the tribolium dorsal gradient - development · tribolium toll(maxton-küchenmeister et al., 1999)...

INTRODUCTION

Drosophila embryos represent an extreme case of long-germdevelopment, a mode of embryogenesis which is likely to bederived since it is found only in other holometabolous insectorders, but is absent in the more primitive hemimetabolousgroups (Sander, 1976). The long-germ type of development ischaracterised by two features. First, all segments along theanteroposterior (AP) axis form more or less synchronously andtheir anlagen are established before gastrulation commences.Secondly, the anlagen of the embryo extend from the anteriorto the posterior tip of the blastoderm. Most of the blastodermgives rise to the embryo proper while only a small portiondevelops into extraembryonic coverings. In short-germ insectson the other hand, only head segments are specified prior togastrulation while more posterior segments emerge later froma posterior growth zone. Additionally, the anlagen of theembryo may occupy only a small region of the blastodermwhile the majority of the blastoderm cells form the serosa, theouter extraembryonic membrane.

The ‘long-germ’ mode of development places highdemands on the amount of positional information required

before gastrulation for metameric and dorsoventral (DV)patterning. Positional information for DV patterning mustexist along the entire egg length since mesoderminvagination, like segmentation, occurs synchronously alongthe anteroposterior axis. In Drosophilathis is accomplishedthrough an extraembryonic signaling cascade, which isactivated along the ventral side of the egg. Ventral activationis triggered by modifications of the extracellular matrix,which can be traced back to the establishment of DV polarityduring oogenesis (Nilson and Schüpbach, 1998; Sen et al.,1998).

The embryo senses the extracellular signal through thetransmembrane receptor Toll which is maternally provided anddistributed uniformly in the plasma membrane (Morisatoand Anderson, 1995). Ventrally activated Toll stimulates thenuclear import of the rel/NF-κB protein Dorsal which, prior tosignaling, is evenly distributed in the cytoplasm where is formsa complex with the IκB-like Cactus protein (Belvin et al., 1995;Bergmann et al., 1996). As a result of ventral signaling, anuclear Dorsal protein gradient forms with peak levels alongthe ventral midline. Thus, the activation pattern which leads toDorsal gradient formation is likely to be a stripe along the

5145Development 127, 5145-5156 (2000)Printed in Great Britain © The Company of Biologists Limited 2000DEV7840

In the long-germ insect Drosophila melanogasterdorsoventral polarity is induced by localized Toll-receptoractivation which leads to the formation of a nucleargradient of the rel/ NF-κB protein Dorsal. Peak levels ofnuclear Dorsal are found in a ventral stripe spanningthe entire length of the blastoderm embryo allowing allsegments and their dorsoventral subdivisions to besynchronously specified before gastrulation. We show thata nuclear Dorsal protein gradient of similaranteroposterior extension exists in the short-germ beetle,Tribolium castaneum, which forms most segments from aposterior growth zone after gastrulation. In contrast toDrosophila, (i) nuclear accumulation is first uniform andthen becomes progressively restricted to a narrow ventralstripe, (ii) gradient refinement is accompanied by changes

in the zygotic expression of the TriboliumToll-receptorsuggesting feedback regulation and, (iii) the gradient onlytransiently overlaps with the expression of a potentialtarget, the Tribolium twist homolog, and does not repressTribolium decapentaplegic. No nuclear Dorsal is seen in thecells of the growth zone of Tribolium embryos, indicatingthat here dorsoventral patterning occurs by a differentmechanism. However, Dorsal is up-regulated andtransiently forms a nuclear gradient in the serosa, aprotective extraembryonic cell layer ultimately coveringthe whole embryo.

Key words: Insect embryogenesis, Evolution of development, Toll,twist, decapentaplegic, zerknüllt, Nuclear concentration gradient,Innate immunity, Tribolium castaneum

SUMMARY

The maternal NF-κ B/Dorsal gradient of Tribolium castaneum : dynamics of

early dorsoventral patterning in a short-germ beetle

Gang Chen 1,‡, Klaus Handel 1,§ and Siegfried Roth 1,*,¶

1Max-Planck-Institut für Entwicklungsbiologie, Spemannstrasse 35/II, D-72076 Tübingen, Germany‡Present address: Universität Tübingen, Zoologisches Institut, LS Entwicklungsphysiologie, Auf der Morgenstelle 28, D-72076 Tübingen, Germany§Present address: Aventis Research & Technologies GmbH & Co KG, Operative Forschung. Industriepark Höchst. Gebäude G 830, D-65926 Frankfurt am Main,Germany¶Present address: Institut für Entwicklungsbiologie, Universität zu Köln, Gyrhofstrasse 17, D-50923 Köln, USA*Author for correspondence (e-mail: [email protected])

Accepted 25 September; published on WWW 2 November 2000

5146

entire ventral side of the egg. How this activation pattern ariseswith such precision is still the subject of ongoing research (forreview see Roth, 1998).

We wondered how much DV positional information existsin a short-germ embryo before gastrulation and how much ofthis information is maternally provided. This question isinteresting not only because the embryo proper forms froma more restricted region of the blastoderm and thereforeglobal DV information along the entire egg length might notbe necessary. In addition, many classical ligation andfragmentation experiments have provided evidence forextensive regulation of DV patterning in short- andintermediate- germ embryos of hemimetabolous as well asholometabolous insects (Sander, 1976). Impressive exampleshave been described in which apparently complete embryosform from a variety of DV egg fragments. Althoughrudimentary forms of DV pattern regulation exist even inDrosophila (Roth and Schüpbach, 1994; Roth et al., 1999), itis unlikely that Drosophila has the regulatory capacityobserved in classical experiments with lower insects sinceimportant aspects of DV axis specification occur early duringDrosophila oogenesis (Nilson and Schüpbach, 1998; Sen etal., 1998). This suggests that during insect evolution majorchanges of the mechanisms of DV axis formation took place.

Besides its function in axis formation the Toll-rel/NF-κBpathway is also required to mediate the innate immuneresponse in Drosophila. Here, pathway activation induces theexpression of potent antimicrobial peptides upon septic injury(Anderson, 2000). Not all pathway components are identical inboth contexts. Thus, for injury-induced signaling Dorsal isreplaced by two other rel/NF-κB proteins, dorsal-relatedimmunity factor (Dif) and Relish (Ip et al., 1993; Dushay etal., 1996). Nevertheless, the employment of Toll signaling bothin axis formation and immunity poses the interesting questionof its ancestral role and the evolutionary path that leads to itsfunctional diversification.

To approach both the evolution of the Toll pathway and themechanistic alterations of DV pattern formation among insectswe have chosen the short-germ beetle Tribolium castaneumforcomparison with Drosophila melanogaster. Molecular andgenetic techniques have been established for Tribolium(Berghammer et al., 1999) and some zygotic DV genes havealready been characterised (Sommer and Tautz, 1994; Falcianiet al., 1996; Sanchez-Salazar et al., 1996). On the basis of theexpression pattern of the snail, twistand dpphomologs ofTribolium the existence of a Dorsal-like activity had beensuggested (Sommer and Tautz, 1994; Sanchez-Salazar et al.,1996). Here, we show that in Tribolium embryos a maternallyexpressed rel/NF-κB protein indeed exists which is highlysimilar to Drosophila melanogasterDorsal (Dm-dl). Like Dm-dl, it forms a nuclear concentration gradient at the blastodermstage. We describe the spatial and temporal dynamics ofgradient formation and compare it to the expression ofTribolium Toll (Maxton-Küchenmeister et al., 1999) as well asto the expression of potential Dorsal target genes of Tribolium.This analysis firstly suggests positive feedback control ofgradient formation involving Dorsal-dependent activation ofToll. Secondly, is shows that the relationship between differentnuclear Dorsal concentrations and the cell fates along thedorsoventral axis is more indirect in Triboliumas compared toDrosophila. Finally, early up-regulation of Dorsal in the serosa

of Tribolium embryos leads us to some speculations about theorigin of the patterning function of the Toll/Dorsal pathway.

MATERIALS AND METHODS

Tribolium and Drosophila stocksThe beetle stock (ecotype San Bernadino) was kept and eggs collectedas described by Beermann (1998). The following Drosophilamelanogasterstocks were used. Wild type: Oregon R, dlT: w; In(2L)cn pr bw/CyO, b, dlI5: w; dlI5 cn bw/CyO DTS, Df(2L)TW119: w;Df(2L)TW119 cn bw/CyO (Roth et al., 1989), and α-tubGal4:VP16:maternal GAL4 driver line on the X chromosome provided by D. StJohnston (Maxton-Küchenmeister et al., 1999).

Cloning of the Tc-dl homologTwo fragments covering the entire rel homology domain (RHD) ofDm-dl were used to screen an embryonic cDNA library of Triboliumcastaneum(Wolff et al., 1995) employing low stringency conditions.One cDNA of 2.2 kb was recovered and used subsequently forscreening under high stringency conditions, which lead to the isolationof 7 additional cDNAs of identical length and the same restrictionmaps. One was sequenced completely and two others were shown tohave identical 5′-ends suggesting that the obtained sequence (2192 bp)represents a complete cDNA. It consists of a 233 bp 5′-untranslatedregion (UTR), an 1671 bp open reading frame and a 288 bp 3′ UTRwith a typical polyadenlylation signal followed by a poly(A) tail.

Rescue constructs Rescue was not obtained using pCaSpeRbcd (Stein et al., 1998) evenafter the 5′- and 3′-UTRs of Tc-dlwere replaced by the correspondingUTRs from Dm-dl. The RNA produced from pCaSpeRbcd [Tc-dl]transgenes appears to be retained in the nurse cells. pUAST[Tc-dl] withα-tubGal4:VP16 as driver also showed no rescue. The final rescueconstruct was pUASp[Tc-dl]. The full-length cDNA of Tc-dlwascloned into the BamHI/ XbaI site of pUASp (Rørth, 1998). pUASp [Dm-dl]: Dm-dl full-length cDNA was cloned into the KpnI/XbaI of pUASp.

RNA injectionsDm-dlor Tc-dlcDNAs were cloned into the EcoRI/XhoI site of pCS2(Rupp et al., 1994) and RNA injections were done as described byJazwinska et al. (1999).

Production and purification of Tc-dl antibodiesTwo fusion protein constructs were made by subcloning the RHD of Tc-dl into the His-tag vector pRSET-A (Invitrogen) and GST-tag vectorpGEX-2T (Pharmacia), respectively. The pRSET-A-derived fusionprotein was used to inject rabbits using a standard immunisation protocol(Harlow and Lane, 1988). The serum from boosted rabbits was affinity-purified using the GST fusion protein. The antibodies were tested bywestern blot analysis using both fusion proteins and embryonic extractsessentially as described previously (Roth et al., 1989).

Immunohistochemistry Immunostaining and in situ hybridisation was done essentially asdescribed previously (Roth et al., 1989; Tautz and Pfeifle, 1989).YOYO-1 (Molecular Probes) and DAPI stainings were donesubsequently.

RESULTS

A rel/NF-κ B protein from Tribolium castaneum withhigh sequence similarity to Drosophila Dorsal Three rel/NF-κB genes have been identified from Drosophilamelanogaster: dorsal (Dm-dl), dorsal-related immunity factor(Dif) and relish (Steward, 1987; Ip et al., 1993; Dushay et al.,

G. Chen, K. Handel and S. Roth

5147The Tribolium Dorsal gradient

1996) which show significant similarity only in an N-terminaldomain of approximately 300 amino acids responsible forDNA binding, dimerisation and IκB/Cactus interaction, calledthe rel homology domain (RHD; Verma et al., 1995). TheRHDs of Dif and relishshow only 42% and 26% identity,respectively to the RHD of dorsal, being on average, as fardiverged from dorsalas the vertebrate members of the rel/NF-κB family (Fig. 1C). Thus, potential dorsal orthologs fromother species are likely to be recognised on the bases of theirRHD sequences. We therefore used probes generated from theRHD of Dm-dl for low stringency screening of an embryonicTribolium castaneum cDNA library and recovered a 2.2 kb

cDNA with a single open reading frame coding for a 556 aminoacid protein (Fig. 1A). The conceptual translation revealed anN-terminal RHD which is highly similar to that of Dm-dl(77%identity) and gambif1(70% identity), a recently identifiedDorsal-like protein form Anopheles gambiaewith a possiblerole in the immune system (Barillas-Mury et al., 1996). Thesequences flanking the RHD show no similarity to knownproteins and contain no recognisable protein motifs. Inparticular, the C-terminal region which by analogy to otherrel/NF-κB proteins is likely to be a transactivation domain,does not contain the polyglutamine, -alanine or -asparaginestretches characteristic of the C-termini of Dm-dl, Dif and

Fig. 1.The Dorsal protein of Tribolium castaneum. (A) Comparison of the amino acid sequences of Dorsal from Tribolium castaneum (Tc-dl),Drosophila melanogaster(Dm-dl) and Gambiae immune factor 1 (Gambif 1) from Anopheles gambiae. Shading of identical amino acidsreveals significant conservation only in the rel homology domain (boxed in red). The GenBank Accession Number for Tc-dl is AY008296.(B) Domain structure of Tc-dl, Dm-dl, Gambif1 and Dif. The numbers refer to the percent identity in the rel homology domain between Tc-dl,Dm-dl, Gambif1 and Dif. Although there is no significant sequence conservation outside the Rel homology domain, Dm-dl, Gambif1 and Difcontain polyglutamine, polyasparagine or polyalanine stretches in the C-terminal, potential transactivation domain which are absent in Tc-dl.(C) Phylogenetic comparison with other known rel/NF-κB family members. EMBL Data Library accession nos: Rel A (p65 human), M62399;Rel B (human), M83221; C-Rel (human), M99576; NF-κB1 (p100 human), X61498; NF-κB2 (p105 human), M55643; Dif, L29015; DmDorsal, M23702; Gambif 1, X95912. The sequences were aligned using the CLUSTAL method (MEGALIN from Lasergene with Gap penalty= 10 and Gap length penalty = 10, Higgins et al., 1989). Tree reconstruction was done with PAUP (Swofford, 1998) using the neighbour-joiningalgorithm (standard settings without transition matrix). The bootstrap values support the close phylogenetic relationship between Tc-dl, Dm-dland Gambif 1.

5148

gambif1 (Fig. 1B). We call this new rel/NF-κB proteinTribolium castaneum-dorsal(Tc-dl) because of the proteindistribution in early Triboliumembryos (see below).

Both mRNA and protein of Tribolium castaneum-dorsal are maternally expressedTc-dl mRNA is present in freshly laid eggs and preblastodermembryos where it is evenly distributed around the embryoniccircumference (Fig. 2A,B). Transverse sections show that themRNA is concentrated in the cortex of the embryo while onlysmall amounts are present between big yolk granules in thecentre. This distribution remains unchanged during blastodermstages (data not shown). However, shortly before gastrulation

starts the mRNA concentrations increase in an anterior cap and,subsequently, high mRNA levels are found in all presumptiveserosa cells while mRNA levels decrease in the germ rudiment(Fig. 2C,D). The RNA continues to be expressed in the serosaduring later stages of embryonic development.

Tc-dl mRNA accumulates in the oocytes of mid and latestage egg chambers (data not shown) suggesting that Tc-dl, likeDm-dl mRNA (Steward et al., 1985) is maternally provided.This assumption is supported by western blot analysis using


Fig. 2.Tribolium dorsalmRNA and protein are maternallyexpressed. (A-D) Tc-dl mRNA distribution. (A,C) Anterior to the leftand dorsal up. (A,B) Preblastoderm. Whole-mount (A) and crosssection (B) show uniform mRNA distribution around the DVcircumference. The RNA is concentrated in the cortex of the egg, butlower amounts are also present in the central yolk-rich cytoplasm.(C,D) Primitive pit formation and serosal segregation. DAPI stainingto visualise the nuclei was performed after RNA in situ hybridisation.The same embryo is shown with bright-field plus fluorescence (C)and fluorescence only (D). The border between presumptive serosaand germ rudiment is indicated by arrows. High levels of Tc-dlmRNA are expressed in serosa cells. (E) Western blot with Triboliumembryonic extracts. The age of the embryos for extract preparation isgiven in hours. Commassie Blue staining was used to normalise theamounts of protein in each lane (data not shown).

Fig. 3.The formation and refinement of the nuclear Tc-dl gradient.(A,C,E-H) Bright-field images of whole embryos stained with anti-Tc-dl antibodies. (B,D) Fluorescence images of the embryos shownin A and C, respectively that were also stained with a DNA dye(YOYO-1) to visualise the cleavage nuclei. The insets in A,B andC,D show magnified energids under bright-field optics (left) andbright-field optics plus fluorescence (right). (A,B) Nuclear cycle 7.The nuclei have not reached the periphery. Tc-dl protein is present inthe cytoplasm surrounding the nuclei. (C,D) Nuclear cycle 8. Thenuclei reach the egg cortex and accumulate Tc-dl protein. (E-H).Consecutive stages of gradient refinement, which occur betweennuclear cycle 9 (E) and the onset of serosal segregation (H). Thearrow points to the anterior ‘cap’ expression of Tc-dl whichcoincides with the presumptive serosa.


antibodies raised against a bacterially produced fusion proteinwhich contained the entire RHD of Tc-dl (Fig. 2E). Inembryonic extracts the antibodies recognise two protein bandswith an apparent molecular mass of 63 kDa and 35 kDa. Theupper band is likely to correspond to the full-length protein,which has a predicted molecular mass of 62 kDa while thelower band might be a degradation product since itsdevelopmental profile mirrors that of the upper band. Tc-dlprotein is produced maternally as it can be detected duringoogenesis (Fig. 2E). During early embryogenesis proteinamounts are low, but they increase significantly when serosalsegregation starts (9 hours after egg deposition; Handel et al.,2000) and high levels of protein can be detected throughoutembryonic development.

Tc-dl protein forms a nuclear concentration gradientin blastoderm embryosAs in Drosophila preblastoderm embryos, early nucleardivisions take place in the yolk-rich centre of the Triboliumegg. The cloud of nuclei expands along the anteroposterior eggaxis and then the nuclei migrate towards the cortex(Grünfelder, 1997). Before they reach the cortex the nuclei aresurrounded by small islands of cytoplasm (energids) whichshow weak anti-Tc-dl staining (Fig. 3A,B). The nuclei appearto exclude the protein (insets of Fig. 3A,B). The cytoplasmicstaining of the energids is even along the DV axis, albeit oftenstronger in the posterior half of the embryo. When theyreach the cortex, all nuclei appear to take up Tc-dl protein(Fig. 3C,D). In contrast to Drosophila, where the very firstnuclei to reach the cortex take up Dorsal protein in agraded fashion (Roth et al., 1989; Rushlow et al., 1989;Steward, 1989), in Tribolium no asymmetry in nuclearconcentrations can be observed at this early stage.

However, two cell cycles later, Tc-dl proteinpreferentially accumulates in nuclei of one half of theembryo (Fig. 3E). A shallow nuclear concentrationgradient forms that spans about 40% of the embryoniccircumference (Fig. 3E). Unlike Drosophila, the shape andcoverings of Triboliumeggs do not allow an unequivocaldetermination of DV polarity, so it is not possible at thisstage to identify the DV position at which nuclearaccumulation begins. However, since the gradient persistsand later overlaps with the mesodermal marker gene twist(see below), we infer that nuclear accumulation, as inDrosophila, is initiated at the ventral side. During the twosubsequent syncytial blastoderm cell cycles this gradientrefines by becoming both steeper and more restricted withregard to the embryonic circumference (Figs 3F,G, 4A-D).Refinement is neither produced by nuclear migration norlinked to the division cycles. Fig. 4A-D show transversesections through embryos of the same nuclear cycle sincethey both have 69 evenly spaced nuclei around thecircumference. The gradient of the upper section spans 23nuclei (33% of the circumference) with 13 nuclei showinghigh concentrations flanked by 5 nuclei with intermediateand low levels. In the lower section, presumably from alater embryo of the same cell cycle, the gradient spreadsover less than half the number of nuclei (11 correspondingto 16% of the circumference) and is steeper, decreasingfrom highest to lowest levels over 3 nuclei. Thus, thenuclear Dorsal gradient is more dynamic in Triboliumas

compared to Drosophilawhere its lateral expansion does notchange during blastoderm stages (Roth et al., 1989).

Tc-dl disappears form the germ rudiment, butbecomes highly expressed in the serosaLater during development, the area that is occupied by thegradient shrinks to a narrow five-cell-wide stripe, whichharbours only a few scattered nuclei with high levels of Tc-dl(Figs 3H and 4E,F). This Tc-dl distribution corresponds to thestage when the prospective serosa cells first become differentfrom the cells of the germ rudiment giving rise to the embryoproper (Handel et al., 2000). The cell density decreases in theprospective serosal region since the serosa cells stop dividing,start to flatten and expand. In contrast, the cells of the germrudiment continue to divide and might also be pushed togetherthrough the flattening of the serosa cells so that here celldensity increases. Since the refinement of the Tc-dl gradient iscomplete before these changes are obvious, we believe thatthey do not significantly contribute to the narrowing of the Tc-dl domain.

While Tc-dl protein expression vanishes from the germrudiment, it is upregulated in the prospective serosa. Theprotein distribution thus parallels that of the mRNA and formsa clearly visible anterior cap of protein expression (arrow inFig. 3H). In this anterior cap the protein is mainly cytoplasmic,with the exception of some ventral nuclei located at the border

Fig. 4. The refinement of the nuclear Tc-dl gradient does not depend oncell movements. Cross sections through blastoderm embryos stained withanti-Tc-dl antibodies showing successive stages of gradient refinement. Allsections are derived from about 65% egg length (0% corresponds to theposterior pole). (A,C,E) Complete sections. (B,D,F) Magnification ofventral portion of sections shown in A, C and E, respectively. The embryosin A and C have a uniform distribution of nuclei around the circumference,but the embryo in E shows cell flattening dorsally.

5150

between germ rudiment and serosa, which maintain high levelsof nuclear Tc-dl (Fig. 3H). By the time the primitive pit forms,the nuclear gradient has disappeared completely from the germrudiment, and only weak cytoplasmic staining remains in thecells of the future ventral furrow and the primitive pit (Figs5A,B, 8D,E). During furrow formation, cytoplasmic Tc-dl alsovanishes from the germ rudiment (Fig. 5F) except in the regionof the primitive pit (data not shown). We have not observed Tc-dl nuclear staining or high levels of cytoplasmic staining in anypart of the embryo during later stages of embryogenesis. Inparticular, no nuclear accumulation was found in the cells ofthe growth zone where together with segment formation DVpatterning also has to continue (data not shown).

However, high Tc-dl protein levels persist in the

differentiating serosa cells. Interestingly, before serosal closureis complete, a stripe of serosa cells facing the ventral side ofthe egg shows nuclear uptake, while the remaining lateral anddorsal serosa cells have strong cytoplasmic staining (Fig. 5C-F). Thus, the asymmetric nuclear uptake, which previously hadtaken place in the embryo, is now restricted to extraembryoniccells facing the perivitelline space. During later stages ofdevelopment a few embryos show strong nuclear accumulationof Tc-dl in circular patches of the serosa (Fig. 5G). Since thesepatches do not always occur at the same location and since theydo not have regularly shaped cells in their centre they mayrepresent scars resulting from injuries of the serosa. Tc-dlnuclear uptake might be a consequence of pathogen stimulationsuggesting that high levels of Tc-dl in the serosa fulfil aprotective function.

Tc-Toll as a potential target gene of Tc-dorsalThe restriction of Tc-dl nuclear import to ventral regionssuggests the existence of a ventrally localized signal that, as inDrosophila, activates a Toll-like plasma membrane receptor.The Tribolium Tollhomolog however, differs from DrosophilaToll by being transcribed only zygotically (Maxton-Küchenmeister et al., 1999). We therefore were interested tocompare in detail Tc-Tollexpression with gradient formation.While during preblastoderm stages no Tc-Toll expression canbe detected (Maxton-Küchenmeister et al., 1999 and data notshown), weak expression becomes visible in early syncytialblastoderm stages when the nuclei reach the cortex. Thisexpression is uniform along the DV axis and parallels the weaknuclear accumulation of Tc-dl (Fig. 6A, data not shown). Whenthe nuclear gradient of Tc-dl forms, Tc-Tollis up-regulatedventrally and becomes strongly expressed in regions of highnuclear Tc-dl concentrations (Fig. 6B). Low Tc-Tolllevels stillremain at the dorsal side (Fig. 6B). During later blastodermstages Tc-Toll expression follows a more complex patternpartially independent of Tc-dl (data not shown). However, inthe posterior half of the germ rudiment, Tc-Toll expressionvanishes in parallel with the refinement and disappearance ofthe Tc-dl gradient (Fig. 6C). These data are consistent with theassumption that at least during early blastoderm stages apositive feedback loop is established. Ventral Toll receptoractivation, through an extraembryonic signal, might enhancethe nuclear import of Tc-dl, which in turn would lead to theactivation of Tolltranscription and thus to the local productionof higher amounts of Toll receptor.

The relationship of the Tc-dl gradient to theexpression of zygotic DV patterning genesIn Drosophila three types of Dorsal target genes can bedistinguished (Rusch and Levine, 1996): type I targets areturned on by high levels of nuclear Dorsal and are thereforeexpressed in a ventral stripe (e.g. twist (twi) and snail(sna));type II targets are activated by low levels of Dorsal beingexpressed in lateral regions (e.g. rhomboid (rho), shortgastrulation (sog), brinker (brk); Jazwinska et al., 1999);type III targets that are uniformly activated by unknownmechanisms are subsequently repressed by Dorsal so that theirexpression is restricted to the dorsal side of the embryo (e.g.zerknüllt(zen) and decapentaplegic(dpp); Rushlow and Roth,1996). Most Dorsal targets also receive inputs from theterminal (torso) system, which modulate their expression


Fig. 5. Tc-dl protein during later development. (A-D) Ventral surfaceviews of whole embryos doubly stained with anti-Tc-dl antibodiesand a DNA dye (YOYO-1). (A,C) Bright-field images.(B,D) Fluorescence images of the embryos shown in A and C,respectively. (A,B) Primitive pit formation (arrows). Note theabsence of nuclear Tc-dl in the region of the presumptive ventralfurrow. (C,D) Early germband extension shortly before the serosalwindow (arrows) closes. Note the presence of nuclear Tc-dl in aventral stripe of serosa cells. (E) Dorsal portion and (F) ventralportion of the same cross section through an embryo slightly olderthan that shown in C, stained with anti-Tc-dl antibodies. s, serosa; a,amnion; vf, ventral furrow. At the position at which the section istaken the serosa has closed ventrally. Note that the serosa in thisparticular embryo is ruptured laterally so that only a small ventralfragment remains. Small amounts of nuclear Tc-dl are still seen inthe amnion while Tc-dl is completely absent from the embryonictissues. High levels of Tc-dl are present in the serosa. The serosalstaining is cytoplasmic at the dorsal side (E) and nuclear at theventral side (F). (G) Accumulation of nuclear Tc-dl in serosal cellssurrounding a region that might consist of necrotic tissue(arrowhead). Out of 50 embryos, 4 exhibited such a patch, which ineach case was located at a different position in the serosa.


pattern at the anterior and posterior extremities of the embryo(Ray et al., 1991). Homologs of type I and III targets have beencloned from Tribolium and their expression patterns havebeen shown to differ from their Drosophilacounterpart inaccordance with the short-germ type of Triboliumembryogenesis (Sommer and Tautz, 1994; Falciani et al., 1996;Sanchez-Salazar et al., 1996). Since the Dorsal gradient ofTribolium resembles that of Drosophilawith regard to itsposition relative to the egg axis,we wondered when and howthe differences in the expression patterns of potential targetgenes arise.

As representative of the type I target genes we studied theexpression of Tc-twiin relation to gradient formation (Sommerand Tautz, 1994; Handel et al., unpublished data). Tc-twibegins to be weakly expressed along the entire region in whichhigh levels of nuclear Tc-dl can be detected (Fig. 7A). Beforea difference between serosa cells and germ rudiment becomesapparent, Tc-twi is repressed in the anterior 20% of the APaxis, and the remaining domain becomes asymmetric alongthe AP axis by increasing its width at the posterior pole(Fig. 7B). These changes are not preceded by correspondinganteroposterior asymmetries in nuclear Tc-dl distribution.Rather, Tc-dl disappears from the germ rudiment as Tc-twilevels rise (Fig. 7C). This is in marked contrast to Drosophilawhere twi expression and high nuclear Dm-dl concentrationsremain tightly coupled during the cellular blastoderm and earlygastrulation. In Tribolium,twi expression might only beinitiated by Tc-dl, while its subsequent regulation becomesindependent from Tc-dl.

In Drosophila, the type III targets, zenand dpp,have earlyexpression patterns composed of a broad dorsal domain andsymmetric terminal caps, which depend on activation by theterminal system (Ray et al., 1991). Terminal cap activation andventral repression occur concomitantly. In Triboliumhowever,the expression of both genes is first initiated in a broad anteriorcap (Falciani et al., 1996; Sanchez-Salazar et al., 1996) whichshows no obvious DV asymmetry, even though the nuclear Tc-dl gradient has already been established at this stage (Figs 7D,8A). For Tc-dppa small posterior cap expression is also visible(Fig. 8A). This suggests that Tc-zen and Tc-dppare firstexclusively under the control of an AP patterning system,presumably the terminal system (Schröder et al., 2000).Subsequently, the Tc-zenexpression domain expands to thedorsal side of the embryo (Fig. 7E). At this stage, the lateralexpansion of Tc-dl has already decreased so that a big gapoccurs between lowest detectable nuclear Tc-dl concentrations

and the sharp border of Tc-zenexpression at the dorsal side.Thus, the shift of the Tc-zendomain to the dorsal side mightnot be a direct consequence of repression by Tc-dl.

The most striking deviation from Drosophilawas found withregard to dpp. After its initial anterior expression, Tc-dppbecomes predominantly expressed in an anterior ventraldomain where high nuclear Tc-dl concentrations are present(Fig. 8B,C). This domain resolves into a stripe separating theanterior cap expression of Tc-dl (presumptive serosa) from theregion where Tc-dl disappears (germ rudiment) (Fig. 8D,E).When ventral furrow formation starts, Tc-dppis weaklyexpressed as a one cell-wide stripe demarcating the serosa (Fig.8F). The transition from the earliest anterior-cap like to

Fig. 6. The nuclear Tc-dl gradient and itsrelationship to Tc-Tollexpression. Allembryos were stained for Tc-dl protein(brown) and Tc-TollmRNA (blue).(A-C) Complete cross sections withmagnified portions of the cortex below.(A) Cycle 8 embryo. Arrows indicate theposition of the nuclei. Tc-dl protein levelsare too low to be detectable in cross sections.Low levels of Tc-TollmRNA are founduniformly around the circumference.(B) Early blastoderm embryo.(C) Blastoderm embryo shortly beforeserosal segregation.

Fig. 7.The nuclear Tc-dl gradient and its relationship to Tc-twiandTc-zenexpression. (A-C) Tc-dl protein (brown) and Tc-twi RNA(blue) distribution in progressively older embryos. (D-F) Tc-dlprotein and Tc-zenRNA distribution in progressively older embryos.For all embryos anterior is to the left. (A,B) Ventral surface views.(C,D,F) Optical midsections. (E) Lateral view of slightly tiltedembryo. (A,D) Early blastoderm. (B,E) Late blastoderm.(C,F) Primitive pit (pp) formation. The arrows point to the posteriormargin of the anterior ‘cap’ expression of Tc-dl.

5152

the subsequent anteroventral expression domain of Tc-dpp,indicates that Tc-dl does not repress, but rather activates Tc-dpp in a direct or indirect manner. Furthermore, since Tc-dppremains confined to the presumptive serosa region, Tc-dl onlymodulates the regulatory input from the AP patterning system.

In summary, despite similarities in the geometry of themorphogen gradients, Dm-dl and Tc-dl show clear differencesin the relationship to their target genes. First, Tc-twiis likelyto be a type I target whose initiation, but not its high levelexpression in the presumptive mesodermal region depends onTc-dl. Second, neither Tc-zennor Tc-dpp represent goodcandidates for type III targets that are directly repressed by Tc-dl with Tc-dpp even being a likely candidate for direct orindirect activation.

Rescue of the Drosophila dorsal mutant phenotypeby Tc dlGiven the more dynamic gradient formation and the alteredrelation to its target genes, it is of interest to determine whetherTc-dl can rescue the Drosophila dlmutant phenotype. In a first

attempt, mRNA encoding Tc-dl was injected into dlmutantembryos. The injected embryos gastrulated with normalpolarity and revealed a rescued DV pattern at the site ofinjection (Fig. 9E,F). Muscle movement indicates that suchembryos formed some mesoderm (data not shown). To morecarefully test the functional similarity between Tc-dl and Dm-dl, constructs for P-element mediated transformation weregenerated. Partial rescue of dlmutant embryos was obtainedwhen a Tc-dltransgene in UASp (Rørth, 1998) was driven bymaternal α-tub Gal4:VP16. One copy of the transgene leads todifferentiation of dorsolateral structures (filzkörper, fk; Fig.9C); two copies promote, in addition, the formation ofventrolateral structures (ventral epidermis, ve; Fig. 9D). Acontrol construct containing Dm-dl leads to complete rescue oreven ventralisation (Fig. 9A,B).

We examined the formation of the Tc-dl gradient and theexpression of DV patterning genes in dl mutant embryosrescued by two copies of the Tc-dl transgene. Tc-dl proteinshows the same early nuclear accumulation at the ventral sideof Drosophila embryos as the endogenous Dorsal (Fig. 9G;Roth et al., 1989). The gradient is maintained untilcellularisation without the signs of dynamic change seen inTribolium embryos (Fig. 9H). A combination of antibodystaining and in situ hybridisation allows the visualisation ofnuclear concentrations of Tc-dl required for repression oractivation of target genes. Activation of twi,although expectedfrom the RNA injection experiment, was not observed (Fig.9G,H). However, in regions of high nuclear Tc-dlaccumulation, zenis repressed and sogis activated (Fig. 9I,K).Only low levels of Dm-dl would be required to achieveequivalent changes in gene expression (Roth et al., 1989;Jazwinska et al., 1999). Together, these findings indicate thatTc-dl binds to Drosophilatarget gene promoters, but that, inthe heterologous situation, it acts both as a weaker repressorand a weaker activator than endogenous Dorsal.

DISCUSSION

Several lines of evidence suggest that Tc-dl is the ortholog ofDm-dl: (1) among all rel/ NF-κB proteins of Drosophila, Dm-dl is by far the closest relative of Tc-dl (the RHDs of bothproteins share 77% identity); (2) Tc-dllike Dm-dl is maternallyexpressed and uniformly distributed in the cytoplasm of earlyembryos; (3) the Tc-dl protein forms a nuclear concentrationgradient in Triboliumblastoderm embryos; (4) Tc-dlpartiallyrescues the dlmutant phenotype of Drosophilaembryos whereit forms a nuclear gradient very similar to the endogenousgradient.

The rescue experiment indicates that Tc-dl correctlyinteracts with the IκB-like Cactus protein of Drosophila(Belvin et al., 1995; Bergmann et al., 1996) and that the Tc-dl/Cactus protein complex is subject to Toll inducedbreakdown at the ventral side. Surprisingly, similarexperiments using maternally expressed Difresulted in an evenbetter restoration of the DV pattern. However, in theseexperiments the nuclear gradient of Dif could not be visualizedand so no data exist about the concentrations of Dif thatinduced a certain transcriptional response (Stein et al., 1998).The rescue capacity of Dif, despite its divergent RHD, pointsto the importance of the C-terminal transactivation domain.


Fig. 8.The nuclear Tc-dlgradient and its relationshipto Tc-dppexpression. Allembryos show both Tc-dlprotein (brown) and Tc-dppmRNA (blue) distributions.The embryos are arrangedaccording to increasingdevelopmental age. Anterioris to the left. (A,C,E) Opticalmidsections. (B,F) Lateralsurface views. The embryoin B is slightly tilted to showthe ventral side. (D) Ventralsurface view. The arrowmarks the border betweengerm rudiment andpresumptive serosa.(A-C) Blastoderm embryos.(D) Serosal segregation.(E) Primitive pit formation.(F) Early gastrulation. s,serosa; pp, primitive pit.


Maybe some features of the transactivation domains of Dm-dland Dif, like the polyglutamine and polyasparagine stretchesabsent in Tc-dl (Fig. 1B), are species-specific adaptations forefficient interaction with the endogenous basal transcriptionmachinery. This might explain why Tc-dl is a weaker activatorand repressor compared to Dm-dl and probably even to Dif.These aspects of protein evolution, however, are less relevantto the main focus of this study concerned with similarities anddifferences in gradient formation and target gene expressionbetween Drosophila and Tribolium, which raise severalinteresting questions regarding the evolution of DV patterningmechanisms in insects.

Dynamics of gradient formation and the regulatorybehaviour of short-germ embryosIn contrast to Drosophila, the Tc-dl gradient undergoes aprocess of dynamic refinement, which leads from weakuniform nuclear up-take via a broad flat gradient to a narrowzone harbouring scattered nuclei with high Tc-dlconcentrations (Figs 3 and 4). This process occurs withremarkable precision along the entire AP axis. Therefore, itsinduction is likely to depend, as in Drosophila, on

extraembryonic cues. Since a Tollhomolog of Tribolium isexpressed at the right stage to mediate gradient formation(Maxton-Küchenmeister et al., 1999) it is highly suggestivethat a Spätzle-like extracellular Toll ligand is produced, as inDrosophila, at the ventral side of the egg. However, since Tollexpression is itself influenced by gradient formation (Fig. 5)the distribution of the extraembryonic ligand might determinethe final shape of the gradient in a less rigid way than inDrosophila(Roth, 1993).

A less rigid control by extraembryonic cues might help tounderstand the high degree of regulation along the DV axiswhich has been observed with many insect embryos. Forexample longitudinal fragmentations of preblastodermembryos of the leaf hopper Euscelislead to the formation ofsegmented germ bands in each egg fragment. This holds trueeven after separation of ventral and the dorsal egg halves(Sander, 1971). Cold treatment of preblastoderm embryos ofthe Chrysomelide beetle Atrachyacan result in up to fourcomplete germ bands forming in one egg (Miya andKobayashi, 1974). In these cases, DV patterning seems to beinitiated from different positions along the embryoniccircumference including even the dorsal side of the egg. To

Fig. 9. Tc-dlpartially rescues the Dm-dlmutantphenotype.(A-F) Dark-field photographs of cuticlepreparations. (A,B) Control rescue with Dm-dl. Thelarvae are derived from dlI5/Df(2L)TW119femalescarrying both tubGAL4:VP16 and UASp-Dm-dltransgenes. (A) Wild-type-like cuticle. de, dorsalepidermis; ve, ventral epidermis; fk,: filzkörper. Thefilzkörper are dorsolateral structures. (B) Ventralizedcuticle. The ventral epidermis (ve) is expanded at theexpense of the dorsal epidermis. This phenotype isidentical to that of cactus(Roth et al., 1991) and mostlikely results from too much Dm-dl produced by thetransgene so that endogenous Cactus is unable to retainDm-dl in the cytoplasm. (C,D) Partial rescue with Tc-dl.The larvae are derived from dlT/Df(2L)TW119femalescarrying tubGAL4:VP16 and either one (C) or two (D)copies of the UASp-Tc-dl transgene. (C) Rescue of onlydorsolateral structures (fk). (D) Rescue of dorsolateraland ventrolateral structures (fk and ve). (E,F) Rescue bymRNA injection. (E) Uninjected control embryo formsonly dorsal epidermis (de). (F) Injected embryo withextensive rescue of ventral epidermis (ve). (G-K) Crosssections through blastoderm embryos having the samegenotype as the embryo shown in D. All embryos werestained with anti-Tc-dl antibodies (brown). (G,H) twiinsitu hybridisation; no twiexpression was detected. (I) Tc-dl protein and zenmRNA (blue) distribution. (K) Tc-dlprotein and sogmRNA (blue) distribution. The arrowsdemarcate regions in which nuclear Tc-dl protein can stillbe detected although zenis not repressed and sogis notactivated. Corresponding nuclear levels of Dm-dl wouldlead to zenrepression and sogactivation.

5154

explain such results a self-organising pattern-forming systemis required, which links local self-enhancement with a lateralinhibition process (Meinhardt, 1982, 1989). In Tribolium, Tolland dorsal could be part of a self-enhancing feedback loop.Small levels of Toll-receptor activation would result in Dorsalnuclear import and in up-regulation of Toll transcription. Thisin turn would locally increase the receptor density and causesequestration of the activating ligand. If, as in Drosophila,theligand is diffusible in the perivitelline space (Stein et al., 1991),this loop can in principle be initiated and maintained at anyregion of the egg.

The molecular nature of the lateral inhibition process thatneeds to accompany the positive feedback loop to preventuncontrolled spreading, is not known. Two principal ways ofinhibition can be envisaged: either the production of adiffusible inhibitor is linked to local self-enhancement or theself-enhancement process depletes its own substrate(Meinhardt, 1982). In Drosophilaremnants of an inhibitoryprocess have been found. In certain mutant backgroundspartial duplication of the DV pattern can be observed (Rothand Schüpbach, 1994). Experiments suggest that Easter, theprotease which activates Spätzle, is subject to pathway-dependent inhibition (Misra et al., 1998). In Triboliumupregulation of the Toll receptor might lead to the depletion ofthe ligand in the surroundings and thereby suppress Tollactivation in the vicinity of an activation centre. Thus, giventhe right dynamic parameters, receptor up-regulation mightcontribute to both the positive feedback and the spatialrestriction required for pattern formation.

Tc-dl plays a less direct role in establishingdorsoventral cell fates compared to Dm-dlAlthough the regulatory behaviour of DV patterning found inperturbation experiments suggests reduced dependence on amaternal prepattern, it is also possible that interactions betweenzygotic DV patterning genes play an important role. In thiscontext, it is interesting that the Tc-dl not only differs from theDm-dl gradient, but also the relationship of the gradients totheir respective target genes and hence to the cell fates alongthe DV axis appears to be different. This is apparent for boththe mesoderm and the ectoderm.

In Drosophila,a ventral stripe of high nuclear Dorsal in thetrunk region of the blastoderm embryo is congruent with themesodermal anlagen since it defines the lateral expansion oftwi expression which, together with sna, promotes ventralfurrow formation. Dm-dl remains present in twi-and sna-expressing cells until the mesoderm has invaginated. InTribolium, in contrast, the early weak Tc-twiexpressiondomain, which is even along the AP axis and coincides withthe highest levels of nuclear Tc-dl, is rapidly replaced by adomain with strong AP asymmetry by becoming repressedanteriorly and broadened towards the posterior pole (Fig.7A,B). When this expression pattern is fully developed, nuclearTc-dl has disappeared from the germ rudiment (Fig. 7C).However, this final Tc-twidomain corresponds to thepresumptive mesoderm since it presages the position and shapeof the ventral furrow (Handel et al., unpublished data). Thisimplies that the shape of the gradient does not fully determinethe mesodermal anlagen and that Tc-twi transcription becomesindependent of activation by Tc-dl at late blastoderm.

The connection between Tc-dl and the patterning of the

ectoderm is even more indirect. In Drosophilalow levels ofnuclear Dorsal activate the expression of type II target geneswhich are required for the formation of neuroectoderm (Ruschand Levine, 1996; Jazwinska et al., 1999). Simultaneously,Dorsal represses the type III target genes of the Dpp groupconfining their expression to the dorsal side which gives riseto the amnioserosa and to the non-neurogenic ectoderm(Rushlow and Roth, 1996). No fate maps have beenconstructed for the Tribolium blastoderm embryo so far.However, the serosa cells seem to derive from a broad anteriordomain which is slightly tilted to the dorsal side (Handel et al.,2000). The serosa is likely to be homologous to theamnioserosa of Drosophila. zenand dppare expressed in bothtissues. While in Drosophilathese are dorsal expressiondomains, in Triboliumthe zenand dppdomains initially havethe shapes of anterior caps, which are symmetric with regardto the DV axis (Falciani et al., 1996; Sanchez-Salazar et al.,1996), even though the Tc-dl gradient has already formed (Figs7D, 8A). Only later Tc-zenshifts to the dorsal side, and Tc-dppshifts to the ventral side (Figs 7E, 8B). The latter is remarkablesince it indicates that a target gene, which is repressed byDorsal in Drosophila, might be activated by Dorsal inTribolium. In Drosophila, zenand dppare also expressed interminal caps, which are not influenced by Dorsal (Rushlow etal., 1987; Roth et al., 1989). However, no function has so farbeen attributed to these terminal caps since all patterningfunctions seem to reside in the dorsal expression domains.It is tempting to suggest that the terminal caps areevolutionary remnants of short-germ development, where theextraembryonic tissues are derived from anterior rather thandorsally located regions of the blastoderm (Schröder et al.,2000).

While the region giving rise to the serosa can be located byfollowing the course of embryonic development, this is harderwith regard to the subdivisions of the ectoderm. Therefore, itis not yet clear how early Tc-dppexpression (Fig. 8E,F) relatesto the later Tc-dppdomain corresponding to the dorsalectoderm (Sanchez-Salazar et al., 1996). There is someindication that the dorsal ectoderm expression is independentlyinitiated in more posterior regions of the germband and is thusnot continuous with the early Tc-dl associated domain (S. Rothunpublished observations).

In summary, a comparison of spatial and temporal aspectsof Tc-dl gradient formation with respect to both the expressionpattern of potential target genes and the likely position of theblastoderm anlagen suggests that Dorsal has a less direct rolein cell fate determination in Triboliumthan it has inDrosophila. It is quite possible that its major role in the germanlage is the initiation of Tc-twiexpression while thepatterning of the embryonic ectoderm is a secondaryconsequence of mesoderm formation initiated by Tc-twi.

The Dorsal gradient as preadaptation for long-germdevelopmentIt has been argued that both very early and late developmentare the major targets of evolutionary change while a certainstage characteristic for each phylum, the ‘phylotypic stage’, isrelatively stable (Slack et al., 1993; for review see Gerhart andKirschner, 1997). Besides vertebrates, insects provide the bestexample for the existence of a stable phylotypic stage. Insectembryos look very similar once segmentation is complete



despite diverse forms of oogenesis and dramatic differences inthe extent to which the body plan is determined beforegastrulation (Sander, 1976). Thus, processes close to thephylotypic stage, such as the final aspects of segmentation, areexpected to be conserved, while early events such as axisspecification under the control of the maternal genome mightbe divergent (Patel, 1994). However, this view of evolutionarychange is based mainly on phenotypic observations since notenough knowledge has accumulated about the molecularmechanisms of early development in diverse insect groups.With regard to maternal anteroposterior gradients several linesof evidence suggest that bicoid, the anterior determinant ofDrosophilamight have arisen late in evolution being unique toDipterans (Stauber et al., 1999). Nevertheless, a Bicoid-likeactivity needs to be invoked in Tribolium to explain theformation of the maternal Caudal protein gradient (Wolff et al.,1998).

The existence of a Dorsal gradient in the short-germ beetleTribolium provides a good example of the conservation of anearly patterning process despite the fact that later aspects ofembryogenesis leading to the phylotypic stage are dramaticallydifferent. The Dorsal gradient in Triboliumhas the same APextension with regard to the egg shell as that of Drosophila.The spatial information it provides however, is not exploited tothe same extent as in the long-germ Drosophilaembryo. Thus,it appears that in early Triboliumembryos a developmentalmechanism exists which is fully used only in embryos withlong-germ development. To find a ‘long-germ’ type Dorsalgradient in a beetle embryo might not be so surprising becauseamong different beetle species transitions between long,intermediate and short-germ types of development have beenobserved (Sander, 1976; Patel et al., 1994). It will however beinteresting to see whether Dorsal gradients exist in moreprimitive short-germ embryos of hemimetabolous insects andif so, how they are positioned within the egg.

Innate immunity and the origins of the DorsalgradientThe immune function of the Toll-rel/NF-κB pathway isevolutionarily very old since it is conserved between insectsand vertebrates (Hoffmann et al., 1999; Anderson, 2000).Although recent data from Xenopussuggest an involvement ofthe Toll pathway in DV axis formation in amphibians, theproblem of the ancestral role has still not been resolved(Armstrong et al., 1998). If the ancestral function was inimmunity, our finding that high amounts of Dorsal areexpressed in the serosa of Tribolium might provide a hint as tohow the system originally devoted to pathogen defence was co-opted for patterning. In primitive hemimetabolous insects mostof the blastoderm cells give rise to the serosa (Sander, 1976).Since the serosa has a protective function for the whole egg,often evident in the formation of serosal cutical (Machida andAudo, 1998), there might have been an evolutionary advantageto supply serosa cells with pathogen defense mechanisms usingthe Toll pathway. Furthermore, the distinction between serosaand germ rudiment is the earliest cell differentiation event inan insect embryo, taking place at blastoderm stage beforegastrulation starts so that serosa specific components have tobe provided very early. Thus, only a small shift in temporal andspatial expression would have been necessary for a shift infunction of the Toll pathway from innate immunity to axis

formation. When, in evolution, might this functional shift haveoccurred? Other arthropods have extraembyonic ectoderm, but,to our knowledge, the distinction between an amnion coveringthe embryo ventrally and a serosa covering the entire egg(embryo plus yolk), is unique to ectognathan insects(Anderson, 1973; Machida and Audo, 1998). Thus, thepathway might have been co-opted for axis formation duringearly insect evolution. To test this hypothesis, it will be crucialto isolate pathway components from more primitive insects andother arthropods.

We thank Anke Beermann, Reinhard Schröder and Martin Klinglerfor introducing us into the secrets of beetle rearing; John Doctor andDiethard Tautz for molecular probes; Reinhard Schröder for thecDNA library; Diethard Tautz for help with the phylogram; Xiang Fanand Oliver Karst for excellent technical assistance; Christian Bökelfor teaching us protein techniques; Manfred Schorpp for help with therabbits; Klaus Sander, Reinhard Schröder, Nicola Berns, Xiang Fan,Maithreyi Narasimha, Thomas Seher and Henry Roehl for valuablecomments on the manuscript. We are grateful to Uli Schwarz (MPIfür Entwicklungsbiologie, Tübingen) and Wolf Engels (University ofTübingen) for their help throughout the course of this study. TeresaNicolson kindly provided space for G. C. and K. H. in her laboratoryduring the last year of the experimental work. The DAAD provided astipend to G. C. The work was supported by grants from the DFG(SPP 1027).

REFERENCES

Anderson, D. T. (1973). Embryology and Phylogeny in Annelids andArthropods.Oxford: Pergamon Press.

Anderson, K. V. (2000). Toll signaling pathways in the innate immuneresponse. Curr. Opin. Immunol. 12, 13-19.

Armstrong, N. J., Steinbeisser, H., Prothmann, C., DeLotto, R. and Rupp,R. A. W. (1998). Conserved Spätzle/Toll signaling in dorsoventralpatterning of Xenopus embryos. Mech. Dev.71, 99-105.

Barillas-Mury, C., Charlesworth, A., Gross, I., Richman, A., Hoffmann,J. A. and Kafatos, F. C. (1996). Immune factor Gambif1, a new rel familymember from the human malaria vector, Anopheles gambiae. EMBO J. 15,4961-4701.

Beermann, A. (1998). Genetische und molekulare Analyse des Distal-lessGens bei Tribolium castaneum. (Dissertation, Tübingen)

Belvin, M. P., Jin, Y. and Anderson, K. V. (1995) . Cactus proteindegradation mediates Drosophiladorsal-ventral signaling.Genes Dev. 9,783-793.

Berghammer, A., Bucher, G., Maderspacher, F. and Klingler, M. (1999).A system to efficiently maintain embryonic lethal mutations in the flourbeetle Tribolium castaneum. Dev. Genes Evol. 209, 382-388.

Bergmann, A., Stein, D., Geisler, R., Hagenmaier, S., Schmid, B.,Fernandez, N., Schnell, B. and Nüsslein-Volhard, C. (1996) . A gradientof cytoplasmic Cactus degradation establishes the nuclear localizationgradient of the dorsal morphogen in Drosophila. Mech. Dev. 60, 109-123.

Dushay, M. S., Asling, B. and Hultmark, D. (1996). Origins of immunity:Relish, a compound Rel-like gene in the antibacterial defense of Drosophila.Proc. Natl. Acad. Sci. USA 93, 10343-10347.

Falciani, F., Hausdorf, B., Schröder, R., Akam, M., Tautz, D., Denell, R.and Brown, S. (1996). Class 3 Hox genes in insects and the origin of zen.Proc. Natl. Acad. Sci. USA 93, 8479-8484.

Gerhart, J. and Kirschner, M. (1997). Cells, Embryos and Evolution.Malden: Blackwell Science.

Grünfelder, C. G. (1997). Vom frisch abgelegten Ei zum Blastoderm:Untersuchungen zur Feinstruktur der frühen Embryogenese desReismehlkäfers Tribolium confusum, Duval (Coleoptera, Tenebrionidae).Doctoral thesis, University of Freiburg.

Handel, K., Grünfelder, C., Roth, S. and Sander, K. (2000). Triboliumembryogenesis: a SEM study of cell shapes and movements from blastodermto serosal closure. Dev. Genes Evol. 210, 167-179.

5156

Harlow, E. and Lane, D. (1988). Antibodies: A Laboratory Manual. ColdSpring Harbor, NY: Cold Spring Harbor Laboratory Press.

Higgins, D. G. and Sharp, P. M. (1989). Fast and sensitive multiple sequencealignments on a microcomputer. CABIOS5, 151-153.

Hoffmann, J. A., Kafatos, F. C., Janeway, C. A. and Ezekowitz, R. A.(1999). Phylogenetic perspective in innate immunity. Science 284, 1313-1318.

Ip, Y. T., Reach, M., Engstrom, Y., Kadalayil, L., Cai, H., Gonzalez-Crespo, S., Tatei, K. and Levine, M. (1993). Dif, a dorsal-related gene thatmediates an immune response in Drosophila. Cell 75, 753-763.

Jazwinska, A., Rushlow, C. and Roth, S. (1999). The role of brinker inmediating the graded response to Dpp in early Drosophila embryos.Development 126, 3323-3334.

Machida, R. and Audo, H. (1998). Evolutionary change in developmentalpotentials of the embryo proper and embryonic membrane along with thederivative structures in Alelocerata, with special reference to Hexapoda(Arthropoda). Proc. Arthropod. Embryol. Soc. Jap. 33, 1-13.

Maxton-Küchenmeister, J., Handel, K., Schmidt-Ott, U., Roth, S. andJäckle, H. (1999). Toll homolog expression in the beetle Tribolium suggestsa different mode of dorsoventral patterning than in Drosophila embryos.Mech. Dev. 83, 107-114.

Meinhardt, H. (1982). Models of Biological Pattern Formation. (London:Academic Press).

Meinhardt, H. (1989). Models for positional signaling with application to thedorsoventral patterning of insects and segregation into different cell types.Development 107 Supplement, 169-180.

Misra, S., Hecht, P., Maeda, R. and Anderson, K. V. (1998). Positive andnegative regulation of Easter, a member of the serine protease family thatcontrols dorsal-ventral patterning in the Drosophilaembryo. Development125, 1261-1267.

Miya, K. and Kobayashi, Y. (1974). The embryonic development of Atrachyamenetriesi Faldermann (Coleoptera. Chrysomelidae). J. Fac. Agric. IwateUniv. 12, 39-55.

Morisato, D. and Anderson, K. V. (1995). Signaling pathways that establishthe dorsal-ventral pattern of the Drosophilaembryo. Ann Rev Genet 29, 371-399.

Nilson, L. A. and Schüpbach, T. (1998). Localized requirements forwindbeutel and pipe reveal a dorsoventral prepattern within the follicularepithelium of the Drosophilaovary. Cell 93, 253-262.

Patel, N. H., Condron, B. G. and Zinn, K. (1994). Pair-rule expressionpatterns of even-skipped are found in both short- and long-germ beetles.Nature 367, 429-434.

Patel, N. H.(1994). Developmental evolution: Insights from studies of insectsegmentation. Science266, 581-590.

Ray, R. P., Arora, K., Nüsslein-Volhard, C. and Gelbart, W. M. (1991). Thecontrol of cell fate along the dorsal-ventral axis of the Drosophilaembryo.Development 113, 35-54.

Rørth, P. (1998). Gal4 in the Drosophilafemale germline. Mech. Dev. 78, 113-118.

Roth, S. (1993) . Mechanisms of dorsal-ventral axis determination inDrosophilaembryos revealed by cytoplasmic transplantations.Development117, 1385-1396.

Roth, S. (1998). The secrets of delayed induction. Curr. Biol. 8, R906-R910.Roth, S., Hiromi, Y., Godt, D. and Nüsslein-Volhard, C. (1991). cactus, a

maternal gene required for proper formation of the dorsoventral morphogengradient in Drosophilaembryos. Development112, 371-388.

Roth, S., Jordan, P. and Karess, R. (1999). Binuclear Drosophilaoocytes:Consequences and implications for dorsoventral patterning in oogenesis andembryogenesis. Development 126, 927-934.

Roth, S. and Schüpbach, T. (1994). The relationship between ovarian andembryonic dorsoventral patterning in Drosophila. Development 120, 2245-2257.

Roth, S., Stein, D. and Nüsslein-Vollhard, C. (1989). A gradient of nuclearlocalisation of the dorsal protein determines dorsoventral pattern in theDrosophilaembryo. Cell 59, 1189-1202.

Rupp, R. A. W., Snider, L. and Weintraub, H. (1994). Xenopus

embryos regulate the nuclear localization of XMyoD. Genes Dev. 8, 1311-1323.

Rusch, J. and Levine, M. (1996). Threshold responses to the dorsal regulatorygradient and the subdivision of primary territories in the Drosophilaembryo.Curr. Opin. Genet. Dev. 6, 416-423.

Rushlow, C., Frasch, M., Doyle, H. and Levine, M. (1987). Maternalregulation of zerknüllt: a homoeobox gene controlling differentiation ofdorsal tissues in Drosophila. Nature 330, 583-586.

Rushlow, C. and Roth, S. (1996). The role of the dpp-group genes indorsoventral patterning of the Drosophilaembryo. Adv. Dev. Biol. 4,27-82.

Rushlow, C. A., Han, K., Hanley, J. L. and Levine, M. (1989). The gradeddistribution of the dorsal morphogen is initiated by selective nucleartransport in Drosophila. Cell 59, 1165-1177.

Sanchez-Salazar, J., Pletcher, M. T., Bennett, R. L., Brown, S. J.,Dandamudi, T. J., Denell, R. E. and Doctor, J. S. (1996). The Triboliumdecapentaplegic gene is similar in sequence, structure, and expression to theDrosophila dppgene. Dev. Genes Evol. 206, 237-246.

Sander, K. (1971). Pattern formation in longitudinal halves of leaf hopper eggs(Homoptera) and some remarks on the definition of embryonic regulation.Wilhelm Roux Arch. Entw. Mech. Org. 167, 336-352.

Sander, K. (1976). Specification of the basic body pattern in insectembryogenesis. Adv. Insect Physiol. 12, 125-238.

Schröder, R., Eckert, C., Wolff, C. and Tautz, D. (2000). Conserved anddivergent aspects of terminal patterning in the beetle Tribolium castaneum.Proc. Natl. Acad. Sci. USA97, 6591-6596.

Sen, J., Goltz, J. S., Stevens, L. and Stein, D. (1998). Spatially-restrictedexpression of pipein the Drosophilaegg chamber defines embryonic dorsal-ventral polarity. Cell 95, 471-481.

Slack, J. M. W., Holland, P. W. H. and Graham, C. F. (1993). The zootypeand the phylotypic stage. Nature361, 490-492.

Sommer, R. J. and Tautz, D. (1994). Expression pattern of twist and snail inTribolium (Coleoptera) suggest a homologous formation of mesoderm inlong and short-germ band insects. Dev. Genet. 15, 32-37.

Stauber, M., Jäckle, H. and Schmidt-Ott, U. (1999). The anteriordeterminant bicoid of Drosophilais a derived Hox class 3 gene. Proc. Natl.Acad. Sci. USA 96, 3786-3789.

Stein, D., Goltz, J. S., Jurcsak, J. and Stevens, L. (1998). The Dorsal-relatedimmunity factor (Dif) can define the dorsal-ventral axis of polarity in theDrosophilaembryo. Development 125, 2159-2169.

Stein, D., Roth, S., Vogelsang, E. and Nüsslein-Volhard, C. (1991) Thepolarity of the dorsoventral axis in the Drosophilaembryo is defined by anextracellular signal. Cell 65, 725-735.

Steward, R. (1987). Dorsal, an embryonic polarity gene in Drosophila, ishomologous to the vertebrate proto-oncogene, c-rel. Science 238, 692-694.

Steward, R. (1989). Relocalization of the Dorsal protein from the cytoplasmto the nucleus correlates with its function. Cell 59, 1179-1188.

Steward, R., Ambrosio, L. and Schedl, P. (1985). Expression of the dorsalgene. Cold Spring Harbor Symp. Quant. Biol. 50, 223-228.

Swofford, D. L. (1998). PAUP. Phylogenetic Analysis Using Parsimony.(Sunderland, MS: Sinauer Associates)

Tautz, D. and Pfeifle, C. (1989). A non-radioactive in situ hybridizationmethod for the localization of specific RNAs in Drosophilaembryos revealstranslational control of the segmentation gene hunchback. Chromosoma98,81-85.

Verma, I. M., Stevenson, J. K., Schwartz, E. M., Van Antwerp, D. andMiyamoto, S. (1995). Rel/NF-kB/IkB family: intimate tales of associationand dissociation. Genes Dev. 9, 2723-2735.

Wolff, C., Schröder, R., Schulz, C., Tautz, D. and Klingler, M. (1998).Regulation of the Triboliumhomologues of caudaland hunchbackinDrosophila: evidence for maternal gradient systems in a short-germ embryo.Development 125, 3645-3654.

Wolff, C., Sommer, R., Schröder, R., Glaser, G. and Tautz, D. (1995).Conserved and divergent expression aspects of the Drosophilasegmentationgene hunchbackin the short-germ band embryo of the flour beetle Tribolium.Development 121, 4227-4236.


the tribolium dorsal gradient - development · tribolium toll(maxton-küchenmeister et al., 1999)...

Documents