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Genetics of morphogen gradients
Author: Tetsuya Tabata
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"620 | AUGUST 2001 | VOLUME 2 www.nature.com/reviews/genetics REVIEWS ?A frog breeds a frog?, or like breeds like, as the Japanese proverb goes. Although organisms develop from a sin- gle undifferentiated cell, the body pattern is genetically programmed in great detail, right down to the shape of the fingers. During the past 20 years, there has been sub- stantial progress in understanding the basic mecha- nisms of development. It has emerged that only a rela- tively small number of genetic networks are essential for designing the body pattern during development. Signalling molecules are key to such networks. Some of these molecules have been shown to function as mor- phogens. Here, the term ?morphogen? (literally ?form- giving?) is used rigorously to indicate a particular type of signalling molecule that sets the positional value of a cell by forming a concentration gradient across the develop- mental field in which the cell resides; the value of the gradient at each point in the field is a function of the distance of the receiving cell from the morphogen- secreting cells 1 . Morphogen gradients are not the only available method used to pattern a developmental field. The same patterning effect could be achieved through the sequential inductive signalling that is relayed between adjacent cells in the field (FIG. 1). However, it is more economical for organisms to use a single sig- nalling system to produce the various cell types that depend on their position in a molecular concentration gradient than to develop a different type of signalling system for each cell type. Indeed, many patterning processes in vertebrates and invertebrates have been attributed to the graded distribution of just one mole- cule. The concept of a morphogen is a very old one; morphogens were speculated to exist before there was any molecular evidence for them 2 . The molecular details of morphogen function have therefore been one of the fundamental issues in the field of developmental biolo- gy ? one that molecular genetic studies, primarily in Drosophila melanogaster, are beginning to unravel. Secreted signalling molecules have been implicated as organizers of pattern and growth in many developing systems, both in embryogenesis and in organogenesis. Some of these signalling molecules, which include members of the transforming growth factor-? (TGF-?) superfamily, and the Hedgehog (Hh) and Wingless (Wg)/Wnt proteins, are thought to function as mor- phogens. Activin and bone morphogenetic protein (BMP), two members of the TGF-? superfamily, are known to specify the MESODERMAL cell fates of early Xenopus embryos in a concentration-dependent man- ner 3?5 . A concentration gradient of Sonic hedgehog (Shh) ? a vertebrate member of the Hh family ? has been shown to organize the ventral half of the develop- ing neural tube 6 (FIG. 2). Shh is also expressed in the pos- terior edge of the developing limb bud, from which it organizes the patterning of the limb bud 6 . Although their mode of action remains elusive, several BMP and Wnt family members are expressed in the dorsal edge of the neural tube ? the floor plate ? which controls the identity and pattern of dorsal neural cell types 7,8 . GENETICS OF MORPHOGEN GRADIENTS Tetsuya Tabata The organization of cells and tissues is controlled by the action of ?form-giving? signalling molecules, or morphogens, which pattern a developmental field in a concentration- dependent manner. As the fate of each cell in the field depends on the level of the morphogen signal, the concentration gradient of the morphogen prefigures the pattern of development. In recent years, molecular genetic studies in Drosophila melanogaster have allowed tremendous progress in understanding how morphogen gradients are formed and maintained, and the mechanism by which receiving cells respond to the gradient. MESODERM The third germ layer in the embryo, formed during the process of gastrulation. Institute of Molecular and Cellular Biosciences, University of Tokyo, Yayoi 1-1-1, Tokyo 113-0032, Japan. e-mail: ttabata@ims.u-tokyo.ac.jp � 2001 Macmillan Magazines Ltd NATURE REVIEWS | GENETICS VOLUME 2 | AUGUST 2001 | 621 REVIEWS ligand and Dpp activity gradients are established and maintained. In this article, I therefore focus on the mechanisms that regulate the Dpp morphogen gradient in Drosophila wing development as a model system and, in less detail, the evidence for how Wg and Hh mor- phogen gradients might arise. Drosophila wing development The Drosophila wing is an ideal model to study pattern formation, as a large body of knowledge exists on the regulatory networks that control gene function in this appendage. The adult wing arises from the larval imagi- nal disc ? a single-layered sac of polarized epithelial cells. Imaginal discs are subdivided into non-intermin- gling sets of cells called compartments 12 : the wing imagi- nal disc is subdivided into anterior (A) and posterior (P) compartments along the A/P axis and is further subdi- vided into dorsal (D) and ventral (V) compartments along the D/V axis 12,13 (FIG. 3a). The source of morphogen lies at the border between adjacent compartments, which is stably maintained because cells in different compartments have a selective affinity for cells within their own compartment 14 . The identity of cells in the P compartment is imparted by the expression of the SELECTOR GENE engrailed (en) 15?17 . As a result, P cells secrete Hh (REF. 18), which acts as a morphogen to signal to A- compartment cells, and both patterns the central domain of the wing blade primordium 19,20 (FIG. 4) and induces dpp in a stripe of cells adjacent and anterior to the A/P compartment boundary 18,21,22 (FIG. 3b).Dpp is essential for the growth of wing cells and is responsible for patterning the wing beyond the central domain 21,23 (FIG. 4), using a concentration-dependent mechanism to induce target genes, such as spalt (sal) and optomotor- blind (omb, also known as bifid), at different distances from the A/P compartment border 24,25 (FIG. 5). Similarly, the expression of fringe 26 (which modulates Notch sig- nalling) by cells in the D compartment, which are pro- grammed by the gene apterous 27 , results in activation of the Notch pathway at the D/V border 28 .Activated Notch induces wg expression at the D/V border 29,30 (FIG. 3c); here, Wg functions as a morphogen and organizes wing patterning by inducing target genes such as Distal-less (Dll) and vestigial (vg) 31,32 . Morphogens in the wing Genetic evidence that Dpp functions as a morphogen comes from elegant experiments using a constitutively active form of the Dpp receptor. The pathway that trans- duces the TGF-? signals involves a combination of two types of serine/threonine kinase receptors (type I and type II) 33 . The activated type I receptor phosphorylates a specific member of cytoplasmic transducers, so-called ?receptor-regulated Smads?, which, upon phosphoryla- tion, translocate into the nucleus and regulate the expression of target genes (FIG. 5). In wing development, Thickveins (Tkv) and Saxophone (Sax) are the type I receptors, and Punt (Put) is the type II receptor. Tkv is crucial for wing development and its constitutively active form (Tkv*) can induce the expression of the target genes sal and omb when ectopically expressed 24,25 . This Another example of a secreted signalling molecule is fibroblast growth factor 8 (FGF8), which is expressed at the junction between the midbrain and the hindbrain, known as the isthmic organizer (IsO). Although FGF8 is thought to mediate, at least in part, the activity of the IsO that patterns the midbrain and rostral hindbrain area 9?11 , it is not yet clear whether FGF8 functions as a morphogen. These are just some of the cases in verte- brate development in which signalling molecules are likely to function as morphogens in various contexts. However, the mode of action of morphogens has never been challenged more rigorously by genetic analysis than in the development of insect appendages. The most notable examples of morphogens that have been extensively illustrated by genetic analysis are Decapentaplegic (Dpp) (of the TGF-? superfamily), Hh and Wg in patterning the adult appendages of the fruit- fly, D. melanogaster. Out of all the secreted signalling molecules, Dpp, which is expressed in the developing fly wing, has been the most extensively studied mor- phogen. Recent work has investigated how both Dpp SELECTOR GENES A class of transcription factor, the products of which control the formation and identity of various morphogenetic fields. Morphogen Signalling relay a b Figure 1 | Patterning a developmental field by long-range signalling. a | Morphogen signalling. A morphogen sets the positional value of a cell by forming a concentration gradient across the developmental field in which the cell resides; the value of the gradient at each point in the field is a function of the distance of the receiving cell from the morphogen-secreting cells (shown in green). b | Relay signalling. The positional value of each cell is set through the sequential inductive signalling events that are relayed between adjacent cells in the field. Roof plate Epidermis Floor plate Somites Notochord Dorsal Ventral Neural- crest cells Neural tube Figure 2 | Inductive signals organize neuronal cell identity along the dorsoventral axis of the neural tube. The expression of Sonic hedgehog (green) by floor-plate cells at the ventral midline patterns the ventral neural tube of vertebrate embryos. Signalling molecules, such as BMPs (red), which are expressed by roof-plate cells at the dorsal midline, pattern the dorsal neural tube. � 2001 Macmillan Magazines Ltd 622 | AUGUST 2001 | VOLUME 2 www.nature.com/reviews/genetics REVIEWS of target genes is autonomously regulated in the mitotic clones of cells (BOX 1) that are mutant either for dishevelled or armadillo, which encode downstream components of the Wg signal transduction pathway 31,32 . Evidence that Hh functions as a morphogen has been provided in a similar way by comparing the activities of the wild-type, secreted form of Hh to a membrane-tethered form of the protein (HhCd2), which was generated by fusing the amino-terminal signalling domain of Hh to rat Cd2,a type I transmembrane protein 20 . Secreted Hh protein activates target genes in nearby cells over a range of ten cells, whereas the membrane-tethered Hh only activates target genes in cells immediately adjacent to the Hh source. This experiment shows that activation of Hh tar- get genes at long range relies on the ability of Hh to move some distance from the cells in which it is expressed. Vertebrate members of the same family of mole- cules probably also function as morphogens. Shh orga- nizes the ventral half of the developing neural tube (FIG. 2); eliminating Shh activity through gene targeting in mice, for example, prevents the differentiation of ventral cells 34 . Moreover, ectopic expression of a mutat- ed form of the Shh receptor, Patched (Ptc), which does information can be used to determine whether Dpp functions as a morphogen. The key to interpreting this experiment lies in determining whether the effect of expressing Tkv* is CELL AUTONOMOUS. If Dpp functions as a morphogen, the effects of Tkv* should be strictly cell autonomous; by contrast, if Dpp triggers a signalling relay mechanism, the effects of overexpressing Tkv* should be non-autonomous, because the second signal emanating from the cells that overexpress Tkv* would also affect surrounding (non-Tkv*-expressing) cells. The unambiguously cell-autonomous effects of expressing Tkv* indicate that Dpp functions directly at a dis- tance 24,25 . Taken together with the observation that Dpp upregulates different sets of target genes at different con- centration thresholds 25 , it is likely that Dpp functions as a morphogen in the fly wing. Similar experiments also indicate that Wg functions as a morphogen. Although the wild-type, secreted Wg acti- vates target genes over a distance, a membrane-tethered form that was generated by fusing Wg to the carboxyl ter- minus of Drosophila Neurotactin (Nrt) ? a type II trans- membrane protein ? upregulates Wg-target genes only in its immediate neighbours 31 . In addition, the expression a Anterior Ventral Ventral Dorsal wg Dorsal Dpp Hh Posterior Anterior Posterior b c hh dpp Figure 3 | Inductive activities during the development of the Drosophila wing. a | The adult Drosophila wing develops from the larval imaginal disc ? a single-layered sac of polarized epithelial cells present in the larva (left). The wing disc is subdivided into anterior (A) and posterior (P) compartments along the A/P axis, and is further subdivided into dorsal (D) and ventral (V) compartments along the D/V axis (dashed line). Cells in the P compartment are programmed by the selector gene engrailed (en, blue). Only part of the imaginal disc, indicated by a bracket, develops into the adult wing (right). The D/V border of the imaginal disc develops into the margin of the adult wing. To visualize this movement, imagine picking up the D/V border in the disc and pulling it towards you, out of the page. b | Secretion of Hedgehog (Hh) by P-compartment cells (green) generates a short-range gradient in the A compartment and both patterns the central domain of the wing and induces the expression of decapentaplegic (dpp, red) in a stripe of cells adjacent anteriorly to the A/P compartment boundary. Dpp patterns the wing beyond the central domain. c | wingless (wg) is expressed along the D/V compartment boundary of the wing imaginal disc. (Reproduced with permission from REF. 70 � (2000) Elsevier Science, and from REF. 101 � (1990) Cold Spring Harbor Laboratory Press.) � 2001 Macmillan Magazines Ltd NATURE REVIEWS | GENETICS VOLUME 2 | AUGUST 2001 | 623 REVIEWS groups dealt successfully with this problem by making a chimeric protein of Dpp and green fluorescent pro- tein (GFP) 38,39 . The expression of Dpp?GFP in the endogenous Dpp expression pattern almost completely rescued the phenotype of a dpp mutant wing, indicat- ing that Dpp?GFP functions in a manner similar to endogenous Dpp. The Dpp?GFP chimeric protein also allows researchers to visualize the functional Dpp gra- dient by monitoring GFP fluorescence. As shown in FIG. 6, fluorescence intensity is highest in cells in which Dpp?GFP is produced and forms a broad, shallow gra- dient on both sides of the endogenous Dpp expression domain. Movement by diffusion alone cannot explain the graded expression pattern, because a secreted GFP fusion protein that is composed of GFP and only the secretory transport domains of Dpp fails to form a gra- dient 39 . Having established that a Dpp gradient exists, the question that remains to be addressed is how the stable gradient is generated and maintained. Generating the Dpp gradient. Several models have been proposed to explain the formation of a stable mor- phogen gradient. These models include the simple diffu- sion of molecules through the extracellular space, PLANAR TRANSCYTOSIS and displacement during growth (FIG. 7a?c). This last model predicts that the gradient might be formed upon cell proliferation: cells receiving Dpp could carry it away from the source as they are displaced by the addition of new cells (their descendants) during prolifer- ation, thereby expanding the gradient 40 . There are two reasons why the displacement model does not explain convincingly how the Dpp gradient is formed. First, the model depends on the high stability of the Dpp mole- cule; however, the secreted Dpp?GFP protein is turned over rapidly (in under 3 h). Second, the time required to form a Dpp gradient in the wing imaginal disc takes only several hours, which is less than the average doubling time (8 h) of cells in the imaginal disc 38,39 . An analysis of the endocytosis requirements of Dpp?GFP supports the planar transcytosis model 39 , although it does not exclude that the molecule moves through the extracellular space by diffusion. Signalling through Notch, the epidermal growth factor (EGF) and Wg requires DYNAMIN-mediated endocytosis 41?43 . That Dpp function requires endocytosis was indicated by the reduced expression domain of a Dpp target gene (sal ) in endocytosis-defective mutants 44 . Dpp?GFP is normally found in the endocytic compartment situated close to the apical surface of the wing disc 39 , but when endocysis is abolished using a Dynamin (shibire, shi) mutant, no Dpp?GFP is internalized in endosomes 39 .Moreover, when a shi ? clone (BOX 1) is made shortly after a short burst of Dpp?GFP expression, Dpp?GFP-positive endo- somes are not present in the area behind the shi ? clone (that is, further away from the Dpp?GFP source) (FIG. 8). Cells that lie behind the shi mutant clone would be expected to receive Dpp?GFP from the upstream mutant cells when the leading edge of the Dpp?GFP wave passes through the clone. The absence of Dpp?GFP in these cells indicates that Dpp transport through the endocytic pathway might be essential for gradient formation. not bind Shh but does antagonize its signalling, causes cell-autonomous V?D switches in neural progenitor identity in the chick neural tube, clearly indicating that Shh functions as a morphogen 35 . Shh is also known to organize the limb bud along the A/P axis and it has been shown recently that a cholesterol modification of Shh is required for the long-range action of Shh in the mouse developing limb bud 36 . During early vertebrate development, members of the TGF-? superfamily and their receptors are implicated in mesoderm formation. Although the extent to which they are essential for cell- type specification has not yet been fully understood, one TGF-? family member, Activin, has a morphogen- like capacity for inducing distinct mesodermal cell fates in Xenopus. The effects of constitutively active Activin receptors are shown to be strictly cell autonomous 4 .In zebrafish, Squint (Sqt) ? another member of the same family ? can induce different target genes at different concentration thresholds. Squint was recently shown to function as a morphogen: single-cell injection of sqt RNA into mutant embryos in which Sqt signalling has been genetically disabled can induce target genes in the wild-type cells that have been implanted far away from the Sqt-injected cell, indicating that Sqt acts directly at a distance 37 . The analysis on Shh and Sqt shown above are almost as rigorous as those done in Drosophila. However, in general, rigorous genetic analyses are not always possible in vertebrates and, therefore, the results in vertebrates should be interpreted with some caution. The Dpp morphogen gradient One important question still remains: does Dpp really form a concentration gradient and, if so, what does it look like? Answering this question relied on being able to detect the Dpp protein in vivo, which, as the available antibody against Dpp fails to detect low levels of Dpp signal in the cells surrounding the Dpp source, meant having to overcome an experimental difficulty. Two CELL AUTONOMOUS If the gene activity causes the effects only in the cells that express it, its function is cell autonomous; if it causes the effects in cells other than (or in addition to) those expressing it, its function is cell non- autonomous. PLANAR TRANSCYTOSIS A mechanism of transcellular transport within the plane of epithelium by which the molecule is internalized by endocytosis, transports intracellularly and is released to signal in the adjacent cells. DYNAMIN A GTPase required for clathrin- mediated endocytosis. ab Figure 4 | Inductive activities of Hedgehog and Decapentaplegic. a | Ectopic expression of Hedgehog in a clone of cells causes a mirror-image duplication of the entire anterior compartment 22 . b | Ectopic expression of Decapentaplegic causes a mirror-image duplication of the anterior compartment that lacks the central domain 23 . Anterior is to the left. � 2001 Macmillan Magazines Ltd 624 | AUGUST 2001 | VOLUME 2 www.nature.com/reviews/genetics REVIEWS dominant-negative mutant of Rab5 is expressed in the wing disc, the expression of a Dpp target gene (sal) is restricted to the cells adjacent to the Dpp source 39 . Conversely, overexpression of Rab5 broadens the expres- sion domain of Dpp target genes. Although Dpp?GFP could not be observed directly in these experiments, the results indicate that endocytic transport is rate limiting for determining the range of Dpp signalling. The authors also speculate that the gradient would remain stable if part of the internalized Dpp was degraded by the endo- cytic pathway 39 . Another small GTPase, Rab7, is known to target endocytic cargo from the early to the late endo- some and then to the lysosome for degradation. Expression of a dominant gain-of-function mutant of Rab7 decreases the levels of GFP?Dpp that are internal- ized and reduces the range of Dpp signalling 39 .Taken together, these experiments indicate that transcytosis might be crucial for forming a long-range Dpp gradient, and that the balance between transcytosis and degrada- tion can regulate that gradient. These conclusions were made despite the fact that, in experiments in which endo- cytosis was disrupted, the distribution of Dpp?GFP could not be observed directly. The experiments described above indicate that Dpp is propagated through the endo- cytic transport pathway. However, it is possible that Dpp is also propagated, in part, by diffusion in the extracellular space, because surface-labelling assays show that most of the Dpp?GFP signal appears to be in the extracellular space. So, more careful genetic and cell-biological studies will be required to determine the extent to which Dpp transport can be ascribed to endocytic mechanisms, to extracellular movement or to other mechanisms. It has been suggested that transcytosis is required for the transport of Wg in embryonic development 41,45 .The lysosomal targeting and subsequent degradation of endocytosed Wg has been shown recently to be involved in generating the asymmetric distribution of Wg in the embryonic epidermis. A new antibody-staining proto- col to detect extracellular Wg protein showed that the Wg protein forms a shallow extracellular gradient 46 . Although Wg was seen in punctate structures within cells, probably in endosomes, the following experiments indicate that endocytosis has no role in transporting the Wg protein in wing development. Wg does not localize to punctate structures in the shimutant clones (as is the case for Dpp), and, in contrast to Dpp, Wg is internal- ized by wild-type cells behind the shi ? clone 46 .Therefore, Wg can move across the shi mutant tissue and is inter- nalized by the adjacent wild-type cells. In fact, Wg levels are at their highest in the shi mutant clones. It should be noted, however, that the Dpp and Wg experiments were done under different conditions: in the case of Dpp, the area lacking Dpp?GFP behind the shi ? clone was only observed under one condition; that is, shortly after the leading edge of the Dpp?GFP wave had passed through disc clones in which pre-existing Dpp?GFP had been replaced by a pulse of Dpp?GFP 39 (FIG. 8). This precau- tion is necessary to prevent Dpp, which travels in all directions, from moving into the area behind the clone from the surrounding wild-type cells. The same model could easily apply to Wg. Therefore, an analysis of the The fact that Dynamin is known to regulate recep- tor internalization and that Dpp?GFP colocalizes with its receptor, Tkv, in endosomes, raises the question of whether Dpp internalization involves receptor-mediat- ed endocytosis. To address this question, Dpp?GFP localization was monitored in tkv ? mutant clones. Dpp?GFP accumulates around the mutant cells in the tkv ? clone that face the Dpp?GFP source, but it is found at much lower levels in both intra- and extracel- lular spaces in mutant cells behind them 39 . This implies that Dpp internalization is Tkv dependent and con- firms that endocytosis is required for the long-range gradient formation of Dpp. If Dpp is propagated through the endocytic transport pathway, then the components that regulate endocytic transport would be expected to control gradient forma- tion. The activity of the small GTPase Rab5 is thought to be rate limiting in the early endocytic pathway. When a Dpp Put Tkv Sax Med Mad MadMed Nucleus Cytoplasm P Mad brk dpp omb sal dad Figure 5 | The Decapentaplegic signalling pathway. The receptor for Dpp consists of a complex of type I (Tkv or Sax) and type II (Put) serine/threonine kinase receptors. Binding of Dpp to this receptor complex results in the activation of the receptor. The activated type I receptor directly phosphorylates Mad, which, upon phosphorylation, translocates into the nucleus along with Med and regulates the transcription of target genes. As a result, sal, omb and dad are upregulated, and brk is downregulated. Dad and Brk function as negative regulators of the pathway: Dad antagonizes Tkv-dependent phosphorylation of Mad, and Brk represses transcription of the target genes sal, omb and dad. The expression pattern of the Dpp target genes in the wing imaginal disc is also shown. brk, brinker; dad, daughters against dpp; Dpp, Decapentaplegic; Mad, Mothers against dpp; Med, Medea; omb, optomotor-blind; Put, Punt; sal, spalt; Tkv, Thickveins. (Reproduced with permission from REF. 82 � (1999) Macmillan Magazines Ltd.) � 2001 Macmillan Magazines Ltd NATURE REVIEWS | GENETICS VOLUME 2 | AUGUST 2001 | 625 REVIEWS way, whereas Ptc constitutively represses Smo activity 47 . Hh is thought to bind directly to Ptc and to liberate Smo from repression by Ptc. Although the underlying mechanism is not understood, the balance between Ptc and Smo proteins is thought to determine the activa- tion state of the pathway (whether it is active or repressed) 48,49 . Hh and Ptc, but not Smo, have been shown to colocalize in endosomes 50 . Although Hh dis- tribution is altered in shi mutant embryos (more Hh is apically localized 18 ), it is not clear if endocytosis is cru- cial for forming a Hh gradient. effects of disrupting and enhancing endocytosis will be required to establish whether transcytosis has a key role in Wg transport. M. Strigini and S. Cohen have shown that shi activity is required for Wg secretion 46 . This con- trasts with the results described above that Shi does not affect Dpp?GFP secretion. Compared with Dpp and Wg, Hh forms a relatively short-range gradient in imaginal development. Two transmembrane proteins, Smoothened (Smo) and Ptc, constitute the receptor complex for Hh: Smo activity is required to activate the Hh signal transduction path- Box 1 | Generating mitotic clones of cells in Drosophila imaginal discs A mitotically dividing cell normally gives rise to daughter cells that are genotypically identical to itself. However, if the exchange of sister chromatids (mitotic recombination) occurs, the resulting daughter cells can be genotypically different provided that the mother cell is heterozygous for the marker under study. Mitotic recombination can occur naturally, or be induced by DNA- damaging agents (X-rays, for example); however, a more controlled way of inducing mitotic recombination is to use site-specific recombination, as shown here. By creating Drosophila strains that contain a yeast (Saccharomyces cerevisiae) site-specific recombinase, FLPase, and its target site, FRT, clones of cells that are mutant for a gene 99,100 can be generated during imaginal development. A modification of this approach can be used to create cells that ectopically express a gene 22 (see figure). The clones can be induced at chosen stages of development by placing the FLPase gene under the control of a heat-shock promoter. The clones can be followed in imaginal discs or in adult structures by using various cellular or morphological markers. Mutant clones After the induction of FLPase by heat shock, homologous chromosome arms undergo site-specific recombination at FRT sites (a). If somatic recombination occurs in a cell that is heterozygous for a mutant gene of interest, then the ensuing cell divisions can give rise to two populations of daughter cells, one homozygous for the mutant gene (mutant clone) and the other homozygous for its wild-type gene (twin-spot clone). If a cellular marker (for example, GFP) is placed in trans to the mutant gene in the parent cell, then the homozygous mutant clone can be identified by the absence of the marker. Creating clones that express a gene Sometimes it is useful to induce the expression of a gene in a cell clone, rather than to remove its function (b). In flies that are transgenic for an FLP-out cassette, the coding sequence of a gene is separated from the promoter sequence by the insertion of a marker gene that is flanked by FRT sequences. In this construct, the coding sequence of the gene is not expressed, whereas the marker gene is expressed. After heat-shock induction, FLPase removes the marker gene by promoting site-specific recombination between the FRT sites and, in so doing, positions the coding sequence immediately downstream to the promoter, from which the coding sequence is expressed. The expressing clones can be identified by the absence of the marker. FRTCentromere Mutation Marker e.g. GFP DNA replication FLPase a FRT recombination b FLP-out expression Proliferation Constitutive promoter FRT FRT FRT + Lost FRTMarker Coding sequence Mitotic recombination Mutant clone FLP-out clone Twin-spot clone Mitosis (one of two possible outcomes) Animated online � 2001 Macmillan Magazines Ltd 626 | AUGUST 2001 | VOLUME 2 www.nature.com/reviews/genetics REVIEWS matrix. HSPGs consist of a protein core to which heparan sulphate glycosaminoglycan (HS-GAG) chains are attached 51 . Genetic studies in Drosophila show clearly that HSPGs ? which have been implicat- ed in several signalling pathways, including those of Wnt, Hh, TGF-? and FGF ? have crucial roles in these signalling pathways, both in embryonic and in imaginal development. The first indication came from the identification of mutations in two genes that are required for HS-GAG biosynthesis: sugarless (sgl) 52?54 and sulfateless (sfl). Mutations in sgl and sfl are defec- tive in FGF and Wg signalling in embryonic develop- ment. Moreover, dally (division abnormally delayed), which encodes Glypican (the protein core of the HSPGs) has been implicated in Wg signalling 55,56 . HSPGs have also been proposed recently to regulate directly Wg distribution in the wing imaginal disc. A decrease in extracellular Wg is observed in sfl mutant cells and, conversely, overexpression of another Glypican, dally-like, sequesters Wg, which indicates that the HSPGs might be involved in the extracellular accumulation of Wg 57 . Hedgehog is unique in that it undergoes autoprote- olysis to yield the functional amino-terminal half (HhNp) 58 , to which cholesterol is covalently attached 59 . The movement of HhNp across the imaginal tissue is more restricted than that of a genetically engineered HhN that does not have a cholesterol moiety, which indicates that cholesterol modification of Hh regulates Hh transport by facilitating its diffusion 60 . Two genes have been identified that regulate Hh transport in a cho- lesterol-dependent manner. One of them encodes a novel Ptc-like transmembrane protein, Dispatched (Disp), which functions in Hh-secreting cells to liberate HhNp from the cells that express it 61 . The second gene, tout-velu (ttv) 62,63 , has significant homology to the verte- brate exostoses (EXT) gene family that encodes the gly- cosyltransferase in heparan sulphate biosynthesis 64 . Ttv- modified HSPG is thought to be required for the proper distribution of HhNp because Hh is not detected in the anterior ttv mutant clones that abut the A/P border 62 and movement of HhNp, but not of HhN, is restricted in ttv mutant embryos 63 . So, movement of Hh is tightly controlled through its cholesterol moiety. Two lines of evidence support the role of EXT in vertebrate Hh transport. First, the EXT gene family is implicated in the inherited bone disorder, human multiple exostoses (EXT) syndrome 65,66 , which is characterized by bone outgrowths and a high incidence of bone tumours. Second, Indian hedgehog (Ihh) ? a member of the ver- tebrate Hh family that has been shown to regulate carti- lage differentiation 67,68 ? does not associate with the surface of cells that are deficient for the Ext1 gene. Although Ihh is present around the surface of visceral endoderm cells of wild-type mice, no signal is detected in the same tissue of gene-targeted mice that are defi- cient for Ext1, despite the fact that Ihh protein and mRNA are expressed in mutant embryos at levels simi- lar to those of wild-type embryos 69 . Although there has been no direct evidence for the extracellular movement of Dpp or the involvement of Regulating morphogen gradients. Considerable progress has been made in recent years to identify the extracellular components that are required for the dis- tribution of morphogen molecules. These molecules are collectively referred to as heparan sulphate proteo- glycans (HSPGs), which form part of the extracellular PosteriorAnterior dpp Tkv Dpp?GFP p-Mad Hh En a b c d Figure 6 | Ligand and activity gradient of the Decapentaplegic morphogen. Confocal microscopy images (left) and schematic drawings (right) of a part of the wing imaginal disc that gives rise to the adult wing. a | Hedgehog (Hh, green), the expression of which is maintained by Engrailed (En) in the posterior (P)-compartment cells, induces decapentaplegic (dpp) expression (red) along the antero(A)/P border. b | Dpp diffuses in both A and P directions and forms a gradient, which can be visualized by the distribution of the chimeric protein Dpp?GFP (green). c | The expression level of Thickveins (Tkv), the Dpp receptor (purple), is very low along the A/P border because Hh downregulates its expression. In the middle of the wing disc, abutting the A/P border, the expression level of Tkv in the P compartment is higher than it is in the A compartment, which causes a steeper Dpp gradient to be present in the P compartment. This dynamic Tkv pattern accounts well for the shape of the activity gradient of Dpp signalling, as shown by d | the levels of phosphorylated Mothers against Dpp (p-Mad, grey), a downstream component of the Dpp signalling cascade. (Reproduced with permission from REF. 38 � (2000) Elsevier Science, and REF. 72 � (2001) Company of Biologists Ltd.) � 2001 Macmillan Magazines Ltd NATURE REVIEWS | GENETICS VOLUME 2 | AUGUST 2001 | 627 REVIEWS Receptor levels shape the morphogen gradient The Dpp activity gradient shown by p-Mad levels differs from its ligand gradient, which raises the possibility that the Dpp activity gradient might be regulated between the Dpp ligand and a cytoplasmic signal transducer, probably at the level of the receptor. Dpp preferentially signals through the Tkv receptor in the wing disc and also negatively regulates tkv expression 40 . The level of tkv expression is higher in cells at the periphery of the wing disc and is lower in the central region (FIG. 6). In addition, a sharp reduction in tkv expression is seen at the A/P border of normal wing discs, a pattern very similar to that of p-Mad. I refer to the level of tkv expression in the area between the periphery and the A/P border as ?basal?. Interestingly, the basal level of tkv expression in the P compartment is higher than in the A compartment (FIG. 6). This might account for the steeper gradient of p-Mad in the P compartment; as high levels of Tkv limit the movement of Dpp, Dpp would not spread as far in the P compartment, leading to a steeper gradient of activity. In fact, the Dpp?GFP gradient is also steeper in the P compartment (FIG. 6). The Hh-dependent reduction of the p-Mad level at the A/P border discussed above has been shown to occur largely by repressing the transcrip- tion of the tkv receptor gene 70 . The higher Tkv level in the P compartment is maintained by the activity of the transcription factor En. Both the Hh and En activities that regulate tkv levels are mediated by the gene master of thickveins (mtv, also known as scribbler), which encodes a putative nuclear protein 72 . The ability of receptor levels to regulate the distribu- tion of receptor ligands is not restricted to the Dpp mor- phogen. The same has also been reported for Hh. Ptc is expressed in the A compartment at low levels ? thus repressing target genes of Hh signalling ? and is highly induced by Hh at the A/P border. So, Hh upregulates the expression of its own repressor. This paradoxical property can be interpreted in the light of the other role of Ptc, HSPGs in Dpp transport, some extracellular molecule might restrict Dpp movement because Dpp cannot move freely across the tissue, even in the absence of the Tkv receptor 39 . Activity gradient of Dpp. Are the observed ligand gra- dients directly reflected in the activity gradient of the receiving cells? To address this question, Dpp sig- nalling activity has been visualized in situ in the Dpp- receiving cells in the wing. As described above, the Dpp signal is transduced by phosphorylating a receptor-regulated Smad, Mothers against Dpp (Mad). Therefore, the phosphorylated version of Mad (p- Mad) can be used as an intracellular marker to moni- tor Dpp morphogen activity using a p-Mad-specific antibody 70 . The relative amount of p-Mad is higher in the cells near the A/P border, as expected; however, it is severely reduced in cells that express dpp (FIG. 6), as a result of the direct repressive action of Hh 70 . It is prob- ably relevant to the significance of this complex regu- latory interaction that Hh also directly organizes pat- terning in the region in which it attenuates Dpp signalling. Hh might need to downregulate Dpp sig- nalling in this region to prevent Dpp signalling from interfering with its own patterning activities. A Dpp target, Daughters against Dpp (Dad), has been shown to regulate the Dpp activity gradient; Dad competes with Mad for binding to the Tkv receptor and antagonizes Mad phosphorylation 71 (FIG. 5). So, the induction of Dad regulates the Dpp activity gradient by creating a negative-feedback loop that limits the domain of p-Mad activation. Apart from the dip in the level of Dpp activity seen at the A/P border, the Dpp activity gradient also differs in the two compartments: it is steeper in the P compart- ment than in the A compartment (FIG. 6). This can be attributed to the differing levels of Dpp-receptor expres- sion, as described below. a b c Proliferation Proliferation Morphogen- expressing cell Morphogen- expressing cell Morphogen- expressing cell Figure 7 | Models for movement of the morphogen molecule. a | Diffusion through the extracellular space. b | Planar transcytosis. c | Displacement during growth. � 2001 Macmillan Magazines Ltd 628 | AUGUST 2001 | VOLUME 2 www.nature.com/reviews/genetics REVIEWS therefore, the expression pattern of Dfz2 is complemen- tary to the distribution of its ligand. This dynamically reg- ulated distribution of Dfz2, and the fact that ectopically expressed Dfz2 leads to the accumulation of Wg, have led to the proposal that the Dfz2 level is crucial in determin- ing the Wg gradient 77 . However, the absence of any phe- notypes in Dfz2 mutants questions the significance of the Dfz2 distribution in shaping the Wg gradient 76 . A further complication is that Dfz3, the third member of the family, is induced by Wg signalling, although its binding affinity to Wg is much less than that of Dfz2 (REF. 75). So, regula- tion of receptor levels might not be crucial for the distribution of all morphogens. Regulation of the receptor levels is poorly under- stood in vertebrates except for Ptc, which is always upregulated by Shh 78,79 and probably functions in the same way as in Drosophila. Regulating target gene expression A morphogen signal is transduced through an intracellu- lar signalling cascade to the nucleus, where it regulates the transcription of target genes in the receiving cells. Because the activity gradient can be modulated at every step of the signal transduction pathway, the ultimate output of the morphogen gradient should be represented by the tran- scriptional activity that it induces. Therefore, to under- stand how the levels of a morphogen are modulated in the receiving cell, we must first understand how tran- scriptional activity is correlated with the morphogen gra- dient. One of the simplest explanations would be that transcription factor activity in target cells is a direct read- out of the activity of the morphogen. Although this seems to be applicable to the Dpp, Hh and Wg mor- phogens, another layer of regulatory mechanism has begun to be elucidated. In Dpp signalling, p-Mad translocates to the nucleus, where it functions as a component of the transcriptional complex and regulates the expression of target genes such as sal, omb and dad. We could, therefore, use the level of p-Mad as a measure of the ultimate determinant. However, there is another complexity in the Dpp mor- phogen system. The transcription factor Brinker (Brk) is known to repress Dpp target genes 80?82 .Furthermore,brk expression is downregulated by the Dpp signal, which leads to the formation of a counter-gradient of repressor to the Dpp morphogen (FIG. 5). So, Dpp can also indirect- ly regulate gene expression by downregulating the expression of brk. The regulatory sequences of several Dpp target genes have many Mad and Brk binding sites of which several overlap. Competition between the bind- ing of Mad and Brk to overlapping sites could determine spatially restricted domains of expression of Dpp target genes 83?85 . Although direct evidence for this is lacking, data indicating that it occurs are now available. By com- paring the Mad/Brk binding sites of Ultrabithorax (Ubx, a target of short-range activation in the visceral meso- derm) and those of vg (a target of long-range activation in the wing disc), a recent report showed that Ubx is more sensitive to repression by Brk. This indicates that Brk binding sites might be crucial in limiting thresholds for activation by Dpp 85 . which is to prevent Hh from spreading too far into the A compartment 73 . The low level of Ptc in the A compart- ment is sufficient to suppress ectopic Hh signalling, but is insufficient to restrict Hh movement. Therefore, Hh induces a high level of Ptc to limit the range of its own distribution gradient. For Wg, the situation is not so straightforward. Three members of the Frizzled (Fz) family of seven-pass trans- membrane proteins ? Fz, Dfz2 (REF. 74) and Dfz3 (REF. 75) ? have been identified as receptors for Wg. Their func- tions are redundant; a single mutation in any of them impairs Wg signalling, whereas in cells that are doubly mutant for fz and dfz2, Wg signalling is abolished 76 . Expression of Dfz2 is negatively regulated by Wg and, Anterior Posterior Anterior Posterior Figure 8 | Evidence for planar transcytosis of Decapentaplegic in the wing imaginal disc. Expression of Decapentaplegic (Dpp)?GFP in Dynamin (encoded by shibire, shi ) mitotic clones in the Drosophila wing imaginal disc indicates that the gradient of Dpp is distributed by planar transcytosis. Mitotic clones of shi ? are marked by the absence of the Nmyc marker (area within the white line in both panels). Dpp?GFP (green) is expressed along the anteroposterior border of the disc and diffuses towards the periphery (arrow). Fewer or no Dpp?GFP vesicular structures are detected in the area (dotted yellow line) distal to the shi mutant clone, indicating that endocytosis is required for Dpp?GFP to reach those cells. (Reproduced with permission from REF. 39 � (2000) Elsevier Science.) � 2001 Macmillan Magazines Ltd NATURE REVIEWS | GENETICS VOLUME 2 | AUGUST 2001 | 629 REVIEWS would not be surprising if additional molecules, with novel functions, were found to have roles in each sig- nalling pathway. It should be noted that the mechanisms described here are based on the assumption that cells closer to the source receive the morphogen and transmit it to the neighbouring downstream cells, regardless of the underlying mechanisms. However, some doubt has been cast over this premise by the discovery of cytonemes ? actin-based long processes that imaginal cells extend towards the A/P border, where Dpp is expressed 98 . By means of cytonemes, even the cells far from the source of morphogen can make direct contact with cells that express the morphogen, thereby removing the need for an intercellular transport system to disperse it. Although we have not so far needed the function of cytonemes to explain the observations or the effects of Dynamin mutant clones on Dpp distribution, further analysis might discover the function of cytonemes and pave the way to understanding the novel mechanisms by which morphogens organize patterning. In both the Hh and the Wg pathways, a component of the signalling cascade translocates into the nucleus on reception of the signal and functions as a subunit of a transcriptional complex. Furthermore, balancing between the activator and repressor is also important in regulating transcription at the end of the Hh 86?89 and the Wg 90?92 signalling cascades. These features have been shown to be in large part conserved in vertebrates 93?96 . Conclusions Is it possible to devise general principles to explain how morphogen gradients are formed and maintained? Our current knowledge is still incomplete. We do not know, for instance, whether endocytosis is a general way of transporting morphogens or whether regulation of receptor levels is a common mechanism for shaping the gradient. A morphogen might behave differently in sepa- rate developmental contexts. A relevant example exists for Dpp signalling: the secreted antagonist of Dpp, Short gastrulation (Sog), is important in embryonic 97 , but not imaginal, development. The mode of action of a mor- phogen might depend on many other factors, such as the type of cell or the size of the field in which it is operating, or the length of time for which it is expected to act. Given that the components of morphogen signalling cascades are highly conserved in evolution, the mechanisms revealed in the Drosophila wing are probably applicable to the action of morphogens in vertebrates. Morphogen gradients are regulated at several levels and are shaped, in part, by feedback loops. Many molec- ular events are involved in the gradient formation, so it 1. Wolpert, L. Positional information and the spatial pattern of cellular differentiation. J. Theor. Biol. 25, 1?47 (1969). 2. Turing, A. M. The chemical basis of morphogenesis. Phil. Trans. R. Soc. Lond. B 237, 37?72 (1952). 3. Gurdon, J. B., Harger, P., Mitchell, A. & Lemaire, P. Activin signalling and response to a morphogen gradient. Nature 371, 487?492 (1994). 4. Jones, C. M., Armes, N. & Smith, J. C. Signalling by TGF-? family members: short-range effects of Xnr-2 and BMP-4 contrast with the long-range effects of activin. Curr. Biol. 6, 1468?1475 (1996). 5. Dosch, R., Gawantka, V., Delius, H., Blumenstock, C. & Niehrs, C. Bmp-4 acts as a morphogen in dorsoventral mesoderm patterning in Xenopus. Development 124, 2325?2334 (1997). 6. Ericson, J., Briscoe, J., Rashbass, P., Van Heyningen, V. & Jessell, T. M. Graded sonic hedgehog signaling and the specification of cell fate in the ventral neural tube. Cold Spring Harb. Symp. Quant. Biol. 62, 451?466 (1997). 7. Lee, K. J. & Jessell, T. M. The specification of dorsal cell fates in the vertebrate central nervous system. Annu. Rev. Neurosci. 22, 261?294 (1999). 8. Lee, K. J., Dietrich, P. & Jessell, T. M. Genetic ablation reveals that the roof plate is essential for dorsal interneuron specification. Nature 403, 734?740 (2000). 9. Irving, C. & Mason, I. Signalling by FGF8 from the isthmus patterns anterior hindbrain and establishes the anterior limit of Hox gene expression. Development 127, 177?186 (2000). 10. Crossley, P. H., Martinez, S. & Martin, G. R. Midbrain development induced by FGF8 in the chick embryo. Nature 380, 66?68 (1996). 11. Martinez, S., Crossley, P. H., Cobos, I., Rubenstein, J. L. & Martin, G. R. FGF8 induces formation of an ectopic isthmic organizer and isthmocerebellar development via a repressive effect on Otx2 expression. Development 126, 1189?1200 (1999). 12. Garcia-Bellido, A., Ripoll, P. & Morata, G. Developmental compartmentalisation of the wing disk of Drosophila. Nature New Biol. 245, 251?253 (1973). 13. Garcia-Bellido, A., Ripoll, P. & Morata, G. Developmental compartmentalization in the dorsal mesothoracic disc of Drosophila. Dev. Biol. 48, 132?147 (1976). 14. McNeill, H. Sticking together and sorting things out: adhesion as a force in development. Nature Rev. Genet. 1, 100?108 (2000). 15. Simmonds, A. J., Brook, W. J., Cohen, S. M. & Bell, J. B. Distinguishable functions for engrailed and invected in anterior?posterior patterning in the Drosophila wing. Nature 376, 424?427 (1995). 16. Guillen, I. et al. The function of engrailed and the specification of Drosophila wing pattern. Development 121, 3447?3456 (1995). 17. Tabata, T., Schwartz, C., Gustavson, E., Ali, Z. & Kornberg, T. B. Creating a Drosophila wing de novo, the role of engrailed, and the compartment border hypothesis. Development 121, 3359?3369 (1995). Shows that the anteroposterior compartment boundary functions as the organizing centre that patterns the complete wing. 18. Tabata, T. & Kornberg, T. B. Hedgehog is a signaling protein with a key role in patterning Drosophila imaginal discs. Cell 76, 89?102 (1994). 19. Mullor, J. L., Calleja, M., Capdevila, J. & Guerrero, I. Hedgehog activity, independent of decapentaplegic, participates in wing disc patterning. Development 124, 1227?1237 (1997). 20. Strigini, M. & Cohen, S. M. A Hedgehog activity gradient contributes to AP axial patterning of the Drosophila wing. Development 124, 4697?4705 (1997). 21. Capdevila, J. & Guerrero, I. Targeted expression of the signaling molecule decapentaplegic induces pattern duplications and growth alterations in Drosophila wings. EMBO J. 13, 4459?4468 (1994). 22. Basler, K. & Struhl, G. Compartment boundaries and the control of Drosophila limb pattern by Hedgehog protein. Nature 368, 208?214 (1994). Landmark paper showing that the Hedgehog protein can induce the complete duplication of the anterior compartment of Drosophila limbs. 23. Zecca, M., Basler, K. & Struhl, G. Sequential organizing activities of engrailed, hedgehog and decapentaplegic in the Drosophila wing. Development 121, 2265?2278 (1995). Shows that Decapentaplegic has an organizing activity that patterns the whole wing except for the central domain. 24. Lecuit, T. et al. Two distinct mechanisms for long-range patterning by Decapentaplegic in the Drosophila wing. Nature 381, 387?393 (1996). 25. Nellen, D., Burke, R., Struhl, G. & Basler, K. Direct and long- range action of a DPP morphogen gradient. Cell 85, 357?368 (1996). With reference 24, this paper showed for the first time that Decapentaplegic (Dpp) functions as a long-range signalling molecule. Reference 25 also shows that Dpp regulates different target genes in a concentration-dependent manner. 26. Irvine, K. D. & Wieschaus, E. Fringe, a boundary-specific signaling molecule, mediates interactions between dorsal and ventral cells during Drosophila wing development. Cell 79, 595?606 (1994). 27. Diaz-Benjumea, F. J. & Cohen, S. M. Interaction between dorsal and ventral cells in the imaginal disc directs wing development in Drosophila. Cell 75, 741?752 (1993). 28. Kim, J., Irvine, K. D. & Carroll, S. B. Cell recognition, signal induction, and symmetrical gene activation at the dorsal?ventral boundary of the developing Drosophila wing. Cell 82, 795?802 (1995). 29. Doherty, D., Feger, G., Younger-Shepherd, S., Jan, L. Y. & Jan, Y. N. Delta is a ventral to dorsal signal complementary to Serrate, another Notch ligand, in Drosophila wing formation. Genes Dev. 10, 421?434 (1996). 30. Neumann, C. J. & Cohen, S. M. A hierarchy of cross- regulation involving Notch, Wingless, Vestigial and Cut organizes the dorsal/ventral axis of the Drosophila wing. Development 122, 3477?3485 (1996). 31. Zecca, M., Basler, K. & Struhl, G. Direct and long-range action of a Wingless morphogen gradient. Cell 87, 833?844 (1996). 32. Neumann, C. J. & Cohen, S. M. Long-range action of Links DATABASE LINKS TGF-? | Hh | Wnt | Activin | BMP | Shh | FGF8 | Dpp | Wg | en | sal | omb | fringe | Notch | apterous | Dll | vg | Tkv | Sax | Put | Nrt | dishevelled | armadillo | Cd2 | Ptc | Sqt | shi | Rab5 | Rab7 | Smo | sgl | sfl | dally | dally-like | Disp | ttv | human multiple exostoses | Ihh | Ext1 | Mad | Dad | mtv | Fz | Dfz2 | Dfz3 | Brk | Ubx | Sog | Med � 2001 Macmillan Magazines Ltd REVIEWS Wingless organizes the dorsal?ventral axis of the Drosophila wing. Development 124, 871?880 (1997). 33. Massague, J. How cells read TGF-? signals. Nature Rev. Mol. Cell Biol. 1, 169?178 (2000). 34. Chiang, C. et al. Cyclopia and defective axial patterning in mice lacking Sonic hedgehog gene function. Nature 383, 407?413 (1996). 35. Briscoe, J., Chen, Y., Jessell, T. M. & Struhl, G. A Hedgehog-insensitive form of Patched provides evidence for direct long-range morphogen activity of Sonic Hedgehog in the neural tube. Mol. Cell 7, 1279?1291 (2001). 36. Lewis, P. M. et al. Cholesterol modification of sonic hedgehog is required for long-range signaling activity and effective modulation of signaling by ptc1. Cell 105, 599?612 (2001). 37. Chen, Y. & Schier, A. F. The zebrafish nodal signal Squint functions as a morphogen. Nature 411, 607?610 (2001). Provides the first unambiguous evidence that a signalling molecule (Squint) functions as a morphogen in vertebrate embryogenesis. 38. Teleman, A. A. & Cohen, S. M. Dpp gradient formation in the Drosophila wing imaginal disc. Cell 103, 971?980 (2000). 39. Entchev, E. V., Schwabedissen, A. & Gonzalez-Gaitan, M. Gradient formation of the TGF-? homolog Dpp. Cell 103, 981?991 (2000). With reference 38, this paper visualizes the Decapentaplegic (Dpp) gradient in vivo by using a Dpp?GFP fusion protein as a marker. Reference 39 also shows that endocytic transport is important in generating the Dpp gradient. 40. Lecuit, T. & Cohen, S. M. Dpp receptor levels contribute to shaping the Dpp morphogen gradient in the Drosophila wing imaginal disc. Development 125, 4901?4907 (1998). 41. Bejsovec, A. & Wieschaus, E. Signaling activities of the Drosophila wingless gene are separately mutable and appear to be transduced at the cell surface. Genetics 139, 309?320 (1995). 42. Seugnet, L., Simpson, P. & Haenlin, M. Requirement for Dynamin during Notch signaling in Drosophila neurogenesis. Dev. Biol. 192, 585?598 (1997). 43. Vieira, A. V., Lamaze, C. & Schmid, S. L. Control of EGF receptor signaling by clathrin-mediated endocytosis. Science 274, 2086?2089 (1996). 44. Gonzalez-Gaitan, M. & Jackle, H. The range of spalt- activating Dpp signalling is reduced in endocytosis-defective Drosophila wing discs. Mech. Dev. 87, 143?151 (1999). 45. Moline, M. M., Southern, C. & Bejsovec, A. Directionality of Wingless protein transport influences epidermal patterning in the Drosophila embryo. Development 126, 4375?4384 (1999). 46. Strigini, M. & Cohen, S. M. Wingless gradient formation in the Drosophila wing. Curr. Biol. 10, 293?300 (2000). 47. Ingham, P. W. Transducing Hedgehog: the story so far. EMBO J. 17, 3505?3511 (1998). 48. Denef, N., Neubuser, D., Perez, L. & Cohen, S. M. Hedgehog induces opposite changes in turnover and subcellular localization of Patched and Smoothened. Cell 102, 521?531 (2000). 49. Alcedo, J., Zou, Y. & Noll, M. Posttranscriptional regulation of smoothened is part of a self-correcting mechanism in the Hedgehog signaling system. Mol. Cell 6, 457?465 (2000). 50. Martin, V., Carrillo, G., Torroja, C. & Guerrero, I. The sterol- sensing domain of Patched protein seems to control Smoothened activity through Patched vesicular trafficking. Curr. Biol. 11, 601?607 (2001). 51. Kjellen, L. & Lindahl, U. Proteoglycans: structures and interactions. Annu. Rev. Biochem. 60, 443?475 (1991). 52. Hacker, U., Lin, X. & Perrimon, N. The Drosophila sugarless gene modulates Wingless signaling and encodes an enzyme involved in polysaccharide biosynthesis. Development 124, 3565?3573 (1997). 53. Binari, R. C. et al. Genetic evidence that heparin-like glycosaminoglycans are involved in wingless signaling. Development 124, 2623?2632 (1997). 54. Haerry, T. E., Heslip, T. R., Marsh, J. L. & O?Connor, M. B. Defects in glucuronate biosynthesis disrupt Wingless signaling in Drosophila. Development 124, 3055?3064 (1997). 55. Tsuda, M. et al. The cell-surface proteoglycan Dally regulates Wingless signalling in Drosophila. Nature 400, 276?280 (1999). 56. Lin, X. & Perrimon, N. Dally cooperates with Drosophila Frizzled 2 to transduce Wingless signalling. Nature 400, 281?284 (1999). 57. Baeg, G. H., Lin, X., Khare, N., Baumgartner, S. & Perrimon, N. Heparan sulfate proteoglycans are critical for the organization of the extracellular distribution of Wingless. Development 128, 87?94 (2001). Together with references 62 and 64, this paper shows that heparan sulphate proteoglycans are involved in regulating morphogen movement. 58. Porter, J. A. et al. The product of Hedgehog autoproteolytic cleavage active in local and long-range signalling. Nature 374, 363?366 (1995). 59. Porter, J. A., Young, K. E. & Beachy, P. A. Cholesterol modification of Hedgehog signaling proteins in animal development. Science 274, 255?259 (1996). 60. Porter, J. A. et al. Hedgehog patterning activity: role of a lipophilic modification mediated by the carboxy-terminal autoprocessing domain. Cell 86, 21?34 (1996). 61. Burke, R. et al. Dispatched, a novel sterol-sensing domain protein dedicated to the release of cholesterol-modified Hedgehog from signaling cells. Cell 99, 803?815 (1999). 62. Bellaiche, Y., The, I. & Perrimon, N. Tout-velu is a Drosophila homologue of the putative tumour suppressor EXT-1 and is needed for Hh diffusion. Nature 394, 85?88 (1998). 63. The, I., Bellaiche, Y. & Perrimon, N. Hedgehog movement is regulated through tout velu-dependent synthesis of a heparan sulfate proteoglycan. Mol. Cell 4, 633?639 (1999). 64. Senay, C. et al. The EXT1/EXT2 tumor suppressors: catalytic activities and role in heparan sulfate biosynthesis. EMBO Rep. 1, 282?286 (2000). 65. Ahn, J. et al. Cloning of the putative tumour suppressor gene for hereditary multiple exostoses (EXT1). Nature Genet. 11, 137?143 (1995). 66. Stickens, D. et al. The EXT2 multiple exostoses gene defines a family of putative tumour suppressor genes. Nature Genet. 14, 25?32 (1996). 67. Vortkamp, A. et al. Regulation of rate of cartilage differentiation by Indian hedgehog and PTH-related protein. Science 273, 613?622 (1996). 68. St-Jacques, B., Hammerschmidt, M. & McMahon, A. P. Indian hedgehog signaling regulates proliferation and differentiation of chondrocytes and is essential for bone formation. Genes Dev. 13, 2072?2086 (1999). 69. Lin, X. et al. Disruption of gastrulation and heparan sulfate biosynthesis in EXT1-deficient mice. Dev. Biol. 224, 299?311 (2000). 70. Tanimoto, H., Itoh, S., ten Dijke, P. & Tabata, T. Hedgehog creates a gradient of DPP activity in Drosophila wing imaginal discs. Mol. Cell 5, 59?71 (2000). The activity gradient of Decapentaplegic (Dpp) is visualized by using an antibody that recognizes a phosphorylated form of the Mothers against Dpp protein, a cytoplasmic signal transducer of Dpp signalling. 71. Tsuneizumi, K. et al. Daughters against Dpp modulates Dpp organizing activity in Drosophila wing development. Nature 389, 627?631 (1997). Shows that the activity gradient of Decapentaplegic (Dpp) is regulated by a negative-feedback loop mediated by the Daughters against Dpp protein. 72. Funakoshi, Y., Minami, M. & Tabata, T. mtv shapes the activity gradient of the Dpp morphogen through regulation of thickveins. Development 128, 67?74 (2001). 73. Chen, Y. & Struhl, G. Dual roles for Patched in sequestering and transducing Hedgehog. Cell 87, 553?563 (1996). Shows that Patched restricts the movement of its ligand, Hedgehog, and regulates its range of action. 74. Bhanot, P. et al. A new member of the frizzled family from Drosophila functions as a Wingless receptor. Nature 382, 225?230 (1996). 75. Sato, A., Kojima, T., Ui-Tei, K., Miyata, Y. & Saigo, K. Dfrizzled-3, a new Drosophila Wnt receptor, acting as an attenuator of Wingless signaling in wingless hypomorphic mutants. Development 126, 4421?4430 (1999). 76. Chen, C. M. & Struhl, G. Wingless transduction by the Frizzled and Frizzled2 proteins of Drosophila. Development 126, 5441?5452 (1999). 77. Cadigan, K. M., Fish, M. P., Rulifson, E. J. & Nusse, R. Wingless repression of Drosophila frizzled 2 expression shapes the Wingless morphogen gradient in the wing. Cell 93, 767?777 (1998). 78. Marigo, V. & Tabin, C. J. Regulation of patched by Sonic hedgehog in the developing neural tube. Proc. Natl Acad. Sci. USA 93, 9346?9351 (1996). 79. Marigo, V., Scott, M. P., Johnson, R. L., Goodrich, L. V. & Tabin, C. J. Conservation in hedgehog signaling: induction of a chicken patched homolog by Sonic hedgehog in the developing limb. Development 122, 1225?1233 (1996). 80. Campbell, G. & Tomlinson, A. Transducing the Dpp morphogen gradient in the wing of Drosophila: regulation of Dpp targets by brinker. Cell 96, 553?562 (1999). 81. Jazwinska, A., Kirov, N., Wieschaus, E., Roth, S. & Rushlow, C. The Drosophila gene brinker reveals a novel mechanism of Dpp target gene regulation. Cell 96, 563?573 (1999). 82. Minami, M., Kinoshita, N., Kamoshida, Y., Tanimoto, H. & Tabata, T. brinker is a target of Dpp in Drosophila that negatively regulates Dpp-dependent genes. Nature 398, 242?246 (1999). 83. Sivasankaran, R., Vigano, M. A., Muller, B., Affolter, M. & Basler, K. Direct transcriptional control of the Dpp target omb by the DNA binding protein Brinker. EMBO J. 19, 6162?6172 (2000). 84. Rushlow, C., Colosimo, P. F., Lin, M. C., Xu, M. & Kirov, N. Transcriptional regulation of the Drosophila gene zen by competing Smad and Brinker inputs. Genes Dev. 15, 340?351 (2001). 85. Kirkpatrick, H., Johnson, K. & Laughon, A. Repression of Dpp targets by binding of brinker to mad sites. J. Biol. Chem. 276, 18216?18222 (2001). 86. Aza-Blanc, P., Ramirez-Weber, F. A., Laget, M. P., Schwartz, C. & Kornberg, T. B. Proteolysis that is inhibited by Hedgehog targets Cubitus interruptus protein to the nucleus and converts it to a repressor. Cell 89, 1043?1053 (1997). 87. Methot, N. & Basler, K. Hedgehog controls limb development by regulating the activities of distinct transcriptional activator and repressor forms of Cubitus interruptus. Cell 96, 819?831 (1999). 88. Chen, C. H. et al. Nuclear trafficking of Cubitus interruptus in the transcriptional regulation of Hedgehog target gene expression. Cell 98, 305?316 (1999). 89. Muller, B. & Basler, K. The repressor and activator forms of Cubitus interruptus control Hedgehog target genes through common generic gli-binding sites. Development 127, 2999?3007 (2000). 90. Brunner, E., Peter, O., Schweizer, L. & Basler, K. pangolin encodes a Lef-1 homologue that acts downstream of Armadillo to transduce the Wingless signal in Drosophila. Nature 385, 829?833 (1997). 91. Van de Wetering, M. et al. Armadillo coactivates transcription driven by the product of the Drosophila segment polarity gene dTCF. Cell 88, 789?799 (1997). 92. Cavallo, R. A. et al. Drosophila Tcf and Groucho interact to repress Wingless signalling activity. Nature 395, 604?608 (1998). 93. Ding, Q. et al. Mouse suppressor of fused is a negative regulator of sonic hedgehog signaling and alters the subcellular distribution of Gli1. Curr. Biol. 9, 1119?1122 (1999). 94. Wang, B., Fallon, J. F. & Beachy, P. A. Hedgehog-regulated processing of Gli3 produces an anterior/posterior repressor gradient in the developing vertebrate limb. Cell 100, 423?434 (2000). 95. Hecht, A. & Kemler, R. Curbing the nuclear activities of ?-catenin. Control over Wnt target gene expression. EMBO Rep. 1, 24?28 (2000). 96. Roose, J. et al. The Xenopus Wnt effector XTcf-3 interacts with Groucho-related transcriptional repressors. Nature 395, 608?612 (1998). 97. Holley, S. A. et al. A conserved system for dorsal?ventral patterning in insects and vertebrates involving sog and chordin. Nature 376, 249?253 (1995). 98. Ramirez-Weber, F. A. & Kornberg, T. B. Cytonemes: cellular processes that project to the principal signaling center in Drosophila imaginal discs. Cell 97, 599?607 (1999). This article reports that cells in Drosophila imaginal discs project cellular extensions called cytonemes to the anteroposterior compartment border, which functions as the organizing centre of the disc. 99. Golic, K. G. & Lindquist, S. The FLP recombinase of yeast catalyzes site-specific recombination in the Drosophila genome. Cell 59, 499?509 (1989). 100. Xu, T. & Rubin, G. M. Analysis of genetic mosaics in developing and adult Drosophila tissues. Development 117, 1223?1237 (1993). 101. Hama, C., Ali, Z. & Kornberg, T. B. Region-specific recombination and expression are directed by portions of the Drosophila engrailed promoter. Genes Dev. 4, 1079?1093 (1990). Acknowledgements I thank A. Kuroiwa, H. Nakamura, S. Noji and K. Tamura for helpful suggestions. Research conducted in the T.T. laboratory was support- ed by grants from the Japan Society for the Promotion of Science (Research for the Future Program) and grants-in-aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan. 630 | AUGUST 2001 | VOLUME 2 www.nature.com/reviews/genetics � 2001 Macmillan Magazines Ltd "
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