Vernon French

Summary

The formation of segments in the Drosophila early embryo is understood in greater detail than any other complex developmental process. Now, by studying other types of insect embryo, we can hope to deduce something of the ancestral mechanism of segmentation and the ways in which it has been modified in evolution. The parasitic wasp, Copidosoma floridanurn, is spectacularly atypical of insects in that the small egg cell divides extensively, with no initial syncytial phase, and forms eventually some 2000 embryos(’). This process raises intriguing questions about the control of embryonic polarity and segmentation.

Background The typical insect egg has a thin layer of cytoplasm that sur- rounds a central core of yolk and remains syncytial during many initial rounds of nuclear division. Cell membranes then intrude, converting the surface syncytium into the blas- toderm cell layer from which the segmented embryo devel- ops. Segmentation can be first detected by the appearance of periodic stripes of engrailed gene expression, and this pattern may arise in very different ways(2). In a ‘long-germ’ insect, such as Drosophila, segmentation of the large embryo is completed around the time that cells are formed(3). In contrast, the embryo of a ‘short-germ’ insect, such as the locust Schistocerca, forms from a small region of the blastoderm and its posterior segments are added sequentially during growth, long after cell~larisation(~).

Short-germ development occurs in many insect orders and was probably the ancestral mode of segmentatiod2), whereas long-germ development only appears in the more recently derived orders, such as Coleoptera (beetles), Hymenoptera (wasps, bees) and Diptera (flies). Consider- ing that the insect orders have been separate for several hundred million years, the mechanisms of short-germ seg- mentation probably differ considerably between, say, locusts and beetled5). Also, long-germ development in, say, the Coleoptera and Diptera may have evolved indepen- dently, and may differ in mechanism(5).

Segmentation in Drosophila Through genetic and molecular analysis, we now broadly understand segmentation in one long-germ dipteran: Dro~ophila(~3~). Embryonic polarity originates way back in oogenesis, principally through the localisation of maternal transcripts of bicoidand nanos in the anterior and posterior

cytoplasm of the oocyte, respectively@). These positional cues are then elaborated through spatial control of ‘seg- mentation gene’ expression in the early embryo. First, with the onset of translation, diffusion in the syncytial cytoplasm results in anterior gradients of Bicoid and (indirectly, through the effect of Nanos) of maternal Hunchback protein. High Bicoid levels activate transcription of hunchbackin the ante- rior nuclei, thereby reinforcing the maternal Hunchback gra- dient, and then other gap genes (e.g. Kruppel, knirps) are activated by specific concentration ranges of Bicoid and Hunchback(7). In this way, transcript localisation during oogenesis leads to protein gradients that locate bands of gap gene transcription in the embryo. Again, the resulting gap proteins diffuse in the syncytial cytoplasm to form a set of overlapping gradients and these establish the spatial periodicity of pair-rule gene expression.

‘Primary’ pair-rule genes such as even-skipped (eve) are activated, through independent enhancers, by different combinations of gap protein concentrations, resulting in seven broad stripes of expression at specific locations along the embryo(7). Expression is then modulated by interactions between pair-rule genes: eve stripes, for example, become narrow and separated by weak secondary stripes, so that expression changes to a segmental periodicity. Around the time of cellularisation, combinations of pair-rule proteins activate the expression of ‘primary’ segment polarity genes (e.g. engraileu) in narrow segmental stripes@. Also, the transient distributions of maternal, gap and pair-rule pro- teins establish initial expression domains of the homeotic genes (e.g. Anfennapedia, Ultrabithorax). The subsequent expression of segment polarity genes underlies pattern for- mation within the segment, and homeotic gene expression specifies the differences between segments.

Segmentation in long- and short-germ insects In Drosophila, the spatial control of segment polarity and homeotic gene expression depends on a sequence of tran- scription factor gradients that apparently result simply from diffusion in the syncytial early embryo. There is consider- able current debate about whether this could be the general (or ancestral) mechanism of insect segmentation, since it seems unsuited to short-germ embryo~(~8~). The segmental expression of engrailed and the domains of homeotic expression are both highly conserved between the different types of insect, but how are they controlled? Expression of some segmentation genes has been studied in a few short- germ embryos and the data are intriguing, but still rather sparse.

In the short-germ locust Schistocerca, nothing is known of gap genes, but the homologues of Drosophila pair-rule genes eve and ftzare not expressed in stripes(iO%li). Short- germ segmentation in the beetle Tribolium, however, does involve bands of gap gene (hunchback, K r ~ p p e 1 ) ( ’ ~ ! ~ ~ ) and broad stripes of pair-rule gene (hairy, eve, ftz)(13r14,15) expression. There are differences, however, between the beetles and Drosophila: in the details of pair-rule expression p a t t e r n ~ ( ’ ~ ! ~ ~ ) and also in the lack of a segmental fusion phenotype after deletion of the Tribolium ftzgene(15). Thus if coleopterans like Tribolium do use pair-rule genes to gener- ate periodic pattern from graded gap protein distributions, the details may differ from those in Drosophila and other dipterand5).

Locusts, as orthopterans, belong to a moderately ancient insect order, so these data hint that the ancestral mecha-

nism of establishing segmental periodicity was by short- range cell interactions(lO), and that ‘gap to pair-rule’ mecha- nisms evolved more recently in some insect orders, facilitat- ing long-germ de~elopment(~). However, this conclusion is somewhat premature and we need data on more genes from more species(5.9).

If segmentation does involve a gap to pair-rule mecha- nism in at least some short-germ insects, how could this operate after cellularisation of the embryo? Gradients could be generated by dilution, if transient gene expression is fol- lowed by cell division at the posterior of the embryo. Alterna- tively, a graded signal could be generated from a region of gap gene expression by a distinct cell interaction system (with secreted small ligands, receptors, etc) subsequently lost in long-germ descendents, like Drosophila, where the gap protein itself can move(5). Perhaps, even in short-germ embryos, the cells may remain connected by junctions that permit diffusion of large segmentation gene p rod~c ts (~8~~) . In one fascinating insect, however, segmentation has been shown to involve the periodic expression of a pair-rule gene, in an embryo (actually, hundreds of them) that definitely is non-syncytial(1).

Polyembryonic development in Copidosoma Almost without exception, insect eggs are large and yolky, remain syncytial until the blastoderm stage and produce single embryos. However, Miodrag Grbic and colleagues(’) have recently studied the egg of a parasitic wasp (Copido- soma floridanurn), which is highly exceptional in having

Fig. 1. Polyembryonic development in the parasitic wasp Copidosoma floridanurn (adapted from ref. 1). (A) Diagrams of early development. (i) The Copidosoma egg is laid inside the egg of the host moth and contains the zygote, plus a persisting polar body (green). (ii) When the host hatches, it contains the parasite primary morula, consisting of about 100 cells surrounded by a syncytial layer (green), derived from the polar body. (iii) In the host third instar larva, the parasite tissue consists of many associated cell groups (and often precocious embryos that develop into larvae and attack male parasites(’8), not shown). (iv) Late in the host fourth instar, some 2000 Copidosoma embryos are formed (one is shown), each initially consisting of about 20 cells, surrounded by syncytium. Embryos undergo cell division, gastrulation and segmentation. They hatch, consume the host while going through larval development, pupate, emerge as wasps and escape. (B) Segmentation gene expression. (i) Embryo at germ band extension stage, showing even-skipped (eve) expression (by antibody recognition of Eve protein). Segmental stripes of Eve are evident in the head (3), thorax (3) and very anterior abdomen, and there is a large posterior expression zone (arrows) from which the other abdominal stripes will resolve in rapid sequence. (ii) Embryo (slightly older than in Bi), double-labelled for Eve (blue) and Engrailed (red) protein. Ten Eve stripes have formed and Engrailed has appeared anteriorly (arrow marks the maxillary segment), in segmental stripes coinciding with eve expression; further Engrailed stripes will appear sequentially down the thorax and abdomen.

complete cell divisions almost from the beginning, and in developing into numerous segmented embryos (Fig. 1).

The wasp egg is laid into a host moth egg, where the zygote develops into a ball of around 100 cells (the primary morula) surrounded by a syncytial layer that is derived from a persisting polar body. After the host egg hatches, the par- asite cells continue to divide inside the larva and form many associated cell groups. Growth continues until late in the host‘s penultimate larval instar, when the tissue forms up to 2000 embryos, each consisting initially of about 20 cells, surrounded by the syncytium. Each embryo develops into a solid ball of cells, undergoes typical morphogenetic move- ments (gastrulation, germ band extension and contraction) and then becomes visibly segmented. The Copidosorna hatch into the unfortunate moth larva, devouring it during their larval growth, and then metamorphose and emerge as adult wasps from the host cuticle.

By cell injection, Grbic et a/.(’) demonstrated that the Copidosoma cells are indeed completely separated after the second division of the zygote. Fluorescently labelled dextran would not pass between cells of the primary morula, the growing cell groups or a developing embryo, nor from an injected cell to the surrounding syncytial layer. Thus exten- sive growth and the formation and segmentation of embryos occurs in a cellular environment within which maternal or gap proteins could not diffuse to give the concentration gra- dients that mediate segmentation in Drosophila. Grbic et

also used antibodies to study segmentation gene expression in Copidosorna. Eve protein first appeared in the pre-gastrulation embryo and then, in rapid anterior-to-pos- terior sequence, expression resolved into 15 thin stripes (in future mandibular to 9th abdominal segments). There was - no indication of the preceding pair-rule expression pattern (broad stripes approximating to alternate segments) that is conspicuous in Drosophila and in beetledi4). Eve was fol- lowed by the appearance of Engrailed protein, first in the posterior head and thorax and then sequentially down the abdomen, in thin stripes corresponding to those cells expressing eve. Another antibody, that recognises both Ultrabithorax and Abdominal-A proteins, revealed a domain of homeotic gene expression from posterior metathorax to 8th abdominal segment.

This study is important as the first demonstration that the familiar segmental engrailed stripes can be formed in an insect embryo that definitely h cellular and it implicates the pair-rule gene eve in the control of engrailed. Unfortunately, nothing is yet known about gap gene transcription or protein distributions that might control eve. In Copidosoma, protein gradients could not form by diffusion. Also, dilution looks unlikely, as segmentation occurs rapidly and the embryo is not extending by posterior growth(’). It is striking that eve precedes engrailed, but not in a pair-rule expression pat- tern. Copidosorna and related polyembryonic wasps have evolved recently from ancestors with typical insect eggs (and probably long-germ so it is possible

that an initial pair-rule phase of eve expression has been lost in these species during adaptation of the mechanism back to operation within a cellular environment(’). The prob- lem is that we know little of segmentation in other hymenopterans, beyond engrailed expression in the long- germ honeybee embryo(17), so it is equally possible that all hymenopterans differ from Drosophila in only expressing eve segmentally (and perhaps not using it to read gap pro- tein concentrations). Further conclusions about the Copido- soma mechanism of segmentation, and its evolution, must await further studies on both this species and its more nor- mal cousins.

The mode of development in Copidosorna also raises intriguing questions about the earliest stages of embryonic patterning. The relationship between oogenesis and embryogenesis is extremely tenuous, as the embryos form after much cell division and apparently in random orienta- tion(’). Although nothing is known of maternal or early zygotic gene expression, it seems improbable that the ante- rior-posterior (and dorsal-ventral) polarity of each embryo could derive directly from molecular localisation in the oocyte, as in Drosophila. As yet, we can only speculate about the mechanism in Copidosorna and how it may have evolved.

Conclusion Analysis of segmentation throughout the insects offers the exciting possibility of tracing the evolution of a developmen- tal mechanism(5). At present, it seems that the ancestral mechanism of establishing segmental periodicity may have differed considerably from that known in Drosophila. Many more species must be studied, however, to establish com- mon (and therefore probably conserved) features of the more ancient orders (e.g. Orthoptera, Odonata), and to compare mechanisms in the more recently derived Coleoptera, Hymenoptera and Di~tera(~3~) . Ultimately, determination of segmentation mechanisms in the different types of insect embryo will require manipulations of gene function, in addition to the description of expression pat- terns.

References 1 Grbic, M., Nagy, L., Carroll, S.B. and Strand, M. (1996). Polyembryonic development: insect pattern formation in a cellularized environment. Development

2 Sander, K. (1976). Specification of the basic body pattern in insect embryogenesis. Adv. lnsectf‘hysiol. 12, 125-238. 3 DiNardo, S., Kuner, J.M., Theis, J. and O’Farrell, P. (1985). Development of embryonic pattern in Drosophila as revealed by accumulation of the nuclear engrailedprotein. Cell43. 59-69. 4 Patel, N.H., Kornberg, T.B. and Goodman, C.S. (1989). Expression of engrailed during segmentation in grasshopper and crayfish. Development 107, 201-212. 5 Patel, N.H. (1994). Developmental evolution: insights from studies of insect segmentation. Science 266, 581 -590. 6 St. Johnston, D. and Nusslein-Volhard, C. (1992). The origin of pattern and polarity in the Drosophilaernbryo. Ce//68,201-219. 7 Pankratz, M.J. and Jackie, H. (1993). Blastoderm Segmentation. In

122,795-804.

Developmenf ofDrosophila (ed. A. Martinez Arias and M. Bate), pp. 467-51 6. Cold Spring Harbor Press, Cold Spring Harbor. 8 Martinez Arias, A. (1993). Development and patterning of the larval epidermis of Drosophila. In Development of Drosophifa (ed. A. Martinez Arias and M. Bate), pp. 517-608. Cold Spring Harbor Press, Cold Spring Harbor. 9 Tautz, D. and Sommer, R. (1995). Evolution of segmentation genes in insects. Trends Genet. 11 I 23-27. 10 Patel, N.H., Ball, E. and Goodman, C.S. (1992). Changing role of even- skippedduring the evolution of insect pattern formation. Nature357,339-342. 11 Dawes, R., Dawson, I., Falciani, F., Tear, G. and Akam, M. (1994). Dax, a locust Hox gene related to fushi-tarazu but showing no pair-rule expression. DevelopmentlZO, 1561-1572. 12 Wolff, C., Sommer, R., Schroder, R., Glaser, G. and Tautz, D. (1995). Conserved and divergent expression aspects of the Drosophila segmentation gene hunchback in the short germ embryo of the flour beetle Tribofium. Developmentl21,4227-4236. 13 Sommer, R. and Tautz, D. (1993). Involvement of an orthologue of the Drosophda pair-rule gene hairy in segment formation of the short germ band embryo of Tribolium (Coleoptera). Nature361,448-450.

14 Patel, N.H., Condron, B. and Zinn, K. (1994). Pair-rule expression patterns of even-skipped are found in both short- and long-germ beetles. Nature 367, 429- 434. 15 Brown, S.J., Hilgenfeld, R.B. and Denell, R.E. (1994). The beetle Tribofium castaneum has a fushi tarazu homolog expressed in stripes during segmentation. Roc. NatlAcad. Sci. USA91,12922-12926. 16 Ivanova-Kasas, O.M. (1 972). Polyembryony in insects. In Deveiopmental Systems: hsecfs (ed. S. J. Counce and C. H. Waddington), pp. 153-186. Academic Press, New York. 17 Fleig, R. (1990). Engrailedexpression and body segmentation in the honeybee Apis mellifera. Roux’s Arch. Dev. Biol. 198, 467-473 18 Grbic, M., Ode, P.J. and Strand, M. (1 993). Sibling rivalry and brood sex ratios in polyembryonic wasps. Nature 360,254-256.

Vernon French is at the Institute of Cell, Animal and Population Biology, University of Edinburgh, Kings Buildings, West Mains Road, Edinburgh EH9 3JT, UK.