patched

DEVELOPMENTAL BIOLOGY

Effects of Mutation or Deletion

This study addresses the question of whether the Hh pathway is distally branched or, in other words, whether the regulation of Ci activity is the sole output of Hh signaling. Putative Ci-independent branches of Hh signaling are explored by analyzing the behavior of cells that lack Ci but have undergone maximal activation of the Hh transduction pathway due to the removal of Patched (Ptc). The analysis of target gene expression and morphogenetic read-outs of Hh in embryonic, larval and adult stages indicates that Ci is absolutely required for all examined aspects of Hh outputs. This is interpreted as evidence against the existence of Ci-independent branches in the Hh signal transduction pathway. It is proposed that most cases of apparent Ci/Gli-independent Hh output can be attributed to the derepression of target gene expression in the absence of Ci/Gli repressor function (Methot, 2001).

The key result of this study is the observation that maximal activation of the Hh pathway (i.e. complete loss of Ptc) has no discernible effect in the absence of Ci. This is taken as evidence against a distal branching in the Hh signal transduction pathway. These results do not exclude the existence of alternative pathways between Smo and Ci, yet all these putative branches must converge at Ci. It is noted that the indispensability of Ci for Hh signaling also explains how developmental compartments are formed and maintained. The essential difference between cells on opposite sides of the anteroposterior compartment boundary is the responsiveness to Hh. Posterior compartment cells do not respond to the Hh signal, even though they are amply exposed to Hh and appear to possess all but one of the components to transduce Smo activity. The lack of Ci, however, precludes any response to Hh and is thus sufficient to create a population of cells that behaves the opposite from that of the anterior, Ci-expressing compartment (Methot, 2001).

Although it is concluded that Hh signaling has no effect in the absence of Ci, it is also concluded that the converse is not the case: Ci does have a function in the absence of Hh signaling. This can be illustrated most effectively by comparing a hh;ci double mutant embryo with a hh single mutant one. Although both animals completely lack the Hh signal, the presence of a functional ci gene considerably increases the segment polarity phenotype of hh mutants. This effect of Ci is brought about by the default state of Ci, which is the repressor function Ci possesses in the absence of Hh input. This function is critical for limb development but not essential for embryogenesis. This is because an uncleavable form of Ci, Ci^U, can substitute for embryonic Ci in spite of the fact that it cannot form detectable amounts of Ci[rep], the repressor form of Ci. The severe phenotype of hh mutant embryos indicates that Ci[rep] activity (although not essential in a wild-type background) can be detrimental in circumstances where Hh signaling is abolished. This situation is reminiscent of the Wg signal transduction pathway, where the nuclear mediator, dTCF/Pangolin, represses Wg target genes in the absence of Wg input. An analogous case has been described for the Notch pathway, where the DNA-binding factor Suppressor of Hairless has a repressive effect on single-minded (sim) transcription in the absence of Notch activity, yet mediates sim activation upon Notch signaling. It may be a general principle that the transcriptional targets of a signaling pathway are repressed in the absence of the signal. Signal-mediated induction, therefore, requires both the abolition of this repression and the concomitant activation of transcription (Methot, 2001).

Based on this analysis, three predictions can be made regarding the Hh pathway in other systems. (1) Loss-of-function mutations in murine Gli genes are likely to cause phenotypes differently from equivalent mutations in Hedgehog genes. In particular, even a triple knockout of the Gli1, Gli2 and Gli3 genes, will presumably behave different from combined mutations in the Sonic, Indian and Desert hedgehog genes. The main reason for postulating this is the Hh-independent repressor function of Gli proteins, which appears to be primarily associated with Gli3. Lack of Shh signaling may lead to an increase of Gli3 repressor activity, while lack of Gli3 expression has the opposite effect. Hence a double Shh Gli3 mutant may have a considerably milder phenotype than a Shh single mutant animal (Methot, 2001).

(2) Given the conservation of the Hh transduction pathway in different species, it is unlikely that the mammalian Hh pathway contains end points other than Gli proteins. The critical but genetically challenging test will be the generation of Gli triple mutant mice and their comparison to animals that lack in addition the Shh or the Ptc gene (Methot, 2001).

(3) These results challenge several previous studies that claim the existence of Ci-independent outputs of the Hh signaling pathway. Some of these studies were conducted with a ci null allele, which removes both activator and repressor functions of Ci. For the wing imaginal disc, lack of Ci[rep] causes the ectopic expression of certain Hh target genes. Genetic evidence is now provided that this is also the case in embryos. It is surmised that the seemingly Ci-independent expression of Hh-induced target genes may reflect transcriptional derepression, owing to removal of Ci[rep] (Methot, 2001).

High Patched levels in the wing imaginal disc, expressed in either normal or ectopic patterns, result in loss of wing vein patterning in both segmental compartments centering at the anterior-posterior border. In addition, patched inhibits the formation of the campaniform sensilla, mechanosensory neurons found in the wing blade. The patched wing vein phenotype is modulated by mutations in hedgehog and cubitus interruptus. patched overexpression inhibits transcription of patched and decapentaplegic and post-transcriptionally decreases the amount of CI protein at the anterior/posterior boundary. In wing discs, which express ectopic hedgehog, CI levels are correspondingly elevated, suggesting that hedgehog relieves patched repression of CI accumulation. Protein kinase A also regulates CI; protein kinase A mutant clones in the anterior compartment have increased levels of CI protein. Thus patched influences wing disc patterning by decreasing CI protein levels and inactivating hedgehog target genes in the anterior compartment (Johnson, 1995).

It is believed that wing veins L3 and L4 do not respond to DPP signaling but instead L3 is determined directly by a threshold response to Hedgehog secreted across the A-P compartment boundary. It has been observed that clones of mutant patched cells in the middle of the anterior compartment are surrounded by an ectopic L3 vein which comprises wild-type cells. Similarly, loss-of-function clones of Protein kinase A, which functions like Ptc to repress ptc expression, are encircled by ectopic veins consisting of wild-type cells. Thus, cells with low levels of ptc may induce adjacent ptc+ cells to assume L3 fates. Since secreted HH is thought to be responsible for inactivating PTC, the position of the L3 primordium might be determined by a threshold response to HH diffusing from the posterior compartment (Sturtevant, 1997 and references).

The absence of ptc gene function causes a transformation of the fate of cells in the middle part of each segment so that they form pattern elements characteristic of cells positioned around the segment border. Mutant phenotypes show that both segment and parasegment borders are included in the duplicated pattern of ptc mutants (Hooper, 1989).

Patched has a role in patterning in the cuticle of the adult fly. Genetic mosaics of a lethal allele of patched show that the contribution of patched varies in a position-specific manner. Analysis of twin clones demonstrates that the reduced clone frequency results from a proliferation failure or cell loss. In the region where clones upset venation, they autonomously fail to form veins and also non-autonomously induce ectopic veins in adjacent wild-type cells. The patched transcript is present throughout the anterior compartment, with a stripe of maximal intensity along the A/P compartment border extending into the posterior compartment (Phillips, 1990).

A new segment polarity gene of Drosophila melanogaster, oroshigane (oro) was identified as a dominant enhancer of Bar (B). oro is a recessive embryonic lethal, and homozygous oro embryos show variable substitution of denticles for naked cuticle. These patterns are distinctly similar to those of hedgehog and wingless mutant embryos, which indicates that oro functions in determining embryonic segment polarity. oro works downstream of hedgehog but upstream of dpp to enhance the Bar phenotypes. Although dpp expression is reduced in oro heterozygotes, hh expression remains the same as that found in wild-type discs. Evidence that oro function is involved in Hh signal transduction during embryogenesis is provided by its genetic interactions with the segment polarity genes patched and fused. ptcIN is a dominant suppressor of the oro embryonic lethal phenotype, suggesting a close and dose-dependent relationship between oro and ptc in Hh signal transduction. oro function is also required in imaginal development. The oro1 allele significantly reduces decapentaplegic (but not hh) expression in the eye imaginal disc. oro enhances the fused1 wing phenotype in a dominant manner. Based upon the interactions of oro with hh, ptc, and fu, it is proposed that the oro gene plays important roles in Hh signal transduction (Epps, 1997).

The photoreceptors within the ommatidia of the Drosophila compound eye form a trapezoid. This occurs in two chiral forms in the dorsal and ventral half of the eye. Ommatidia in the dorsal half of the compound eye are oriented with the R3 photoreceptor cell dorsal and anterior, the R7 photoreceptor being ventral. Ommatidia in the ventral half of the eye are inverted. This asymmetry is established during the progression of the morphogenetic furrow as it moves across the epithelium of the eye imaginal disc from posterior to anterior. As the furrow moves it lays down a new column of ommatidial clusters roughly once every 2 hours. However, the ommatidial clusters in one column are not initiated at the same moment, i.e. the first cluster is formed at the center of the furrow (the midline or future equator); subsequent clusters are formed dorsal and ventral to this at about 10-min intervals. This point at the center of the furrow is known as the firing center, an inductive node which transmits information in two directions, i.e. induction of new ommatidial columns towards the anterior and induction of new ommatidial clusters towards the dorsal and ventral poles (Reifegerste, 1997 and references).

Two manipulations were used to induce ectopic ommatidia, in combination with molecular markers for specific positions in the retinal field. Ectopic furrows were generated by shift of winglessl-12 homozygotes to a nonpermissive temperature for 48 hours. Loss of function patched clones were used to induce ectopic furrows, because patched functions as a negative regulator of furrow initiation. Ectopic morphogenetic furrows induced on the eye field margin (or midline) and those induced in the body of the field have different consequences for the establishment of retinal polarity. Ectopic clones on the midline or margin is associated with ectopic expression of early markers of retinal field polarity, while ectopic expression of clones that do not lie on the margin or midline are not associated with such markers. In cases where clones fail to induce ectopic furrows, such clones can re-specifiy polarity field markers if they lie on the margin or midline. Photoreceptor cells in the ectopic ommatidia formed by patched clones produce axons that do not always follow the normal polarity field toward the posterior and the optic stalk. In cases in which a field of ectopic ommatidial clusters is still disconnected from those formed by the endogenous field, the ectopic clusters do not find a path to the optic stalk, but converge on the center of their local field. This phenomenon may be similar to the development of axon tracts in the insect central nervous system and is consistent with a homophilic axon guidance model (Reifegerste, 1997).

An early equatorial model for retinal polarity is proposed. In this model, early events establish the dorsal/ventral polarity of the retinal field and establish the midline/equator; only later does the furrow initiate and then the firing center follows the midline, but does not form it. This idea is derived from the observation that markers of polarity are expressed in specific parts of the retinal field before furrow initiation. Thus events that initiate furrow movement on the margin or the midline re-specify the field markers, while those that lie off the margin or the midline do not. Evidence for a preexisting field of positional information comes from the characterization of the homeoprotein mirror, which seems to be involved in the establishment of retinal polarity. The gene four-jointed shows a graded expression in equatorial-polar direction along the equator in third instar eye imaginal discs. Four jointed is a putative cell surface or secreted protein. Another candidate for an equatorial signal is Wingless itself. Wingless could act early to signals from the margins inwards. A second signal from the midline could be induced by early Wingless. Mosaic clones for frizzled affect retinal polarity; these have a domineering non-autonomy on adjacent wild type tissue. Proteins similar to Frizzled have been shown to act as Wnt receptors (Reifegerste, 1997 and references).

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 Hh receptor Ptc, a multiple-pass membrane protein, and the cAMP-dependent protein kinase (PKA) normally maintain the Hh signal transduction pathway in a repressed state. Loss-of-function mutations in either of these genes mimic Hh signal reception and result in the cell autonomous activation of Hh target genes in many tissues. LPCs harboring mutations for either pka or ptc undergo differentiation cell-autonomously and independently of retinal innervation. Mutant cells anterior to the furrow do not differentiate precociously. This observation is consistent with the consequences of ectopic Hh expression in an the lamina in mutants lacking retinal innervation of the lamina. Hh expression in regions anterior to the lamina furrow does not induce precocious lamina differentiation, as though competence to respond to Hh is acquired by G1-phase LPCs at the anterior margin of the lamina furrow. Within the lamina target field, wild-type cells neighboring the pka or ptc mutant cells are never observed to express Dac. Thus activation of the Hh pathway by loss-of-function in either gene results in a strictly autonomous induction of LPC maturation. These results permit the conclusion that the terminal cell division and differentiation of LPCs both require the direct reception of the Hh signal (Huang, 1998).

Like the Drosophila embryo, the abdomen of the adult consists of alternating anterior (A) and posterior (P) compartments. However the wing is made by only part of one A and part of one P compartment. The abdomen therefore offers an opportunity to compare two compartment borders (A/P is within the segment and P/A intervenes between two segments), and ask if they act differently in pattern formation. In the embryo, abdomen and wing P compartment cells express the selector gene engrailed and secrete Hedgehog protein while A compartment cells need the patched and smoothened genes in order to respond to Hedgehog. Clones of cells were produced with altered activities for the engrailed, patched and smoothened genes. The results confirm (1) that the state of engrailed, whether 'off' or 'on', determines whether a cell is A or P type and (2) that Hedgehog signaling, coming from the adjacent P compartments across both A/P and P/A boundaries, organizes the patterning of all the A cells. Four new aspects of compartments and the expression of engrailed in the abdomen have been uncovered. (1) engrailed acts in the A compartment: Hedgehog leaves the P cells and crosses the A/P boundary where it induces engrailed in a narrow band of A cells. engrailed causes these cells to form a special type of cuticle. No similar effect occurs when Hedgehog crosses the P/A border. (2) The polarity changes induced by the clones were examined, and a working hypothesis was generated, as follows: polarity is organized, in both compartments, by molecule(s) emanating from the A/P but not the P/A boundaries. (3) It has been shown that both the A and P compartments are each divided into anterior and posterior subdomains. This additional stratification makes the A/P and the P/A boundaries fundamentally distinct from one another. (4) When engrailed is removed from the P cells (of segment A5, for example) the P cells transform not into A cells of the same segment, but into A cells of the same parasegment (segment A6) (Lawrence, 1999).

The cells of the dorsal epidermis of the adult abdomen in Drosophila exhibit two properties: (1) a scalar property, shown by the identity of the cuticle they secrete, and (2) a vectorial property, indicated by the orientation of hairs and bristles. The scalar properties are represented by the presence of subdomains within both the A and P compartments. ptc-;en- cells at the front and the back of the A compartment give different transformations, confirming that there are two domains in A. These domains correspond largely to the territories of a1, a2 (no bristles) and a3, a4, a5 cuticle (with bristles). Removal of the Notch (N) gene from these two regions gives different outcomes: N- clones in a2 cuticle make epidermal cells, while those in a3 do not. It follows that the cells composing a2 (non-neurogenic) and a3 (neurogenic) are fundamentally distinct. The P compartment is also subdivided. Thus, the loss of en from posterior P cells converts them from making p1 cuticle to either a1 or a2, depending on whether they can receive the Hh signal. The removal of en from anterior P cells causes them to make either a5 or a3 cuticle, again depending on whether they can receive Hh (Lawrence, 1999).

Why should there be such a subdivision of the compartments? Perhaps it is connected with making a distinction between A/P and the P/A borders, for if both were simply an interface between A and P cells, they would differ only in their orientation. It is not known what agent discriminates between the two domains in either compartment; perhaps one regulatory gene would be sufficient for both: its expression could flank the segment boundary, redefining nearby regions of the A and P compartments. The domains are not maintained by cell lineage. Analogous domains are found in the legs, where A compartment cells respond to Hh by expressing high levels of either Decapentaplegic or Wingless, depending on whether they are located dorsally or ventrally in the appendage. This dorsoventral bias in response is established early in development, and then maintained, not by lineage, but by feedback between Wg- and Dpp-secreting cells (Lawrence, 1999).

The vector property of the epidermis is represented by the orientation of adult hairs. A model has been proposed where Hh crosses over from P to A and elicits production of a `diffusible Factor X' that grades away anteriorly from the A/P border, and has a long range; the cells are oriented by the vector of this gradient. For simplicity, this discussion will be restricted to the posterior domain of the A compartments. The A/P boundaries cannot be unique sources of X, for polarity changes also occur when cells from one level of A confront those from another (e.g. when a5 and a3 cells meet at the edge of ptc-;en- clones). This suggests that away from the compartment boundaries, cells also produce X, the quantity depending on the amount of Hh received. It is therefore imagined that a gradient of X would be formed both by the graded production of X (high near the A/P boundary, low further away) and also by its further spread into territory (a3) where Hh is low or absent. Note that this model fits with most of the results for it makes the A/P boundaries the organisers: whenever ectopic A/P boundaries are generated by the clones, their orientation correlates with the polarity of territory nearby; this is most clearly seen at the back of en-expressing clones. The line where polarity switches from normal to reversed does not occur at a fixed position in the segment but rather appears to be related to the position of nearby A/P borders (Lawrence, 1999).

en- clones in the P compartment make A cuticle. In the anterior part of P these clones have normal polarity. In the posterior part of P the whole clone displays reversed polarity, as do some cells outside the clone. In order to understand this (at least, in part), consider the behaviour of ptc- clones in the A compartment: they behave differently depending on their distance from the A/P border, the presumed source of X. At the back of the A compartment they are near that border and have little or no effect on polarity, but when closer to the front of A, they repolarize several rows of cells in the surround. This is explained as follows: near the source of X, where the ambient level is high, limited production of X might not much affect the concentration landscape. But far from the source, where the local concentration of X would be low, any effects would appear greater. Likewise, if there were a polarizing factor similar to X in the P compartment, then clones of en minus cells that produce complete or partial borders might become ectopic sources of this factor: they would produce altered polarities only in an environment where the level of the factor were low. This argument suggests that a polarising factor 'Y' for the P compartment might emanate from the A/P border and spread backward. Thus the evidence is consistent with the idea that polarizing signals spread in both directions from the A/P boundaries. The P/A (segment) boundaries might act to stop these factors trespassing into the next segment, just as they appear to block the movement of Wingless protein (Lawrence, 1999).

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).

The Hedgehog (Hh) signal has an inductive role during Drosophila development. Patched is part of the Hedgehog-receptor complex and shows a repressive function on the signaling cascade, which is alleviated in the presence of Hh. The first dominant gain-of-function allele of patched has been identified: Confused (patched^Con). Analysis of the patched^Con allele has uncovered novel features of the reception and function of the Hh signal. At least three different regions of gene expression were identified and a gradient of cell affinities was established in response to Hh. A new state of Cubitus interruptus activity, responsible for the activation of araucan and caupolican genes of the iroquois complex, is described. This state has been shown to be independent of Fused kinase function. In the disc, patched^Con behaves like fused mutants and can be rescued by Suppressor of fused mutations. However, fused mutants are embryonic lethal while patched^Con is not, suggesting that Patched could interpret Hedgehog signaling differently in the embryo and in the adult (Muller, 2000).

Thus ptc^Con has partially impaired Hh-signaling transduction, interpreting the surrounding Hh concentration that reaches the cell as lower than it really is. Changes in Hh concentration alter Hh target gene expression in ptc^Con cells and, subsequently, the ptc^Con phenotype, indicating that ptc^Con affects the interpretation of Hh levels. The lesion of the ptc^Con protein is located in the first extracellular loop of the Ptc protein, which, in vertebrates, is involved in binding Shh. A putative explanation for this would be that ptc^Con binds Hh less efficiently, impeding the proper transduction of the signal. The transduction of the Hh signal can be interpreted as a balance between Ptc protein interacting with Hh to open the pathway and Ptc protein interacting somehow with Smo to block the pathway. The interaction between Ptc and Hh and between Ptc and Smo could take place inside the cell in distinct subcellular compartments. Hh could sequester Ptc to avoid the negative, direct or indirect, interaction with Smo. If this were the situation, given that ptc^Con binds Hh less efficiently, the result would be more Ptc protein interacting with Smo. The increase in Ptc-Smo interaction could impede the release or modification of Smo to transduce the signal. This explanation also accounts for the dominant effect of ptc^Con. In a heterozygotic fly, both forms of Ptc would be present. One of them, ptc^Con, would have less affinity for Hh, which would reduce the reception of Hh at the A-P border. Thus, A cells would receive less Hh because ptc^Con competes with the wild-type protein for the reception of Hh. The high Hh levels that induce some responses such as anterior En expression would not be read, provoking the dominant phenotype of ptc^Con (Muller, 2000).

Depending on the domain where a ptc^Con clone is located, the results of blocking the Hh signal are different. The specification of vein 3 has been a subject of debate due to its morphogenetic implications. Some lines of evidence suggest that vein 3 differentiation depends upon the presence of high levels of Dpp. Nevertheless, ectopic expression of Dpp does not affect vein 3 or promote differentiation in a genetic background in which Hh signaling is impaired. In ptc^Con and fu clones, dpp is not expressed and yet both types of clones differentiate vein 3 when the Hh concentration is sufficient to induce a response. When a dose of hh is removed, ptc^Con mutant cells do not differentiate vein 3. It follows that Hh, and not Dpp, specifies the location of vein 3, and Dpp has a permissive role in establishing a broad, competent domain for vein 3 differentiation. The results presented here confirm that Hh forms a concentration gradient in the A compartment and strongly suggest that Hh acts as a morphogen in the wing disc to pattern the central region of the wing (Muller, 2000).

In the abdomen, most morphogenetic functions are mediated by Hh, and although other morphogenetic molecules might exist, Dpp does not seem to have a role in patterning the abdomen. In ptc^Con discs, dpp is not expressed and this may account for the lack of growth in these discs. Nevertheless, the larvae reach the third larval instar stage and the discs are similar in size to those from the second larval instar. Thus, Dpp activation in response to Hh seemed to function only after the second larval instar to promote growth and patterning of the discs. Hh may have evolved as the primary morphogen of adult structures and it was not until the advent of appendages during evolution that Dpp was recruited for long-range patterning of structures. This may be due to a need for a higher diffusion capacity to pattern the new structures (wings, antennae, and legs) (Muller, 2000).

Hh is also responsible for inducing a change in cell affinity. Lack of Smo completely abolishes Hh signaling and, consequently, impedes the change in A-cell affinity. Although the involvement of Hh and Smo in this process has been clear, that of the Hh-receptor Ptc has not. There is the possibility of a second signaling pathway, dependent on Smo but not on Ptc, which would mediate the responses for changing cell affinity. In this study it is concluded that the establishment of the lineage restriction border (LRB) depends upon the correct Ptc perception of the Hh signal. The mechanism by which the LRB arises raises a further question: why do A cells responding to Hh not form a restriction border with A cells not responding to Hh? ptc^Con clones close to the P compartment present straight boundaries with both A and P cells, indicating that the cell affinity of ptc^Con cells is different from that of both populations of cells. In ptc^Con cells, there is a weak response to Hh, which may be responsible for a discrete change in cell affinities in ptc^Con cells, making them different from both the P cells, which do not respond to Hh, and the adjacent A cells, which do respond to Hh. When a copy of hh is removed, ptc^Con clones take up more posterior positions and adopted more wiggly boundaries with P cells, indicating that their cell affinity is more similar to that of P cells. Changes in cell affinities seem to form in a gradient fashion, with different changes in response to different concentrations of Hh. Adjacent A cells receiving the Hh signal may have such similar cell affinities that no restriction border forms between A cells. A similar mechanism has been suggested to occur in the abdomen of Drosophila (Muller, 2000).

In ptc^Con clones, a unique experimental situation is presented in that reception of Hh signaling is severely impaired, allowing the accumulation of Ci in the cytoplasm without the activation of dpp. ptc^Con clones in the wing differentiate vein 3 when close to the P compartment and substitute vein 4 for vein 3. This is in accordance with the activation of Caup in ptc^Con clones, which is involved in determining vein 3 in the wing imaginal disc. When lowering the concentration of Hh by removing a copy of hh, vein 3 is not induced in ptc^Con clones and the levels of cytoplasmic Ci are low, similar to smo clones that do not differentiate vein 3. In the same line, ptc^Con clones close to but not touching the A-P border do not develop vein 3 nor express Caup. Since ptc^Con cells interpret high Hh levels as low, these results ascribe the role of determining the position and differentiation of vein 3 to low levels of Hh. Furthermore, Ci accumulation in the cytoplasm indicates the activation of Ci to induce expression of Caup and differentiation of vein 3 (Muller, 2000).

The fact that ptc^Con imaginal discs reach second larval instar suggests that it is not until this stage that the responses to Hh affected by ptc^Con are needed. However, there is still a paradox: if fu clones behave like ptc^Con clones, why are smo and fu mutants embryonic lethal while ptc^Con is not? It is proposed that ptc^Con affects a function of Ptc that is needed only in larval stages, perhaps to interact with another protein, providing further refinements to Hh-signaling interpretation. Alternatively, in the embryo, another protein may participate in the Hh-receptor complex (so far formed by Ptc and Smo) by binding to Ptc through a domain not affected by the ptc^Con mutation. Evidence for the existence of other proteins involved in receiving the Hh signal is provided by the embryonic ptc;hh double-mutant phenotype, which is not identical to that of ptc, indicating that Ptc alone does not receive the Hh signal in the embryo. A putative candidate, Hip, has been recently found in vertebrates. Hip is a membrane protein that binds Hh with the same affinity as that of Ptc and, similar to Ptc, is expressed and modified by Hh. Since ptc^Con would affect a domain of Ptc needed only in larval stages, Ptc function in embryos would be unaltered (Muller, 2000).

Ci is involved in controlling the transcription of Hh target genes. It has been recently proposed that Hh controls both the repressing and the activating functions of Ci. Apart from negatively regulating the generation of a repressor form of Ci (Ci-75), Hh controls the activation of Ci. Only two forms of Ci are detected in a Western blot: a 75-kDa form which bears repressor activity and a 155-kDa form which seems to act as an activator. Two activation states for Ci have been described, both of which are probably modifications of the Ci-155 form. One is responsible for inducing en and the other for inducing ptc and dpp. Neither of these responses is produced in the absence of fu or in ptc^Con cells (Muller, 2000).

The unmasking of a third level of apparent Ci activity is reported that is independent of the other two levels. This new state of Ci activity is responsible for the activation of iro and the differentiation of vein 3 in the wing. The other two levels of Ci activity arise from high levels of Hh and depend on Fu activity. The new state of Ci is activated by low levels of Hh and is Fu independent. Thus, Hh signaling activates two different pathways through inhibition of Ptc function. Fu would be involved in mediating transduction of the signal in one of these pathways. The second pathway would modify Ci to activate it in a Fu-independent manner. It has been suggested that low levels of Hh activate a new form of Ci, named 'Ci default', which does not depend on Fu activity (Muller, 2000).

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 ptc^S2 are fully viable. The ptc^S2 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).

Patched controls the Hedgehog gradient by endocytosis in a dynamin-dependent manner, but this internalization does not play a major role in signal transduction

The Hedgehog (Hh) morphogenetic gradient controls multiple developmental patterning events in Drosophila and vertebrates. Patched (Ptc), the Hh receptor, restrains both Hh spreading and Hh signaling. Endocytosis regulates the concentration and activity of Hh in the wing imaginal disc. Ptc limits the Hh gradient by internalizing Hh through endosomes in a dynamin-dependent manner, and both Hh and Ptc are targeted to lysosomal degradation. The ptc¹⁴ mutant does not block Hh spreading, because it has a failure in endocytosis. However, this mutant protein is able to control the expression of Hh target genes as does the wild-type protein, indicating that the internalization mediated by Ptc is not required for signal transduction. In addition, both in this mutant and in those not producing Ptc protein, Hh still occurs in the endocytic vesicles of Hh-receiving cells, suggesting the existence of a second, Ptc-independent, mechanism of Hh internalization (Torrioja, 2004).

Through the analysis of ptc¹⁴ (a mutant that does not internalize Hh but is able to perform Hh signal transduction) this study shows that both proposed Ptc functions are genetically uncoupled. Ptc limits the Hh gradient by internalizing Hh in a dynamin-dependent manner, and this Hh-Ptc complex is targeted to the degradation pathway. These findings strongly suggest that internalization mediated by Ptc shapes the Hh gradient and also leads to the challenging suggestion that Hh signaling can occur in the absence of Ptc-mediated Hh internalization. The two functions of Ptc in Hh signal transduction are discussed in the light of these results (Torrioja, 2004).

Hh and Ptc sorting to the endocytic membrane-bound compartment plays a crucial role in modulating Hh levels during development. A strong support of the conclusions in this work comes from the analysis of the ptc¹⁴ allele. Although ptc¹⁴ mutants are lethal with a strong ptc^- embryonic phenotype, ptc¹⁴ mutant cells in the imaginal discs show an effect only when the clone touches the AP compartment border but not in any other part of the disc. This result indicates that the presence of Hh is required to reveal a defect in Ptc¹⁴ function. This Hh requirement has been probed by the lack of activation of the Hh targets in ptc¹⁴ cells in the absence of Hh, either in the embryos or in the imaginal discs. The complementation of ptc¹⁴ with ptc^S2 allele, which is considered as null for blocking Hh signal transduction and acts as dominant negative, indicates that Ptc¹⁴ does not have a greater sensibility to Hh than the Ptc wild-type protein. Conversely, it has been shown in this study that there is a decrease of internalization of Hh in ptc¹⁴ mutant clones compared with wild-type Ptc territory and an extension of the range of Hh gradient. Therefore, it can be concluded that Ptc¹⁴ is unable to sequester Hh efficiently in either the embryo or imaginal discs and that the ptc¹⁴ embryonic phenotype would be the result of greater spreading of Hh and not due to the constitutive activation of the Hh pathway (Torrioja, 2004).

Ptc¹⁴ responds to Hh as does the wild-type Ptc protein and activates the signaling pathway indicating that the interaction of Ptc¹⁴ and Hh is probably normal. However, this Hh-Ptc interaction does not necessarily imply sequestration. Although Ptc¹⁴ occurs at the plasma membrane, no internalization of Hh or extracellular Hh accumulation occurs in ptc¹⁴ mutant clones. These results, therefore, suggest that Hh-Ptc interaction is not sufficient to sequester Hh and that an active internalization process of Hh mediated by Ptc to control Hh gradient is required. This Hh internalization mediated by Ptc is Dynamin-dependent, based on the membrane accumulation of Hh and Ptc in shi mutant clones and the lack of accumulation of Hh in shi^ts1; ptc¹⁶ double mutant clones. However, the initiation of the internalization process is not blocked in shi mutants because Dynamin is required for fission of clathrin-coated vesicles after the internalization process has already started. This fact would explain why Hh gradient and signaling is not extended when endocytosis is blocked in shi mutant cells. Since Ptc¹⁴ seems to have a problem in entering the endocytic compartment and no Hh accumulation is found in shi^ts1; ptc¹⁴ double mutant clones, it is concluded that the initiation of the internalization process does not occur in Ptc¹⁴. Taken together, these data indicate that only when Ptc forces Hh to the endocytic pathway Hh is sequestered in the receiving cells (Torrioja, 2004).

To block the degradative pathway, deep orange (dor) mutants were used. dor, one of the mutations that affects eye pigmentation in Drosophila, is required for normal delivery of proteins to lysosomes. The behavior of Hh and Ptc in dor^- cells indicates that after sequestration, Ptc internalizes Hh, and both Hh and Ptc are degraded. Thus, controlling both endocytosis and degradation of Hh modulates its gradient. Similar mechanisms have been described for controlling the asymmetric gradient of Wg in embryonic segments. It is possible that additional factors may contribute to shaping the Hh gradient, because in large ptc^- clones close to the AP border, which lack Ptc protein to sequester Hh, an Hh gradient in endocytic vesicles is also observed, although the range of this gradient is more extended than in wild-type cells. This is consistent with two mechanisms of Hh internalization in Hh receiving, one mediated by Ptc and another not mediated by Ptc (Torrioja, 2004).

From studies in both vertebrates and Drosophila, it was thought that Hh protein binds to Ptc. Ptc is then internalized and traffics Hh to endosomal compartments where both are degraded, the entire process triggering activation of the Hh pathway. It is shown in this study that Ptc¹⁴ responds to Hh as would the wild-type Ptc protein in activating the pathway. However, Ptc¹⁴ does not internalize Hh to the endocytic compartment because it is defective in endocytosis. It is therefore suggested that the massive Hh internalization by Ptc to control the gradient is not a requirement for Hh pathway signal transduction (Torrioja, 2004).

In Hh signal transduction, the cellular mechanisms that regulate Smo function remain unclear, although the distribution of Ptc/Smo suggests that Ptc destabilizes Smo levels. It has also been proposed that Ptc-mediated Hh internalization changes the subcellular localization of Ptc preventing Smo downregulation. Furthermore, in cultured cells, Shh induces the segregation of Ptc and Smo in endosomes, allowing Smo signaling, independently of Ptc. It is known, however, that binding of Shh to Ptc is not sufficient to relieve the repression of the Hh pathway (Torrioja, 2004).

As in wild-type cells, in the absence of Hh, Ptc¹⁴ downregulates both Smo levels and Smo activity, while in the presence of Hh, the normal upregulation of Smo occurs. Consequently, Ptc¹⁴ levels are high at the AP border because upregulation of Ptc by Hh occurs in the absence of internalization of Hh to the degradative pathway. It might then be expected that the high levels of Ptc¹⁴ not targeted to the degradative pathway would block Smo activity. However, against all predictions, the presence of Hh is still able to release Smo activity in mutant ptc¹⁴ cells. Thus, there must be a positive mediator of Smo activity to overcome the repressive effect of Ptc¹⁴ and allow Hh pathway activation in response to Hh. Alternatively, if Ptc¹⁴ is located at the plasma membrane, it could control Smo activity without entering the endocytic compartment by regulating the entrance of small molecules, as has been proposed. In fact, Ptc is similar to a family of bacterial proton-gradient-driven transmembrane molecule transporters known as RND proteins. Accordingly, as a membrane transporter, Ptc could indirectly inhibit Smo through translocation of a small molecule that conformationally regulates the active state of Smo. The inter-allelic complementation of Ptc suggests that Ptc has the oligomeric structure needed for this type of transporter (Torrioja, 2004).

Although one of the normal functions of Ptc is to mediate Hh internalization, the data demonstrate the presence of internalized Hh vesicles in the absence of Ptc protein. It is therefore suggested that another receptor mediates Hh internalization in Hh-receiving cells. This molecule could act as a positive mediator of Hh signaling. Several observations have been published that cannot easily be reconciled with the idea of Ptc acting as the only receptor for Hh. For example, it was found that Hh activates signal transduction in both A and P compartment cells of wing imaginal discs, despite the absence of Ptc in P cells. Furthermore, it has been reported that some neuroblasts in Drosophila embryos, the maturation of which is dependent on Hh, do not express or require Ptc. This suggests that a receptor other than Ptc mediates Hh signaling. Recently, the glypican protein Dally-like, which belongs to the heparan sulfate proteoglycan protein family, was found to be required for Hh signal transduction and probably for the reception of the Hh signal in Drosophila tissue culture cells. Dally-like could act as co-receptor for Hh and it would be interesting to know if Dally-like is required for Hh endocytosis. In addition, the large glycoprotein 'Megalin' has recently been identified as a Shh-binding protein. Megalin is a multi-ligand-binding protein of the low-density lipoprotein (LDL) receptor family whose function is to mediate the endocytosis of ligands. The finding that megalin-mediated endocytosed N-Shh is not efficiently targeted to lysosomes for degradation suggests that N-Shh may also traffic in complexes with Megalin and thus be recycled and/or transcytosed. In the Wg pathway, specific LDL receptor-related proteins are essential co-receptors for Wnt ligands. Further investigation will determine whether LDL receptor-related proteins could function as co-receptors that internalize Hh in the absence of Ptc. Alternatively, these proteins could be required for endocytosis and further delivery of Hh to Ptc in intracellular vesicles, perhaps facilitating the transcytosis of Hh. A future challenge will be to find other molecules that internalize Hh and to understand how Hh interacts with Smo to activate the Hh pathway (Torrioja, 2004).

Hedgehog pathway induces segmental grooves in the Drosophila epidermis

The formation of segmental grooves during mid embryogenesis in the Drosophila epidermis depends on the specification of a single row of groove cells posteriorly adjacent to cells that express the Hedgehog signal. However, the mechanism of groove formation and the role of the parasegmental organizer, which consists of adjacent rows of hedgehog- and wingless-expressing cells, are not well understood. This study reports that although groove cells originate from a population of Odd skipped-expressing cells, this pair-rule transcription factor is not required for their specification. It was further found that Hedgehog is sufficient to specify groove fate in cells of different origin as late as stage 10, suggesting that Hedgehog induces groove cell fate rather than maintaining a pre-established state. Wingless activity is continuously required in the posterior part of parasegments to antagonize segmental groove formation. These data support an instructive role for the Wingless/Hedgehog organizer in cellular patterning (Mulinari, 2009).

It has been reported that segmental groove formation requires the activity of engrailed (en) and hh and that en has a function that is independent of its role in hh activation. More recently, it was been found that en is not expressed in groove cells, thus creating a non-cell-autonomous requirement for en. To address this issue, the role of hh and en in segmental groove formation was reinvestigated (Mulinari, 2009).

It was found that segmental grooves do not form in hh mutants. When hh was overexpressed, the four to five cell rows posterior to the Hh source constricted apically, elongated their apical-basal axis and took on a shape characteristic of segmental groove cells. Very similar cell behavior was observed in patched (ptc) mutants or when activated Ci, which mediates hh activity, was expressed. These observations suggest that Hh can organize segmental groove formation. No cell constrictions were observed in the ventral epidermis, indicating that a different mechanism might regulate cell shape there (Mulinari, 2009).

To address the proposed hh-independent function of en, en, invected (inv) double mutants were investigated in which hh expression was maintained using prd-Gal4. Segmental grooves were rescued in these mutants, suggesting that en is not required for segmental groove formation independent of its role in hh activation. By contrast, it was found that en represses groove cell behavior when ectopically expressed together with hh. A previous study that reported a requirement of en in groove formation was based on the analysis of en, inv, wg triple mutants, in which hh expression was maintained but did not rescue groove formation. This result was confirmed, but it is proposed that wg may be required in en mutants to allow the morphological differentiation of grooves (Mulinari, 2009).

Analysis of ptc mutants, or embryos overexpressing hh, reveals that a broad region of cells posterior to the en expression domain are specified as groove cells. However, groove-like invaginations form only at the edges of these regions. This is even more obvious in double mutants of ptc and the segment polarity gene sloppy paired 1 (slp1), which is required for maintained wg expression. In slp1, ptc mutants, wg expression fades prematurely and Hh signaling is constitutively active. This results in a substantial expansion of the number of groove cells. However, furrows differentiate only at the edges of groove cell populations. It is proposed that the morphological differentiation of segmental grooves can occur only at the interface between groove and non-groove cells (Mulinari, 2009).

To test this, wg, ptc double mutants were used in which Hh signaling is active throughout the epidermis and all cells take on a groove fate. Interestingly, these embryos did not differentiate grooves. A similar observation has been reported in en, inv, wg mutants, in which hh expression is sustained, leading to the suggestion that en might be required for groove specification (Mulinari, 2009).

Analysis of cell behavior in wg, ptc mutants showed, however, that cells throughout the tissue constrict their apices but fail to form invaginating furrows. The failure of wg, ptc mutants and en, inv, wg; UAS-hh embryos to differentiate grooves might be due to the absence of non-groove cells in the epidermis and the concomitant absence of an interface with groove cells (Mulinari, 2009).

The pair-rule gene odd is initially expressed in 4- to 5-cell wide stripes in even-numbered parasegments. At early gastrulation, odd expression expands to segmental periodicity and is subsequently refined to a single row of prospective groove cells located posterior to en. Continued expression of odd in these cells requires hh. In odd⁵ mutant embryos, grooves are unaffected in odd-numbered parasegments, but partially missing in even-numbered parasegments, and residual grooves coincide with regions in which odd expression is detectable (Vincent, 2008). These observations have been interpreted as indicating that groove fate might be specified prior to the requirement of Hh and differentiation of the groove. Thus, the later activity of Hh might not induce, but merely maintain, groove cell identity that has been pre-established in the odd-expressing cell population (Vincent, 2008). However, this hypothesis is based on the presumption that odd has a function in groove cell specification and this has not been demonstrated (Mulinari, 2009).

Residual grooves in odd⁵ mutants have been attributed to the hypomorphic nature of the odd⁵ allele; however, the molecular lesion in odd⁵ is unknown. Therefore the nucleotide sequence of odd⁵ was determined and a substitution was found that mutates codon 84 from CAG to a TAG stop codon. The resulting truncated peptide, which lacks all four putative zinc fingers encoded by wild-type odd, is no longer restricted to the nucleus but uniformly distributed in the cell. Thus, odd⁵ is likely to be a null allele (Mulinari, 2009).

To exclude the possibility that groove formation may be rescued by read-through of the stop codon in odd⁵ mutants, or that odd may be required redundantly, segmental grooves were investigated in Df(2L)drm^P2 mutants, in which odd and its sister genes drumstick (drm) and sister of odd and bowl (sob) are entirely deleted. In these embryos, normal grooves formed in odd-numbered parasegments in the complete absence of odd function (Mulinari, 2009).

Next even-numbered parasegments were investigated in which grooves are partially missing. odd encodes a transcriptional repressor that regulates the expression of other segmentation genes in the early embryo. In odd mutants, derepression of the en activator fushi-tarazu in even-numbered parasegments results in the formation of an ectopic en stripe posterior to the normal stripe. Simultaneously, wg expands anteriorly and becomes expressed adjacent to the ectopic en-expressing cells. This results in the formation of an ectopic parasegment boundary with reversed polarity. Thus, the outward-facing edges of both en stripes are genetically anterior and lined by wg-expressing cells that do not form grooves. The inward-facing edges of the normal and ectopic en stripes fuse in some areas, and these corresponded to areas in which grooves were missing, as cells that were genetically posterior to en and could respond to the Hh signal had been replaced by en-expressing cells. The fusion of normal and ectopic en stripes was more severe in Df(2L)drm^P2 mutants; however, islands of invaginating groove cells could still be observed, demonstrating that groove fate is specified in the absence of odd, drm and sob function in all parasegments. It is concluded that all cells that are genetically posterior to en are specified as groove cells in the absence of odd function and the partial absence of grooves in even-numbered parasegments in odd mutants is a secondary consequence of the pair-rule phenotype of these embryos. The slightly more severe pair-rule phenotype seen in Df(2L)drm^P2 mutants might be due to a contribution from one of the odd sister genes, most likely sob, to pair-rule function, or could be caused by low-level read-through of the stop codon in the odd⁵ allele (Mulinari, 2009).

Finally, to investigate whether odd is sufficient to trigger cell shape changes, a UAS-odd transgene was expressed either alone or together with hh in the epidermis. No induction of groove cell behavior other than that triggered by hh was observed. Together, the data show that odd plays no essential role in groove cell specification and that odd paralogs are unlikely to act redundantly in this process (Mulinari, 2009).

The identification of odd as a groove cell marker led Vincent to suggest that groove fate might be specified prior to Hh requirement and that Hh may merely maintain groove fate instead of having an inducing role (Vincent, 2008). This study demonstrate that grooves are specified in the absence of odd function; however, this could be due to an odd-independent, early-acting mechanism present in the cells from which grooves arise (Mulinari, 2009).

In order to address whether groove fate is pre-established in the odd-expressing cell population, it was asked if groove fate could be induced in cells of a different origin at a later point in time. lines (lin) mutants were used in which late wg expression is altered resulting in the formation of an ectopic segment boundary at the anterior edge of the en domain in the dorsal epidermis. Importantly, the early expression of pair-rule or segment polarity genes is not affected (Mulinari, 2009).

In lin mutants, ectopic expression of the groove marker odd was initiated at stage 12 in a single row of groove-forming cells anterior to en that are derived from a previously non-odd-expressing cell population that does not contribute to grooves in the wild type. Ectopic grooves require hh as they were not induced in hh, lin double mutants, and ectopic odd expression was not induced in this background. An increase in hh levels in lin mutants resulted in the specification of groove fate in all cells except those expressing en. These results suggest that hh is sufficient, late in development, to specify groove cell fate in cell populations of different origins and that earlier-acting factors present in the population of odd-expressing cells posterior to en are not required. Very similar results have been reported by Piepenburg (2000), who showed that segment border cells form solely in response to the Hh signal that emanates from the en domain (Mulinari, 2009).

The findings are consistent with the role of Hh in the regulation of cell shape in other systems. Thus, during Drosophila eye development, Hh has been shown to control cell shape in the morphogenetic furrow, and Hh activation in other tissues is sufficient to induce apical constriction and groove formation. It is likely that Hh plays a similar role in tissue morphogenesis in other organisms. During neural tube closure in vertebrates, cells undergo similar shape changes involving apical-basal elongation and apical constriction, which is likely to be in response to Hh sources in the notochord and floor plate. Accordingly, knockout of sonic hedgehog is associated with defects in neural tube closure in mice. These observations suggest that Hh might be a principal inducer of cell shape across species (Mulinari, 2009).

It has previously been established that wg antagonizes the activity of hh in the specification of segment border cells (Piepenburg, 2000). However, it is not clear whether wg has a similar role in segmental groove formation, and a late requirement of wg to antagonize Hh-mediated groove specification has been questioned (Vincent, 2008). To investigate a direct role of wg in groove specification, a dominant-negative form of the transcription factor pan (pan^DN), which suppresses Wg signaling, was expressed. For this, pnr-Gal4, which initiates expression in the dorsal epidermis at stage 10-11 and thus does not affect early wg function, was used. Embryos that express pan^DN formed a single row of ectopic groove cells anterior to the en domain, confirming the results in lin mutants. Strikingly, inactivating Wg signaling and increasing Hh levels at the same time by co-expression of pan^DN and hh resulted in the expansion of groove fate to all cells except those expressing en. These results show that Wg signaling is required after stage 10 to repress groove specification anterior to en, thus making the activity of Hh asymmetric. These results also confirm observations that Hh is sufficient to induce groove fate in cells from different positions along the anterior-posterior axis and suggest that groove fate is not determined before stage 10 (Mulinari, 2009).

To confirm the ability of wg to repress groove fate, wg was expressed posterior to en in cells that normally take on groove fate. This resulted in the loss of Odd from many cells, suggesting that wg indeed antagonizes hh activity. Interestingly, these cells still formed grooves. However, these grooves appeared much earlier than segmental grooves, suggesting that they are ectopic parasegmental grooves caused by ectopic wg expression, as recently suggested (Larsen, 2008). Together, these data therefore support the contention that Wg signaling is required to repress Hh-mediated induction of groove fate after stage 10, thus permitting the formation of segmental grooves posterior, but not anterior, to en in the wild type (Mulinari, 2009).

Effects of Mutation: Patched and ovary development

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).

Hh signaling in Drosophila generally regulates the abundance and activity of Ci proteins without altering CI mRNA levels. By contrast, vertebrate Hh homologs frequently regulate transcription of the Ci-related GLI family of transcriptional effectors. The induction of CI RNA in ptc mutant follicle cells provides the first evidence that this circuitry can also be found in Drosophila. Other consequences of altering the activity of Hh signaling components in ovarian somatic cells substantiate the hypothesis that Hh signaling activates at least two distinct intracellular pathways. One pathway, involving protection of Ci-155 from proteolysis and perhaps also release from cytoplasmic anchoring, is phenocopied by PKA and cos2 mutations. In the ovary, cos2 mutations elicit stronger phenotypes than PKA mutations, perhaps because cos2 mutations preferentially disrupt cytoplasmic anchoring of Ci-155. The second pathway increases the specific activity of Ci-155 in opposition to the inhibitory effects of Su(fu). This pathway is elicited by ptc, but not by PKA mutations and requires Fu kinase activity. In accordance with this model, PKA Su(fu) double mutant cells produce phenotypes almost as strong as for ptc mutants in ovaries, whereas ptc fu double mutant cells exhibit minimal phenotypes and PKA mutant phenotypes are not greatly altered by additional loss of Fu kinase activity. In imaginal discs high level Hh signaling to nearby cells is phenocopied by ptc mutations and requires Fu kinase activity, whereas only low level Hh signaling to more distant cells can be phenocopied by PKA mutations and does not require Fu kinase activity. PKA mutations in somatic ovarian cells can effectively substitute for Hh activity: Fu kinase activity is not essential for somatic cell proliferation and ptc mutations engender excessive Hh signaling phenotypes even in the absence of Hh activity. Hence, it is surmised that ovarian somatic cells normally undergo only low levels of Hh signaling, in keeping with the observation that the source of Hh in the germarium is separated from its target cells by several cell diameters (Zhang, 2000).

The rescue of apparently normal oogenesis in hh^ts 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).

In adult Drosophila females, egg chambers are produced continuously in the germarium of each of the 15-18 ovarioles that are bundled together to form an ovary. In regions 1 and 2a of the germarium, 16-cell germline cysts develop from germline stem cells. Each cyst is enveloped by a monolayer of follicle cells in region 2b and separated from the next cyst by a short stalk as it buds from region 3 to form an egg chamber. Follicle and stalk cells derive from somatic stem cells that reside at the region 2a/2b border. When a somatic stem cell divides, one daughter retains a stem cell identity and continues to divide as a stem cell for several days. The other 'pre-follicle cell' daughter proliferates for about eight cycles as its progeny associate with germline cysts, pass posteriorly down the ovariole over a five to six day period, and differentiate into multiple specialized cell types. Hedgehog (Hh) is expressed selectively in specialized non-proliferating, somatic 'terminal filament' and 'cap' cells at the anterior tip of the germarium. Inactivation of Hh, using conditional hh alleles, arrests egg chamber budding, and causes germline cysts to accumulate in swollen germaria, suggesting that too few follicle cells are being produced. Conversely, excessive Hh signaling in germarial region 2 can be induced by temporally controlled activation of an hh transgene or by inactivation of the Hh receptor Patched (Ptc), and causes marked overproliferation of somatic cells, which accumulate between egg chambers (Zhang, 2001).

The proliferative response to excessive Hh signal transduction was investigated further by using antibodies against phospho-histone H3, which stains cells only during mitosis. High levels of Hh signal transduction were induced by inactivating patched (ptc) in marked somatic cell clones generated by heat-shock induced mitotic recombination. Ovaries were examined 8 d later to ensure that all proliferating somatic cells assayed derived from stem cells that were present at the time of clone induction. As each ovariole contains more than one somatic stem cell, this procedure generates some ovarioles containing only ptc mutant somatic cells and others that are mosaic for wild-type and ptc mutant cells. The number of mitotic somatic cells in germarial region 2 in ovarioles containing only ptc mutant somatic cells was twice that in wild-type ovarioles. A similar ratio was observed in region 3 of the germarium and in newly budded (stage 1-2) egg chambers, suggesting an early increase in proliferation of ptc mutant cells followed by wild-type rates of proliferation of a twofold enlarged cell population. Accordingly, the follicular monolayers surrounding stage 6 egg chambers in wild-type and ptc mutant ovarioles contained almost identical numbers of mitotic cells within numerically equivalent cell populations. In ovarioles mosaic for ptc mutant and wild-type somatic cells, the number of cells in mitosis was increased roughly 1.5-fold in region 2; again, this ratio was maintained in region 3 and in the earliest egg chambers. This is consistent with a cell-autonomous effect of ptc inactivation, affecting roughly half of the somatic cells in a mosaic germarium (Zhang, 2001).

To establish whether excessive Hh signaling accelerates somatic stem cell cycles or increases the number of stem cells in an ovariole, stem cells were counted by using mitotic recombination. Thus, in almost every ovariole examined, a loss of ptc activity led to a cell-autonomous doubling of somatic stem cell number. In 19 of these instances, the two ptc mutant stem cells were directly adjacent, suggesting that excessive Hh signal transduction allowed local expansion of a stem cell niche. In the remaining 12 cases the additional ptc mutant stem cell had migrated away from its presumed sister cell (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 effects of Hh signaling on cell fate determination in Drosophila are mediated largely by altering the activity of the transcription factor Cubitus interruptus (Ci). The role of Ci in somatic stem cell proliferation was examined by inducing somatic clones lacking ci activity. As with smo, very few clones were recovered 8-10 d after clone induction and these clones occupied only a small proportion of the ovariole, indicating that stem cells cannot proliferate normally in the absence of ci activity. When the expression of a constitutively active derivative of Ci was induced by heat-shock-induced excision of a transcriptional terminator, ovarioles were recovered showing massive overproliferation of somatic cells, which accumulated between egg chambers as observed for ptc mutant ovarioles. Thus, the activity of Hh as a stem cell factor seems to depend on Ci-mediated regulation of transcription (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 ptc^mat-zyg+ and smo^mat-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 ptc^mat- (and to a lesser extent pka^mat-) 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).

Effects of Mutation: Patched and Head Development

The function of the Dpp and Hh signaling pathways in partitioning the dorsal head neurectoderm of the Drosophila embryo has been analyzed. This region, referred to as the anterior brain/eye anlage, gives rise to both the visual system and the protocerebrum. The anlage splits up into three main domains: the head midline ectoderm, protocerebral neurectoderm and visual primordium. Similar to their vertebrate counterparts, Hh and Dpp play an important role in the partitioning of the anterior brain/eye anlage. Dpp is secreted in the dorsal midline of the head. Lowering Dpp levels (in dpp heterozygotes or hypomorphic alleles) results in a 'cyclops' phenotype, where mid-dorsal head epidermis is transformed into dorsolateral structures, i.e. eye/optic lobe tissue, which causes a continuous visual primordium across the dorsal midline. Absence of Dpp results in the transformation of both dorsomedial and dorsolateral structures into brain neuroblasts. Regulatory genes that are required for eye/optic lobe fate, including sine oculis (so) and eyes absent (eya), are turned on in their respective domains by Dpp. The gene zerknuellt (zen), which is expressed in response to peak levels of Dpp in the dorsal midline, secondarily represses so and eya in the dorsomedial domain. Hh and its receptor/inhibitor, Patched (Ptc), are expressed in a transverse stripe along the posterior boundary of the eye field. Hh triggers the expression of determinants for larval eye (atonal) and adult eye (eyeless) in those cells of the eye field that are close to the Hh source. Eya and So, which are induced by Dpp, are epistatic to the Hh signal. Loss of Ptc, as well as overexpression of Hh, results in the ectopic induction of larval eye tissue in the dorsal midline (cyclopia). The similarities between vertebrate systems and Drosophila are discussed with regard to the fate map of the anterior brain/eye anlage, and its partitioning by Dpp and Hh signaling (Chang, 2001).

Hh signaling is negatively regulated by Ptc, a membrane linked protein that, by binding to Hh ligand, becomes inactivated in cells receiving high levels of Hh. Ptc expression in the head resembles hh expression at an early stage. A wide antennal/pre-antennal stripe traverses the head in front of the cephalic furrow. During germband extension, this domain splits into two stripes. At the late extended germ band stage, ptc remains expressed in a large domain that corresponds to the anterior optic lobe (Chang, 2001).

Loss of hh results in a strong reduction of the head midline epidermis, a reduction in the size of the brain and optic lobe, and the total absence of the larval and adult eye primordium. Temperature-sensitive shift experiments of hh^ts2 embryos indicate that the phenocritical period for Hh function in Bolwig's organ development is between 4 and 7 hours. Aside from the larval eye, the primordium of the compound eye, which is marked from stage 12 onward by the expression of eyeless (ey), is also affected by the loss of hh. Heatshock induced overexpression of hh, as well as loss of ptc, causes an increase in larval eye neurons and optic lobe precursors. Interestingly, ectopic Hh activity is able to induce optic lobe and Bolwig's organ tissue in the head midline and thereby generate a cyclops phenotype similar to the condition described above for partial reduction of dpp. Applying heatshocks at different times of development indicates that the phenocritical period for the Hh induced cyclops is early, between 2.5 and 5 hours. Thus, heat pulses administered during this time cause fusion of the optic lobe and, at a lower frequency, of the larval eye without significantly increasing the number of optic lobe and larval eye cells. By contrast, later heat pulses (after 5 hours) lead to larval eye/optic lobe hyperplasia but no concomitant cyclops phenotype (Chang, 2001).

The finding that both loss of Hh and Dpp cause the absence of visual structures, and ectopic expression of Hh and partial loss of Dpp cause transformation of head midline epidermis into visual primordium, begs the question of how the two signaling pathways interact. In Drosophila compound eye development, hh expression is required to turn on dpp expression. To establish whether a regulatory relationship exists between Hh and Dpp signaling, the expression of dpp and pMAD was examined in the background of hh loss of function, as well as hh, ptc and Cubitus interruptus (Ci) expression in the background of dpp loss of function. Cells in which Dpp signaling is activated can be visualized by an antibody against phosphorylated MAD (pMAD) protein. Dpp RNA expression and pMAD are normal in a stage 5-9 hh-null background, indicating that Hh is not required to activate Dpp signaling in the embryonic head (Chang, 2001).

A model is proposed to explain the phenotypes resulting from manipulating Dpp, Hh and Ptc expression:

1. In wild type, Hh can activate larval eye only in cells expressing so and eya. No larval eye develops in the dorsal midline because so is down regulated in this region rapidly, and Hh 'has no opportunity' to overcome the ptc mediated inhibition and induce visual system at an early stage when so is still present in the dorsal midline (Chang, 2001).
2. In ptc^-, Hh is able to induce larval eye fate in the dorsal midline because it is not inhibited at the early stage when so is still expressed dorsomedially (Chang, 2001).
3. Heatshock-induced Hh expression at an early stage (stage 5; around 3 hours) has the same effect, overcoming the ptc-mediated inhibition and inducing larval eye dorsomedially (Chang, 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/+, ptc^gal4/ptc^null), 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, ptc^S2, the mutation is an amino acid change from charged to neutral in the sterol-sensing domain. ptc^S2 fully complements ptc^hdl and the transheterozygotes have no head capsule defects. Moreover, in ptc^hdl/ptc^null 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).

Hedgehog signaling in the Drosophila eye and head: an analysis of the effects of different patched Trans-heterozygotes

Characterization of different alleles of the Hedgehog receptor patched (ptc) indicates that they can be grouped into several classes. Most mutations result in complete loss of Ptc function. However, missense mutations located within the putative sterol-sensing domain (SSD) or C terminus of ptc encode antimorphic proteins that are unable to repress Smo activity and inhibit wild-type Ptc from doing so, but retain the ability to bind and sequester Hh. Analysis of the eye and head phenotypes of Drosophila in various ptc/ptc^tuf1 heteroallelic combinations shows that these two classes of ptc alleles can be easily distinguished by their eye phenotype, but not by their head phenotype. Adult eye size is inversely correlated with head vertex size, suggesting an alteration of cell fate within the eye-antennal disc. A balance between excess cell division and cell death in the mutant eye discs may also contribute to final eye size. In addition, contrary to results reported recently, the role of Hh signaling in the Drosophila head vertex appears to be primarily in patterning rather than in proliferation, with Ptc and Smo having opposing effects on formation of medial structures (Thomas, 2003).

Antimorphic alleles of ptc were initially identified by the severity of their wing phenotype when heteroallelic with ptc^tuf1. Such trans-heterozygotes exhibit large outgrowths of the anterior wing, consistent with activation of dpp due to ectopic Hh signaling, whereas in the hemizygous condition ptc^tuf1 shows only a mild anterior outgrowth. By contrast, the eye phenotype appears, at least at first sight, to be much more severe in LF/ptc^tuf1 mutants than in AN/ptc^tuf1 mutants. However, closer analysis reveals that the two classes of alleles have distinct effects, the eyes of AN/ptc^tuf1 adults being significantly larger than those of wild type, rather than simply less reduced than those of LF/ptc^tuf1 mutants. However, the head defects typical of AN/ptc^tuf1 trans-heterozygotes do appear to be mild versions of those seen in LF/ptc^tuf1 mutants. Because antimorphic Ptc proteins are distinguished by their ability to sequester the Hh ligand, this implies that excessive Hh diffusion, rather than ectopic pathway activation due to Smo derepression, is the principal cause of the head phenotypes such as ectopic ocelli. In summary, it appears that the difference between allele class phenotypes in wing, eye, and head reflects the different relative impact of cell-autonomous ectopic pathway activation vs. excess Hh diffusion in the three different structures (Thomas, 2003).

In the canonical Hh signaling pathway, Ptc functions as a negative regulator both by sequestering Hh ligand and by inhibiting Smo activity. By contrast, evidence has been presented suggesting that Ptc and Smo act in concert to promote growth in the head, a function that is opposed by the activity of Hh. Reexamination of this issue does not support such an atypical interaction between Ptc and Smo in the head; moreover, it suggests that the predominant role of Hh signaling in the head is in patterning rather than in proliferation (Thomas, 2003).

The eye disc contains two domains of apoptosis: one immediately anterior to the MF that is regulated by Eya and one posterior to the MF whose function is unknown. The latter domain of cell death is promoted by Hh signaling from photoreceptors. The milder increase in cell death seen in AN/ptc^tuf1 trans-heterozygotes perhaps reflects the efficient sequestration of Hh in such mutants, suggesting that the range of Hh diffusion may be important in influencing the proportion of cells that die behind the MF. Normally, a large amount of cell death, necessary for the elimination of two to three excess pigment cells per ommatidium, occurs in pupal eye discs. Cell death behind the MF may have a similar function, perhaps in removing cells mis-specified during differentiation. It is possible that Hh could regulate apoptosis through activation of a molecule at short range behind the MF. Alternatively, cell death may not depend directly upon Hh, but rather upon the presence of increased numbers of mis-specified cells in ptc mutants that may result in cell death occurring to regulate differentiation effectively (Thomas, 2003).

The earliest known role of Hh in the eye imaginal disc is to help specify the dorso-ventral organizer. Incorrect DV specification compromises the function of N in promoting growth, resulting in small eyes. However, the small-eye phenotype seen in LF/ptc^tuf1 appears to be unconnected to this process. Although some disorganization of the equator is seen in ptc trans-heterozygotes, it is not significant, and its presence in both classes of mutant indicates that the effect, if any, on eye size is small (Thomas, 2003).

The two major targets of Hh signaling during MF progression are dpp and ato. The data indicate that although dpp is ectopically activated in ptc trans-heterozygotes, ato is not. This is unexpected, since Hh signaling activates the initial expression of ato, so an increase might be anticipated to expand the ato expression domain into more anterior regions, while maintaining or even increasing the level of expression. Conversely, reducing the activity of ptc in the hh¹ mutant rescues the expression of ato, but not that of dpp. There are several possible explanations for these findings. (1) ato is an indirect target of Hh signaling and the mediators of Hh activity in this context are unclear. It is likely that other factors, in addition to those directly induced by Hh, are necessary for ato expression, any one of which may be limiting. (2) dpp may respond to lower levels of Hh pathway activation more than genes upstream of ato. In the wing disc, dpp is activated by relatively low levels of Hh anterior to the AP border, whereas other Hh target genes such as collier require a higher level of pathway activation. In ptc trans-heterozygotes some Ptc activity is retained and hence the very highest level of Hh signaling cannot be reached. (3) Dpp in its role as an inducer of the preproneural state can actually inhibit the expression of ato through activation of the proneural repressors h and emc. In ptc trans-heterozygous discs, the domain of h expression does appear to be expanded, suggesting a possible explanation for the observed downregulation of ato expression. A fourth possibility that has not been tested is that the increased level of Hh signaling in ptc trans-heterozygote discs results in an expansion of the domain of rough (ro) expression. Ro is induced by high-level Hh signaling at the posterior margin of the MF, but, if expressed at excessive levels (as in the ro^Dom mutant), causes a downregulation of ato expression. Although a severe reduction in ato expression such as that caused by ro^Dom can result in furrow arrest, the significance of a mild downregulation of expression is unknown (Thomas, 2003).

The two classes of mutants both show an increase in dpplacZ expression relative to wild type. However, the domain of ectopic expression differs significantly between allele types, suggesting a difference in the way in which the pathway is activated in the two classes of mutant. Because LF ptc alleles cannot sequester Hh efficiently, the broad band of ectopic dpplacZ seen ahead of the MF may be caused by excessive diffusion of Hh anteriorly. In contrast, the AN/ptc^tuf1 trans-heterozygotes can sequester Hh efficiently and consequently demonstrate only phenotypes caused by autonomous ectopic pathway activation (Thomas, 2003).

Despite the rescue of adult-eye phenotype observed in ptc/ptc^tuf1;hh¹ double mutants, the expression of dpplacZ was not restored in the center of the disc. Since Dpp does not play a major role in MF progression, the lack of expression in this situation may not have a significant effect. Alternatively, excessive dpp expression at the margins may allow the protein to diffuse medially into the disc, thus aiding MF progression in an unconventional way (Thomas, 2003).

Dpp is known to have several functions in the eye disc, all of which, when modified, can influence the final size and shape of the adult eye. However, despite the disparity between the patterns of dpplacZ expression in the two types of trans-heterozygote, surprisingly little difference is detected downstream of Dpp. Although ectopic dpp expression has been shown to induce ectopic MFs, this does not occur in the ptc trans-heterozygotes, presumably because the ectopic Dpp is either not high enough or not expressed in the right place (Thomas, 2003).

In addition to its effect on furrow initiation, Dpp is also responsible for defining the eye field via inhibition of Wg and for inducing cell cycle arrest ahead of the furrow. Small-eye mutants do show an increased head vertex size, suggesting that an eye-to-vertex fate change has occurred. However, ptc trans-heterozygotes do not display critical differences either from wild type or between allele classes in the distribution of Wg in second instar eye discs. In addition, dpp expression is actually expanded in eye discs of small-eye mutants, indicating that processes other than Wg/Dpp antagonism must be involved in specification of eye vs. head domains (Thomas, 2003).

Ectopic Dpp ahead of the furrow does not appear to induce premature cell cycle arrest and therefore cannot explain the reduced-eye phenotypes observed in LF/ptc^tuf trans-heterozygotes. However, when compared to wild type, ptc mutants do show an increase in cell divisions ahead of the furrow. It is suggested that in addition to an eye/head vertex specification defect, LF/ptc^tuf1 trans-heterozygotes may exhibit a small-eye phenotype due to excessive cell death, despite some increase in cell divisions ahead of the furrow. Conversely, in DN/ptc^tuf1 trans-heterozygotes, increased proliferation could overcompensate for increased cell death, leading to larger eyes. This suggests that the adult-eye phenotype is at least partially dependent upon a balance between cell division and cell death in the disc, in addition to an eye-to-head fate change (Thomas, 2003).

Hedgehog-stimulated stem cells depend on non-canonical activity of the Notch co-activator Mastermind

Normal self-renewal of follicle stem cells (FSCs) in the Drosophila ovary requires Hedgehog (Hh) signaling. Excess Hh signaling, induced by loss of patched (ptc), causes cell-autonomous duplication of FSCs. A genetic screen was used to identify Mastermind (Mam), the Notch pathway transcriptional co-activator, as a rare dose-dependent modifier of aberrant FSC expansion induced by excess Hh. Complete loss of Mam activity severely compromises the persistence of both normal and ptc mutant FSCs, but does not affect the maintenance of ovarian germline stem cells. Thus, Mam, like Hh, is a crucial stem cell factor that acts selectively on FSCs in the ovary. Surprisingly, other Notch pathway components, including Notch itself, are not similarly required for FSC maintenance. Furthermore, excess Notch pathway activity alone accelerates FSC loss and cannot ameliorate the more severe defects of mam mutant FSCs. This suggests an unconventional role for Mam in FSCs that is independent of Notch signaling. Loss of Mam reduces the expression of the Hh pathway reporter ptc-lacZ in FSCs but not in wing discs, suggesting that Mam might enhance Hh signaling specifically in stem cells of the Drosophila ovary (Vied, 2009).

Drosophila follicle stem cells (FSCs) provide a paradigm for stem cell behavior that includes the basic attributes of visualizing a defined stem cell and its environment, coupled with the potential to manipulate stem cell genotypes extensively and measure stem cell function. Nevertheless, only a limited number of factors have so far been defined as being essential to FSC function. Among these are the Hh, Wnt and BMP signaling pathways, adhesion molecules, a chromatin-remodeling factor and a histone ubiquitin protease. This study defines Mastermind as an essential FSC factor. Mam is not generally required for cell proliferation or survival in follicle cells or other Drosophila tissues. Mam is also not required for GSC function, assayed under exactly the same conditions and in the same animals that reveal its role in FSCs. However, in the absence of Mam, FSCs are lost as rapidly from adult ovaries as FSCs that cannot transduce a Hh signal (Vied, 2009).

These experiments show that Mam function is required cell-autonomously within the FSC lineage and it is assumed that this reflects a function in the FSC itself. More evidence of increased apoptosis of mam mutant FSCs or of prolonged quiescence of such cells (positive-marking studies) was seen, suggesting that mam FSCs are prematurely lost from their characteristic position in the germarium, taking on the fate of non-stem FSC daughter cells. Whether loss of Mam primarily affects a fundamental stem-daughter cell decision or adhesive properties contributing to niche retention is, as for most other FSC factors, unknown (Vied, 2009).

Mam has long been considered to be a dedicated co-activator in the Notch signaling pathway, because genetic analyses in Drosophila and other model organisms generally show a congruence between Notch and Mam loss-of-function phenotypes. Biochemically, Mam binds to a composite surface contributed by the cleaved intracellular domain of activated Notch and the DNA-binding protein Su(H), and provides an essential transcriptional activation function that includes the recruitment of CREB-Binding Protein (CBP). In ovaries, characteristic Notch mutant phenotypes were seen in response to mam mutations, showing that Mam does indeed act as an essential co-activator for Notch signaling in the follicle cell lineage. However, that function of Mam cannot account for its role in FSCs because FSC function is not impaired by null mutations affecting Notch and Su(H), the direct binding partners of Mam. That assertion is also consistent with the finding that expression of a dominant-negative Mam derivative inhibited the Notch-dependent behaviors of FSC derivatives without impairing FSC maintenance. To determine whether loss of Notch signaling contributed even partially to the mam mutant FSC phenotype, attempts were made to ameliorate the mam phenotype by activating Notch signaling in a Mam-independent manner. It was found that a synthetic Su(H)-VP16 activator could not rescue mam or ptc mam mutant FSC loss. Furthermore, increased activity of the Notch pathway by itself [using Su(H)-VP16 or N^intra] caused moderate FSC loss. Thus, the essential activity of Mam in FSCs appears to be entirely independent of the well-known role of Mam as a co-activator for Notch signaling (Vied, 2009).

The finding that FSC maintenance is not markedly impaired by the elimination of Notch signaling is notable in itself, because it contrasts with a requirement for each of the three other pathways (Hh, BMP and Wnt) that have been investigated to date. Notch signaling is important in the germarium for the earliest known decision of FSC progeny to adopt polar and stalk cell fates, and for the specification and maintenance of cap cells, which are themselves essential for maintaining GSCs (Vied, 2009).

The loss of FSCs in response to increased Notch activity might also be informative, although the heightened Notch activity induced by N^intra or Su(H)-VP16 is likely to be beyond physiological levels. The FSC loss induced by N^intra cannot be explained by titration of Mam away from other essential partners, because Su(H)-VP16 induces a similar phenotype but cannot bind to Mam in the absence of activated Notch (Vied, 2009).

The Notch ligand Delta is known to be expressed in terminal filament, cap, follicle and germline cells, and a clear increase in Delta signaling from the germline to overlying follicle cells at stage 6 triggers a switch from mitosis to follicle cell endocycles. Interestingly, that switch is still imposed on ptc mutant follicle cells that contact the germline, and is accompanied by Notch-dependent inhibition of ci expression and Hh pathway activity, which is mediated by the transcription factors Hindsight (Pebbled in FlyBase) and Tramtrack. FSC loss induced by Notch hyperactivity is also seen for ptc mutant cells and might conceivably involve an analogous mechanism, although Hindsight expression is not normally observed prior to stage 6. Moreover, it is possible that FSCs normally evade Notch-induced repression of Hh signaling by minimizing contact with the germline, while non-stem cell daughters embrace passing germline cysts (Vied, 2009).

There are currently very few reports of Notch-independent roles of Mam proteins (McElhinny, 2008). In two cases, the mammalian Mam homolog MAML1 was shown to bind and collaborate with DNA-binding proteins (p53 and MEF2C) other than those of the Su(H) family. In the third case, MAML1 was shown to bind β-catenin and to contribute to TCF-dependent induction of Wnt target genes. It seems likely from these examples, and from the established role of Mam in recruiting Mediator and histone acetyltransferase complexes, that the essential action of Mam in stem cells is as a transcriptional co-activator (Vied, 2009).

Mam function in FSCs has a notable dosage-sensitive interaction with the Hh signaling pathway. Mam was first identified in this context because a heterozygous mam mutation strongly suppressed ovarian somatic cell overproliferation induced by excess Hh signaling. This suppression was partially reproduced by controlling the level of Mam expression from a UAS-Mam transgene and was shown under those circumstances to suppress the duplication of FSCs normally induced by excessive Hh pathway activity. The initial genetic screen suggests that dose-dependent suppressors of Hh-induced FSC expansion are rare. Complete loss of mam was fully epistatic over ptc mutations with regard to FSC duplication and FSC maintenance (Vied, 2009).

Several mechanisms might theoretically account for the observed interactions of Mam with the Hh pathway. However, it was also seen that loss of Mam inhibited expression of the Hh pathway reporter ptc-lacZ in FSCs, focusing attention on the idea that Mam might act as a co-activator in the Hh pathway. Some further observations are relevant to this hypothesis (Vied, 2009).

First, no evidence was found of Mam affecting Hh signaling output in wing discs. Thus, any effect of Mam on FSC Hh signaling is tissue specific. Very little is known of the mechanisms underlying tissue-specific responses to Hh signaling, but tissue-specific interactions of Ci with other transcription factors and co-activators are likely conduits. Second, loss of Mam limited the induction of ptc-lacZ in ptc mutant FSC clones, but only to levels seen in normal FSCs. Thus, if Mam does indeed act in FSCs to potentiate Hh signaling, it could only be crucial for target genes induced by strong Hh pathway activity, for which ptc-lacZ is an insufficient marker. There is a precedent for exactly this situation in wing discs. There, loss of Fu kinase activity in ptc mutant clones completely eliminates the expression of Engrailed (which responds only to strong pathway activity) and substantially alters the resulting wing phenotype without reducing ptc-lacZ expression (Vied, 2009).

In summary, epistasis of mam over ptc and the specific requirement for Mam in FSCs, which experience higher Hh signaling than their progeny, are consistent with a role for Mam as a co-activator of crucial FSC target genes induced only by strong Hh pathway activity. However, it is also possible that Mam contributes to FSC function independently of the Hh pathway, affecting ptc-lacZ expression in FSCs only indirectly. Further investigation would benefit greatly from the identification of crucial FSC Hh target genes and detailed examination of the chromatin localization of Mam, Ci and other transcription factors in the FSC lineage (Vied, 2009).

A neurodegenerative disease affecting synaptic connections in Drosophila mutant for the tumor suppressor morphogen Patched

The tumor-suppressor morphogen, Patched (Ptc), has extensive homology to the Niemann-Pick-C 1 (NPC1) protein. The NPC disease is a paediatric, progressive and fatal neurodegenerative disorder thought to be due to an abnormal accumulation of cholesterol in neurons. This study reports that patched mutant adults develop a progressive neurodegenerative disease and their brain contains membranous and lamellar inclusions. There is also a significant reduction in the number of synaptic terminals in the brain of the mutant adults. Interestingly, feeding cholesterol to wild type flies generates inclusions in the brain, but does not cause the disease. However, feeding cholesterol to mutant flies increases synaptic connections and suppresses the disease. These results suggest that sequestration of cholesterol in the mutant brain in the form of membranous material and inclusions affects available pool of cholesterol for cellular functions. This, in turn, negatively affects the synaptic number and contributes to the disease-state. Consistent with this, in ptc mutants there is a reduction in the pool of cholesterol esters, and cholesterol-mediated suppression of the disease accompanies an increase in cholesterol esters. It was further shown that Ptc does not function directly in this process since gain-of-function for Hedgehog also induces the same disease with a reduction in the level of cholesterol esters. It could be that loss of function for ptc causes neurodegeneration via two distinct ways: de-repression of genes that interfere with lipid trafficking, and de-repression of genes outside of the lipid trafficking; the functions of both classes of genes ultimately converge on synaptic connections (Gazi, 2009).

Ptc has extensive homology to the Niemann Pick C 1 (NPC1) protein. For example, both Ptc and NPC1 proteins are seven-pass membrane proteins of similar sizes, both have sterol-sensing domains located approximately at the middle of the protein, the two proteins also have two of the what are known as permease domains (present in MexD and AcrB bacterial proteins), placed at the C-terminal portion of the protein; the two proteins have significant homology in these domains, including the transmembrane domains. However, any functional sharing between these proteins has not been explored and it remains unknown if this structural similarity translates into and functional sharing in developmental processes or disease or it is just a coincidence (Gazi, 2009).

This study shows that loss of function for ptc causes a neurodegenerative disease that has similarity to the NPC disease in vertebrates. This involvement of Ptc in a neurodegenerative disease defines a novel role for Ptc. The similarity to the NPC disease is consistent with the fact that NPC1 and Ptc share significant sequence homology. It was also shown that the loss of function for Ptc results in a reduction in the levels of cholesterol esters in the brain. Loss of Ptc function also progressively affects the number of Syt-positive synaptic connections in such structures as the dendritic-rich calyx of the brain. The fact that levels of cholesterol esters, and the number of connections in the mutant brain can be restored by feeding cholesterol, and that the disease can be suppressed by feeding cholesterol argues a primary role for cholesterol esters and synaptic connections in the progression of the disease. These results are also supported by the EM data that the number of connections are reduced in the mutant brain, which can be restored by feeding cholesterol. These results also reveal a role for cholesterol esters in forming and or maintaining synaptic connections, either directly or indirectly. Moreover, it was also shown that inclusions per se are not the problem since wild type flies can have the inclusion but that does not cause the disease. These could be novel results which shed new insight into the basic science of neurodegenerative diseases in general. The study also demonstrates new tools to analyze the adult brain that should generally be applicable in adult brain development and functional studies (Gazi, 2009).

There are two NPC1 genes in Drosophila, NPC1a and NPC1b. The Ptc protein shares significant homology to the NPC1 proteins in Drosophila or to the vertebrate NPC1 protein. Two previous studies show that null mutants for NPC1a die as first instar larvae, but these mutant larvae can be rescued to adulthood by feeding them cholesterol. Also, that while the brain in these rescued mutants are normal, the malphigian tubule (the kidney equivalent in flies) has the highest sterol accumulation and contains large multi-lamellar inclusions. These inclusions are strikingly similar to what was found in the brain of ptc mutants in this study. Moreover, in NPC disease, it is thought that unesterified cholesterol accumulates in the E/L system. It was found that there is a reduction in the pool of esterified cholesterol. These results suggest that the cholesterol metabolism is affected in both NPC disease and ptc mutants (Gazi, 2009).

These results further suggest that NPC1 genes in flies may play a smaller role in the brain. This possibility is consistent with another study which reports that null mutants for dNPC1a gene mimic human NPC patients with progressive motor defects and reduced life span; the brains of these mutants reportedly contain higher levels of cholesterol and multi-lamellar inclusions. What is the relationship between ptc and dNPC1? It was observed that ptc mutant individuals that are also heterozygous for NPC1a (ptc/ptc; NPC1a/+) do not show an enhancement of the disease. While it was not examined if the double mutants between ptc and dNPC1 mutants show an enhanced neurodegenerative phenotype, it may be that one needs to eliminate also both NPC1 genes (dNPC1a and dNPC1b) in order to observe a strong brain phenotype. Nonetheless, it seems likely that the ptc-neurodegenerative disease in flies is closest to the NPC-disease in vertebrates. In vertebrates, since loss of function for ptc die as embryos, it is not known if Ptc has any role in preventing a neurodegenerative disease; however, at least in Drosophila, Ptc may function in the same pathway as the NPC1 proteins. Additional work is needed to fully determine the relationship between dNPC1a and dNPC1b as well as between dNPC1 genes and ptc (Gazi, 2009).

The role of Hh-Ptc-Smo signaling in development and disease has been examined in many different studies over the last several years. This study reveals that Ptc has a role in preventing a neurodegenerative disease. This disease is not due to a strange allele of ptc but due to the loss of function for ptc. The disease was observed in several different allelic combinations of ptc, including combinations of known loss of function alleles and deficiencies (which also rules out a background-mediated locomotor defect). The phenotype can also be rescued by a ptc transgene. Ptc is widely expressed in the adult brain, and at the same time, the expression of Hh is restricted to mostly olfactory lobes, suggesting that Ptc plays a repressive role in the development of the disease. This is also consistent with the result that a gain of function for Hh causes the same disease as the loss of function for ptc. Moreover, the fact that inducing Hh in the brain at any point in the adult life will cause the disease indicates that the disease in the loss of function for ptc is not due to some developmental abnormality. Results with Hs-hh also indicate that Ptc functions indirectly via repressing Smo. This conclusion is consistent with the result that reducing the dosage of smo alleviates the disease (Gazi, 2009).

It is thought that in NPC disease, formation of inclusions in neurons causes neuronal death, which results in a disease-state. However, no direct evidence exists to link the inclusions to disease-state. A previous study suggests that in the case of Huntington’s disease, formation of inclusion bodies is a cellular response to decrease the mutant protein and prevent the poisoning of other neurons. Therefore, formation of such inclusions is a good thing for neurons in slowing down the disease. It was shown that the inclusions in the brain of ptc mutants are due to cholesterol: feeding a large amount of cholesterol to wild type flies causes formation of inclusions in the brain. This also indicates that the presence of inclusions in the brain of ptc mutants is indeed due to an aberrant metabolism or accumulation of cholesterol in neurons. Quantifying the number of inclusions in the entire brain in EM experiments was found to be a difficult proposition for several reasons. First is the fact that there are no serial section EM maps of the fly brain and it is nearly impossible to pinpoint the precise location within the brain to be able to compare between brains of different genotypes, conditions and age. Second, the inclusions are not uniformly spread in the brain. The only way to avoid these two problems and be able to give a precise quantification of inclusions between brains is to do serial EM sections of the entire brain and count the number of non-overlapping inclusions in each brain (Gazi, 2009).

To perform serial EM sections of an entire brain, the following calculations were made. Each EM section is about 100 nm thick and one needs to cut about 1,500 sections to cover the entire brain. The area of the each section is about 150,000 μm2. In order to reliably examine the section for lamellar inclusions, one needs to examine the sections at a magnification of 5,000 times. At this magnification each image will cover 300 μm2 of an area. This means that one needs to take 500 images to cover one entire section at this magnification. Thus, the total number of images one would have to take to examine an entire brain is 1,500 × 500 = 750, 000. This is not practical given that one will have to section a minimum of 3 brains for each sample. Therefore, quantification of inclusions reported in this study should be considered approximations and may or may not be representative of the entire brain. However, it is to be noted that quantification of synaptic connections from EM photomicrographs is somewhat of a different scenario since the synaptic connections are generally spread in a uniform fashion within the neuropile (and the brain overall). Therefore, the error due to imprecision of the location, though still exists, it is at a much lower level. Nevertheless, the quantification of the synaptic connections using EM should be again treated as an approximation. Thus, the number of Syt-positive connections in a discrete structure such as the calyx were examined using the Apotome microscopy between different samples of the brain, a much more accurate analysis (Gazi, 2009).

Because of the technical difficulty with quantifying inclusions between brain samples, this study focused on examining synaptic connections in a discrete structure such as the calyx, where one can quantify the number of Syt-positive terminals using the newly developed method and draw meaningful conclusions. It was found that loss of synaptic terminals is a major contributor to the disease-state. In ptc mutants, there is a significant reduction in the Syt-positive synaptic connections. One can argue that the loss of synaptic connections is due to the degenerative disease. However, it was shown that loss of synaptic connections and the disease-state in ptc mutants can be suppressed by feeding cholesterol; cholesterol appears to prevent the loss of the synaptic connections and also the progression of the disease (Gazi, 2009).

Normally, esterified cholesterol (LDL) is taken up inside the cell via endocytosis by the endosomal/lysosomal (E/L) system and de-esterified in lysosomes; it is then re-esterified in the cytoplasm and then stored or sent to other cellular components. For instance, the most prominent characteristic of the NPC disease at the cellular level is the blockade of intracellular transport of LDL-derived cholesterol between the E/L compartment and the plasma membrane resulting in the accumulation of unesterified cholesterol in the E/L system. This blockade of cholesterol transport appears to be the cause for the formation of inclusion bodies in neurons of NPC human patients or NPC mice. Additionally, the finding that the gene corresponding to NPC2 encodes an ubiquitously-expressed cholesterol binding lysosomal protein, known as HE1, further supports a role for cholesterol in the disease. On the other hand, in human NPC patients, a combination of diet and cholesterol-lowering drugs has been found to have no beneficial effect. Therefore, the nature of cholesterol involvement in the genesis of the disease is not known (Gazi, 2009).

Results from this study suggest that one of the consequences of loss of function for ptc is the accumulation of cholesterol in the cytoplasm leading to the formation of inclusions and membranous material. Furthermore, there is a progressive loss of synaptic connections in the mutant brain. Thus, one possibility is that in ptc mutants, cholesterol becomes limiting, and this leads to a loss of synaptic connections. This is supported by the fact that feeding cholesterol to wild type or mutant flies increases the connections and in mutants, feeding cholesterol suppresses the disease. Results from a few previous studies are also consistent with this possibility. For example, an in vitro study suggests that cholesterol promotes formation of synapses. This was also observed in the adult fly brain. Moreover, apoE has long been suspected to be involved in neurodegenerative loss of synaptic plasticity in the Alzheimer’s disease. Furthermore, the e4 mutant in the gene for ApoE, an important cholesterol transport protein, is associated with an increased risk of late-onset Alzheimer’s disease; this mutant isoform of the protein is less able to promote neurite outgrowth than other apoE isoforms. It is not clear, however, if cholesterol promotes synapse formation by directly influencing the membrane property (i.e., fluidity), as a structural component, via regulating gene activity, or via the synthesis of neurosteroids. A decrease in the available cholesterol may increase the fluidity of the plasma membrane of synaptic terminals and make synaptic connections structurally unstable. Alternatively, limiting amounts of cholesterol may affect gene expression or the synthesis of neurosteroids, contributing to the disease-state. It should also be noted that the suppression of the disease with cholesterol is not complete. Thus, it canbe speculated that loss of function for ptc causes neurodegeneration via two distinct ways: 1) de-repressing genes that interfere with lipid/cholesterol trafficking, and 2) de-repressing genes outside of the lipid trafficking; the functions of both classes of genes ultimately converge on synaptic connections (Gazi, 2009).

Establishment of global patterns of planar polarity during growth of the Drosophila wing epithelium

Epithelial tissues develop planar polarity that is reflected in the global alignment of hairs and cilia with respect to the tissue axes. The planar cell polarity (PCP) proteins form asymmetric and polarized domains across epithelial junctions that are aligned locally between cells and orient these external structures. Although feedback mechanisms can polarize PCP proteins intracellularly and locally align polarity between cells, how global PCP patterns are specified is not understood. It has been proposed that the graded distribution of a biasing factor could guide long-range PCP. However, epithelial morphogenesis has been identified as a mechanism that can reorganize global PCP patterns; in the Drosophila pupal wing, oriented cell divisions and rearrangements reorient PCP from a margin-oriented pattern to one that points distally. This study used quantitative image analysis to study how PCP patterns first emerge in the wing. PCP appears during larval growth and is spatially oriented through the activities of three organizer regions that control disc growth and patterning. Flattening morphogen gradients emanating from these regions does not reduce intracellular polarity but distorts growth and alters specific features of the PCP pattern. Thus, PCP may be guided by morphogenesis rather than morphogen gradients (Sagner, 2012).

To study the emergence of polarity in the wing disc, the subcellular distribution of the PCP proteins Flamingo (Fmi) and Prickle (Pk) were quantified. Planar cell polarity (PCP) nematics were calculated based on Fmi staining and PCP vectors based on the perimeter intensity of EGFP::Pk clones. At 72 hr after egg laying (hAEL), the wing pouch has just been specified and is small. EGFP::Pk localizes to punctate structures at the cell cortex that are asymmetrically distributed in some cells, but PCP vectors exhibit no long-range alignment. By 96 hAEL, PCP vector magnitude increases and a global pattern emerges. Later, PCP vector magnitude increases further and the same global polarity pattern is clearly apparent. It is oriented with respect to three signaling centers: the dorsal-ventral (DV) boundary (where Wingless [Wg] and Notch signaling occur), the anterior-posterior (AP) compartment boundary (where Hedgehog [Hh] and Decapentaplegic [Dpp] signaling occur), and with respect to the hinge fold (where levels of the atypical Cadherin Dachsous [Ds] change sharply) (Sagner, 2012).

PCP vectors in the wing pouch near the hinge fold point away from it toward the center of the pouch. Within the Wg expression domain at the DV boundary, PCP vectors parallel the DV boundary and point toward the AP boundary. Just outside this domain, PCP nematics and vectors turn sharply to point toward the DV boundary in central regions of the wing pouch. However, where the DV boundary intersects the hinge-pouch interface, they remain parallel to the DV boundary over larger distances such that PCP vectors orient away from the hinge around the entire perimeter of the wing pouch (Sagner, 2012).

The AP boundary is associated with sharp reorientations of PCP. First, PCP vectors that parallel the DV boundary point toward the AP boundary in both anterior and posterior compartments. Second, although PCP vectors in the central wing pouch are generally orthogonal to the DV boundary, they deflect toward the AP boundary where Hh signaling is most active (as defined by upregulation of the Hh receptor Patched [Ptc]). On either side of this region, PCP vectors turn sharply to realign parallel to the AP boundary. Third, PCP vectors in the hinge point away from the AP boundary and align parallel to the hinge fold (Sagner, 2012).

The atypical Cadherins Fat (Ft) and Ds limit disc growth and orient growth perpendicular to the hinge. Their loss perturbs the PCP pattern in pupal wings and alters hair polarity. To investigate whether they influence the larval pattern, PCP was was quantified in ft and ds mutant discs. The PCP pattern is similar to wild-type (WT) in the central wing pouch but altered in proximal regions close to the hinge fold. Polarity vectors deviate from their normal orientation (away from the hinge fold) in many regions of the proximal wing pouch. This is especially clear near the intersection of the DV boundary with the hinge - here, PCP vectors orient toward the DV boundary rather than away from the hinge. Furthermore, near the AP boundary, vectors form a reproducible point defect, with vectors pointing away from the defect center (Sagner, 2012).

After pupariation, morphogenesis reshapes the wing disc, apposing its dorsal and ventral surfaces such that the DV boundary defines the margin of the wing blade. During reshaping the PCP pattern evolves, but specific local features are retained through pupal development. Consistent with this, hair polarity in ds adult wings proximal wing near the anterior wing margin orient toward the margin rather than away from the hinge. Near the AP boundary, hairs form swirling patterns. Thus, Ft and Ds are required during larval growth to ensure that PCP vectors in the proximal wing orient away from the hinge (Sagner, 2012).

Notch and Wg signaling at the DV boundary organize growth and patterning in the developing wing. These pathways maintain each other via a positive feedback loop; Notch induces transcription of Wg at the DV interface, and Wg signaling upregulates expression of the Notch ligands Delta (Dl) and Serrate (Ser) adjacent to the Wg expression domain, further activating Notch signaling at the DV boundary. To study how the DV boundary organizer affects PCP, Ser was ectopically expressed along the AP boundary with ptc-Gal4 (ptc > Ser). In the ventral compartment, Ser induces two adjacent stripes of Wg expression, which then upregulate Dl expression in flanking regions (dorsally, Fringe prevents Notch activation by Ser. The posterior Wg and Dl stripes are distinct, but the anterior stripes are broader due to the graded activity of ptc-Gal4. In these discs, the ventral compartment overgrows along the AP boundary, parallel to the ectopic 'organizers'. PCP nematics and vectors near the posterior Wg/Dl stripes are organized similarly to those flanking the normal DV boundary, running parallel to the stripe and turning sharply outside this region to orient toward the ectopic organizer). PCP nematics anterior to the ectopic Ser stripe run parallel to it over larger distances before turning sharply, consistent with the broader Wg/Dl expression in this region. In resulting adult wings, hairs orient toward the ectopic wing margin that forms along the AP boundary. Ectopically expressing Wg along the AP boundary also generates an ectopic organizer that reorients growth and PCP (Sagner, 2012).

To ask how loss of the DV boundary organizer affected PCP, a temperature-sensitive allele of wg was used that blocks Wg secretion (wgTS), or wings were populated with wg null mutant clones. Loss of Wg signaling severs the feedback loop with Notch such that both decay. PCP nematics were quantified in wgTS discs shifted to the restrictive temperature shortly after the second to third-instar transition (earlier, Wg is required to specify the wing pouch). wgTS discs have smaller wing pouches than WT and are missing a large fraction of the central region of the pouch where polarity orients perpendicular to the DV boundary. Polarity still orients away from the hinge, thus the PCP pattern in wgTS discs appears more radial (i.e., oriented toward the center of the wing pouch). Analogously, adult wings populated by wg null clones are missing those regions of the distal wing blade where hairs normally point perpendicular to the wing margin. The remaining proximal tissue is normally polarized except at its distal edges. Here, polarity deflects from the proximal-distal axis to parallel the edge of the wing. Normally, hair polarity in the wing blade parallels the margin only in proximal regions, where Ft/Ds influences polarity. Thus, the DV organizer is needed to orient PCP in distal regions perpendicular to the margin. Ft/Ds is required for a complementary subset of the PCP pattern in the proximal wing. Their influences largely reinforce each other (i.e., away from the hinge and toward the DV boundary or wing margin) except where the hinge and wing margin intersect. Here, loss of one signaling system expands the influence of the other. Wg is distributed in a graded fashion and is a ligand for Frizzled (Fz). Thus, it could bias the PCP pattern directly, e.g., by asymmetrically inhibiting interactions between Fz, Strabismus (Stbm), and Fmi or causing Fz internalization. If so, uniform Wg overexpression should prevent intracellular polarization or reduce cortical localization of PCP proteins. To investigate this, Wg was overexpressed uniformly (C765 > wg::HA). Uniform Wg expression elongates the wing pouch parallel to the AP boundary. It broadens the pattern of Dl expression, such that sharp Dl stripes at the DV boundary are lost, but Dl expression remains excluded from the Hh signaling domain anterior to the AP boundary. Fmi and EGFP::Pk polarize robustly in these discs; thus, the Wg gradient does not act directly on PCP proteins to induce or orient polarity. However, the pattern of PCP vectors and nematics is altered. PCP points away from the hinge (rather than perpendicular to the DV boundary) over larger distances compared to WT and then turns sharply to face theDV boundary in the middle of the wing pouch. Because specific alterations in the PCP pattern are induced by uniform Wg overexpression, Wg protein distribution does not directly specify the new PCP pattern (Sagner, 2012).

To identify signals that influence the PCP pattern near the AP boundary, the effects of uniform high-level expression of Dpp and Hh, two morphogens that form graded distributions near the AP boundary, were examined. Uniform Dpp expression does not influence the magnitude of PCP or the range over which PCP deflects toward the AP boundary. Interestingly, uniform Hh expression dramatically increases the range over which PCP deflects toward the AP boundary, suggesting that Hh is important for this aspect of the pattern. However it clearly indicates that PCP vectors are not oriented directly by the graded distribution of Hh or by the graded activity of Hh signaling, because both are uniformly high in the anterior compartment of Hh overexpressing discs. Whether the apposition of cells with very different levels of Hh signaling might produce sharp bends in the PCP pattern was therefore considered. In WT discs, Hh signaling levels change at two interfaces: one along the AP boundary and one along a parallel line outside the region of highest Hh signaling where Ptc is upregulated. PCP vectors orient parallel to the AP boundary in the cells posterior to it, deflect toward the boundary anteriorly, and then reorient sharply outside of this region to align parallel to the AP boundary. Discs uniformly overexpressing Hh have only one signaling discontinuity (at the AP boundary), because Hh signaling is high throughout the anterior compartment. This could explain why PCP in these discs remains deflected toward the AP boundary over longer distances (Sagner, 2012).

To test this, clones mutant for the Hh receptor Ptc, which constitutively activate signaling in the absence of ligand, were generated. Quantifying PCP nematics in these discs reveals reproducible patterns of polarity reorientation at interfaces between WT and ptc^- tissue. In WT tissue adjacent to ptc^- clones, PCP aligns parallel to the clone interface. Due to the typical clone shape, this orientation is often consistent with the normal PCP pattern. However, PCP also aligns parallel to ptc^- clones in regions where this is not so. Thus, ptc^- clones exert a dominant effect on adjacent WT tissue. In contrast, on the mutant side of the clone interface, polarity tends to orient perpendicular to the interface. Thus, apposition of high and low levels of Hh signaling causes a sharp bend in the PCP pattern. Corresponding polarity reorientation by ptc^- clones is also seen in adult wing. Thus, Hh signaling has two effects in WT discs: within the Hh signaling domain, it deflects PCP toward the AP boundary, and just outside the Hh signaling domain, it orients PCP parallel to the AP boundary. In this region, the tendency for polarity to align parallel to Hh signaling interfaces is consistent with the orientation of polarity toward the DV boundary and away from the hinge. Thus, these three polarity cues reinforce each other throughout much of the wing pouch, making the global PCP pattern robust (Sagner, 2012).

Simulations have highlighted the difficulty of establishing long-range polarity alignment in a large field of cells from an initially disordered arrangement. The pattern typically becomes trapped in local energy minima, forming swirling defects. Introducing a small bias in each cell removes such defects - this has been an attractive argument for the involvement of large - scale gradients in orienting PCP. The graded distribution of Ds along the proximal-distal axis (orthogonal to the hinge-pouch interface) suggested a plausible candidate for such a signal. Strikingly, the Ds expression gradient gives rise to intracellular polarization of both Ft and Ds, and the recruitment of the atypical myosin Dachs to the distal side of each cell. Nevertheless, most of the PCP defects in ft mutants can be rescued by uniform overexpression of a truncated Ft version that cannot interact with Ds, and PCP defects in ds mutants can be rescued by uniform overexpression of Ds. Moreover, blocking overgrowth through removal of dachs also suppresses PCP phenotypes in both mutants. The remaining disturbances in PCP in each of these backgrounds are restricted to very proximal regions, both in adult wings and the wing disc. Thus, the graded distribution of Ds does not provide a direct cue to orient PCP over long distances; rather, it appears to be important only locally near the hinge. Furthermore, this study shows that the two other key signaling pathways that contribute to the global PCP pattern in the disc do not act directly through long-range gradients. How do these signals specify the PCP pattern, if not through gradients (Sagner, 2012)?

Simulations in the vertex model have suggested that long-range polarity can be established in the absence of global biasing cues if PCP is allowed to develop during growth. PCP easily aligns in a small system, and globally aligned polarity can then be maintained as the system grows. Such a model obviates the necessity of long-range biasing cues like gradients, at least to maintain long-range alignment of PCP domains. The finding that a global PCP pattern develops early during growth of the wing makes this idea plausible. It may be that a combination of local signals at the different organizer regions specifies the vector orientation of PCP when the disc is still small, and that the pattern is maintained during growth. This may explain why loss-of-function studies have failed to identify the signaling pathways at the AP and DV boundaries as important organizers of the PCP pattern (Sagner, 2012).

In addition to local signals, the orientation of growth may provide additional cues that help shape the PCP pattern. Simulating the interplay between PCP and growth in the vertex model showed that oriented cell divisions and cell rearrangements orient PCP either parallel or perpendicular to the axis of tissue elongation, depending on parameters. Interestingly, each of the signaling pathways that influence PCP in the disc also influences the disc growth pattern. Wg/Notch signaling at the DV boundary drives growth parallel to the DV boundary, consistent with the pattern of clone elongation at the DV boundary. Growth near the AP boundary, where Hh signaling is most active, is oriented parallel to the AP boundary. This behavior probably reflects oriented cell rearrangements rather than oriented cell divisions. Finally, Ft and Ds orient growth away from the hinge. Suppressing overgrowth in ft or ds mutant wings by altering downstream components of the Hippo pathway rescues normal PCP except in the most proximal regions of the wing. Thus, altered growth orientation may contribute to the PCP defects seen in ft and ds mutants (Sagner, 2012).

Growth orientation reflects the orientation of both cell divisions and neighbor exchanges, and these can each exert different effects on the axis of PCP. Understanding the influence of local growth patterns on PCP will require a quantitative description of the patterns of cell divisions and rearrangements in the disc. More refined simulations incorporating local differences in the orientation of cell divisions and rearrangements will allow exploration of how planar polarity patterns can be guided by different growth patterns (Sagner, 2012).

Ligand-independent activation of the Hedgehog pathway displays non-cell autonomous proliferation during eye development in Drosophila

Deregulation of the Hedgehog (Hh) signaling pathway is associated with the development of human cancer including medullobastoma and basal cell carcinoma. Loss of Patched or activation of Smoothened in mouse models increases the occurrence of tumors. Likewise, in a Drosophila eye model, deregulated Hedgehog signaling causes overgrowth of eye and head tissues. Surprisingly, cells with deregulated Hh signaling do not or only little contribute to the tissue overgrowth. Instead, they become more sensitive to apoptosis and may eventually be eliminated. Nevertheless, these mutant cells increase proliferation in the adjacent wild-type tissue, i.e., in a non-cell autonomous manner. This non-cell autonomous effect is position-dependent and restricted to mutant cells in the anterior portion of the eye. Precocious non-cell autonomous differentiation was observed in genetic mosaics with deregulated Hh signaling. Together, these non-cell autonomous growth and differentiation phenotypes in the Drosophila eye model reveal another strategy by which oncogenes may generate a supportive micro-environment for tumor growth (Christiansen, 2012).

Hh signaling is known to regulate proliferation. Consistently, this study shows that deregulated, ligand-independent Hh signaling due to loss of the negative regulators cos2 and ptc causes overgrowth phenotypes of mosaic eyes and heads. Paradoxically, however, cos2 and ptc mutant cells have a growth-disadvantage and are eventually eliminated by apoptosis. In mosaic discs, proliferation is increased at the border to adjacent cos2⁺ tissue suggesting that the overgrowth is mediated through induction of non-cell autonomous proliferation. This effect is position-dependent and restricted to cos2 clones in or anterior to the MF. Finally, it was demonstrated that cos2 clones not only cause non-cell autonomous precocious proliferation, but also non-cell autonomous differentiation (Christiansen, 2012).

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).

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