Development 1994 Supplement, 217-223 (1994)Printed in Great Britain @ The Company of Biologists Limited 1994

Developmental regulatory mechanisms in the evolution of insect diversity

Sean B. Carroll

Howard Hughes Medical lnstitute, and Laboratory of Molecular Biology, University of Wisconsin-Madison , 1525 Linden Drive,Madison, Wl 53706, USA

SUMMARY

The major architectural differences between mostArthropod classes and orders involve variations in thenumber, type and pattern of body appendages. We haveutilized the emerging knowledge of appendage formationin fruit flies to begin to address the developmental andgenetic basis of morphological diversity among insects.Butterflies, for example, differ from fruit flies in possessinglarval abdominal limbs, two pairs of adult wings, and asophisticated system of wing color pattern formation. Wehave found that the genetic bases for these three majormorphological features involve differences between fliesand butterflies at three levels of genetic regulation duringdevelopment.

First, we show that the presence of abdominal limbs inbutterflies is associated with striking changes in the regu-

lation of specific homeotic genes in the abdominal segmentsof the butterfly embryo. Second, w€ suggest that the two-winged state of the fruit fly and the distinct pattern of thebutterfly hindwing are the consequence of many accurru-Iated changes in the target genes regulated by the Ultra-bithorax homeotic gene. And finally, w€ demonstrate that anew genetic program, involving many of the same genesthat specify the conserved global patterning coordinates offruit fly and butterfly wings, has been superimposed ontothe butterfly wing to create their unique color patterningsystem. These findings demonstrate how morphologicaldiversity arises from the different ways in which conservedsets of regulatory genes are deployed during development.

Key words: appendages, homeotic genes, butterflies, color patterns

217

"...remember that we are still at the beginning, that thecomplexity of the problem of Specific Difference is hardlyless now than it was when Darwin first shewed thatNatural History is a problem and no vain riddle."

William Bateson, preface to Materials for theStudy of Variation (1894)

William Bateson's book, in which he catalogued and inter-preted a dazzling variety of nature's oddities and coined theterm "homoeosis", marked one of the first post-Darwinianinquiries into the relationship between heredity, development,and morphological evolution. Bateson, and later biologistssuch as Goldschmidt ( 1940) who attempted similar syntheses,were lacking crucial information about mechanisms of inheri-tance and animal development. Now, a century later, with ourmastery of genetics and rapidly expanding knowledge ofembryology, the long anticipated integration of these disci-plines with evolution appears within reach. Molecular geneticshas provided the means to understand both gene functionduring development and to explore evolutionary relationships(Raff, 1992). We are now in a position to expand beyond thestandard model organisms, which represent just a few phyla,in order to ask specific questions about the developmentalmechanisms underlying morphological evolution.

The past decade's progress in dissecting animal develop-ment in genetically or experimentally favorable model speciesand recent forays into comparative molecular embryology have

uncovered two general trends. First, the types of moleculesinvolved in cell interactions, signal transduction, and gene reg-ulation have been conserved among most metazoans. Andsecond, many of the molecular pathways connecting theseprocesses during pattern development in diverse organismshave been remarkably conserved. These sometimes startlingsimilarities between animals are satisfying, in terms of identi-fying common elements of animal development, but also agreat challenge in that we now must ask, 'how are thesecommon batteries of gene families and signalling pathwaysused to create animal diversity?' To Allan Wilson, the answerwas plainly evident two decades ago. Namely, that the essenceof morphological diversity is based on changes in the mecha-nisms controlling the expression of genes rather than sequencechanges in proteins (Wilson et &1., 1974; King and Wilson,1975; Wilson, 1985). Therefore, not only should we identifywhich genes function in morphogenesis, but also decipher howtheir regulation and function contributes to diversity.

The fruit fly Drosophila melanogaster has yielded criticalinformation about the genetic basis of morphogenesis. The pastdecade's work on this animal has allowed us to draw the crucialconnections between phenotype and gene function and regula-tion during development. The visualization of the chemicalchanges (i.e. gene expression) in cells that precede the overtformation of pattern has revolutionized our perspective andapproach to embryogenesis. While the discovery of particularconserved sequences (e.g. homeoboxes) has provided animportant dimension to understanding animal relationships, the

218 S. B. Carroll

direct inspection and perturbation of gene function duringdevelopment has painted a rich picture that forms the founda-tion of a new comparative embryology. Now that we have con-siderable genetic, molecular and developmental understandingof oogenesis, segmentation, neurogenesis, homeosis, organo-genesis, appendage formation and cell differentiation fromfruit flies, evolutionary aspects of these processes can beaddressed.

However, given the emerging picture of molecular conser-vation, the tricky question becomes how to study diversity.Forinstance, we know a fair amount about bristle formation andpatterning in flies. However, the Dipteran bristle pattern is so

static it offers little hope of finding any meaningful mecha-nisms of diversity. The same may be said largely of the insecteye, the development of which in Drosophila is becomingincreasingly well understood, but what little variation theremay be in insect retinal cell architecture is of dubious signifi-cance. It follows then that we need some criteria for choosingtopics and organisms to study. First, the specific difference inquestion must be of obvious functional consequence. Second,we should hav e a reasonably deep grasp of the formal geneticlogic and identity of the molecules involved in the formationof the structure,, trant, or pattern in a reference species (for ourpurposes here - fruit flies). Finally, we should focus on animalsfor comparison whose molecular genetics and embryologypresent no obvious barriers or better yet, afford distinct exper-imental advantages (e.g. husband.y, some genetics, generationtime, all relevant stages of development easy to obtain, etc.).

We have chosen to work with butterflies for precisely thesereasons. Several major body characters differ in obvious waysbetween flies and butterflies, particularly with respect to theirappendages and the cell types found in them (Table 1).

Compared with other insects, they are fairly closely related toflies, and therefore gene homologs may be readily obtained.Finally, there is a reasonable knowledge of Lepidopteranembryology (Nagy et al., 1994).

We were also drawn to butterflies out of interest in the devel-opment and evolution of appendages, especially insect wings.The analysis of wing morphogenesis in Drosophila is one ofthe few genetically accessible models for understandingpattern formation in a growing cellular field and is providinga general framework for the analysis of wing development andevolution (Williams and Carroll , 1993). Given that the wingevolved in the course of insect evolution (see Kukalova-Peck,1978 for a fascinating discussion), comparative analysis ofwing patterning genes will allow us to explore the origin ofthis adaptation. In addition, butterflies differ greatly in theirwing patterns. Analysis of the development of these wing colorpatterns may both shed light on an important but unsolveddimension of pattern formation (coloration) and create the

Table 1. Order-specific differences between fruit flies andbutterflies

Character Drosophila Precis coenia

Limb number No abdominal limbs 4 pairs of larval abdominalprolegs, anal prolegs

Four, hindwing largeElaborate color patternScales (modified bristle?),

epithelia

opportunity to investigate patterning differences at the specieslevel. Here, I will review the results of our initial investigationof butterfly appendage formation and patterning, in which wehave found that three order-specific differences between but-terflies and fruit flies can be correlated with three types ofgenetic regulatory differences in development.

GENERAL APPROACH

To investigate specific differences between fruit flies and but-terflies, we had to choose a suitable butterfly species for initialstudy. We decided on the Buckeye (Precis coenia: Nymphali-dae) because there has been considerable investigation into thedevelopment of its wing color pattern (Nijhout, l980a,b) andthis species can be mass-reared in population cages on a simplediet. As a start, we constructed wing and embryonic cDNAlibraries and developed embryonic and imaginal in situ hybrid-rzatron and immunohistochemical procedures for Precis coenia.It is expected that most butterfly groups that will be of com-parative interest in the future have been separated by less than100-150 Myr, with some genera such as the tropical Heliconiusrepresenting much more recent radiations (Sheppard et aI.,

1985). Based upon the experiences of many investigators,including ourselves, in analyzing gene expression in differentDiptera that are 80-100 Myr divergent (often utilizing anti-bodies to D. melanogaster proteins), we are hopeful that nucleicacid probes and/or antibodies for P. coenia gene products willprovide tools for exploring butterfly relationships.

With libraries constructed, we cloned various butterflyhomologs of Drosophila genes involved in appendageformation, segment identity, segment polarity, and intercellu-lar signalling (Table 2). Of the genes shown in Table 2, allbutwingless (cloned by PCR) were cloned by cross-hybrrdrzatronwith Drosophila probes. In only one instance (rhomboid), havewe not yet been able to clone a P. coenia homolog, howeverthis protein lacks homology to other known proteins or motifsthat would help to target our approach. In the next threesections, I will describe what the analysis of the expression ofthese genes has taught us about the formation of the butterflybody pattern relative to that of Drosophila.

DIFFERENCES IN APPENDAGE NUMBER:REGULATION OF HOMEOTIC GENES

P. coenialarvae possess prolegs on the third through sixth andterminal abdominal segments (Fig. 1A). There is not fullagreement as to the evolutionary relationships between prolegsand other appendages or indeed their status as limbs (Birket-Smith, 1984). Since it has been established in Drosophila thatthe Distal-less (Dll) homeobox gene is the earliest marker ofthe limb primordia (Fig. 1B; Cohen et al., 1989) and is requiredfor the elaboration of the proximodistal axis of adult limbs(Cohen and Jiirgens, 1989), we reasoned that the P. coenia Dllgene could reveal new information about proleg formation.The P. coenia Dll gene is expressed in all appendages (exceptthe mandible), including the abdominal and anal prolegs (Fig.lC; Panganiban et al., 1994). This suggests that the proxi-modistal axis of the prolegs is elaborated in a fashion similarto cephalic and thoracic limbs.

Wing number, type Two, halteres on T3Wing pattern UnpigmentedWing cell types Bristles, epithelia

Unlike P. coenict, D. melanogaster lacks abdominal Dllexpression and larval limbs. The lack of abdominal Dllexpression has been shown to be due to the direct repressive

action of the bithorax complex (BX-C) homeotic gene productson the Dll gene (Vachon et al., 1992). The abdominal expressionof Dll in P. c'oeniu indicates that either the P. coenia BX-C gene products do not repress Dll in the same way orthat they are deployed differently during embryonicdevelopment. To address this question we cloned Sex

comhs reduced (Sc'r), Antennapedia (Antp), Ultrabitho-rax (Ubx), and abdominol-A (abd-A) cDNAs from P.

coenia and examined their expression during embryo-genesis (Warren et al., unpublished data). These genes are

initially expressed in similar segmental registers as theirDrosophila counterparts (Warren et al., unpublisheddata). However, after the initial deployment of Ubx and

abcl-A in the abdomen, these two genes are selectivelyshut off in the abdominal proleg primordia (Fig. lD;Warren et al., unpublished data).

This observation that abdominal proleg formation is

enabled by a striking alteration in homeotic gene regula-tion was unexpected. Dll de-repression in the abdomencould have easily occurred through alteration of c'is-reg-

ulatory elements of the Dll gene without any changes inhomeotic genes. There are many potential molecularmechanisms for the selective repression of BX-C genes

in the prolegs (Warren et al., unpublished data), however,the fact that this feature, specific to the Lepidopteranorder, is developmentally regulated at the homeotic levelindicates that limb number can be regulated differently inthe presence of the same complement of homeotic genes.

It is possible that the suppression of limbs in insectancestors was due to the evolution of a regulatory linkbetween BX-C genes and limb-patterning genes, and notthe evolution of BX-C genes.

DIFFERENCES IN APPENDAGE TYPE:REGULATION OF HOMEOTIC TARGET GENES

The flight appendages of flies and butterflies differ inthat the metathoracic (T3) segment in flies bears veryreduced structures, the halteres (Fig. 2A), which are

used in balancing the animal during flight. Butterfliespossess two well-developed pairs of wings (Fig. 2B) and

pattern formation on the two wing pairs is often coordi-nated So as to present a continuous overall pattern whenthe animal's wings are at rest. The existence ofmutations in the Ubx gene that revert the fly to itsancestral four-winged state has led to the suggestion thathaltere formation was related to the evolution of the Ubxgene (Lewis, 1978).

It is possible, then, that if the presence of Ubx is suf-ficient to suppress the posterior flight appendages, thedeveloping hindwings of butterflies might lack Ubxexpression (to permit their growth as full-sized wings).To investigate this possibility, we examined Ubxexpression in P. coenia wing imaginal discs. Like the

Drosophila metathoracic haltere disc (Fig. 2C), the P.

coenia hindwing imaginal disc expresses high levels ofUbx protein (Fig . 2D). This demonstrates that the

Evolution of diversity in insects 219

presence of Ubx is not sufficient to promote reduction of wingsize. The deployment of Ubx in the hindwings of a four-winged insect and the identification of the Ubx gene in otherinsects (Ueno et al., 1992), crustacea (Averof and Akam,1993) and even annelids (Wysocka-Diller et al., 1989) demon-

''!h\

Fig. 1. Insect limb number and the regulation of Dll and homeotic gene

expression (A) P. coenia larva displays four pairs of abdominal prolegs (a3-

a6) and one pair of anal prolegs (ap). (B) Dll expression in a Drosophilaembryo beginning germband retraction. The antennal, maxillary, labial, and

thoracic (tl-t3) limb primordia express D// (C). P. coenia embryo at 407o ofembryogenesis , Dll RNA is expressed in a proximal ring and large distalsection of each thoracic limb and in the abdominal (a3-a6) and anal prolegs(ap). (D) Ubx and abd-A protein expression in P. coenie embryo at about25Vo of development, detected with a monoclonal antibody that recognizesboth proteins (Kelsh et al ., 1994). Both proteins are selectively shut off inthe abdominal proleg primordia.

220 S. B. Carroll

Table 2. P. coenia homologs of D. melanogaster genes

Protcin l'unctiorr Cortttttc-rtts

Homeotic^tt'.t' r 't tttt lt.s t'r'rlttr'(tl'i'A t t I t' t t t t tt 1t t' d i t fi'U I t ru lt i t I t ( ) t'( t.\''i'

ttlxlr tttt irtu l-A'i'

Appendage formationI)i.stul-lc,s.s'i'

ul)l ( t'ott.\':;

,scu I lttltcrl -i.

ctt,qruilcdlittvt't'lcd.i.rli ll.q/t'.r.r:i:

d c <' tt 1t c t t t u 1t I e,q i t':i.

rln/--l\lttttchcr|:ll

Others604 $

t'.\'I t'tt tttttt' t't t<'II tt t'lrtc N

Horncodornai n. DN A -bi nd i n-u

Honrcodornain. DNA-bindingHorncodornain. DNA-bi ndi ng

Horttcodolnai n. DNA-bindi ng

H ontcodont:li n. DN A -bi nd i n-u

LI M -typc horttcodortrai rr

TEA Ianrily transcription lhctorHorrtcodonain. DNA-bi ndi ng

Hortrcoclornai n. DNA -bi ndi ngSccrctcd sisnalling rnolcculcTGF-B typc signalIing rnoleculcW i n,q I c ss rclatccl protci rr

Trarr snrcnrbranc rcccptor'l

TGF-p typc prote irr

Hclix-loop-lrcl i x negati ve reuulatoryprotcin

Initial scgnrental rcgister conscrvedHigh lcvcl cxprcssion in thoraxRepresscd in prolegsRcpresscd in pnrlegs

Expresscd in all linrb prirnordia except mandible.also irt wing eyespots

Dorsal only wing cxprcssion consL-rved

Irr all cclls ol'thc wingMarks antcrirlr/postcrior boundaryMarks antcrior/postcrior boundaryMarks wing nrarginDepkryed irr wing cellExprcssion pattern unknownA/P houndary conserved

Pattcrn unknownPattcrn unknown

L. and M.P. Scott. unpublished.

Fig. 2. The role of theUb.r gene indetermining the typeof flight appendage.(A) The adultDrosophila wing andhaltere. Photocourtesy of JuanPablo Couso. (B) Theadult P. coenicrforewing andhindwing. (C) TheUbx protein isexpressed throughoutthe third instarDrosophila haltereimaginal disc.(D) Ubx protein isalso expressedthroughout the fifthinstar P. c'oenict

hindwing imaginaldisc. Ubx expressionin both halteres andhindwings indicatesthat the mere presenceof Ubx is notsufficient to reducethe posterior flightappendages.

strates that the Ub.r gene existed well betore the evolution ofthe haltere.

Since no mutations are known outside of the BX-C and its

regulators that transform halteres to wings, it is likely that thehaltere is the product of multigenic regulation by Ubx. Asflies evolved from a four-winged ancestor (Riek, 1977),,

evolution ofUbx-regulated

gene set

FOUR-WINGED ANCESTORwing pairs identical

gradualreductlon

of hindwing

Ubx mutatlon, entlre Ubx-regulatedgene set expressed as in forewing

Evolution of diversity in insects 221

Ubx mutant

Fig. 3. The role of the Ubx gene

in the evolution of the haltere inDiptera . Ubx was probablyexpressed in the developinghindwings of the four-wingedancestor of the Diptera (as it is inLepidopteran hindwings). Theconstellation of Ubx-regulatedgenes evolved such that thehindwing was gradually reduced.Loss of Ubx by mutations does

not simply reverse the course ofevolution, it eliminates the Ubx-dependent regulation of a largegene set in the posterior flightappendages.

the eyespot takes place in the late larval and early pupalimaginal wing disc. This is at a time well before the scale cellsdifferentiate and produce pigment. Therefore, we have soughtto identify molecules that might be involved in a prepattern inthe imaginal wings.

Our bias was that genes involved in appendage patternformation and cell-cell signalling might play a role in butterflywing patterns. We cloned butterfly homologs of the Drosophilagenes that play key roles in dorsal/ventral (apterous),anterior/posterior (invected/ engrailed/ decapentaplegic), andproximal/distal (scalloped, Distal-less) organization of the wingand in formation of the wing margin (wingless; Carroll et al.,1994). The expression of each of these genes reflects a commonorganization of the wings of P. coenia and Drosophila. Specif-ically, the spatial coordinates in the Drosophila wing are

marked by the expression of particular pattern-regulating genes.

Comparable coordinates in the butterfly wing are marked byhomologs to the Drosophila pattern-regulating genes. Unex-pectedly, however, we found that most of the P. coenia genes

displayed a second pattern of transcription during the key fifthlarval instar that was reiterated within each wing cell. Thesepatterns included rays of wingless transcription in cells flankingthe midline at the distal end of each wing cell (Fig. 4B), stripedaccumulations of scalloped transcripts along the veins (Fig.4C), and chevrons of apterous transcription at the distal end ofdorsal wing cells (Fig. 4D). While none of these patterns neatlyconespond to color pattern elements in P. coenia, midlinestripes, chevrons, and venous stripes occur in many butterflies(Nijhout, 1991). We suggest that these transcription patterns

reflect a fundamental dynamic patterning system within eachwing cell, and that the interpretation of this landscape is species-specific (Carroll et al., 1994).

The best evidence to date for a dynamic butterfly wing cellpatterning system comes from analysis of P. coenia DUexpression. In P. coenia, Dll is initially transcribed in a distalband along the wing with higher levels of transcript accumu-lating along the midline of each wing cell. At the proximal

DIPTERA

numerous genes have probably fallen under control of theUbx gene. The regulation of this network of genes by Ubxduring imaginal disc formation and morphogenesis results inthe transformation of the potential wing into a haltere (Fig.3). The reduction of the posterior wings may have been a

gradual process, since fossil ancestors of modern Dipteraexhibited reduced hindwings (Riek, 1977). The dramaticfour-winged phenotype brought about by mutations in theUbx region (Lewis , 1978) is not the simple reverse of thepathway followed by evolution; it represents a complete lossof the Ubx-regulated gene network in the posterior flightappendages (Fig. 3).

DIFFERENCES IN APPENDAGE PATTERN:CREATING NEW PATTERNS FROM OLD CIRCUITS

Wings of different butterfly species sport a dazzling variety ofcolors and patterns, organized on a similar framework. Never-theless, all patterns can be interpreted as variations on a basicgroundplan (Nijhout, l99l). The evolution of the patterningsystem that gives rise to this groundplan appears to be uniqueto butterflies (see Nijhout, this volume), while other insects and

Lepidoptera have evolved pigmentation, none possess a systemas robust as that found in butterflies. The fundamental units ofpattern formation in the butterfly wing are the subdivisionsbounded by veins and the wing margin, termed "wing cells".Pattern formation within each wing cell is largely independentof that in other wing cells. The overall wing pattern consistsof a serial repetition of the pattern elements of each wing cell,although not all elements present in one wing cell are neces-

sarily present in the others. Among the more striking patternelements are the eyespots, concentric rings of pigmentationsurrounding a center or focus. Transplantation and cauteryexperiments suggest that the focus is an org anizer capable ofinducing surrounding cells into eyespot formation (Nijhout,1980b, 1985; French and Brakefield, '1992). Determination of

222 S. B. Carroll

Fig. 4. Utilization of appendage-patterning genes in novel wingpatterning circuit in the butterfly. (A-E) In all panels, anterior istoward the top and the distal wing margin to the left. (A) P. coeniahindwing displaying one anterior and one posterior eyespot. (B-E) Insitu hybridizations of P. coenia gene probes to fifth instar hindwingimaginal discs. (B) The P. coenia wg gene is transcribed in pairs ofrays emanating from the margin of each wing cell. (C) The P. coeniasd gene is transcribed at higher levels in cells lying along the veins ofeach wing cell. (D) The P. coenia ap gene is modulated in a chevron-like pattern at the distal end of each wing cell. (E) The P. coenia Dllgene is transcribed at high levels in cells that will give rise to theposterior eyespot. The wing cell transcription patterns in B-E suggestthat a dynamic patterning system, involving some of the same globalwing patterning genes, operates in each wing cell.

boundary of these midline rays, Dll transcription expands tran-siently into small circles, and in the posterior hindwing andforewing cells that develop eyespots, the Dll circles expandfurther to form spots comprising about 200 cells by the late

fifth instar whereas Dll expression is lost from the centers ofother wing cells (Fig. 4E). These features of Dll expressionsuggest that a wing cell patterning system creates the initialproximodistal restriction, the midline rays, the proximalenlargements and finally the posterior eyespot prepattern.Since Dll plays a crucial role in appendage formation and itsexpression is spatially regulated along the primary body axisand within limb fields, we propose that this appendage-pat-terning gene has been recruited into another regulatory circuitthat is devoted to generating patterns within the wing cells. Thespecific transcription patterns of the other appendage-pattern-ing genes noted above support the idea that many elements ofa proximodistal patterning circuit are being deployed to patternthese tertiary wing cell fields.

THREE LEVELS OF REGULATORY EVOLUTION

Our studies have illustrated how order-specific differences inappendage number, type, and pattern involve the evolution ofregulatory systems at three levels. First, we showed thatabdominal limb formation is enabled by a major difference inthe way BX-C homeotic genes are regulated in the abdomen.Second, we have deduced that haltere formation in the Dipterais probably the product of many accumulated changes in thenetwork of genes regulated by the Ubx protein. And third,we've presented evidence that a second regulatory circuit,involving many of the same genes required for global organ-ization of insect wings, operates specifically within butterflywings to generate their unique groundplan.

The clear implication of this work is that much morpholog-ical diversity is derived from the way conserved batteries ofdevelopmental genes are regulated and does not require theexpansion of existing gene families or the invention of newones. While the latter have certainly occurred, the chemicalevolution of animals has not been nearly as great as their mor-phological evolution. Changes in the number, type, position,and pattern of structures, as illustrated here, are likely toinvolve changes in the timing and spatial regulation of existinggenes. It is, as Francois Jacob put it, a matter of "tinkering" -using the same elements and adjusting, altering, and arrangingcombinations to produce new morphologies (Jacob ,1977). Thechallenge then for comparative embryology is to decipherwhere and what kind of tinkering has taken place.

Further studies of the evolution of appendage number, type,and pattern may help to address the developmental and geneticroots of morphological evolution at both greater and lessertaxonomic distances than those illustrated here with flies andbutterflies. For example, since wings arose within the insects, itwould be interesting to look at the deployment of wing pattern-ing genes in primitive wingless insects. What role do apterous,vestigial, scalloped, etc. play in an animal that never had wings?We would predict that these genes exist in all insects, but theirdeployment could shed light into the origins of the geneticcircuitry guiding wing formation or to a morphogenetic programthat may have been co-opted into wing formation.

Second, it may be possible to study morphological evolutionwithin an order. Clearly, some of the features that distinguishbutterflies from fruit flies are the product of regulatoryevolution; what about family, genus, or species-level differ-ences? Of the 15,000 or so butterfly species, at what genetic

and developmental level may we expect to explain differencesin wing patterns or other traits? The simplest model is thatmore related species possess similar wing ground plans and

developmental prepatterns and that the interpretation of thepatterning landscape by downstream genes, such as the pig-mentation genes, may differ. It is also possible that elementsof the prepattern differ according to which pattern elements arepresent. For example, the number as well as the coloration ofeyespots differs between species. While a mechanistic grasp ofcolor pattern formation is still just a distant hope, we can usemolecular probes to ask at what level of butterflies tinker, i.e.how the patterning landscape of the butterfly wing and/or itsinterpretation differs between species.

The goal of this comparative molecular embryology, as withmost new areas of investigation, is to identify a few first prin-ciples about the developmental and genetic mechanisms ofmorphological change. As Garcia-Bellido (1993) points out,we are going to have to trade the precision of mechanisticdetails in individual model species for deeper understanding ofanimal relationships and the process of change. Our under-standing of and appreciation for the functions and regulationof Drosophila genes that have been at the center of attentionfor decades, such as the homeotic genes, is greatly enriched bythis comparative approach. The shut-off of BX-C genes inspecific butterfly limb primordia and the role (or more accu-rately, lack thereof) for Ub.r gene evolution in the origin of thehaltere are new chapters in the homeotic story.

There are important lessons in the haltere example in thatwhile genetics can demonstrate a pathway back to a wing froma haltere via a simple mutation, this is clearly not the reverseof the course followed by evolution. Neither genetics nor com-parative embryology will tell us the exact sequence of steps inevolution. Nevertheless, we have, in Jacob's (1982) phraseol-ogy, distinguished the possible (the evolution of the Ubx gene

driving haltere formation) from the actual (genes downstreamof Ubx determine haltere character). The evolution of the Ubxgene provided the opportunity for distinguishing insect flightappendages but the course of fly evolution is only one of manypaths by which this was achieved. Butterflies, on the otherhand, probably use this same gene to distinguish hindwingfrom forewing patterns. Our Drosophila-centric view mighthave led us to believe that Ubx was invented by the Diptera tomake halteres. The deeper understanding is available onlythrough a comparative lens.

I thank J. Williams, J. Selegue, L. Nagy, J. GateS, B. Warren, D.Keys, G. Panganiban, and S. Paddock who have gotten the embryol-ogy and molecular biology of P. coenia off the ground and who haveprovided the data for the illustrations in this review; Steve Paddockand Leanne Olds for preparation of the figures; Juan Pablo Couso forFigure 2A; Peter Carroll, Steve Paddock, Lisa Nagy, Bob Warren and

Grace Panganiban for comments on the manuscript; and Jamie Wilsonfor its preparation. I am very grateful to the NSF, the Shaw ScientistProgram of the Milwaukee Foundation, and the Howard HughesMedical Institute whose generous and flexible support made these

studies possible.

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