The evolving of Hox arthropods - Development

Development 1994 Supplement, 209-215 (1994)Printed in Great Britain @ The Company of Biologists Limited 1994

The evolving role of Hox genes in arthropods

Michael Akam, Michalis Averof, James Castelli-Gair, Rachel Daw€s, Francesco Falciani and David FerrierWellcome/CRC lnstitute and Department of Genetics, Tennis Court Road, Cambridge, CB2 1QR, UK

SUMMARY

Comparisons between Hox genes in different arthropodssuggest that the diversity of Antennapedia-class homeoticgenes present in modern insects had already arisen beforethe divergence of insects and crustaceans, probably duringthe Cambrian. Hox gene duplications are thereforeunlikely to have occurred concomitantly with trunk 'segment diversification in the lineage leading to insects.Available data suggest that domains of homeotic geneexpression are also generally conserved among insects, butchanges in Hox gene regulation may have played a signifi-cant role in segment diversification. Differences that havebeen documented alter specific aspects of Hox gene regu-Iation within segments and correlate with alterations insegment morphology rather than overt homeotic transfor-mations.

The Drosophila Hox cluster contains several homeoboxgenes that are not homeotic genes - bicoidrfushi-tnrazu and

209

zen. The role of these genes during early development hasbeen studied in some detail. It appears to be withoutparallel among the vertebrate Hox genes. No wellconserved homologues of these genes have been found inother taxa, suggesting that they are evolving faster than thehomeotic genes. Relatively divergent Antp-class genesisolated from other insects are probably homologues offushi-tarazu, but these are almost unrecognisable outside oftheir homeodomains, and have accumulated approximately10 times as many changes in their homeodomains as havehomeotic genes in the same comparisons. They showconserved patterns of expression in the nervous system, butnot during early development.

Key words: homeobox, homeotic gene, tagmosis, insect developmentevolution

INTRODUCTION

In Drosophila, the differences between the trunk segments arecontrolled by the homeotic genes of the Antennapedia andBithorax gene complexes (Lewis, 1978; S6nchez-Herrero etal., 1985; Kaufman et a1., 1990). This paper is concerned withthe extent to which changes affecting these Hox genes mayhave contributed to the evolution of body form and develop-mental mechanism in the arthropods, and in particular theinsects. We begin by considering the timing of gene duplica-tions that gave rise to the Insect Hox cluster*, in relation to theorigin of invertebrate phyla and arthropod classes. Then weconsider how changes in the regulation of Hox genes may havecontributed to the diversification of the insects. Finally, wepresent evidence that the selective constraints on some genesin the Hox clusters appear to have been relaxed in some insectlineages, allowing them to diverge relatively rapidly, both interms of sequence and role in development.

*Here we shall use the term Hox genes to refer generically to the homeobox genescontained within the arthropod homeotic gene clusters, as well as their vertebrate homo-logues. The term Hom/Hox, never euphonic, is now redundant. The epithet Hox shouldno longer be applied to other classes of homeobox genes (Scott, 1992). We include withinthe 'Hox' designation the non-homeotic genes of the DrosophilaHox clusters (bcd, ftz,zen). The final section of this manuscript provides some justification for this usage. Weuse the term Antennapedia-class (Antp-class) Hox genes more restrictively, to refer tothat subset of the Hox genes that have a homeobox conforming to the Antp-classconsensus defined by Scott et al (1989). This includes all the central genes of the Hoxclusters, but excludes the labial, proboscipedia, Deformed and Abdominal-B classes (ver-tebrate paralogy groups l-4, 9-13).

HOX GENE DUPLICATIONS AND THE ORIGIN OFARTHROPOD BODY PLANS

An explicit hypothesis relating Hox genes to evolutionarychange was articulated by E. B. Lewis in the openingparagraph of his classic paper (Lewis , 1978):

"During the evolution of the fly, two major groups of genesmust have evolved: 'leg suppressing' genes which removedlegs from abdominal segments of millipede like ancestors,followed by 'haltere-promoting' genes which suppressed thesecond pair of wings of four-winged ancestors...During evolution a tandem ar-ray of redundant genes pre-sumably diversified by mutation to produce this complex."(the Bithorax Complex).

In essence, Lewis proposed that the evolutionary history ofthe homeotic gene clusters would be related to the evolutionof morphology in the arthropod lineage, with increasingcomplexity of the gene cluster underlying increasing com-plexity of the body plan. No doubt he had in mind a schemeproposed by Snodgrass (1935), where the archetypal insect isderived from a superficially myriapod-like ancestor in whichall the segments between the mouthparts and the analsegment were similar. In the parlance of arthropod morphol-ogists, such an animal is homonomous; all the segments ofthe trunk form a single tagma, or body region. This contrastswith the regional specialisation of segment form and function

210 M. Akam and others

that constitutes tagmosis, and characterises most arthropodclasses.

Snodgrass' scheme assumes that in the proposedinsect/myriapod lineage, a homonomous trunk is the primitivestate, and tagmosis a derived character. At first sight thisconflicts with the observation that most representatives of theother major group of mandibulate arthropods - the Crustacea

- also display clear trunk tagmosis, and typically have distinctthorax and abdomen (Schram, 1986). However, conventionalphylogenies argue that these analogous states have beenacquired independently in the two lineages (Brusca andBrusca, 1990): homonomous Crustacea exist (the remipedes),and are assumed to reflect the condition ancestral to this wholeclass (Fig. 1).

It is not obvious why a homonomous, myriapod-like animalwould need the array of homeotic genes that specify trunksegment diversity in Drosophila, most particularly the set ofdistinct Antp-class Hox genes that specify the thoracic andabdominal segments to be different from one another (Anten-napedia (Antp), Ultrabithorax (Ubx) and abdominal-A (abd-A)). Indeed, we previously argued that the duplication leadingto these particular genes may have occuffed relatively late, inassociation with the origin of the thorax/abdomen distinctionin insects (Akam et al., 1988).

One test of this model is to compare the structure andexpression of Antp-class Hox genes in crustaceans and insects.If the mechanism to specify thorax and abdomen arose inde-pendently in these two lineages, we should see signs of thiswhen we examine the molecular machinery. The first step inthis comparison has been achieved (Averof and Akam, 1993).In a study of Hox gene sequences in the branchiopod crus-tacean Artemia, we have shown that specific homologues ofAntp, Ubx and abd-A, as well as Sex Combs Reduced (Scr) andDeformed @fA are present in Crustacea. Other, moredivergent Hox gene classes (labial, proboscipedia, Abdominal-B) are known to be of even more ancient origin (Fig. 2;Schubert et al., 1993). We conclude that the existing diversityof homeotic genes in insects (or at least Drosophila) derivesfrom gene duplications that occurred prior to the insect/crus-tacean split.

Arthropods recognisable as crustaceans are well docu-mented among Cambrian fossils (Robison and Kaesler, 1987),including probable Branchiopods from the lower Cambrian,550 Myr before present (Butterfield, 1994). In contrast, theearliest insect fossils appear significantly later, during theDevonian, 400 Myr before present (Jeram et al., 1990). Theseinsects are likely to derive from an early crustacean-mandibu-late clade, in which case the final diversification of the Antp-class Hox genes may have occurred within this arthropodanlineage. However, at least one of the relevant gene duplicationevents occurred earlier, before the separation of annelid andarthropod lineages: leeches possess a Ubx/abd-A type gene(Lox2), distinct from other Antp class sequences (Lox I, Lox5;Wysocka-Diller et &1., 1989; Nardelli-Haefliger andShankland, 1992).

These findings argue against the strong version of the Lewishypothesis - that the acquisition of trunk tagmosis in insectswas directly correlated with the origin of new Hox genes.However, they do not rule out the possibility that significantchanges in the regulation of pre-existing Hox genes may haveplayed a crucial part in this transition. Further work will be

W

Crustaceanswffi

Crustaceansw_aF

Fig. 1. 'Conventional' and 'alternative' hypotheses concerning theorigin of trunk tagmosis in relation to arthropod phylogeny.(A) Conventional view depicting the common ancestor of insects andcrustaceans with a homonomous trunk. Thorax/abdomen tagmosis isassumed to have arisen independently in the two lineages.Myriapods, assumed to constitute a sister group to the insects, andremipedes within the Crustacea, retain a homonomous trunkreflecting the primitive condition for this whole lineage (B) Analternative scenario in which insects and Crustacea derive from acommon ancestor that already displays well differentiated trunksegments (perhaps controlled by the differential expression ofmultiple Antp-class Hox genes, which we infer to have been presentin this ancestor). Those myriapods (some or all) that constitute a

sister group to the insects, and remipedes among the Crustacea,would then show a secondarily simplified trunk. Note that themonophyly of the myriapods is uncertain, and the position of somemyriapods within this lineage has recently been challenged:Sequence data (Turbeville et al., l99l; Ballard et al., 1992; Averof,1994) and patterns of neural development (Whitington et al., l99l)suggest that many myriapods may form an outgroup to the wholelineage depicted here.

required to establish if and how the Antp class Hox genes are

used to define trunk segment identities in Crustacea, and inhomonomous arthropods (e.g. myriapods).

Even before these data are available, it is perhaps worthquestioning the validity of one assumption on which the Lewismodel is based, for the argument illuminates one contributionthat developmental data may make to evolutionary discussions.This is the assumption that homonomy is always primitive. Asdevelopmental geneticists, we would emphasise that there is an

enorrnous asymmetry between the invention of a complexfeature (e.g. the transition from homonomy to heteronomy) andits loss (heteronomy+homonomy). An animal that has no

Insects

@

Insects#*

ffi

tIt123

rll4 (6-8)(e-13)

IIIIIIIINIt z 3 4 Scr

fi:*on, ubx abA

[l[,

Hox genes in arthropods 211

ChOrdateS (Amphioxus, human, mouse)

t 2 3 4 5 6 7 8 9 l0 il t2 13

Annelids (Helobdeua, Hirudo)

TTTIIIMTI-;i t- I Lx5 Lx2

Crustaceans (Ailemia)

trtrtrIIIIINTDfd Scr Hx I Ant Ubx abA(4)

InSeCtS (o"osophila, Tribolium, Schistocerca)

lab pb zen Dfd Scr ftz Ant Ubx abA AbB( I ) (2) (3?) (4) Dax (9- 13)

IIIt23

Fig.2.The minimal inferred complexity of Hox gene clusters in the lineage leading to the insects. Panels on the right summarise the diversityof Hox genes described from insects, Crustacea, annelids (Class Hirudinea, leeches) and chordates(Amphioxus, and several vertebrates; a

single 'complete' chordate cluster is illustrated, based on Amphioxus data; Garcia-Fernandez and Holland, 1994). Genes characterised by atleast the full homeobox sequence are shown as coloured boxes. Open boxes in the annelid and crustacean panels are used for genes that areassumed to exist in these taxa, but have not yet been characterised adequately. Identical colours are used for genes that can be assigned uniquehomologues (orthologues) within the clusters of different taxa. Similar but non-identical colours are used for genes where orthologyrelationships are unclear. Boxes are joined only where linkage has been established in some member of the taxon. The assumed phylogeneticrelationships of these four taxa are not disputed. Panels on the left show the minimal complexity of the Hox cluster in each of the stem groupsthat is implied by considering this phylogeny together with the assumed orthology relationships. A single Antp-class ancestor is shown for thebasal stem group (though Scr and the genes of vertebrate paralogy group 5 may derive from a second ancestral Antp-class sequence present atthe base of this lineage. The origin of these genes is not clear; Btirglin,1994). DistinctAntp-like and Ubx/abd-A-like genes are shared byarthropods and annelids. All homeotic gene classes are shared by insects and Crustacea. The inclusion of a group 3 homologue in the arthropodlineages is based on unpublished data (see text), and on the isolation of a homeobox PCR fragment that has residues diagnostic for class 3 Hoxgenes from Limulus, a chelicerate arthropod (Cartwright et al., 1993). Gene symbols have been abbreviated as follows: Lx, Lox (leech Hox)genes; Ant, Antp; abd, Ubx/abd-Arelated; ab-A, abd-A; Ab-B, Abd-B. (References: Chordates: Biirglin, 1994; Garcia-Fernandezand Holland,1994. Annelids: Loxl, Aisemberg and Macagno, 1994); Inx2,Wysocka-Diller et al., 1989; Nardelli-Haefliger and Shankland, 1992; Lox 5,6,7 ,

Shankland et al., 1991. Crustaceans: Averof and Akam, 1993; see also legend to Fig.4. Insects: For a summary of Drosophila gene sequences,

see Biirglin, 1994; for other insects, see legend to Fig. 4.)

Fig. 3. Segment specification involves precise spatial regulation ofhomeotic genes in Drosophila. A region of the Drosophila embryo atextended germ band stage (maxillary to second abdominal segments)has been stained for protein produced by the Ubx gene (black). Atthis stage, homeotic genes are regulated in parasegmentaldomains.Ubx plays no role in segment specification in parasegment 4(PS4; posterior first thoracic segment/anterior second). Nearubiquitous expression of Ubx characterises parasegment 6 (PS6,T3p/A I a), and can be shown to specify PS6 by experimentalmanipulation (Gonzalez-Reyes et al., 1990; Mann and Hogness,1990). Spatially regulated expression of Ubx characterisesparasegment 5 (PS5, T2ptT3A), and is necessary for its correctspecification. In this preparation, counter staining for the product of adistal-less reporter gene (Vachon et al., 1992) marks the developingleg discs of the thoracic segments (brown). Scale, l0 pm.


developmental mechanism to make segments different mustinvent both the mechanism to specify the differences, and alsothe machinery to elaborate distinct, adaptive segment types.Loss of heteronomy can be achieved by what seem to us to bemuch simpler processes - by turning off the mechanism thatspecifies segments to be different, or simply by eliminatingsome tagma from the adult body plan altogether.

We do not seriously doubt the hypothesis that the firstmetamerically segmented ancestors of the arthropods werehomonomous - though there is at present no way of knowingwhere to place that ancestor on the phylogenetic tree. We doquestion whether homonomy is generally a primitive traitamong extant arthropods. In an environment where it is usefulfor all segments to have legs, this adaptive change may be rel-atively easy to achieve. A single Bx-C mutation will make a'myriapod' of a fly (albeit not a viable one; Lewis, 1978), butwe cannot conceive of the reverse transition occurring in a

single step.An analogous situation pertains to the origin and loss of

insect wings. It takes many genes to make a wing (Williamsand Carroll , 1993) yet, in Drosophila, we know of severalsingle gene mutations that will suppress wing formation in anotherwise viable fly (wingless, apterous, vestigial; Lindsleyand Zimm, 1992). Among the insects, where the phylogeneticcontext is much less ambiguous, wings are believed to haveevolved only once; we do not hesitate to invoke convergencerepeatedly to explain the origin of wingless taxa at many phy-logenetic levels among the pterygotes, from whole orders (e.g.

fleas, lice) to individual species (Kristensen, I99l).If heteronomy is hard to achieve de novo, but useful and

easy to modify once invented, we should expect to see two verydifferent modes of evolution: clades that generate a diversityof taxa which retain essentially homonomous body plans, andclades that exploit heteronomy to filI many niches. Trilobitesmay exemplify the first mode; despite their abundance andtaxonomic diversity throughout the Palaeozorc, virtually alltrilobite families retain largely homonomous trunk segments(Eldridge, 1977). The Crustacea present a very differentpicture, with a Palaeozoic radiation giving rise to a hugediversity of body plans (Cisne, 1974; Schram, 1986; Briggs et&1., 1992).

HOMEOTIC GENE REGULATION ANDEVOLUTIONARY CHANGE WITHIN INSECTS

Hox gene duplications may have been a necessary precondi-tion for segment diversification within the insects, but theresults summarised above suggest that they were not animmediate driving force for its appearance. There is someevidence, however, that changes in the regulation of the Hoxgenes have occurred within insects, and may have contributedto morphological evolution. These changes are to be seen

against a broadly conserved role for many Hox genes, at leastin so far as can be inferred from the phenotypes of mutationsin the Hox clusters of beetles and moths (Beeman et a1., 1993;Tazima, 1964; Booker and Truman, 1989; Ueno et al. , 1992),and from the patterns of expression of Hox genes in these andother insects (see below).

Most data are available for abd-A. In each of four specieswhere the expression of abd-A has been examined

(Drosophila, Karch et al., 1990; Macias et aI.,1990; Manduca,Nagy et al. , I99I; Tribolium, Stuart et al. , 1993; Schistocerca,Tear et al., 1990), the gene is expressed throughout much ofthe abdomen, with no evidence for expression in the head orthorax. Moreover, in each case, the gene shows an abruptanterior boundary of expression within the first abdominalsegment. Where tested, this lies at the parasegmental boundarydefined by engrailed expression, suggesting that, at least in thiscase, the precise co-ordination of segmentation and homeoticgene expression is maintained in insects as diverse as

Orthopteru and Diptera (Tear et al., 1990).In some other cases, the domains of homeotic gene

expression are broadly conserved, but the precise limits ofexpression are not the same as those in Drosophila. Forexample, the Abd-B genes in Tribolium and Schistocercaappear to be expressed in the epidermis of only the mostposterior abdominal segments (from A8p back; Kelsh et al.,

1993; J. He & R. Denell, personal communication), whereasin Drosophila Abd-B is also expressed in the more anteriorabdominal segments, A5p-A8a. This anterior expression of thegene, which is necessary for normal development, is regulatedquite independently of that in the more posterior abdomen: Itderives from a separate promoter and is activated throughdifferent regulatory elements (Boulet et a1., 199I). Altered reg-ulation of the Abd-B gene may have played a role in theevolution of the modified abdomen that is characteristic of thehigher Diptera.

Comparisons between Schistocerca and Drosophila revealone clear case of 'molecular heterochrony'. In Drosophila,abd-A is normally never expressed in the ninth or moreposterior abdominal segments (parasegment l4).All of thesesegments fuse with A8 to form the 'terminalia'. In Schisto-cerca, abd-A is expressed at high levels in A.9 and A10 duringearly embryogenesis, but this expression is rapidly lost, at thesame time that Abd-B expression extends anteriorly (Tear et&1., 1990; Kelsh et al., 1994). It seems most likely that this isdue to the direct repression of the abd-A gene by Abd-Bprotein, for this same interaction has been demonstrated inDrosophila. In Abd-B mutants of Drosophila,, abd-Aexpression extends posteriorly into ,A.9 (Macias et al., 1990),and this segment now develops characteristics of the moreanterior abdomen, including a denticle belt (S6nchez-Herreroet &1., 1985). Thus a regulatory interaction that occurs as atemporal sequence after segment formation in Schistocerca,appears to be 'hard wired' at an earlier stage in Drosophiladevelopment, and is only apparent in mutants.

The two cases above suggest that alterations in the timingor extent of homeotic gene expression may be used to modifysegment development even though they do not result instriking 'homeotic' transformations. However, there ate anumber of observations in the entomological literature sug-gestive of homeotic transformations that have been fixed in thecourse of evolution. One that we are unlikely to be able to studyin detail concerns the Monura - an extinct group of insects thatflourished in the Carboniferous (Kukalova-Peck, 1991). Theseinsects, in common with a number of other early groups, hadreduced but well-formed legs (with terminal claws) on eachabdominal segment A1 to A10: thorax-abdomen tagmosis wasnot complete. However, in contrast with all other knowninsects, in the Monura, these legs were also present on theterminal abdominal segment, in place of the filiform cerci that

are the usual appendages of A1 1. The presumed sister group(Paleodictyoptera) and outgroups (Thysanura, Symphyla) tothe Monura had cerci, so it is possiUte that a homeotic trans-formation of cerci to leglets occurred in this particular order.Alternatively, of course, this 'simple' character state mayrepresent a retained primitive feature that has been lost severaltimes in other groups.

The case of the Strepsipteran haltere is more amenable toexperimental analysis. The Strepsiptera ate a small order ofparasitic insects. The adult females are neotenrc, retaining larvalmorphology. It is the males that are interesting for our pu{poses(Whiting and Wheeler, 1994). They have only a single pair ofwings, but these-are developed from the third thoracic segment,not the second, as in the Diptera. The dorsal appendages of thesecond thoracic segment are reduced to halteres, which in manydetails resemble the halteres derived from the third thoracicsegment of Diptera. Until recently, this resemblance has beenascribed to convergence, for the Strepsiptera have beenassumed to be only distantly related to the Diptera. RibosomalDNA sequence data now provides strong evidence that theStrepsiptera are in fact the sister group to the Diptera. Thisprompted Whiting and Wheeler to suggest that the Strepsipteranhaltere is indeed homologous to the haltere of Diptera, and thatthis developmental pathway has been adopted in T2 of theStrepsiptera, presumably by a change in the segmental regula-tion of Ubx. This hypothesis is open to test, using a recentlydescribed antibody that cross-reacts with Ubx protein in a widerange of insect species (Kelsh et al., 1994).

However, we should be cautious in making such predictions.This dramatic modification of adult segment morphology maydepend entirely on the regulation of genes acting downstreamof the homeotics. Even if it does involve modification ofhomeotic gene expression, it may involve only their regulationin a subset of tissues at a particular stage in development (e.g.in the developing imaginal discs). It is not necessary to assumethat evolutionary change affecting the homeotic genes mustinvolve major, saltatory change in the morphology of wholesegments. It may much more frequently proceed by incremen-tal changes affecting levels of expression, or by the alterationof those enhancer elements within their promoters that controlactivity in particular structures within a segment.

This view is supported by careful studies of the role of onehomeotic gene, Ubx, rn Drosophila (J. Castelli-Gair and M.Akam, unpublished data). The Ubx gene is responsible fordefining the identities of two quite different (para)segments,parasegment 5 (T3) and parasegment 6 (A1). This differentialfunction does not appear to depend on the segment-specificexpression of different protein variants (Busturia et al., 1990).Rather it seems to involve the precise temporal and spatial reg-ulation of the gene in parasegment 5 (Fig. 3). Examples of howchanges in the precise pattern of within-segment regulationmay have contributed to insect segment diversity are providedby the work of Carroll et al. (this volume). They show, forexample, that the loss of expression of abd-A specificallywithin some limb buds may allow the development of larvalabdominal prolegs in Lepidoptera.

HOX GENES THAT GOT AWAY

Up to this point, we have taken for granted that Hox genes


cloned from any insect can be identified without question asthe speciflc homologue (orthologue) of one of the Hox clustergenes defined rn Drosophila. In general, this is the case. Withfew exceptions, the homeodomains are almost identical, andgene-specific sequences around the homeodomain andextending upstream to the hexapeptide motif (Biirglin, 1994)are sufficiently similar to make identification unambiguous.

This is true, however, only for homologues of the 'homeotic'genes within the Drosophila clusters (labial, proboscipedia,Dfd, Scr, Antp, Ubx, abd-A and Abd-B). Well-conserved homo-logues have not been isolated for the several homeobox genesof the Antp complex that have 'anomalous' roles in develop-ment. There are four such genes rn Drosophila melanogaster

- bicoid, fushi-taruzu and the two zen genes.Bicoid is only expressed maternally. It encodes a gradient

morphogen in the early (syncytial) embryo (Driever andNiisslein-Volhard, 1988). Fushi-taruzu (ftz) is a 'pair-rule' seg-mentation gene (Kaufman et al., 1990). It is re-used duringneurogenesis to specify the identity of certain neurons in eachtrunk segment (Doe et al., 1988). Zenl is required very earlyfor the specification of the dorsal pattern elements in theembryo (Ferguson and Anderson, 1991). None of these rolesis analogous to that of the typical homeotic genes, or to thoseof the Hox genes in vertebrates, so far as we know. Theexistence of these genes within the insect Hox clusters has beensomething of an enigma, but isolation of several 'divergent'Hox genes from other insects throws some light on their origin.

The best studied of these are putative homologues of fushi-taraztt, isolated from Tribolium (Brown et al.,1994) and Schis-tocerca (Dawes et al., 1994). Neither of these genes showsextensive similarity to ftz (or any other Hox gene) outside ofthe homeobox, and even within the homeobox they are muchmore divergent than is typical for the other Hox genes (Fig. a).However, both of these genes are closely linked to the Hoxcluster, and both show a pattern of expression in the nervoussystem very similar to that of the fushi-tarazu gene inDrosophila.

Outside of the homeodomain, short conserved peptides areshared uniquely between Drosophila ftz and the Tribolium ftz-like gene, and between the Tribolium and Schistocerca genes.In the latter case, the conserved region is centred on a YPWMsequence just upstream of the homeodomain. This so-calledhexapeptide motif is characteristic of the homeotic Hox genes,and generally well conserved between distant species, oftenshowing extended similarity between insect and vertebrategenes of the same class. Its presence in the Schistocerca andTribolium genes strongly suggests that, on structural grounds,these should be regarded as 'typical' Hox genes. However,note that no such motif is conserved in the Drosophila ftz gene.

In contrast to the conserved expression of ftz in the nervoussystem, these putativ e ftz homologues show different patternsof expression during early development. Drosophila ftz isexpressed in parasegmental stripes at two-segment intervals,and is needed for the formation of alternate segment bound-aries (Lawrence and Johnston, 1 989). ln Tribolium, the ftzgeneis expressed in less well resolved stripes, and is apparently notneeded for making the correct number of segments (Stuart etdI., I99l; Brown et al., 1994). The Schistocerca gene isexpressed in a posterior domain of the early embryo, but neverin stripes (Dawes et al., 1994); its function in early develop-ment is unknown.


A homeotic

O ftz-related

+ bicoid

amlnoaciddiffsin

Hbox

VS

D. mel.

A

AA A

AA

A

Fig. 4. Relative divergence of homeotic and other Hox clusterhomeodomains within Arthropods. Species for which Hox gene

sequences are available have been grouped according to approximatephylogenetic distance from Drosophila melanogaster. Roughestimates of divergence times are given below, based onimmunological similarity (within Diptera; Beverley and Wilson,1984)) or on the first appearance of other taxonomic groups in thefossil record (Kukalova-Peck, 1991 ). Other Drosophila subgenera,(40-60 My): D. hydeiftz (Jost and D. Maier, personalcommunication; EMBL accessio n X7 9494); other Dipteranfamlly/suborder, (100-200My): Musca bicoid (n.b.6 changes in 44residues of homeodomain sequenced (Sommer and Tautz, I99l);Aedes abd-A (Eggleston, 1992); other Endopterygote insects(250My): Bombyx (Lepidoptera) Ubx, abd-A (Ueno et al. , 1992);Manduca (Lepidoptera) abd-A (Nagy et al. , I99I); Apis(Hymenoptera) Antp, Scr, Dfd (Fleig et al., 1988; Walldorf et al.,1989); Tribolium (Coleoptera) abd-A (Stuart et al. , 1993), ftz (Brownet al. , 1994). Schistocerca (Orthoptera, approx. 300My) abd-A (Tearet aI.,1990), Abd-B (Kelsh et al., 1993), Scr (Akam et al., 1988),Dax (ftz-related gene (Dawes et al., 199D; crustacean: Artemia(Anostraca,550 My; Averof and Akam, 1993). The assignment ofthe divergent Artemia gene AfiIxl to theftz homology group istentative, but is consistent with the fast divergence of these genes.

For comparison, the divergence between some annelid or vertebrateHox sequences and their closest Drosophila match are shown on theright.

We believe that all of these ftz-related genes derive from asingle Antp-class Hox gene that acquired novel functions inarthropod development. They are not involved in the mainte-nance of segmental identity in the mesoderm or the epidermis.They may have a role in very early patterning of the embryoin all insects, but this role appears to be different in differentinsects. In Schistocerca and Triboliuffi, whatever constraintsmaintain the integrity of the YPWM motif and its flankingsequences are retained, but the function of ftz tn Drosophilaappears no longer to impose the same sequence requirement.

Because the changes in the homeobox are confined to regionsother than those that contact DNA, we suggest that it may bethe lower complexity of protein-protein interactions that allows

ftz to diverge more rapidly than the canonical homeotic genes.The other 'non-homeotic' genes of the Drosophila Ant-C are

in some ways analogous to ftz. Their homeoboxes are verydivergent, with respect to all other non-Dipteran homeoboxgenes; they lack any recognisable hexapeptide motif; compar-ison between Dipteran species shows that the bicoid homeoboxis diverging rapidly (Sommer and Tautz, l99I; Schroder andSander , 1993). Activity capable of rescuing bicoid mutants canbe recovered from the eggs of other cyclorapphan Diptera, butwas not detected in honeybee, or even lower Diptera (Schroderand Sander, 1993). Searches for bicoid genes in other insectshave been unsuccessful (I. Dawson, S. Frenk and M. A.,unpublished results).

We think that these genes may be derived, Iike ftz, from Hoxgenes which, in the lineage leading to Drosophila, haveescaped from the conservative selection that characteriseshomeotic genes. We have recently isolated from Schistocercaa cDNA that lends some support to this hypothesis. It encodesa homeodomain protein that shows some similarities to theproboscipedia and Zen genes, but has no close homologue inDrosophila. It resembles most closely Hox class 3 vertebratesequences (F. F., unpublished results). As yet we know nothingof the function or expression of this gene tn Schistocerca, butwe guess that it may derive from an ancestral sequence thatgave rise, after considerable divergence, to the zen and/orbicoid genes of Drosophila.

Work from the authors laboratory was supported by the WellcomeTrust. We thank Nick Butterfield, Dieter Maier, Sue Brown and RobDenell for communicating results prior to publication; Geoffrey Fryerand Hans-Georg Frohnhofer for their advice at the early stages of ourCrustacean work, Peter Holland and David Stern for valuablecomments on the manuscript.

REFERENCES

Aisemberg, G. O. and Macagno, E. R. (1994). Loxl, an Antennapedia-Classhomeobox gene is expressed during leech gangliogenesis in both transientand stable central neurons. Dev. Biol. 161,455-465.

Akam, M., Dawson,I. and Tear, G. (1988). Homeotic genes and the control ofsegment identity. Development 104 Supplement , 123-133.

Averof, M. (1994). HOM/Hox Genes of a Crustacean; EvolutionaryImplications. Ph.D. thesis, University of Cambridge.

Averof, M. and Akam, M. ( 1993). HOM/Hox genes of Artemia: rmplicationsfor the origin of insect and crustacean body plans. Current Biology 3,73-78.

Ballard, J. W. O., Olse[, G. J., Faith, D. P., Odgers, \ry. A., Rowell, D. M.and Atkinson, P. \ry. 0992). Evidence from 12S ribosomal RNA sequences

that onychophorans are modified arthropods. Science 258, 1345-1348.Beeman, R. \ry., Stuart, J. J., Brown, S. J. and Denell, R. E. (1993). Structure

and function of the homeotic gene complex (HOM-C) in the beetle Triboliumc as tane um. B io E s s ay s 15, 439 -444.

Beverley, S. M. and Wilson, A. C. (1984). Molecular evolution in Drosophilaand the higher Diptera.z. A time scale for fly evolution. J. Mol. Evol.2l, l-13.

Booker, R. B. and Truman, J. \ry. (1989). Octopod, a homeotic mutation ofthe moth Manduca sexta, influences the fate of identifiable pattern elementswithin the CNS . Development 105, 621-629.

Boulet, A. M., Lloyd, A. and Sakonju, S. (1991). Molecular definition of themorphogenetic and regulatory functions and the cis-regulatory elements ofthe Drosophila Abd-B homeotic gene. Development 11.L, 393-406.

Briggs, D. E. G., Fortey, R. A. and Wills, M. A. (1992). Morphologicaldisparity in the Cambrian. Science 256, 1670-1673.

A

A

A

A

5(D)4(D(a

;rts.Vo.ts+CD

cocoo+==:1_=EEqds-D9uugltsoHS(DCDo'oo.:1 FrA6_Tg3

6;;!-'=rli]H. -F! v)

Fo Oc iioF(D

Brown, S. J., Hilgenfeld, R. B. and Denell, R. E. (1994). The beetle Triboliumcastaneum has a fushi tarazu homolog expressed in stripes duringsegmentation. Proc. Nat. Acad. Sci. USA (in press).

Brusca, R. C. and Brusca, G. J. (1990). Invertebrates. Massachusetts:Sinauer.

Biirglin, T. R. (1994). A comprehensive classification of homeobox genes. InGuidebook to the Homeobox Genes (ed. D. Duboule), pp. 25-71. Oxford:Oxford University Press.

Busturifl, A., Vernosr I., Macias, A., Casanovfl, J. A. and Morata, G. (1990).Different forms of Ultrabithorax proteins generated by alternative splicingare functionally equivalent. EMBO J. 9,3551-3555.

Butterfield, N. J. (1994). Burgess Shale-type fossils from an early Cambrianshallow-shelf sequence, northwestern Canada. Nature 369, 477 -479 .

Carroll, S. B. (1994). Developmental regulatory mechanisms in the evolutionof insect diversity . Development 1994 Supplement,

Cartwright, P., Dick, M. and Buss, L.'W. (1993). Hom/Hox type homeoboxesin the Chelicerate Limulus polyphemus. Mol. Phylogenet. Evol. 2,185-192.

Cisne, J. L. (1974). Evolution of the world fauna of aquatic free-livingarthropo ds . Evolution 28, 337 -366 .

Dawes, R., Dawson, I., Falciani, F., Tear, G. and Akam, M. ( 1994). Dax, aLocust Hox gene related tofushi-tarazu but showing no pair-rule expression.Development 120, I 56 I -157 2.

Doe, C. Q., Hiromi, Y., Gehring, W. J. and Goodman, C. S. (1988).Expression and function of the segmentation gene fushi tarazu duringDrosophila neurogenesis. Science 239, 17 0-17 5 .

Driever, W. and Niisslein-Volhard, C. (1988). The bicoid protein determinesposition in the Drosophila embryo in a concentration-dependent manner.Cell 54,95-104.

Egglestor, P. (1992).Identification of the abdominal-Ahomologue from Aedesaegypti and structural comparisons among related genes. Nucleic AcidsRe s e arch 20, 4095 -4096.

Eldridge, N. (1977). Trilobites and evolutionary patterns. In Patterns ofevolution, as illustrated by the fossil record (ed. A. Hallam), pp. 305-332.Amsterdam: Elsevier.

Fergusor, E. L. and Andersor, K. Y. ( 199 I ). Dorsal-ventral pattern formationin the Drosophila embryo - the role of zygotically active genes. CurrentTopics Dev. Biol. 25, 17 -43.

Fleig, R., Walldorf, U., Gehring, \ry. J. and Sander, K. (1983). In situlocalisation of the transcripts of a homeobox gene in the honeybee Apismellifura L. (Hymenoptera). Roux's Arch. Dev. Biol. 197,269-276.

Garcia-Fernandez, J. and Holland, P. 'W. H. (1994). Archetypal organization

of the Amphioxus Hox gene cluster. Nature 370,563-566.Gonzalez-Reyes, A., Urquia, N., Gehring, W., Struhl, G. and Morata, G.

( 1990). Are cross regulatory interactions between homeotic genesfunctionally significant? Nature 344, 78-80.

Jeram, A. J., Selden, P. A. and Edwards, D. (1990). Land animals in theSilurian: arachnids and myriapods from Shropshire, England. Science 658-669.

Karch, F., Bender, W. and Weiffenbach, B. (1990). abdominal-A expressionrn Drosophila embryos. Genes Dev. 4, 1573-1588.

Kaufmar, T. C., Seeger, M. A. and Olsen, G. (1990). Molecular and geneticorganisation of the Antennapedia complex of Drosophila melanogaster.InGenetic Regulatory Hierarchies in Development (ed. T. R. F. Wright), pp.Academic Press.

Kelsh, R., Dawson, I. A. and Akam, M. (1993). An analysis of Abdominal-Bexpression in the locust Schistocerca gregaria. Development ll7, 293-305.

Kelsh, R., Weinzierl, R. O. J., White, R. A. H. and Akam, M. (1994).Homeotic gene expression in the Locust Schi stocerca: an antibody thatdetects conserved epitopes in Ultrabithorax and abdominal-A proteins.Developmental Genetics 15, 19-31.

Kristensen, N. P. (1991). Phylogeny of extant hexapods. In The Insects ofAustralia (ed. CSIRO. Div. of Entomology), pp. 125-140. Melbourne:Melbourne University Press.

Kukalova-Peck, J. (1991). Fossil history and the evolution of hexapodstructures. In The Insects of Australia (ed. CSIRO Div. of Entomology), pp.l4l-179. Melbourne: Melbourne Univ. Press.

Lawrence, P. A. and Johnstor, P. (1989). Pattern formation in the Drosophilaembryo: allocation of cells to parasegments by even-skipped and fushitarazu. Development 105, 7 6l-7 69 .


Lewis, E. B. (1978). A gene complex controlling segmentation rn Drosophila.Nature 276,565-570.

Lindsley, D. L. and Zimm, G. G. (1992). The genome of Drosophilamelanogaster. San Diego: Academic Press.

Macias, A., Casanova, J. and Morata, G. (1990). Expression and regulationof the abd-A gene of Drosophila. Development 110, ll97 -1207 .

Mann, R. S. and Hogness, D. S. (1990). Functional dissection of Ultrabithoraxproteins tn D . melano gaster. Cell 60, 597 -6 10.

Nagy, L. M., Booker, R. and Riddiford, L. M. (1991). Isolation andembryonic expression of an abdominal-A-llke gene from the lepidopteran,Manduca sexta. Development ll2, lI9-I31.

Nardelli-Haefliger, D. and Shankland, M. (1992). Lox2, a putative leechsegment identity gene, is expressed in the same segmental domain indifferent stem cell linea ges. Development 116, 697 -7 10.

Robison, R. A. and Kaesler, R. L. (1987). Phylum Arthropoda. In FossilInvertebrates (ed. R. S. Boardman, A. H. Cheetham and A. J. Rowell), pp.205-269. Palo Alto, CA: Blackwell Scientific.

S6nchez-Herrero,8., Vern6s, f., Marco, R. and Morata, G. (1985).Genetic organisation of the Drosophila bithorax complex . Nature 313,108-l 13.

Schram, F. R. (1986). Crustacea. Oxford: University Press.

Schriider, R. and Sander, K. (1993). A comparison of transplantable bicoidactivity and partial bicoid homeobox sequences in several Drosophila andblowfly species (Calliphoridae). Roux's Arch. Dev. 9ioL203,34-43.

Schubert, F. R., Nieselt-Struwe, K. and Gruss, P. (1993). The Antennapedia-type homeobox genes have evolved from three precursors separated early inmetazoan evolution. PNAS 90, 143-147 .

Scott, M. P. (1992). Vertebrate Homeobox gene nomenclature. Cell7l,55l-553.Scott, M. P., Tamkun, J. W. and Hartzell, G.'W. I. (1989). The structure and

function of the homeodomain. Biochim. Biophys. Acta 989, 25-48.Shankland, M., Martindale, M. Q., Nardelli-Haefliger, D., Baxter, E. and

Price, D. J. (1991). Origin of segmental identity in the leech nervous system.D ev e lo p me nt Supplem ent 2,, 29 -38 .

Snodgrass, R. E. (1935). Principles of Insect Morphology. London and NewYork: McGraw-Hill.

Sommer, R. and Tautz, D. (1991). Segmentation gene expression in thehousefly Musca domestica. Development 113, 419-30.

Stuart, J. J., Brown, S. J., Beeman, R. \ry. and Denell, R. E. (1991). Adeficiency of the homeotic complex of the beetle Tribolium. Nature 350,72-74.

Stuart, J. J., Brown, S. J., Beeman, R. \ry. and Denell, R. B. (1993). TheTribolium homeotic gene Abdominal rs homologous to abdominal-A of theD r o s o p hil a bithorax complex. D ev e I o p me nt ll7, 233 -243 .

Tazima, Y. (1964). The Genetics of the Silkworm. London: Logos Press.Tear, G., Akam, M. and Martinez-Arias, A. (1990). Isolation of an

abdominal-A gene from the locust Schistocerca gregaria and its expressionduring early embryogenesis. Development 110, 915-926.

Turbeville, J. M., Pfeifer, D. M., Field, K. G. and Raff, R. A. (1991). Thephylogenetic status of arthropods as inferred from 18S rRNA sequences.Mol. Biol. Evol.8, 669-686.

Ueno, K., Hui, C.-C., Fukuta, M. and Suzuki, Y. (1992). Molecular analysisof the deletion mutants in the E homeotic complex of the silkw orm Bombyxmori. D ev elopment ll4, 555-563.

Vachon, G., Cohen,8., Pfeifl€, C., McGuffin, M. E., Botas, J. and Cohen, S.M. (1992). Homeotic genes of the bithorax complex repress limbdevelopment in the abdomen of the Drosophila embryo through target geneDistal-less. Cell Tl, 437 -450.

Walldorf, U., Fleig, R. and Gehring, W. J. (1989). Comparison of homeobox-containing genes of the honeybee and Drosophila. Proc. Natl. Acad. Sci.usA 86,9971-997 5.

Whiting, M. F. and Wheeler, W. C. (1994). Insect homeotic transformation.Nature 368, 696.

Whitington, P. M., Meier, T. and King, P. (1991). Segmentation,neurogenesis and formation of early axonal pathways in the centipede,Ethmostigmus rubripes (Brandt) . Roux's Arch Dev Biol 199,349-363.

Williams, J. A. and Carroll, S. B. (1993). The origin, patterning and evolutionof insect appenda ges. BioEssays l5, 567 -577 .

Wysocka-Diller, J. W.o Aisemb€rg, G. O., Baumgarten, M., Levine, M. andMacagno, E. R. (1989). Characterrzatron of a homologue of bithoraxcomplex genes in the leech Hirudo medicinalis. Nature 341,760-763.

Documents

The evolving of Hox arthropods - Development