci^rep (Pappu, 2003).

Co-expression of dpp, ey and eya using the 30A-Gal4 driver induces photoreceptor differentiation in both wing compartments, albeit with low penetrance. This effect becomes stronger and more penetrant when dpp, ey, eya and so are misexpressed in a ring around the wing pouch. These results demonstrate that providing ey, dpp and eya from an exogenous source is sufficient to bypass the requirement for Hh signaling during initiation of ectopic photoreceptor differentiation. In addition, these results implicate eya as a key target for Hh signaling during the initiation of normal retinal morphogenesis, most likely by blocking Ci^rep (Pappu, 2003).

The data from ectopic expression analyses in the wing disc suggest that Ci^rep has a major role in blocking eya expression in areas that are not exposed to Hh signaling. However, Ci^act also plays an important role in patterning the anterior compartment of the wing disc. For example, adult wings that contain ci mutant clones develop with defects in the anterior compartment. In the Drosophila eye disc, ci is expressed uniformly but Ci protein expression follows a dynamic pattern. It has been proposed that in regions anterior to the furrow Ci is subject to PKA-dependent phosphorylation and SCF^Slimb-dependent processing into Ci^rep. Cells in the MF, however, receive and transduce the Hh signal and prevent the proteolytic processing of Ci, therefore blocking production of Ci^rep. Furthermore, it has been proposed that cells that are posterior to the MF do not accumulate Ci^rep in a PKA-dependent manner. Instead, these cells use a smo- and cullin3- dependent proteolytic process leading to the complete degradation of Ci. Therefore, the role for Ci in the eye appears to be limited only to cells that are part of, and anterior to, the MF. However, these studies do not establish separate functional roles for Ci^act and Ci^rep in the developing eye (Pappu, 2003).

Surprisingly, Eya expression and photoreceptor differentiation are not perturbed in Drosophila eye discs that contain large ci-null mutant clones. Similarly, adult eyes containing large ci mutant clones appear normal both externally and in internal sections. These results, coupled with ectopic expression analysis in the wing disc, suggest that Ci^act plays little or no role during normal photoreceptor differentiation. Furthermore, these results support a model in which the major role for Hh signaling during the initiation of photoreceptor differentiation is to prevent the production of Ci^rep (Pappu, 2003).

Interestingly, ci-null mutant clones that span the furrow do not hasten furrow progression. Although ectopic activation of the Hh pathway is sufficient to induce precocious furrow advancement and photoreceptor differentiation, loss of Ci is not. A likely explanation for this apparent contradiction may be found in the distinction between loss- and gain-of-function experiments. Specifically, although Ci^act normally plays little or no role in eye development, ectopic production of Ci^act is sufficient to induce precocious furrow advancement. Intriguingly, vertebrate homologs of Drosophila ci have evolved to carry out either activator (Gli1 and Gli2) or repressor (Gli3 and perhaps Gli2) functions independently. These findings demonstrate that in the absence of gene duplication, tissue-specific separation of these functions has also occurred in Drosophila (Pappu, 2003).

It is proposed that Hh signaling acts as a binary switch during Drosophila eye development to control the timing of initiation of photoreceptor differentiation. Specifically, the data suggest that during early larval development Ci^rep normally inhibits retinal morphogenesis by blocking eya and dpp expression. Hh signaling in late second instar larvae blocks production of Ci^rep, which in turn allows dpp and eya expression, MF initiation, progression and photoreceptor differentiation. Rather than regulating the differentiation of multiple cell types in a concentration-dependent manner, the data suggest that Hh signaling acts as a molecular switch that is sufficient to initiate dpp and eya expression and retinal morphogenesis. This model also explains the seemingly contradictory phenotypes of loss of smo (blocks MF initiation) and loss of ci (no effect) during Drosophila eye development. Loss of ci creates a permissive environment for eya and dpp expression and photoreceptor differentiation, rendering eye development Hh independent. By contrast, Ci^rep persists in the absence of smo function and thus photoreceptor morphogenesis does not occur in smo clones. Since ci null mutant clones in the eye develop normally, other Hh independent mechanisms must also act to control the initiation of retinal morphogenesis in Drosophila (Pappu, 2003).

Posterior margin smo mutant clones lack Eya expression and photoreceptor differentiation. The lack of eya expression in these cells is attributed to their inability to block the production of Ci^rep. Furthermore, the data demonstrate that co-expression of dpp and eya in these posterior smo mutant clones rescues photoreceptor differentiation. In addition, dpp and eya co-expression is sufficient to rescue delayed furrow progression in smo clones. However, the precise temporal and spatial order of photoreceptor recruitment may not be rescued in these clones. Thus, the requirement for Hh signaling in the eye can be circumvented by the expression of the downstream targets dpp and eya. These results demonstrate that eya is a crucial eye-specific target of Hh signaling during the initiation of retinal differentiation and has led to a new model for the initiation of retinal morphogenesis. In this model, Hh signaling blocks the proteolytic degradation of Ci^act into Ci^rep, thus allowing initiation of dpp expression. Once dpp expression is established, the absence of Ci^rep allows dpp to act in parallel with ey to initiate eya expression, which in turn leads to so expression. Furthermore, dpp cooperates with eya and so to initiate the expression of dac and extensive feedback regulation among these genes leads to consolidation of retinal cell fates (Pappu, 2003).

Deciphering synergistic and redundant roles of Hedgehog, Decapentaplegic and Delta that drive the wave of differentiation in Drosophila eye development

In Drosophila, a wave of differentiation progresses across the retinal field in response to signals from posterior cells. Hedgehog (Hh), Decapentaplegic (Dpp) and Notch (N) signaling all contribute. Clones of cells mutated for receptors and nuclear effectors of one, two or all three pathways were studied to define systematically the necessary and sufficient roles of each signal. Hh signaling alone is sufficient for progressive differentiation, acting through both the transcriptional activator Ci155 and the Ci75 repressor. In the absence of Ci, Dpp and Notch signaling together provide normal differentiation. Dpp alone suffices for some differentiation, but Notch is not sufficient alone and acts only to enhance the effect of Dpp. Notch acts in part through downregulation of Hairy; Hh signaling downregulates Hairy independently of Notch. One feature of this signaling network is to limit Dpp signaling spatially to a range coincident with Hh (Fu, 2003).

The development of cells mutant for all three transcription factors, Mad, Su(H), and ci is a helpful starting point, since they may reflect a 'ground state' of eye development that requires extracellular signals to differentiate. Mad Su(H) ci cells fail to express the atonal or senseless genes that initiate R8 differentiation, and, consequently, fail to support retinal differentiation. This shows that the absence of Ci75 is not sufficient for differentiation. Dpp alone can induce Ato [e.g., in Su(H) ci clones], but N and Dpp signaling together are required to activate Atonal with normal kinetics, as occurs in ci-mutant cells. N signaling alone (in tkv ci clones) is insufficient. In the presence of Ci, prompt differentiation requires Hh to downregulate Ci75, and differentiation is delayed in Smo clones that lack this input. The normal role of Hh is not just to remove Ci75 thus permitting Dpp and N to work, because Atonal is turned on normally in Mad Su(H) clones that do not respond to Dpp or N signals. Such differentiation depends exclusively on Hh yet progresses normally, except that a neurogenic phenotype reflects dependence of lateral inhibition on Su(H). Hh depends positively on ci to drive differentiation in Mad Su(H) cells and, therefore, requires Ci155. The positive role of ci can also be inferred from the delayed differentiation of Su(H) ci clones in comparison with Su(H) clones (Fu, 2003).

Hairy is downregulated redundantly by Hh and N signaling. Prolonged Hairy expression is not sufficient to block differentiation completely but it does antagonize it (e.g., in Su(H) ci clones). Downregulation of Hairy in response to Hh as well as N explains why both ci and Su(H) mutant clones can differentiate promptly, and why N enhances differentiation in response to Dpp but is not required for differentiation in response to Hh (Fu, 2003).

Comparison between Mad Su(H) ci cells and Su(H) ci cells shows that Dpp signaling is sufficient to initiate eye differentiation in its normal location in the absence of Hh or N signals, but such differentiation is delayed. The normal timing of differentiation is restored by combined Dpp and N signals (in ci clones). This is the basis for the ectopic differentiation on co-expression of Dpp and Dl ahead of the furrow (Fu, 2003).

Superficially, these results differ from previous ectopic expression studies that concluded that Dpp signaling alone was not sufficient to induce ectopic differentiation in all locations. This discrepancy is probably explained by the baseline repressor activity of Su(H) protein. Previous work shows that without N signaling, repressor activity of Su(H) protein retards differentiation. Dpp signaling is sufficient for differentiation in the experiments where the Su(H) gene has been deleted. In the presence of the Su(H) gene, Dpp may be most effective at locations where there is little Su(H) repressor activity, such as close to the morphogenetic furrow where N signaling is active (Fu, 2003).

Comparison between Mad Su(H) ci cells, which do not differentiate, and Mad ci or tkv ci cells, which differentiate slowly or not at all, shows that Notch signaling alone is insufficient for differentiation. Premature differentiation reported when N is activated ectopically ahead of the furrow must reflect endogenous Dpp signaling at such locations (Fu, 2003).

These experiments reveal an outline of the mechanisms of Hh, Dpp and N redundancy. First, the results show that Mad and Ci independently reinforce differentiation, presumably through the transcription of target genes because Mad is sufficient for differentiation in the absence of Ci, and vice versa. The results show unequivocally that the transcriptional activator Ci155 activates differentiation in addition to Ci75 antagonizing differentiation (Fu, 2003).

It was surprising to find that Dpp stabilizes Ci155 in the absence of Smo, which suggests Dpp input into Hh signal transduction. Although the requirement for smo-dependent input through fused makes it unlikely that Ci155 is functional in smo clones, Ci155 accumulation might be associated with reduced Ci75 levels. Ci75 is shown to repress differentiation in smo clones because smo ci clones differentiation normally. Ci155 stabilization cannot be due to an indirect effect of Dpp signaling on Hh, Ptc or Smo expression levels because the effect is detected in the absence of smo, and, therefore, reflects an effect on Hh signal transduction components downstream of Smo. One idea is that Dpp signaling (or Dpp-induced differentiation) may replace SCF^Slimb processing of Ci (which cleaves Ci155 to Ci75) with Cullin3-mediated Ci degradation, just as normally occurs posterior to the morphogenetic furrow. In a smo clone, Ci155 would accumulate because Smo is required for Cullin3 to degrade Ci. However, the SCF^Slimb-to-Cullin3 switch may not be the only effect of Dpp on Ci processing, because Tkv slightly enhances Ci155 accumulation even when smo is present (Fu, 2003).

Finally, downregulation of Hairy by N requires the Su(H) gene. N also overcomes baseline repressor activity of Su(H) protein to promote progression of differentiation. This role of N must be independent of Hairy (Fu, 2003).

Dl, Hh and Dpp are generally thought to signal over very different distances. How can signals of such different range substitute for one another to permit normal eye development? Dpp is transcribed in response to Hh signaling and is produced where Ci155 levels are highest. Dl is regulated by Hh indirectly through Ato and Ato-dependent Egfr activity in differentiating cells. Hh is expressed most posteriorly of the three, in differentiating photoreceptors (Fu, 2003).

Eye differentiation uses Hh to progress through cells unable to respond to Dpp (tkv, Mad) or N (Su(H)). The range of Hh diffusion depends in part on the shape of the morphogenetic furrow cells. The Dpp that drives differentiation through ci-mutant cells unable to respond to Hh must diffuse from outside the ci clones because Dpp synthesis is Hh dependent. Large ci clones develop normally so Dpp diffusion cannot be limiting (dpp-mutant clones offer no information about the range of Dpp because they express and differentiate in response to Hh). Instead, the rate of progression in response to Dpp is controlled by Dl. Dl signals over (at most) one or two cell diameters at the morphogenetic furrow (Fu, 2003).

The previous view of eye patterning was influenced by the morphogen function of Hh and Dpp in other discs. It was thought that domains of Ato and Hairy expression reflected increasing concentrations of Hh and Dpp. The data shows that, in the eye, the combination of signals is important. Differentiation is triggered where Dl and/or Hh synergize with Dpp, regardless of where the source of Dpp is. The additional requirements limit Dpp to initiating differentiation at the same locations that Hh does (Fu, 2003).

Extracellular signals responsible for spatially regulated proliferation in the differentiating Drosophila eye

Spatially and temporally choreographed cell cycles accompany the differentiation of the Drosophila retina. The extracellular signals that control these patterns have been identified through mosaic analysis of mutations in signal transduction pathways. All cells arrest in G1 prior to the start of neurogenesis. Arrest depends on Dpp and Hh, acting redundantly. Most cells then go through a synchronous round of cell division before fate specification and terminal cell cycle exit. Cell cycle entry is induced by Notch signaling and opposed in subsets of cells by EGF receptor activity. Unusually, Cyclin E levels are not limiting for retinal cell cycles. Rbf/E2F and the Cyclin E antagonist Dacapo are important, however. All retinal cells, including the postmitotic photoreceptor neurons, continue dividing when rbf and dacapo are mutated simultaneously. These studies identify the specific extracellular signals that pattern the retinal cell cycles and show how differentiation can be uncoupled from cell cycle exit (Firth, 2005).

The EGFR holds R2-R5 cells in G1 phase and promotes G2/M progression of other cells during the second mitotic wave (SMW). Earlier regulation is now found to depend on longer-range signaling by the Hh, DPP, and N signals already known to drive the progression of the morphogenetic furrow. These studies exclude other models that show that Hh, Dpp, or N act indirectly by releasing other, cell cycle-specific signals from differentiating cells, or that patterned cell cycle withdrawal or reentry occur independent of extracellular signals, such as by synchronized growth. Instead, specific signals are necessary or sufficient for each aspect of cell cycle patterning (Firth, 2005).

G1 arrest ahead of the morphogenetic furrow depends on posterior-to-anterior spread of Hh and Dpp. Hh is secreted from differentiating cells, starting at column 0 in the morphogenetic furrow. Dpp is transcribed in ~6 ommatidial columns in the morphogenetic furrow in response to Hh. Cells accumulate in G1 about 16-17 cell diameters anterior to column 0, suggesting an effective range of ~13-17 cells for Hh and Dpp (Firth, 2005).

The contribution of Dpp to this cell cycle arrest is known already, but that of Hh was not suspected. Both Dpp and Hh signaling can promote proliferation in other developmental contexts (Firth, 2005).

S phase entry in the SMW depends on another signal, N. Expression of the N ligand Dl begins at the anterior of the morphogenetic furrow. The first S phase cells are detected 6-8 cell diameters more posteriorly, just behind column 0. The transmembrane protein Dl must act more locally or more slowly than the secreted Hh and Dpp proteins, to explain gaps between S phases (Firth, 2005).

Although N activity has been associated with growth through indirect mechanisms involving the release of other secreted growth factors and also regulates endocycles, this appears to be the first report of a specific role of N in G1/S in diploid Drosophila cells. Notably, deregulated N signaling contributes to at least two human cancers and is oncogenic in mice (Firth, 2005).

At the same time that N promotes S phase entry in the SMW, EGFR activity ensures that R2-R5 cells remain in G1. N is still required in the absence of EGFR, so N activity is a positive signal and is not required only to counteract EGFR activity. Instead, EGFR activity interferes with S phase entry in response to N (Firth, 2005).

Ligands for the EGF receptor are thought to be released from R8 precursor cells, although EGFR-dependent MAPK phosphorylation is detected one ommatidial column before the column where R8 precursor cells can be identified, which is in column 0. This means that EGFR activation begins after Dl expression but before S phase DNA synthesis starts. Later, ligands released from differentiating precluster cells activate EGFR in surrounding cells to permit SMW mitosis around columns 3-5 (Firth, 2005).

Hh and Dpp together promote expression of Dl and of EGFR ligands; in part, this occurs indirectly through Atonal and the onset of differentiation. EGF receptor activity also promotes Dl expression (Firth, 2005).

At least three genetic mechanisms arrest distinct retinal cells in G1. Arrest ahead of the morphogenetic furrow depends on Dpp and Hh. During the SMW, R2-R5 cells are held in G1 by EGFR, which counteracts the SMW-promoting N activity. In addition, R8 cells, which are defined by the proneural gene atonal, remain in G1 independent of EGFR. After the SMW, all cells remain in G1 indefinitely, independent of EGFR. Although cell cycle withdrawal roughly correlates with differentiation, many of the cells that arrest after the SMW are still unspecified (Firth, 2005).

Loss of rbf and dap together overcome all cell cycle blocks, even though cell differentiation continues. This redundancy indicates that Cyclin E/Cdk2 targets other than Rbf are needed for proliferation, consistent with many other studies. Dap may be regulated by EGFR in R2-R5 cells. If rbf regulates the normal SMW, where Cyclin E expression seems not to be limiting, then other E2F targets may be involved. Some cell cycle arrest can also be overridden by forced expression of Cyclin E, E2F/DP, dRef, and ORC1, or by mutation of the Cyclin A antagonist rux (Firth, 2005).

The results show that mechanisms that assure both short- and long-term arrest of retinal cells must operate upstream of (or parallel to) Rbf and Cyclin E activities. They might resemble the barriers to transformation and regeneration that exist in mammals (Firth, 2005).

Hedgehog and Decapentaplegic pathway function in the developing Drosophila compound eye: smoothened, thickveins and the genetic control of cell cycle and cell fate

The Hedgehog and Decapentaplegic pathways have several well-characterized functions in the developing Drosophila compound eye, including initiation and progression of the morphogenetic furrow. Other functions involve control of cell cycle and cell survival as well as cell type specification. This study used the mosaic clone analysis of null mutations of the smoothened and thickveins genes (which encode the receptors for these two signals) both alone and in combination, to study cell cycle and cell fate in the developing eye. It is concluded that both pathways have several, but differing roles in furrow induction and cell fate and survival, but that neither directly affects cell type specification (Vrailas, 2006).

Interestingly, though Hedgehog signaling is required for Decapentaplegic expression, the two pathways are not completely redundant. The data demonstrate that for some aspects of eye development, the two pathways have separable and independent functions, such as Hedgehog signaling regulation of rough expression and S phase of the second mitotic wave. However, both pathways have redundant roles in the apical constriction of the actin cytoskeleton and proper expression of elements of the Egfr/Ras and Notch/Delta signaling pathways as well as in cell fate specification, though neither pathway is required for differentiation. Finally, the Decapentaplegic pathway is epistatic to the Hedgehog pathway for G1 arrest in the furrow and G1, G2 and M phases of the second mitotic wave. These various ways in which the Hedgehog and Decapentaplegic pathways work together (or not) demonstrate the complexity of pathway integration for proper eye development (Vrailas, 2006).

A strong effect of loss of Hedgehog signaling was seen on the morphology of cells in the furrow, and it is suggested consequentially, in the distribution of the Egfr and Notch receptors. This disruption of the localization of elements of other signaling pathways, which is enhanced by the additional loss of thickveins, may explain some of the phenotypes observed. For example, cells at the edges of smoothened and double mutant clones near wild type tissue are still able to enter S phase. The Notch/Delta pathway has been shown to regulate the G1/S transition of the second mitotic wave with loss of pathway activity leading to a loss of S phase. Therefore, it may be that Notch/Delta signaling between cells in the wild type tissue and in the clone, allows for the S phases seen at the edges of the clones, while in the center of clones, where the Notch/Delta pathway is disrupted, S phase is lost. Cell fate specification can still occur at the edges of smoothened thickveins double mutant clones. It may be that the furrow does not really pass through the double mutant clones, but some signal from outside the clone can still induce photoreceptor cell fate, at least close to the clone margins. This is likely to be Spitz/Egfr signaling, which is present but disrupted in smoothened clones, since this signal can induce photoreceptor fate ectopically even anterior to the furrow and without the formation of R8/founder cells (Vrailas, 2006).

This study reports the roles of Hedgehog and Decapentaplegic signaling in eye development, however, these pathways are also instrumental for patterning and proliferation in the developing wing. Studies in the wing have shown that as in the eye, decapentaplegic expression is downstream of hedgehog, suggesting that these pathways may also rely on each other for proper wing development. Though smoothened and thickveins have no role in ommatidial cell fate, Hedgehog signaling is required for specification of intervein and vein territories in the central region of the wing, and Decapentaplegic signaling has been shown to be required for vein cell fate in the developing pupal wing (Vrailas, 2006).

As in the eye, Hedgehog and Decapentaplegic signaling have been implicated in cell cycle regulation in the developing wing. Studies in the wing found that overexpression of the Hedgehog signal induces proliferation through upregulation of Cyclin D and Cyclin E, as well as specifically promotes S phase in the wing margin. FACS analysis of wing discs revealed that thickveins loss of function clones (tkv⁷) have a reduced number of cells in S phase and an increase in the number of cells in G1 phase. Additionally, inhibition of the Hedgehog signal results in decreased growth and cell proliferation rates, and loss of Decapentaplegic pathway signaling results in small clones, suggesting that these pathways are important in cell survival and/or proliferation in the wing (Vrailas, 2006).

It appears that both tissues use Hedgehog signaling to promote S phase and possibly cell survival, since inhibiting Hedgehog signaling results in cell death in the eye and decreased growth in the wing. Additionally, the two tissues may use Hedgehog signaling to regulate the G1 phase, though this regulation may have subtle differences. In addition, Decapentaplegic signaling also appears to be necessary for proliferation in the developing eye and wing, though these tissues may use this signal to regulate the cell cycle differently. This is not surprising, since the developing third instar eye and wing discs may have fundamental differences in cell cycle regulation; the eye has a coordinated second mitotic wave and the wing does not. For example, the eye may utilize some factors that are not present in the wing disc to prevent the build up of too much Cyclin E. Therefore, Cyclin E levels are decreased in the eye but not in the wing. Additionally, thickveins appears to be responsible for G1 arrest in the furrow, while in the wing, G1 arrest in the zone of nonproliferating cells is mediated by Wingless signaling. However, it may be that the eye and wing regulate the cell cycle using Hedgehog and Decapentaplegic signaling in much the same way, but the techniques used to examine this phenomenon in the different tissues do not allow for a direct comparison of results. For example, it may be that FACS analysis is a more sensitive technique than immunohistochemistry, and thus subtle changes in the cell cycle that were observed in the wing were not observed in the eye. Alternatively, the FACS analysis was performed on wing discs that contained thickveins clones in a Minute background in order to achieve a larger sample of thickveins mutant cells. However, dying cells, such as those homozygous for Minute mutations, have been shown to have non-autonomous effects on the biology of the surrounding cells in the wing. Indeed, one study has reported that Minute mutations can non-autonomously affect pattering of photoreceptors in the developing eye. It may be that the Minute background partially masked the thickveins cell cycle phenotypes and the eye and wing may not be as different as it initially appears (Vrailas, 2006).

The data also shows that the Hedgehog and Decapentaplegic pathways are only partially redundant in the eye, which has also been shown in the wing. Hedgehog signaling alone is required for specification of veins 3 and 4 and the sensory organ precursors (SOPs) near the anterior/posterior boundary of the developing wing, whereas Decapentaplegic signaling mediated by Hedgehog promotes some SOP formation in the notum and some other regions of the wing (Vrailas, 2006).

In some instances, the data contrasts with previous reports from others. In one case, in which different alleles of smoothened (smo³ versus smo^D16) were examined, phenotypic variation may be a result of allele specific effects. However, in another case, the same allele was used by two groups, and it may be that some other aspect of the genetic background of the stocks differed that influenced the results observed. The effects of removing a receptor (Smoothened) may also differ in some cases from those of removing a downstream element (Ci). It was also observed that clones the remove thickveins or smoothened and thickveins together often appear to be re-specified as other structures, resembling appendage discs. This may be due to other functions of the Decapentaplegic pathway on the disc margins and in defining the limits of the eye field. The interpretations of others may have been confounded by such re-specification in some cases. Indeed, in the developing wing, cells lacking Decapentaplegic pathway function actually leave the epithelium. Some care was taken to analyze only those small clones near the center of the eye field that do not have these characteristics. Indeed, the fact that photoreceptor specific markers were observed in some cells that lack both smoothened and thickveins demonstrates that even the double mutant clones do not always re-specify (Vrailas, 2006 and references therein).

In summary, it is concluded that the Hedgehog pathway has important roles in inducing furrow initiation and progression. The Hedgehog and Decapentaplegic pathways have redundant roles in actin constriction in the morphogenetic furrow, expression of Egfr, Notch and Delta, and differentiation with neither pathway essential for cell type specification. Likewise, no role was found for either Hedgehog or Decapentaplegic signaling in ommatidial rotation or chirality. It is also suggested that the Hedgehog pathway alone is required for rough expression and the G1/S transition in the second mitotic wave and provides a protective function against apoptosis. In contrast, the Decapentaplegic pathway appears critical for furrow initiation at the disc margins (but not progression in the center). In addition, the Decapentaplegic pathway is epistatic to Hedgehog signaling for maintenance of G1 arrest in the furrow and regulation of G1 phase and the G2/M transition in the second mitotic wave (Vrailas, 2006).

Novel function of the class I bHLH protein Daughterless in the negative regulation of proneural gene expression in the Drosophila eye

Two types of basic helix-loop-helix (bHLH) family transcription factor have functions in neurogenesis. Class II bHLH proteins are expressed in tissue-specific patterns, whereas class I proteins are broadly expressed as general cofactors for class II proteins. The Drosophila class I factor Daughterless (Da) is upregulated by Hedgehog (Hh) and Decapentaplegic (Dpp) signalling during retinal neurogenesis. The data suggest that Da is accumulated in the cells surrounding the neuronal precursor cells to repress the proneural gene atonal (ato), thereby generating a single R8 neuron from each proneural cluster. Upregulation of Da depends on Notch signalling, and, in turn, induces the expression of the Enhancer-of-split proteins for the repression of ato. It is proposed that the dual functions of Da--as a proneural and as an anti-proneural factor--are crucial for initial neural patterning in the eye (Lim, 2008).

Da is upregulated in the furrow region. Surprisingly, however, it was found that there are two distinct patterns of Da upregulation. The first pattern is a broad, low-level upregulation in the furrow (hereafter referred to as basal level). The second pattern is a stronger expression of Da (hereafter referred to as high level) selectively in the non-neural cells surrounding the Ato-positive R8 cells between proneural clusters. Tests were perfomed to see whether this previously unrecognized pattern of expression of Da is specific by examining eye discs containing da loss-of-function (LOF) clones. Both the basal and high-level expressions of Da in the furrow were lost in the LOF clones of da³, a null allele, showing the specificity of the pattern of Da expression (Lim, 2008).

The basal level of Da upregulation overlaps with the domain of Ato expression near the furrow, where they function together to regulate neurogenesis. As the furrow progression and expression of Ato are controlled by Hh and Dpp signalling, it was reasoned that regulation of Da expression in the furrow might be linked to these signalling pathways (Lim, 2008).

To test whether Hh signalling is required for the expression of Da, Da expression was examined in hh¹ mutant eye discs in which the production of Hh ceases after the mid-third instar stage, resulting in reduced expression of Ato and arrest of furrow progression. The expression of Da was downregulated in hh¹ mutant eye discs. LOF clones of smoothened (smo), a crucial component for Hh signal transduction, were generated. Da expression was significantly reduced in smo mutant clones spanning the furrow, suggesting that Hh signalling is required for the expression of Da. However, the expression of Da was not completely eliminated in hh¹ mutant eye discs or in smo LOF clones. As Dpp signalling is partly required for the expression of Ato, whether Dpp signalling is also necessary for the expression of Da was tested by analysing LOF clones of mad (mothers against dpp), an essential factor for Dpp signalling transduction. Da expression showed little reduction in mad mutant clones, indicating that Dpp signalling by itself is not essential for Da expression. By contrast, the expression of Da was almost completely abolished in LOF clones of smo and mad double-mutant cells in the furrow region. Thus, the Hh and Dpp signalling pathways are crucial but partly redundant for the expression of Da. It was also found that loss of function of Ato reduced the level of Da expression in the furrow. Therefore, several factors, including Ato, coordinate the accumulation of Da in the furrow (Lim, 2008).

To test whether the upregulation of Da in the furrow has a function in neurogenesis, da³ LOF clones were generated and the effects of da mutation on the expression of Ato and neuronal differentiation were examined. Loss of da resulted in ectopic expansion of Ato expression in the mutant clone, suggesting that Da is crucial for repressing the expression of Ato (Lim, 2008).

Despite ectopic expression of Ato, most of the cells in da LOF mutant clones could not differentiate into photoreceptor cells, as indicated by the lack of neuronal markers such as Senseless (R8 marker) and Elav (pan-neural marker). Hence, the expression of ectopic Ato is insufficient to induce retinal differentiation in the absence of Da. However, local differentiation was occasionally detected near the posterior end of some clones. This might be due to the perdurance of Da in LOF clones, although other possibilities, such as partial non-autonomy or partial independence of photoreceptor differentiation from Da in the posterior region of the eye disc, cannot be excluded (Lim, 2008).

To support the idea that a high level of Da expression is required for the repression of Ato, a temperature-sensitive allele of da (da^ts) was examined that causes conditional partial loss of function of Da at the restrictive temperature. In da^ts mutant eye discs, Ato was expressed in several cells rather than a single R8 cell per proneural cluster. In addition, the effects of conditional expression of Da was tested by temperature shifts of heat-shock (hs)-da flies. Ato was repressed by the overexpression of Da after a longer heat shock but not after a shorter heat shock. These observations support the idea that enriched Da expression in the cells surrounding each R8 cell is required for generating a single R8 cell by the inhibition of Ato expression (Lim, 2008).

The expanded expression of Ato in da mutant clones might, in part, be due to the failure of da mutant cells to induce lateral inhibition of Ato expression. It is also possible that Da might be involved in the cell-autonomous repression of Ato expression. To test this possibility, Da was overexpressed in the dorsoventral margin of the eye disc using the optomotor blind (omb)-Gal4 driver. The overexpression of Da downregulated Ato expression in the expression domain of omb. Furthermore, the overexpression of Da in the antenna disc using the dpp-Gal4 driver resulted in Ato repression in the expression domain of dpp. Taken together, these data from LOF and overexpression analyses suggest that the high-level expression of Da is necessary and sufficient for the cell-autonomous repression of Ato during the selection of R8 (Lim, 2008).

Both Da and Notch (N) are essential for the selection of R8 by repressing Ato expression in non-R8 precursors within proneural clusters. Hence, Da might be involved in N-dependent lateral inhibition. Furthermore, the overexpression of ASC proneural factors, together with Da, can synergize with Suppressor of hairless and N to activate the expression of Enhancer-of-split (E(spl)) in cultured cells. Since E(spl) is expressed complementary to the expression of Ato in the same cells expressing a high level of Da, whether Da alone could regulate the expression of E(spl) was tested in vivo. The expression of E(spl) proteins was reduced in da³ mutant cells, showing that Da is required for the expression of E(spl) in vivo. Furthermore, the overexpression of Da with dpp-Gal4 could induce the expression of ectopic E(spl) in the dpp domain of the antenna disc. These results indicate that a high level of Da expression is necessary and sufficient for the activation of E(spl) expression (Lim, 2008).

Since E(spl) is the main mediator of N signalling, Ato repression by a high level of Da might be dependent on the expression of E(spl). To test this possibility, the MARCM method was used to generate E(spl) LOF clones in which the expression of Da is induced by tubulin (tub)-Gal4. Da overexpression in E(spl) LOF clones did not show a significant repression of Ato. Similarly, overexpression of E(spl)mδ in da LOF clones did not show noticeable repression of Ato. These data suggest that both Da and E(spl) are required for positive feedback regulation and for repression of Ato during lateral inhibition. However, it is also possible that other bHLH family genes of the E(spl) complex loci might be required, or that the overexpression of E(spl) or Da by tub-Gal4 in MARCM assays might not be strong enough to repress the expression of ato. By contrast, Da expression by dpp-Gal4 induces the expression of E(spl), even in the proximal sector of the antenna disc where Ato is not expressed. amos, the proneural gene for olfactory sensilla, is not expressed in the antenna disc at this time. Thus, a high level of Da can induce E(spl) in the absence of Ato, although Da might act with other class II proteins to promote the expression of E(spl) (Lim, 2008).

Since N signalling is activated in the same cells surrounding R8 founder neurons, whether Da expression is affected was examined by removing the function of N using a temperature-sensitive allele, N^ts. The loss of function of N at the restrictive temperature resulted in several Ato-positive cells per proneural cluster. Furthermore, the transient loss of N activity abolished the high-level of Da expression between the proneural clusters but did not eliminate the basal level of Da expression in the same cells. This suggests that N signalling is essential for the high-level upregulation of Da expression. Since the expression of da is regulated by Hh and Dpp signalling, as well as Ato, it is possible that the regulation of Da by Hh and Dpp might be mediated by Ato-dependent N signalling in the non-R8 precursor cells (Lim, 2008).

To investigate further the role of N signalling in the expression of Da, whether E(spl) proteins mediate the function of N in inducing a high level of Da expression was examined. Loss of E(spl) caused ectopic expression of Ato in E(spl) mutant clones because of the lack of N-mediated lateral inhibition. Interestingly, the high level of Da expression was suppressed, but the basal level of Da expression was still detected in E(spl) mutant clones, as seen in N^ts mutant eye discs. Thus, E(spl) is required for the high level but not for the basal level of Da expression. In contrast to da³ LOF mutant cells that fail to differentiate in spite of ectopic Ato expression, E(spl) LOF mutant cells not only expressed ectopic Ato but also differentiated into ectopic photoreceptors. Thus, the basal level of Da expression remaining in E(spl) LOF clones is sufficient for the formation of a functional complex with Ato to induce neural differentiation (Lim, 2008).

On the basis of the above observations, a model is proposed in which Da has dual functions as a proneural and as an anti-proneural factor depending on the expression level during early retinal neurogenesis . The anti-proneural function of Da proposed in this model provides an explanation for the abnormal upregulation of Ato in da mutant cells in the furrow, although the LOF experiments are also consistent with the pre-existing view that Da promotes the function of Ato. In Ato-positive neural precursors, low levels of Da expression are sufficient to form heterodimers with Ato to function as a proneural factor. In neighbouring cells, the N-E(spl) pathway further upregulates the expression of Da, which, in turn, induces more expression of E(spl). This putative feedback regulation might provide a mechanism for more effective lateral inhibition of Ato expression for the selection of R8. Interestingly, Da can form a homodimer and bind to DNA in vitro. Thus, in Ato-negative cells surrounding the R8 precursors, a high level of Da expression might enforce the formation of Da homodimers and/or heterodimers with other unknown bHLH proteins to repress the expression of ato. It would be interesting to see whether mammalian type I bHLH proteins such as E proteins might also be specifically regulated to have distinct developmental functions as seen in the case of Da (Lim, 2008).

Regulation of Hh signal transduction as Drosophila eye differentiation progresses

Differentiation of the Drosophila retina occurs as a morphogenetic furrow sweeps anteriorly across the eye imaginal disc, driven by Hedgehog secretion from photoreceptor precursors differentiating behind the furrow. A BTB protein, Roadkill, is expressed posterior to the furrow and targets the Hedgehog signal transduction component Cubitus interruptus for degradation by Cullin-3 and the proteosome. Clonal analysis and conditional mutant studies establish that roadkill transcription is activated by the EGF receptor and Ras pathway in most differentiating retinal cells, and by both EGF receptor/Ras and by Hedgehog signaling in cells that remain unspecified. These findings outline a circuit by which Hedgehog signal transduction is modified as Hedgehog signaling initiates retinal differentiation. A model is presented for regulation of the Cullin-3 and Cullin-1 pathways that modifies Hedgehog signaling as the morphogenetic furrow moves and the responses of retinal cells change (Baker, 2009).

As the morphogenetic furrow crosses the eye disc, Ci155 accumulates most highly just anterior to the morphogenetic furrow, even though Hh is secreted posterior to the morphogenetic furrow. The sharp reduction in Ci155 as the furrow passes is associated with a switch from Cul1-dependent processing to Cul3-dependent degradation (Ou, 2002). The posterior eye expresses rdx, encoding a BTB protein that couples Ci 155 to the Cul3 pathway (Kent, 2006; Zhang, 2006). This study identified the signals that induce rdx and that process Ci155 in the posterior eye (Baker, 2009).

The induction of rdx transcription couples Ci155 processing to Cul3 (Kent, 2006; Zhang, 2006). rdx transcription is regulated by both Hh signaling and Ras signaling, and there were distinctions between cell types. The smo mosaic and hh^ts2 experiments show that Hh signaling is continuously required for rdx transcription in unspecified cells with basal nuclei. In the absence of smo, EGFR-dependent rdx transcription occurs in differentiating photoreceptor cells only, not in unspecified cells. The egfr mosaics show that EGFR is essential for rdx transcription in all cells except the R8 photoreceptor class. Thus, EGFR-dependent differentiation was sufficient to induce rdx in photoreceptors even without Hh signaling, but Hh was not sufficient to induce rdx anywhere without EGFR signaling, except for the R8 cells. Undifferentiated cells might require both the Ras and Hh signaling pathways to induce rdx because the level of Ras signaling is lower in unspecified cells than in differentiating cells of the ommatidia. Alternatively, there may be a combinatorial requirement for both pathways in unspecified cells (Baker, 2009).

There has been some discussion of whether proteolysis of Ci155 by Cul-3 is regulated directly by Hh, as is Cul-1 dependent Ci processing. The current studies provide no support for this idea. In all the genotypes examined, Ci proteolysis correlates with the expression of rdx, and the simplest explanation is that the only effect of Hh on the Cul-3 pathway is through rdx transcription, directly in unspecified cells, and indirectly via EGFR-mediated differentiation in most specified cells (Baker, 2009).

Two mechanisms, acting in different cells, appear to reduce Hh responses through Ci155 after the furrow passes. One also occurs in wing development, where rdx is transcribed only by cells experiencing high Hh signaling levels close to the source of Hh. In wing development, rdx and the Cul3-pathway modulate the amount of Ci155 available for Cul1-dependent processing, lowering the maximum level of Ci155 activity at high Hh levels. Rdx could lower Ci155 levels in unspecified eye cells posterior to the furrow by this mechanism, in which an equilibrium between Hh-dependent induction of rdx, and rdx- and Cul3-dependent degradation of Ci155, leads to a lower level of Ci155 protein than anterior to the furrow. By contrast, in the specified, differentiating eye cells, rdx transcription becomes independent of Hh signaling, and Ci155 is degraded more completely (Baker, 2009).

If there is Hh signaling posterior to the furrow, as these studies find maintains rdx transcription in unspecified retinal cells, why are genes such as atonal that are induced by Hh signaling ahead of the furrow not also expressed posterior to the furrow? There are at least three possible explanations. First, rdx may dampen Ci155 accumulation in unspecified cells such that the threshold necessary for ato expression is not achieved posterior to the furrow. This is unlikely to be the sole explanation, since mutating rdx or cul3 permits Ci155 accumulation but does not lead to ectopic R8 specification, but it could contribute in conjunction with other mechanisms. Secondly, other genes may interfere posterior to the furrow. This could include egfr induction of Bar gene expression, since Bar genes antagonize ato expression. There seem to be multiple respects in which EGFR-dependent differentiation renders cells unable to continue anterior responses to Hh, and it is also envisaged that egfr might play a role in further mechanisms that modulate the response to Dpp signaling posterior to the furrow, should such mechanisms exist. Finally, recent evidence suggests that induction of ato by Hh is not so simple as the induction of a target gene above a threshold in a morphogen gradient, but depends indirectly on Hh repressing Eyeless and activating Sine Oculis, so that these transcription factors are coexpressed and turn on ato only in a domain ahead of the furrow. In this case, persistent Hh signaling would not be expected to activate ato expression once Ey had been repressed (Baker, 2009).

Recently, Hh has been discovered to induce compensatory proliferation in response to eye disc cell death, a further example of post-furrow Hh function. The current results now suggest the model that loss of EGFR-dependent rdx expression elevates Ci155 locally to permit Hh responses when photoreceptor cells that secrete EGFR ligands are lost. Consistent with this idea, loss of rdx or cul3 also result in proliferation of eye disc cells (Baker, 2009).

The regulation of rdx expression and thus degradation of Ci by Cullin-3 may not be sufficient to explain Ci regulation posterior to the furrow. In order for Ci155 to be stable, as observed in cul3 mutant clones and egfr mutant clones, Ci155 must escape processing to Ci75 by Cul-1. Ahead of the furrow, and in most other tissues, rdx is not expressed, Ci is not coupled to Cul3, and Ci155 is stabilized wherever Hh inhibits Smo and the Cul1 pathway. The observation that Ci155 is stable in cul3 clones, or in the genotypes where rdx is not expressed, shows that Ci155 escapes processing by the Cul1 pathway in the posterior eye as well, but this is not due to Hh. Ci155 accumulates in smo egfr mutant clones that do not express rdx and cannot respond to Hh (Baker, 2009).

One model would be that once rdx is induced, Ci155 is sequestered and not available to be processed by Cul1. This model cannot explain why Ci155 accumulates in egfr clones that lack rdx expression, where Ci155 should be available for Cul1. Therefore Ci155 must escape Cul1-mediated processing in the posterior eye by a distinct mechanism. This could be explained by the induction of a component distinct from Rdx that inhibits the processing of Ci155 by Cul1, or sequesters Ci155. It is equally possible that a component essential for processing of Ci155 by Cul1 is repressed posterior to the morphogenetic furrow (Baker, 2009).

Previous studies show that Ci155 never accumulates in smo tkv clones that are unable to respond to either Hh or Dpp signaling. Clones of cells unable to respond to Dpp, but able to respond to Hh and Ras, show only a subtle change in Ci155 labeling. These previously published observations suggest that Ci155 remains a target of Cul1 in the absence of both Dpp and Hh signaling, perhaps through failure to transcribe or repress transcription of a gene that modulates Ci155 proteolysis by Cul1 posterior to the furrow (Baker, 2009).

It is now possible to account for why smo clones affect Ci155 levels differently from cul3 clones, a previously puzzling observation. In cul3 clones, or egfr clones that do not express rdx, the Cul3 pathway cannot degrade Ci155 and the Cul1 pathway is inactivated posterior to the furrow exactly as in wild type discs, so Ci155 accumulates. In smo clones, Ci155 transiently accumulates in those cells in which processing by Cul1 has been lost but rdx not yet induced. In such cells, Ci155 is not coupled to any cullin, and is stable. Eventually, differentiation spreads into the posterior of smo clones, leading to rdx expression, and Cul3-dependent Ci degradation. If differentiation and rdx expression are prevented, as in smo egfr clones, then Ci155 remains stable. Because there is a delay in expressing rdx in smo clones compared to wildtype, Ci155 is not subject to Cul3-mediated processing as soon as in wild type, and there is a period when Ci155 has been uncoupled from Cul1-processing but not yet coupled to the Cul3 pathway. It is during this period that Ci155 accumulates in smo mutant cells (Baker, 2009).

These findings help explain how a wave of differentiation moves across the eye disc uni-directionally. Hh, secreted from differentiating photoreceptor cells, must be present at highest concentrations posterior to the furrow. Indeed, ahead of the furrow Ci155 is stabilized in a decreasing posterior-to-anterior gradient, consistent with a gradient of Hh protein coming from a source posterior to the furrow. Yet, the cell-autonomous responses to Hh signaling that are seen ahead of the furrow, such as cell cycle arrest and atonal expression, do not occur posterior to the furrow, where Ci is rendered unstable by Rdx and Cul3, induced both directly by Hh itself, and indirectly by the photoreceptor differentiation that is largely induced by EGFR posterior to the furrow (Baker, 2009).

There are other examples where Hh-secreting tissues are not the targets of Hh signaling. For, example, in Drosophila wing development, anterior compartments respond to Hh secreted by posterior compartments, but posterior compartment cells do not respond because ci transcription is repressed by the posterior-specific protein Engrailed. In vertebrate development, notochord cells express Shh but the responses seen in the nearby spinal cord are not seen in notochord. Such segregation of Hh-producing cells from fields competent to respond to Hh makes sense, if the purpose of Hh signaling in development is to pattern new body regions. Hh signaling is also deregulated in many tumors. Whether any of these tumors activate Hh signaling by affecting GLI protein stability, or other normal down-regulatory mechanisms, remains to be seen. In any case, mechanisms that render cells unresponsive to Hh by coupling Ci155 to the proteosome might prove useful in the treatment of cancers that depend on Hh signaling (Baker, 2009).

Branchless and Hedgehog operate in a positive feedback loop to regulate the initiation of neuroblast division in the Drosophila larval brain

The Drosophila central nervous system is produced by two rounds of neurogenesis: one during embryogenesis to form the larval brain and one during larval stages to form the adult central nervous system. Neurogenesis caused by the activation of neural stem division in the larval brain is essential for the proper patterning and functionality of the adult central nervous system. Initiation of neuroblast proliferation requires signaling by the Fibroblast Growth Factor homolog Branchless and by the Hedgehog growth factor. The Branchless and Hedgehog pathways form a positive feedback loop to regulate the onset of neuroblast division. This feedback loop is initiated during embryogenesis. Genetic and molecular studies demonstrate that the absolute level of Branchless and Hedgehog signaling is critical to fully activate stem cell division. Furthermore, over-expression and mutant studies establish that signaling by Branchless is the crucial output of the feedback loop that stimulates neuroblast division and that Branchless signaling is necessary for initiating the division of all mitotically regulated neuroblasts in the brain lobes. These studies establish the molecular mechanism through which Branchless and Hedgehog signaling interface to regulate the activation of neural stem cell division (Barretta, 2008).

These studies have demonstrated that Hh and Bnl act in a positive feedback loop in the larval brain to control the onset of neuroblast proliferation. The feedback loop acts at the transcriptional level, such that Hh signaling activity is essential to control the level of bnl expression and vice versa. Double mutant analyses showed that an absolute level of signaling by both Bnl and Hh are required to maintain normal neuroblast activation, rather than other possible models that would suggest a certain balance of signaling activity (for example more Bnl than Hh) is sufficient regardless of the exact magnitude of signaling activity. The discovery that Bnl signaling is the critical output of the feedback loop suggests that the main function of Hh signaling is to maintain the proper level of Bnl production and signaling. Furthermore, the observation that only the mushroom body and ventral lateral neuroblasts continue to divide in bnl null mutants regardless of the level of Hh signaling indicates that all the regulated neuroblasts, both optic lobe and central brain sets, require the input of the Bnl pathway to enter S phase. Thus the Hh-Bnl feedback loop appears to control cell cycle progression in all the mitotically arrested neuroblasts that begin cell division in first instar (Barretta, 2008).

Other developmental events that require Hedgehog and FGF signaling have used those pathways in different manners to achieve their goals. For example, in the mouse ventral telencephalon, Hedgehog and FGF/MAPK signaling operate as two independent pathways. FGF signaling is independent of Sonic Hedgehog (SHH) and does not affect expression of either SHH itself or its target gene and effector GLI1. Other systems have shown a linear dependence of FGF expression on SHH signaling and vice versa. During budding morphogenesis in the mouse lung Hedgehog signaling inhibits expression of FGF10 but up-regulates FGF7. In the Xenopus eye, expression of Banded Hedgehog increases expression of FGF8. In the zebrafish forebrain inhibition of Hh signaling decreases expression of FGF3, FGF8 and FGF19. Hedgehog also regulates FGF expression in the zebrafish mid/hindbrain. However, in the zebrafish forebrain HH expression requires FGF signaling. Inhibition of both FGF3 and FGF8 expression resulted in a downregulation of SHH. Alternatively, the HH and FGF pathways can integrate at the level of intracellular components. FGF has been shown to induce expression of GLI2, a transcription factor and HH signaling effector in ventroposterior development in zebrafish (Barretta, 2008).

Of course the classic example of FGF and SHH interplay is the development of the chick limb bud. In this system, several FGFs set up a signaling center at the tip of the bud that turns on expression of SHH in the posterior limb mesenchyme. In turn, SHH signaling is required for maintenance of FGF4, FGF9 and FGF17 expression in the bud tip. This function of SHH occurs through the expression of Gremlin, an inhibitor of Bone Morphogenetic Protein signaling. Gremlin inhibition of Bone Morphogenetic Protein signaling prevents down-regulation of the FGFs. Thus a positive feedback loop exists between SHH and FGFs, mediated by Gremlin (Barretta, 2008).

The model of the Hh-Bnl feedback loop proposed in this study is most similar to the classic SHH-FGF feedback loop described in the vertebrate limb bud. In is not yet known whether the regulation of bnl expression by Hh signaling is direct or if it is mediated by another signaling pathway such as the Gremlin/Bone Morphogenetic Protein connection that operates in the limb bud. However, like the distinct domains of FGF and SHH in the limb bud, bnl and hh expression also occur in distinct regions of the brain lobe. The fact that the Hh-Bnl feedback loop is activated during embryogenesis, but that the first regulated neuroblasts do not enter S phase until 8-10 h after larval hatching also suggests that additional events must take place downstream of Bnl signaling to permit mitotically arrested stem cells to transit through G1 to S phase. One such possibility is exposure to the steroid hormone ecdysone, which is necessary during first larval instar for the initiation of neuroblast division a few hours later. Both SHH and FGF2 have been shown to be necessary for the division of different subsets of neural stem cells in many different vertebrate and mammalian models and in multiple contexts. This is the first time that the interactions between these two pathways necessary to stimulate the reactivation of stem cell division in quiescent neural stem cells have been elucidated. The next challenge will be to determine whether different molecular mechanisms tying these two signaling pathways are used for different developmental decisions such as progeny cell fate, initiation of cell division and maintenance of stem cell identity (Barretta, 2008).

Hedgehog signaling acts with the temporal cascade to promote neuroblast cell cycle exit

In postembryonic neuroblasts, transition in gene expression programs of a cascade of transcription factors (also known as the temporal series) acts together with the asymmetric division machinery to generate diverse neurons with distinct identities and regulate the end of neuroblast proliferation. However, the underlying mechanism of how this 'temporal series' acts during development remains unclear. This study shows that Hh signaling in the postembryonic brain is temporally regulated; excess (earlier onset of) Hh signaling causes premature neuroblast cell cycle exit and under-proliferation, whereas loss of Hh signaling causes delayed cell cycle exit and excess proliferation. Moreover, the Hh pathway functions downstream of Castor but upstream of Grainyhead, two components of the temporal series, to schedule neuroblast cell cycle exit. Interestingly, Hh is likely a target of Castor. Hence, Hh signaling provides a link between the temporal series and the asymmetric division machinery in scheduling the end of neurogenesis (Chai, 2013).

This study shows that Hh signaling functions during later postembryonic development and acts together with the NB temporal transcription factor cascade to regulate NB cell cycle exit. It was further demonstrated that hh is a downstream target of Cas, a member of temporal series that determines the time at which NBs terminate proliferation via down-regulation of Grh. While increased Hh signaling results in increased cell cycle length and premature NB cell cycle exit, loss of Hh signaling decreases NB cell cycle length and also prolongs the duration of NB proliferation (Chai, 2013).

Hh family proteins can act as short- or long-range morphogens covering distances as few as ten cell diameters (20 µm), or as far as a field containing many more cell diameters (200 µm). In the postembryonic brain, hh is expressed predominantly in the NBs and the newborn GMCs, whereas the expression of its target gene reporter, ptc-lacZ is observed in a narrow area covering the adjacent NB and the sibling GMCs, indicating a limited response to and suggesting a limited spread of Hh ligand. In addition, Hh protein is always found to be enriched and clustering around the NBs in a punctuated form rather than forming a gradient. These data, together with the lineage autonomous phenotype of hh mutant NB clones, strongly suggest that Hh acts locally at short range in the larval brain. This is consistent with the structural arrangement of the larval brain, where each NB lineage comprising of the NB itself, GMCs, and neurons, is encapsulated by a meshwork of glial processes that form a three-dimensional scaffold that potentially acts as a stem cell niche. Such a spatial arrangement may serve as a barrier to restrict spread of the ligand and confine signaling events within a particular lineage so that an individual NB lineage can development with considerable independence from its neighbouring lineages. Indeed, a NB clone derived from a hh null allele exhibits the GMC pool expansion phenotype even though GMCs from its neighbouring lineages are competent in producing Hh ligand (Chai, 2013).

While it is tempting to assume that Hh can also act on the GMCs in an autocrine mode of action judging from the presence of ptc-lacZ expression, no noticeable GMC fate transformation or change in their proliferative capability was seen in ptc^S2 and smo^IA3 clones. The higher mitotic rate in hh loss-of-function NBs could largely explain the amplification of the GMC pool and enlarged clone-size; however, a possible delay in GMC differentiation cannot be ruled out. The proposition that Hh ligand, which is produced by the NB and daughter GMCs, feeds back on the NB to control its own proliferative capacity and the timing of cell cycle exit is interesting but not totally unfamiliar. Similar feedback signalling mechanism has been demonstrated in the mouse brain in which post-mitotic neurons signal back to the progenitor to control cell fate decisions, as well as the number of neurons and glia produced during corticogenesis (Chai, 2013).

Hh signal reception is detectable in NBs as early as in L2 and persists throughout larval life and in early pupae when NBs undergo Pros-dependent cell cycle exit. This delay of approximately 96 h between the start of Hh reception and the ultimate outcome of cell cycle exit may be due to a requirement for cumulative exposure of NBs to increasing local concentrations of Hh. Such a graded response will enable the wt postembryonic NBs to progress from high to low proliferative stages before ceasing division, in line with the development of the larva. Evidence supporting this notion includes gradual accumulation of Hh on the NBs, lengthening of NB cell cycle time, as well as the necessity of high levels of Hh signaling to trigger cell cycle exit. It is worthwhile to note that even at pre-pupal stage during which most NBs are starting to undergo cell cycle exit, fewer than 20% of them are associated with Hh puncta at any point of time. One likely explanation is that not all the NB lineages within the larval central brain respond synchronously to Hh-mediated temporal transition. However, unlike the embryonic central nervous system in which hh expression is localized to rows 6-7 of the neuroectoderm, this study found it difficult to pinpoint a specific expression pattern in the postembryonic central brain due to the disorganized array of NB lineages. It is equally possible that different NBs exit cell cycle progression at different time points. This is also consistent with the structural organization of individual NB into different 'trophospongium' or stem cell niches (Hoyle, 1986). Nevertheless, the possiblility cannot be ruled out that Hh signal activation primes another yet-to-be-identified developmentally regulated signal/event to schedule NB cell cycle exit (Chai, 2013).

Interestingly, a recently proposed 'cell cycle length hypothesis' postulates that cell cycle length, particularly the length of G1 phase in neural stem cells acts as a switch to trigger the transition from proliferative to neurogenesis mode (Salomoni, 2010). In fact, experiments have shown that manipulation of cdk4/cyclinD1 expression and cdk2/cyclinE activity that result in the lengthening of G1 is sufficient to induce precocious neurogenesis; while inhibition of physiological lengthening of G1 delays neurogenesis and promotes expansion of intermediate progenitors. The curren results show that Drosophila postembryonic NBs in the central brain exhibit a comparable trend of cell cycle lengthening from young to old larval stages. Interestingly, NBs with excess Hh signaling have an extended cell cycle time, consistent with the idea that there is a forward shift of the 'perceived' age, leading to premature cell cycle exit. In contrast, Hh loss-of-function NBs have a shorter cell cycle time compared to their wt counterparts of the same actual age; hence, they have a younger 'perceived' age and are able to maintain their proliferative phase over a longer period of time. Consistent with this, it was shown that persistent NB proliferation in smo^IA3 clones as well as the early termination of ptc^S2 NBs proliferation, are always associated with the presence and absence of CycE expression, respectively. However, loss of Hh signaling in NBs merely extends their proliferative phase but is not sufficient to ensure perpetual proliferation as no mitotic NB is observed in the adult brain. It is also noted that a previous report suggested that the cell cycle time of the larval NBs reduced during their growth and reached a peak at late third instar with a minimum cell cycle time of 55 min. However, this study was conducted on thoracic NBs from the neuromeres T1 to T3, which have a very distinctive proliferative profile to the central brain NBs assayed in the current study. Indeed, has been shown in that abdominal NBs exhibit significantly different cell cycle times compared to their thoracic counterparts (Chai, 2013).

In Drosophila, the precise timing of NB cell cycle exit is governed by a highly regulated process that involves sequential expression of a series of transcription factors: Hb->Kr->Pdm1->Cas, known as the temporal series. It is known that the temporal series probably utilizes Grh in the postembryonic NBs to regulate Pros localization or apoptotic gene activity, thus determining the time at which proliferation ends. In addition, the temporal series also regulates postembryonic Chinmo->Br-C neuronal switch, which specifies the size and the identity of the neurons. The current data show that Hh signaling does not regulate early to late neuronal transition as Chinmo and Br-C expression timings appear unaffected in both ptc and smo mutant clones. In contrast, excess Hh signaling leads to a variety of features associated with NB cell cycle exit: (1) premature down-regulation of Grh, (2) nuclear localization of Pros (in NBs), and (3) reduction of NB size. Taken together with the extended proliferative duration of Hh loss-of-function NBs, it is apparent that Hh signaling is a potent effector of the temporal series and functions late to promote NB cell cycle exit (Chai, 2013).

The results from the current genetic interaction assays with Hh pathway components and grh reaffirmed the conclusions from previous studies that Grh is necessary to maintain the mitotic activity of the postembryonic NBs. The loss of Hh signaling keeps the central brain type I NBs in their proliferative state and this is largely contributed by persistent grh expression past their normal developmental timing at around 24 h APF. Even though Grh is necessary to extend the proliferative phase of these NBs, it is not sufficient to rescue all aspects of the premature cell cycle exit phenotype seen in ptc mutant NBs. Hence, down-regulation of grh by over-activating Hh signaling is not solely responsible for NB proliferative defects, and this implies that Hh signaling may terminate NB cell cycle via other mechanisms in addition to Grh (Chai, 2013).

The expression of hh appears to be dependent on the pulse of Cas expression at the transition between L1 and L2, as induction of cas mutant clones after that stage does not significantly affect hh expression. Moreover, ChIP assays suggest that Cas binds the hh genomic region, thereby placing Hh as a direct downstream target of the temporal series. However, it is intriguing to speculate on how the early pulse of Cas can mediate hh expression, which only comes on later during larval development. One possible explanation involves a relay mechanism in which that pulse of Cas activates an (or a cascade of) unknown components, which persist and eventually turns on the later hh expression. Yet, in such a model, Cas need not interact directly with the hh locus as the ChIP assay clearly suggests. Moreover, there are at least two pulses of hh expression during larval brain development, and the earlier, shorter pulse that is required for the activation of quiescent NBs appear to be independent from Cas regulation as Cas is only switched on in the larval NBs upon reactivation. Most importantly, the data show that mis-expression of cas abolishes, rather than triggers ectopic hh expression. Thus, the findings do not favour the continuous expression of a hh activator downstream of Cas. Alternatively, Cas may be involve in the epigenetic modifications of the hh locus such that it is primed for expression at a much later stage. This may also explain why saturating the system with Cas for prolonged period of time via mis-expression can negatively affect subsequent hh expression because of to its potential aberrant association with the chromatin. Although such a function has not been reported for Cas, previous studies have postulated that components of the temporal series, such as Hb (or mammalian homolog Ikaros) and Svp (or mammalian homolog COUP-TFI/II), play a role in modulating chromatin structure, hence modifying the competency of downstream gene expression subsequently (Chai, 2013).

The relationship between svp and Hh signaling within the postembryonic temporal series cascade is interesting yet unexpected. svp was thought to be a downstream component of cas on the basis of studies in postembryonic NBs in the thoracic segment of the ventral nerve. This is supported by the observations that the pulse Svp occurs at 40-60 h ALH following the pulse of Cas at 30-50 h ALH. Moreover, both svp and cas mutant clones affect Chinmo/Br-C neuronal target transition, apart from causing NBs' failure to exit the cell cycle at early pupal stage. However, examinations of Svp and Cas expression patterns in the central brain region in this study reveal that the Cas expression window overlaps with the peak of the Svp expression window, even though the latter has a much wider expression window in which low expression levels can still be detected in the NBs at 96 h ALH. Moreover, the data show that abolishment of cas function starting from the embryonic stage does not reduce Svp expression in the NBs at 24 h ALH. Hence, previous interpretation that svp functions downstream of cas in the thoracic postembryonic NBs may not be easily extrapolated to NBs in other brain regions. On the basis of the current results, it is tempting to postulate that Cas and Svp constitute two parallel pathways within the temporal series and Hh signaling is regulated by Cas but not Svp. Nevertheless, such a hypothesis warrants more in depth studies (Chai, 2013).

The precise generation of diverse cell types with distinct function from a single progenitor is important for the formation of a functional nervous system during animal development. It has been shown that, in Drosophila, the developmental timing mechanism (the temporal series) is tightly coupled with the asymmetric machinery. However, the underlying mechanism of this coordination remains elusive. The current data suggest that on the one hand, Hh signaling is under the control of the temporal series (hh expression is directly regulated by Cas), while on the other hand, Hh signaling participates in asymmetric segregation of Mira/Pros during NB division. Introduction of ectopic/premature Hh signaling (in ptc mutant clones) during developmental stages in which NBs are proliferating results in cytoplasmic localization of Mira/Pros during mitosis, reduction of NB size, and slow-down of NB cell cycle progression, reminiscent of the final division of NBs in early pupa just before cessation of proliferation. Consequently, these NBs exit the cell cycle prematurely. It is speculated that Pros may be a direct or indirect target of Hh signaling as elevated pathway activity invariantly leads to increased pros expression in the NBs. Furthermore, reducing the level of Pros protein by removing one copy of function pros is able to rescue the Mira delocalization phenotype seen in ptc mutant NBs. Thus, it is plausible that Hh signaling impinges on the asymmetric division apparatus, likely through Pros, to diminish NB fate gradually (as seen with the absence of Dpn and Mira delocalization) prior to the final cell cycle exit. Despite the results indicating a tight correlation between nuclear entry of Pros into the NBs and the eventual cell cycle exit of these NBs during pupal stage, it should be considered that Pros may not be the direct causative agent in controlling NB cell cycle exit. Therefore the actual role of Pros in this process is purely speculative as far as this study is concerned (Chai, 2013).

In contrast, loss of Hh signaling (e.g., in Smo mutant clones) maintains NBs in their 'younger' proliferating stage far beyond the time when they normally exit the cell cycle. Thus, Hh signaling couples the developmental timing mechanism (the temporal series) with the NB intrinsic asymmetric machinery for the generation of a functional nervous system (Chai, 2013).

In vertebrates, constitutive activation of the Sonic hedgehog (SHH, a homologue of Drosophila Hh), signaling pathway through inactivation mutations in PTCH1, activating mutations in SMO, as well as other mutations involving SHH, IHH, GLI1, GLI2, GLI3, and SUFU, has been implicated in a vast array of malignancies. The proven association of Hh signaling pathway with tumourigenesis and tumour cell growth fuel the view that Hh constitutes a mitogenic signal that promotes pro-proliferative responses of the target cells. Moreover, Hh acts as a stem cell factor in somatic stem cells in the Drosophila ovary, human hematopoietic stem cells, and mouse embryonic stem cells, possibly by exerting its effects on the cell cycle machinery (Chai, 2013).

This report provides an opposing facet of Hh signaling where it is required for timely NB cell cycle exit in the postembryonic pupal brain. This may sound astonishing, but the essential roles of Hh signaling as a negative regulator of the cell cycle has been eclipsed by the common bias that it stimulates proliferation, given the many examples of malignancies with the Hh pathway dysregulation. Indeed, studies have indicated that cell cycle exit and differentiation of a number of cell types, such as absorptive colonocytes of the mammalian gut, zebrafish, and Drosophila retina, require Hh activities. SHH signaling pathway is highly activated in human embryonic stem cell (hESC) and such activity is crucial for hESC differentiation as embryoid bodies. The opposing functions of Hh signaling pathway in different cell types reveal that the ultimate effect of this pathway is likely to be tissue specific, depending on its interaction with other regulatory pathways. The current data indicate that in Drosophila postembryonic NBs of the brain this does indeed appear to be the case, because in this system, Hh signaling pathway interacts with NB-specific temporal series and likely the asymmetric cell division machinery to promote pros nuclear localization to trigger cell cycle exit (Chai, 2013).

Hedgehog signaling and the development of the genital disc

In both sexes, the Drosophila genital disc comprises three segmental primordia: the female genital primordium derived from segment A8, the male genital primordium derived from segment A9 and the anal primordium derived from segments A10-11. Each segmental primordium has an anterior (A) and a posterior (P) compartment, the P cells of the three segments being contiguous at the lateral edges of the disc. Hedgehog (Hh) expressed in the P compartment differentially signals A cells at the AP compartment border and A cells at the segmental border. As in the wing imaginal disc, cell lineage restriction of the AP compartment border is defined by Hh signalling. There is also a lineage restriction barrier at the segmental borders, even though the P compartment cells of the three segments converge in the lateral areas of the disc. Lineage restriction between segments A9 and A10-11 depends on factors other than the Hh, En and Hox genes. The segmental borders, however, can be permeable to some morphogenetic signals. Furthermore, cell ablation experiments show that the presence of all primordia (either the anal or the genital primordium) during development are required for normal development of genital disc. Collectively, these findings suggest that interaction between segmental primordia is required for the normal development of the genital disc (Gorfinkiel, 2003).

The three segmental primordia of the genital disc are contiguous. This means that the P compartment of one primordium is adjacent to A cells of the corresponding primordium and to A cells of the following primordium. In addition, the P compartment cells of the three segments converge in lateral areas of the genital disc. Hh activates target genes in the receiving cells both behind and in front. These target genes are different on each side of its expression domain. Particularly, Hh at the posterior compartment of the male genital primordium (A9 segment) signals anteriorly, inducing Wg and/or Dpp expression in anterior cells of this primordium, and posteriorly, inducing Ptc expression. Hh also posteriorly signals anterior cells of the anal primordium (segments A10-11), inducing En expression in a narrow band of cells. Interestingly, Cad expression is reduced in these cells. A similar situation has been described in embryonic segments in which Hh activates wg at the AP border and rhomboid at the segmental border. Hh controls Wg and EGF signalling pathways on each side of its expression domain in embryos (Gorfinkiel, 2003).

Hh has a pivotal role in the morphogenesis of all imaginal discs. Ectopic Hh gives rise to duplications of parts of, or whole, appendages in the imaginal discs of the fly. In wild-type genital discs, such as in the leg and antenna, Hh induces the expression of wg and dpp in A compartment cells close to the AP border of each of the three primordia. The ectopic expression of hh in the anal primordium induces complete duplication of the genital disc with the corresponding expression of these genes in their normal expression domains. The repressed male and female primordia also seemed to be duplicated in the female and male genital discs, respectively. These results indicate again that Hh diffuses across the border between the genitalia and analia, although this border acts as a cell lineage restriction barrier. It should be noted that Hh also diffuses across the border between the embryonic segments and between the abdominal segments of adult flies. The results presented here also show that the ectopic expression of either dpp or wg in the analia also affects the development of the male and female genitalia (Gorfinkiel, 2003).

The non-autonomous effect that ectopic Dpp in the analia has on the development of the genitalia is due to diffusion of Dpp itself from the analia to the genitalia, and not to the non-autonomous effect of Dpp downstream genes. By contrast, the same effect on the development of the genitalia is observed when Wg itself or any of the downstream components of the Wg-pathway, Tcf or Arm, are ectopically expressed in the analia. Ectopic expression of Wg in the analia can either recover structures of the genitalia or can prevent the development of both genitalia and analia. These results together with the observation that no Wg protein is detected in the genitalia when it is ectopically expressed in the analia indicate that the non-autonomous effect of ectopic Wg is due to an unknown signal activated by the Wg-pathway (Gorfinkiel, 2003).

Wg spreads and acts within the embryonic epidermis of Drosophila in different ranges in anterior and posterior directions. Transport or stability is reduced in engrailed-expressing cells, and further posterior Wg movement is blocked at the presumptive segmental boundary. hh function is involved in the formation of this barrier. If Wg diffusion across the genitalia-analia border is limited, it might be established very early in development by a similar mechanism to that observed in the embryo (Gorfinkiel, 2003).

The lack of development of the analia gives rise to a lack of genital structures, consistent with the outcome of genetic ablation of the analia by Ricin A. This result indicates that morphogenetic signals diffuse from the analia to the genitalia, and that this diffusion is needed for the normal development of the genital disc. However, does diffusion also occur between the male and female genital primordia of the genital disc? The results obtained in the clonal analysis of transformer (tra) in the female genital disc are relevant to this question. It has been found that male tra^- clones could give rise to male genital structures associated with a loss of female genital structures (vaginal plates and tergite eight). These data suggest a communication system between both genital primordia. Results presented here support this contention. Ablation of cells of the repressed female genital primordium of a male genital disc by Ricin protein causes a reduction of proliferation of the genital disc (Gorfinkiel, 2003).

In summary, the development of the genital disc, as that of other imaginal discs, requires interaction among the compartments forming each of its three primordia. The results presented here indicate that cell communication among different segmental primordia is also required for the development of the genital disc (Gorfinkiel, 2003).

A study of cell lineage in the male and female genital discs has revealed that there is a cell lineage restriction between the analia and the female or the male genitalia. No cell lineage restriction between the male and female genitalia could be determined since each of these does not develop in the opposite sex and consequently does not produce adult structures. For this reason, a clonal analysis was performed in intersexual flies in which both genital primordia develop. It was found that there is a cell lineage restriction barrier between female (A8) and male (A9) primordia, and between male and anal (A10-11) primordia. Even though the P compartment cells of the three segments converge in the lateral areas of the disc, there is still a cell lineage restriction among the P cells of different segments. In other imaginal discs, such as those of the wing and leg discs — composed of an A and P compartment — the cell lineage restriction between A and P cells is a consequence of the different affinity of posterior En/Hh expressing cells and anterior non-En/Hh expressing cells (Gorfinkiel, 2003).

The present results indicate that Hh signalling is not responsible for the cell lineage restriction between segmental primordia between A9 and A10-11. The question arises as to how this cell lineage restriction is achieved. The Hox gene cad, which is expressed only in the anal primordium, is not required for the restriction at the segmental border between the male genital primordium (A9) and anal primordium (A10-11). The gene Abd-B, which produces two different Abd-Bm and Abd-Br proteins, is expressed in the genital primordia. Abd-Bm is only present in the female genital primordium (A8), whereas Abd-Br is only found in the male genital primordium (A9). This suggests that the Hox genes might not only have a role in defining the identity of each segment of the genital disc but may also be involved in establishing the cell lineage restriction among segmental boundaries. However, smo; cad double mutant clones are still unable to straddle the segmental boundary between the A9 and A10-11 segments. Moreover, the overlapping of Abd-B and Cad expression domains was observed at the border between A9 and A10-A11. Thus, this segmental boundary does not behave like other segmental boundaries where the Hox expression domains are exclusive. Nevertheless, it is still possible that the interface between Abd-Bm and Abd-Br expressing cells forms the A8-A9 lineage restriction barrier (Gorfinkiel, 2003).

Sex lethal is part of the Hedgehog signaling complex: Rate of nuclear entry of full-length Cubitus interruptus are enhanced by Sxl

Sex-lethal (Sxl), the Drosophila sex-determination master switch, is on in females and controls sexual development as a splicing and translational regulator. Hedgehog (Hh) is a secreted protein that specifies cell fate during development. Sxl protein has been shown to be part of the Hh cytoplasmic signaling complex and Hh promotes Sxl nuclear entry (Vied, 2001; Horabin, 2003). In the wing disc anterior compartment, Patched (Ptc), the Hh receptor, acts positively in this process. This study shows that the levels and rate of nuclear entry of full-length Cubitus interruptus (Ci), the Hh signaling target, are enhanced by Sxl. This effect requires the cholesterol but not palmitoyl modification on Hh, and expands the zone of full-length Ci expression. Expansion of Ci activation and its downstream targets, particularly decapentaplegic the Drosophila TGFß homolog, suggests a mechanism for generating different body sizes in the sexes; in Drosophila, females are larger and this difference is controlled by Sxl. Consistent with this proposal, discs expressing ectopic Sxl show an increase in growth. In keeping with the idea of the involvement of a signaling system, this growth effect by Sxl is not cell autonomous. These results have implications for all organisms that are sexually dimorphic and use Hh for patterning (Horabin, 2005) (Horabin, 2005).

Drosophila Hh is synthesized as a 45 kDa precursor that is shortened to a mature form with two lipid modifications; palmitic acid at the N terminus and cholesterol at the C terminus. Maturation involves autoproteolytic processing under the control of the C-terminal domain of Hh. To test whether either of the lipid modifications plays a role in Hh promoted Sxl nuclear entry, female wing discs expressing Hh with only a single modification were examined. HhN encodes the N-terminal region of Hh that is palmitoylated but, because it does not undergo autoproteolytic processing, does not contain the cholesterol moiety. This form of Hh is functional for Ci activation and full-length Ci is detected distantly anterior of the AP boundary. Where HhN levels are maximal, there is a reduction of full-length Ci, most likely from the activation of en, which inhibits Ci transcription. HhN does not increase Sxl nuclear levels, however. The normal high nuclear levels in the posterior compartment and graded nuclear localization in the anterior compartment (Horabin, 2003) are detected, with no change in the cells expressing HhN (Horabin, 2005).

The alternative single modification [cholesterol without palmitoyl (C84S-Hh)], by contrast, is active with respect to Sxl. The dppGAL4 driver was used to drive expression of C84S-Hh. Relative to endogenous Hh, about threefold less of the nuclear export inhibitor Leptomycin B (LMB) was required to detect nuclear Sxl in these discs, suggesting that the nuclear Sxl is effected primarily by the ectopic Hh (Horabin, 2005).

C84S-Hh has been shown to dominantly destabilize Ci, decreasing the expression of Hh target genes. Patterning of the wing is compromised and the size of the region between veins L3 and L4 is reduced. C84S-Hh is also unable to rescue the embryonic segmentation phenotype caused by loss of Hh. C84S-Hh destabilizes Ci, but only in males. Females show the opposite effect, increasing the levels of full-length Ci (Horabin, 2005).

This sex specificity, coupled with the observation that Sxl is present in the Hh cytoplasmic complex, suggests that Sxl may be acting to stabilize Ci on Hh signaling. If this is the case, expressing Sxl in males should increase the levels of full-length Ci. Indeed, male discs expressing Sxl (MS3 isoform), as well as C84S-Hh under the control of dppGAL4, now show higher levels of full-length Ci and the protein is more nuclear, as seen in females. Taken together, these results suggest that when the cholesterol moiety is present on Hh, Sxl enhances the production of full-length Ci (Horabin, 2005).

Curiously, the presence of Sxl does not temper the wing patterning defect caused by the ectopic expression of C84S-Hh; the reported narrowing between wing veins L3 and L4 is the same in the two sexes. The form of Ci that Sxl stabilizes through C84S-Hh must not be the form responsible for Hh patterning (Horabin, 2005).

The data presented here show that when Sxl is present, the Hh signal is augmented. This is seen as an increase in full-length Ci in whole-mount tissue, and in Western blots which give a more quantitative sense of protein levels. In addition to elevating the levels of full-length Ci, several of the Hh downstream targets, including ptc, dpp and some of the downstream targets of Dpp, show an increase in expression. Conversely, removal of Sxl in female cells shows a reduction in the strength of the Hh signal (Horabin, 2005).

Sxl also enhances the nuclear entry rate of Ci, with either endogenous Hh or Hh that has only the cholesterol modification. In females, when Sxl is co-expressed with Hh with only the cholesterol modification, the amount of LMB required to detect nuclear Ci is reduced (by almost sixfold), further supporting the idea that Sxl affects Ci nuclear entry rate on Hh signaling (Horabin, 2005).

Hh enhancement of Sxl nuclear entry also depends on the cholesterol and not the palmitoyl modification. Given that Ci and Sxl are in a complex in the cytoplasm and both respond to the Hh cholesterol modification, it is tempting to speculate, although the data presented does not address this issue, that the two proteins may also enter the nucleus as a complex. This may be the method by which Sxl stabilizes Ci, diverting it from rapid proteolysis, particularly the highly activated form that is functionally detectable but has not been identified biochemically (Horabin, 2005).

Stabilization of full-length Ci by Hh with only the cholesterol modification in females is in contrast to what occurs in males. this form of Hh can destabilize Ci as well as compromise the Hh signal, but only in males. The effect of the cholesterol moiety contrasts with the palmitoyl that potentiates Hh in activating Ci for patterning. This is generally also true in vertebrates, where the cholesterol modification appears to have less of a role in patterning and a more significant role in the release and extracellular transport of the Hh ligand (Horabin, 2005).

In both sexes, ectopic expression of Sxl shows an increase in intensity of ptc expression, indicating it is possible to further elevate the Hh response. Other than en, which was difficult to score in these experiments, ptc requires the highest levels of Ci activation for its transcription (Horabin, 2005).

In females, the ectopic Sxl elevates ptc expression in the cells near the AP boundary, but the depth of the cells showing this highest level of Ci activation is reduced. A reduction in the number of cells transcribing ptc, when compared with the wider but less intense width of ptc transcription in the control half of the disc, suggests a restriction in Hh diffusion. Elevated ptc transcription is expected to produce more Ptc at the membrane, which should sequester more Hh close to the AP boundary. This result shows that Sxl can both enhance the Hh response and effectively alter the Hh gradient (Horabin, 2005).

In males, the increase in ptc transcription induced by Sxl both intensifies and widens the ptc expression zone. This suggests that the activation of Ci is at a lower peak in males than in females, and its enhancement by ectopic Sxl does not reach the same maximum that additional Sxl in females produces (Horabin, 2005).

Ectopic expression of Sxl in the dpp expression zone has been shown to adversely affect female wing development, narrowing the region between veins L3 and L4. This defect was taken to suggest that the relative concentrations of both Ci and Sxl are important for their normal function (Horabin, 2003). The data presented in this study support this conclusion while providing an explanation for the apparent decrease in effectiveness of the Hh signal. When additional Sxl is expressed, the slope of the Hh gradient becomes steeper. Since Hh directly patterns the L3 to L4 wing vein region, a steeper gradient of Hh will reduce the area patterned because the normal Hh patterning minimum is reached more rapidly. The L3 to L4 intervein region should correspondingly become narrower. No adult males expressing Sxl were recovered (presumably because of upsets in dosage compensation) so their wings could not be scored (Horabin, 2005).

Depending on the expression driver used, ectopic Sxl is not only lethal to males but also females. This is perhaps not altogether surprising given that Sxl can modulate the signal strength of a molecule crucial to the development of numerous tissues. The in vivo concentration of Sxl is, most likely, tightly controlled. It has been shown that Sxl negatively regulates translation of its own mRNA. Combined with its positive autoregulatory splicing feedback loop, which ensures that essentially all of the Sxl mRNA is spliced in the productive female mode in females, this dual negative and positive autoregulation implies a homeostasis that keeps the concentration of Sxl in a predetermined fixed range. The potent effect of Sxl on the Hh signal makes the requirement for this dual regulation more readily understood (Horabin, 2005).

Mutations in Sxl that produce sex transformed females generally result in animals that are small and male-like in size. Females transformed by mutations in tra appear as males but maintain the female size, indicating that sexual dimorphic body size is controlled by Sxl (Horabin, 2005).

The enhanced levels of full-length Ci suggest that Sxl promotes disc growth. Indeed, when ectopic Sxl is being expressed in the dorsal half, many of the discs, both male and female, show an overgrowth phenotype with the dorsal half of the wing pouch frequently expanded and distorted. This growth effect is non autonomous, indicating that it is affected by a system that signals beyond the cells expressing Sxl. This is consistent with the idea that Hh signaling is augmented to result in the overgrowth. The experiments described here do not rule out the possibility that Sxl may additionally regulate growth autonomously (Horabin, 2005).

Hh with only the cholesterol modification has the greater impact on Sxl and its stabilization of full-length Ci. However, the Ci that is stabilized does not appear to accomplish Hh patterning. This raises the mechanistic question of how Sxl achieves growth of the entire disc (Horabin, 2005).

Simply reducing the levels of the repressor form of Ci (which is accomplished by increasing the levels of full-length Ci) should increase the expression of the growth factor dpp. This is because dpp is affected by Ci at two levels: absence of the Ci repressor ameliorates repression to give low levels of dpp expression, while activated full-length Ci further elevates dpp transcription. Indeed, while the wing patterning defect caused by the ectopic expression of C84S-Hh narrows the region between wing veins L3 and L4 equally in the two sexes (due to its dominant-negative effect on endogenous Hh), the overall sexual dimorphic size difference is maintained. Consistent with this idea, co-expressing Sxl and Hh with only the cholesterol modification produces an overgrowth phenotype in discs, indicating Sxl can promote disc growth through this form of Hh (Horabin, 2005).

The growth induced by Dpp has been described as 'balanced', involving both mass accumulation as well as cell cycle progression. The net effect is that cell size does not change, nor does the ploidy. This is in contrast to growth induced by hyperactivation of Ras, Myc or Phosphoinositide 3 kinase, which increase growth but do not induce a progression through the G2/M phase of the cell cycle and, as a result, increase cell size (Horabin, 2005).

It is proposed that in the wild-type gradient of Hh with both its lipid modifications, Sxl augments the overall Hh signal to increase both full-length as well as activated full-length Ci. The two Hh targets (Ci and Sxl) respond differentially to the various components of the pathway (Horabin, 2003). Since Sxl is able to alter signal strength, the final outcome of the Hh signal must reflect the balance in activities of the components, modulated by the lipid moieties recognized, the membrane proteins used (Ptc versus Smo) and the proteins present in the Hh cytoplasmic complex. The studies reported here provide a strong rationale for why Sxl resides within the Hh cytoplasmic complex (Horabin, 2005).

Sxl not only elevates expression of dpp and its downstream targets to induce growth, but is able to elevate ptc expression. Enhancing ptc suggests that the Hh signal is 'corrected' for the enlarged patterning field, since short-range patterning has to be controlled by Hh. By enhancing dpp, Sxl indirectly also enhances the long-range patterning system of the disc. Augmenting the Hh signal would thus appear an elegant solution for increasing overall size without changing the basic body plan or pattern. Since Sxl is expressed in all female tissues from very early in development and this expression is maintained for the rest of the life cycle, Sxl is constantly available to upregulate the Hh signal. This augmentation must be kept within check, however, because, as argued above, too high an increase can change the overall slope of the Hh gradient, effectively changing the final patterning of the tissue (Horabin, 2005).

The Hh pathway can also control body size in mammals. ptc1 mutations in mice provide an overgrowth phenotype with large body size, while increasing ptc1 expression decreases body size. Humans with basal cell nevus syndrome, an autosomal-dominant condition caused by the inheritance of a mutant ptc allele, have been reported to have multiple developmental abnormalities and, relevant to this study, larger body size. Whether the mechanism described in this study is global to sexually dimorphic organisms that use Hh for patterning remains to be seen (Horabin, 2005).

Cross regulation of intercellular gap junction communication and paracrine signaling pathways during organogenesis in Drosophila

The spatial and temporal coordination of patterning and morphogenesis is often achieved by paracrine morphogen signals or by the direct coupling of cells via gap junctions. How paracrine signals and gap junction communication cooperate to control the coordinated behavior of cells and tissues is mostly unknown. This study found that Hedgehog signaling is required for the expression of wingless and of Delta/Notch target genes in a single row of boundary cells in the foregut-associated proventriculus organ of the Drosophila embryo. These cells coordinate the movement and folding of proventricular cells to generate a multilayered organ. hedgehog and wingless regulate gap junction communication by transcriptionally activating the innexin2 gene, which encodes a member of the innexin family of gap junction proteins. In innexin2 mutants, gap junction-mediated cell-to-cell communication is strongly reduced and the proventricular cell layers fail to fold and invaginate, similarly as in hedgehog or wingless mutants. It was further found that innexin2 is required in a feedback loop for the transcriptional activation of the hedgehog and wingless morphogens and of Delta in the proventriculus primordium. It is proposed that the transcriptional cross regulation of paracrine and gap junction-mediated signaling is essential for organogenesis in Drosophila (Lechner, 2007).

In both vertebrates and invertebrates, the posterior foregut constitutes a center of organogenesis from which gut-associated organs such as the lung in vertebrates or the proventriculus in Drosophila develop. Proventriculus development involves the folding and invagination of epithelial cell layers to generate a multiply-folded organ. Two cell populations, the anterior and the posterior boundary cells, were shown previously to control cell movement and the folding of the proventriculus organ. In the posterior boundary cells, which organize the endoderm rim of the proventriculus, the JAK/STAT signaling cascade cooperates with Notch signaling to control the expression of the gene short stop encoding a cytoskeletal crosslinker protein of the spectraplakin superfamily. Thereby the Notch signaling pathway is connected to cytoskeletal organization in the posterior boundary cells, which have to provide a stiffness function to enable the invagination of the ectodermal foregut cells. The findings in this paper provide evidence that hedgehog is essential for the Notch signaling-dependent allocation of the anterior boundary cells. In amorphic hedgehog mutants, evagination and the formation of the constriction at the ectoderm/endoderm boundary are not affected, however, the inward movement of the anterior boundary cells is not initiated at the keyhole stage. The lack of cell movement of the ectodermal proventricular cells is consistent with the finding that hedgehog specifically controls Notch target gene activity in the anterior boundary cells. Genetic experiments further identify wingless as a target gene of hedgehog in the anterior boundary cells. wingless, in turn, controls the transcription of the innexin2 gene, which is expressed in the invaginating proventricular cells. When wingless is re-supplied in the genetic background of hedgehog mutants, innexin2 expression is rescued, providing further evidence that innexin2 is a target gene of wingless in the proventriculus primordium. Innexin2 encodes a member of the innexin family of gap junction proteins and is essential for the development of epithelial tissues. In the proventriculus, innexin2 mRNA is initially expressed in the early evagination stage in a broad domain covering both the ectodermal and endodermal precursor cells of the proventriculus primordium. When the ectodermal cells start to invaginate into the proventricular endoderm, innexin2 expression is upregulated in the ectodermal cell layer. Invagination of the ectodermal cells fails in hedgehog, wingless and kropf mutant proventriculi and dye tracer injection experiments demonstrate that hedgehog and kropf mutants show a strong reduction of gap junction communication. These data suggest that the direct coupling of cells via Innexin2-containing gap junctions, which are induced in response to hedgehog and wingless activities, is important for the coordinated movement of the ectodermal cell layer. It is known from extensive studies in mammals that the coupling of cells and tissues via gap junctions enables the diffusion of second messengers, such as Ca2+, inositol-trisphosphate (IP3) or cyclic nucleotides to allow the rapid coordination of cellular behavior during morphogenetic processes such as cell migration and growth control. Cell movement and folding involves a modulation of cell adhesion and of cytoskeletal architecture of the proventricular cells. A functional interaction of innexin2 with the cell adhesion regulator DE-cadherin, which is a core component of adherens junctions has been shown recently by co-immunoprecipitation, yeast two-hybrid studies, and genetic analysis. In mutants of DE-cadherin, Innexin2 is mislocalized and vice versa suggesting that the regulation of cell adhesion and gap junction-mediated communication may be linked. Similar evidence for a coordinated regulation of connexin activity and N-cadherin has been obtained in mammals during migration of neural crest cells (Lechner, 2007).

In kropf mutants or innexin2 knockdown animals, hedgehog, wingless and Delta transcription is strongly reduced as shown by in situ hybridization and by quantitative RT PCR experiments using mRNAs isolated from staged embryos. Furthermore, hedgehog, wingless and Delta are ectopically expressed and their mRNA is upregulated in embryos in which innexin2 is overexpressed. In summary, these experiments provide strong support that the gap junction protein Innexin2 plays an essential role enabling or promoting transcriptional activation of hedgehog, wingless and Delta. These data point towards an essential requirement of gap junction communication for the transcriptional activation of morphogen-encoding genes activating evolutionary conserved signaling cascades essential for patterning in animals. It is of note that gap junctions are established at very early stages of embryonic development, correlating with a maternal and zygotic expression of innexin2 and other innexin family members. kropf mutant animals, which are devoid of maternal and zygotic innexin2 expression are early embryonic lethal and develop no epithelia, consistent with a fundamental role of gap junctions in development, on top of which pattern formation of tissues and organs may occur. It has been shown previously that gap junctions are essential for C. elegans, Drosophila, and vertebrate embryogenesis from early stages onwards (Lechner, 2007 and references therein).

In the nematode C. elegans, a transient network formed by the innexin gap junction protein NSY-5 was recently shown to coordinate left-right asymmetry in the developing nervous system. Previous findings in chick and Xenopus laevis embryos have suggested an essential role of connexin43-mediated gap junction for the determination of the left-right asymmetry of the embryos. Treatment of cultured chick embryos with lindane, which results in a decreased gap junctional communication, frequently unbiased normal left-right asymmetry of Sonic hedgehog and Nodal gene expression, causing the normally left-sided program to be recapitulated. An important role of connexin43 (Cx43)-dependent gap junction communication for sonic hedgehog expression was also observed in limb patterning of the chick wing. Additionally, modulation of gap junctions in Xenopus embryos by pharmacological agents specifically induced heterotaxia involving mirror-image reversals of the heart, gut, and gall bladder. These data in combination with the current findings indicate that the transcriptional regulation of hedgehog and other morphogen-encoding genes by gap junction proteins may be evolutionary conserved between deuterostomes (vertebrates) and protostomes (Drosophila), although the Drosophila innexin gap junction genes share very little sequence homology with the connexin genes. The molecular mechanism underlying innexin2-mediated transcriptional regulation of hedgehog, wingless and Delta is not clear. It has been proposed that the nuclear localization of the carboxy-tail of connexin43 may exert effects on gene expression and growth in cardiomyocytes and HeLa cells. This would infer a cleavage of connexin43 to release the C-terminus, however, in vivo evidence for this event is still lacking. Sequence analysis reveals a nuclear receptor recognition motif within the C-terminus of Innexin2. It has been demonstrated that this recognition motif mediates the interaction of coactivators with nuclear receptors. However, there is no immunohistochemical evidence for a nuclear localization of Innexin2 or the Innexin2 C-terminus in Drosophila embryonic cells indicating that a direct involvement of Innexin2 in regulating transcription of target genes may not occur. The direct association of a transcription factor with gap junctions has been recently proposed for the mouse homolog of ZO-1-associated nucleic acid-binding protein (ZONAB). This transcription factor binds to ZO-1, which is associated with oligodendrocyte, astrocyte and retina gap junctions. It is possible that innexin2-dependent transcriptional regulation may involve a similar type of mechanism: a still unknown transcriptional regulator associated with the C-terminus of innexin2-containing gap junctions could be released upon modulation of gap junction composition thereby modulating the transcription of innexin2-dependent target genes (Lechner, 2007).

Hedgehog signaling regulates the ciliary transport of odorant receptors in Drosophila

Hedgehog (Hh) signaling is a key regulatory pathway during development and also has a functional role in mature neurons. This study shows that Hh signaling regulates the odor response in adult Drosophila olfactory sensory neurons (OSNs). This is achieved by regulating odorant receptor (OR) transport to and within the primary cilium in OSN neurons. Regulation relies on ciliary localization of the Hh signal transducer Smoothened (Smo). This study further demonstrates that the Hh- and Smo-dependent regulation of the kinesin-like protein Cos2 acts in parallel to the intraflagellar transport system (IFT) to localize ORs within the cilium compartment. These findings expand knowledge of Hh signaling to encompass chemosensory modulation and receptor trafficking (Sanchez, 2016).

This study demonstrates that the Hh pathway modulates the magnitude of the odorant response in adult Drosophila. The results show that the Hh pathway determines the level of the odorant response because it regulates the response in both the positive and negative directions. Loss of Ptc function increases the odorant response and the risk for long sustained responses, which shows that the Hh pathway limits the response potential of the OSNs and is crucial for maintaining the response at a physiological level. In addition, it was shown that the OSNs produce Hh protein, which regulates OR localization, which is interesting because autoregulation is one of the prerequisites for an adaptive mechanism. It was further shown that Hh signaling regulates the responses of OSNs that express different ORs, which demonstrates that the regulation is independent of OSN class and suggests that Hh signaling is a general regulator of the odorant response. It has been shown previously that Hh tunes nociceptive responses in both vertebrates and Drosophila (Babcock, 2011). It is not yet understood how Hh regulates the level of nociception. However, the regulation is upstream of the nociceptive receptors, which indicates that the Hh pathway is a general regulator of receptor transport and the level of sensory signaling (Sanchez, 2016).

The results show that OSN cilia have two separate OR transport systems, the Hh-regulated Cos2 and the intraflagellar transport complex B (IFT-B) together with the kinesin II system. The results show that Cos2 is required for OR transport to or within the distal cilium domain and suggest that the IFT system regulates the inflow to the cilium compartment. The two transport systems also are required for Smo cilium localization (Kuzhandaivel, 2014). This spatially divided transport of one cargo is similar to the manner in which Kif3a and Kif17 regulate distal and proximal transport in primary cilia in vertebrates. However, Cos2 is not required for the distal location of Orco or tubulin (Kuzhandaivel, 2014), indicating that, for some cargos, the IFT system functions in parallel to Cos2 (Sanchez, 2016).

Interestingly, the vertebrate Cos2 homolog Kif7 organizes the distal compartment of vertebrate primary cilia (He, 2014). Similar to the current results, Kif7 does so without affecting the IFT system, and its localization to the cilia is dependent on Hh signaling. However, the Kif7 kinesin motor function has been questioned (He, 2014). Therefore, it will be interesting to analyze whether Kif7-mediated transport of ORs and other transmembrane proteins occurs within the primary cilium compartment and whether the ciliary transport of ORs is also regulated by Hh and Smo signaling in vertebrates. To conclude, these results place the already well-studied Hh signaling pathway in the post-developmental adult nervous system and also provide an exciting putative role for Hh as a general regulator of receptor transport to and within cilia (Sanchez, 2016).

Larval - Hedgehog signals travel from the eye to the brain

Continued: Hedgehog Developmental biology - Larval part 2/2

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