document

REVIEWS

Drosophila has a complicated life cycle that includes anembryonic phase, three larval periods (termedinstars), a pupal stage and finally adulthood. Unlike inmore primitive insects, these stages do not representdevelopmental forms that gradually evolve towardsthe adult stage, but are more like polymorphic formsof the same organism. During the pupal stage there isa complete metamorphosis in which nearly all larvalstructures disintegrate and are replaced by the struc-tures of the adult fly. The latter are differentiated bygroups of ‘imaginal’ cells that were present and hadproliferated in the larva but did not contribute to thelarval patterns. The best-characterized imaginal cellsare those of the IMAGINAL DISCS — sac-like structuresthat form the adult cuticular structures (the hard,external skeleton of arthropods). It is from these imag-inal discs that appendages arise, in a SEGMENT-specificmanner, along with the cells that form the trunk cor-responding to the segment. Different imaginal discshave their particular size and shape, and are namedafter the part of the body they form: wing disc, legdisc, eye-antennal disc, genital disc and so forth (FIG. 1).

It is not always obvious where to draw the borderbetween appendage and trunk. In the leg, for example,there is no clear morphological landmark separatingthe two parts, either in the differentiated structure orin the imaginal disc. Also, the trunk and appendagesdo not originate from different LINEAGES of cells: CLONAL

ANALYSES1,2 have shown that at least until late in devel-opment the descendants of a single cell can contributeto both structures. So what, exactly, defines anappendage? Some structures are not easily recognizedas appendages at first sight (FIG. 1). It is obvious that a

leg, a wing or an antenna are appendages, but to callthe mouthparts or the external analia appendagesrequires some elaboration. Ultimately, the distinctionbetween appendage and trunk rests on different genet-ic and developmental mechanisms operating in theappendages and in the trunk. Some of the geneticmechanisms involved in the development ofDrosophila appendages seem also to function in verte-brate limbs, suggesting that there is a universalappendage design common to all animals.

Homeobox genes and morphogensTo appreciate how appendages subdivide and howthey develop from the imaginal cells, it is necessary toconsider some general processes that are not specificto appendages but have critical roles in their develop-ment: the process of compartmentalization and thefunction of two groups of molecular players — HOMEOBOX

genes and morphogens. These molecules orchestratethe developmental signalling pathways that both dif-ferentiate limb from trunk, and make one limb differ-ent from another.

Compartments. Originally defined in the wing disc3,compartments are basic components of theDrosophila body plan. They are parts of the body thatoriginate from the same lineage of cells. From thebeginning, the disc primordia in all segments containtwo separate cell lineages, which form the anteriorand posterior compartments3–5 of each adult segment.This is a consequence of the function of the home-obox gene engrailed (en), which segregates anteriorand posterior compartment cells6. In the imaginal

HOW DROSOPHILA APPENDAGES DEVELOPGinés Morata

Just a glance at the body of the fruitfly Drosophila reveals that it has a main body part — thetrunk — and a number of specialized appendages such as legs, wings, halteres andantennae. How do Drosophila appendages develop, what gives each appendage its uniqueidentity, and what can the fruitfly teach us about appendage development in vertebrates?

NATURE REVIEWS | MOLECULAR CELL BIOLOGY VOLUME 2 | FEBRUARY 2001 | 89

Centro de BiologíaMolecular, ConsejoSuperior de InvestigacionesCientificas, UniversidadAutónoma de Madrid,Madrid 28049, Spain.e-mail:[email protected]

IMAGINAL DISCS

Sac-like structures present inthe larvae and composed ofcells destined to form thedifferent adult cuticularstructures. They are namedafter the adult part that theymake: for example, wing, leg,eye-antennal, genital.

SEGMENT

The insect body is divided alongthe anteroposterior axis into anumber of individual units orsegments. This organization isvisible in embryos, larvae andadult insects.

LINEAGE

The cellular ancestry of a givenstructure. Two structures sharethe same lineage if the progenyof a single cell (a clone) cancontribute to both. Otherwisethey have different lineages.

© 2001 Macmillan Magazines Ltd

90 | FEBRUARY 2001 | VOLUME 2 www.nature.com/reviews/molcellbio

R E V I E W S

protein can move across the anterioposterior (A/P)border to the anterior compartment (FIG. 2). In theanterior cells close to the border, Hh induces the activi-ty of the signalling genes wingless (wg, which encodes amember of the Wnt family of proteins) and/ordecapentaplegic (dpp, which encodes a transforminggrowth factor-β homologue), which are largelyresponsible for patterning the appendages (see below).

In addition, the activity of en in the posterior cellsconfers on them specific adhesion properties thatmake them immiscible with anterior cells. Expressionof en sets up the expression of a series of subsidiarygenes, some of which are presumed to encode specificcell-adhesion molecules. There is recent evidence thatthis function is mediated in part by the signalling pro-tein Hh10–12 in cells close to the A/P border, as indicatedin FIG. 2. This suggests that there might be an abruptdiscontinuity of affinities at the A/P border, a usefulproperty to keep the two compartments separate.

In the wing disc, and probably also in the HALTERE

disc, a second compartment subdivision appears dur-ing larval development1,3 separating the dorsal fromthe ventral regions of the pre-existing anterior andposterior compartments. This subdivision was ignoredfor a long time until it was found that the LIM-homeoboxgene apterous (ap) is expressed in the dorsal compart-ment as precisely defined by the dorsoventral (D/V)border13,14. ap, like en, has the features that are normal-ly associated with selector genes. It is involved in abinary switch that selects alternative pathways: the ONstate specifies dorsal and OFF specifies ventral identity.

ap activity is also important for the growth and pat-terning of the wing. It activates the gene fringe (fng),which modulates the ability of two ligands, Delta andSerrate, to activate their receptor Notch15,16. In turn,Notch induces vestigial (vg, which encodes a nuclearprotein presumed to be a transcription factor17) andwg activity at the D/V border. Wg acts as an organizerof wing development18,19, expanding the vg domainand activating other response genes. Notch activationby ap is also necessary for the maintenance of the D/Vlineage segregation; cells lacking either Notch or fngactivity fail to recognize the D/V boundary20,21, eventhough they contain normal ap activity. Thus, thereseems to be a selector-gene-driven signalling mecha-nism necessary for maintenance of the A/P and theD/V compartment borders.

The A/P and D/V subdivisions are of great develop-mental importance as they establish fixed developmen-tal borders — sources of the morphogenetic signalsHh, Dpp and Wg (see below) — that are largelyresponsible for patterning the appendages8,22.

Homeobox genes. The terms homeobox and HOMEOTIC

are often, erroneously, used interchangeably, so someclarification is required. Homeotic genes control thedevelopment of entire body regions along the A/P axis.Also, the borders of homeotic gene expression oftencorrespond to the borders of compartments23. InDrosophila, homeotic genes are arranged in two sepa-rate clusters of tightly linked genes: the Antennapedia

discs, en behaves as a classical SELECTOR GENE6,7; it speci-fies the identity of the posterior compartment cells byselecting between anterior or posterior developmentalprogrammes. The activity of en in the posterior com-partment cells triggers a genetic and signalling cascadethat patterns the appendage (reviewed in REFS 8,9). enactivates the hedgehog (hh) gene, which encodes asecreted protein. The effect of the Hh protein in theposterior compartment cells is blocked by en, but the

Figure 1 | Imaginal discs and the structures that developfrom them. a | A third-instar larva showing the position of allimaginal discs (colour coded according to the appendagesthat they will develop into). Photographs of wing, haltere andleg discs are shown. In the leg discs the presumptive regionscorresponding to trunk and appendage are indicated. b | Thebody of the adult Drosophila, indicating the trunk and thedifferent appendages. In addition to wings, halteres,antennae and legs, the mouthparts and analia can also beconsidered as appendages. Appendages are segment-specific structures: the second thoracic segment (II) developsthe wing in the dorsal and the second leg in the ventralregion. The third thoracic segment (III) develops the haltereand the third leg.

Wing

Labial

ClypealProthoracic

Wing HaltereLeg 3

Leg 1 Leg 2Genital

a

b

Wing Haltere Leg

Antenna

Mouthparts

Leg 1 Leg 2

Leg 3

Haltere Analia

III III

CLONAL ANALYSIS

A type of study based onexperiments in whichindividual cells are markedgenetically duringdevelopment. In the finalstructure the progeny of each ofthese cells will appear as a groupof marked cells — a clone.

HOMEOBOX

A 180-bp sequence present inmany developmental genes ofanimals and plants. It encodes aDNA-binding helix–turn–helixmotif, indicating thathomeobox-containing geneproducts function astranscription factors.

SELECTOR GENES

The concept of selector genes isintimately linked to that ofcompartments — body regionsof fixed lineage. Selector genesbecome activated preciselywithin compartments, wherethey ‘select’ specificdevelopmental routes. Classicalexamples of selector genes areengrailed, apterous and the BX-C genes.

HALTERE

A small dorsal appendage in thethird thoracic segment, thoughtto be involved in flight control.

LIM DOMAIN

A repeat of about 60 aminoacids containing cysteine andhistidine residues. It is thoughtto be involved inprotein–protein interactions.

HOMEOTIC MUTATIONS

A class of mutations in which agiven organ or a segmentdevelops in the same way as onenormally present in anotherpart of the body.



R E V I E W S

In the absence of homeotic gene activity, all segmentsdevelop the same ‘ground’ pattern, a mixture of tho-racic and cephalic pattern elements; no morphologicaldiversity is generated along the A/P body axis.

The ANT-C and BX-C genes encode transcriptionfactors that contain a 180-base-pair (bp) stretch ofsequence homology — the homeobox25,26 — coding fora helix–turn–helix DNA-binding motif, and are con-served throughout metazoans. In vertebrates, the num-ber of homeotic genes is greater than in flies owing tocascade duplications that occurred during vertebrateevolution, but homologues of Drosophila’s ANT-C andBX-C complexes form single clusters, known as the Hoxgene clusters. This name is now also used to describe theDrosophila ANT-C and BX-C genes.

Drosophila and vertebrates also have homeobox-containing genes outside the Hox gene cluster that arenot Hox genes (reviewed in REF. 27). There are morethan 100 homeobox sequences in the Drosophilagenome28. Of these, at least 25 represent true geneticfunctions as their mutations give rise to mutant phe-notypes29. Many are involved in development andsome, such as en, Distal-less (Dll), ap, extradenticle(exd) and homothorax (hth), are critical duringappendage development.

hth and exd deserve special mention as they areinvolved in several important developmental processes(see BOX 1). These two genes are highly conserved ininsects and vertebrates30,31. exd is transcribed andtranslated all over the body but it is regulated at thesubcellular level: the Exd protein is functional onlywhen it localizes to the cell nucleus32,33. Hth behaves asa positive regulator of exd by promoting the transportof Exd to the cell nucleus31. Conversely, in the absenceof exd activity, the Hth protein seems to be degraded34.Therefore hth and exd functions are always associatedand can be considered as a single functional unit.

Morphogens. The extracellular signalling moleculesHh, Dpp and Wg have critical functions in theprocesses of gene activation and pattern formation ofthe embryo, larvae and adult of Drosophila — and pre-sumably throughout the animal kingdom. These mol-ecules are morphogens (that is, carriers of positionalinformation) that are secreted from a fixed source,establishing a concentration gradient from the origin.During the development of the appendages thesources are positioned along the A/P and D/V com-partment boundaries. In their target cells, mor-phogens induce a cascade of events (reviewed in REFS

8,9) that, ultimately, are resolved in the nucleus of thereceiving cell by transcription factors that regulate theactivity of distinct response genes. The identity ofthese response genes often depends on the local con-centration of morphogen, which is a measure of thephysical distance from the source. Several develop-mentally important genes — many encoding tran-scription factors — have been identified that are acti-vated by Wg and/or Dpp. There is good evidence thatWg and Dpp function as long-range signals; that is,they influence the gene activity of cells located many

Complex (ANT-C, named after the Antp mutation,which converts antennae to legs), which contains fivehomeotic genes, and the Bithorax Complex (BX-C,named after a mutation that converts halteres towings), which contains three. The only homeotic genethat does not fall into one of these clusters is caudal(cad), which is responsible for analia development24.

Figure 2 | Generation of cell affinity differences betweenanterior and posterior compartment cells in the wingdisc. The activity of engrailed (en) in the posterior cellsinduces hedgehog (hh), but the Hh pathway (vertical arrows ingrey) cannot be activated in the posterior cells (probablybecause en represses cubitus interruptus (ci ), thetranscription factor that activates hh target genes in the cellnucleus). However, the Hh protein can travel across theanterioposterior (A/P) border to the anterior cells where the Hhpathway can be triggered. The end result of the Hhtransduction cascade is that cleavage of the Ci protein isprevented. The uncleaved form Ci(act) becomes active. Ci(rep)represents the cleaved form, which acts as a repressor. TheCi(act) protein is thought to activate anterior-specific cell-adhesion genes. In the posterior compartment, en activatesposterior-specific cell-adhesion genes, although it is possiblethat both en and ci induce different levels of expression in thesame cell-adhesion gene12. Hh can only travel a shortdistance so the Ci(act) protein is only produced in the anteriorcells close to the A/P border. This ensures that the maximaldifferences in cell affinities appear in the vicinity of the border.

Hh hh

Ci(rep)Ci(act)ci

Anterior-specificcell adhesion gene(s)

Posterior-specificcell adhesion gene(s)

en

Anterior compartment Posterior compartment

Box 1 | The diverse functions of Hth and Exd

In addition to their interaction with Hedgehog, Decapentaplegic and Winglesssignalling, the exd/hth genes have several important functions during development,some of which have not been considered in this review as they are not directly relevantto appendage development. A principal one is to confer in vivo specificity to Hox genesby acting as cofactors of Hox proteins: Hth and Exd modulate the affinity andspecificity of the Hox proteins to their DNA target sites. The vertebrate homologue ofExd, Pbx, also associates with Hox proteins and presumably has a similar role inconferring Hox specificity (reviewed in REF. 96).

hth and exd also have a Hox-like gene function to promote antennal development75;they participate in a binary switch that determines segment identity; the OFF stateresults in leg development, whereas the ON state promotes antennal development.Ectopic hth/exd activity can also induce antennal development, indicating thatHth/Exd can trigger a whole developmental programme.

hth and exd are also involved in head development by repressing eye formation in theventral head97, in the distinction of the dorsal and ventral identity in the adultabdomen, and in the positioning of thoracic bristles97,98.

BITHORAX COMPLEX (BX-C)

GENES

A group of three adjacenthomeotic genes responsible forthe identity of part of thethorax and the abdomen of thefly. Together with theantennapedia complex (ANT-C), it forms the Hox genecluster in Drosophila.



R E V I E W S

The initial leg disc is subdivided into central andperipheral domains (FIG. 3), with different patterns ofgene expression that correspond to appendage andbody trunk in adult flies. The cells in the centraldomain express Dll and those in the peripheral oneexpress escargot37 (esg), which encodes a zinc-finger-containing transcription factor41, and the two relatedhomeobox genes hth and exd (FIG. 3). This probablyreflects a genetic diversification that occurs during theearly phase of disc development. During early embryo-genesis, hth and exd are expressed highly and uniform-ly in the thorax, including the cells that originallyexpress Dll. Later, Dll represses hth transcription (andas a consequence exd becomes inactive), thus establish-ing a distinction between hth/exd-expressing and non-expressing cells. The existence of these two distinctdomains is also indicated by the observation that Dllmutant cells can contribute only to ventral body walland proximal leg structures42, that is, the hth/exd/esg-expressing domain. All these observations indicate thatby the time it is set apart from other embryonic cells,the leg disc shows a complex pattern of gene expres-sion: en, esg, Dll and hth are expressed in restricteddomains (FIG. 3).

The wing (and the haltere) disc originates from asubset of Dll-expressing cells that migrate dorsally.Three genes encoding putative transcription factorsbecome transcriptionally active in these cells43: vg, snail(sna) and esg. Sna and Esg are related proteins thatcontain similar zinc-finger domains41,44. It is likely thatthese three genes are originally activated independentlyas a response to an unidentified induction, but later vgexpression falls under the control of snail and esg43.Thereafter vg becomes a marker of wing and halteredevelopment. Indeed, vg activity is essential to formthe wing and haltere: in vg mutants these structures arenot formed17. The wing and haltere are organized moresimply than the leg disc at this stage (FIG. 3). For exam-ple, hth and exd, are uniformly expressed in all cells inthe early discs45,46.

Distinguishing trunk from appendageThe trunk and appendage regions do not derive fromdifferent lineages of cells but, from the beginning, theyshow different patterns of gene expression. How is thetrunk/appendage distinction achieved? The criticalelement seems to be antagonism between theHh/Wg/Dpp signalling pathways and hth/exd func-tion (BOX 1).

In the developing leg, the hh gene is activated by enin the posterior compartment cells47, but the secretedHh protein induces the activation of dpp in anteriordorsal cells and of wg in anterior ventral cells close tothe A/P boundary48. It is not clear how this differencearises, but wg is initially expressed in the ventral regionof the early disc34,49 and this asymmetry might bemaintained in later stages by the mutual antagonismbetween the Wg and Dpp pathways described below.The diffusion of Dpp and Wg from their origin inanterior and posterior directions organizes the leg pat-tern48. However, although Wg and Dpp are present in

cell diameters away from the origin, whereas Hh, atleast in the appendages, is a short-range morphogen.

Allocation of appendage cellsThe specification of the embryonic cells destined toform appendages occurs during embryogenesis. Theprimordia of the wing, haltere and leg imaginal discsoriginate in a lateral position of the embryo, at theintersection between two ‘stripes’ expressing the Wgand Dpp morphogens35. A critical event is the activa-tion of Dll36, which becomes a developmental andmolecular marker of embryonic primordia. Dllexpression is restricted to a lateral position in theembryo by the combined effects of Wg, Dpp and theDrosophila epidermal growth factor homologuespitz37. Studies on the expression of Dll homologues inmany animal groups indicate that it has a basic func-tion in the formation of body outgrowths throughoutthe animal kingdom38. In principle, all segments havethe potential to develop appendages: in the absence ofthe BX-C genes, which repress Dll transcription39, Dllis activated in an equivalent site in all abdominal seg-ments and appendage primordia are formed. This fitsnicely with the idea that insects derive from multi-legged ancestors40 that subsequently lost legs in theirabdominal segments.

Figure 3 | Gene activity in the leg and wing imaginal discprimordia in a late embryo (stage 14). Both primordiapossess engrailed (en) and hedgehog (hh) activity (black lines)restricted to the posterior compartments. The leg primordiumshows Distal-less (Dll ) activity (red) restricted to the centralregion and coincidental expression of homothorax(hth)/extradenticle (exd) and escargot (esg) (green) in a ring ofcells surrounding the Dll domain. The wing primordiumshows less diversity of gene expression. All cells expressvestigial (vg), hth/exd , esg and snail (sna) (blue). The dottedline from the dorsal to the ventral of the embryo separatesanterior and posterior compartments.

Wingprimordium

Legprimordium

vg & hth/exdesg/sna

Dll hth/exdesg

en/hh

Dorsal

Ventral

Anterior Posterior



R E V I E W S

which, in turn, repress hth/exd activity34,54,55. In theperipheral region, the levels of Dpp and Wg are lowerand cannot activate either Dll or dac — but they couldstill activate other genes of the Hh cascade — and sohth/exd remains active. Where they are active, hth/exdcompletely blocks the response of Dpp and Wg targetgenes, in effect functionally eliminating Hh signalling.The antagonism between exd function and Hh sig-nalling can be readily shown in experiments in whichhth/exd function is forced in the Dll domain, whichresults in the loss of the appendage51. This mutualantagonism segregates two domains (FIG. 4): a centralregion where Dpp and Wg response genes are activeand that will form the appendage, and a peripheralregion where appendage development is preventedand that retains trunk features.

In the wing disc the situation is more complicated.Whereas the disposition and function of Hh, Dpp andWg remain constant during leg development, this isnot the case during wing disc development, owing toadditional regulatory mechanisms. For example, wg isexpressed in the early wing disc in ventral anteriorcells, just like in the leg disc, but later it falls under thecontrol of Notch signalling56. This generates a newdomain of wg expression in the D/V boundary. Thissecondary tier of Wg regulation is critical for establish-ing vg expression and the development of theappendage. Additionally, Hh has two distinct (dpp-mediated and non-dpp-mediated) functions57,58, acomplication that does not occur in the leg.

However, in the wing disc, as in the leg disc, there isantagonism between hth/exd and the Dpp and Wg sig-nals45,46. In this case, the Dpp and Wg target gene thatis involved in the repression of hth/exd seems to be vg,which fulfils a function similar to that of Dll in the legdisc. The involvement of Wg in the trunk/appendagesegregation in the wing disc is illustrated by the muta-tion wg1, which duplicates the thorax at the expense ofthe wing. In this mutation the early wg function in thewing disc is defective56.

Subdividing the appendagesDifferential responses to Dpp and Wg determine fur-ther genetic diversification during appendage develop-ment. Whereas early discs only show abutting Hth andDll domains, in mature discs the non-hth part con-tains a region (FIG. 4) that does not express Dll butexpresses dac. This gene is activated by lower Dpp andWg levels than Dll, but is repressed by high signal lev-els53. The localization of the sources of Wg and Dppdictate that the central region of the disc will containDll activity whereas dac will be activated in a moreperipheral ring that overlaps with the Dll domain.This subdivides the appendage part of the leg intothree domains of gene expression along the proxi-modistal axis (FIG. 4). Thus the polarity along the proxi-modistal axis is a consequence of the localization ofWg and Dpp sources in the disc. The process of genet-ic diversification along the proximodistal axis is fur-ther refined by the activity of Dll and dac target genesthat define smaller subdomains. Several genes have

both the presumptive appendage and trunk regions,they seem to be functional only in the appendage: theloss of activity of hh, wg or dpp results in the loss of thedistal part of the leg (the tarsal segments, basitarsus,tibia and femur, FIG. 4), but the proximal segments(coxa and trochanter) and the body wall are unaffect-ed50,51. This indicates a subdivision of the leg into tworegions, an Hh-dependent and an Hh-independentone (FIG. 4). The Hh-dependent region is characterizedduring early disc development by the expression ofDll. In more advanced discs, another response gene,dachshund (dac), which encodes a nuclear protein nec-essary for eye and leg development52, is activated.Together, the expression domains of Dll and dac coverthe entire Hh-dependent region in mature legdiscs53,54. The Hh-independent region is geneticallycharacterized by the activity of hth and exd, whichbehave as a single functional unit.

How are the Hh-dependent and the hth/exddomains developmentally segregated? The currentmodel is as follows34,54: the topology of the disc and thelocalization of the sources of Dpp and Wg ensure thatcells in the central portion of the disc, which are des-tined to differentiate into distal structures, receivemoderate to high levels of both Dpp and Wg. Theselevels are required for expression of Dll and dac31

Figure 4 | Subdivision of the mature leg disc and the adult leg into distinct geneticsubdomains. a | The disposition of the hedgehog (hh) (yellow), decapentaplegic (dpp) (darkblue) and wingless (wg) (red) domains. The thick arrows illustrate that the Dpp and Wg signalsdiffuse from their source to organize the pattern of the anterior and posterior compartments. b | Depending on their concentration they may activate Distal-less (Dll ) alone (red), Dll anddachshund (dac) (purple) or dac alone (blue). These appendage subdomains are furthersubdivided into smaller ones by the activity of genes that lie downstream of Dll or dac. TheDll/dac-expressing region would be the genuine appendage and the homothorax(hth)/extradenticle (exd )-expressing region an expansion of the trunk. c | The differentsubdomains in an adult leg. The dotted line extending from the Dll to the hth/exd domain is toillustrate that in young discs Dll and hth/exd define complementary domains, but this is latermodified by the activation of dac.

en

hh

Dpp

Wg

Pleura

Coxa

Trochanter

Femur Tibia Tarsalsegments

hth/exd

hth/exd

dac

dac&Dll

Dll

Dll

dac

Posterior

Ventral

Dorsala

c

b



R E V I E W S

ence between the more central and the more peripher-al regions of the disc. Wg diffuses from the D/Vborder15,16 and presumably subdivides the wing alongthe proximodistal axis.

Specifying appendage identityDrosophila appendages, then, fall into two categorieswith broadly similar developmental mechanisms: theventral ones, exemplified by the leg, and the dorsalones, exemplified by the wing (FIG. 5). However, super-imposed on these are mechanisms to discriminate theidentity of the different ventral or dorsal appendages(FIG. 5). Two main components seem to act in combina-tion: a segment-specific property provided by the Hoxgenes and a general ventral or dorsal property providedby Dpp and Wg response genes such as Dll or vg.

The identity of segments along the A/P body axis isestablished by the Hox genes (reviewed in REF. 21). Asparts of thoracic and cephalic segments, theappendages are also specified by the Hox genes. Thelegs provide the best example74: the identity of the firstleg is specified by the Hox gene Sex combs reduced, thesecond leg is specified by Antp and the third leg by Ubx(FIG. 5). The identity of the antennal segment is speci-fied by hth/exd 75 (BOX 1). Finally, the other appendage-like structures, the analia, are specified by the Hox genecaudal (cad)24.

Dll has a critical role in the specification of ventralappendages. It seems to confer a general ‘ventralappendage’ property, which is qualified combinatorial-ly by Hox genes (FIG. 5). Dll activity is necessary for thegrowth and identity of ventral appendages: legs, anten-nae, mouthparts and analia2,35,41,76. Moreover, ectopicexpression of Dll in dorsal discs induces the formationof distinct ventral appendage structures depending onthe pattern of Hox gene expression2.

The specification of the identity of the dorsalappendages seems to follow similar rules. vg seems tohave a principal role, homologous to that of Dll in theventral appendages. Its activity is necessary for the for-mation of wings and halteres and its ectopic expressioninduces wing or haltere tissue depending on the seg-ment77,78. The fact that it induces wing development inthe head, and fore- and midleg segments, but halteredevelopment in the hindleg segment suggests that, likeDll, vg specifies distinct appendage identity in combi-nation with Hox genes.

It is less clear how Hox genes contribute to the spec-ification of dorsal appendages. The classical view79–81 isthat the development of the haltere is specified by Ubx,whereas wing identity is a default situation that resultsfrom no Hox gene activity. Some genetic data supportthis view. For example, in the homeotic mutationContrabithorax the wing is transformed into a haltere,and the transformation is accompanied by inappropri-ate expression of Ubx in the wing cells82. Several Ubxtarget genes have been identified that are expressed inthe wing but not in the haltere83. These genes are prob-ably responsible for some of the differences betweenwing and haltere. However, some results question thespecificity of Ubx in haltere development84. Using new

been identified that are associated with individualsegments of the leg. These genes (ap, aristaless, Bar,bric a brac, spineless)59–65 are initially expressed in over-lapping domains but later establish exclusive domainsthrough transcriptional repression63. Interestingly, allthese genes also encode transcription factors, suggest-ing the existence of several layers of transcriptionalregulation during leg development. One outcome ofthis process of genetic subdivision is the localized acti-vation of Notch ligands Delta and Serrate, which leadsto rings of restricted activation of Notch that result inthe formation of joints between leg segments66.

Other response genes encoding transcription fac-tors such as optomotor-blind (omb) and H15 areinduced specifically by either Dpp or Wg and con-tribute to the identity of the dorsal and ventral regionsrespectively67,68. The Wg and Dpp pathways antagonizeeach other68–70 and these antagonistic interactions areprobably responsible for the developmental segrega-tion of dorsal and ventral GENETIC DOMAINS in the leg.

In the wing disc, diffusion of the Hh-dependentlong-range Dpp morphogen from the vicinity of theA/P border patterns the anterior and posterior com-partments71,72. As a result, the genes omb and spalt(both of which encode transcription factors) are acti-vated in different domains and presumably affect dif-ferent sets of target genes. Hh also has a short-rangedpp-independent patterning function in the anteriorcompartment57,58. These functions define distinctgenetic domains in the anterior wing. Moreover, theantagonistic function of brinker73, a transcriptionalrepressor of dpp target genes, also establishes a differ-

Figure 5 | Specification of appendage identity by the combinatorial contributions of Hoxgenes and Distal-less and vestigial. In the ventral appendages, the general activity of Distal-less (Dll ) is qualified in each segment by the resident Hox gene (proboscipedia (pb), homothorax(hth)/extradenticle (exd ), Sex combs reduced (Scr ), Antennapedia complex (Antp), Ultrabithorax(Ubx) and caudal (cad )), to determine the identity of the appendage. For the two dorsalappendages — wings and halteres — vestigial (vg) and Ubx seem to specify halteredevelopment, but no Hox gene activity is found in the wing, which may be specified solely by vg.

Dll

vg

pb

hth/exd

Scr

Antp

Ubx

cad

vg target genes

Ubx

Antenna

Mouthparts

Leg 1 Leg 2

Leg 3

Haltere Analia

III III

Wing

GENETIC DOMAINS

A general term referring to theregion of the body where aparticular gene is expressed.They are usually visualizedusing specific antibody, DNAor RNA probes.



R E V I E W S

evidence that Hh signalling is required in mouselimbs, for these do not develop properly in Sonichedgehog (Shh) knockouts89. The mode of action ofShh in chick limb development also resembles the sit-uation in Drosophila: it acts as a short-range activatorof bone morphogenetic protein (Bmp2), a vertebratehomologue of dpp, and much of the effect of Shh ismediated by Bmp2 and other Bmps90.

Similarly, the role of Dll in appendage formationseems to be highly conserved. Studies done in manyanimal groups38 have shown that Dll homologues aregenerally expressed in body outgrowths, be thesearthropod appendages, tube feet of sea urchins ormouse and chicken limb buds. These observations indi-cate that Dll might derive from an ancient homeoboxgene involved in the production of body wall out-growths in the entire animal kingdom38. Thus, the keygenes involved in distinguishing trunk from appendagein Drosophila, hth/exd and Dll, have been highly con-served during evolution. The patterning signals Hh, Wgand Dpp also have comparable functions in differentanimals. Together these observations indicate that thereis a universal mechanism to form a limb, shared by allanimal groups38.

Future goalsThe appendages represent specialized forms of devel-opment, likely to derive from primitive body wall out-growths. The formation of these outgrowths requiresthe identification and developmental isolation ofgroups of cells in the trunk, and the triggering of theiradditional proliferation. During this process, Hh sig-nalling seems to function through the activation ofspecific targets such as Dll or vg. These genes are notonly needed for appendage growth, but also to con-tribute to appendage identity, acting in combinationwith the Hox genes. Because the Hox genes, Dll and vgencode transcription factors, it is clear that betterunderstanding of appendage development will requirethe identification of new target genes of both Hoxgenes and Hh/Dpp/Wg signalling. Moreover, it isexpected that there must be molecular interactions

methods to target gene expression85 it has been foundthat the wing-to-haltere transformation can beinduced not only by Ubx, but also by the other BX-Cgenes abdominal-A and Abdominal-B, the normalfunction of which is to specify abdominal identities86.This indicates a lack of specificity of Hox genes in thedetermination of appendage identity. This might bedue to the fact that the appendages do not havehth/exd function, which is necessary for Hox genespecificity. One possibility is that the differencesbetween wings and halteres are mediated by Dpp andWg response genes (in addition to vg). Several of thesetarget genes, including the transcription factors spalt,achaete-scute, and serum response factor, are active inthe wing but not in the haltere83 and are likely to con-tribute towards making a haltere different from awing. Their response in the haltere is blocked by Ubx,which is the BX-C gene that is normally expressed inthe haltere, but the other BX-C genes might also havethe same ability. In this view the differences betweenwings and halteres would be due to a partial blockagein the Hh/Dpp/Wg signalling pathway in halteres.

A general principle for making an appendage?A principal feature of the organization of Drosophilaappendages is the distinction between the distal part(the appendage proper) that contains full activity ofthe Hh/Dpp/Wg cascade, and the proximal part thatcontains hth/exd activity (see TABLE 1 for simalaritiesand differences in the various appendages). The lattercan be considered as an expansion of the trunk.

Recent work comparing Drosophila appendageswith mouse and chicken limbs54,87,88 has shown astriking degree of conservation of this configuration:the vertebrate homologues of hth and exd — meisand Pbx1 respectively — are functional in the proxi-mal but not in the distal limb. Even the subcellularregulation of exd by hth in Drosophila is alsoobserved for Pbx1 and meis in mouse limb develop-ment. In both Drosophila and chicken, ectopicexpression of hth or meis in the distal componentblocks limb development87. Furthermore, there is

Table 1 | Comparing the development of different appendages

Antennae (compared Analia (compared Mouthparts (compared Halteres (comparedwith leg) with leg) with leg) with wing)

Similarities • Similar disposition of the Hh, • hh, wg and dpp expression patterns resemble • Labial disc expresses DII • Disposition of the Hh,Dpp and Wg signals76 those in the leg disc103,104 in early development76 Dpp and Wg signals is

• Dll is activated as a response to • The Dll domain (distal) is not segregated • Proboscipedia mutations very similar in the wing Wg and Dpp signals by cell lineage from the proximal domain, transform the mouthparts and haltere discs

• Legs and antennae can be where Dll expression is repressed into legs105 • Homeotic transformed one into another by (characterized, in analia, by transformations homeotic mutations75,97–100 brachyenteron expression)24 between halteres and

• Can be homeotically transformed into wings can be inducedantennae by ectopically expressing hth75

Differences • Anteroposterior compartment • Proximal region does not express hth/exd, • Some Dpp target border is established much later so restriction of Wg and Dpp signalling is genes in the wing are than in leg101 not achieved by antagonism from hth/exd22 not expressed

• Areas of hth/exd and DII • No (or less) wgexpression overlap75; hth/exd expression in the D/Vand DII pathways seem to border in the posteriorco-operate in antennal haltere83

specification101



R E V I E W S

1. Garcia-Bellido, A., Ripoll, P. & Morata, G. Developmentalcompartmentalization in the dorsal mesothoracic disc ofDrosophila. Dev. Biol. 48, 132–147 (1976).

2. Gorfinkiel, N., Morata, G. & Guerrero, I. The homeoboxgene Distal-less induces ventral appendage developmentin Drosophila. Genes Dev. 11, 2259–2271 (1997).

3. Garcia-Bellido, A., Ripoll, P. & Morata, G. Developmentalcompartmentalization of the wing disk of Drosophila.Nature New Biol. 245, 251–253 (1973).First demonstration of the existence ofcompartments in Drosophila.

4. Steiner, E. Establishment of compartments in thedeveloping imaginal leg discs of Drosophila. Wilhelm RouxArch. Dev. Biol. 180, 31–46 (1976).

5. Lawrence, P. A. & Morata, G. The early development ofmesothoracic compartments in Drosophila. An analysis ofcell lineage and fate mapping and an assessment ofmethods. Dev. Biol. 56, 40–51 (1977).

6. Morata, G. & Lawrence, P. A. Control of compartmentdevelopment by the engrailed gene in Drosophila. Nature255, 614–617 (1975).Provides evidence for the role of engrailed inmaintaining the A/P compartment border in the wingdisc.

7. Garcia-Bellido, A. in Cell Patterning, Ciba Found. Symp.Vol. 29 (ed. S. Brenner) 161–182 (Associated ScientificPublishers, New York, 1975).

8. Lawrence, P. A. & Struhl, G. Morphogens, compartments,and pattern: lessons from Drosophila? Cell 85, 951–961(1996).A lucid review on patterning mechanisms inDrosophila relating the classical ideas ofcompartments and homeotic genes with thefunction of the Hh, Wg and Dpp morphogens.

9. Strigini, M. & Cohen, S. Formation of morphogen gradientsin the Drosophila wing. Semin. Cell Dev. Biol. 10, 335–344(1999).

10. Rodriguez, I. & Basler, K. Control of compartmental affinityboundaries by hedgehog. Nature 389, 614–618 (1997).

11. Blair, S. S. & Ralston, A. Smoothened-mediatedHedgehog signalling is required for the maintenance of theanterior–posterior lineage restriction in the developing wingof Drosophila. Development 124, 4053–4063 (1997).References 10 and 11 provide evidence that the roleof engrailed in keeping anterior and posterior cellsseparate is mediated by the signalling genehedgehog.

12. Dahman, C. & Basler, K. Opposing transcriptional outputsof Hedgehog signaling and engrailed controlcompartmental cell sorting at the Drosophila A/P

boundary. Cell 100, 411–422 (2000).13. Diaz-Benjumea, F. J. & Cohen, S. M. Interaction between

dorsal and ventral cells in the imaginal disc directs wingdevelopment in Drosophila. Cell 75, 741–752 (1993).

14. Blair, S. S. Mechanisms of compartment formation:evidence that non-proliferating cells do not play a criticalrole in defining the D/V lineage restriction in the developingwing of Drosophila. Development 119, 339–351 (1993).References 13 and 14 provide evidence for the roleof the gene apterous in establishing the D/Vcompartment border in the wing disc and in actingas a selector gene.

15. Panin, V. M., Papayannoupolos, V., Wilson, R. & Irvine, K. D.Fringe modulates Notch-ligand interactions. Nature 387,908–912 (1997).

16. Fleming, R. J., Gu, Y. & Hukriede, N. A. Serrate-mediatedactivation of Notch is specifically blocked by the product ofthe gene fringe in the dorsal compartment of theDrosophila wing imaginal disc. Development 124,2973–2981 (1997).

17. Williams, J. A., Bell, J. B. & Carroll, S. B. Control ofDrosophila wing and haltere development by the nuclearvestigial gene product. Genes Dev. 5, 2481–2495 (1991).

18. Zecca, M., Basler, K. & Struhl, G. Direct and long rangeaction of a Wingless morphogen gradient. Cell 87,833–844 (1996).

19. Neumann, C. & Cohen, S. M. A hierarchy of cross-regulation involving wingless, vestigial and cut organizesthe dorsal/ventral axis of the Drosophila wing.Development 122, 3477–3485 (1996).

20. Rauskolb, C., Correia, T. & Irvine, K. D. Fringe-dependentseparation of dorsal and ventral cells in the Drosophilawing. Nature 401, 476–480 (1999).

21. Micchelli, C. A. & Blair, S. S. Dorsoventral lineagerestriction in wing imaginal discs requires Notch. Nature401, 473–476 (1999).

22. Dahmann, C. & Basler, K. Compartment boundaries: atthe edge of development. Trends Genet. 15, 320–326(1999).

23. Lawrence, P. A. & Morata, G. Homeobox genes: theirfunction in Drosophila segmentation and patternformation. Cell 78, 181–189 (1994).

24. Moreno, E. & Morata, G. caudal is the Hox gene thatspecifies the most posterior Drosophila segment. Nature400, 873–877 (1999).

25. McGinnis, W., Levine, M. S., Hafen, E., Kuroiwa, A. &Gehring, W. J. A conserved DNA sequence in homeoticgenes of the Drosophila Antennapedia and bithoraxcomplexes. Nature 308, 428–433 (1984).

26. Scott, M. P. & Weiner, A. J. Structural relationships among

genes that control development: sequence homologybetween Antennapedia, Ultrabithorax and fushi tarazu lociof Drosophila. Proc. Natl Acad. Sci. USA 81, 4115–4119(1984).References 25 and 26 report the discovery of thehomeobox, arguing for a common origin of homeoticgenes. They also provided a molecular marker ofhomeotic genes that eventually led to their discoveryin other animal species.

27. Burglin, T. R. in Biodiversity and Evolution (eds Arai, R.,Kato, M. & Doi, Y.) 291–336 (The National ScienceMuseum Foundation, Tokyo, 1995).

28. Adams, M. D. et al. The genome sequence of Drosophilamelanogaster. Science 287, 2185–2195 (2000).

29. Duboule, D. (ed.) Guidebook to the Homeobox Genes(Sambrook & Tooze Publications, Oxford Univ. Press,Oxford, 1994).

30. Rauskolb, C., Peifer, M. & Wieschaus, E. extradenticle, aregulator of homeotic gene activity, is a homolog of thehomeobox-containing human proto-oncogene pbx1. Cell74, 1–20 (1993).

31. Rieckhof, G., Casares, F., Ryoo, H. D., Abu-Shaar, M. &Mann, R. S. Nuclear translocation of Extradenticle requireshomothorax, which encodes an Extradenticle-relatedhomeodomain protein. Cell 91, 171–183 (1997).

32. Mann, R. & Abu-Shaar, M. Nuclear import of thehomeodomain protein Extradenticle in response toDecapentaplegic and Wingless signalling. Nature 383,630–633 (1996).

33. Aspland, S. & White, R. A. H. Nucleocytoplasmiclocalisation of extradenticle protein is spatially regulatedthroughout development of Drosophila. Development 124,741–747 (1997).

34. Abu-Shaar, M. & Mann, R. S. Generation of multipleantagonistic domains along the proximodistal axis duringDrosophila leg development. Development 125,3821–3830 (1998).

35. Cohen, B., Simcox, A. & Cohen, S. M. Allocation ofthoracic imaginal primordia in the Drosophila embryo.Development 117, 597–608 (1993).

36. Cohen, S. M., Bronner, G., Kuttner, F., Jurgens, G. &Jackle, H. Distal-less encodes a homeodomain proteinrequired for limb development in Drosophila. Nature 338,432–434 (1989).

37. Goto, S. & Hayashi, S. Specification of embryonic limbprimordium by graded activity of Decapentaplegic.Development 124, 125–132 (1997).

38. Panganiban, G. et al. The origin and evolution of animalappendages. Proc. Natl Acad. Sci. USA 94, 5162–5166(1997).

between the Hox gene and the signalling cascades, asillustrated by the gene spalt, which is a target gene ofboth Dpp and the Hox gene Ubx83. Another aspectworth mentioning concerns the function of the long-range signals Dpp and Wg. These signals are presentboth in the adult trunk and in the appendages, but verylittle is known about their function in the trunk, where,for example, no target gene has yet been identified.

Another issue that remains to be explored is themolecular basis of the antagonistic roles of hth/exdand the Hh, Dpp and Wg pathways in growth control.The observation in Drosophila and vertebrates thatforcing ectopic hth/exd expression in the domains ofHh, Dpp and Wg blocks appendage development45,46,87

suggests that the proliferative response to the signals issuppressed by hth/exd. How this is achieved remainsto be elucidated.

Studies in the mechanisms of appendage develop-ment will be of interest, not only because of the generalconservation of the process, but also because of theirimportance for understanding the general mechanismscontrolling growth and proliferation in higher animals.Mutations of human homologues of exd and hth (Pbx1and meis) give rise to leukaemias91–93 in humans and

mutant forms of genes of the Hh signalling pathway94,95

are involved in tumorigenesis. Fully understandingappendage development in Drosophila therefore hasimplications that go beyond developmental and evolu-tionary biology, to biomedicine.

Links

DATABASE LINKS engrailed | hedgehog | wingless |decapentaplegic | LIM-homeobox | apterous | fringe |Delta | Serrate | Notch | vestigial | AntennapediaComplex | Bithorax Complex | caudal | helix–turn–helix| Distal-less | extradenticle | homothorax | Wnt family |Spitz | escargot | zinc-finger | dachshund | aristaless | Bar | bric a brac | spineless | H15 | spalt | brinker | Sexcombs reduced | Antp | Ubx | abdominal-A | Abdominal-B | achaete-scute | serum response factor | meis | Pbx1FURTHER INFORMATION Interactive Fly: imaginal discsand tissues | Interactive Fly: the Drosophila leg |Interactive Fly: wing | Homeobox Genes Database ENCYCLOPEDIA OF LIFE SCIENCES Vertebrate embryo:limb development | Evolutionary developmentalbiology: Hox gene evolution | Drosophila embryo: cellsignalling and segmental patterning



R E V I E W S

39. Vachon, G., Cohen, B., Pfeifle, C., McGuffin, M. E., Botas,J. & Cohen, S. M. Homeotic genes of the bithoraxcomplex repress limb development in the abdomen of theDrosophila embryo through the target gene Distal-less.Cell 71, 437–450 (1992).

40. Lewis, E. B. A gene complex controlling segmentation inDrosophila. Nature 276, 565–570 (1978).

41. Whiteley, M., Noguchi, P. D., Sensabaugh, S. M.,Odenwald, W. F. & Kassis, J. A. The Drosophila geneescargot encodes a zinc finger motif found in snail-relatedgenes. Mech. Dev. 36, 117–127 (1992).

42. Cohen, S. M. & Jurgens, G. Proximal-distal patternformation in Drosophila: cell autonomous requirement forDistal-less gene activity in limb development. EMBO J. 8,2045–2055 (1989).

43. Fuse, N., Hirose, S. & Hayashi, S. Determination of wingcell fate by the escargot and snail genes in Drosophila.Development 122, 1059–1067 (1996).

44. Boulay, J. L., Dennefeld, C. & Alberga, A. The Drosophiladevelopmental gene snail encodes a protein with nucleicacid binding fingers. Nature 330, 395–398 (1987).

45. Casares, F. & Mann, R. S. A dual role for homothorax ininhibiting wing blade development and specifying proximalwing identities in Drosophila. Development 127,1499–1508 (2000).

46. Azpiazu, N. & Morata, G. Function and regulation ofhomothorax in the wing imaginal disc of Drosophila.Development 127, 2685–2693 (2000).

47. Tabata, T. & Kornberg, T. Hedgehog is a signalling proteinwith a key role in patterning Drosophila imaginal discs. Cell76, 89–102 (1994).

48. Basler, K. & Struhl, G. Compartment boundaries and thecontrol of Drosophila limb pattern by hedgehog protein.Nature 368, 208–214 (1994).A classic study demonstrating the patterning role ofthe Hh/Wg/Dpp pathway in appendagedevelopment.

49. Campbell, G., Weaver, T. & Tomlinson, A. Axis specificationin the developing Drosophila appendage: the role ofwingless, decapentaplegic, and the homeobox genearistaless. Cell 74, 1113–1123 (1993).

50. Diaz-Benjumea, F. J., Cohen, B. & Cohen, S. M. Cellinteractions between compartments establish theproximal-distal axis of Drosophila legs. Nature 372,175–179 (1994).

51. Gonzalez-Crespo, S. & Morata, G. Genetic evidence forthe subdivision of the arthropod limb into coxopodite andtelopodite. Development 122, 3921–3930 (1996).

52. Mardon, G., Solomon, N. M. & Rubin, G. M. dachshundencodes a nuclear protein required for normal eye and legdevelopment in Drosophila. Development 120, 3473–3486(1994).

53. Lecuit, T. & Cohen, S. M. Proximal-distal axis formation inthe Drosophila leg. Nature 388, 139–145 (1997).Wg and Dpp activate different target genesdepending on the local concentration of bothmorphogens along the proximodistal axis.

54. Gonzalez-Crespo, S., Torres, M., Martinez-A, C., Mann, R.S. & Morata, G. Antagonism between extradenticlefunction and Hedgehog signalling in the developing limb.Nature 394, 196–200 (1998).Provides evidence both in Drosophila and the mouselimb that the formation of the appendage requiresthe suppression of extradenticle function.Suppression is achieved by preventing the nucleartransport of the Exd protein.

55. Wu, J. & Cohen, S. M. Proximodistal axis formation in theDrosophila leg: subdivision into proximal and distaldomains by Homothorax and Distal-less. Development126, 109–117 (1999).

56. Ng, M., Diaz-Benjumea, F., Vincent, J. P., Wu, J. & Cohen,S. M. Specification of the wing by localised expression ofwingless protein. Nature 381, 316–318 (1996).

57. Mullor, J. L., Calleja, M., Capdevila, J. & Guerrero, I.Hedgehog activity, independent of Decapentaplegic,participates in wing disc patterning. Development 124,1227–1237 (1997).

58. Strigini, M. & Cohen, S. M. A Hedhehog activity gradientcontributes to AP axial patterning of the Drosophila wing.Development 124, 4697–4705 (1997).

59. Cohen, B., McGuffin, M. E., Pfeifle, C., Segal, D. & Cohen,S. M. apterous, a gene required for imaginal discdevelopment in Drosophila encodes a member of the LIMfamily of developmental regulatory proteins. Genes Dev. 6,715–729 (1992).

60. Scheitz, K., Spielman, S. & Noll, M. Molecular genetics of

aristaless, a prd-type homeobox involved in themorphogenesis of proximal and distal pattern elements ina subset of appendages in Drosophila. Genes Dev. 7,114–129 (1993).

61. Campbell, G., Weaver, T. & Tomlinson, A. Axis specificationin the developing Drosophila appendage: the role ofwingless, decapentaplegic, and the homeobox genearistaless. Cell 74, 1113–1123 (1993).

62. Campbell, G. & Tomlinson, A. The roles of the homeoboxgenes aristaless and Distal-less in patterning the legs andwings of Drosophila. Development 125, 4483–4493(1998).

63. Kojima, T., Sato, M. & Saigo, K. Formation andspecification of distal leg segments in Drosophila by dualBar homeobox genes, BarH1 and BarH2. Development127, 769–778 (2000).

64. Godt, D., Couderc, J. L., Cramton, S. E. & Laski, F. A.Pattern formation of limbs of Drosophila: bric a brac inboth a gradient and a wave-like pattern and is required forspecification and proper segmentation of the tarsus.Development 119, 799–812 (1993).

65. Duncan, D. M., Burgess, E. A. & Duncan, I. Control ofdistal antennal identity and tarsal development inDrosophila by spineless-aristapedia, a homolog of themammalian dioxin receptor. Genes Dev. 12, 1290–1303(1998).

66. de Celis, J. F., Tyler, D. M., de Celis, J. & Bray, S. J. Notchsignalling mediates segmentation of the Drosophila leg.Development 125, 4617–4626 (1998).

67. Grimm, S. & Pflugfelder, G. O. Control of the geneoptomotor-blind in Drosophila wing development bydecapentaplegic and wingless. Science 271, 1601–1604(1996).

68. Brook, W. J. & Cohen, S. M. Antagonistic interactionsbetween Wingless and Decapentaplegic responsible fordorsal-ventral patterning in the Drosophila leg. Science273, 1373–1377 (1996).

69. Jiang, J. & Struhl, G. Complementary and mutuallyexclusive activities of Decapentaplegic and Winglessorganise axial patterning during Drosophila legdevelopment. Cell 86, 401–409 (1996).

70. Penton, A. & Hoffmann, F. M. Decapentaplegic restrictsthe domain of wingless during Drosophila limb patterning.Nature 382, 162–165 (1996).

71. Nellen, D., Bruke, R., Struhl, G. & Basler, K. Direct andlong-range action of a DPP morphogen gradient. Cell 85,357–368 (1996).

72. Lecuit, T. et al. Two distinct mechanisms for long-rangepatterning by Decapentaplegic in the Drosophila wing.Nature 381, 387–393 (1996).References 71 and 72 provide direct evidence thatDpp acts at a distance from its origin and in a dose-dependent manner.

73. Jazwinska, A., Kirov, N., Wieschaus, E., Roth, S. &Rushlow, C. The Drosophila gene brinker reveals a novelmechanism of Dpp target activation. Cell 96, 563–573(1999).

74. Struhl, G. Genes controlling segmental specification in theDrosophila thorax. Proc. Natl Acad. Sci. USA 79,7380–7384 (1982).A classic paper showing that Hox genes act as acombinatorial code.

75. Casares, F. & Mann, R. S. Control of antennal versus legdevelopment in Drosophila. Nature 392, 723–726 (1998).

76. Cohen, S. M. in The Development of Drosophilamelanogaster Vol. 2 (eds Bate, M. & Martinez-Arias, A.)747–842 (Cold Spring Harbor Laboratory Press, NewYork, 1993).

77. Kim, J. et al. Integration of positional signals and regulationof wing formation and identity by Drosophila vestigial gene.Nature 382, 133–138 (1996).

78. Maves, L. & Schubiger, G. A molecular basis fortransdetermination in Drosophila imaginal discs:interactions between wingless and decapentaplegicsignaling. Development 125, 115–124 (1998).

79. Lewis, E. B. Genes and developmental pathways. Am.Zool. 3, 33–56 (1963).

80. Lewis, E. B. in The Role of Chromosomes in Development(ed. Locke, M.) 231–252 (Academic, New York, 1964).

81. Morata, G. & Garcia-Bellido, A. Developmental analysis ofsome mutants of the bithorax system of the Drosophila.Wilhelm Roux Arch. Dev. Biol. 179, 125–143 (1976).

82. White, R. A. H. & Akam, M. E. Contrabithorax mutationscause inappropriate expression of Ultrabithorax productsin Drosophila. Nature 318, 567–569 (1985).

83. Weatherbee, S. D., Halder, G., Kim, J., Hudson, A. &

Carroll, S. Ultrabithorax regulates genes at several levels ofthe wing-patterning hierarchy to shape the development ofthe Drosophila haltere. Genes Dev. 12, 1474–1482 (1998).

84. Casares, F., Calleja, M. & Sanchez-Herrero, E. Functionalsimilarity in appendage specification by the Ultrabithoraxand abdominal-A Drosophila Hox genes. EMBO J. 15,3934–3942 (1996).A striking result showing that the three BX-C genesUbx, abd-A and Abd-B can induce halteredevelopment in the wing. It argues for a rethinking ofBX-C gene function in the appendages.

85. Brand, A. & Perrimon, N. Targeted gene expression as ameans of altering cell fates and generating dominantphenotypes. Development 118, 401–415 (1993).A very useful method to express a cloned gene inspecific tissues or body regions.

86. Sanchez-Herrero, E., Vernos, I., Marco, R. & Morata, G.Genetic organization of Drosophila bithorax complex.Nature 313, 108–113 (1985).

87. Mercader, N. et al. Conserved regulation of the proximo-distal limb axis development by Meis/Hth. Nature 402,425–429 (1999).

88. Capdevila, J., Tsukui, T., Rodriguez-Esteban, C.,Zappavigna, V. & Izpizua-Belmonte, J. C. Mol. Cell 4,839–849 (1999).

89. Chiang, C. et al. Cyclopia and defective axial patterning inmice lacking Sonic Hedgehog gene function. Nature 383,407–413 (1996).

90. Drosospolou, G. et al. A model for anteroposteriorpatterning of the vertebrate limb based on the sequentiallong- and short-range Shh signalling and Bmp signalling.Development 127, 1337–1348 (2000).

91. Kamps, M. P., Murre, C., Sun, X. & Baltimore, D. A newhomeobox gene contributes to the DNA binding domain ofthe t(1;19) translocation protein in pre-B ALL. Cell 60,547–555 (1990).

92. Nourse, J. et al. Chromosomal translocation t(1;19) resultsof the synthesis of a homeobox fusion mRNA that codesfor a potential chimeric transcription factor. Cell 60,535–545 (1990).

93. Moskow, J., Bulrich, F., Juebner, K., Daar, I. & Buchberg, A.Meis1, a PBX1-related homeobox gene involved inmyeloid leukemia in BXH-2 mice. Mol. Cell. Biol. 15,5434–5443 (1995).

94. Hahn, H. et al. Mutations of the human homolog ofDrosophila patched in the nevoid basal cell carcinomasyndrome. Cell 85, 841–851 (1996).

95. Jonhson, R. L. et al. Human homolog of patched, acandidate gene for the basal cell nevus syndrome. Science272, 1668–1672 (1996).

96. Mann, R. S. & Chan, S. -K. Extra specificity fromextradenticle: the partnership between HOX and exd/pbxhomeodomain proteins. Trends Genet. 12, 258–262(1996).

97. Gonzalez-Crespo, S. & Morata, G. Control of Drosophilaadult pattern by extradenticle. Development 121,2117–2125 (1995).

98. Rauskolb, C., Smith, K., Peifer, M. & Wieschaus, E.extradenticle determines segmental identities throughoutdevelopment. Development 121, 3663–3671 (1995).

99. Struhl, G. A homeotic mutation transforming leg toantenna in Drosophila. Nature 292, 635–638 (1981).

100. Schneuwly, S., Klemenz, R. & Gehring, W. J. Redesigningthe body plan of Drosophila by ectopic expression of thehomeotic gene Antennapedia. Nature 325, 816–828(1987).

101. Morata, G. & Lawrence, P. A. Anterior and posteriorcompartments in the head of Drosophila. Nature 274,473–474 (1978).

102. Dong, P. D., Chu, J. & Panganiban, G. Co-expression ofthe homeobox genes Distal-less and homothoraxdetermines Drosophila antennal identity. Development127, 209–216 (2000).

103. Chen, E. & Baker, B. S. Compartmental organization of theDrosophila genital imaginal discs. Development 124,205–218 (1996).

104. Sanchez, L., Casares, F., Gorkinkiel, N. & Guerrero, I. Thegenital disc of Drosophila melanogaster II. Role of thegenes hedgehog, decapentaplegic and wingless. Dev.Genes Evol. 207, 229–241 (1997).

105. Lindley, D. & Zimm, G. The Genome of Drosophilamelanogaster (Academic, San Diego, California, 1992).

AcknowledgementsI thank E. Sanchez-Herrero, J. F. de Celis, M. Calleja and D.Duboule for discussions and their comments on the manuscript.


document

Documents

Transcript of document