- Email: [email protected]
Signalling by hedgehog family proteins in Drosophila and vertebrate development
Signalling by hedgehog family proteins in Drosophila and vertebrate development Philip W Ingham Imperial Cancer Research Fund, London, UK Members of the hedgehog gene family encode a novel class of secreted proteins and are expressed in embryonic cells known to possess important signalling activities in organisms as diverse as flies and chickens. Proteins of the hedgehog family act in these different developmental contexts as both permissive and instructive signals. How this signalling activity is transduced is (as yet) poorly understood, but recent studies point to the involvement of protein kinase A in both Drosophila and vertebrates.
Current Opinion in Genetics and Development 1995, 5:492-498 Introduction
I discuss recent studies presenting some interesting and unexpected answers to this question.
The hedgehog (hh.) gene was identified originaUy through the segment polarity phenotype caused by its mutation in Drosophila [1]. Genes of the hh family have now been isohted fi'om several vertebrate species, including mouse [2,3"], chicken [4], zebrafish [5,6"'], rat [6"'], Xenopus [7",8"] and human [9"]. These genes not only show a high degree of structural homology both within and between species, but in addition exhibit some remarkable similarities in the ways in which they function in various embryonic processes [10",11"]. The paradigm, for hh signalling was first estabfished by the genetic analysis of segmental patterning in the Drosophila embryo [12]. Embryos lacking hh activity die, revealing changes in their cuticular patterns suggestive of a requirement for the gene throughout each segment [1,13]. This requirement contrasts with the spatially restricted expression of hh [14-17], but could be explained if such localized expression establishes a source of hh protein that diffuses across each segment to generate a gradient of hh activity. This scenario is consistent with the postulated role of hh in the dorsal ectoderm, where the fates of individual cells appear to be determined directly by the levels of hh activity to which they are exposed [18"']. In the ventral ectoderm, however, several lines of evidence suggest instead that hh acts at short range to control the localized expression of another signalling molecule, encoded by the segment polarity gene wingless (wg) [19,20]. These contrasting roles of hh present a conundrum that is also raised by the effects of other hh family genes in vertebrate embryos: how does a single protein act as both a short-range and a long-range signal? In this review,
Sonic hedgehogand midline signalling in vertebrates The Sonic hedgehog (Shh) gene is expressed in the notochord and floorplate of vertebrate embryos [2,4,5,6"'], two midline structures implicated in inductive interactions that control the patterning of the neural tube [21] and somites [22]. The differentiation of floorplate is it.serf induced by the notochord, a process that can be reproduced in vitro, but only if the inducing and responding tissues are in direct contact [23]. By contrast, the ability of both notochord and floorplate to induce sclerotome differentiation in somitic mesoderm can be reproduced in vitro in the absence of ceU contact [24"]. Moreover, whereas floorplate differentiation is restricted to cells immediately adjacent to the inducing notochord, sclerotome differentiation can extend for over 200 mm in explants of presomitic mesoderm [24"]. Two fines of evidence suggest that Shh encodes the activity mediating both of these processes. First, ectopic expression of Shh in vivo leads to the activation of floorplate markers in inappropriate regions of the neural tube [2,5,6"',7"] and of sclerotome markers in inappropriate regions of the somites [25"'] Second, COS cells expressing Shh can induce floorplate differentiation in neural tube explants [6"',26"] and sclerotome differentiation in exphnts of presomitic mesoderm [24"], only the former effect depending upon direct contact of the COS cells with the responding tissue
Abbreviations AIER--apical ectodermal ridge; ci~cubitus interruptus; dpp--decapentaplegic hk~hedgehog; HNF--hepatocyte nuclear factor; PKA~protein kinase A; ptc--patched; 5kh~5onic hedgehog; w#--wingtess; ZPA--zone of polarizing activity. 492
© Current Biology Ltd ISSN 0959-437X
Signalling by hedgehog family proteins in Drosophilaand vertebrate development Ingham 493 Do different forms of hedgehog/Sonic hedgehog mediate distinct signalling activities? Both hh and Shh undergo autoproteolytic cleavage to generate two distinct subspecies of unequal size [15,27",28"], the smaller of which (-19kDa in both cases) is amino-terminally derived (hhN and ShhN) and represents the portion of the protein most highly conserved between different species. The hrger carboxyterminal portion (hhC and ShhC) varies in size between species and exhibits significantly less sequence identity, although certain regions, including domains required for autoproteolysis [27",29"], are highly conserved. The generation of these distinct protein forms suggests a possible molecular basis for the different modes of signalling exhibited by hh family members [27"',28"']. What makes this scenario even more attractive is the finding that the carboxy-terminal portion is readily secreted when expressed in Xenopus oocytes [28"'] or tissue culture cells [27"'-29"'], whereas the aminoterminal portion remains closely associated with the cell surface [27"--29"'], being displaced only by the addition ofheparin or suramin to the medium. That this behaviour reflects the properties of the proteins in vivo is indicated by immuuohistochemical analyses: antibodies specific for the amino-terminal portion detect protein predominantly around the periphery of cells expressing hh or Shh [27",30,31"'], whereas, in Drosophila at least, the carboxy-terminal portion appears to diffuse away from the expressing cells [27"°]. Thus, hhC and ShhC have the properties expected of a long-range signalling molecule and hhN and ShhN display characteristics consistent with a short-range or contact-dependent signal. Despite this strong prima facie case, we now have conclusive evidence that all the signalling activity of both hh and Shh resides exclusively within their amino-terminal domains. Thus ShhN, but not ShhC, is equally effective at inducing floorplate [31"',32"'] or sclerotome [33"'] differentiation in in vitro assays. Similarly, in Drosophila, overexpression of hhN is sufficient to respecify the pattern of both the ventral and dorsal epidermis, whereas the ectopic expression o f h h C has no effect on either pattern [29",34"']. The distinction between 'contact-dependent' and 'longrange' signalling activities seems, at least in vertebrates, to simply reflect the fact that different cellular responses can be invoked by different threshold levels of Shh activity. Elucidation of this phenomenon has been greatly facilitated by the discovery that ShhN generated artificially by expressinga truncated Shh cDNA is readily secreted by tissue culture cells [28",29"']. Apart from the interesting implication that autoproteolytic cleavage of the full-length protein results in a modification of the amino-terminal ~agment promoting its association with the ceil surface [28"',29"'], this finding has the important practical consequence that cells expressing the construct produce conditioned medium with significant inducing
activity. Medium containing ShhN at concentrations of 5 nM or higher can induce expression of the floorplate marker hepatocyte nuclear factor (HNF)3~ in neural plate explants [31"'], and concentrations of 1.25nM, although ineffective with respect to HNF3~ induction [31"], are sufficient to induce expression of Pax1, a sclerotome-specific marker, in presomitic mesoderm explants [33"]. Such experiments, and similar ones employing bacterially produced ShhN protein, have also provided compelling evidence that Shh mediates a second 'contact-independent' inducing activity of the notochord, the induction of motor neurons in the ventral neural tube [31",32"°]. Analysis of this phenomenon was complicated initially because floorplate cells are also effective motor neuron inducers, so any assay in which floorplate differentiation is induced by Shh could not discriminate between a direct or indirect effect of Shh on motor neuron differentiation [6"']. This problem has now been circumvented, because concentrations of bacterially produced ShhN and ShhN conditioned medium below the threshold required for floorphte induction are sufficient to induce motor neuron differentiation [3 I",32"']. Thus, what had previously appeared to be a qualitative difference between notochord-derived signals that induce floorplate and those that induce motor neurons is now taken to reflect a quantitative difference in the requirements of the two cell types for the same Shh-encoded inducing activity. Although it is possible that the form in which the Shh protein is presented to cells may influence their response to its activity [32"'], it seems more likely that the preferential retention of ShhN on the surface of cells from which it is secreted serves to generate a steep gradient of Shh protein [31"]; thus, only cells in direct contact with the notochord are induced to form floorplate, whereas cells some distance from the notochord experience levels of Shh compatible with motor neuron differentiation. Whether or not the long-range effects of hh in the dorsal epidermis of the Drosophila embryo are similarly direct is (as yet) unclear; however, raising the level of hh expression in a manner that should change the profile of the putative hh protein gradient across the parasegment has no effect on dorsal patterning [34"'], implying that, as in the ventral epidermis, hh acts indirectly by regulating the expression of some (as yet) unidentified signalling molecule, rather than in a direct concentration-dependent manner analogous to Shh.
hedgehog and imaginal disc patterning in Drosophila In Drosophila, hh continues to be required a~er ~mbryogenesis for the patterning ofimaginal discs, sheets of cells that give rise to the structures of the adult fly. Loss o f hh function or ectopic hh expression alike have fairly
494
Patternformation and developmental mechanisms
global effects on imaginal disc development, but, as in the embryo, these effects seem to reflect a role for hh in regulating the expression of other signalling factors. The hh gene is expressed throughout the posterior compartment of each disc [15,16], maintained by a lineage-based mechanism most likely mediated by the engrailed homeodomain protein. Across the compartment boundary, the signal-encoding genes wg and decapentaplegic (dpp) are, by contrast, expressed in restricted domains in close proximity to the hh-expressing cells. This spatial relationship is significant, because in the absence of hh activity, transcription of both wg and dpp is lost [35"'], whereas ectopic expression of hh in the anterior compartment of leg or wing discs leads to the inappropriate activation of wg and/or dpp around the hh-expressing cells [27",35"'-38",39",40"]. Moreover, ectopic expression of either the full-length hh protein [27"',35"'-38",39",40"], or of its amino-terminal portion [34"'], causes the respecification of cell identities and duplication of structures, effects that are produced by ectopic expression of wg [41,42"] or dpp [36"--38"'] alone. These findings have led to the conclusion that hh acts at short range to maintain localized sources of wg and/or dpp protein at each compartment boundary; it is these proteins that act, either in a direct dose-dependent manner [38",41] or in conjunction with other factors [42"], to specify the positional identity of surrounding cells, In an analogous manner, hh and dpp also interact to control the differentiation of the compound eye. The transition of retinal precursors from an actively dividing state to a terminally differentiated state is initiated as they enter the so-called morphogenetic furrow, a transient groove that sweeps across the retinal field during differentiation (also see the review in this issue by N M Bonini and K-W Choi [pp 507-515]). As cells enter the furrow, they activate transcription of dpp, a process that is dependent upon hh expression in cells just posterior to the furrow [43,44]. Inactivation of either hh or dpp blocks progression of the furrow [43,44], whereas ectopic expression ofhh results in the precocious activation of dpp and the inappropriate initiation of differentiation and furrow migration [45"'].
Sonic hedgehogand limb patterning in vertebrates The discovery that Shh is also expressed in the posterior mesenchyme of limb buds in mouse, chick and fish embryos [2,4,5] suggests a remarkable parallel between the patterning of limbs in Drosophila and vertebrates [10"]. The posterior mesenchyme is known to be the source of a signalling activity that polarizes the developing limb bud; thus, Shh appears an attractive candidate for the molecular basis of this so-called zone of polarizing activity (ZPA).
Consistent with this suggestion, ectopic expression of Shh can induce the generation of mirror-image duplication of digits and other structures similar to those caused by classic ZPA grafts or by the localized application of retinoic acid [3",4]. In addition, digit duplications caused by the ectopic expression of the murine Hox-b8 gene are invariably presaged by the inappropriate activation of Shh in the anterior limb mesenchyme of transgenic mice [46"]. Although Shh expression is initiated adjacent to the flank at the onset of hb bud development, this domain progressively shifts distally as the bud grows out, mirroring the distal displacement of the ZPA. This dynamic expression pattern contrasts with the static lineage-restricted expression of hh in imaginal discs and implies the operation of a regulatory mechanism that maintains Shh expression around the progress zone that is patterned by the ZPA. In fict, Shh expression is maintained by two distinct signals emanating from the dorsal ectoderm and the apical ectodermal ridge (AEK). Experiments in which Shh transcription is rescued by the expression of specific factors in limbs denuded of either tissue indicate that Wnt7a mediates the effects of the dorsal ectoderm [47"'], and FGF4 is responsible for the AEK-derived signal [48"',49"']. FGF4 is expressed specifically in the AER, whereas expression of Wnt7a is normally restricted to the dorsal ectoderm of the limb; moreover, in mouse embryos homozygous for a null allele of Wnt7a, Shh expression is initiated normally in the early hmb bud, but rapidly disappears as the bud develops [50"], confirming its involvement in this process. As well as maintaining Shh expression, FGF4 predisposes cells to respond to its activity, as pattern duphcations are normally induced by ectopic Shh only when it is expressed in close proximity to the AER. In the proximal part of the anterior mesenchyme, ectopic Shh has no effect unless accompanied by the simultaneous application of FGF4 protein [48°']. As the range of FGF4 is fairly restricted [49"'], it follows that Shh itself acts over only a limited range. This implies that in the vertebrate limb, Shh may act in an analogous manner to its counterpart in Drosophila imaginal discs, exerting a long-range effect by regulating the expression of some other signalling molecule. One potential candidate for such a mediator of Shh activity in the limb is BMP2; transcription of Bmp2 is localized in and around the Shh domain in the posterior limb mesenchyme [48",51"] and is activated in response to Shh and FGF4 [47"',48"]. ILemarkably, BMP2 is the vertebrate TGF~3 family member most closely related to Drosophila dpp. Although the inference that the same signalling pathway has been conserved between vertebrates and invertebrates is appealing, there is no evidence that BMP2 itself has limb polarizing activity [51"]. It may be that BMP2 acts together with some other factor, perhaps even as a heterodimer with another BMP (see the review by C Tickle in this issue [pp 478-484]).
Signalling by hedgehog family proteins in Drosophila and vertebrate development Ingham Transduction
o f t h e hedgehog s i g n a l
Transduction of signals encoded by hh family genes is (as yet) poorly understood, but most of what is known comes from studies in Drosophila. To date, the best candidate for a hh receptor is the novel multipass transmembrane protein encoded by the segment polarity gene patched (ptc) [52,53]. The spatial regulation of ptc expression is reciprocal to that of hh in embryos and imaginal discs [40°°,52-54], and in both contexts, ptc functions to repress the transcription of wg and/or dpp [19,55•], the genes activated by hh. This observation led to the proposal that hh activates its targets by antagonizing the activity of ptc [56], a model that is supported by the finding that expression of both wg and dpp genes is independent of hh activity in the absence of pro. At present, however, no direct evidence exists for such a physical interaction between the two proteins. Other genes involved in hh signal transduction have been identified largely on the basis of their epistatic relationships with ptc [57,58"]. Three genes with mutant phenotypes similar to hh are absolutely required for wg transcription, even in the absence of ptc activity. Two of these, fused and smoothened, are expressed both zygotically and maternally ([59]; M van den Heuvel, PW Ingham, unpublished data), and the third, cubitus interruptus (cO, is required only zygotically. The zinc finger protein encoded by ci has strong homology with the DNA-binding domain of the vertebrate GLI family of transcription factors [60]; thus, ci is an attractive candidate for a transcription factor that regulates u,g expression in response to hh activity [57]. Consistent with this interpretation, d, like ptc, is expressed in a reciprocal pattern to that of hh, such that it is present in cells that respond to ectopic hh activity as well as in those that normally activate wg and ptc transcription in response to hh [61°]. This suggests that ci is maintained in an inactive form, except in cells that receive the hh signal. The fused gene acts downstream of ptc and hh and encodes a putative serine/threonine kinase [62], the immediate targets of which are (as yet) unknown. Epistasis analysis suggests that fused acts upstream of a fourth member of the segment polarity class, costal-2 [59], which like ptc is required to repress wg transcription in the embryo [57]. Unlike ptc, however, maternally supplied costaf-2 is sufl%ient to support normal embryogenesis, suggesting that the costal-2 product is ubiquitously distributed in the embryo. All four of the genes implicated in hh signalling in the embryo are also involved in the patterning of imaginal discs. Viable mutations of pro and costal-2 cause outgrowths and mirror-image duplication of structures in the anterior compartment of the wing [54,55 °] similar to those induced by ectopic hh expression. The same kinds of duplications are also caused by viable hypomorphic or antimorphic mutations of the catalytic subunit of protein kinase A (PKA) [63••,64••], and clones
of cells completely lacking PKA activity in the anterior compartments of wing and leg imaginal discs or the retina of the eye disc autonomously activate u~g or dpp transcription in a hh-independent manner [63"°-67"]. These findings suggest that PKA, like ptc and costal-2, functions to suppress the hh response. Although this could imply that PKA is a direct downstream effector of ptc activity, this seems unlikely, because expression of a dominant active form of PKA at levels that rescue plea mutants is unable to suppress the ptc mutant phenotype [65"']. Instead, it seems that PKA acts by keeping the effectors ofhh activity inactive. As the ci protein contains multiple consensus PKA phosphorylation sites in its carbox-y-terminal region [60], it represents a likely target for this activity of PKA; according to this scenario, ci would be maintained in an inactive phosphorylated state by PKA activity unless activated by de-phosphorylation of these sites (or phosphorylation of other sites or both) in response to the transduction of the hh signal. Intriguingly, induction of sclerotome differentiation by Shh can be inhibited by drugs that up-regulate PKA activity, suggesting that the involvement of PKA in hh signalling may also be conserved between vertebrates and invertebrates [33"'].
Conclusions Members of the hh gene family have been highly conserved through evolution and play remarkably similar roles in the development of vertebrates and invertebrates. Despite these similarities, some important differences are seen in the way these roles are executed in different contexts. In Drosophila and in the vertebrate limb, hh proteins act as local signals that induce the expression of other signalling molecules, whereas in the trunk of the vertebrate embryo, Shh appears to act as a 'true' morphogen.
References and recommended
reading
Papers of particular interest, published within the annual period of review, have been highlighted as: • or" specia) interest •of outstanding interest 1.
N0ssfein-VolhardC, Wieschaus E: Mutations affecling menl number and polarity in Drosophila, Nature 1980, 287:795-801.
2.
Echelard Y, Epstein DJ, St Jacques B, Shen L, Mohler J, McMahon JA: Sonic hedgehog, a member of a family of putative signaling molecules is implicated in the regulation of CNS and limb polarity. Cell 1993, 75:1417-1430.
3. •
Chang DT: Products, genetic linkage and limb palleming activity of a murlne hedge/ioB gene. D4evetopment 1994, 120:3339-3353. Describes cloning, characlerization and functional analysisof a mouse hh homotoguethat is identical to Sbh. Like the zebrafish shh,the mouse gene is active in Drosophilaand also has potarizing activity in the chick limb.
495
496
Pattern formation and developmenta| mechanisms 4.
Riddle RD, Johnson RL, Laufer E, Tabin C: Sonic hedgehog mediates the polarizing activity of the ZPA. Cell 1993, 75:1401-1416.
and expression of hedgeho~ a Drosophila segment-polarity gene required for cell-cell communication. Gene 1993, 124:183-189.
5.
KraussS, Concordet J-P, Ingham PW: A functionally conserved Immolog of the Ormophi/a segment polarity gene hedgehog is expressed in tissues with polarisiog activity in zebrafish embryos, Cell 1993, 75:1431-1444.
18. Heemskerk J, Dinardo S: Drosophila hedgehog ads as a •. morphogen in cellular pattemiog. Cell 1994, 76:449460. This paper presents an analysis of the role of hh in the patterning of the dorsal cuticle of the Drosophila embryo. The authors show thai this is temporally distinct from its role in regulating wg transcription and adduce evidence, based on the manipulation of temperature-sensitive hh alleles and a heat-shock hh construct, that in this context hh acts directly to specify cell fate in a concentration-dependent manner.
6. •.
Roelink H, Augsburger A, Heemskerk J, Korzh V, Norlin S, Ruiz i Ahaba A, Tanabe Y, Placzek M, Edlund 1, Jessell TM et al.: Floor plate and motor neuron induction by ~ h - l , a vertebrate homoleg of hedgehog expressed by the notochord. Cell 1994, 76:761-775. An indepehclent search for homologues of hh in rat and zebrafish yielded genes that appear to be identical to Shh on the basis of their sequence, expression patterns and biological activity. This study also shows that COS cells expressing the vhh-f {Shh) gene can induce floorplate and motor neuron differentiation in neural plate explants in vitro.
7. •
Ruiz i Altaba A, Roelink H, Jessell TM: Restrictions to floorpbte induction by hedgehog and winged helix genes in mc',ural tube of frog embryos. Mol Cell Neurosci 1995, in press. This paper describes the sequence and expression pattern of the Xenopus Shh gene. The induction of floorplate by ectopic 5hh expression is temporally and spatially constrained. B. •
Yokota C, Mukasa T, Higashi M, Odaka A, Muroya K, Uchiyama H, Eto Y, Asahima M, Momoi "I-: Activin induces the expression of the Xenopus homolog of sonic hedgehogduring mesoderm formation in.Xenq~s explants. Biochem Biophys Res Commun 1995, 207:i-7. Describes the PCR cloning of a fragment of the Xenopus 5hh gene and demonstrates that its exp~ssion can be induced by activin in isolated animal caps in Ihe absence of protein synthesis. 9. •
Marigo V, Roberts DI, Lee SMK, Tsukurov O, Levi T, Gastier )M, Epstein DJ, Gilbert D), Copeland NG, Seidman CE et aL: Cionin~ expression and chromosomal location of shh and i,Sh two human homolognes of the Drosophila segment polarity get~e ~ , Genomics 1995, 27:in press.. Northern analysis shows thai human Ihh is expressed in adult kidney and liver, whereas Shh is expressed in these o~ans and the lung and intestine of the fetus. 5hh is closely linked to, but dislinct from, the polysyndactyly locus. 10. Ingham PW: Hedgehog points the way. Curt Biol 1994, • 4:347-350. Mini-review comparing and contrasting the likely roles of Shh in vertebrate embryos with the function of hh in Drosophila. 11. •
Fietz MJ, Concordet J-P, Barbosa R, Johnson R, Krauss S, McMahon AP, Tabin C, lngham PW: The hedgehog gene family in Drosophilaand vertebrate development. Development 1994,
Supph43-51.
Reviews the function of hh in the Drosophila embryo and imaginal discs and compares the spatial and temporal deployment of Shh in chick, mouse and fish embryos.
12.
|ngham PW: ~ e n t polarity genes and cell patterning within the Drosophila body segment. Curt Opin Genet Dev 1991, 1:261-267.
13.
Mohler J: Recluiremenls for hedgeho~ a segment polarity geue, in pattemiJq~ larval and adult cuticle of Drosophila. Genetics 1988, 120:1061-1072.
t4.
Mohler J, Vani K: expre~ion of the communication in Development 1992,
1S.
Lee JJ, Von Kessler DP, Parks S, Beachy PA: SeCretion and Iocalised transcription su~esls a role in positional signaUing for products of the segmentation gone hedgehog. Cell 1992, 70:777-789.
Molecular orgonlsation and embryonic hedgehog gene involved in cell-cell segmental patterning in Drosophila, 115:957-971.
16.
Tabata T, Eaton S, Kornberg TB: The Dt~ldlila hedgehog gene is expressed specifically in posterior compartment cells and is a target of engrailed regulation. Genes Dev 1992, 6:2635-2645.
17.
Tashiro S, Michiue T, Higashijima S, Zenno 5, lshimam S, Takahashi F, Orihara M, Koiima T, Saigo K: Structure
19.
Ingham PW, Hidalgo A: Regulation of wingless transcription in the Drosophila embryo. Development 1993, 117:283-291.
20.
Ingham PW: Localised hedgehog activity controls spatially restricted transcription of wingless in the Drosophila embryo. Nature 1993, 366:560-562.
21.
Jesse[I TM, Dodd J: Midline signals that control the dorso-ventral polarity of the neural tube. Semin Neurosci 1992, 4:317-325.
22.
Pourquie O, Cohey M, Teille! MA, Ordahl C, Le Douarin NM" Control of dorsoventral patterning of somitic derivatives by notochord and floor plate. Proc Nail Acad Sci USA 1993, 90:5242-5246.
23.
Placzek M, Jessell TM, Dodd J: Induction of floor plate differentiation by contact dependent, homengenetic signals. Development 1993, 117:205-218.
24. ,..
Fan C-M, Tessier-Lavigne M: Patterning of mammalian somites by the surface ectoderm and the notochord: evidence for scleretome induction by Sonic hedgehog/Vhh.l. Cell 1994, 79:1175-1186. Establishment of an in vitro assay for sclerotome induction "m presomitic mesoderm demonstrates thal notochord produces a long-range diffusible activity that can be mimicked by COS cells expressing Shh. 25. Johnson RL: Ectopic expression of Sonic hedgehog alters •. dotal-ventral patterning of fc0mnites.Cell 1994, 79:1165-1173. Localized expression of Shh mediated by recombinant retroviruses causes the dorsal expansion of Pax1, a marker of ventral fate, and elimination of Pax3 expression, a marker of dorsal fate, in developing somites, suggesting that 5hh is the endogenous inducer ot sclerotome differentiation. 26. •
lanabe Y, Roelink H, Jessell TM: Induction of motor neurons by Sonic hedgehog is independent of floor plate differentiation. Curt Biol 1995, 5:651-658. COS cells expressing Shh can induce both floorplate and motor neuron differentiation in neural lube explants, the latter, but not the fowner, occurring when a filter is interposed between the COS cells and the tissue. When Shh is expressed ectopically in vivo, floorplale and motor neurons differentiate simultaneously, suggesting that they are independently induced by Shh activity. 27. =.
Lee JJ, Ekker 5C, Von Kessler DP, Porter JA, Sun BI, Beachy PA: Autoproleolysis in hedgehog protein biogenesis. Science 1994, 266:1528-I 537. Analysis of the protein products of certain hh mutant alleles and of deletion derivatives generated in vitro reveals that the proteolytic cleavage of the hh protein is autocatalyzed and depends exclusively upon sequences within the carboxy-terminal portion of the protein. At least Iwo vertebrate hh family proteins are processed in a similar manner. Antisera specific for either the amino-lerminal or carboxy-terminal fragments of the Drosophila protein reveal different distributions in embryos, suggesting thai the two products of autoproteolysis may mediate short-range and long-range effects of hh, respectively. 28. ••
Bumcrot DA, Takada R, McMahon AP: Proteolyti¢ processing yields two secreted forms of Sonic hedgehog, Mol Cell Biol 1995, 15:2294--2303. Analysis of chick and mouse Shh expressed in Xenopus oocytes and tissue culture cells reveals that they are processed similarly to Drosophila hh. The carboxy-terminai derivative is readily secreted into the medium, whereas the amino-terminal derivative remains closety associated wilh the cell surface except in the presence of heparin or when derived from a truncated cDNA lacking the carboxy-terminal portion of the coding region.
Signalling by hedgehog family proteins in Drosophila and vertebrate development Ingharn 29. • ,,
Porter JA, Von Kessler DP, Ekker SC, Young KE, Lee II, MosesK, Beachy PA: The product of hedgehog auloproteolytlc cleavage active in local and long-range signalling. Nature 1995, 374:363-366. Identifies the precise site of autoproleolytic cleavage of hh by direcl sequencing of the cleavage product; deletion derivatives that produce only one or other of the normal cleavage products reveal that the amino-terminal portion mediates both 'short-range' and 'long-range' activities of hh in Drosophi/a. The association of the amino-terminal portion with the cell surface depends upon autoproteolysis. AH hh mutant alleles examined affect either the generation or structure of the amino-terminal portion. 30.
Taylor AM, Nakano Y, Mohter J, Ingham PW: Contrasling dislributions of patched and hedgehog proteins in the Drosophila embryo. Mech Dev 1993, 43:89-96.
31. .t
Roelink H, Porter J, Chiang C, Tanabe Y, Chang DT, Beachy PA, }essellTM: Floor plate and motor neuron induction by different concentrations of the amino terminal cleavage product of Sonic hedgehog autoproteolysis. Cell 1995, B1:445-455. Bacterially produced amino-terminal Shh or medium conditioned by ceils expressing a truncated eDNA encoding only this portion of the protein can act in a concentration-dependent manner on neural tube explanls Io induce both floorplate and motor neuron differentiation. in rat, mouse and chick embryos, as in Drosophila, the amino-terminal portion of the protein is closely associated with the surface of cells that secrete it. Marti E, Bumcrot 19A, Takada R, McMahon AP: Requirement of 19kDallon form of Sonic hedgehog for induction of distinct ventral cell types in vertebrate CNS explants. Nature 1995, 375:322-325. Recombinant amino-terminal, but not carboxy-terminal, Shh can induce floorplate and motor neuron differentiation in neural tube explants. Floorplate is induced only when the protein is presented coupled to a bead, whereas motor neurons are induced by protein dissolved in the medium. An antibody specific for the amino-terminal portion of Shh blocks motor neuron induction by nolochord in vitro.
32. •-
33. •*
Fan C-M, Porter )A, Chiang C, Chang DT, Beachy PA, Tessier-Lavigne M: Long-range sclerotome induction by Sonic hedgehog: direct role of the amino terminal cleavage product and modulation by the cyclic AMP signaling pathway. Cel/ t 995, 81:4S7-46S. Recombinant amino-terminal Shh can induce expression of the sclerotome-specific marker Paxl in presomitic mesoderm in vitro. This effect is efficiently suppressed by drugs that increase PKA activity. 34. •.
Fietz MJ, lacinto A, Taylor AM, Alexandre C, Ingham PW: Secretion of the amino-terminal fragment of the Hedgehog protein is necessary and sufficient for hedgehog signalling in Drosophila. Curt giol 1995, 5:643-650. A mutation in Drosophila hh blocking signal peptide cleavage does not affect autoproteolysis, but the protein lacks all signalling activity, whereas deletion of the carboxy-terminal portion of the protein has little effect on signalling activity in either embryos or imaginal discs. Increasing the levels of hh expression within its normal domain has no effect on dorsal cuticle pattern, arguing against a direct dependence on hh concentration. Basler K, Struhl C: Compartment boundaries and the control of Drosophila limb Pattem by hedgehog protein. Nature 1994, 368:208-214. Clones of cells constitutively expressing hh have no effect if located in the posterior compartment of imaginal discs, but in the anterior comparlment induce the generation of large duplications of structures; these effects on patterning are preceded by ectopic induction of wg and dpp expression, suggesting that hh acts via these two signalling proteins. 35. -,
36. •.
Capdevila J, Cuerrero I: Targeted expression of the signalling
molecule decapentaplegic induces pattern duplications and
growth alterations in Drosophila wings. EMBO ] 1994, 13:4459-4468. Eclopic expression of hh driven by GAL4 enhancer trap lines causes pattern duplications and ectopic activation of dpp. Expression of dpp driven by similar GAL4 enhancers has comparable effects on patterning, implying that hh acts exclusively via dpp in the wing. 37. ,*
Diaz-Benjumea FJ, Cohen SM: Cell-interaction between comParlments establishes the proximal-distal axis of Drosophila legs. Nature 1994, 372:175-179.
Expression of the Distallessgene in leg imaginal discs requires hh activity. Ectopic expression of dpp in the ventral compartment of the disc induces the same kinds of leg bifurcations generated by ectopic hh expression. Ingham PW, Fielz MI: Quantitative effects of hedBeheg and decapentaplegic activity on the Palteming of the Drosophila wing. Curr Biol 1995, 5:432-441, Ectopic expression of hh or dpp driven by the same GAL4 enhancer results in almost identical pattern defects. The effects of both genes vary with temperature, indicating a dose dependence of both proteins. Very high levels of hh activate ptc, but not dpp. 38. •.
39.
Kojima T, Michiue T, Orihara M, Saigo K: Induction of
•
a mirror-image duplication of anterior wing slructures by
localized hedge/rag expression in the anterior compartment of Drosophila melanogasterwing irnaginal discs. Gene 1994, 148:211-217. Ectopic expression of hh in the wing imaginal discs because of random insertion of a hh transgene results in wing duplications and inappropriate expression of dpp. 40. ••
Tabala T, Kornberg TB: Hedgehog is a signalling protein with a key role in patterning Drosophila imaginal discs. Cell 1994, 76:89-102. The hh protein is found associated with engraileoLexpressing cells and their neighbours, indicating that it is secreted. This distribution is modified by treatment with monesin or by a mutant thai blocks endocytosis. Overexpression of hh has similar effects to mutations in ptc in both the embryo and the imaginal discs. 4I.
Struhl G, Basler K: Or~lnlsing activity of wingless protein in Drosophila. Cett 1993, 72:527-540.
42. •
Wilder EL, Perrimon N: Dual functiom of wingless in the Drosophila leg imaginal disc. Development 1995, 121:477~188. High-level expression of wg by GAL4 enhancers results in partial ventralization of cells in the leg discs and increased proliferation of dorsal cells. These results argue agains~ a simple morphogen model for . wg action, 43.
Heberlein U, Wolff 1", Rubin GM: The TGFp homolng dpp and the segment polarity ~ hedgehog are required for propagation of a morpho~metic wave in the Drosophila retina. Ceil 1993, 75:913-926.
44.
Ma C, Zhou Y, Beachy PA, Moses K: The segment polarity gene hedb~hog is required for progression of the morphogenetic furrow in the developing Drosophila eye. Cell 1993, 75:927-938.
45. •,
Heberlein U, Singh CM, Luk AY, Donohoe TI; Growth and differentiation in the Drosophila eye coordinated by hedgehog. Nature 1995, 373:709-711. Clones of cells constitutively expressing hh in the Drosophila retinal field induce new foci of differentiation, presaged by the induction of dpp and hairy expression in cells surrounding the clone. 46.
Charite l, De Graaff W, Shen S, Deschamps J: Ectopic expressionof Hoxb-8 causes duplication of the ZPA in the forelimb and homeotie transformation of axial structures, Cell 1994, 78:589-601. Transgenic mouse embryos in which the Hox-b8 gene is misexpressed exhibit ectopic expression of Shh in the anterior mesenchyme of their hindlimbs and a concomitant duplication of digits.
•
47. •,
Yang YZ, Niswander L: Interaction between the s i ~ i n g molecules wnt7a and shh during vertebrate limb development dorsal signals regulate anteroposlerior Patterning. Cell 1995, 80:939-947. Recombinant FGF4 can maintain 5hh expression in proximal limb buds and induces BMP2, 8MP4 and Hox-c113expression. Removal of dorsal ectoderm results in down-regulation of 5hh transcription, an effect that can be reversed by expression of WntTa. 48. •.
Laufer E, Nelson CE, Johnson RL, Morgan BA, Tabin C: Sonic hedgehog and Fgf-4 act through a signalling cascade and feedback loop to integrate growth and pattemin8 of the developing limb bud. Cell 1994, 79:993-1003. Ectopic Shh expression in the chick limb induces BMP2 and Hox-dll and Hox-dl3 expression. The effects of Shh on these genes depend upon FGF4 activity; in addition, FGF4 expressed in Ihe AER controls the maintenance of Shh transcription. FGF4 expression in the AER also depends upon Shh.
497
498
Pattern formation and developmenta| mechanisms Niswander L, Jeffrey S, Martin GR, Tickle C: A positive feedback loop coordinates growtlh and PaUeming in the vertebrate limb. Nature 1994, 371:609-612. Removal of the AER leads to loss of Shh expression, an effect that can be reversed by recombinant FGF4. FGF4 and retinoic acid act in concert to induce Shh transcription.
interruptu5 Dominant of Drosophila. Genes Dev 1990,
49. •-
50. oe
Parr BA, McMahon AP: Dorsalizing signal wnt-7a required for normal polarity of ( I v and a-p axes of mouse limb. Nature 1995, 374:350-353. Mouse embryos lacking Wnt7a activity fail to maintain Shh expression in posterior limb mesenchyme. BMP2 is similarly affected. Francis PH, Richardson MK, Brickell PM, Tickle C: Bone moq~m~metlc proteins and a signalling pathway Ihat controls PatteminB in Ihe developing chick limb. Development 1994, 120:209-218. The germ encoding BMP2 is expressed in the posterior AER and in the posterior limb mesenchyme. Expression is ectopically induced by ZPA grafts, but recombinant BMP2 does not have polarizing activity.
4:1053-1067. 61. •
51usarski DC, Motzny CK, Holmgren R: Mutations thai aller the timing and paltern of cubitus interruptus gene expression in Drosophila melanosaster. Genetics 1995, 139:229-240. Presents an analysis of the spatial distribution of ci protein in Drosophila embryos and imaginal discs and the effects of various mutations on ils deployment and stability. 62.
51. •
52.
Hooper J, Scott MP: The Drosophila patched gene encodes a putative membrane protein required for segmental patteming. Cell 1909, 59:751-765.
53.
Nakano Y, Guerrero I, Hidalgo A, Taylor AM, Whittle IRS, Ingham PW: lhe Drosophila segment polarity gene patched encodes a protein with multiple potential membrane spanning clomains. Nature 1989, 341:508-51 3.
54.
Phillips
R,
Roberts I, Ingham
PW, Whinle
JRS; The
Drosophila se~eni polarity gene patched is involved in a position-si~alling mechanism in imaginal discs. Development 1990, 110;105-114. 55.
CapdeviLa J, Estracla MP, Sanchez Herrero E, Cuerrero I: • The Drosophila segmenl polarity gene Palched interacts with decaix,ntapleBic in wing developmenl. EMBO J 1994, 13:71-82. Viable mutations of the pro gene cause ectopic expression of dpp in the anterior compartments of wing imaginaI discs. 56.
Iogham I~V, Taylor AM, Nakano Y: Role of the Drosophila positional signalling. Nature 1991,
Patched Bene in 353:184-I 87. 57.
Forbes AJ, .Nakano Y, Taylor AM, Ingham PW: Genetic analysis of hedgehog signalling in the Drosophila embryo. Development 1993, SU1~1:115-124.
58. •
Hooper j: Distinct Pathways for aulocrlne and paracrine Wingless signalling in Drosophila embryos. Nature 1994, 372:461-464. Genetic eplstasis analyses suggest that fused, ci and smoothened act downstream of ptc to aclivate wg transcription. 59.
Prdat T, Th~rond P, Limbourg-Bouchon B, Pham A, Tricoire H, Busson O, Lamour-lsnard C: Segmental polarity in Dlosoph#a melanoBaste~ ~metic dissection of fused in a suppressor of fused bad(Bround reveals interaction with costal-2. Genetics 1993, 135:1047-1062.
60.
Orenic TV, Slusarski DC, Kroll KL, Holmgren RA: Cloning and characterization of the segment polarity gene cubitus
Prdat T, The%end P, Limbour~-Bouchon B, Pham A, Tricoire H, Busson D, Lamour-lsnard C: A putalive serine/threonine protein klnase encoded by the segment-polarity fused germ of Drosophila. Nature 1990, 347:87-89.
63. o-
Lepage T, Cohen SM, Diaz-Benjumea Fj, Parkhus! SM: Signal transduction by cAMP-depeodenl protein kinase A in Drosophila limb patterning. Nature 1995, 373:711-715. A viable allele of pka causes wing duplications similar to those induced by ectopic hh, preceded by ectopic dpp and ptc expression. Clones of cells lacking pka or ptc activity cause wing and leg duplications. 64. •-
Pan D, Rubin GM: cAMP-dependent protein klnase and hedgehog act antagonistically in regulating decapentapleBic transcription in Drosophila imaginal discs. Cell 1995, 80:543-552. Clones of cells lacking PKA activate dpp inappropriately in Ihe anterior compartment of the wing disc and anlerior to the morphogenelic furrow in the eye disc, inducing wing duplications or ectopic furrows. A dominant-negative PKA allele has a similar effect in the wing and suppresses the hh mutant phenotype in the eye. 65.
Jiang J, 5truhl G: Protein kinase A and hedgehog signaling in Drosophila limb development. Cell 1995, 80:563-572. Clones of cells lacking PKA activity in the anterior compartments of wings and legs cause pattern duplications similar to those induced by ectopic hh and induce ectopic expression of dpp and wg. Constitutively active pica, has no effect on normal dpp expression, nor does it suppressthe ptc mutant phenolype,
•.
66. •°
Li W, Ohlmeyer .IT, Lane ME, Kalderon D: Function of prolein kinase A in hedgehog signal transduction and Drosophila imaginal disc development. Cell 1995, 80:553-562. Similar analysis to [65"'], but these authors show that high levels of a constitutively active PKA can rescue the ptc mutant phenotype. 67. --
Strult DI, Wietsdorff V, Mlodzik M: Regulation of furrow progression in the Drosophila eye by cAMP-dependent protein kinase A. Nature 1995, 373:705-709. Clones of cells lacking PKA activity in the developing retina have similar effects to clones expressing hh in Ihat they initiate morphogenetic furrow migration ectopically. In contrast to those expressing hh, however, the PKA clones themselves initially activate dpp expression.
PW Ingharn, Molecular Embryology Laboratory, Imperial Cancer Research Fund, 44 Lincoln's Inn Fields, London WC2A 3PX, UK.
Current Opinion in Genetics and Development 1995, 5:492-498 Introduction
I discuss recent studies presenting some interesting and unexpected answers to this question.
The hedgehog (hh.) gene was identified originaUy through the segment polarity phenotype caused by its mutation in Drosophila [1]. Genes of the hh family have now been isohted fi'om several vertebrate species, including mouse [2,3"], chicken [4], zebrafish [5,6"'], rat [6"'], Xenopus [7",8"] and human [9"]. These genes not only show a high degree of structural homology both within and between species, but in addition exhibit some remarkable similarities in the ways in which they function in various embryonic processes [10",11"]. The paradigm, for hh signalling was first estabfished by the genetic analysis of segmental patterning in the Drosophila embryo [12]. Embryos lacking hh activity die, revealing changes in their cuticular patterns suggestive of a requirement for the gene throughout each segment [1,13]. This requirement contrasts with the spatially restricted expression of hh [14-17], but could be explained if such localized expression establishes a source of hh protein that diffuses across each segment to generate a gradient of hh activity. This scenario is consistent with the postulated role of hh in the dorsal ectoderm, where the fates of individual cells appear to be determined directly by the levels of hh activity to which they are exposed [18"']. In the ventral ectoderm, however, several lines of evidence suggest instead that hh acts at short range to control the localized expression of another signalling molecule, encoded by the segment polarity gene wingless (wg) [19,20]. These contrasting roles of hh present a conundrum that is also raised by the effects of other hh family genes in vertebrate embryos: how does a single protein act as both a short-range and a long-range signal? In this review,
Sonic hedgehogand midline signalling in vertebrates The Sonic hedgehog (Shh) gene is expressed in the notochord and floorplate of vertebrate embryos [2,4,5,6"'], two midline structures implicated in inductive interactions that control the patterning of the neural tube [21] and somites [22]. The differentiation of floorplate is it.serf induced by the notochord, a process that can be reproduced in vitro, but only if the inducing and responding tissues are in direct contact [23]. By contrast, the ability of both notochord and floorplate to induce sclerotome differentiation in somitic mesoderm can be reproduced in vitro in the absence of ceU contact [24"]. Moreover, whereas floorplate differentiation is restricted to cells immediately adjacent to the inducing notochord, sclerotome differentiation can extend for over 200 mm in explants of presomitic mesoderm [24"]. Two fines of evidence suggest that Shh encodes the activity mediating both of these processes. First, ectopic expression of Shh in vivo leads to the activation of floorplate markers in inappropriate regions of the neural tube [2,5,6"',7"] and of sclerotome markers in inappropriate regions of the somites [25"'] Second, COS cells expressing Shh can induce floorplate differentiation in neural tube explants [6"',26"] and sclerotome differentiation in exphnts of presomitic mesoderm [24"], only the former effect depending upon direct contact of the COS cells with the responding tissue
Abbreviations AIER--apical ectodermal ridge; ci~cubitus interruptus; dpp--decapentaplegic hk~hedgehog; HNF--hepatocyte nuclear factor; PKA~protein kinase A; ptc--patched; 5kh~5onic hedgehog; w#--wingtess; ZPA--zone of polarizing activity. 492
© Current Biology Ltd ISSN 0959-437X
Signalling by hedgehog family proteins in Drosophilaand vertebrate development Ingham 493 Do different forms of hedgehog/Sonic hedgehog mediate distinct signalling activities? Both hh and Shh undergo autoproteolytic cleavage to generate two distinct subspecies of unequal size [15,27",28"], the smaller of which (-19kDa in both cases) is amino-terminally derived (hhN and ShhN) and represents the portion of the protein most highly conserved between different species. The hrger carboxyterminal portion (hhC and ShhC) varies in size between species and exhibits significantly less sequence identity, although certain regions, including domains required for autoproteolysis [27",29"], are highly conserved. The generation of these distinct protein forms suggests a possible molecular basis for the different modes of signalling exhibited by hh family members [27"',28"']. What makes this scenario even more attractive is the finding that the carboxy-terminal portion is readily secreted when expressed in Xenopus oocytes [28"'] or tissue culture cells [27"'-29"'], whereas the aminoterminal portion remains closely associated with the cell surface [27"--29"'], being displaced only by the addition ofheparin or suramin to the medium. That this behaviour reflects the properties of the proteins in vivo is indicated by immuuohistochemical analyses: antibodies specific for the amino-terminal portion detect protein predominantly around the periphery of cells expressing hh or Shh [27",30,31"'], whereas, in Drosophila at least, the carboxy-terminal portion appears to diffuse away from the expressing cells [27"°]. Thus, hhC and ShhC have the properties expected of a long-range signalling molecule and hhN and ShhN display characteristics consistent with a short-range or contact-dependent signal. Despite this strong prima facie case, we now have conclusive evidence that all the signalling activity of both hh and Shh resides exclusively within their amino-terminal domains. Thus ShhN, but not ShhC, is equally effective at inducing floorplate [31"',32"'] or sclerotome [33"'] differentiation in in vitro assays. Similarly, in Drosophila, overexpression of hhN is sufficient to respecify the pattern of both the ventral and dorsal epidermis, whereas the ectopic expression o f h h C has no effect on either pattern [29",34"']. The distinction between 'contact-dependent' and 'longrange' signalling activities seems, at least in vertebrates, to simply reflect the fact that different cellular responses can be invoked by different threshold levels of Shh activity. Elucidation of this phenomenon has been greatly facilitated by the discovery that ShhN generated artificially by expressinga truncated Shh cDNA is readily secreted by tissue culture cells [28",29"']. Apart from the interesting implication that autoproteolytic cleavage of the full-length protein results in a modification of the amino-terminal ~agment promoting its association with the ceil surface [28"',29"'], this finding has the important practical consequence that cells expressing the construct produce conditioned medium with significant inducing
activity. Medium containing ShhN at concentrations of 5 nM or higher can induce expression of the floorplate marker hepatocyte nuclear factor (HNF)3~ in neural plate explants [31"'], and concentrations of 1.25nM, although ineffective with respect to HNF3~ induction [31"], are sufficient to induce expression of Pax1, a sclerotome-specific marker, in presomitic mesoderm explants [33"]. Such experiments, and similar ones employing bacterially produced ShhN protein, have also provided compelling evidence that Shh mediates a second 'contact-independent' inducing activity of the notochord, the induction of motor neurons in the ventral neural tube [31",32"°]. Analysis of this phenomenon was complicated initially because floorplate cells are also effective motor neuron inducers, so any assay in which floorplate differentiation is induced by Shh could not discriminate between a direct or indirect effect of Shh on motor neuron differentiation [6"']. This problem has now been circumvented, because concentrations of bacterially produced ShhN and ShhN conditioned medium below the threshold required for floorphte induction are sufficient to induce motor neuron differentiation [3 I",32"']. Thus, what had previously appeared to be a qualitative difference between notochord-derived signals that induce floorplate and those that induce motor neurons is now taken to reflect a quantitative difference in the requirements of the two cell types for the same Shh-encoded inducing activity. Although it is possible that the form in which the Shh protein is presented to cells may influence their response to its activity [32"'], it seems more likely that the preferential retention of ShhN on the surface of cells from which it is secreted serves to generate a steep gradient of Shh protein [31"]; thus, only cells in direct contact with the notochord are induced to form floorplate, whereas cells some distance from the notochord experience levels of Shh compatible with motor neuron differentiation. Whether or not the long-range effects of hh in the dorsal epidermis of the Drosophila embryo are similarly direct is (as yet) unclear; however, raising the level of hh expression in a manner that should change the profile of the putative hh protein gradient across the parasegment has no effect on dorsal patterning [34"'], implying that, as in the ventral epidermis, hh acts indirectly by regulating the expression of some (as yet) unidentified signalling molecule, rather than in a direct concentration-dependent manner analogous to Shh.
hedgehog and imaginal disc patterning in Drosophila In Drosophila, hh continues to be required a~er ~mbryogenesis for the patterning ofimaginal discs, sheets of cells that give rise to the structures of the adult fly. Loss o f hh function or ectopic hh expression alike have fairly
494
Patternformation and developmental mechanisms
global effects on imaginal disc development, but, as in the embryo, these effects seem to reflect a role for hh in regulating the expression of other signalling factors. The hh gene is expressed throughout the posterior compartment of each disc [15,16], maintained by a lineage-based mechanism most likely mediated by the engrailed homeodomain protein. Across the compartment boundary, the signal-encoding genes wg and decapentaplegic (dpp) are, by contrast, expressed in restricted domains in close proximity to the hh-expressing cells. This spatial relationship is significant, because in the absence of hh activity, transcription of both wg and dpp is lost [35"'], whereas ectopic expression of hh in the anterior compartment of leg or wing discs leads to the inappropriate activation of wg and/or dpp around the hh-expressing cells [27",35"'-38",39",40"]. Moreover, ectopic expression of either the full-length hh protein [27"',35"'-38",39",40"], or of its amino-terminal portion [34"'], causes the respecification of cell identities and duplication of structures, effects that are produced by ectopic expression of wg [41,42"] or dpp [36"--38"'] alone. These findings have led to the conclusion that hh acts at short range to maintain localized sources of wg and/or dpp protein at each compartment boundary; it is these proteins that act, either in a direct dose-dependent manner [38",41] or in conjunction with other factors [42"], to specify the positional identity of surrounding cells, In an analogous manner, hh and dpp also interact to control the differentiation of the compound eye. The transition of retinal precursors from an actively dividing state to a terminally differentiated state is initiated as they enter the so-called morphogenetic furrow, a transient groove that sweeps across the retinal field during differentiation (also see the review in this issue by N M Bonini and K-W Choi [pp 507-515]). As cells enter the furrow, they activate transcription of dpp, a process that is dependent upon hh expression in cells just posterior to the furrow [43,44]. Inactivation of either hh or dpp blocks progression of the furrow [43,44], whereas ectopic expression ofhh results in the precocious activation of dpp and the inappropriate initiation of differentiation and furrow migration [45"'].
Sonic hedgehogand limb patterning in vertebrates The discovery that Shh is also expressed in the posterior mesenchyme of limb buds in mouse, chick and fish embryos [2,4,5] suggests a remarkable parallel between the patterning of limbs in Drosophila and vertebrates [10"]. The posterior mesenchyme is known to be the source of a signalling activity that polarizes the developing limb bud; thus, Shh appears an attractive candidate for the molecular basis of this so-called zone of polarizing activity (ZPA).
Consistent with this suggestion, ectopic expression of Shh can induce the generation of mirror-image duplication of digits and other structures similar to those caused by classic ZPA grafts or by the localized application of retinoic acid [3",4]. In addition, digit duplications caused by the ectopic expression of the murine Hox-b8 gene are invariably presaged by the inappropriate activation of Shh in the anterior limb mesenchyme of transgenic mice [46"]. Although Shh expression is initiated adjacent to the flank at the onset of hb bud development, this domain progressively shifts distally as the bud grows out, mirroring the distal displacement of the ZPA. This dynamic expression pattern contrasts with the static lineage-restricted expression of hh in imaginal discs and implies the operation of a regulatory mechanism that maintains Shh expression around the progress zone that is patterned by the ZPA. In fict, Shh expression is maintained by two distinct signals emanating from the dorsal ectoderm and the apical ectodermal ridge (AEK). Experiments in which Shh transcription is rescued by the expression of specific factors in limbs denuded of either tissue indicate that Wnt7a mediates the effects of the dorsal ectoderm [47"'], and FGF4 is responsible for the AEK-derived signal [48"',49"']. FGF4 is expressed specifically in the AER, whereas expression of Wnt7a is normally restricted to the dorsal ectoderm of the limb; moreover, in mouse embryos homozygous for a null allele of Wnt7a, Shh expression is initiated normally in the early hmb bud, but rapidly disappears as the bud develops [50"], confirming its involvement in this process. As well as maintaining Shh expression, FGF4 predisposes cells to respond to its activity, as pattern duphcations are normally induced by ectopic Shh only when it is expressed in close proximity to the AER. In the proximal part of the anterior mesenchyme, ectopic Shh has no effect unless accompanied by the simultaneous application of FGF4 protein [48°']. As the range of FGF4 is fairly restricted [49"'], it follows that Shh itself acts over only a limited range. This implies that in the vertebrate limb, Shh may act in an analogous manner to its counterpart in Drosophila imaginal discs, exerting a long-range effect by regulating the expression of some other signalling molecule. One potential candidate for such a mediator of Shh activity in the limb is BMP2; transcription of Bmp2 is localized in and around the Shh domain in the posterior limb mesenchyme [48",51"] and is activated in response to Shh and FGF4 [47"',48"]. ILemarkably, BMP2 is the vertebrate TGF~3 family member most closely related to Drosophila dpp. Although the inference that the same signalling pathway has been conserved between vertebrates and invertebrates is appealing, there is no evidence that BMP2 itself has limb polarizing activity [51"]. It may be that BMP2 acts together with some other factor, perhaps even as a heterodimer with another BMP (see the review by C Tickle in this issue [pp 478-484]).
Signalling by hedgehog family proteins in Drosophila and vertebrate development Ingham Transduction
o f t h e hedgehog s i g n a l
Transduction of signals encoded by hh family genes is (as yet) poorly understood, but most of what is known comes from studies in Drosophila. To date, the best candidate for a hh receptor is the novel multipass transmembrane protein encoded by the segment polarity gene patched (ptc) [52,53]. The spatial regulation of ptc expression is reciprocal to that of hh in embryos and imaginal discs [40°°,52-54], and in both contexts, ptc functions to repress the transcription of wg and/or dpp [19,55•], the genes activated by hh. This observation led to the proposal that hh activates its targets by antagonizing the activity of ptc [56], a model that is supported by the finding that expression of both wg and dpp genes is independent of hh activity in the absence of pro. At present, however, no direct evidence exists for such a physical interaction between the two proteins. Other genes involved in hh signal transduction have been identified largely on the basis of their epistatic relationships with ptc [57,58"]. Three genes with mutant phenotypes similar to hh are absolutely required for wg transcription, even in the absence of ptc activity. Two of these, fused and smoothened, are expressed both zygotically and maternally ([59]; M van den Heuvel, PW Ingham, unpublished data), and the third, cubitus interruptus (cO, is required only zygotically. The zinc finger protein encoded by ci has strong homology with the DNA-binding domain of the vertebrate GLI family of transcription factors [60]; thus, ci is an attractive candidate for a transcription factor that regulates u,g expression in response to hh activity [57]. Consistent with this interpretation, d, like ptc, is expressed in a reciprocal pattern to that of hh, such that it is present in cells that respond to ectopic hh activity as well as in those that normally activate wg and ptc transcription in response to hh [61°]. This suggests that ci is maintained in an inactive form, except in cells that receive the hh signal. The fused gene acts downstream of ptc and hh and encodes a putative serine/threonine kinase [62], the immediate targets of which are (as yet) unknown. Epistasis analysis suggests that fused acts upstream of a fourth member of the segment polarity class, costal-2 [59], which like ptc is required to repress wg transcription in the embryo [57]. Unlike ptc, however, maternally supplied costaf-2 is sufl%ient to support normal embryogenesis, suggesting that the costal-2 product is ubiquitously distributed in the embryo. All four of the genes implicated in hh signalling in the embryo are also involved in the patterning of imaginal discs. Viable mutations of pro and costal-2 cause outgrowths and mirror-image duplication of structures in the anterior compartment of the wing [54,55 °] similar to those induced by ectopic hh expression. The same kinds of duplications are also caused by viable hypomorphic or antimorphic mutations of the catalytic subunit of protein kinase A (PKA) [63••,64••], and clones
of cells completely lacking PKA activity in the anterior compartments of wing and leg imaginal discs or the retina of the eye disc autonomously activate u~g or dpp transcription in a hh-independent manner [63"°-67"]. These findings suggest that PKA, like ptc and costal-2, functions to suppress the hh response. Although this could imply that PKA is a direct downstream effector of ptc activity, this seems unlikely, because expression of a dominant active form of PKA at levels that rescue plea mutants is unable to suppress the ptc mutant phenotype [65"']. Instead, it seems that PKA acts by keeping the effectors ofhh activity inactive. As the ci protein contains multiple consensus PKA phosphorylation sites in its carbox-y-terminal region [60], it represents a likely target for this activity of PKA; according to this scenario, ci would be maintained in an inactive phosphorylated state by PKA activity unless activated by de-phosphorylation of these sites (or phosphorylation of other sites or both) in response to the transduction of the hh signal. Intriguingly, induction of sclerotome differentiation by Shh can be inhibited by drugs that up-regulate PKA activity, suggesting that the involvement of PKA in hh signalling may also be conserved between vertebrates and invertebrates [33"'].
Conclusions Members of the hh gene family have been highly conserved through evolution and play remarkably similar roles in the development of vertebrates and invertebrates. Despite these similarities, some important differences are seen in the way these roles are executed in different contexts. In Drosophila and in the vertebrate limb, hh proteins act as local signals that induce the expression of other signalling molecules, whereas in the trunk of the vertebrate embryo, Shh appears to act as a 'true' morphogen.
References and recommended
reading
Papers of particular interest, published within the annual period of review, have been highlighted as: • or" specia) interest •of outstanding interest 1.
N0ssfein-VolhardC, Wieschaus E: Mutations affecling menl number and polarity in Drosophila, Nature 1980, 287:795-801.
2.
Echelard Y, Epstein DJ, St Jacques B, Shen L, Mohler J, McMahon JA: Sonic hedgehog, a member of a family of putative signaling molecules is implicated in the regulation of CNS and limb polarity. Cell 1993, 75:1417-1430.
3. •
Chang DT: Products, genetic linkage and limb palleming activity of a murlne hedge/ioB gene. D4evetopment 1994, 120:3339-3353. Describes cloning, characlerization and functional analysisof a mouse hh homotoguethat is identical to Sbh. Like the zebrafish shh,the mouse gene is active in Drosophilaand also has potarizing activity in the chick limb.
495
496
Pattern formation and developmenta| mechanisms 4.
Riddle RD, Johnson RL, Laufer E, Tabin C: Sonic hedgehog mediates the polarizing activity of the ZPA. Cell 1993, 75:1401-1416.
and expression of hedgeho~ a Drosophila segment-polarity gene required for cell-cell communication. Gene 1993, 124:183-189.
5.
KraussS, Concordet J-P, Ingham PW: A functionally conserved Immolog of the Ormophi/a segment polarity gene hedgehog is expressed in tissues with polarisiog activity in zebrafish embryos, Cell 1993, 75:1431-1444.
18. Heemskerk J, Dinardo S: Drosophila hedgehog ads as a •. morphogen in cellular pattemiog. Cell 1994, 76:449460. This paper presents an analysis of the role of hh in the patterning of the dorsal cuticle of the Drosophila embryo. The authors show thai this is temporally distinct from its role in regulating wg transcription and adduce evidence, based on the manipulation of temperature-sensitive hh alleles and a heat-shock hh construct, that in this context hh acts directly to specify cell fate in a concentration-dependent manner.
6. •.
Roelink H, Augsburger A, Heemskerk J, Korzh V, Norlin S, Ruiz i Ahaba A, Tanabe Y, Placzek M, Edlund 1, Jessell TM et al.: Floor plate and motor neuron induction by ~ h - l , a vertebrate homoleg of hedgehog expressed by the notochord. Cell 1994, 76:761-775. An indepehclent search for homologues of hh in rat and zebrafish yielded genes that appear to be identical to Shh on the basis of their sequence, expression patterns and biological activity. This study also shows that COS cells expressing the vhh-f {Shh) gene can induce floorplate and motor neuron differentiation in neural plate explants in vitro.
7. •
Ruiz i Altaba A, Roelink H, Jessell TM: Restrictions to floorpbte induction by hedgehog and winged helix genes in mc',ural tube of frog embryos. Mol Cell Neurosci 1995, in press. This paper describes the sequence and expression pattern of the Xenopus Shh gene. The induction of floorplate by ectopic 5hh expression is temporally and spatially constrained. B. •
Yokota C, Mukasa T, Higashi M, Odaka A, Muroya K, Uchiyama H, Eto Y, Asahima M, Momoi "I-: Activin induces the expression of the Xenopus homolog of sonic hedgehogduring mesoderm formation in.Xenq~s explants. Biochem Biophys Res Commun 1995, 207:i-7. Describes the PCR cloning of a fragment of the Xenopus 5hh gene and demonstrates that its exp~ssion can be induced by activin in isolated animal caps in Ihe absence of protein synthesis. 9. •
Marigo V, Roberts DI, Lee SMK, Tsukurov O, Levi T, Gastier )M, Epstein DJ, Gilbert D), Copeland NG, Seidman CE et aL: Cionin~ expression and chromosomal location of shh and i,Sh two human homolognes of the Drosophila segment polarity get~e ~ , Genomics 1995, 27:in press.. Northern analysis shows thai human Ihh is expressed in adult kidney and liver, whereas Shh is expressed in these o~ans and the lung and intestine of the fetus. 5hh is closely linked to, but dislinct from, the polysyndactyly locus. 10. Ingham PW: Hedgehog points the way. Curt Biol 1994, • 4:347-350. Mini-review comparing and contrasting the likely roles of Shh in vertebrate embryos with the function of hh in Drosophila. 11. •
Fietz MJ, Concordet J-P, Barbosa R, Johnson R, Krauss S, McMahon AP, Tabin C, lngham PW: The hedgehog gene family in Drosophilaand vertebrate development. Development 1994,
Supph43-51.
Reviews the function of hh in the Drosophila embryo and imaginal discs and compares the spatial and temporal deployment of Shh in chick, mouse and fish embryos.
12.
|ngham PW: ~ e n t polarity genes and cell patterning within the Drosophila body segment. Curt Opin Genet Dev 1991, 1:261-267.
13.
Mohler J: Recluiremenls for hedgeho~ a segment polarity geue, in pattemiJq~ larval and adult cuticle of Drosophila. Genetics 1988, 120:1061-1072.
t4.
Mohler J, Vani K: expre~ion of the communication in Development 1992,
1S.
Lee JJ, Von Kessler DP, Parks S, Beachy PA: SeCretion and Iocalised transcription su~esls a role in positional signaUing for products of the segmentation gone hedgehog. Cell 1992, 70:777-789.
Molecular orgonlsation and embryonic hedgehog gene involved in cell-cell segmental patterning in Drosophila, 115:957-971.
16.
Tabata T, Eaton S, Kornberg TB: The Dt~ldlila hedgehog gene is expressed specifically in posterior compartment cells and is a target of engrailed regulation. Genes Dev 1992, 6:2635-2645.
17.
Tashiro S, Michiue T, Higashijima S, Zenno 5, lshimam S, Takahashi F, Orihara M, Koiima T, Saigo K: Structure
19.
Ingham PW, Hidalgo A: Regulation of wingless transcription in the Drosophila embryo. Development 1993, 117:283-291.
20.
Ingham PW: Localised hedgehog activity controls spatially restricted transcription of wingless in the Drosophila embryo. Nature 1993, 366:560-562.
21.
Jesse[I TM, Dodd J: Midline signals that control the dorso-ventral polarity of the neural tube. Semin Neurosci 1992, 4:317-325.
22.
Pourquie O, Cohey M, Teille! MA, Ordahl C, Le Douarin NM" Control of dorsoventral patterning of somitic derivatives by notochord and floor plate. Proc Nail Acad Sci USA 1993, 90:5242-5246.
23.
Placzek M, Jessell TM, Dodd J: Induction of floor plate differentiation by contact dependent, homengenetic signals. Development 1993, 117:205-218.
24. ,..
Fan C-M, Tessier-Lavigne M: Patterning of mammalian somites by the surface ectoderm and the notochord: evidence for scleretome induction by Sonic hedgehog/Vhh.l. Cell 1994, 79:1175-1186. Establishment of an in vitro assay for sclerotome induction "m presomitic mesoderm demonstrates thal notochord produces a long-range diffusible activity that can be mimicked by COS cells expressing Shh. 25. Johnson RL: Ectopic expression of Sonic hedgehog alters •. dotal-ventral patterning of fc0mnites.Cell 1994, 79:1165-1173. Localized expression of Shh mediated by recombinant retroviruses causes the dorsal expansion of Pax1, a marker of ventral fate, and elimination of Pax3 expression, a marker of dorsal fate, in developing somites, suggesting that 5hh is the endogenous inducer ot sclerotome differentiation. 26. •
lanabe Y, Roelink H, Jessell TM: Induction of motor neurons by Sonic hedgehog is independent of floor plate differentiation. Curt Biol 1995, 5:651-658. COS cells expressing Shh can induce both floorplate and motor neuron differentiation in neural lube explants, the latter, but not the fowner, occurring when a filter is interposed between the COS cells and the tissue. When Shh is expressed ectopically in vivo, floorplale and motor neurons differentiate simultaneously, suggesting that they are independently induced by Shh activity. 27. =.
Lee JJ, Ekker 5C, Von Kessler DP, Porter JA, Sun BI, Beachy PA: Autoproleolysis in hedgehog protein biogenesis. Science 1994, 266:1528-I 537. Analysis of the protein products of certain hh mutant alleles and of deletion derivatives generated in vitro reveals that the proteolytic cleavage of the hh protein is autocatalyzed and depends exclusively upon sequences within the carboxy-terminal portion of the protein. At least Iwo vertebrate hh family proteins are processed in a similar manner. Antisera specific for either the amino-lerminal or carboxy-terminal fragments of the Drosophila protein reveal different distributions in embryos, suggesting thai the two products of autoproteolysis may mediate short-range and long-range effects of hh, respectively. 28. ••
Bumcrot DA, Takada R, McMahon AP: Proteolyti¢ processing yields two secreted forms of Sonic hedgehog, Mol Cell Biol 1995, 15:2294--2303. Analysis of chick and mouse Shh expressed in Xenopus oocytes and tissue culture cells reveals that they are processed similarly to Drosophila hh. The carboxy-terminai derivative is readily secreted into the medium, whereas the amino-terminal derivative remains closety associated wilh the cell surface except in the presence of heparin or when derived from a truncated cDNA lacking the carboxy-terminal portion of the coding region.
Signalling by hedgehog family proteins in Drosophila and vertebrate development Ingharn 29. • ,,
Porter JA, Von Kessler DP, Ekker SC, Young KE, Lee II, MosesK, Beachy PA: The product of hedgehog auloproteolytlc cleavage active in local and long-range signalling. Nature 1995, 374:363-366. Identifies the precise site of autoproleolytic cleavage of hh by direcl sequencing of the cleavage product; deletion derivatives that produce only one or other of the normal cleavage products reveal that the amino-terminal portion mediates both 'short-range' and 'long-range' activities of hh in Drosophi/a. The association of the amino-terminal portion with the cell surface depends upon autoproteolysis. AH hh mutant alleles examined affect either the generation or structure of the amino-terminal portion. 30.
Taylor AM, Nakano Y, Mohter J, Ingham PW: Contrasling dislributions of patched and hedgehog proteins in the Drosophila embryo. Mech Dev 1993, 43:89-96.
31. .t
Roelink H, Porter J, Chiang C, Tanabe Y, Chang DT, Beachy PA, }essellTM: Floor plate and motor neuron induction by different concentrations of the amino terminal cleavage product of Sonic hedgehog autoproteolysis. Cell 1995, B1:445-455. Bacterially produced amino-terminal Shh or medium conditioned by ceils expressing a truncated eDNA encoding only this portion of the protein can act in a concentration-dependent manner on neural tube explanls Io induce both floorplate and motor neuron differentiation. in rat, mouse and chick embryos, as in Drosophila, the amino-terminal portion of the protein is closely associated with the surface of cells that secrete it. Marti E, Bumcrot 19A, Takada R, McMahon AP: Requirement of 19kDallon form of Sonic hedgehog for induction of distinct ventral cell types in vertebrate CNS explants. Nature 1995, 375:322-325. Recombinant amino-terminal, but not carboxy-terminal, Shh can induce floorplate and motor neuron differentiation in neural tube explants. Floorplate is induced only when the protein is presented coupled to a bead, whereas motor neurons are induced by protein dissolved in the medium. An antibody specific for the amino-terminal portion of Shh blocks motor neuron induction by nolochord in vitro.
32. •-
33. •*
Fan C-M, Porter )A, Chiang C, Chang DT, Beachy PA, Tessier-Lavigne M: Long-range sclerotome induction by Sonic hedgehog: direct role of the amino terminal cleavage product and modulation by the cyclic AMP signaling pathway. Cel/ t 995, 81:4S7-46S. Recombinant amino-terminal Shh can induce expression of the sclerotome-specific marker Paxl in presomitic mesoderm in vitro. This effect is efficiently suppressed by drugs that increase PKA activity. 34. •.
Fietz MJ, lacinto A, Taylor AM, Alexandre C, Ingham PW: Secretion of the amino-terminal fragment of the Hedgehog protein is necessary and sufficient for hedgehog signalling in Drosophila. Curt giol 1995, 5:643-650. A mutation in Drosophila hh blocking signal peptide cleavage does not affect autoproteolysis, but the protein lacks all signalling activity, whereas deletion of the carboxy-terminal portion of the protein has little effect on signalling activity in either embryos or imaginal discs. Increasing the levels of hh expression within its normal domain has no effect on dorsal cuticle pattern, arguing against a direct dependence on hh concentration. Basler K, Struhl C: Compartment boundaries and the control of Drosophila limb Pattem by hedgehog protein. Nature 1994, 368:208-214. Clones of cells constitutively expressing hh have no effect if located in the posterior compartment of imaginal discs, but in the anterior comparlment induce the generation of large duplications of structures; these effects on patterning are preceded by ectopic induction of wg and dpp expression, suggesting that hh acts via these two signalling proteins. 35. -,
36. •.
Capdevila J, Cuerrero I: Targeted expression of the signalling
molecule decapentaplegic induces pattern duplications and
growth alterations in Drosophila wings. EMBO ] 1994, 13:4459-4468. Eclopic expression of hh driven by GAL4 enhancer trap lines causes pattern duplications and ectopic activation of dpp. Expression of dpp driven by similar GAL4 enhancers has comparable effects on patterning, implying that hh acts exclusively via dpp in the wing. 37. ,*
Diaz-Benjumea FJ, Cohen SM: Cell-interaction between comParlments establishes the proximal-distal axis of Drosophila legs. Nature 1994, 372:175-179.
Expression of the Distallessgene in leg imaginal discs requires hh activity. Ectopic expression of dpp in the ventral compartment of the disc induces the same kinds of leg bifurcations generated by ectopic hh expression. Ingham PW, Fielz MI: Quantitative effects of hedBeheg and decapentaplegic activity on the Palteming of the Drosophila wing. Curr Biol 1995, 5:432-441, Ectopic expression of hh or dpp driven by the same GAL4 enhancer results in almost identical pattern defects. The effects of both genes vary with temperature, indicating a dose dependence of both proteins. Very high levels of hh activate ptc, but not dpp. 38. •.
39.
Kojima T, Michiue T, Orihara M, Saigo K: Induction of
•
a mirror-image duplication of anterior wing slructures by
localized hedge/rag expression in the anterior compartment of Drosophila melanogasterwing irnaginal discs. Gene 1994, 148:211-217. Ectopic expression of hh in the wing imaginal discs because of random insertion of a hh transgene results in wing duplications and inappropriate expression of dpp. 40. ••
Tabala T, Kornberg TB: Hedgehog is a signalling protein with a key role in patterning Drosophila imaginal discs. Cell 1994, 76:89-102. The hh protein is found associated with engraileoLexpressing cells and their neighbours, indicating that it is secreted. This distribution is modified by treatment with monesin or by a mutant thai blocks endocytosis. Overexpression of hh has similar effects to mutations in ptc in both the embryo and the imaginal discs. 4I.
Struhl G, Basler K: Or~lnlsing activity of wingless protein in Drosophila. Cett 1993, 72:527-540.
42. •
Wilder EL, Perrimon N: Dual functiom of wingless in the Drosophila leg imaginal disc. Development 1995, 121:477~188. High-level expression of wg by GAL4 enhancers results in partial ventralization of cells in the leg discs and increased proliferation of dorsal cells. These results argue agains~ a simple morphogen model for . wg action, 43.
Heberlein U, Wolff 1", Rubin GM: The TGFp homolng dpp and the segment polarity ~ hedgehog are required for propagation of a morpho~metic wave in the Drosophila retina. Ceil 1993, 75:913-926.
44.
Ma C, Zhou Y, Beachy PA, Moses K: The segment polarity gene hedb~hog is required for progression of the morphogenetic furrow in the developing Drosophila eye. Cell 1993, 75:927-938.
45. •,
Heberlein U, Singh CM, Luk AY, Donohoe TI; Growth and differentiation in the Drosophila eye coordinated by hedgehog. Nature 1995, 373:709-711. Clones of cells constitutively expressing hh in the Drosophila retinal field induce new foci of differentiation, presaged by the induction of dpp and hairy expression in cells surrounding the clone. 46.
Charite l, De Graaff W, Shen S, Deschamps J: Ectopic expressionof Hoxb-8 causes duplication of the ZPA in the forelimb and homeotie transformation of axial structures, Cell 1994, 78:589-601. Transgenic mouse embryos in which the Hox-b8 gene is misexpressed exhibit ectopic expression of Shh in the anterior mesenchyme of their hindlimbs and a concomitant duplication of digits.
•
47. •,
Yang YZ, Niswander L: Interaction between the s i ~ i n g molecules wnt7a and shh during vertebrate limb development dorsal signals regulate anteroposlerior Patterning. Cell 1995, 80:939-947. Recombinant FGF4 can maintain 5hh expression in proximal limb buds and induces BMP2, 8MP4 and Hox-c113expression. Removal of dorsal ectoderm results in down-regulation of 5hh transcription, an effect that can be reversed by expression of WntTa. 48. •.
Laufer E, Nelson CE, Johnson RL, Morgan BA, Tabin C: Sonic hedgehog and Fgf-4 act through a signalling cascade and feedback loop to integrate growth and pattemin8 of the developing limb bud. Cell 1994, 79:993-1003. Ectopic Shh expression in the chick limb induces BMP2 and Hox-dll and Hox-dl3 expression. The effects of Shh on these genes depend upon FGF4 activity; in addition, FGF4 expressed in Ihe AER controls the maintenance of Shh transcription. FGF4 expression in the AER also depends upon Shh.
497
498
Pattern formation and developmenta| mechanisms Niswander L, Jeffrey S, Martin GR, Tickle C: A positive feedback loop coordinates growtlh and PaUeming in the vertebrate limb. Nature 1994, 371:609-612. Removal of the AER leads to loss of Shh expression, an effect that can be reversed by recombinant FGF4. FGF4 and retinoic acid act in concert to induce Shh transcription.
interruptu5 Dominant of Drosophila. Genes Dev 1990,
49. •-
50. oe
Parr BA, McMahon AP: Dorsalizing signal wnt-7a required for normal polarity of ( I v and a-p axes of mouse limb. Nature 1995, 374:350-353. Mouse embryos lacking Wnt7a activity fail to maintain Shh expression in posterior limb mesenchyme. BMP2 is similarly affected. Francis PH, Richardson MK, Brickell PM, Tickle C: Bone moq~m~metlc proteins and a signalling pathway Ihat controls PatteminB in Ihe developing chick limb. Development 1994, 120:209-218. The germ encoding BMP2 is expressed in the posterior AER and in the posterior limb mesenchyme. Expression is ectopically induced by ZPA grafts, but recombinant BMP2 does not have polarizing activity.
4:1053-1067. 61. •
51usarski DC, Motzny CK, Holmgren R: Mutations thai aller the timing and paltern of cubitus interruptus gene expression in Drosophila melanosaster. Genetics 1995, 139:229-240. Presents an analysis of the spatial distribution of ci protein in Drosophila embryos and imaginal discs and the effects of various mutations on ils deployment and stability. 62.
51. •
52.
Hooper J, Scott MP: The Drosophila patched gene encodes a putative membrane protein required for segmental patteming. Cell 1909, 59:751-765.
53.
Nakano Y, Guerrero I, Hidalgo A, Taylor AM, Whittle IRS, Ingham PW: lhe Drosophila segment polarity gene patched encodes a protein with multiple potential membrane spanning clomains. Nature 1989, 341:508-51 3.
54.
Phillips
R,
Roberts I, Ingham
PW, Whinle
JRS; The
Drosophila se~eni polarity gene patched is involved in a position-si~alling mechanism in imaginal discs. Development 1990, 110;105-114. 55.
CapdeviLa J, Estracla MP, Sanchez Herrero E, Cuerrero I: • The Drosophila segmenl polarity gene Palched interacts with decaix,ntapleBic in wing developmenl. EMBO J 1994, 13:71-82. Viable mutations of the pro gene cause ectopic expression of dpp in the anterior compartments of wing imaginaI discs. 56.
Iogham I~V, Taylor AM, Nakano Y: Role of the Drosophila positional signalling. Nature 1991,
Patched Bene in 353:184-I 87. 57.
Forbes AJ, .Nakano Y, Taylor AM, Ingham PW: Genetic analysis of hedgehog signalling in the Drosophila embryo. Development 1993, SU1~1:115-124.
58. •
Hooper j: Distinct Pathways for aulocrlne and paracrine Wingless signalling in Drosophila embryos. Nature 1994, 372:461-464. Genetic eplstasis analyses suggest that fused, ci and smoothened act downstream of ptc to aclivate wg transcription. 59.
Prdat T, Th~rond P, Limbourg-Bouchon B, Pham A, Tricoire H, Busson O, Lamour-lsnard C: Segmental polarity in Dlosoph#a melanoBaste~ ~metic dissection of fused in a suppressor of fused bad(Bround reveals interaction with costal-2. Genetics 1993, 135:1047-1062.
60.
Orenic TV, Slusarski DC, Kroll KL, Holmgren RA: Cloning and characterization of the segment polarity gene cubitus
Prdat T, The%end P, Limbour~-Bouchon B, Pham A, Tricoire H, Busson D, Lamour-lsnard C: A putalive serine/threonine protein klnase encoded by the segment-polarity fused germ of Drosophila. Nature 1990, 347:87-89.
63. o-
Lepage T, Cohen SM, Diaz-Benjumea Fj, Parkhus! SM: Signal transduction by cAMP-depeodenl protein kinase A in Drosophila limb patterning. Nature 1995, 373:711-715. A viable allele of pka causes wing duplications similar to those induced by ectopic hh, preceded by ectopic dpp and ptc expression. Clones of cells lacking pka or ptc activity cause wing and leg duplications. 64. •-
Pan D, Rubin GM: cAMP-dependent protein klnase and hedgehog act antagonistically in regulating decapentapleBic transcription in Drosophila imaginal discs. Cell 1995, 80:543-552. Clones of cells lacking PKA activate dpp inappropriately in Ihe anterior compartment of the wing disc and anlerior to the morphogenelic furrow in the eye disc, inducing wing duplications or ectopic furrows. A dominant-negative PKA allele has a similar effect in the wing and suppresses the hh mutant phenotype in the eye. 65.
Jiang J, 5truhl G: Protein kinase A and hedgehog signaling in Drosophila limb development. Cell 1995, 80:563-572. Clones of cells lacking PKA activity in the anterior compartments of wings and legs cause pattern duplications similar to those induced by ectopic hh and induce ectopic expression of dpp and wg. Constitutively active pica, has no effect on normal dpp expression, nor does it suppressthe ptc mutant phenolype,
•.
66. •°
Li W, Ohlmeyer .IT, Lane ME, Kalderon D: Function of prolein kinase A in hedgehog signal transduction and Drosophila imaginal disc development. Cell 1995, 80:553-562. Similar analysis to [65"'], but these authors show that high levels of a constitutively active PKA can rescue the ptc mutant phenotype. 67. --
Strult DI, Wietsdorff V, Mlodzik M: Regulation of furrow progression in the Drosophila eye by cAMP-dependent protein kinase A. Nature 1995, 373:705-709. Clones of cells lacking PKA activity in the developing retina have similar effects to clones expressing hh in Ihat they initiate morphogenetic furrow migration ectopically. In contrast to those expressing hh, however, the PKA clones themselves initially activate dpp expression.
PW Ingharn, Molecular Embryology Laboratory, Imperial Cancer Research Fund, 44 Lincoln's Inn Fields, London WC2A 3PX, UK.