Development 105, 621-628 (1989)Printed in Great Britain © The Company of Biologists Limited 1989

621

Octopod, a homeotic mutation of the moth Manduca sexta, influences the

fate of identifiable pattern elements within the CNS

RONALD BOOKER* and JAMES W. TRUMAN

Department of Zoology, University of Washington, Seattle, Washington 98195, USA

* Present address: Cornell University, Division of Biological Sciences, Section of Neurobiology and Behavior, Seelely G. Mudd Hall, Ithaca,New York 14853-2702, USA

Summary

Octopod (Octo) is a mutation of the moth Manduca sexta,which results in the homeotic transformation of theventral surface of the first (Al) and less often the second(A2) abdominal segments in the anterior direction. Theextent of the transformation ranges from a slight defor-mation of the ventral cuticle, up to the formation ofminiature thoracic legs on Al. The extent of the trans-formation is always less within A2 as compared to Al. Agenetic analysis revealed that Octo is an autosomalmutation which shows incomplete dominance. The effectof this mutation on the central nervous system (CNS)was assessed by examining the distribution and fate ofthe postembryonic neuroblasts in the segmental gangliaof Octo larvae. In each of the thoracic ganglia of wild-type larvae, there is a set of 45-47 neuroblasts; areduced but homologous array of 24 and 10 neuroblasts

are found in Al and A2, respectively. Ganglion Al ofOcto larvae had 1 to 6 supernumerary neuroblasts, and20 % of the A2 ganglia showed a single ectopic neuro-blast. The supernumerary neuroblasts corresponded toidentifiable neuroblasts normally found in more anteriorganglia. The Octo mutation also influenced the mitoticactivity of stem cells normally present in Al. In this case,the neuroblasts generated a lineage of cells that weretypical of a thoracic location rather than Al. These datademonstrate that homeotic mutations can influence thefate of identifiable pattern elements within the CNS of anbisect.

Key words: insect CNS, neurogenesis, homeotic mutant,Octopod, Manduca sexta.

Introduction

The central nervous system (CNS) of adult insects ischaracterized by dramatic regional differences in neur-onal numbers (Bate, 1976; Thomas et al. 1984; Booker& Truman 1987; Truman & Bate, 1988). Typically 4-7times as many neurones are found in the adult thoracicganglia compared to the abdominal ganglia. Among theinsects there are two strategies employed in generatingthese regional differences in neuronal number. In thoseinsects that go through an incomplete metamorphosis,such as crickets and grasshoppers, all of the neurones ineach segmental ganglion are generated during embryo-genesis from a nearly identical array of neuroblasts(Bate, 1976; Thomas et al. 1984). Differences in neur-onal numbers in the thoracic versus abdominal gangliaresult from two different mechanisms. First, neuro-blasts in the abdominal ganglia generate fewer progenythan their thoracic homologues. Second, segment-specific differences in the pattern of neuronal cell deathresult in higher levels of cell death within abdominal ascompared to thoracic ganglia (Loer et al. 1983).

Insects that go through a complete metamorphosis,such as flies and moths, utilize a different strategy togenerate the dramatic segmental differences in neur-onal numbers observed in adults. The embryonic phasein these insects is similar to that seen in grasshoppers inthat the thoracic and abdominal ganglia are generatedby nearly identical arrays of embryonic neuroblasts(Hartenstein & Campos-Ortega, 1984; Thomas et al1984). However, by the end of embryogenesis, there areonly small differences in the number of neurones foundin the thoracic and abdominal ganglia (Booker &Truman, 1987; Truman & Bate, 1988). The dramaticregional differences in neuronal numbers found in thesegmental ganglia of adult moths and flies are producedduring the postembryonic period and are due to seg-mental differences in the number of neuroblasts foundwithin the various regions of the larval CNS. Forexample, in larvae of Manduca sexta there are 45 to 47(22 to 23 paired and 1 unpaired) neuroblasts in thethoracic ganglia, while in the typical abdominalganglion (A3 to A8) there are only 8 neuroblasts (4paired). The thoracic set of neuroblasts add 3000 to

622 R. Booker and J. W. Truman

4000 new neurones to each thoracic ganglion while inthe abdominal segments about 100 new cells are added.

Present evidence suggests that the development ofboth the CNS and the epidermis of insects is influencedby segmental determination and that the genetic controlof these processes at least partly overlap. Regions of thebithorax complex (BX-C) and the antennapedia com-plex are expressed in the CNS at both the level ofRNAs, based on in situ hybridization (Akam, 1983;Hafen et al. 1983) and proteins, as shown through theuse of antibodies (White & Wilcox, 1984). However,our knowledge of the role of such segmentation genes inthe development of the CNS lags behind that of theepidermis. Much of the analysis has been carried out inthe CNS of Drosophila melanogaster. In the fruitfly, ithas been clearly shown that the branching patternwithin the CNS of sensory neurones and the identifiablegiant fibre can be altered in a number of homeoticmutations that affect the determined state of thoracicand abdominal segments (Palka et al. 1979; Thomas &Wyman, 1984). Moreover, staining patterns within theCNS can be transformed by certain homeotic mutations(Jimenez & Campos-Ortega, 1981; Teugels & Ghysen,1983). The report by Green (1981) used identifiablemotomeurones within affected regions to examine therole of homeotic mutations on CNS differentiation.Such studies, however, have only begun to scratch thesurface. Essentially nothing is known about the influ-ence of the homeotic genes on the fate of the neuronalprecursors that generate the dramatic regional diversityin neuronal number and specificity observed within theCNS of holometabolous insects.

Here, we report the isolation of the first homeoticmutation of the moth Manduca sexta. This mutation,named Octopod (Octo), results in the transformation ofthe ventral surface of the first, and less often thesecond, abdominal segments in the anterior direction.The mutation is characterized by the appearance ofectopic thoracic legs in both larvae and adults. Thearray of postembryonic neuroblasts within the segmen-tal ganglia of Manduca larvae provide a nearly ideal setof pattern elements for examining the influence of Octoin the segmental determination of the adult CNS ofManduca. Reconstructions of the segmental ganglia ofOcto animals reveal that the distribution and fate ofspecific neuroblasts within the segmental ganglia areselectively influenced by the Octo mutation.

Materials and methods

Experimental animalsLarvae of the tobacco hornworm, Manduca sexta, werereared on an artificial diet under conditions of 26°C and a17L:7D photoperiod as described by Bell & Joachim (1978).Under these rearing conditions approximately 18 days elapsefrom hatching to pupal ecdysis and 20 days from pupal ecdysisto the emergence of the adult. The ecdyses to the 4th and the5th larval instars and pupal ecdysis were used as references instaging the insects (4+0, 5+0 and P+0, respectively).

The Octo mutation was a spontaneous mutation first notedin our laboratory stock of Manduca sexta in 1985 by Dr

Kiyoshi Hiruma. Selection of fifth instar larvae for strongexpression of the Octo trait (ectopic leg on the first abdominalsegment) coupled with inbreeding for approximately 12 gen-erations has resulted in a stock that shows 100% expression.

As a wild-type stock for the genetic experiments, crosseswere made to aiaboratory strain carrying the recessive black(bl) mutation (Safranek & Riddiford, 1975). Larvae of thisstrain show melanization of the cuticle in the fourth and fifthlarval stage as a result of abnormally low circulating levels ofjuvenile hormone. This strain has been inbred for over 17years and shows no expression of the Octo phenotype.

For the genetic crosses, two to six pairs of freshly emergedfemales and males of the appropriate genotype were placed ina mating cage with a tobacco plant in a 17L: 7D photoperiod at20-21°C in a humid environment. Eggs were collected dailyand transferred to 26 °C.

Histological techniquesGanglia were removed from CO2-anaesthetized animals andfixed overnight in alcoholic or aqueous Bouin's fixative atroom temperature, then dehydrated through a graded ethanolseries and embedded in paraffin. Ganglia were serially sec-tioned at lOjan, and the sections were stained with haema-toxylin and eosin. Estimates of progeny number were deter-mined as reported previously (Booker & Truman, 1987).

Results

The Octo phenotypeOcto larvae show a homeotic transformation of theventral surface of Al in the anterior direction. In themost extreme cases, the transformation was manifestedin the appearance of complete but miniature thoraciclegs on segment Al (Fig. 1). The expression of the Octomutation was typically assessed in newly moulted fifthinstar larvae. A five point scoring system was devised asfollows: 0 = wild-type; 1 = slight deformation of theventral surface of the first abdominal segments oftenwith a few bristles that are usually found on the thoraciclegs; 2 = raised region of cuticle (coxa); 3 = coxa withpatches of melanized cuticle; 4 = at least two well-formed distal leg segments. Often the appendages onthe two sides of Al showed different levels of ex-pression. In such cases, the larva was assigned thehigher of the two scores received. In most instances,there was no more than a one point difference in thescore received by the two ectopic structures. In thehomozygous condition, the penetrance of the pheno-type is complete with the majority of the animals(~85%) receiving a score of 3 or 4. In a group ofanimals who were tracked from hatching through themoult to the fifth larval instar, there was no change inthe level of expression of the Octo phenotype through-out larval life. The extent of the transformation of adultstructures mirrored that seen in the larvae.

A small percentage (~10 %) of the homozygous Octoindividuals also showed transformation of the secondabdominal segment (A2). As was the case for Al, thetransformation of A2 was manifested by the formationof thoracic leg structures on the ventral surface of thesegment. At the highest level of expression, the ectopicstructures included only the most proximal leg segment,

Influence of a homeotic mutant on the insect CNS 623

the coxa. For example, among the animals that showedexpression in A2, the level of expression was scored aseither 1 (98%) or 2 (2%). Animals with ectopicstructures on A2 were only seen among those Octoanimals in which the A l transformation received ascore of 3 to 4.

The dorsal surface of A l and A2 were unaffected bythe Octo mutation. The dorsal cuticular markings ofthese segments were normal in mutant animals andshowed no thoracic characteristics. Also, there were nosigns of wing imaginal discs in segments Al and A2.

When Octopod females and males were mated,relatively few eggs were laid. The mating of 4 mutantpairs resulted in a total of only 244 eggs(mean = 58 ± 39-6 S.E.M.) being laid over a six-dayperiod, with two of the. females laying no eggs. Wild-type females by comparison usually lay more than 300eggs over the same time period. Crosses of homozygousOcto animals at 21 °C resulted in 40 to 60 % of the eggsproducing viable larvae compared to over 95 % for

crosses between wild-type animals. The remaining eggsappeared to be unfertilized eggs (such as from unmatedfemales) or they produced islands of white, flocculentmaterial dispersed in the yolk rather than a viableembryo. When crosses between mutant animals weremade at 27 °C, as opposed to 21 °C, fewer than 1 % ofthe eggs were viable. It is not known if this aberrantdevelopment is indeed related to the Octo gene. Therewas no lethality apparent during later stages of develop-ment. Viable Octo animals were indistinguishable fromwild-type animals in terms of the rate of their embry-onic, larval and adult development.

The Octo genotypeThe results of a series of crosses suggest that Octo is anautosomal dominant mutation (Fig. 2). When homo-zygous Octo males or females were crossed to blanimals approximately 60 % of the progeny appeared aswild-type, while the remaining 40 % expressed the Octophenotype. Among the latter group, nearly all the

Fig. 1. Series of photomicrographs of the ventral surface of the first abdominal segment (Al) illustrating the range ofexpression of the epidermal phenotype of Octo larvae. The larvae were scored using a 5-point scale (0 to 4).(A) Photomicrograph of the smooth ventral surface of Al of a wild-type animal; scored as 0. (B) Ventral Al of an Octoanimal scored as 1, showing the disruption of the cuticle (arrow). (C) Photomicrograph revealing the phenotype of a mutantanimal which received a score of 2, showing the ectopic coxa and a hair (arrow) normally associated with the thoracic legs.(D) In mutants receiving a score of 3, there is a well-formed coxa as well as signs of melanized tissues which are normallyassociated with the more distal segments of the thoracic legs. (E) The small, but obviously segmented, leg of an Octo mutantreceiving a score of 4. (F) A photomicrograph of a normal, larval thoracic leg.

624 R. Booker

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Fig. 2. A summary of the results of a series of genetic crosses. (A) The range of expression in the offspring of crossesbetween Octo males and females. (B) Results of crosses between Octo males and wild-type (+) females. (C) Results ofcrosses between wild-type (+) males and Octo females. In each case, the values represent the per cent of offspring receivinga score of 0—4 for expression of the Octo phenotype.

scores were in the range of 1 to 3 with no evidence oftransformation of A2. Among the FjS, males andfemales expressed the Octo trait at an equal rate.Backcrosses to the parental strains confirmed that theOcto trait is an autosomal dominant mutation (data notshown).

Influence of Octo on the neuroblast arraysWhile the influence of homeotic mutations on the fateof epidermal tissues has been extensively studied(Lewis, 1978; Akam, 1987), their role in the develop-ment of the CNS of insects is less well understood.Consequently, we examined the effect of the Octomutation on two postembryonic processes that areimportant in establishing segmental differences withinthe CNS; namely, segment-specific differences in thenumber of neuroblasts and the size of specific lineages.Fig. 3A shows the complete array of neuroblasts foundin ganglion T2 of wild-type larvae. Sets of neuroblastsare segmentally deleted from this airay as one pro-gresses posteriorly to Al and A2. Reconstructions ofthe neuroblast arrays found in Al of Octo larvaerevealed the presence of supernumerary neuroblasts.The number of extra neuroblasts ranged from 1 to 6(mean = 3-0 ± 0-26 S.E.M. n = 27) and they were locatedonly in the anterior half of Al (Figs 3B and 4). Theposition of these cells and the point of entry of theaxons of their progeny into the neuropile indicated thatthese neuroblasts were members of the set of stem cellsnormally found only in thoracic ganglia (Figs 3 and 4).In the 27 Al ganglia that were examined in detail, only3 of the thoracic specific neuroblasts were representedectopically in mutant larvae; the B, K and L neuroblasts(Fig. 3B). The frequency with which these 3 neuro-blasts were found in Al of mutant larvae ranged from26% to 72%. In many instances, only one of theneuroblast pair was found. In no instance did we findextra neuroblasts that were not homologous to thosefound in the more anterior ganglia.

Ganglion A2 possesses only 10 of the neuroblasts thatare found in Al. Since the epidermis of Octo animalsoccasionally shows partial transformation of ventralA2, we examined the pattern of neuroblasts present inganglion A2 of mutant larvae (Fig. 3). Approximately15 % of the Octo animals examined possessed at least

one supernumerary neuroblast in A2. As was true ofAl, the supernumerary neuroblasts were found only inthe anterior half of the ganglion. In all cases, the extraneuroblasts were identified as the A neuroblast, whichis normally found only in ganglia T2-A1 (Fig. 4). In allbut one case, only one of the A neuroblast pair wasfound.

Size of neuronal lineages in Octo larvaeSegmental differences in neuronal number arise notonly from differences in the number of neuroblasts, butalso from variations in the number of progeny gener-ated by homologous stem cells in different segments(Bate, 1976; Booker & Truman, 1987). Within thesegmental ganglia of Manduca larvae, all the neuro-blasts begin to divide at approximately the same time,the mid-second to early third instar. Yet, by the onset ofmetamorphosis there are dramatic differences in thenumber of progeny associated with each of the stemcells. Indeed, the number of progeny generated by aparticular neuroblast is characteristic for that stem cell,but varies according to its segmental location (Fig. 5).The thoracic neuroblasts typically generate a signifi-cantly greater number of progeny by the end of thefeeding larval stage than do their homologues located inabdominal ganglia A2 to A8. The number of progenyproduced by an Al neuroblast is intermediate betweenthat seen for its thoracic and abdominal homologues.

We counted the number of progeny associated witheach Al neuroblast in both wild-type and mutantlarvae. For the majority of neuroblasts normally foundin Al, the number of progeny produced by a given stemcell by the end of the feeding period was the same inboth wild-type and Octo larvae. The only exception wasthe M neuroblast, in which about half of the lineageswere in the range normally seen for thoracic M neuro-blast lineages (Fig. 5). None of the A2 neuroblastsshowed lineages that were larger than normal.

Of the ectopic neuroblasts found in the Al (B, K andL) all produced the number of cells characteristic of therespective thoracic stem cell (e.g. Fig. 5C). The ectopicA neuroblast in A2 generated the number of progenyusually associated with the A neuroblast of Al, notthose in the thorax (data not shown).

Influence of a homeotic mutant on the insect CNS 625

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Fig. 3. (A) Illustrates the distribution of neuroblasts in T2, Al and A2 of wild-type animals. The circles represent theposition of the identifiable neuroblasts. The filled circles in T2 and Al represent the neuroblasts which drop out of the arrayin the next ganglion. In those instances where there is uncertainty as to which neuroblasts drop out of the array (the I-J andN-T clusters) they are shaded. (B) Represents the frequency of occurrence (in per cent) of the identifiable ectopicneuroblasts found in Al and A2 of Octo larvae (n = 27). (C) Reconstructions of Al and A2 from two fifth instar Octo larvaeshowing the distribution of the postembryonic neuroblasts. Ectopic neuroblasts are identified with the letter of their thoraciccounterparts.

Discussion

The Octo mutation results in the transformation of theventral epidermis of the first, and to a lesser extent thesecond, abdominal segments in the anterior directionresulting in the formation of supernumerary thoraciclegs. The penetrance of the Octo phenotype in homo-zygous animals was complete and the level of ex-pression was relatively high. A genetic analysis of thismutation revealed that it is an autosomal mutationwhich exhibits incomplete dominance.

The phenotype of Octo is similar to several of themutations of the E locus of the moth Bombxy mori

(Tajima, 1964). Mutations in the E locus alter segmen-tal identity and have properties similar to mutants of theBX-C of Drosophila. The Octo phenotype is alsosimilar to bithoraxoid (bxd), a mutation within the BX-C (Lewis, 1978). In bxd mutants, there is a transform-ation of the first abdominal segment to metathoracicresulting in the appearance of supernumerary thorax-like legs. Like Octopod, most bxd alleles when homo-zygous have little effect on dorsal structures. UnlikeOcto, alleles of bxd do not result in the appearance ofthoracic legs on the second abdominal segment. How-ever, for some alleles when hemizygous there is evi-dence of a slight transformation of more posterior

626 R. Booker and J. W. Truman

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Fig. 4. Set of photomicrographs showing lineages from ectopic neuroblast within the segmental ganglia of Octo larvae.(A) Section through the anterior region of Al revealing a mitotic F neuroblast and a portion of the C nest. In addition, aportion of an ectopic B nest can be seen. (B) Section through A2 of a mutant animal revealing an ectopic A nest. Bars,20 tan.

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Fig. 5. Counts of the number of progenyassociated with the neuroblasts normallyfound in both T3 and Al of wild-type (+)and mutant larvae. In each instance, thetop panel represents the progeny associatedwith the T3 neuroblast; the middle panel,its Al homologue; and the bottom panel,the Al homologue in Octo larvae. (A) TheG neuroblast; (B) the M neuroblast;(C) the B neuroblast.

abdominal segments (Lewis, 1978). For example, sen-sory structures normally associated with the ventralsurface of the thoracic segments appear in all theabdominal segments in such hemizygotes. Whether theOcto mutation lies within Manduca's equivalent of theBX-C has yet to be determined. Recently, a Manducagenomic clone was isolated which, based on sequenceanalysis, contains a sequence similar to the homeo-domain within the abdominal-A locus of Drosophila'sBX-C (L. Nagy, R. Booker and L. M. Riddiford,unpublished data). This suggests that at least someportion of the BX-C is conserved in Manduca and thatthis approach may provide a handle for a more detailedanalysis of the Octo mutation.

Studies on Drosophila have given some insight intothe role of homeotic mutations in determining the fateof central neural elements. In embryos deficient for theentire BX-C (Jimenez & Campos-Ortega, 1981), theirCNS fails to condense and all of the segmental gangliadisplay a staining pattern normally observed only in the

thoracic ganglia. Similarly, in adult flies carrying thebxd mutation, a supernumerary leg neuromere is oftenformed, suggesting transformation of Al to a thoracicneural segment (Teugels & Ghysen, 1983; Ghysen &Lewis, 1986). Importantly, in this latter example, thepresence of this transformed neuromere does not re-quire the obvious transformation of epidermal struc-tures. BX-C mutations also influence the segment-specific pattern of commissures within the segmentedventral nerve cord (Teugels & Ghysen, 1983). Thereport of Green (1981) using the triple mutant abx bx3

pbx is the only one to examine the CNS of mutant fliesusing an identifiable class of central neurones, the legmotorneurones. These results support a role forhomeotic mutations in determining the fate of centralneurones within their domain of influence. However,many of the differences observed between the motor-neurones of wild-type and mutant individuals are subtleand difficult to interpret. Taken together, the resultsargue that the homeotic genes of Drosophila play an

Influence of a homeotic mutant on the insect CNS 627

important role in shaping the segmental differencesobserved within its CNS.

Analysis of postembryonic neuroblasts and theirmitotic activity provides a new dimension for examiningthe role of homeotic genes in CNS development. Oneeffect of the Octo mutation is to increase the number ofneuroblasts found in Al and, to a lesser extent, in A2.In all cases, the supernumerary neuroblasts were hom-ologous to stem cells that are normally found only inmore anterior segments. Interestingly, the extra neuro-blasts only appeared in the anterior half of the ganglion.Whether this limited distribution of the supernumeraryneuroblasts is suggestive of compartments within theCNS of Manduca cannot be determined from theanalysis of this single mutation.

Only 3 out of 11 thoracic specific neuroblasts thatcould possibly be present were found ectopically in theganglia of mutant larvae; the B, K and L neuroblasts. InA2 only a single ectopic neuroblast, the A neuroblast,was found. The reason for this selectivity of the Octomutation is not known at present. One speculation isthat these particular lineages may deal with sensoryprocessing or motor activity associated with the ventralregion of a thoracic segment, and might therefore bemore susceptible to the influences of a gene controllingthe segmental fate of ventral structures.

While the relationship between the embryonic andpostembryonic neuroblasts of Manduca has not beendetermined, analysis of another holometabolous insect,the tsetse fly, Glossina palidipes, shows that the neuro-blasts found in larval ganglia are surviving embryonicneuroblasts (Truman, unpublished results). Accord-ingly, in Manduca the various segments of the embryostart with essentially identical arrays of stems cells(Thomas et al. 1984). Cell death late in embryogenesispresumably establishes the segment-specific sets thatone finds in the larva. Consequently, the Octo mutationpresumably is involved in determining which of theembryonic neuroblasts survive the round of cell deathto go on to contribute a postembryonic lineage withinthe affected segments.

In terms of neurogenesis, segmental differences areseen not only in the number of stems cells, but also inthe number of progeny generated by a given neuroblastin different segments. Our analysis of the segmentsaffected by the Octo mutation revealed only a singleinstance where a stem cell normally present in thatsegment had been transformed in terms of the size ofthe lineage it produced. In the case of the M(A1)neuroblast, about half of those examined had generatedthe number of progeny normally produced by theirthoracic homologues. In many mutant larvae, the fateof the two M(A1) neuroblasts was mixed with onegenerating a thoracic-sized lineage and the other thenumber typical for Al.

All of the ectopic neuroblasts in Al (B, K and L)generated the number of progeny typically associatedwith their respective thoracic homologues. At this levelof analysis, there is no means for determining whichparticular thoracic segment they represent since theseneuroblasts generate similar numbers of progeny in all 3

thoracic segments. Interestingly, the ectopic A neuro-blasts in A2 did not produce a thoracic-sized lineage,but generated the same number of progeny that istypical for Al. Thus, the nervous system takes on acharacteristic of Al, while the epidermis assumes tho-racic characters with the formation of leg parts.

While care has to be taken in interpreting the resultsfrom the analysis of a single mutation, they suggest thathomeotic genes can influence at least two aspects ofneurogenesis. First, they can influence the number ofneuroblasts present in the ganglia during the larvalstage, thereby altering the array of postembryoniclineages. Second, they appear capable of altering thesegment-specific number of progeny generated by aparticular neuroblast. Taken together this suggests thatsegmental determination plays a critical role in shapingthe regional differences in neuronal number observedin the segmental ganglia of adult insects.

We thank Drs David Morton, Jane Witten, Susan Fahrbachand Shirley Reis for a critical reading of the manuscript. Thiswork was supported by a grant from NIH (ROI-NS-13079) toJWT.

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THOMAS, J. B., BASTIANI, M., BATE, M. & GOODMAN, C. G. (1984). bithorax complex in Drosophila. Cell 39, 163-171.From grasshopper to Drosophila: A common plan for neuronaldevlopment. Nature, Lond. 310, 203-207. (Accepted 1 December 1988)