The human Smoothened inhibitor PF-04449913 induces exit from quiescence and loss of multipotent Drosophila hematopoietic progenitor cells

The efficient treatment of hematological malignancies as Acute Myeloid Leukemia, myelofibrosis and Chronic Myeloid Leukemia, requires the elimination of cancer-initiating cells. In mammals, inhibition of Smoothened, the key mediator of the Hedgehog signaling pathway, reduces Chronic Myeloid Leukemia progression and propagation. These findings make Smo a candidate target to inhibit maintenance of leukemia-initiating cells. In Drosophila the same pathway maintains the hematopoietic precursor cells of the lymph gland, the hematopoietic organ that develops in the larva. This study investigated in Drosophila the mode of action of PF-04449913, a small-molecule inhibitor of the human Smo protein. Drosophila larvae fed with PF-04449913 showed traits of altered hematopoietic homeostasis. These include the development of melanotic nodules, increase of circulating hemocytes, the size increase of the lymph gland and accelerated differentiation of blood cells likely due to the exit of multi-potent precursors from quiescence. Importantly, the Smo inhibition can lead to the complete loss of hematopoietic precursors. It is concluded that PF-04449913 inhibits Drosophila Smo blocking the Hh signaling pathway and causing the loss of hematopoietic precursor cells. Interestingly, this is the effect expected in patients treated with PF-04449913: number decrease of cancer initiating cells in the bone marrow to reduce the risk of leukemia relapse. Altogether these results indicate that Drosophila comprises a model system for the in vivo study of molecules that target evolutionary conserved pathways implicated in human hematological malignancies (Giordani, 2016).

Effects of Mutation or Deletion

smo mutants manifest a classic segment polarity cuticle phenotype but with considerable variability in the phenotype (Alcedo, 1996).

Rather convincing genetic arguments maintain that SMO does not function as the yet-to-be identified Wingless receptor, but instead functions as a HH receptor. In smo mutant embryos, wg and en expression are initiated normally, at the cellular blastoderm stage. WG protein subsequently decays during stage 9 [Images], before EN protein begins to decay. The temporal loss of EN protein in smo mutant embryos resembles that in hh mutant embryos. It differs from that in wg mutants, where EN decays earlier than in eithersmo or hh mutants due to the direct control of en by WG (Alcedo, 1996 and references).

Ectopic wg drives ectopic expression of EN even in smo mutants, suggesting that SMO is not a wingless receptor. Ubiquitously expressed hh results in the ectopic expression of wg in anteriorly expanded stripes. In smo mutants, ubiquitously expressed hh does not induce ectopic expression of wg. Instead, WG stripes decay and wg expression is indistinguishable from that of smo mutants in the absence of ectopic hh. Since etopic HH has little or no effect in smo mutants, wild type smo is required for HH signal transduction (Alcedo, 1996).

It is thought that the posterior expression of the 'selector' genes engrailed and invected control the subdivision of the growing Drosophila wing imaginal disc into anterior and posterior lineage compartments. In the selector-affinity model, cells in one compartment do not adhere to or recognize cells in the adjacent comparment and minimize contact with them, creating a sharp, smooth boundary between selector-expressing and non-expressing cells. At present, the cellular mechanisms by which separate lineage compartments are maintained are not known. Most models have assumed that the presence or absence of selector gene expression autonomously drives the expression of compartment-specific adhesion or recognition molecules that inhibit intermixing between compartments. Nevertheless, adhesion assays have provided little evidence for gross adhesive differences between cells in different compartments and no good candidate compartment-specific adhesion or recognition molecules has yet been described. However, the present understanding of Hedgehog signaling from posterior to anterior cells raises some interesting alternative models based on a cell's response to signaling. Anterior cells that lack smoothened, and thus the ability to receive the Hedgehog signal, no longer obey a lineage restriction in the normal position of the anterior-posterior boundary. Rather, these clones extend into anatomically posterior territory, without any changes in engrailed/invected gene expression. These cells of anterior origin retain anterior-like features in the posterior territory such as their neuronal character. Such smo- cells of anterior origin do not associate normally with posterior cells in that the mutant cells have abnormally smooth boundaries and do not interdigitate normally with neighboring posterior cells. Clones lacking both en and inv were also examined; these too show complex behaviors near the normal site of the compartment boundary, and do not always cross entirely into anatomically anterior territory. These double mutant cells do not associate normally with anterior cells; instead, they exhibit abnormally rounded boundaries. These results suggest that compartmentalization is a complex process that cannot be explained by simple affinity models (Blair, 1997).

This study reports the expression pattern of Dll in the genital disc, the requirement of Dll activity for the development of the terminalia and the activation of Dll by the combined action of the morphogenetic signals Wingless (Wg) and Decapentaplegic (Dpp). In Drosophila, the terminalia comprise the entire set of internal and external genitalia (with the exception of the gonads), and includes the hindgut and the anal structures. They arise from a single imaginal disc of ventral origin which is of complex organization and shows bilateral symmetry. The genital disc shows extreme sexual dimorphism. Early in development, the anlage of the genital disc of both sexes consists of three primordia: the female genital primordium (FGP); the male genital primordium (MGP), and the anal primordium (AP). In both sexes, only two of the three primordia develop: the corresponding genital primordium and the anal primordium. These in turn develop, according to the genetic sex, into female or male analia. The undeveloped genital primordium is the repressed primordium (either RFP or RMP, for the respective female and male genital primordia) (Gorfinkiel, 1999).

During the development of the two components of the anal primordium -- the hindgut and the analia -- only the latter is dependent on Dll and hedgehog (hh) function. The hindgut is defined by the expression of the homeobox gene even-skipped. The lack of Dll function in the anal primordia transforms the anal tissue into hindgut by the extension of the eve domain. Meanwhile targeted ectopic Dll represses eve expression and hindgut formation. The Dll requirement for the development of both anal plates in males and only for the dorsal anal plate in females, provides further evidence for the previously held idea that the analia arise from two primordia. In addition, evaluation was made of the requirement for the optomotor-blind (omb) gene which, as in the leg and antenna, is located downstream of Dpp. These results suggest that the terminalia show similar behavior as the leg disc or the antennal part of the eye-antennal disc, consistent with both the proposed ventral origin of the genital disc and the evolutive consideration of the terminalia as an ancestral appendage (Gorfinkiel, 1999).

Hh signal is required to form the genital and anal structures but not the hindgut. In the leg and antennal discs, the expression of Dll depends on the Hh signaling pathway. Using the hh ts2 allele, it was observed that in the genital disc, Hh is also required for Dll activation: after 4 days at the restrictive temperature, the genital discs are very small and show no Dll expression. In the same hh ts2 larvae, residual Dll expression can be detected in the trochanter region of the leg disc. However, eve expression in the anal primordia is maintained and occupies most of the reduced genital disc. This result indicates that Dll, but not eve expression, depends on Hh and that all the terminalia with the exception of the hindgut require Hh function. To further analyze this hh requirement for Dll activation, the effect of smoothened (smo) lack of function was examined. In smo2 cells, Hh reception is impeded because smo is a component of the Hh receptor complex. In the genital disc, Dll expression only disappears in smo2 clones when the clone is large enough to cover most of the Dll expression domain. Accordingly, eve expression is also ectopically activated in smo2 mutant cells; although in Dll2 cells eve cannot be activated in certain regions of the clones. These results indicate once again that Dll is dependent on Hh function while eve is not (Gorfinkiel, 1999).

Large smo2 clones close to the A/P compartment transform some structures of the external genitalia and analia. In the female genitalia, smo2 clones duplicate the long bristle of the vaginal plates and clones in T8 to produce tissue overgrowth with y2 bristles. Large smo2 clones reduce the female dorsal anal plate, whereas the female ventral anal plate is rarely affected. Some clones produce segregated tissue in the female analia labelled with y bristles in the perianal region. However, small clones or clones located outside the A/P compartment border have no effect. In the male genitalia, smo2 clones duplicate the genital arc, part of the claspers and the hypandrium bristle. All these structures are located close to the A/P compartment border. As in Dll2 clones, large smo2 clones delete the anal plate in males. In both males and females, only when the clone is large enough can Dll expression not be activated in the disc primordia, giving rise to the Dll2 phenotype. This result suggests that only in large smo2 clones both wg and dpp are not activated and therefore are unable to induce Dll expression (Gorfinkiel, 1999).

Very little information is available about gene expression during the larval period, a developmental interval critical to the formation of the adult. To what extent does gene expression during this period resemble that in the embryonic stages, and how does gene expression during the larval period contribute to segment polarity in the adult? In fact, all the genes expressed during embryonic segment polarity also play a similar role in the formation of the adult. Cells destined to form the cuticle of the adult abdomen are present as clusters of small, non-dividing diploid cells (the anterior dorsal, posterior dorsal and ventral histoblast nests) located at stereotyped postions in the larval epidermis. These cells, just as do their embryonic counterparts, express engrailed, hedgehog, wingless, patched, cubitus interruptus and sloppy paired in a stereotyped manner dependent on their positions within each segment. Each segment is subdivided into an anterior (A) and posterior (P) compartment, distinguished by activity of the selector gene engrailed (en) in P but not A compartment cells. The ventral epidermis of each abdominal segment forms a flexible cuticle, the pleura, with a small plate of sclerotised cuticle, the sternite, centered on the ventral midline. The pleura is covered with a uniform lawn of hairs, all pointed posteriorly, whereas the sternite contains a stereotyped pattern of bristles. Posterior compartments are to a large degree devoid of hairs and bristles, while the sternite cuticle of the A compartment consists of an anterior-to posterior progression of six types of cuticle distinguished by ornamentation and pigmentation. Just anterior to the posterior compartment, A6 is unpigmented, with hairs and none of the larger ornaments called bristles. A5 is darkly pigmented with hairs and bristles of large size. A4 and A3 are darkly and lightly pigmented respectively with moderately sized hairs and bristles. A2 is lightly pigmented with hairs, and A1, adjacent to the next more anteriorly located "posterior" compartment is unpigmented without hairs (Struhl, 1997a).

A second paper (Struhl, 1997b) deals directly with the instances in which cell polarity does not correspond to the presumed concentration gradient of Hh and considers whether Hh acts directly or by a signal relay mechanism. In some cases various manipulations cause non-autonomous effects on cell polarity, a vectorial property. For example, PKA mutant clones in the A3 and A4 region alter polarity of hairs and bristles both within the clone and outside it. In general, wild-type cells positioned laterally and posteriorly to the mutant clone form hairs and bristles that point centripetally towards the clone; thus, behind the clone, cells form hairs and bristles that point anteriorly. The region of wild-type tissue showing this reversed polarity can be up to 4 cell diameters wide. Of great interest is the effect of PKA and smo mutant clones in the anterior portion of A compartment. PKA and smo mutant clones in the anterior region of the A comparment alter cell type much as they do in the posterior portion, but some clones of smo cells in the A1 region form hairs that have reversed polarity and these hairs point forward. Consequently, it is surmised that Hh influences cell polarity indirectly, possibly by inducing other signaling factors (Struhl, 1997b).

Evidence is presented that Hh does not polarize abdominal cells by utilizing either Decapentaplegic or Wingless, the two morphogens through which Hh acts during limb development. If Hh were to work through Wg to influence polarity, removal of wg from clones of cells that are activated in the Hh pathway should eliminate that influence. Neither the change in cell type nor the alterations in cell polarity cause by the loss of PKA activity appear to be due to the ectopic expression of wg. Likewise, eliminating dpp from PKA mutant clones fails to alter the polarity phenotype (Struhl, 1997b).

How might Hh polarize cells via a signal-relay mechanism? One clue is that within and surrounding some PKA mutant clones the hairs and bristles point inwards, towards the center. A simple model is that the loss of PKA activity in these cells mimics reception of Hh and hence induces them to secrete a diffusible polarising factor, 'X'. Because mutant cells in the center of the clone would be surrounded by X-secreting cells, they might be exposed to higher levels of X than mutant cells at the periphery. If cells were oriented by the direction of maximal change (the vector) in the concentration of X, cells both inside and outside of the clone would point towards the center of the clone. Such a propagation model does not demand that X be diffusible, because polarity could be organized by local cell-cell interactions, which spread as in a game of dominoes (Struhl, 1997b).

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, 1999 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, 1999).

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, 1999).

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, 1999).

In the Drosophila leg disc, wingless and decapentaplegic are expressed in a ventral-anterior and a dorsal-anterior stripe of cells, respectively. This pattern of expression is essential for proper limb development. While the Hedgehog (Hh) pathway regulates dpp and wg expression in the anterior-posterior (A/P) axis, mechanisms specifying their expression in the dorsal-ventral (D/V) axis are not well understood. Evidence is presented that supernumerary limbs (slimb) mutant clones in the disc deregulate wg and dpp expression in the D/V axis. This suggests for the first time that their expression in the D/V axis is actively regulated during imaginal disc development. Furthermore, slimb is unique in that it also deregulates wg and dpp in the A/P axis. The misexpression phenotypes of slimb- clones indicate that the regulation of wg and dpp expression is coordinated in both axes, and that slimb plays an essential role in integrating A/P and D/V signals for proper patterning during development. Genetic analysis further reveals that slimb intersects the A/P pathway upstream of smoothened (smo) (Theodosiou, 1998).

The abdomen of adult Drosophila consists of a chain of alternating anterior (A) and posterior (P) compartments which are themselves subdivided into stripes of different types of cuticle. Most of the cuticle is decorated with hairs and bristles that point posteriorly, indicating the planar polarity of the cells. This study has focused on a link between pattern and polarity. Previous studies have shown that the pattern of the A compartment depends on the local concentration (the scalar) of a Hedgehog morphogen produced by cells in the P compartment. Evidence is presented in this study that the P compartment is patterned by another morphogen, Wingless, which is induced by Hedgehog in A compartment cells and then spreads back into the P compartment. Both Hedgehog and Wingless appear to specify pattern by activating the optomotor blind gene, which encodes a transcription factor. A working model that planar polarity is determined by the cells reading the gradient in concentration (the vector) of a morphogen 'X' which is produced on receipt of Hedgehog, is re-examined. Evidence is presented that Hedgehog induces X production by driving optomotor blind expression. X has not yet been identified and data is presented that X is not likely to operate through the conventional Notch, Decapentaplegic, EGF or FGF transduction pathways, or to encode a Wnt. However, it is argued that Wingless may act to enhance the production or organize the distribution of X. A simple model that accommodates these results is that X forms a monotonic gradient extending from the back of the A compartment to the front of the P compartment in the next segment, a unit constituting a parasegment (Lawrence, 2002).

It has been concluded that Hh acts indirectly via another system (a gradient of 'X') to effect polarity. The evidence was based on clones that lacked such downstream genes as patched (ptc) or cAMP-dependent protein kinase 1 (Pka). In the A compartments, Ptc and Pka proteins act within cells to prevent the Hh pathway from being activated inappropriately; if either protein is removed the Hh pathway becomes constitutively activated within the mutant cells themselves. With respect to the type of cuticle (the scalar output of Hh) the results fit the model; the mutant cells make the cuticle normally made by cells responding strongly to Hedgehog and all the cells outside the clone make the normal type of cuticle (a cell-autonomous effect). However, with respect to polarity (the vectorial output of Hh), the results are different; polarity is altered in the wild-type cells up to several cell diameters away from the clone (a cell non-autonomous effect). Although it has been argued that these effects were not due to Hh itself, the possibility was not eliminated that low levels of ectopic Hh might be produced by the clone and diffuse out, being sufficient to repolarize the cells without changing the scalar. This study now disproves this possibility by making clones that lack both effective Ptc protein and the hh gene. These clones still cause repolarization in the back half of the clone and behind it arguing strongly that the Hh protein is a component of 'X' and raising again the question, what is X? X should be engendered downstream of Hh receipt, which is where the search is started (Lawrence, 2002).

If the production of X depends at least in part on omb, then ptc- clones, in which the Hh pathway has been constitutively activated, should produce little or no X if they also lack omb. Clones were made that were both ptc- and omb-; these clones form a6 cuticle as do ptc- clones. However, in the middle of the A compartment and unlike ptc- clones in that position, they fail to repolarize behind, but reverse their polarity in front -- as do omb- cells. Similarly, omb- ptc- clones situated at the back of the A compartment behave like omb- clones, the whole being reversed in polarity (and not like ptc- clones in the same location, that have normal polarity). Thus in terms of the type of the cuticle (the scalar), omb- ptc- behave as ptc- clones, but in terms of the vector they behave as omb- clones. These results confirm that Hh induces X production through the action of omb (Lawrence, 2002).

The model for X suggests that, if omb were ectopically activated in cells at the front of the A compartment, those cells could become a source of X. Indeed omb-expressing clones can repolarize the cells behind them -- as if there were a local peak in the X distribution (Lawrence, 2002).

smoothened (smo), is an essential component of Hh transduction; without it the cells cannot see Hh protein. As regards polarity, one would expect neither omb- nor smo- clones to produce X and for their phenotype to be the same. Although this is generally the case, the effects of smo- and omb- differ for clones located at the back of the A compartment. Polarity within these omb- clones is completely reversed, consistent with the model, whereas the corresponding smo- clones are reversed only within the anterior portion of the clone, polarity returning to normal at the very back of the A compartment. The preferred explanation for this discrepancy is that Smo protein perdures in smo- clones, allowing partial rescue of the smo mutant phenotype, particularly at the back of the A compartment, where Hh is most abundant. This rescue could allow production of X, enough to restore normal polarity at the back of the clone, but not enough to specify a4 cuticle or to upregulate ptc.lacZ. For both smo- and omb- clones, some Hh would be expected to move forward across the clone and induce an ectopic peak of X production in more anterior, wild-type cells, accounting for the polarity reversals that are observed in both cases (Lawrence, 2002).

To test this explanation Hh receipt was blocked by a different method that is not so subject to perdurance: a marked clone was made that contained no wild-type Ptc, but provided instead a mutant form of Ptc that is ineffective at transducing the Hh signal. Such clones behave like smo- clones in most respects, including making a3 cuticle instead of a4, a5 or a6 cuticle in the back half of the A compartment, and causing polarity reversals both within and anterior to the clone. However, unlike smo- clones, the polarity at the back of these clones does not return to normal. Instead, in the majority of cases, polarity remains reversed all the way to the back edge of the clone, and sometimes beyond, as observed for omb- clones in the same position. These results support the perdurance explanation for the smo- clones and are consistent with the working model, which is based mainly on the results with omb (Lawrence, 2002).

slimb was identified in a mutant screen. To identify recessive overproliferation mutations in genes that are lethal in homozygous mutant animals, genetic screens were performed in mosaic flies containing homozygous mutant patches in otherwise wild-type backgrounds. Two classes of recessive overproliferation mutations have been identified. Mutations of the first group cause mutant cells to undergo extensive proliferation and form unpatterned, tumorous outgrowths in mosaic adults. Mutations of the second group induce both patterned and irregular outgrowths. slimb affects developmental signals that regulate cell proliferation and pattern organization. The slimb transcript encodes a Cdc4-related protein containing F-box and WD-40 motifs. Jiang (1998) has independently reported the identification of this gene. Using a Drosophila slimb cDNA, a human homolog (H-slimb) has been isolated. The fly and human proteins share 78% amino acid identity throughout, suggesting that slimb is functionally conserved (Theodosiou, 1998).

To further explore how slimb regulation and function correlates with A/P signaling, double mutant analysis was carried out with slimb mutants and with mutants of hh and smo. No reduction of outgrowths was observed in slimb-, hh- double mutant clones. Furthermore, slimb mutant clones have no effect on hh expression. This indicates that slimb acts downstream or independent of Hh signaling. In contrast, slimb-, smo- double mutant clones almost completely suppress slimb induced outgrowths. Consistent with the adult phenotype, discs carrying slimb- , smo- clones fail to ectopically express either dpp or wg. These data suggest that slimb intersects the A/P signal upstream of smo. Jiang (1998) suggested that slimb acts downstream of smo. This difference may be explained by the use of different alleles for smo and slimb. The Slimb product contains WD-40 repeats believed to act as a scaffold for the binding of multiple proteins. It is possible that this structure may allow for proteins such as Smo and components of a D/V pathway to converge. The Slimb-related protein Cdc4 from Saccharomyces cerevisiae along with Cdc53, and Cdc34 are part of the ubiquitin proteolysis machinery. The current data that Slimb acts upstream of Smo, together with its sequence homology with Cdc4, suggests that Slimb could be involved in the regulation of Smo protein degradation (Theodosiou, 1998).

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).

During Drosophila eye development, Hedgehog (Hh) protein secreted by maturing photoreceptors directs a wave of differentiation that sweeps anteriorly across the retinal primordium. The crest of this wave is marked by the morphogenetic furrow, a visible indentation that demarcates the boundary between developing photoreceptors located posteriorly and undifferentiated cells located anteriorly. Evidence is presented that Hh controls progression of the furrow by inducing the expression of two downstream signals. The first signal, Decapentaplegic (Dpp), acts at long range on undifferentiated cells anterior to the furrow, causing them to enter a 'pre-proneural' state marked by upregulated expression of the transcription factor Hairy. Acquisition of the pre-proneural state appears essential for all prospective retinal cells to enter the proneural pathway and differentiate as photoreceptors. The second signal, presently unknown, acts at short range and is transduced via activation of the Serine-Threonine kinase Raf. Activation of Raf is both necessary and sufficient to cause pre-proneural cells to become proneural, a transition marked by downregulation of Hairy and upregulation of the proneural activator, Atonal (Ato), which initiates differentiation of the R8 photoreceptor. The R8 photoreceptor then organizes the recruitment of the remaining photoreceptors (R1-R7) through additional rounds of Raf activation in neighboring pre-proneural cells. Dpp signaling is not essential for establishing either the pre-proneural or proneural states, or for progression of the furrow. Instead, Dpp signaling appears to increase the rate of furrow progression by accelerating the transition to the pre-proneural state. In the abnormal situation in which Dpp signaling is blocked, Hh signaling can induce undifferentiated cells to become pre-proneural but does so less efficiently than Dpp, resulting in a retarded rate of furrow progression and the formation of a rudimentary eye (Greenwood, 1999).

Hh, secreted by maturing photoreceptor cells, is normally responsible for inducing cells within and ahead of the morphogenetic furrow to initiate photoreceptor differentiation. Nevertheless, cells that lack Smoothened (Smo) function, and hence the ability to transduce Hh, can form normal ommatidia. These findings suggest that Hh can induce photoreceptor differentiation in Smo-deficient cells through the induction of other signaling molecules in neighboring wild-type tissue. As a first step toward identifying such secondary signals and analyzing their roles, the consequences of creating clones of cells homozygous for smo3, an amorphic mutation, have been examined on two early markers of retinal development, the expression of Ato and Hairy, which are expressed in adjacent dorso-ventral stripes within and anterior to the morphogenetic furrow. Ato expression has two prominent phases in the developing eye. In the first phase, Ato is expressed uniformly in a narrow dorso-ventral swath of cells that demarcates the anterior edge of the furrow. This uniform swath then breaks up into small clusters of Ato expressing cells and resolves into the second phase, a spaced pattern of single Ato expressing cells (the future R8 photoreceptor cells). The first phase of Ato expression is severely reduced or absent in clones of smo3 cells, similar to large clones that lack Hh. However, the second phase of expression still occurs, even though it is displaced posteriorly, indicating that it is delayed. This displacement is more severe in the middle of the clone than along the dorsal and ventral borders, producing a crescent shaped distortion of the line of spaced single cells that express Ato. It is concluded that cells within smo mutant clones can be induced to express Ato even though they cannot receive Hh, provided that they are located near to wild-type cells across the clone border. Equivalent effects have been observed for Hairy expression. Hairy is normally expressed at peak levels in a dorso-ventral stripe positioned immediately anterior to the Ato stripe, but is abruptly downregulated in more posteriorly situated cells. Clones of smo3 cells have only a modest effect on Hairy expression anterior to the furrow, causing a slight, but consistent, posterior displacement of the anterior edge of the stripe. However, they are associated with a pronounced failure to repress Hairy expression in some, but not all, posteriorly situated smo3 cells. As in the case of Ato expression, the exceptional mutant cells that retain the normal downregulation of Hairy are those positioned close to the lateral and posterior borders of the clones. Just within the lateral border, a line of cells is typically observed, one or two cell diameter lengths wide, where Hairy expression is repressed. Along the posterior border, the zone of mutant cells in which Hairy expression is repressed is usually wider (Greenwood, 1999).

These results are interpreted to indicate that (1) Hh normally induces cells to express a secondary signal (or signals) that can activate Ato expression and repress Hairy expression; (2) this signal acts non-autonomously, allowing it to move from wild-type cells where it is induced by Hh to nearby smo3 cells where it regulates Ato and Hairy expression; and (3) the range of this signal is short, restricting its action to only one or two cells across the lateral borders of smo3 mutant clones. A somewhat greater range of action is apparent along the posterior borders of such clones, perhaps because the adjacent wild-type cells were induced by Hh to send this signal at an earlier time than those along the lateral (more anterior) borders of the clone, allowing the signal more time to accumulate to higher levels and to move deeper into mutant tissue (Greenwood, 1999).

To examine how the posterior displacements in Ato and Hairy regulation in smo3 clones influence subsequent ommatidial development, the expression of the protein Elav, a marker of photoreceptor differentiation was examined. Clones of smo3 cells are capable of differentiating as photoreceptors, in agreement with previous findings. However, there is a significant delay. In wild-type tissue, Elav expression initiates immediately posterior to the morphogenetic furrow with the specification of the R8 cell and continues as other photoreceptors are recruited into the ommatidial cluster. In clones of smo3 cells, there is a clear posterior displacement in the onset of photoreceptor differentiation in mutant cells: photoreceptor differentiation is first seen at the posterior, and occasionally lateral, edges of the clone, correlating with the effects of neighboring wild-type tissue on Hairy and Ato expression and indicating a general delay in photoreceptor differentiation. However, as seen in more posteriorly situated clones, most or all of the smo3 tissue eventually differentiates as normally patterned ommatidia. Thus, Hh signal transduction is not autonomously required for presumptive eye cells to express Ato, downregulate Hairy, or differentiate as photoreceptors. This is in contrast to the general requirement for Hh signaling revealed by experiments in which Hh signaling is blocked throughout the entire disc using temperature-sensitive hh mutations. In the latter case, loss of Hh signaling causes a rapid and complete block in photoreceptor differentiation and furrow progression. Hh signaling appears to induce at least two secondary signals that are essential for the normal recruitment of undifferentiated cells to form the R8 photoreceptors. One of them appears to be the short-range signal that can induce Ato expression and repress Hairy in clones of smo minus cells. The second, Decapentaplegic (Dpp), appears to act at longer range to prime cells to receive this short range signal (Greenwood, 1999).

The arrival of retinal axons in the Drosophila brain triggers the assembly of glial and neuronal precursors into a neurocrystalline array of lamina synaptic cartridges. Retinal axons arriving from the eye imaginal disc trigger the assembly of neuronal and glial precursors into precartridge ensembles in the crescent-shaped lamina target field. In the eye disc, photoreceptor cells assemble into ommatidial clusters behind the morphogenetic furrow (mf) as it moves to the anterior. The ommatidial clusters project their axon fascicles into the crescent-shaped lamina. Neuronal precursor cells of the lamina (LPCs) are incorporated into the axon target field at its anterior margin, which is demarcated by a morphological depression known as the lamina furrow. Glia precursor cells (GPCs) are generated in two domains that lie at the dorsal and ventral anterior margins of the prospective lamina. These glial precursors migrate into the lamina along an axis perpendicular to that of LPC entry. Postmitotic LPCs within the lamina axon target field express the nuclear protein Dac, as revealed by anti-Dac antibody staining. Like the eye, lamina differentiation occurs in a temporal progression on the anterioposterior axis. Axon fascicles from new ommatidial R-cell clusters arrive at the anterior margin of the lamina (adjacent to the lamina furrow) and associate with neuronal and glia precursors in a vertical lamina column assembly. At the anterior of the lamina, at the trough of the lamina furrow, LPCs await a retinal axon-mediated signal in G1-phase and enter their terminal S-phase at the posterior margin of the furrow. Postmitotic (Dac-positive) LPCs assemble into columns at the posterior margin of the furrow. In older columns at the posterior of the lamina, a subset of postmitotic LPCs express definitive neuronal markers as they become specified as the lamina neurons L1-L5. Lamina neurons L1-L4 form a stack in a superficial layer, while L5 neurons reside in a medial layer near the R1-R6 axon termini. These neurons arise at cell-type specific positions along the column's vertical axis. Lamina glial cells take up cell-type positions in the precartridge assemblies. Epithelial (E-glia) and marginal (Ma-glia) glia are located above and below the R1-R6 termini, respectively. Satellite glia are interspersed among the neurons of the L1-L4 layer. The Ma-glia and E-glia layers, both located ventral to the neuronal precursor column, sandwich the R1-R6 axon termini. The medulla neuropil serves as the target for R7/8 axons and is separated from the lamina by the medulla glia, situated just below the Ma-glia (Huang, 1998 and references).

Hedgehog, a secreted protein, is an inductive signal delivered by retinal axons for the initial steps of lamina differentiation. In the development of many tissues, Hedgehog acts in a signal relay cascade via the induction of secondary secreted factors. Lamina neuronal precursors respond directly to Hedgehog signal reception by entering S-phase, a step that is controlled by the Hedgehog-dependent transcriptional regulator Cubitus interruptus. The terminal differentiation of neuronal precursors and the migration and differentiation of glia appear to be controlled by other retinal axon-mediated signals. Thus retinal axons impose a program of developmental events on their postsynaptic field utilizing distinct signals for different precursor populations (Huang, 1998).

The activities of a number of Hh signal transduction pathway components are now well characterized. Mutations at these loci have been shown to either mimic or block Hh signal reception in a cell-autonomous fashion. Examining the cellular requirements for these genes in mosaic animals should help illuminate the cellular circuitry that mediates the Hh-dependent events of lamina development. The seven-pass transmembrane protein encoded by smoothened (smo) acts as a positive effector of Hh signal reception, downstream of the Hh receptor Patched. If Hh exerts its effects directly on LPCs, it would be expected that loss of smo function should block the entry of G1-phase LPCs into S-phase and/or prevent the expression of Hh-dependent markers of lamina differentiation such as Dac. Inducing smo mutant clones reveals that with respect to lamina differentiation, smo acts cell autonomously. smo clones that extended to the posterior of the lamina are rare. It is possible that LPCs that cannot respond to Hh are not readily incorporated into the lamina and displaced by smo+ LPCs. LPCs that are unable to respond to Hh might be eliminated by cell death (Huang, 1998).

A hallmark of Hh signal reception in many Drosophila tissues is an increase in immunoreactivity to the C-terminal portion of the protein Ci, a transcriptional mediator of Hh signaling. This enhanced Ci immunoreactivity is due to inhibition of Ci proteolytic processing, a cellular response to Hh signal reception. LPCs posterior to the lamina furrow display the enhanced Ci immunoreactivity that would be predicted for Hh signal reception by LPCs. In animals in which hh- retinal axons innervate the lamina target field, cells posterior to the lamina furrow display a level of Ci immunoreactivity equivalent to the basal level detected anterior to the furrow, indicating that the increased Ci observed in the wild type is Hh-dependent. In smo mosaic animals, smo cells either anterior or posterior to the lamina furrow display a basal level of Ci immunoreactivity, while smo + cells immediately adjacent to the portion of smo clones within the lamina display the high Hh-dependent level. The initial response of LPCs to the arrival of Hh-bearing retinal axons would appear to be entry into S-phase at the lamina furrow. To determine whether cell cycle progression is directly dependent on Hh signal reception, the incorporation of bromodeoxyuridine (BrdU) into S-phase cells was examined in smo mosaic animals. In the wild type, LPCs that have entered their terminal S-phase form a discrete and continuous band at the posterior margin of the lamina furrow. In animals lacking photoreceptor innervation (due to defective hh expression in the eye disc) or animals in which photoreceptor axons lacking functional Hh enter the lamina target field, only a low background of scattered S-phase cells are detected. It is unclear whether the products of these scattered divisions are incorporated into the lamina (i.e., that these cells are indeed LPCs). In smo 3 mosaic animals, mutant clones that include the posterior margin of the lamina furrow lack S-phase LPCs. In contrast, the scattered S-phase cells anterior to the lamina furrow, and the distribution of S-phase cells in other proliferation centers, such as the OPC, are unaffected by the loss of smo function. At the lamina furrow, smo+ cells bordering smo clones are often found in S-phase. Thus, in sum, smo+ behaves as a cell-autonomous requirement for LPCs to initiate the Hh-dependent steps of lamina differentiation (Huang, 1998).

The localized expression of Hedgehog (Hh) at the extreme anterior of Drosophila ovarioles suggests that it might provide an asymmetric cue that patterns developing egg chambers along the anteroposterior axis. Ectopic or excessive Hh signaling disrupts egg chamber patterning dramatically through primary effects at two developmental stages. (1) Excess Hh signaling in somatic stem cells stimulates somatic cell over-proliferation. This likely disrupts the earliest interactions between somatic and germline cells and may account for the frequent mis-positioning of oocytes within egg chambers. (2) The initiation of the developmental programs of follicle cell lineages appears to be delayed by ectopic Hh signaling. This may account for the formation of ectopic polar cells, the extended proliferation of follicle cells and the defective differentiation of posterior follicle cells, which, in turn, disrupts polarity within the oocyte. Somatic cells in the ovary cannot proliferate normally in the absence of Hh or Smoothened activity. Loss of protein kinase A activity restores the proliferation of somatic cells in the absence of Hh activity and allows the formation of normally patterned ovarioles. Hence, localized Hh is not essential to direct egg chamber patterning (Zhang, 2000).

The rescue of apparently normal oogenesis in hhts animals at the restrictive temperature by PKA mutations in somatic stem cells implies that there is no essential role for spatially graded Hh levels in the germarium. However, the level of Hh signaling must fall within certain bounds for oogenesis to proceed normally. Normal rates of somatic cell proliferation require some Hh signaling but also require that Ptc limits Hh signaling. Ptc must also restrain Hh signaling in order to allow somatic cells to enter the developmental program appropriate to their lineage in a timely fashion. It is not clear at this stage whether Hh signaling has any essential function in oogenesis other than stimulating cell proliferation. In one case, normal egg chambers can include smo mutant cells in a variety of positions. In particular, polar cells can form in normal numbers and at the correct position from within a group of smo mutant cells, which are presumed to be unable to transduce any Hh signal. Alternatively, in smo mutant ovarioles, egg chamber budding is sometimes arrested or defective, and normal egg chambers completely enveloped by smo mutant follicle cells have never been seen. These phenotypes might derive solely from an insufficient supply of somatic cells, resulting directly from impaired proliferation of smo mutant cells. However, the possibility cannot be dismissed that Hh signaling has a more direct role in germline cyst encapsulation, promoting egg chamber budding, or delaying somatic cell lineage decisions until the appropriate developmental stage (Zhang, 2000).

Although Hedgehog proteins most commonly affect cell fate, they can also stimulate cell proliferation. In humans several distinctive cancers, including basal-cell carcinoma, result from mutations that aberrantly activate Hh signal transduction. In Drosophila, Hh directly stimulates proliferation of ovarian somatic cells. Hh acts specifically on stem cells in the Drosophila ovary. These cells cannot proliferate as stem cells in the absence of Hh signaling, whereas excessive Hh signaling produces supernumerary stem cells. It is deduced that Hh is a stem-cell factor and it is suggested that human cancers due to excessive Hh signaling might result from aberrant expansion of stem cell pools (Zhang, 2001).

Whether Hh is required for somatic stem cell maintenance or proliferation was investigated by using conditional hh alleles and by generating somatic cell clones lacking smoothened (smo) activity. Inactivation of smo universally blocks Hh signal transduction cell-autonomously. The results demonstrate that a cell that is unable to transduce an Hh signal cannot proliferate as a somatic stem cell. It is suspected that smo mutant somatic stem cells remain abnormally quiescent for up to 7-8 d and at some point during this period acquire the characteristics of a pre-follicle cell, proliferating normally in that capacity to produce a clone occupying roughly one-third of an egg chamber (Zhang, 2001).

The primitive gonad of the Drosophila embryo is formed from two cell types, the somatic gonad precursor cells (SGPs) and the germ cells, which originate at distant sites. To reach the SGPs the germ cells must undergo a complex series of cell movements. While there is evidence that attractive and repulsive signals guide germ cell migration through the embryo, the molecular identity of these instructive molecules has remained elusive. Evidence is presented suggesting that hedgehog (hh) may serve as such an attractive guidance cue. Misexpression of hh in the soma induces germ cells to migrate to inappropriate locations. Conversely, cell-autonomous components of the hh pathway appear to be required in the germline for proper germ cell migration (Deshpande, 2001).

Known cell-autonomous components of the Hh signaling pathway also appear to be required in germ cells for normal migration behavior. Germline clones were used to test four different hh pathway genes -- ptc, pka, smo, and fu. For all four, abnormalities in germ cell migration were observed in the progeny. In the case of both the ptc and smo germline clones, eggs fertilized by wild-type sperm developed into completely normal adults. Moreover, there are no apparent defects in the formation of the somatic gonad or in the pattern of Clift expression. These findings would support the view that the migration defects seen in ptcmat-zyg+ and smomat-zyg+ embryos arise from cell-autonomous deficiencies in the response to Hh by the germ cells. However, it should be pointed out that there could be some undetected nonautonomous problem in somatic hh signaling in these embryos that induces abnormalities in germ cell behavior (Deshpande, 2001).

As would be expected from the known properties of these four genes in other well characterized hh pathways, the phenotypes produced by ptc and pka germline clones are similar and quite distinct from those observed for smo and fu. Moreover, the migration defects observed in ptc/pka and smo/fu germline clones can be explained by the antagonistic role of these genes in the hh signaling pathway. In the absence of maternal ptc or pka, smo and its downstream effectors in the hh pathway are activated in the germ cells independent of the Hh ligand. As a consequence, many of the germ cells clump together as they begin passing through the midgut, and then remain in place instead of migrating toward the SGP cells. Additionally, the mitotic cycle in ptcmat- (and to a lesser extent pkamat-) germ cells is inappropriately activated. Up regulation of cell division has been observed in somatic tumors that lack ptc function and in ptc mutant C. elegans germ cells. In the case of smo and fu, the germ cells can't respond to the Hh ligand, and they are unable to detect or associate with the SGP cells, and instead migrate randomly through the mesoderm (Deshpande, 2001).

Patched regulates Drosophila head development by promoting cell proliferation in the eye-antennal disc. During head morphogenesis, Patched positively interacts with Smoothened, which leads to the activation of Activin type I receptor Baboon and stimulation of cell proliferation in the eye-antennal disc. Thus, loss of Ptc or Smoothened activity affects cell proliferation in the eye-antennal disc and results in adult head capsule defects. Similarly, reducing the dose of smoothened in a patched background enhances the head defects. Consistent with these results, gain-of-function Hedgehog interferes with the activation of Baboon by Patched and Smoothened, leading to a similar head capsule defect. Expression of an activated form of Baboon in the patched domain in a patched mutant background completely rescues the head defects. These results provide insight into head morphogenesis and reveal an unexpected non-canonical positive signaling pathway in which Patched and Smoothened function to promote cell proliferation as opposed to repressing it (Shyamala, 2002).

Thus, a novel pathway has been uncovered by which Ptc promotes proliferation of cells in the eye-antennal disc to generate the Drosophila head capsule. Ptc, together with the enigmatic transmembrane protein Smo, promotes activation of Babo, the Activin type I receptor, to stimulate cell proliferation. Previous studies have shown that Ptc is a repressor of Smo, and the interaction of Hh and Ptc relieves this repression on Smo, allowing Smo to activate downstream genes. Ptc signaling is also known to be a suppressor of cell proliferation and loss of function for Ptc in vertebrates, for example, leads to nevoid basal carcinomas. The results described here show that Ptc signaling, in concert with Smo, can also promote cell proliferation and that this is via activation of downstream genes. Thus, these results reveal an intriguing and non-canonical mode of action by this pathway during head morphogenesis (Shyamala, 2002).

The loss of the head capsule in ptc mutants is not due to cell death, no inappropriate and massive cell death has been observed in the eye-antennal disc by the TUNEL assays. However, a lack of BrdU incorporation is observed as well as fewer phospho-histone-positive cells in the eye-antennal disc. Lack of differentiation of cells of the eye-antennal discs can also give rise to similar head capsule defects. For example, pharate adults mutant for the headcase gene show severe head capsule defects with resemblance to ptc mutants. However, in headcase mutants, the morphology, the size and the shape of the eye-antennal discs are normal and the head capsule defects appear to be due to a failure in the differentiation of cells of the eye-antennal disc. In ptc mutants, the morphology, organization, and size of the eye-antennal disc are severely affected by late 3rd instar larvae and the primary cause for the head capsule defects is loss of cell proliferation. This conclusion is further supported by the fact that an activated form of Babo completely rescues the head capsule defects in ptc mutants. babo is a known player in promoting cell proliferation and is required only for cell proliferation but not for cell differentiation in the imaginal discs. Moreover, in vitro culture of eye-antennal discs indicate that the differentiation per se is not affected in ptc mutants. Therefore, it is concluded that Ptc promotes cell proliferation in the eye-antennal disc during head development (Shyamala, 2002).

Previous studies indicate that Ptc is likely to complex with Smo and repress Smo from activating downstream target genes. Binding of Hh to Ptc frees Smo from Ptc repression, which then goes on to activate downstream target genes. Thus, Ptc has been always viewed as a suppressor of gene activity via suppressing Smo. For example, during the development of the embryonic nerve cord, loss of ptc activity leads to missing RP2 neurons. This is due to the ectopic activation of Gsb in the neuroectoderm from which the RP2 precursor neuroblast (NB4-2, a row 4 NB) delaminates; ectopic Gsb prevents Wingless signaling from specifying NB4-2 identity and therefore the loss of RP2 neurons. Consistent with the possibility that Smo is downstream of Ptc, ectopic expression of Gsb in row 4 in ptc mutants and the consequent loss of RP2 neurons is rescued in ptc, smo double mutants. If this signaling also occurs during the head development, loss of Ptc will lead to inappropriate activation of Smo, leading to the head capsule defects; loss of Smo activity in a ptc mutant background, therefore, should suppress the head capsule defects. However, reducing the dose of Smo in a ptc mutant background (smo/+, ptcgal4/ptcnull), instead of suppressing the head defects (or at least reducing the severity), enhances the head capsule defects. Moreover, loss of Smo activity leads to the same head capsule defects as in ptc mutants (Shyamala, 2002).

Previous results have indicated that Ptc might negatively regulate levels of Smo via vesicular trafficking of Smo from the cell surface. Thus, in ptc mutants it has been inferred that the level of Smo on the membrane is high, leading to the inappropriate activation of downstream target genes. That a similar mechanism might operate during head capsule development is unlikely for the following reasons: (1) reducing the dose of smo in ptc mutant background enhances the phenotype; (2) in one of the ptc alleles, ptcS2, the mutation is an amino acid change from charged to neutral in the sterol-sensing domain. ptcS2 fully complements ptchdl and the transheterozygotes have no head capsule defects. Moreover, in ptchdl/ptcnull mutant eye-antennal disc, the level of Smo is not upregulated. Based on these results, it is concluded that a positive signaling by Ptc and Smo regulates cell proliferation during head development (Shyamala, 2002).

In the conventional Ptc-signaling, interaction of Hh with Ptc relieves the repression on Smo, thus allowing Smo to function. When Hh is ectopically expressed, it interacts with Ptc to relieve the repression on Smo. This in turn is thought to cause phenotypes in hh gain-of-function situations. Thus, in the CNS, for example, loss of Ptc activity from the RP2 neuronal precursor cell leads to missing RP2 neurons; ectopic expression of Hh in adjacent rows of cells leads to loss of RP2 neuron via inappropriate activation of Gsb in the neuroectoderm from which NB4-2 is delaminated. The results described in this paper, that during head development gain-of-function Hh mimics a loss of function ptc phenotype, are not inconsistent with the finding that Ptc, together with Smo, promotes cell proliferation. That is, ectopic expression of Hh will bind to Ptc and this will interfere with the positive signaling by Ptc and Smo. One possibility is that Ptc and Smo are physically associated with one another, and binding of Hh to Ptc will break this physical association, rending Ptc or Smo unable to positively regulate cell proliferation in the eye-antennal disc (Shyamala, 2002).

The results indicate that Ptc-Smo signaling leads to the activation of Babo. During Activin signaling, Activin binds to Activin type II receptor, which promotes physical interaction between type II and type I receptors and the phosphorylation of type I receptor. Both type I and type II receptors are transmembrane serine/threonine kinases. Phosphorylation of the type I receptor results in the activation of its kinase activity and the phosphorylation of downstream transcription activators such as the Smad proteins, resulting in their nuclear localization. In Drosophila, analysis of null mutants for the type I receptor babo, as well as analysis of babo germline clones, indicates that babo is not required during embryogenesis but is essential during pupal development and adult viability. The major defect in babo mutants is a reduction of cell proliferation in the imaginal discs and brain tissue. It has also been shown that in tissue culture experiments, a constitutively active form of Babo can signal to vertebrate TGF-ß/Activin, but not to BMP-responsive promoters. The activated Babo then interacts with Drosophila Smad2 to effect the nuclear localization of this transcription factor (Shyamala, 2002).

These results, that expression of an activated form of Babo in the ptc-expression domain in the eye-antennal disc of ptc mutants completely rescues the head capsule defects, indicates that Ptc-Smo signaling ultimately leads to activation of Babo and promotes cell proliferation in the eye-antennal disc. Since babo and ptc show transheterozygous interaction, it is tempting to speculate that the interaction between Ptc and Babo might be direct. A transheterozygous interaction is generally observed in several cases where the two proteins associate with one another, in cases such as the receptor-ligand pairs Notch and Delta. However, it is also possible that Ptc-Smo signaling and Babo signaling represent parallel pathways that converge at the point of cell cycle control. In this scenario, partial reduction in each could have a synergistic negative affect on cell proliferation, while overexpression of one (i.e. activated Babo) could compensate for loss of the other. Yet another possibility would be that the Pt-Smo pathway activates one of the Activin-like ligands. While the results indicate that there is no transheterozygous genetic interaction between ptc and punt (the inferred type II receptor for Activin), the possibility cannot be ruled out that the Ptc-Smo pathway does not interact with Punt. This is due to the fact that a lack of transheterozygous interaction does not mean that the two players do not interact, as it actually depends on what is limiting. Nonetheless, the finding that Ptc, together with Smo stimulates cell proliferation and the interfacing of Ptc-signaling with Babo-signaling in this process provides new insight into the process of head development (Shyamala, 2002).

The development of multicellular organisms requires the establishment of cell populations with different adhesion properties. In Drosophila, a cell-segregation mechanism underlies the maintenance of the anterior (A) and posterior (P) compartments of the wing imaginal disc. Although engrailed (en) activity contributes to the specification of the differential cell affinity between A and P cells, recent evidence suggests that cell sorting depends largely on the transduction of the Hh signal in A cells. The activator form of Cubitus interruptus (Ci), a transcription factor mediating Hh signaling, defines anterior specificity, indicating that Hh-dependent cell sorting requires Hh target gene expression. However, the identity of the gene(s) contributing to distinct A and P cell affinities is unknown. A genetic screen based on the FRT/FLP system has been to search for genes involved in the correct establishment of the anteroposterior compartment boundary. By using double FRT chromosomes in combination with a wing-specific FLP source, 250,000 mutagenized chromosomes were screened. Several complementation groups affecting wing patterning have been isolated, including new alleles of most known Hh-signaling components. Among these, a class of patched (ptc) alleles was identified exhibiting a novel phenotype. These results demonstrate the value of this setup in the identification of genes involved in distinct wing-patterning processes (Végh, 2003).

A total of 250,000 mutant chromosomes covering the X chromosome and both major autosomes were screened. Four complementation groups were identified that affected wing patterning similar to mutations in smo. The largest of these groups represents alleles in smo itself. Two groups exhibiting a subset of smo phenotypes represent new alleles of fused and collier/knot. Fused is a positive regulator of Hh signaling, and collier/knot is an Hh target gene required for the formation of the L3/L4 intervein region. Surprisingly, the remaining complementation group turned out to consist of novel ptc alleles with striking characteristics. Molecularly, they represent point mutations causing an amino acid substitution in either the first or the second large extracellular loop. In contrast to ptc null alleles, homozygous mutant clones failed to upregulate Hh target genes even in the presence of Hh. Together these findings suggest that the mutant proteins repress Smo constitutively, most likely because they fail to bind Hh. Animals mutant for trans-heterozygous combinations of these new ptc alleles with ptcS2 are fully viable. The ptcS2 product lacks the ability to repress Smo but is able to sequester, and hence bind to, Hh. The intragenic complementation that was observed suggests that both functions of Ptc, binding of Hh and repression of Smo, can be provided by individual proteins that possess only one of each. Recently, it was shown that a combination of two proteins, one consisting of the N- and the other the C-terminal half of Ptc, reconstitutes Ptc function. Although these experiments cannot be directly compared with the findings in this study, together they do suggest that Ptc function can be separated intramolecularly into independent modules of N- vs. C-terminal and extra- vs. intracellular domains. One possible scenario that could explain the intragenic complementation would be if Ptc proteins act in a multimeric complex (Végh, 2003).

Induction and autoregulation of Bar during retinal neurogenesis

Neurogenesis in the Drosophila eye imaginal disc is controlled by interactions of positive and negative regulatory genes. The basic helix-loop-helix (bHLH) transcription factor Atonal (Ato) plays an essential proneural function in the morphogenetic furrow to induce the formation of R8 founder neurons. Bar homeodomain proteins are required for transcriptional repression of ato in the basal undifferentiated retinal precursor cells to prevent ectopic neurogenesis posterior to the furrow of the eye disc. Thus, precise regulation of Bar expression in the basal undifferentiated cells is crucial for neural patterning in the eye. Evidence is shown that Bar expression in the basal undifferentiated cells is regulated by at least three different pathways, depending on the developmental time and the position in the eye disc. (1) At the time of furrow initiation, Bar expression is induced independent of Ato by Hedgehog (Hh) signaling from the posterior margin of the disc. (2) During furrow progression, Bar expression is also induced by Ato-dependent EGFR (epidermal growth factor receptor) signaling from the migrating furrow. (3) Once initiated, Bar expression can be maintained by positive autoregulation. Therefore, it is proposed that the domain of Bar expression for Ato repression is established and maintained by a combination of non autonomous Hh/EGFR signaling pathways and autoregulation of Bar (Lim, 2004).

Hh expression is dynamic, depending on the time and the position in the developing eye disc. In the early third instar eye disc, Hh is expressed in the posterior margin and is required for the furrow initiation. During furrow progression, Hh is also produced in the differentiating photoreceptor cells generated posterior to the furrow and secreted anteriorly to promote furrow progression. During this process, Bar is specifically expressed in the basal undifferentiated cells posterior to the furrow and inhibits ectopic retinal neurogenesis by repressing proneural gene ato expression (Lim, 2004).

Hh signaling is required for Bar expression in the basal undifferentiated cells during initial eye development because Bar expression is strongly reduced or absent within smo LOF clones generated near the furrow or close to the posterior margin of the disc. Prior to the photoreceptor differentiation, Hh expressed in the posterior margin of the disc is responsible for Bar expression at specific distances from the posterior region of the eye disc proper. A graded expression of Bar near the posterior region in ato1 mutant eye disc might be the effects of Hh secreted by the posterior margin (Lim, 2004).

During furrow progression, Hh signaling is required for Ato expression in the furrow, and Ato-mediated EGFR signaling is required for Bar activation. Therefore, it is possible that the loss of Bar expression near the furrow in smo LOF clones might be caused by indirect effects of reduced Ato expression rather than by direct effects of Hh signaling on Bar expression. Hh may partially contribute to Bar expression by activating normal levels of Ato expression in the furrow. Thus, the Hh-Ato-EGFR cascade activates Bar expression just posterior to the furrow. Alternatively, since Hh signaling may also affect furrow progression, it is possible that the loss of Bar expression near the furrow in smo LOF clones might be caused by indirect effects of slow furrow migration rather than by direct effects of Hh signaling on Bar expression (Lim, 2004).

Functional domains and sub-cellular distribution of the Hedgehog transducing protein Smoothened in Drosophila

The Hedgehog signalling pathway is deployed repeatedly during normal animal development and its inappropriate activity is associated with various tumours in human. The serpentine protein Smoothened (Smo) is essential for cells to respond to the Hedeghog (Hh) signal; oncogenic forms of Smo have been isolated from human basal cell carcinomas. Despite similarities with ligand binding G-protein coupled receptors, the molecular basis of Smo activity and its regulation remains unclear. In non-responding cells, Smo is suppressed by the activity of another multipass membrane spanning protein Ptc, which acts as the Hh receptor. In Drosophila, binding of Hh to Ptc has been shown to cause an accumulation of phosphorylated Smo protein and a concomitant stabilisation of the activated form of the Ci transcription factor. This study identifies domains essential for Smo activity and investigates the sub-cellular distribution of the wild type protein in vivo. Deletion of the amino terminus and the juxtamembrane region of the carboxy terminus of the protein result in the loss of normal Smo activity. Using Green Fluorescent Protein (GFP) and horseradish peroxidase fusion proteins it was shown that Smo accumulates in the plasma membrane of cells in which Ptc activity is abrogated by Hh but is targeted to the degradative pathway in cells where Ptc is active. It was further demonstrated that Smo accumulation is likely to be a cause, rather than a consequence, of Hh signal transduction (Nakano, 2004).

The Smo protein is an essential component of the Hh signal transduction pathway: in all contexts analysed to date, inactivation of Smo renders cells incapable of responding to Hh. Reciprocally, gain of function mutations have been isolated in human Smo that are sufficient to activate the Hh pathway in the absence of ligand (Nakano, 2004).

Hh signalling regulates Smo activity via Ptc. In the absence of Hh ligand, Ptc represses Smo activity whereas when Hh binds to Ptc, Smo becomes active. Immunoprecipitation studies of vertebrate Smo from tissue culture cells co-expressing Ptc and Smo have suggested that this Ptc-dependent modulation of Smo activity is mediated by a direct interaction between the two proteins, a view consistent with the effects of high level Smo expression in cultured cells carrying a Shh-responsive reporter gene. Studies in Drosophila, however, have suggested that Ptc suppresses Smo activity by regulating the sub-cellular distribution and stability of the protein in a non-stoichiometric manner. The relatively weak effects of ectopic Smo expression in the wing imaginal disc, presumably reflect this sub-stoichiometric regulation of the exogenously supplied protein by endogenous Ptc activity. Consistent with this, it was found that ectopically expressed Smo protein is clearly subject to Ptc-mediated destabilisation, as revealed by the distribution of the GFP and YFP tagged forms of Smo. Nevertheless, when expressed at very high levels, as occurs for instance using the 30A GAL4 driver line, the Smo protein can escape the effects of Ptc activity and accumulate in anterior compartment cells where it activates Hh target gene activity. Even under these circumstances, however, the phenotypic effects of its over-expression are relatively mild (Nakano, 2004).

Smo shares limited sequence homology with members of the Frizzled family of Wnt receptors, in particular in the Cys rich domain (CRD) of the putative N-terminal ECD that in Fz has been shown to be necessary and sufficient for Wnt binding. By contrast with Fz, there is no evidence that the ECD of Smo binds Wg (or other Wnts) or indeed Hh, consistent with the finding that Smo activity is independent of Hh in the absence of the inhibitory effects of Ptc. Nevertheless, of the five Smo point mutants identified in this study, three affect residues in the N-terminal ECD, including two Cys residues that are highly conserved in all Fz family members, suggesting ECD and the CRD in particular play some critical role in Smo function. In line with this, it was found that deletion of the ECD results in a loss of function of the protein when assayed both in mutant rescue and imaginal disc ectopic expression systems. One explanation for this requirement could be that the N-terminus binds an as yet unidentified ligand that is required even in the absence of Ptc repressive activity, or indeed the secretion of which is inhibited by Ptc. Alternatively, the ECD may itself function as an activating ligand. The protease-activated G-protein coupled receptors (PARs) provide a precedent for such intramolecular receptor activation. PARs are activated by proteolytic cleavage of the ECD, the tip of the new amino terminus created by this cleavage acting as a tethered ligand by binding to the receptor core. It is emphasised, however, that there is no evidence that Smo undergoes a similar proteolytic activation. In contrast to these findings, it has previously been demonstrated that an N-terminally deleted form of human Smo retains activity when over expressed in 10T1/2 cells. The disparity between this result and the current findings may reflect differences in the levels of over-expression achieved in the in vivo versus the in vitro systems; it may be that at high enough levels, the mutant form of Smo can saturate the repressive activity of endogenous Ptc in the 10T1/2 cells, thus leading to the activation of the endogenous wild type Smo protein (Nakano, 2004).

Since no mutations have been identified in the CTD of Smo, the requirement for this region was also investigated by deletion analysis. In contrast to the results of in vitro studies of human Smo suggesting that the terminal domain is dispensable, this study found that deletion of the entire domain or even 80% of it completely inactivates the protein. Only deletions that preserve the juxtamembrane half of the C-terminal tail retain any activity; notably it is this part of the C-terminus that has been highly conserved between Drosophila and vertebrates (Nakano, 2004).

Although these results demonstrate a requirement for carboxy terminal regions of the protein, they do not give any direct indications as to their molecular function. For example, structures in the carboxy terminus could be needed to keep other parts of Smo in the correct conformation. Alternatively, the carboxy terminus could bind directly to another protein or proteins that transduce the Hh signal to the multimeric protein complex that regulates Ci stability and activation, or indeed to the multimeric complex itself. Amongst the new smo mutant alleles that were isolated, one, smo2A, is a missense mutation that converts a highly conserved arginine to a cysteine at amino acid position 474 in the third intracellular loop. Residues at the C-terminal end of i3 in GPCRs have been shown to be critical for appropriate coupling to heterotrimeric G-proteins: loss of charged residues in this region of the angiotensin II receptor type I abolishes such coupling. While it has been shown that Frizzled proteins can transduce Wnt signals via heterotrimeric G-proteins, the evidence for G-protein involvement in Smo activity remain equivocal (Nakano, 2004).

Both smo4DI and smo2A hypomorphic mutations reside in the heptahelical domain of the protein, the structural integrity of which has been implicated in Smo activity. Small molecule agonists and antagonists of vertebrate Smo have been shown to bind to this domain, implying its involvement in regulating the activity of the protein. Exactly how these small molecules influence Smo activity is unclear, but one suggestion is that they may alter the conformation of the protein, respectively, towards a more active or inactive state. In this view, the normal regulation of Smo by Ptc might involve the transport of a cellular small molecule agonist/antagonist that similarly binds to Smo, altering its conformation. Alternatively, Ptc might act to redistribute Smo to a membrane compartment where it is susceptible to such an antagonist ligand or inaccessible to an agonist (Nakano, 2004).

Analysis of the sub-cellular distribution of tagged forms of the Smo protein in Drosophila tissues is consistent with a role for Ptc in the intracellular trafficking of Smo. Specifically it was found that Smo accumulates in the plasma membrane of cells that lack Ptc activity, whereas when co-expressed with Ptc, the same protein localises exclusively intracellularly, accumulating in distinct punctate structures. Similar results using the salivary gland as an assay system have been reported. Significantly, electron microscopy analysis of the HRP::Smo fusion proteins reveals high levels of HRP activity in the lysosomes of cells in which Ptc is active whereas in Ptc negative cells, the protein accumulates predominantly in the plasma membrane as well as in endosomes and unidentified presumptive transport vesicles (Nakano, 2004).

Analysis of the localisation and activity of putative hypermorphic forms of Smo might afford an insight into the mechanism of Smo function. Interestingly, when driven with ap-GAL4, the putative hypermorphic form of Smo, GFP::SmoA479, accumulates in the plasma membrane irrespective of Ptc activity and yields a stronger phenotype than GFP::Smo expressed under the same conditions, as assayed by dpp-lacZ expression. Surprisingly, however, the resulting adult phenotype appears to be no more severe in the case of the putative hypermorph than the wild type form. Furthermore, the over-expression of SmoA479Y as well as two other putative gain of function forms of Smo, SmoK580Q and SmoW553L, directed by the 30A and 71B GAL4 drivers result in lower levels of ectopic dpp-lacZ activation than those induced by wild type Smo. Consistent with this, the resulting adult phenotypes are also weaker in the case of the mutant forms of Smo than their wild type counterpart. It has been reported, however, that the over-expression of certain putative hypermorphic forms of Smo results in stronger phenotypes than wild type Smo. The reason for these disparities is currently unclear (Nakano, 2004).

Finally, observations that the cellular distribution of Smo is unaffected in imaginal disc clones lacking the activity of PKA or Cos2 (and hence constitutively transducing the Hh signal) has suggested Hh signalling is not sufficient to stabilise Smo and that Smo stabilisation is therefore more likely to be a cause, rather than an effect, of Hh signal transduction. However, it has been observed that over-expression of Cos2 in salivary gland cells can result in Smo destabilisation, suggesting that Cos2 activity is capable of influencing Smo levels. Taken together with the current data, this suggests that antagonising Cos2 activity might be a necessary, but not sufficient, prerequisite for Smo stabilisation at the cell surface (Nakano, 2004).

In conclusion, the data favour a mechanism whereby Ptc regulates Smo activity through inhibition of its accumulation in the plasma membrane, targeting it instead to the lysosomal pathway. In this view, Ptc might regulate Smo activity simply by modulating the levels of protein present in the cell. Alternatively, it may be that the sub-cellular location of Smo is critical for its activation, the plasma membrane perhaps providing an environment in which Smo is accessible to an intracellular agonist (Nakano, 2004).

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, 2006a).

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, 2006a).

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, 2006a).

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, 2006a).

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, 2006a).

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, 2006a).

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, 2006a).

In some instances, the data contrasts with previous reports from others. In one case, in which different alleles of smoothened (smo3 versus smoD16) 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, 2006a 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, 2006a).

smoothened and thickveins regulate Moleskin/Importin 7-mediated MAP kinase signaling in the developing Drosophila eye

The Drosophila Mitogen Activated Protein Kinase (MAPK) Rolled is a key regulator of developmental signaling, relaying information from the cytoplasm into the nucleus. Cytoplasmic MEK phosphorylates MAPK (pMAPK), which then dimerizes and translocates to the nucleus where it regulates transcription factors. In cell culture, MAPK nuclear translocation directly follows phosphorylation, but in developing tissues pMAPK can be held in the cytoplasm for extended periods (hours). This study shows that Moleskin antigen (Drosophila Importin 7/Msk), a MAPK transport factor, is sequestered apically at a time when lateral inhibition is required for patterning in the developing eye. It is suggested that this apical restriction of Msk limits MAPK nuclear translocation and blocks Ras pathway nuclear signaling. Ectopic expression of Msk overcomes this block and disrupts patterning. Additionally, the MAPK cytoplasmic hold is genetically dependent on the presence of Decapentaplegic (Dpp) and Hedgehog receptors (Vrailas, 2006b).

Early in eye development, all cells anterior to the furrow (phase 0) are primed for Ras-induced neural differentiation; ectopic activation of the pathway causes all cells to differentiate as photoreceptors, even without atonal. Normally these cells are thought to receive only low levels of Egfr-mediated Ras signaling, supporting proliferation but not differentiation. Later, in the furrow (phase 1), Delta-induced, Notch-mediated lateral inhibition progressively restricts Atonal expression to single founder cells. Suspension of Ras signaling is required for this inhibition in order to avoid premature neuronal differentiation, and it has been proposed that this inhibition is mediated by MAPK cytoplasmic hold. However, this block to the Ras pathway must be released in phase 2 (posterior to the furrow) to allow for developmental induction by the R8 cell. To better understand how MAPK cytoplasmic hold is maintained in phase 1, the role was examined of the pMAPK nuclear transport factor Drosophila Importin 7/Msk, in eye development (Vrailas, 2006b).

It is suggested that in wild-type eye discs, the level of pMAPK antigen is a very misleading reporter of Egfr/Ras pathway activity, because cytoplasmic hold in phase 1 allows even a relatively low level of pathway activity to build up high levels of pMAPK antigen. A system has been developed to reveal MAPK nuclear translocation without the use of an antibody (MG-driven reporter gene expression that reveals MAPK nuclear translocation). [Note: MG (Mapk-Gal4vp16) contains the entire sequence of Rolled, followed by the yeast GAL4 DNA binding domain (which is not known to contain a nuclear localization signal) with an acidic activation domain from herpes simplex virus protein 16]. However, it has been since found that under all conditions tested, MG-driven reporter expression does not reveal nuclear MAPK in phase 0, where Ras pathway activation is required. MG-driven reporter expression is reliably see in phase 2, where there is thought to be high (or sustained) levels of Ras pathway activity. In phase 1, the level of pathway signaling may be insufficient for expression, and thus MG-driven reporter expression may reveal only high (or sustained) levels of nuclear MAPK. Alternatively, this could be caused by a technical limitation: the hsp70 promoter drives the expression of only low levels of MG protein. Therefore, two less direct assays were used, that together, are interpreted as revealing the loss of MAPK cytoplasmic hold in the furrow: (1) loss of Atonal expression (as previously demonstrated by fusing an SV40 NLS to MAPK and by the ectopic expression of Rasv12); and (2) loss of pMAPK antigen, which may be due to exposure to a nuclear phosphatase/protease (Vrailas, 2006b).

The MAPK nuclear transport factor Drosophila Importin 7/Msk is apically sequestered in phase 1, the time when pMAPK nuclear access is blocked. Furthermore, ectopic Msk is sufficient to break the cytoplasmic hold in the furrow, as seen by loss of pMAPK antigen and suppression of the early stages of Atonal expression. However, this transient expression of Msk is unable to promote the precocious neural differentiation or the increase in rough expression, as has been seen with hs:rasv12 or nuclear-directed MAPK. Because ectopic rasv12 produces an increase in pMAPK, and the phosphorylation state of nuclear-directed MAPK is not required for nuclear translocation, it may be that the available pool of pMAPK that can be imported into the nucleus by Msk is enough to affect Atonal expression, but not to affect Elav or Rough expression. Genetic evidence shows that the MAPK cytoplasmic hold depends on the Hedgehog receptor Smo and is enhanced by the loss of the Dpp receptor Tkv. smo loss-of-function clones reduce Atonal and pMAPK expression, whereas tkv clones have much weaker effects. However, the loss of smo and tkv together completely abolishes both pMAPK and Atonal expression in the furrow. This is consistent with a previous report of the loss of Atonal expression in smo tkv clones. Additionally, MAPK cytoplasmic hold in smo tkv clones is rescued by the additional loss of msk. Thus, msk genetically antagonizes pMAPK levels in the morphogenetic furrow: msk gain-of-function reduces pMAPK and msk loss-of-function (in smo tkv clones) increases it (Vrailas, 2006b).

Hedgehog signaling has also been reported as a positive regulator of Atonal on the anterior side of the furrow and as a negative regulator (perhaps through Rough or Bar) on the posterior side. However, the inductive effect of Hedgehog on Atonal appears to be independent of the Hedgehog pathway transcription factor Ci, which is consistent with an indirect effect through the MAPK cytoplasmic hold. smo tkv msk triple mutant clones were used to show that msk is genetically epistatic to smo and tkv in the furrow, and suggest that Msk sequestration in the furrow is required for MAPK cytoplasmic hold, and that smo and tkv are genetically upstream of this sequestration of Msk. Indeed, loss of smo and tkv results in a disruption of the actin cytoskeleton in the furrow, as well as of expression of Egfr and other signaling molecules. The loss of apical constriction may therefore disrupt Msk apical sequestration in such a way as to allow precocious Msk-mediated pMAPK nuclear import (Vrailas, 2006b).

What is more surprising is that differentiation and ommatidial assembly, which are known to require Ras signaling and MAPK nuclear translocation, occur normally in the absence of Msk in phase 2. It may be that cytoplasmic MAPK targets are important for ommatidial assembly or that pMAPK can translocate into the nucleus by some Ran-independent mechanism. However, the possibility is favored that, in phase 2, other (possibly redundant) transport factors are expressed (Vrailas, 2006b).

Like the Ras pathway, msk plays a role in ommatidial rotation but not chirality. It may be that in the absence of Msk, enough pMAPK can translocate into the nucleus for ommatidial assembly, but not enough for proper rotation. Additionally, in phase 0, Msk is found to be required for proliferation, which also requires Ras signaling. Therefore, Msk is required for some pMAPK nuclear translocation in phase 0 and phase 2, but is not necessary in phase 1, in order to allow for the initial specification of the Atonal-positive R8 (Vrailas, 2006b).

To conclude, the apical sequestration of Drosophila Importin 7/Msk in the morphogenetic furrow has been identified and it is suggested that this may be required for the MAPK cytoplasmic hold in the developing eye. Cytoplasmic hold is required to allow initial patterning through lateral inhibition and the focusing of the proneural factor Atonal. It is further suggested that this is mediated by the combined action of Hedgehog and Dpp (Vrailas, 2006b).

A screen for modifiers of Hedgehog signaling in Drosophila melanogaster identifies swm and mts

Signaling by Hedgehog (Hh) proteins shapes most tissues and organs in both vertebrates and invertebrates, and its misregulation has been implicated in many human diseases. Although components of the signaling pathway have been identified, key aspects of the signaling mechanism and downstream targets remain to be elucidated. An enhancer/suppressor screen was performed in Drosophila to identify novel components of the pathway and 26 autosomal regions were identified that modify a phenotypic readout of Hh signaling. Three of the regions include genes that contribute constituents to the pathway: patched, engrailed, and hh. One of the other regions includes the gene microtubule star (mts) that encodes a subunit of protein phosphatase 2A. mts is necessary for full activation of Hh signaling. A second region includes the gene second mitotic wave missing (swm). swm is recessive lethal and is predicted to encode an evolutionarily conserved protein with RNA binding and Zn+ finger domains. Characterization of newly isolated alleles indicates that swm is a negative regulator of Hh signaling and is essential for cell polarity (Casso, 2008).

This screen identified twenty-six autosomal regions that modified a smo hypomorphic phenotype in a dosage-sensitive manner. Two aspects of its design were key to its success. First, its two-generation crossing scheme eliminated background effects by homogenizing the genetic backgrounds of both experimental and control flies. It also generated reasonably large numbers of both classes of progeny so that a good estimate of an average phenotype could be obtained. These features allowed monitoring of subtle variations in wing vein morphology, despite the significant strain differences among the many lines tested. Second, its high scoring threshold rendered it relatively insensitive to changes in Hh signaling strength, thereby helping to submerge weak influences. Key to this property was the ptcGAL4 driver that was used to express smo RNAi; it functioned in part as a 'genetic buffer.' Since ptcGAL4 is itself responsive to Hh, a modifier that increased Hh signaling would also be predicted to increase the expression of ptcGAL4 and smo RNAi, while a modifier that decreased Hh signaling might be expected to decrease the expression of the ptcGAL4 and smo RNAi. ptcGAL4 therefore buffered against changes in signaling strength and decreased the effects of genetic factors that enhance or suppress signaling; as a consequence, only highly penetrant and consistent phenotypes were scored (Casso, 2008).

The screen netted many of the known core components of the Hh signaling pathway, including smo, ptc, hh, and en. mts and swm were two genes whose haplo-insufficiency phenotypes were sufficiently strong to score above the threshold set by the genetic tests. Many other known regulators of Hh signaling were not identified in this screen. There are perhaps multiple reasons, including the high scoring threshold of the smo RNAi screen, or the possibility that not all pathway regulators have haplo-insufficiency phenotypes. skinny hedgehog or suppressor of fused were not included among those identified in the screen, despite the fact that deficiencies that removed them interacted with smo RNAi. The reason is that mutant alleles of these genes that were tested did not yield similar interaction phenotypes. Since many examples were observed of interaction between null alleles of Hh pathway regulators and smo RNAi but consistent failure of hypomorphic alleles to interact, lack of interaction is not viewed as evidence against a gene being a smo RNAi enhancer/suppressor. The possibility that stronger alleles might interact cannot be discounted. It was surprising that hemizygosity of cos2 did not show an interaction with smo RNAi. This could be because it is not haplo-insufficient in the particular assay or because of the complex positive and negative roles cos2 plays in Hh signaling. Finally, there was no apparent overlap between the regions identified and the mutant lines that were identified in previous screens for modifiers of Hh phenotypes; the smo RNAi assay may be less sensitive but more specific (Casso, 2008).

mts lies within 1 of 16 regions that enhanced the smo RNAi phenotype, suggesting that its wild-type function augments the Hh response. mts encodes the catalytic (C) subunit of PP2A, a heterotrimeric phosphatase that has two regulatory subunits, B and B'. It was previously identified as a Hh pathway regulator in CL8 cells (Nybakken, 2005); the current study provides in vivo evidence for a role in Hh signaling during development. Three proteins in Hh signal transduction have been shown to be functionally phosphorylated. Phosphorylation of the Smo C terminus is induced by Hh and is required for surface accumulation of Smo and normal activation of the pathway. Thus, reduction of PP2A activity and increased phosphorylation of Smo would not be expected to decrease Hh signaling and enhance the smo RNAi phenotype. Other possible targets of Mts are Ci and Cos2. Phosphorylation of Ci by PKA, casein kinase 1α, and GSK3β is required to convert Ci from its full-length form to its transcriptional repressor form, Ci-75. Hh signaling blocks this proteolytic transformation and also promotes conversion of Ci to an activator form. A decrease in phosphatase activity might increase levels of phosphorylated Ci to effect enhanced conversion to Ci-75 and reduced levels of Ci activator. Levels of Hh signaling would be predicted to decrease. Alternatively, Mts might control phosphorylation of Cos2 by Fu. Phosphorylation of Cos2 prevents its binding to Smo and release of Smo from Cos2 increases the cell surface accumulation of Smo that is necessary for pathway activation. Therefore, a reduction of Smo on the plasma membrane due to loss of PP2A activity might attenuate Hh pathway activation (Casso, 2008).

While the catalytic subunit of PP2A carries enzymatic phosphatase activity, the substrate specificity of PP2A is directed by its regulatory subunits. The phenotypes of mutants in genes that encode the B and B' regulatory subunits of PP2A, twins and widerborst (wdb), respectively, are interesting to consider in the context of Hh signaling. Wing discs in the twinsP mutant have mirror symmetrical posterior compartment duplications that are associated with ectopic compartment borders. Symmetric wing duplications have also been observed after ectopic expression of Hh or Dpp, or after loss of en/inv induces an ectopic compartment border. Since loss of PP2A function should reduce Hh signaling, it is not obvious how loss of the B twins regulatory subunit leads to an ectopic signaling center. Understanding this interesting aspect of the twins phenotype warrants further investigation (Casso, 2008).

Misexpression of PP2A can cause cell planar polarity defects in the wing. Misexpression of mts, wdb, or mutant alleles of these genes disrupted wing hair polarity. Like mts, reducing wdb expression with RNAi reduced Hh signaling in CL8 cells (Nybakken, 2005). This evidence, as well as the wing hair polarity phenotype of swm mutants, raises the possibility that PP2A links Hh signaling with cell polarity. The PCP and Hh pathways may be parallel and independent if PP2A activity is simply common to both, but evidence that Hh is required to establish PCP in the Drosophila embryonic and adult epidermis has recently been described. The phenotype of swm mutants provides additional evidence for an association of Hh signaling with cell polarity (Casso, 2008).

swm was first identified as l(2)37Dh in a screen for recessive lethal alleles within Df(2L)E55 (37D2-38A1). It was shown to exhibit synthetic lethality as an enhancer of Minutes. Among the mutant chromosomes from the current screen that failed to complement swm, one had a Minute-like phenotype. No changes in the swm coding sequence were found in this mutant; rare escapers that eclosed as heterozygotes with the verified swm alleles had a variety of phenotypes including loss of ocelli, thin macrochetae, and deformed legs. In contrast to swm mutant escapers, however, both their eyes and wings were phenotypically normal (not shown) (Casso, 2008).

swm was identified as a suppressor of the roughex eye phenotype. Alleles of ptc were also isolated in this screen. These interactions between rux, ptc, and swm were confirmed. Since Ptc is a negative regulator of the Hh pathway and ptc mutations are therefore likely to elevate Hh signaling, and since Hh plays a key role in eye morphogenesis, the rux phenotype is apparently sensitive to Hh levels. Therefore the identification of both ptc and swm mutants as rux suppressors is interpreted as a consequence of the same mechanism -- an increase in Hh signaling caused by a decrease in the level of a negative regulator (Casso, 2008).

The results provide several additional lines of evidence that swm negatively regulates Hh signaling. swm mutants dominantly suppress smo hypomorphic phenotypes (smo RNAi and smo5A, enhance a Hh gain-of-function phenotype (hhMrt), and increase targets of Hh signaling such as Ptc and Ci. These effects on Hh signaling seem to occur through swm activity in the anterior compartment since swm RNAi expressed in these cells is sufficient to suppress smo RNAi. Although these interactions implicate Swm, it has not been determined how and where Swm impacts signal transduction or what its molecular function might be. Swm protein has features suggestive of a function in nucleic acid metabolism -- it has a putative RRM RNA binding domain and a CCCH Zn+ finger, and a GFP-Swm fusion that was examined localized to nuclei in cultured cells. Presumably, Swm affects expression, production, or presentation of proteins involved in Hh signaling or signal transduction. However, swm function is not specific to Hh signaling, since many aspects of the phenotype (e.g., ectopic venation, wing hair polarity, cell size, and interaction with Minutes) are not attributable to defects in Hh signaling (Casso, 2008).

swm is expressed broadly in both embryos and larvae, and in wing discs, it appears to be required in all cells. Null alleles, which are cell lethal in a swm/+ background, share some, but not all characteristics of Minute ribosomal protein mutants. Although swm mutants do not have thin bristles as is characteristic of Minutes, they are recessive lethal and developmentally delayed, and they interact genetically with Minutes and Minute-like loci. The wings of the Minute locus RpL38 have defects which are similar to swm wings -- extra venation, expanded distance between veins 3 and 4, wing hair polarity abnormalities, and increased cell size. Although RpL3845-72, Df(2R)M41A10, and M41A4 suppressed hhMrt, they did not interact with smo RNAi or smo5A (Casso, 2008).

While the interactions between swm and Minutes, as well as the similar phenotypes of swm and the RpL38 genes, might indicate a direct role in ribosome function, both Drosophila Swm and one of its two vertebrate homologs (RBM-27) are nuclear. The presence of RRM sequences in Swm and its homologs might suggest a role in RNA binding or metabolism, and the RRM of RBM-27 binds RNA. However, RRMs can have a structural role in protein-protein interactions independent of RNA binding, so the molecular function of Swm and its homologs cannot be determined by genetic methods alone. The fact is intriguing that the other vertebrate homolog, RBM-26, was identified as se70-2, an autoantigen that is recognized by sera of cutaneous T-cell lymphoma patients and has been used as a diagnostic marker for this tumor. In addition, the mouse RBM-26/se70-2 locus was identified as one of four genes deleted in a region required for normal murine skeletal, cartilage, and craniofacial development. Perhaps the roles of Hh that extend beyond pattern formation to cell cycle regulation, growth control, and cell polarity signify that Hh signal transduction integrates inputs from all three pathways. The pleiotropy of swm and mts may reflect these multiple inputs (Casso, 2008).

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 Gli3Xt 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).

Hh signalling is essential for somatic stem cell maintenance in the Drosophila testis niche

In the Drosophila testis, germline stem cells (GSCs) and somatic cyst stem cells (CySCs) are arranged around a group of postmitotic somatic cells, termed the hub, which produce a variety of growth factors contributing to the niche microenvironment that regulates both stem cell pools. This study shows that CySC but not GSC maintenance requires Hedgehog (Hh) signalling in addition to Jak/Stat pathway activation. CySC clones unable to transduce the Hh signal are lost by differentiation, whereas pathway overactivation leads to an increase in proliferation. However, unlike cells ectopically overexpressing Jak/Stat targets, the additional cells generated by excessive Hh signalling remain confined to the testis tip and retain the ability to differentiate. Interestingly, Hh signalling also controls somatic cell populations in the fly ovary and the mammalian testis. These observations might therefore point towards a higher degree of organisational homology between the somatic components of gonads across the sexes and phyla than previously appreciated (Michel, 2012).

Hh thus provides a niche signal for the maintenance and proliferation of the somatic stem cells of the testis. CySCs that are unable to transduce the Hh signal are lost through differentiation, whereas pathway overactivation causes overproliferation. Hh signalling thereby resembles Jak/Stat signalling via Upd. Partial redundancy between these pathways might explain why neither depletion of Stat activity nor loss of Hh signalling causes complete CySC loss (Michel, 2012).

This study has shown that loss of Hh signalling in smo mutant cells blocks expression of the Jak/Stat target Zfh1, whereas mutation of ptc expands the Zfh1-positive pool. Overexpression of Zfh1 or another Jak/Stat target, Chinmo, is sufficient to induce CySC-like behaviour in somatic cells irrespective of their distance. By contrast, Hh overexpression in the hub using the hh::Gal4 driver only caused a moderate increase in the number of Zfh1-positive cells relative to a GFP control. Ectopic Hh overexpression in somatic cells under c587::Gal4 control increased this number further. However, unlike in somatic cells with constitutively active Jak/Stat signalling, the additional Zfh1-positive cells remained largely confined to the testis tip, although their average range was increased threefold. Thus, Hh appears to promote stem cell proliferation, in part, also independently of competition (Michel, 2012).

It is tempting to speculate that further stem cell expansion is limited by Upd range. Consistently, cells with an ectopically activated Jak/Stat pathway remain undifferentiated, whereas ptc cells can still differentiate. Future experiments will need to formally address the epistasis between these pathways. However, the observations already show that Hh signalling influences expression of the bona fide Upd target gene zfh1, and therefore presumably acts upstream, or in parallel to, Upd in maintaining CySC fate (Michel, 2012).

In addition, the reduction in GSC number following somatic stem cell loss implies cross-regulation between the different stem cell populations that presumably involves additional signalling cascades, such as the EGF pathway (Michel, 2012).

In recent years, research has focused on the differences between the male and female gonadal niches. This paper instead emphasizes the similarities: in both cases, Jak/Stat signalling is responsible for the maintenance and activity of cells that contribute to the GSC niche, and Hh signalling promotes the proliferation of stem cells that provide somatic cells ensheathing germline cysts. In the testis, both functions are fulfilled by the CySCs, whereas in the ovary the former task is fulfilled by the postmitotic escort stem cells/escort cells and the latter by the FSCs. Finally, male desert hedgehog (Dhh) knockout mice are sterile. Dhh is expressed in the Sertoli cells and is thought to primarily act on the somatic Leydig cells. However, the signalling microenvironment of the vertebrate spermatogonial niche is, as yet, not fully defined. Future experiments will need to clarify whether these similarities reflect convergence or an ancestral Hh function in the metazoan gonad (Michel, 2012).

smoothened: Biological Overview |Evolutionary homologs | Regulation | Developmental Biology | References

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