Sabbi Lall and Nipam H. Patel · curs well before gastrulation in many short and long germ embryos....

19 Oct 2001 10:54 AR ar144-15.tex ar144-15.sgm ARv2(2001/05/10)P1: GJB

Annu. Rev. Genet. 2001. 35:407–37Copyright c© 2001 by Annual Reviews. All rights reserved

CONSERVATION AND DIVERGENCE IN MOLECULAR

MECHANISMS OF AXIS FORMATION

Sabbi Lall and Nipam H. PatelHoward Hughes Medical Institute, University of Chicago, Chicago, Illinois 60637;e-mail: [email protected]

Key Words axis formation, insects,Drosophila, evolution

■ Abstract Genetic screens inDrosophila melanogasterhave helped elucidatethe process of axis formation during early embryogenesis. Axis formation in theD.melanogasterembryo involves the use of two fundamentally different mechanisms forgenerating morphogenetic activity: patterning the anteroposterior axis by diffusion ofa transcription factor within the syncytial embryo and specification of the dorsoventralaxis through a signal transduction cascade. Identification ofDrosophilagenes involvedin axis formation provides a launch-pad for comparative studies that examine the evo-lution of axis specification in different insects. Additionally, there is similarity betweenaxial patterning mechanisms elucidated genetically inDrosophilaand those demon-strated for chordates such asXenopus. In this review we examine the postfertilizationmechanisms underlying axis specification inDrosophila. Comparative data are thenused to ask whether aspects of axis formation might be derived or ancestral.

CONTENTS

INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 408Axis Formation inDrosophila melanogaster. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 409Are Models of Axis Formation Formulated inDrosophilaTenable in Other Insects?. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 409

THE DORSOVENTRAL SYSTEM . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 410DV Axiation Processes inD. melanogaster . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 410Dpp Signaling May Play a Conserved Rolein Axis Formation Across Phyla. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 413

twist Is Involved in Mesoderm Patterning in Many Animals. . . . . . . . . . . . . . . . . . 417TheToll/dorsalSignaling Pathway May Have Been Co-Opted intoAxis Formation from the Immune System. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 418

Conservation of the DV Axis FormationCassette in Its Entirety?. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 419

THE ANTERIOR SYSTEM . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 421AP Patterning inDrosophila melanogaster. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 421caudalMay Play a Conserved Role in Posterior Patterning Across Phyla. . . . . . . . 421hbMay Play a Conserved Role in AP Patterning Within the Insects. . . . . . . . . . . . 422bcdFunction May Be a Recent Novelty in AP Patterning. . . . . . . . . . . . . . . . . . . . 422

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EVOLUTION OF AXIS FORMATION MECHANISMS . . . . . . . . . . . . . . . . . . . . . . 423REDUNDANCY IN AXIS FORMATION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 424LINKING MORPHOLOGICAL CHANGES INEMBRYOGENESIS TO EVOLUTION OF AXIS FORMATION. . . . . . . . . . . . . . . 426

CONCLUDING REMARKS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 427

INTRODUCTION

Many organisms manifest polarity at some level; indeed, asymmetry seems essen-tial for promoting meaningful interaction with the environment. Even the simplestunicellular organisms display temporary polarity in response to their environment;for example, in reception and response to chemotactic signals (for example, see11). In higher animals the body plan is arranged along two axes: the anteroposterior(AP) axis, and the broadly perpendicular dorsoventral (DV) axis.

Breaking symmetry to specify these axes is one of the most basic and earliestprocesses in the development of higher animals. The early nature of axis forma-tion is clear inDrosophila melanogasterwhere the process has been geneticallydissected, and the cues for AP and DV axiation are established during oogenesis.Animals can utilize diverse environmental cues as the basis of axiation, suggestingthat axis formation is a variable, plastic process. For instance, the physical processof axis formation can vary between closely related nematodes (46). Furthermore,Xenopus laeviscan be induced to use gravity rather than sperm entry point as a cuefor axis formation; indeed, the dorsalizing organizer forms 180◦ to sperm entrypoint only 70% of the time (45). How can an event that provides basic informationfor patterning the entire body plan be so inconstant?

In this review, we use knowledge about embryonic axis formation in the dipteranDrosophila melanogaster(D. melanogaster) to address the contention that axisformation is variable, and then to ask how this can be true of such a fundamentalprocess. Variation in axis formation probably has an intimate relationship withalterations in embryonic morphology and changes in life-history. Hence we discussdata suggesting that variation in axis formation within the dipterans correlatesphylogenetically with variation in embryonic morphology.

D. melanogasterhas four maternal coordinate systems that specify the majorbody axes (reviewed in 165). Three specify positional information along the APaxis (the anterior, posterior, and terminal systems), whereas the fourth is involvedin DV axis formation. We describe the molecular nature of the anterior and DVpatterning systems, addressing which of the following might be true:

1. Does a given molecular process play a conserved role in axis formationacross phyla, and could it therefore be ancient?

2. Might a particular molecular process have been co-opted into axis formationfrom another context prior to the origin of insects?

3. Might a given molecular process be a very recent evolutionary innovation?


EVOLUTION OF INSECT AXIS FORMATION 409

Examples of all three scenarios can be drawn out of the axis formation mecha-nisms elucidated inD. melanogaster, suggesting that the process of axis formationmay well have been elaborated upon multiple times. We discuss the fact thatD.melanogasterhas been shown genetically to display redundancy between two ma-ternal coordinate systems, and suggest that this may facilitate radical changes inaxis formation. Finally, we examine recent data suggesting that rapid molecularevolution of axiation can be correlated with gross morphological rearrangementswithin the dipterans.

Axis Formation in Drosophila melanogaster

Genetic screens and subsequent molecular analyses have led to a detailed under-standing of the earliest embryonic events in axis formation inD. melanogaster(165). All four of the maternal coordinate systems are set up during oogenesis,and recognition of and elaboration upon axial maternal cues are among the earliestevents after egg activation and fertilization. The anterior system involves cyto-plasmic diffusion of morphogens within the syncytialD. melanogasterembryo.In contrast the terminal and dorsal patterning systems require signaling from theextracellular perivitelline space to the embryo. In this review, we focus on thedorsoventral patterning system, as an example of morphogenetic activity set upby extracellular signaling, and the anterior system, as an illustration of cytoplas-mic diffusion to form a morphogen gradient. We begin by asking whether suchmechanisms are likely to be conserved among other insect orders.

Are Models of Axis Formation Formulated in DrosophilaTenable in Other Insects?

D. melanogasterdisplays a mode of development designated long germ embryo-genesis, in which the presumptive head, thoracic, and abdominal cells are presentat blastoderm stage in the same proportion as in the hatching larva (reviewed in140).D. melanogasteralso has a relatively prolonged syncytial stage. The cuesfor AP axis formation are initially elaborated upon within a syncytial environmentduring the first 2.5 h of embryonic development. Short germ insects, on the otherhand, have only specified the most anterior segments at the end of blastodermstage, the posterior segments being generated by subsequent growth [for reviewsaddressing short and long germ development, see (140, 144, 177)]. Although mostinsects display superficial cleavage and have a syncytial phase, cellularization oc-curs well before gastrulation in many short and long germ embryos. For example,grasshopper nuclei cellularize almost as soon as they reach the embryonic cortex,i.e., at the onset of blastoderm stage (55).

Both short germ embryogenesis and early cellularization have profound con-sequences for axis formation models involving morphogen diffusion. Short germembryogenesis suggests that patterning of posterior segments may be substantiallydelayed, whereas early cellularization makes it difficult to envision specification bythe diffusion of a morphogen such as thebicoid transcription factor inDrosophila


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(see below). In contrast, systems that elaborate axes via a signaling pathway mightmore obviously be conserved. The insect orders discussed in this review and theirapproximate relationships are illustrated in Figure 1b (based on 82, 195).

Axis formation mechanisms inD. melanogasterare even harder to apply toanimals that undergo holoblastic (complete) rather than superficial cleavage afterfertilization. In embryos undergoing complete cleavage, diffusion or segregation ofa cytoplasmic morphogen must occur during the first embryonic cleavages. Thus,if aspects of axial patterning inDrosophilaare conserved across species, they willat the very least have been modified in the lineage leading to flies.

THE DORSOVENTRAL SYSTEM

DV Axiation Processes in D. melanogaster

Our understanding of DV patterning during early embryogenesis inD. melanogasteris based on the genetic dissection of theToll/dorsalpathway (Figure 2) (reviewedin 100). Upon mutation, 12 genes show maternal-effect dorsoventral patterningdefects in the embryo, but have normal eggshell patterning (5, 21, 152). Thesegenes are (in putative order of action within the pathway)windbeutel, pipe, nudel,gastrulation defective, snake, easter, spatzle, Toll, pelle, tube, dorsal, andcactus(4, 22, 53, 99, 133). Normal DV development is ultimately evident by the stereo-typical arrangement of denticles on the larval cuticle, and more immediately bythe correct expression of molecular markers indicating specification of territo-ries along the dorsoventral axis during embryogenesis. The strongest recessivemutations in 11 of the dorsal group genes lead to a larval cuticle that is cov-ered in dorsal-type denticles and lacks ventral tissue such as the mesoderm. The12th gene,cactus, gives the opposite phenotype: a ventralized cuticle (133, 152).Readouts of theToll/dorsalpathway display dose sensitivity genetically, and de-pend upon differential levels of Dorsal nuclear localization along the DV axis(110, 125, 134, 136, 138, 171, 172). Such evidence suggests that fate along the DVaxis depends upon the activity of thedorsaltranscription factor.

During oogenesis, a molecular cue localized around the oocyte nucleus deter-mines follicle cells lying on one side of the oocyte as dorsal. This leads to thedevelopment of follicle cells on the other side of the oocyte as ventral, via the ex-pression of thepipegene.pipeencodes a heparan sulfate 2-O-sulfotransferase andhas been suggested to make a ventral extracellular modification, or perhaps modifynudelventrally, as the latter behaves nonautonomously and may thus potentiallymediate an extracellular signal (109, 155). These cues, set up during oogenesis, ini-tiate a proteolytic cascade, mediated by the proteasesnudel, gastrulation defective,snake, andeaster, in the perivitelline space outside the fertilized embryo (Figure2a) (see 20, 58, 79, 86, 169). Interestingly,pipe is not essential for initiating theproteolytic activity of Gastrulation-defective, implying that the situation is morecomplicated than a simple spatial cue locally activating the protease at the top of aproteolytic hierarchy (86). The proteolytic cascade results in the ventral processing



Figure 1 Phylogenetic trees illustrating the positions of the species and phyla dis-cussed in this review. The trees provide a rough framework of (a) the positions ofthe phyla discussed (based on 1), and (b) the positions of the insect orders and par-ticular dipterans (flies) used as examples in the text. The asterisk indicates the originof bilaterians, essentially animals with a body plan arranged along two perpendicularaxes. Arthropod species are:Schistocerca americana, Tribolium castaneum, Nasoniavitripennis, Bombyx mori, Clogmia albipunctata, Empis livida, Megaselia abdita, andDrosophila melanogaster.


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Figure 2 DV patterning inDrosophila melanogaster. The pathways illustrate theinteractions of genes that act to pattern the DV axis of the embryo without influencingeggshell morphology (a). (a) shows maternal effect genes that act, within the periv-itelline space (pv space) to control the nuclear localization of Dorsal protein alongthe DV axis. (b) Dorsal is found in a nuclear gradient. At high nuclear concentrationsDorsal represses zygotic genes that pattern the dorsal regions of the embryo while ac-tivating ventral development throughtwistandsnailactivation. At intermediate levelsthe Dorsal transcription factor promotes lateral fates (b). (c) Factors regulating Dpp sig-naling are illustrated, showing basic interactions.sogboth antagonizes and potentiatesDpp signaling.



of Spatzle protein to a 23-kD form by Easter (99, 147). When injected into the periv-itelline space, cleaved Sp¨atzle activates ventral development in both a site- andconcentration-specific fashion (99). Genetic and biochemical evidence suggeststhat the cleaved and active form of Sp¨atzle then acts as a ligand for the Toll recep-tor, which is immediately upstream ofToll but downstream ofeaster(Figure 2a)(see 22). Localized activation of the Toll receptor leads to the stimulation of anintracellular pathway involvingtubeandpelle, the end result of which is the phos-phorylation and degradation of the IkB orthologuecactus(10, 53, 126).

Cactus physically interacts with and thereby inhibits a key gene in dorsoventralaxis formation, the morphogenetic transcription factor Dorsal (aNFκB/rel homo-logue; 170). Degradation of Cactus allows Dorsal to enter the nucleus (12, 186).Since Toll is activated ventrally, Dorsal protein enters the nucleus at highest concen-tration ventrally. Immunostaining against Dorsal protein allows direct and elegantvisualization of its nucleocytoplasmic gradient, running ventral to dorsal acrossthe embryo (134, 138, 171).

Activation of the maternalToll/dorsalpathway leads to the expression of zy-gotic genes at different DV levels of the embryo (Figure 2b). At highest nuclearconcentration (ventral), Dorsal activatestwist andsnail, which are required forspecification of ventral fate (mesoderm) and for the inhibition of lateral fatessuch as neurectoderm (48, 66, 80, 87, 104, 124, 125, 180–182). Laterally, interme-diate nuclear concentrations of Dorsal activate a second group of targets includingrhomboidandshort gastrulation(sog). These genes specify lateral neurectoder-mal territories and influence the activity of Decapentaplegic (Dpp), respectively(see below; 42, 65). Dorsal also acts as a transcriptional repressor of genes such asdppandzen, which specify dorsal fates (32, 64, 68, 125, 136). Thus, higher Dorsalnuclear activity in ventral and lateral regions restrictsdppexpression to the dor-sal side of the embryo.dpp acts to pattern ectoderm, specifying different tissueterritories such as amnioserosa and dorsal epidermis (39, 187).

Which aspects of these processes are conserved across species? We focuson components with known homologues in other species that can therefore bediscussed in an evolutionary context. In particular, we discussdpp and twistand examine their potential regulation by theToll signaling pathway outsideD.melanogaster.

Dpp Signaling May Play a Conserved Rolein Axis Formation Across Phyla

dpp is expressed at blastoderm stage, in a longitudinal stripe restricted by Dorsalprotein to the dorsal 40% of the embryo (166). Dpp protein (enhanced by the ligandScrew; 106) is responsible for the transcriptional activation of a number of tar-gets in specific dorsal territories includingzerknullt (zen) (8, 39, 68, 125, 137, 187).Dpp is a ligand for the Tkv/Punt receptor complex (17, 88, 105, 116, 135). Genera-tion of a Dpp activity gradient is also dependent on thedpp antagonist,sog.Genetic mosaic analysis suggests thatsog is required ventrolaterally and acts


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non-cell autonomously. Moreover, genetic experiments addressing the interactionof dpp andsogsuggest thatsogantagonizes Dpp function dorsolaterally but in-tensifies Dpp activity in the extreme dorsal region of the embryo (Figure 2c) (see7, 13, 40, 42, 92, 199). Therefore an antagonistic gradient of Sog emanating fromlateral regions of the embryo may help to grade the activity of Dpp in dorsalterritories.

Regulation of Dpp signaling is also mediated by thetolloid gene.tolloid em-bryos have a ventralized cuticle phenotype, and are missing the most dorsal struc-tures (amnioserosa) as well as some dorsal epidermis (74, 156).tolloid is expresseddorsally, encodes a metalloprotease, and has been shown genetically to be upstreamof dpp(Figure 2c). Epistasis analysis inDrosophilaand second axis induction as-says inXenopussuggest thattolloid functions upstream ofsogas an antagonist (92).Tolloid cleaves Sog protein, as evidenced by the fact that Sog cleavage productscan be detected in the embryo uponsogoverexpression, or when Sog and Tolloidare co-incubated in vitro (92, 196). Sog destruction by Tolloid may allow Dpp tobind to its receptor, perhaps by reducing the affinity of the Sog/Dpp interactionand thereby freeing Dpp to function independently of Sog. The isolation of inter-acting antimorphic mutations was used to suggest a physical interaction betweenthe tolloid anddpp gene products. These phenotypes have not been reconciledwith the current view thattolloid acts onsog(24, 40, 41, 123). Further regulationof Dpp activity is suggested by recent data concerning thetwisted gastrulationgene, which though originally proposed to antagonize Sog might actually be anantagonist of Dpp via Sog (for more details, see 132, 196).

Components downstream of the Tkv/Punt receptor complex have also beenidentified. Mad and Medeamutants are enhancers of a weakdpp phenotype,and mediate the transcriptional response to Dpp signaling (107, 108, 123, 154).Between them, theMad/Medeaand schnurri DNA binding transcription fac-tors mediate activation and repression ofdpp-responsive transcriptional targets(27, 61, 77, 93, 107, 108, 123, 154, 184).Mad family genes seem to collaboratewith a variety of transcriptional cofactors and are more generally required forDpp signaling thanschnurri, which functions in part by antagonizing the generalrepression ofdpp transcriptional targets bybrinker (71, 93). Some of the abovecomponents have been isolated not only from other insect species, but also fromacross phyla, suggesting thatdpp may have played an ancient role in dorsoven-tral axis formation. We first discuss comparative insect studies which are distinctfrom chordate studies in focusing on Dpp expression, not activity. We then dis-cuss data from chordates, where the regulation of Dpp signaling activity has beensuccessfully dissected biochemically and genetically.

dppandzenhomologues have been isolated from more basal insects, includingthe beetleTribolium castaneum(Tc-dppandTc-zen) and the grasshopperSchis-tocerca americana. Analysis of the genes has been used to determine whetherthey are expressed in dorsal tissue (as inDrosophila), and whetherdppexpressionis regulated by Dorsal protein. Expression ofTc-dppandTc-zenis observed inserosal cells (23, 37, 139). Serosa is an extraembryonic membrane with a putative



protective function during insect embryogenesis (3). In the higher flies extraem-bryonic membranes are reduced and referred to as the amnioserosa, a dorsallyplaced tissue that, inDrosophila, also expressesdpp andzen(2). In more basalinsects such asTribolium andSchistocerca, the serosa is anteriorly placed in theegg. At first this may suggest thatdpp and zendo not play a role in DV axisformation in basal insects but expression ofdpp in the serosa of basal insectsmay still play a role in patterning dorsal tissue, given the position of serosa rela-tive to the dorsal ectoderm.Tc-dppis expressed in serosal cells surrounding thegerm anlage [i.e., closest to dorsal ectoderm (72, 139)]. Positionally, this is essen-tially the same, relative to the dorsal ectoderm, asdppand zen expression inD.melanogasterand may therefore constitute “dorsal” expression. Thus the serosaldppdomain might still be involved in patterning “dorsal” tissue in theTriboliumembryo. Later in development, bothTriboliumandSchistocerca dppare expressedin the dorsal ectoderm in the abdominal field of the embryo (72, 139). Thus thereis potentially a more compelling argument for DV patterning bydppduring laterdevelopment.

Evidence for repression ofTc-dppby Dorsal protein can be inferred from thefact thatTc-dpp is not co-expressed in most cells with nuclear Tc-Dorsal (23).Although one could argue against repression ofTc-dppby Tc-Dorsal as theyare co-expressed in terminal cells, bothdpp and nuclear Dorsal are also foundin terminal cells of theD. melanogasterembryo suggesting differences indppregulation at the termini in both species. Here we should point out the limits ofexpression data, in thatTribolium dppexpression also potentially overlaps withthe anterior factorhunchbackand with the terminal system gene producttailless(23, 148, 193). Thus expression data could just as well be used to argue thatTc-dppis regulated by the anterior or terminal systems. The growing inventory of toolsavailable for gene expression manipulation inTriboliummay confirm whether thetranscriptional regulation ofdppby Dorsal is conserved from flies to beetles.

dpp, one of theD. melanogasterhomologues of the TGFβ superfamily, ismost similar to the BMP2/4 group. Indeed, thedpp phenotype can be rescuedusing a Dpp-BMP4 fusion product (112, 113). Manipulations inXenopusas wellas zebrafish genetics suggest that the Dpp signaling pathway plays a conservedrole in axial patterning (reviewed in 29, 56). As inDrosophila, regulation of Dppprotein activity may be the key to generating positional information along the DVaxis in chordates. This has been shown inXenopusby the injection of BMP4, whichaffects DV patterning in a concentration-dependent fashion: Injection of BMP4 canventralize mesoderm (promoting fates such as blood, and having antineurogeniceffects on ectodermal tissue). Similarly, injection of a dominant negative BMPreceptor shows that BMP signaling is required for the development of ventralfates (47). The role of BMPs in DV patterning of zebrafish has been demonstratedgenetically, where theswirl (BMP2) mutant has a dorsalized phenotype [broadenednotochord and expanded somites (51, 78)].

Regulation of BMP signaling is also conserved in chordates in that antag-onists of BMPs operate during DV axial patterning. Classical embryological


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experiments demonstrate that dorsal fates are promoted by organizer tissue, whichupon transplantation leads to dorsalization of ventral host tissue, i.e., an ectopic DVaxis (163). TheXenopusorganizer expresses BMP antagonists includingchordin,the functional homologue ofshort gastrulation(57, 141).Xenopus chordininjec-tions induce a second axis, rescue UV ventralized embryos, and can dorsalizemesoderm by antagonizing BMPs (118, 141). Conversely, the effect ofchordinin-jections alone can be mitigated by the co-injection of high concentrations of BMP4,suggesting a competitive binding interaction (189). Physical Chordin/BMP4 inter-action has been confirmed by in vitro binding studies that demonstrate interactionwith a binding constant sufficient to interfere with receptor binding (118). Gen-etic data from zebrafish confirm that the interaction of BMP/Chordin mirrors theDrosophila Dpp/Sog interaction in patterning the DV axis. Thus thechordino(chordin) mutant leads to ventralization, a phenotype that is suppressed by theswirl (BMP2) mutant (51, 78, 149).

Further levels of conservation are revealed upon examining Chordin regulation.A Xenopus tolloidhomologue is capable of cleaving Chordin, thereby overridingChordin inhibition of BMP4 (117). More recently, conservation of thetwisted gas-trulation gene has also been demonstrated. In contrast to initial data,twisted gas-trulation may be a conserved inhibitor of BMP function. Some evidence indicatesthat it is responsible for producing a differential cleavage product of Sog/Chordinthat may have increased anti-Dpp/BMP activity (19, 111, 132, 153, 196).

Downstream effectors of Dpp signaling also appear to be conserved in chor-dates, such that signal transduction is mediated byXenopushomologues of theMad transcription factor (for example, see 89). A number of zebrafish mutationsleading to DV phenotypes lie in genes encoding members of the BMP signal-ing pathway, once again suggesting a conserved role for this signal transductionpathway in DV axis formation (for examples, see 9, 14, 54).

Thus thedppligand, its antagonist (sog), potentiator (tolloid ), and downstreamcomponents (Mad homologues) all play a role in dorsoventral axis formationin Drosophila, Xenopus, and zebrafish, suggesting a potentially ancient role forthis pathway in DV axis formation. Moreover, injection ofD. melanogaster dppandsoginto Xenopusshows that they behave functionally as BMP4 andchordin(57, 144). However, not all aspects of DV axis formation are conserved fromflies to chordates. Although the protein Noggin binds and antagonizes BMP4in Xenopus, an orthologue has yet to be found in the genomic sequence of the fly(103, 198).

Since Dpp signaling is involved in providing axial information in bothXenopusandD. melanogaster, one might argue that it is an ancient component in dorsoven-tral axis formation and perhaps ancestral. However, there is evidence that the axisclassically regarded as the dorsoventral axis inXenopusmay not actually be so.Reassessment of the position of primitive blood on theXenopusfate map has beenused to argue that the classical embryonic DV axis can actually be characterizedas an anteroposterior axis (83). This does not necessarily argue for nonconser-vation, only that the BMP signaling pathway may also control some aspects of



anteriorization along the “DV” axis inXenopus. One could also argue that asdppgenetically behaves morphogenetically, it could have been recruited into axisspecification in multiple lineages, given that morphogenetic activity is clearly anefficient means of generating positional information.

However, the BMP pathway is tightly coupled to the dorsoventral axis, asdemonstrated by the facts that the deuterostome dorsoventral axis seems mor-phologically inverted when compared to protostomes, but that this inversion oc-curs with appropriate alterations in the expression patterns ofdpp/BMP4 andsog/chordin (56). More specifically, tissue closest to the blastopore and centralnervous system expresseschordin/sog, whereas tissue further away (be it am-nioserosa or blood) expresses BMP4/dpp. This suggests a tight coupling of thedpphomologue to distinct tissue fates, wherever they lie along the DV body axis(dorsal in flies and ventral in frogs), and may reflect an ancestral role of thepathway in restricting neural fates. The intimate relationship betweendpp/BMPexpression and dorsoventral territories across phyla argues that the Dpp signal-ing pathway played an ancestral role in DV patterning. However, systems in-volved in the activation ofdpp/BMP4 transcription have not been shown to beconserved across species in the context of axis formation, and thus may be derived(see below). This may also be true of the system for setting up ventral fates inDrosophila.

twist Is Involved in Mesoderm Patterning in Many Animals

twistis essential for specifying the ventral-most territory of theDrosophilaembryo[fated as mesoderm; (Figure 2b)]. Genetic analysis and dissection of thetwistpromoter suggest that it is directly activated by nuclear Dorsal (182). Loss-of-function alleles oftwist lead to loss of mesoderm, suggesting thattwist is essentialfor specification of ventral tissue in flies (180). This role fortwist seems to beconserved between flies and beetles as theTribolium twistorthologue is expressedat blastoderm stage in a narrow ventral stripe overlapping the ventral embryonicdomain of nuclear Dorsal (23, 160). Slightly later in embryogenesis, thetwistexpression domain retracts anteriorly and widens posteriorly in an apparentlyTc-Dorsal-independent fashion (23). Thus inTribolium, the role of twist as a specifierof ventral fate seems conserved, but its early expression pattern may not be asdependent ondorsalas it is inDrosophila.

However, unlikedpp, twist does not appear to play a role in specifying axialfate in the chordates, as evidenced by its lack of involvement in pan-mesodermalspecification. Although multipletwist orthologues have been isolated from chor-dates including mouse andXenopus, they are expressed in and seem to activatespecific mesodermal derivatives, and may also inhibit some myogenesis (for ex-ample, see 59, 191).twist may therefore control submesodermal fates rather thanacting as a mesoderm or ventral specification factor in the chordates. Interestingly,the jellyfishtwistorthologue is expressed in nonmesodermal tissue (164). Jellyfishare considered to be diploblastic (i.e., have only two germ layers, lacking true


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mesoderm). Thus the expression oftwist in muscle-like cells in the jellyfishmight suggest thattwist is involved in specification of a mesodermal-like layer.Alternatively, twist may play a role in specification of muscle-like cell fate thatpredates the origin of mesoderm.twist may have been independently recruited asan axial patterning output in the arthropod lineage, a likely hypothesis as the datafrom jellyfish and chordates suggest that the ancestral role oftwist is not generalmesoderm specification. As withdpp, regulation oftwist by the Dorsal transcrip-tion factor may be a recent innovation, as theToll signaling pathway may havebeen recently recruited into DV axis formation in the insects.

The Toll/dorsal Signaling Pathway May Have Been Co-Optedinto Axis Formation from the Immune System

The role of theToll signaling pathway in dorsoventral axis formation may not beancestral. This is particularly interesting as theD. melanogaster Tollpathway isdirectly responsible for restricting transcriptional activation ofdpp to the dorsalside of the embryo, and the latter appears to be ancestral for axis formation (seeabove). As with thedpppathway, the components of theToll signaling pathwayhave orthologues within the insects and across phyla. ThusToll homologues havebeen isolated fromTribolium castaneum, as well as from mammals (52, 94, 130).The key downstream effector ofToll receptor homologues seems functionallyconserved across species (dorsal in the insects,NFκB in mammals).

Expression data indicate that bothToll anddorsalplay a conserved role in DVpatterning ofTribolium (23, 94). BothToll anddorsalare found in gradients in theventral region of the embryo during earlyTribolium embryogenesis. Indeed, im-munostaining indicates that theTribolium Dorsal protein forms a ventral nuclearlocalization gradient during blastoderm stage. Interestingly, theTribolium Tollreceptor seems at first sight to differ in some respects from itsD. melanogastercounterpart (94). Rather than being maternally provided and ubiquitously ex-pressed,Tribolium Toll is found in a ventral gradient in cells with nuclearly lo-calizedTc-Dorsal. This finding was used as the basis of the proposition that theToll gradient may form zygotically in response to nuclear localization of Dorsal(23, 94). Local (as opposed to ubiquitous) injections ofD. melanogaster Tollcanrescue theDrosophila Tollphenotype (52). Thus, although it seems important tohave enoughDrosophilaToll receptor to sequester ligand,Toll transcript need onlybe applied (and presumably expressed) relatively locally to form a normal axis.The localized expression ofToll in Tribolium does not necessarily imply a func-tional difference to ubiquitousToll expression inDrosophila. Furthermore, it hasbeen argued that there has been a shift toward maternal control of axis formationin higher insects such asDrosophila, which is consistent with a shift from zygoticto maternal expression of factors such asToll in flies (115, 121). Essentially, theventral increase inToll levels in putative response to nuclear Dorsal may indicateextensive zygotic refinement of pattern in response to axial signaling. Thus, al-though the transcriptional regulation ofTribolium Tollappears to be different, the



Toll/dorsal pathway may play a fundamentally conserved role in axis formationfrom beetles to flies.

It is unknown whether these factors play a role in axis formation in otherphyla. The mammalianToll anddorsalhomologues were identified for their rolein the immune system. Interestingly,Toll anddorsal in D. melanogasterand theIL-1R/NFκB pathway in mammals both play a role in stimulation of the in-nate immune response during pathogenic aggression (84, 85, 95). AlthoughD.melanogaster Toll,spatzle, anddorsalcan stimulate dorsalization in UV-ventralizedXenopusembryos, there is no evidence that the chordate homologues of these genesfunction, or are expressed, at the correct developmental time to play a role in DVaxis formation in these animals (6). Thus one can only hypothesize that the an-cestral role of theToll/dorsalsignaling pathway was in the immune system. Thepathway appears to have become involved in axis formation in the lineage leadingto insects (Figure 3).

Conservation of the DV Axis FormationCassette in Its Entirety?

Although the Dpp signaling pathway appears to play a highly conserved role in DVaxis formation, this is not necessarily true of eithertwistor dorsal. twistmay haveplayed an ancestral role in some form of mesoderm development (either general

Figure 3 A simplified model for the evolution of DV patterning inD. melanogaster.The model considers an ancestral state (bubbled boxes, top row) where Dpp signalingwas involved in DV patterning,twist in mesoderm fate specification, andToll signalingin the innate immune response. In the lineage leading to flies (big arrow), dppandtwistfell under the regulation ofToll signaling during early development, and thus a singlematernal axis formation system.


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mesoderm specification or, more likely, specification of muscle percursors) intriploblastic lineages, and perhaps even in more primitive animals. At some point inthe lineage leading to higher insects, very earlydppandtwistexpression may havefallen under the regulation of theToll signaling pathway. TheToll/dorsalpathwaycould have been recruited from an ancestral role in the immune system for DV axisspecification (as suggested by conserved usage of this signaling system in innateimmunity fromDrosophilato mammals). Assumption bydorsalof transcriptionalregulation of factors such asdppandtwist would have led to the modern cascadeof pathways specifying regions along the DV axis inD. melanogaster(Figure 3).

However, the above genes may already form a regulatory cassette in the chick,suggesting that regulation oftwistanddppbydorsalis more ancient than implied inthe above scenario (18, 75). During outgrowth of the chick limb bud,NFκB(dorsal)is expressed in mesenchyme underlying the apical ectodermal ridge (AER). TheAER is a morphological ridge along the developing limb bud, which is known fromclassical embryological experiments to be required for limb outgrowth (142, 175).Decreasing the activity of NFκB using viral overexpression of anIκB (cactus)mutant that cannot be targeted for degradation leads to defects in limb outgrowth(18, 75). Interestingly,twist mutants in mouse and humans lead to similar limbphenotypes, suggesting conservation of this role in limb development across ver-tebrate species (15, 60).twist, which is expressed in chick limb mesenchyme, isdownregulated under these experimental conditions, implying that it is under pos-itive regulation byNFκB (18, 75). Furthermore, in this experiment the expressionof BMP4 is upregulated, suggesting that adpphomologue is negatively controlledby NFκB in the context of chick limb development.

Thus the entire regulatory cassette (dpp and twist under control of adorsalhomologue) may actually be an ancient network that in extant animals has becomecritical for limb development in chordates and DV axiation inD. melanogaster.Whether this cassette had a function in axis formation in the chordates is not knownsince vestiges ofdorsal-mediateddppregulation during early embryogenesis havenot been found outside the arthropods. There is as yet no indication that aNFκBhomologue is expressed in mouse during early embryogenesis [although not alldorsal homologues have been tested, and the transgenic reporter approach usedto analyze expression may not recapitulate the complete expression pattern ofthe gene (see 146)]. The fact thatD. melanogaster spatzleandToll can inducedorsalization in ventralizedXenopusembryos is compelling in this context (6).However, expression analysis and loss-of-function studies are required to showthat theXenopus Tolland putativespatzleorthologues are expressed at the correcttime and can also generate DV phenotypes when overexpressed.

Thus far, we have discussed conservation of signal transduction systems thatcan easily be envisioned as being conserved in animals with very different embryo-genesis from that ofD. melanogaster. We now examine the anterior system, whichis based on the diffusion of molecules in the syncytial environment of the earlyDrosophilaembryo, and ask whether such an extreme mechanism for generatinga morphogen gradient can be conserved.



THE ANTERIOR SYSTEM

AP Patterning in Drosophila melanogaster

Our understanding of the anterior system centers around the archetypal morphogen,bicoid (bcd) (Figure 4). During oogenesisbcdmRNA becomes localized to theanterior of the oocyte in a process depending upon various factors (96). Diffusion ofBcd protein from its anterior location sets up a gradient that determines positionalinformation. The homeoprotein Bcd regulates gene activity through transcriptionaland translational control. An example of the former is zygotichunchback(hb),which bcd transcriptionally activates in an anterior domain (33). Like Bcd,hbprovides information for anterior patterning through gap, pair-rule, andhoxgenes(reviewed in 127, 157, 173). Bcd is also responsible for the anterior translationalrepression of ubiquitous maternalcaudalmRNA leading to a posterior gradientof the homeoprotein transcription factor Caudal (34, 129).caudal is involved inposterior patterning through the gap genes and behaves as a homeotic gene inthe posterior-most segment of the fly (90, 97, 98, 128, 151). The anterior systeminvolves two factors,caudalandhb, that are conserved to varying extents in otherspecies.

caudal May Play a Conserved Role inPosterior Patterning Across Phyla

caudalappears to play a role in posterior patterning throughout arthropods andacross to the chordates. Within the arthropods,caudal has been cloned from,amongst other insects,Tribolium castaneumand the silkmothBombyx mori(150,194). BothTribolium andBombyx caudal(RNA and protein) form posterior gra-dients, reminiscent of those observed in the flies, and suggestive of conservedregulation. Furthermore, heterologous expression of theTribolium caudalgene inD. melanogastershows that abcd-dependent gradient can be generated, suggest-ing conserved translational regulation ofcaudalin the beetle, though aTriboliumbicoid homologue has not yet been identified (192).caudal is also expressedposteriorly in the grasshopper embryo, suggesting a conserved role in posteriorpatterning in a more basal insect (31).caudalmay also play a conserved role inthe waspNasonia vitripennis, where thehead onlymutation greatly resemblesthe phenotype of theD. melanogaster caudalmutant (121). Strikingly,caudalhomologues may play conserved roles in vertebrate development, as well as in anascidian and nematode (for example, see 36, 43, 44, 69, 73, 76, 91, 119). For exam-ple, cdxA-Cin chick are expressed in a spatially and temporally graded fashionin posterior neural plate and midline during primitive streak stages, suggesting aconserved role in posterior patterning. Loss ofC. elegans caudal( pal-1) functionaffects posterior blastomere and adult male tail development (36, 63). Similarly,thecaudalhomologue in an ascidian has been shown to play a functional role intail development (76). Thuscaudallikely played a role in posterior patterning inthe ancestral insect and perhaps even in the ancestral bilaterian.


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hb May Play a Conserved Role in APPatterning Within the Insects

The status ofhunchback(hb) as an ancestral AP patterning factor is less clear-cut. Orthologues have been cloned across phyla, but there is no evidence for arole in AP patterning outside the insects. As described above, maternal and zy-gotichbare involved in AP patterning during earlyD. melanogasterdevelopment(Figure 4). Flies closely related toD. melanogaster(Drosophila virilis), as wellas basal insects such as the grasshopperSchistocerca americana, havehb homo-logues (115, 131, 158, 159, 168, 183, 193). The fact that insecthbhomologues areexpressed maternally and zygotically in an anterior domain suggests a conservedrole in AP patterning within the insects. Interestingly, the grasshopper,S. ameri-cana, expresseshb in a cellular environment in blocks of different concentrationalong the AP axis (115). This may reflect how positional information is conveyedin a cellular environment, as opposed to the gradients observed in the syncytialenvironment of theDrosophila blastoderm. However, outside the insects thereis no clear evidence for a role in AP patterning from expression data, althoughhb orthologues are found in conserved domains in the nervous system (and aretherefore probably true orthologues). For example, neitherH. triserialis (annelid)norC. elegans(nematode)hb is expressed in an early anterior domain that wouldindicate a role in AP patterning (38, 70, 143). Perhapshb was co-opted relativelyrecently from the nervous system into its role as an anterior morphogen withinthe arthropods. Thushunchbackmay have played a role in AP axis formation inthe lineage leading to insects, but probably not in other phyla.hbhas orthologuesoutside the insects. Expression data from the chelicerates and crustaceans shouldindicate whetherhbplays a role in AP patterning in all arthropods.

bcd Function May Be a Recent Novelty in AP Patterning

The status ofbcd in ancestral AP patterning is markedly different. Despite itsimportance in AP patterning inDrosophila, bcdhomologues have been isolatedonly from higher dipterans (the cyclorrhaphan flies; 158, 168). There is evidencethatMegaselia abdita bicoid(Ma-bcd) does play a functional role in axis forma-tion: Ma-bcdis expressed anteriorly, and dsRNA interference experiments lead toa phenotype that resembles that of theD. melanogaster bcdmutant, except thatthe range of its action extends more posteriorly (168). These data suggest that incontrast toDrosophila, bcd’s sphere of influence may extend further posteriorlyalong the AP axis ofMegaselia. Indirect evidence for the conservation ofbcd ina more basal insect comes fromTribolium. As noted above,Tc-caudaltranscriptcan be regulated in abcd-dependent fashion when introduced into flies (192). Thissuggests the existence of an as yet unidentifiedbicoid-like activity in Tribolium.Such data would imply thatbcd is involved in AP patterning from flies to beetles.

Molecular data, however, suggest that thebcd gene arose relatively recently.Phylogenetic analysis of theMegaselia abdita bicoidandzenorthologues suggeststhat they are products of a Hox3 duplication in an ancestor of the higher dipterans(167). bcd and zenreside together in the part ofD. melanogasterHox cluster



in the location where one should find a Hox3 orthologue. This adds support tothe idea that the two genes are products of a Hox3 duplication. Furthermore, theregion of theTriboliumhox cluster in which one would expect to findbcdhas nowbeen sequenced and nobcd orthologue was found (16). Although theTriboliumorthologue could have moved via a recombination event, the inability to isolatebcd from lower insects, as well as the sequence analysis ofMegaselia bcdandzen, are evidence for a relatively recent origin ofbcd in the lineage leading tohigher flies.bcdis thus an interesting case in which a gene has arisen recently andyet become a major molecular player in axis formation (whether the ancestor ofbcdplayed a role in axis formation is interesting but unresolved). The mechanismby which bcd might have become involved in AP axis formation is discussedbelow and by Schmidt-Ott (145). The Hox3 orthologue has been cloned from twochelicerates, a mite and a spider (28, 179). Hox3 in chelicerates is expressed ina discrete domain of the AP axis, but its anterior border coincides with the Hoxgeneproboscopedia( pb). Thus, although Hox3 in chelicerates is found in a hox-like expression domain, it is expressed more anteriorly than expected, suggestinga breakdown in colinear expression of this gene within the arthropods. Overlapwith pband the breakdown of colinearity are hypothesized to have allowed Hox3to lose hox function and take on novel roles in the lineage leading to insects(28, 179).

What does this tell us about the way in which the anterior patterning systemin D. melanogasterevolved? The expression ofhb outside the insects suggeststhat its role in nervous system development is highly conserved. The ancestralpatterning system in insects may have involved a posteriorcaudalgradient and ananteriorhb gradient. The duplication of an ancestral Hox3 gene may have givenrise tobcd, which in the modern fly plays a dual role as translational regulatorof maternalcaudal, and transcriptional activator of zygotichb. In this scheme anovel gene,bcd, has become a major organizer of the AP axis (Figure 5) (reviewedin 30). Thus anterior patterning inD. melanogasterdisplays the same propertiesas the DV system: Potentially ancient (caudal) and more recently recruited (hb)regulatory factors have fallen under the global coordination of a novel gene (theanteriorbcdgradient) (Figure 5).

EVOLUTION OF AXIS FORMATION MECHANISMS

The maternal coordinate systems inDrosophilaappear to be a melange of molec-ular processes elaborated upon multiple times in evolutionary history. ModernD. melanogasterutilizes evolutionarily ancient factors such asdpp andcaudal,which play a conserved role in axial patterning in chordates.Drosophilaalso usemolecules such aszenandbicoid, which may have arisen relatively recently, inthe lineage leading to higher dipterans. This suggests that axis formation inD.melanogasteris a plastic and rapidly evolving process at the molecular level andis constantly taking advantage of new cues and innovations. However, innova-tion does not necessarily mean the displacement of pathways, although the cues


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Figure 5 A potential, simplified scheme for the origin of theD. melanogasterante-rior system. The scheme envisages an ancestral state (bubble boxes, top row) wherehunchbackwas involved in neural patterning,caudal in posterior patterning, and aHox3 gene in segmental identity along the AP axis. In the lineage leading to insects,hunchbackandcaudalmay both have been involved in axial patterning, whereas Hox3became expressed in a spatial pattern that is no longer colinear with other Hox genes(middle row). In the lineage leading to the higher dipterans, the duplication of Hox3may have led tobicoid, which acts in the modern fly as a major player in AP axisformation through the regulation ofhunchbackandcaudal.

regulating them may have changed, as exemplified by the deep-set link betweenDpp signaling and DV axiation. Also, it is not simply axial cues that change, butdownstream specification factors can also fall under the control of axial patterningsystems. This is exemplified bytwist, which plays a role in the development ofmesoderm and its derivatives in all systems studied, but may only constitute aglobal mesoderm specification factor within the insects.

REDUNDANCY IN AXIS FORMATION

How can an event as important as axis formation evolve rapidly? One mechanismwould be to retain old pathways in a redundant fashion.bcdmay still be rapidlychanging at the sequence level, and its role in AP axiation might have arisenrelatively recently. Interestingly, the anterior system has long been known to show



redundancy with the posterior maternal coordinate system. Such large-scale re-dundancy may be crucial to plasticity in axis formation.

Both the anterior and posterior systems lead to the same outcome, althoughthis outcome is slightly temporally displaced: Both systems result in a Hb proteingradient with highest levels at the anterior (Figure 4) (see 33, 62, 67, 173, 176).Hb protein is detectable as a plateau until about 50% egg length, where it beginsto taper off (176). As mentioned above, the Bcd gradient is crucial to generatingthe zygotichb pattern. The earliest differentialhb expression is, however, due tothe translational repression of a uniform maternal pool ofhb transcript (Figure 4)(see 176, 178). This translational repression is mediated bynanos, and its cofactorpumilio (62, 67, 102). Pumilio recognizes a sequence (NRE) in the 3′UTR of hbtranscript, and forms a multiprotein complex in vitro, which includes the NRE,Nanos protein, and Pumilio itself (161, 162, 188). Sincenanostranscript is ma-ternally localized to the posterior of the developing oocyte and translationallyrepressed anteriorly, a Nanos protein gradient forms emanating from the posterior(Figure 4) (26, 185). Hence, maternalhb transcript is translationally repressed atthe posterior, leading to an anterior gradient of Hb protein.

Thus two systems, an anterior and a posterior system, independently gener-ate an anterior Hb gradient. Furthermore, elegant genetic data indicate that thetwo gradients ofhb are redundant. Genetically, thenanosphenotype, in whichabdominal segments are entirely lost, can be rescued by eliminating maternalhbtranscript (62, 67). Thenanosaxis formation phenotype is therefore due to theectopic activity of maternalhb in the posterior of the embryo. Furthermore, zy-gotic hb compensates entirely for loss of the maternal transcript. Therefore, theposterior maternal coordinate system can be eliminated and AP axis formationoccurs normally, with only the anterior system operating.

More recent evidence suggests the converse may also be true. Elimination ofbcdactivity cannot be compensated for by the posterior system as it stands (190).Phenotypes are still observed in T2/T3 (parasegment 4), so that levels of maternalhbseem to be unable to compensate for the lack of zygotically activatedhb in thisdomain. However, if extra copies ofhb are supplied, and the levels ofhb furtherincreased by reducing levels of a transcriptional repressor of zygotichb (knirps),the need forbcd in thoracic development is abrogated (190). Thushb can almostentirely compensate for the anterior system during early AP axis formation (almostentirely, since the extreme anterior of thebcdcuticle phenotype cannot be rescuedin this way).

Why doesD. melanogasterhave two virtually redundant systems carrying outsuch similar functions? The answer may be that one system was derived morerecently than the other. Nanos response elements are found inhb orthologues inmany insects (for example, see 158, 183, 193). Furthermore,nanosandpumiliohave been cloned from multiple phyla including chordates, nematodes, and an-nelids (e.g., 25, 81, 101, 120, 174, 197).nanosmay even play a role in axis speci-fication in comparatively basal insects (S.L. & N.H.P., unpublished observation).Thus while thebcd system seems a recent innovation in axis formation, settingup a gradient of Hb using translational repression by Nanos seems to be a more


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ancestral mechanism. What we may be observing inD. melanogasteris an inter-mediate in the displacement of an ancient AP patterning system (nanosregulated)by a more recently innovated one (bcdregulated). Redundancy of the two systemscould constitute a safety net that protects the integrity of AP patterning as axisformation rapidly evolves.

LINKING MORPHOLOGICAL CHANGES INEMBRYOGENESIS TO EVOLUTION OF AXIS FORMATION

Recent data from dipterans suggest a correlation between the origin ofbcdas ananterior patterning factor and a profound and potentially linked change in devel-opmental morphology. Less derived insects have two extraembryonic membranes,the amnion and the serosa, that have distinct morphologies and derivations duringembryogenesis (3). However,D. melanogasterhas a fused and reduced single ex-traembryonic tissue called the amnioserosa (2). The link between the amnioserosain D. melanogasterand the amnion and the serosa in less derived insects is clearfrom the expression of extraembryonic markers such aszen, as well as from thefate of these tissues in later embryogenesis. However, whereasD. melanogasteramnioserosa lies in the DV axis of the egg and is clearly specified by the DV axis for-mation pathway (under the control ofdpp), serosa in basal insects originates fromtissue anterior to the embryo proper. A major change may thus have occurred inspecification of extraembryonic material since it has moved into a different eggaxis.

Examination of the status of extraembryonic membranes within dipterans sug-gests that the fused and reduced amnioserosa is a character of the higher dipterans(cyclorrhaphan flies). Thus the basal cyclorrhaphan flyMegaselia abditahas asingle extraembryonic membrane, whereas a representative species from a sistertaxon, theEmpidoidea(E. livida), has a separate amnion and serosa (145, 167).These data place the origin of amnioserosa and its DV axis location in the samephylogenetic position as the hypothesized duplication of a Hox3 group gene togive bcd andzen(167). This gene duplication may also have been essential forrelocation of extraembryonic material from the anterior region of the egg to the DVaxis under control of theToll signaling system. The appearance of a novel anteriorpatterning factor,bcd, may have been key to allowing full spatial repositioning ofamnioserosa, along withzenexpression, into the DV axis of the egg (145).

Interestingly, there are also major changes in head morphology at the base ofthe cyclorrhaphan flies. In particular, the cyclorrhaphan flies have reduced feedingstructures (the cephalopharyngeal skeleton) and larvae hatch with involuted heads(195). Although the morphology of head characters is variable amongst the cyclor-rhaphan flies, displacement of an ancestral anterior patterning system bybcdmayhave been essential for (or even responsible for) these changes. Early patterningby bcd may have a profound effect on later head morphology, and the fact thatMegaseliahas a markedly different range ofbcdactivity may indicate that evenwithin the cyclorrhaphan fliesbcdhas varying roles or effects (168).



Thus the molecular evolution of axis formation systems in the higher fliesappears to correlate with major changes in morphology. This would suggest thatrapid evolution of axis formation is potentially tied to the diversity of morphologybefore and perhaps even after the phylotypic stage, i.e., the stage where embryosof different species within a phylum converge on a very similar morphology.

CONCLUDING REMARKS

Recent data, such as those examiningbicoidandzenin more basal insects, suggestthat some factors involved in axis formation inD. melanogasterare derived (167).Along with data examining the morphology of axis specification in nematodes,these findings suggest that axis formation is a variable and plastic process (46). Thisis consistent with models that view developmental processes as an hourglass withvariability at the base leading to a highly conserved phylotypic stage, followed byincreasing diversity in adult body plan (see 35, 122). This analogy may understatethe remarkable underlying conservation of axis formation (for example in the useof Dpp signaling in DV axis formation andcaudalin AP patterning).

The recent data that correlate changes in morphology with molecular evolutionof axis formation are particularly exciting, as they indicate the types of morpholog-ical change that rapid evolution of axis formation might allow. Early developmentalprocesses have been investigated in the light of extreme modifications in life his-tory, for example, in the context of the endoparasitic waspCopidosoma(reviewedin 49).Copidosomaundergoes polyembryonic development, where early cleavageevents give rise to 2000 randomly oriented embryos. Here axis formation maydiffer from the mechanisms elucidated inD. melanogaster(50). Future data fromCopidosomamay shed light on the molecular evolution of axis formation duringa derived and fundamentally different form of embryogenesis.

ACKNOWLEDGMENTS

We thank William Browne, Gregory Davis, James McClintock, and Steven Podosfor reading and helpful discussion of the manuscript. NHP is an HHMI investigator.

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29 Oct 2001 16:42 AR AR144-15-COLOR.tex AR144-15-COLOR.SGM ARv2(2001/05/10)P1: GDL

Figure 4 AP patterning inD. melanogaster. The diagram illustrates the interconnec-tions between the anterior and posterior maternal co-ordinate systems. The anteriorsystem (key genebicoid) leads to the localized activation of zygotichb transcriptionand maternalcaudaltranslational control. The posterior system involves the transla-tional repression of maternalhunchbacktranscript. The resulting Hunchback proteingradient has an effect on zygoticcaudaltranscription. Anterior is to the left, posteriorto the right, and shading indicates gradient vs ubiquitous localization.

Sabbi Lall and Nipam H. Patel · curs well before gastrulation in many short and long germ embryos....

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