Brown, S.

Denell, R.

Fellers, J.

Schmidt-Ott, U.

Shippy, T.

Stauber, M.

English

MPG.PuRe

CorrespondenceA strategy formapping bicoid on thephylogenetic treeS. Brown*, J. Fellers†,T. Shippy*, R. Denell*,M. Stauber‡ andU. Schmidt-Ott‡In Drosophila, a gradient of Bicoidprotein (BCD) originates fromprelocalized mRNA at the anteriorpole of the egg and establishesdevelopmental programs includingthose for the larval head and thorax ina concentration-dependent manner[1,2]. Orthologous proteins have beenreported only from cyclorrhaphan flies[3,4]. However, a BCD-likedeterminant has been postulated for alarge variety of insects includingleafhopper, beetles and midges [5–7].It therefore remains to be determinedwhether diverged bicoid (bcd)orthologs exist in other insect orders.bcd encodes a homeodomain-containing transcription factor, and islocated immediately upstream ofzerknüllt (zen) in the Hox genecomplex (Hox-C) of variousdrosophilids [8,9]. Based on thesefindings M. Akam (in [10]) suggestedthat bcd originated as a result of agene duplication involving zen.However, it has been difficult to testthis hypothesis due to rapidsequence evolution of bcd and zen[3]. Only recently, sequence analysisof a bcd homolog from the basalcyclorrhaphan fly Megaselia(Phoridae) provided direct supportfor a sister-gene relationship of bcdand zen, and implicated the positionof bcd upstream of zen in the Hox-Cas ancestral [11].We tested for linkage of bcd andzen in the blowflies Calliphoraerythrocephala and Lucilia sericata(Calliphoridae, Diptera) where bcdhomologs have been identifiedpreviously [4]. Using degenerate PCRprimers we isolated zen homeoboxesfrom both species (Figure 1a).Specific nested primer pairs, whenused in long range PCR, amplifysingle genomic DNA fragments of12 kb (Calliphora) and 16 kb (Lucilia),linking the homeoboxes of the bcdand zen homologs (Figure 1b). Theidentity of each fragment was verifiedby Southern-blot hybridization andterminal sequencing . Thus, bcd islinked to a Hox class 3 gene inblowflies (Calyptratae) as it is indrosophilids (Acalyptratae), and the 5′to 3′ orientation of bcd and zen withrespect to one another is conserved.This linkage was probably inheritedfrom the common ancestor of themonophyletic Schizophora(Acalyptratae and Calyptratae) [12],which comprise the majority offamily-level diversity of Diptera [13].Our observations strongly support thehypothesis that bcd arose as a tandemduplication of zen within the Hox-C(Figure 2). We conclude that analysisof the relevant Hox-C portion ofselected species provides a means tomap the origin of bcd on thephylogenetic tree.In the red flour beetle Triboliumcastaneum, a holometabolous insectdistantly related to flies, orthologs ofthe eight arthropod homeotic genes[14], as well as ftz [15] and zen [16]are arrayed in the same order as theirDrosophila counterparts (Figure 2),an order that has been maintained forover 300 million years. To determinewhether a highly diverged bcdortholog is located in its predictedposition in the Tribolium Hox-C wesequenced a BAC clone spanning theregion from the 5′ exon of mxp/Hox2to Tcftz (Genbank accessionAF321227). We analyzed thissequence using BLAST (NCBI), andthe BCM genefinder program(Baylor College of Medicine Website) to predict open reading framesand putative transcription units. Wefound a second zen gene (see thehomeodomain comparisons inFigure 1a) immediately downstreamof the one previously identified.These genes are most likely theresult of an independent duplicationin the lineage leading to Tribolium.Although known transcription unitswere faithfully predicted, no otherhomeodomain-encoding sequenceswere found. The Tribolium Hox-Cdoes not, therefore, contain a bcdortholog in the interval betweenmxp/Hox2 and TcDfd/Hox4.Conservation of the relativepositions of zen and bcd in blowfliesMagazine R43Figure 1bcd–zen linkage in blowflies. (a) Homeo-domain sequence alignments of BCD[4,11,17] and ZEN from Drosophilamelanogaster (Dm) [18], Megaselia abdita(Ma) [11], Tribolium castaneum (Tc),Calliphora erythrocephala (Ce) and Luciliasericata (Ls) with internal primer positions(arrows) used for long range DNAamplification experiments and sequencing.The partial zen homeoboxes of Calliphoraand Lucilia were amplified with degenerateprimers as described previously [11].(b) Amplified DNA fragments from Calliphora(12 kb) and Lucilia (16 kb) that were used astemplate for terminal sequencing.        1                                                          60Dm-ZEN  LKRSRTAFTSVQLVELENEFKSNMYLYRTRRIEIAQRLSLCERQVKIWFQNRRMKFKKDIMa-ZEN  TKRSRTAFTSIQLLELENEFKKNKYLNRPRRIEISLRLSLSERQVKIWFQNRRMKSKKDRTc-ZEN  GKRARTAYTSAQLVELEREFHHGKYLSRPRRIQIAENLNLSERQIKIWFQNRRMKHKKEQTc-ZEN2 GKRARTAYTSSQLVELEREFHRSKYLCRPRRIQMAQNLNLTERQIKIWFQNRRMKFKKEECe-ZEN  LKRSRTAFTSSQLVQLESEFKRLs-ZEN  LKRSRTAFTSSQLVQLESEFKRNMYLYRTRRIEIAQRLSLCERQVKIWFzenbcd 12 / 16 kbCe Ls8,610,112,215,017,0kb5’5’ 3’3’        1                                                          60Dm-BCD  PRRTRTTFTSSQIAELEQHFLQGRYLTAQRLADLSAKLALGTAQVKIWFKNRRRRHKIQSMa-BCD  RRRTRTTFTSSQIAELEEYFRQGKYLNNIRLSELTGRLNLGQAQVKIWFKNRRRRFKIEQCe-BCD      RTTFTSAQIAELEQHFLQGRYLTSSRLAQLSAKLALGTAQVKIWFKNRRRRHKIQALs-BCD      RTTFTSAQIAELEQHFLQGRYLTSSRLAQLSAKLALGTAQVKIWFKNRRRRHKIQS(a)  Current Biology(b)NMYLYRTRRIEIAQRLSLCERQVKIWFand drosophilid fruitflies, combinedwith the absence of a bcd gene in thecorresponding position in theTribolium Hox-C (Figure 2), providedirect support for the hypothesis thatbcd originated recently [11],presumably after the basal radiationof holometabolous insects. As thehomeotic complexes of additionalholometabolous insects are analyzedfor bcd, our understanding of theorigin of bcd will be refined.Our conclusion that bcd emergedafter the basal radiation ofholometabolous insects does notnecessarily imply that beetles andmore primitive insects developwithout an anterior determinant. Infact, several observations have beentaken as evidence for such a factor inbeetle and leafhopper development[5,6]. If these inferences are correctour results suggest that BCDfunctionally replaced an ancestralanterior determinant.References1. St Johnston D, Nüsslein-Volhard C: Theorigin of pattern and polarity in theDrosophila embryo. Cell 1992,68:201-220.2. Rivera-Pomar R, Jäckle H: From gradientsto stripes in Drosophila embryogenesis:filling in the gaps. Trends Genet 1996,12:478-483.3. Sommer R, Tautz D: Segmentation geneexpression in the housefly Muscadomestica. Development 1991,113:419-430.4. Schröder R, Sander K: A comparison oftransplantable bicoid activity and partialbicoid homeobox seqences in severalDrosophila and blowfly species(Calliphoridae). Roux´s Arch Dev Biol1993, 203:34-43.5. Wolff C, Schröder R, Schulz C, Tautz D,Klingler M: Regulation of the Triboliumhomologues of caudal and hunchback inDrosophila: evidence for maternalgradient systems in a short germ embryo.Development 1998, 125:3645-3654.6. Sander K: Specification of the basic bodypattern in insect embryogenesis. AdvInsect Physiol 1976; 12:125-238.7. Kalthoff K: Analysis of a morphogeneticdeterminant in an insect embryo (SmittiaSpec., Chironomidae, Diptera). InDeterminants of Spatial Organization.Edited by Subtelny S, Konigsberg IR. NewYork: Academic Press; 1979:79-126.8. Randazzo FM, Seeger MA, Huss CA,Sweeney MA, Cecil JK, Kaufman TC:Structural changes in the Antennapediacomplex of Drosophila psuedoobscura.Genetics 1993, 133:319-330.9. Terol J, Perez-Alonso M, de Frutos R:Molecular characterization of thezerknüllt region of the Antennapediacomplex of D. subobscura. Chromosoma1995, 103:613-624.10. Sander K: The evolution of insectpatterning mechanisms: a survey ofprogress and problems in comparativemolecular embryology. Development1994, Suppl:187-191. 11. Stauber M, Jäckle H, Schmidt-Ott U: Theanterior determinant bicoid of Drosophilais a derived Hox class 3 gene. Proc NatlAcad Sci USA 1999, 96:3786-3789.12. McAlpine JF: Phylogeny and classificationof the Muscomorpha. In Manual ofNearctic Diptera. Edited by McAlpine JF.Hull: Canadian Government PublishingCentre, 1989:1397-1518.13. Yeates DK, Wiegmann BM: Congruenceand controversy: toward a higher-levelphylogeny of Diptera. Annu Rev Entomol1999, 44:397-428.14. Denell RE, Brown SJ, Beeman RW:Evolution of the organization andfunction of insect homeotic complexes.Sem Cell Dev Biol 1996, 7:527-538.15. Brown SJ, Hilgenfeld RB, Denell RE: Thebeetle Tribolium castaneum has a fushitarazu homolog expressed in stripesduring segmentation. Proc Natl Acad SciUSA 1994; 91:12922-12926.16. Falciani F, Hausdorf B, Schröder R, Akam M,Tautz D, Denell R, Brown S: Class 3 Hoxgenes in insects and the origin of zen. ProcNatl Acad Sci USA 1996, 93:8479-8484.17. Berleth T, Burri M, Thoma G, Bopp D,Richstein S, Frigerio G, et al.: The role oflocalization of bicoid RNA in organizingthe anterior pattern of the Drosophilaembryo. EMBO J 1988, 7:1749-1756.18. Rushlow C, Doyle H, Hoey T, Levine M:Molecular characterization of thezerknüllt region of the Antennapediagene complex in Drosophila. Genes Dev1987, 1:1268-1279.19. Kristensen NP: Phylogeny of extanthexapods. In Insects of Australia. Edited byC.S.a.I.R.O.D.o. Entomology. Melbourne:University Press; 1991:125-140.20. Kaufman TC, Seeger MA, Olsen G:Molecular and genetic organization ofthe Antennapedia gene complex ofDrosophila melanogaster. In Advances inGenetics. Edited by Wright TRF. AcademicPress, New York; 1990:309-362. 21. von Allmen G, Hogga I, Spierer A, Karch F,Bender W, Gyurkovics H, Lewis E: Splits infruitfly Hox gene complexes. Nature1996, 380:116.22. Beeman RW: A homeotic gene cluster inthe red flour beetle. Nature 1987,327:247-249.AcknowledgementsSJB and RED thank Barbara Van Slyke forexpert technical assistance. This work wassupported by NIH and NSF (SJB and RED),and by the Max-Planck-Gesellschaft and theDeutsche Forschungsgemeinschaft (US-O).Addresses: *Division of Biology, KansasState University, Manhattan, Kansas 66506,USA. †USDA-ARS, Department of PlantPathology, Kansas State University,Manhattan, Kansas 66506, USA. ‡Max-Planck-Institut für biophysikalische Chemie,Abteilung Molekulare Entwicklungsbiologie,Am Fassberg 11, 37077 Göttingen,Germany.R44 Current Biology Vol 11 No 2Figure 2Simplified phylogenetic tree of holometabolousinsects, adapted from [19]. Linkage of Hoxgenes in Drosophila [8,9,20,21] Calliphora,Lucilia and Tribolium [14–16,22] is indicatedto the right (not to scale). Note that a secondzen gene (not shown) has been reportedfrom D. melanogaster but not from otherdrosophilids. The extent of the Tribolium BACclone is shown by a line above the TriboliumHox-C; the sequenced region is marked by athick line. Abbreviations: lab, labial; pb,proboscipedia; zen, zerknüllt; bcd, bicoid;Dfd, Deformed; Scr, Sex-combs reduced; ftz,fushi-tarazu; Antp, Antennapedia; Ubx,Ultrabithorax; abdA, abdominal-A; AbdB,Abdominal-B; mxp, maxillopedia; Cx,Cephalothorax; ptl, prothoraxless; Utx,Ultrathorax; A, Abdominal and eu,extraurogomphri.Hox1 Hox2 Hox4 Hox5 Hox6-8 Hox9-13AbdBpb zenlab Dfd   Scr   ftz   Antp   Ubx   abdAHox3Hox3Hox3Hox1 Hox2 Hox9-13   eumxp TcDfd   Cx    Tcftz   ptl   Utx   ATclabHox6-8Hox4  Hox5TriboliumDrosophilaCalliphoraLuciliaHOLOMETABOLATczen2 TczenHox3bcdCebcdCezenLsbcdLszen   Current Biology   COLEOPTERALEPIDOPTERADIPTERAHYMENOPTERA(Antennapedia-subgroup)

A strategy for mapping bicoid on the phylogenetic tree.

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A strategy for mapping bicoid on the phylogenetic tree.

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