Gradients and insect segmentation

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1 Development 104 Supplement, 3-1,6 (1988) Printed in Great The Company of Biologists Limited 1988 Gradients and insect segmentation VERNON FRENCH Department of Zoology, University of Edinburgh, Kings Buildings, West Mains Road, Edinburgh, EHg 3JT, UK Summary 'Morphogen' gradients have long been invoked as a means of specifying spatial patterns of developmental fate, and it has now been demonstrated that they are indeed involved in the early steps of insect segmentation. n many insects, including Drosophilar ligature and transplantation experiments have shown that the segment pattern develops through interactions between the ends of the egg. These results, plus those from irradiation and centrifugation of chironomid eggs, suggest that specific maternally synthesized RNAs are localized at the ends of the oocyte, and act as sources of opposing anterior and posterior gradients in the early egg. n Drosophila, different groups of maternal 'segmentation' genes are required for depositing within the oocyte terminal, anterior and posterior spatial cues. njection of wild-type cytoplasm into mutant eggs which lack the anterior (bicoifi or posterior (oskar) cue suggests that these are normally distributed as gradients from strictly localized sources. t has now been shown directly that bicoid RNA passes into the oocyte from the nurse cells, remains localized in the anterior tip, and is later translated into protein which forms an exponenti al concentration gradient down the early egg. Genes required for posterior spatial information have not yet been cloned, so a posterior gradient (most likely to consist of n&nos product) has yet to be directly demonstrated. Analysis of zygotic tsegmentation' genes has shown that the different segment primordia are not directly specified by small changes in the anterior or (postulated) posterior gradient. t seems likely that the maternal cues specify a few bands of expression of zygotic gap genes such as hunchback, Kriippel and knirps, and that the pattern is then elaborated through interactions between these. The anterior gradient seems to form by diffusion of bicoid protein, but the posterior signal seems to be capable of reorganization in some injection experiments. This could imply a diffusion/reaction mecharism, or could result simply from the \ryay in which the terminal, anterior and posterior cues act via gap gene activity. Hence the segment pattern formed after injection (and after irradiation of chironomid eggs) will not always correspond to the gradient profile. Other types of insect egg develop with no nurse cells or external anterior source of RNA and, in these, there is some evidence of a posterior gradient but not of a similar signal from the anterior end. t is now clear from the analysis of segmentation in Drosophila that the determinants and gradients inferred from earlier studies do provide a positional framework within which the segment pattern is gradually elaborated. nvestigation of segmentation in other eggs will be greatly assisted if the molecular techniques can be transferred from Drosophila. Key words: gradient, insect embryo, segment, pattern formation. lntroduction n most animals, the spatial organtzation of the future embryo originates as the oocyte is developing within the ovary. t acquires a visible polarity and often localized regions of cytoplasm (determinants) which will subsequently confer specific properties on the embryonic cells that come to contain them. These initial differences are elaborated by interactions within the egg and developing embryo until the developmental fate of a cell eventually becomes determined in relation to those of its neighbours and in accordance with position in the embryo. Since the idea was first proposed by Morgan (1901)

2 V. French and others, it has become widely accepted that the 'interactions' often occur by the establishment of a gradient of some unknown 'morphogen'. The gradient is then interpreted to give the morphological pattern through cells responding in specific ways to different morphogen concentrations (Wolpert, L969). A gradient could be formed by the simple diffusion of morphogen from a localized source (Crick, 1970), or by diffusion and reaction between two components (activator and inhibitor) in the absence of any predetermined source (Gierer & Meinhardt, 1972). For almost all developing patterns, however, the gradient is only a plausible model, supported by indirect evidence in the form of abnormal patterns resulting from embryological manipulations. There is considerable evidence of this kind for the involvement of gradients in the early development of insects, but in Drosophila there is now also direct molecular evidence for cytoplasmic determinants and gradients in A (i) fat the egg and early embryo. We are also beginning to understand the steps by which simple graded information in the egg eventually leads to the formation of the complex segmented body pattern. nsects vary considerably in the details of their oogenesis and early embryogenesis. This review will concentrate on relating the embryological, genetic and molecular evidence that the interpretation of morphogen gradients underlies segmentation in Drosophila, and will briefly consider the extent to which these processes may also occur in other types of insect. Early development The formation of insect eggs and embryos has been extensively studied (see reviews of Mahowald, L972; Gutzeit & Sander, 1985; Anderson, 1973; Sander, 1976; Sand er et al. 1984) and will be described only briefly. The oocyte originates at the proximal end of the ovary and matures gradually as it passes out, surrounded by the follicle cells which eventually secrete onto it the vitelline membrane and chorion. n most insects, the egg is then fertilized by stored sperm and is laid. Large amounts of RNA are accumulated during oogenesis, and insect ovaries differ fundamentally in the source of this RNA (Fig. 1A). n the panoistic ovary, active transcription within the oocyte nucleus provides all (or nearly all) of the RNA for the egg. n meroistic ovaries, however, the germ line also gives rise to nurse cells (whose number and arrangement varies with species) which remain connected to the anterior end of the developing oocyte by cytoplas- Ant Post (iii) Fig. L. Oogenesis and early embryogenesis in insects. (A) The development of the oocyte in (i) panoistic and (ii) meroistic types of ovary (adapted from Mahowald, 1972). The oocyte (Oo) passes down the ovariole (or) surrounded by a layer of follicle cells (fl and, in the meroistic ovary, associated with a group of nurse cells (n). The major source of RNA for the oocyte is indicated by arrows. (B) The origin of the embryo from the blastoderm of the early egg (adapted from Sander, L976). The eggs have the same orientation (Ant, anterior; Post, posterior; V, ventral; D, dorsal), but are not drawn to the same scale. n short germ eggs (i, Tachycines) the small embryo consists only of procephalon (Pr) and a posterior growth zone (black), while the rest of the blastoderm (unshaded) forms extraembryonic membranes. n intermediate germ eggs (ti, Acheta; iri, Euscelis) the early embryo seems to consist of the procephalon and gnathocephalon (G) of the head, and the thorax (T), plus a growth zone. n long germ eggs (iv, Drosophila) al of the segments, including those of the abdomen (Ab) are represented on the blastoderm (see Fig. 3).

3 Gradients and insect segmentation mic bridges. The nurse cells synthesize RNA and pass it into the oocyte, whose own nucleus usually remains completely inactive. t seems that ancestral insects had panoistic ovaries and these have been retained by some orders (e.g. Orthoptera), while the meroistic ovary has evolved in others (e.g. Homoptera, Coleoptera, Diptera), and is now associated with much more rapid oogenesis and embryonic development. The fusion of the egg and sperm nuclei is followed by numerous rounds of nuclear division (inappropriately term ed 'cleavage'), giving rise to many nuclei within the yolky core of the egg. Most nuclei migrate out to the peripheral cytoplasm where they eventually become enclosed by folds of the surface membrane, forming the blastoderm cell layer surrounding the yolk. The embryo is formed only from a ventral region, the germ anlage, while the rest of the blastoderm forms temporary extraembryonic membranes. The germ anlage develops into the visibly segmented germ band, comprising the procephalon (traditionally regarded as an anterior nonsegmental acron and three indistinct segments - see Anderson, L973), the typical segments of the gnathocephalon (3) and thorax (3) and abdomen (8-11), and a posterior nonsegmental telson. The extent of the germ anlage and the fate of the cells within it have been difficult to establish precisely as, so far, a reliable technique of marking and tracing cells has been developed only for Drosophila (Hartenstein et al. 1985). The fate maps of other species derive from descriptive studies and from mapping the segmental defects caused by local damage, and must be regarded as provisional. Even with this qualification, it is clear that there are major differences between insects in the extent of the germ anlage and the way in which it gives rise to the germ band (Krause, L939 - reviewed by Sander, 1976). n long germ eggs, the germ anlage covers much of the blastoderm (Fig. 18) and develops directly into the germ band, without any major change in proportions. n short germ eggs, however, the germ anlage seems to consist only of the prospective procephalon plus a small posterior region which gradually elongates and within which the segments appear sequentially. Between these extremes, there is a range of intermediate (or semi-long) germ eggs where the prospective head and thorax may be represented in proportion on the germ anlage, but segments of the abdomen are added sequentially during growth at the posterior end. t is striking that long germ eggs (e.g. Drosophila) are developed only in meroistic ovaries, intermediate germ eggs can come from either type of ovary, while most short germ eggs result from panoistic oogenesis (Fig. L). Because of the polarized source of RNA in meroistic ovaries, it is possible that the two forms of oogenesis produce eggs differing in their degree of spatial organization and hence in the interactions involved in segmentation. Embryological evidence fo, determinants and gradients Over the last 70 years ('the days of experimental teratology'- see Sander, this volume!) the traditional methods of the embryologist (cautery, irradiation, ligature, grafting, centrifugation) have been used to study segmentation by eliminating or displacing parts of the insect egg (for extensive review and interpretation see Sander, 1976, 1984). n many insects, a distinct 'pole plasm' is localized at the posterior end of the oocyte, and the germ line develops only from the cells that incorporate that part of the egg cytoplasm. llmensee & Mahowald (L974) demonstrated the presence of a germ cell determinant by injecting pole plasm into a more anterior site in the early Drosophila egg, and showing that the blastoderm cells subsequently forming in that position could develop into germ cells. f this were a general mechanism and the segments were already located within the oocyte by an arcay of such local determinants, each should develop autonomously, regardless of the presence or the position of other regions. This is not the case, &s development of the full segmental pattern depends on interactions occurring between regions of the egg. Sander (1959) ligated the intermediate germ eggs of the leaf hopper, Euscelis, at early stages and found that each half forms less than normal, leading to a gap in the pattern of segments formed by the complementary anterior and posterior fragments (Fig.2A). The position of the gap depends on the level at which the egg was ligated, and the size of the gap falls as the ligature is made at later and later stages, disappearing if the egg is left intact until germ band stage. Ligature has now been performed on many other long and intermediate germ eggs, including beetles and both lower and higher dipterans (see Sander, 1976). All these studies gave similar results: a gap of 6-8 segments after ligature in cleavage stages, falling to 0-1 segment if ligature is delayed until cellular blastoderm (in dipterans) or germ band (in beetles) stage. Vogel (1977) obtained only small gaps in Drosophila and argued that large gaps result from extensive damage, but this is not supported by histological studies (e.9. Newman & Schubiger, 1980). Also, temporary ligature of the early Drosophila egg results in a transverse membrane and a gap in the later segment pattern but, if this membrane is punctured in a previously ligated egg, continuity is reestablished and a complete pattern can form (Schubiger et al. 1977). Hence the gap in the pattern does

4 V. French result from blocking interaction between opposite ends of the egg. Sander (1960) suggested that the egg contains anterior (A') and posterior (P') determinants which are localized during oogenesis and act as sources of \] t{ s A Post opposing diffusion gradients whose relative levels specify the array of segments (Fig. 2B). Further evidence for the P' determinant was obtained by displacing posterior cytoplasm into a more anterior position and later ligating behind it (Sander, 1960). (iii) Fig. 2. Ligature experiments on the Euscelis egg (after Sander, 1959, 1960, 1976). (A) Eggs in schematic ventral view, showing (i) the approximate position on the blastoderm of cells normally forming parts of the embryo (abbreviations as in Fig. 1) and (ii-iv) changes in fate following operations. The black spot indicates a ball of symbiotic bacteria. After early ligature (ii), partial embryos are formed, but the six segments between procephalon (Pr) and first abdominal segment (A) are missing. After ligature at blastoderm stage (iii), the three segments between mandibular (Gl) and mesothoracic Q2) are absent. f the posterior symbionts and cytoplasm are translocated early and the egg is ligated at blastoderm (iu), a small complete embryo forms anteriorly and a reversed partial pattern may form in the posterior fragment. (B) Double-gradient model for specification of segments (see Sander, 1960). (i) Normal gradient profiles are maintained by diffusion from localized determinants (A', P') which act as sources. A' is in the anterior part of the egg (at 60 % of egg length from the posterior pole - 60 %EL) and forms the anterior gradi ent (A, dashed line), and P' (at the posterior pole - 0%EL) forms the posterior gradient (P). The concentration (conc) ratio Al P varies continuously along the egg axis and directs blastoderm cells to form the successive segment primordia. (ii) After ligature (double line) at 30%EL, gradient profiles change (e.g. in the anterior fragment A levels will rise and P levels will fall), the AP ratio becomes discontinuous across the ligature and some segments will not be formed. The later the ligature, the less gradient profiles will change before segment determination, and the smaller will be the gap. (iii) After translocation and ligature (as in Aiv) opposing A and P gradients will form in the anterior fragment but, to obtain the full pattern (the full range of AP ratios), the gradients cannot be independent (as shown) but must be antagonistic, with high A suppressing P and vice versa (see Sander, 1976). The pattern formed in the posterior fragment will be reversed but its extent will depend on exactly when and where the ligature is made. A'

5 Gradients and insect segmentation The transplant causes a complete set of segments to form in the anterior fragment, and also causes polarity reversal in the partial pattern formed in the posterior fragment (Fig. 2Aiv). The results of more recent deletion and transplantation experiments suggest that the Drosophila egg contains both A' and P' determinants, which have antagonistic long-range effects on the segment pattern and could correspond to gradient sources (Frohnhofer et al. 1986). Puncturing the egg to remove a small amount of anterior or posterior cytoplasm has a major effect on the resulting segment pattern, deleting most of the head or abdominal segments, respectively. njection of posterior cytoplasm into the anterior pole suppresses head development and may cause the formation of a symmetrical double abdomen pattern, while the reciprocal injection suppresses abdomen formation and can produce a symmetrical double head-and-thorax pattern (Ntisslein-Volhard et al. 1987). Further evidence for antagonistic A' and P' determinants is provided by the effects of irradiation and centrifugation on the eggs of the dipteran midges, Smittia and Chironomus (reviewed by Kalthoff, 1983). U.v. irradiation restricted to the anterior pole of the early egg (oviposition to syncytial blastoderm stage) can cause the formation of a symmetrical double-abdomen pattern (Yajima, 1964; Kalthoff & Sander, 1968; Kalthoff, 1978). A similar u.v. irradiation of the posterior pole can cause a doublecephalon pattern, consisting of a duplication of the anterior part of the head. This occurs very rarely in Smittia (Kalthoff, 1983) but much more readily in Chironomus (Yajima, 1964, 1983). Variation in the stage, the dose or the site of irradiation caused great changes in the frequency but not the extent of the double-abdomen pattern (Kalthoff, 97B), and this also seems true of the double cephalon (Yajima' Lgg3). The double-abdomen pattern arises through u.v. inactivation of anterior RNA, since it can be reversed by subsequent exposure to visible light, and this is correlated with the disappearance of the pyrimidine dimers induced by u.v. (see Kalthoff, 1983). Also, double abdomens can be induced by anterior treatment with RNAse (but not other enzymes), and the irradiated Chironomus egg can be rescued by anterior injection of RNA extracted from unirradiated eggs (Elbetieha & Kalthoff, 1988). Posterior irradiation has not been so extensively studied but the effect will also photorevert (Yajima, 1983), suggesting that the double cephalon also results from inactivation of ocalized RNA. Centrifugation of these eggs also has major effects, producing double cephalons, double abdomens and complete but inverted embryos (Yajima, 1960, 1983; Rau & Kalthoff, 1980). From experiments involving centrifugation plus local irradiation, Kalthoff et al. (1982) have suggested that u.v.-sensitive A' and P' determinants are normally located principally at opposite poles of the egg but may be redistributed by centrifugation, So that either end may have A' > P' (and become anterior) or A' < P' (and become posterior), giving rise to the four classes of segment pattern. The A' and P' determinants in the midge egg are likely to be maternally synthesized RNA, but they cannot be equated simply with the anterior and posterior gradient sources of Sander's (1960) model, since there is no reason why the inactivation of one source should result in a symmetrical segment pattern. Similarly, even if sources can be moved by centrifugation, there is no reason why only the normal, the inverted and the two forms of symmetrical embryo should result. Kalthoff (1975, 1983) has suggested that segmentation involves two distinct processes, one subdividing the axis into the correct number of equivalent units and the other labelling those units sequentially from the anterior (A' > P') and posterior (P' > A') ends. Ligature would disrupt gradients involved in the subdivision (causing a gap), while the terminal determinants could be inactivated by u.v. and moved away from the ends by centrifugation (causing mislabelling). This model was founded on the Smittia double abdomen reliably having almost the same number of segments as the normal embryo, but it can explain neither the more variable pattern in Chironomus nor the double cephalons, which contain far fewer segments. Meinhardt (1977, 1982) proposed a model involving posterior gradients of reacting activator and inhibitor. This can explain in detail the ligature and trartsplantation results (see Fig. 2A), and also the formation of Smittia double abdomens (although there are problems with photoreversion - see Kalthoff, 1978). t is a property of this class of model that peaks will form at the ends of the egg (see Meinhatdt, this volume), accounting for the formation of normal, reversed or double gradients after the disturbance of centrifugation. However, with segmentation depending only on posterior gradients, the model cannot explain the formation of the double-cephalon pattern. Operations that damage, move or isolate parts of the newly laid egg indicate that determinants, which must be synthesized and localized during oogenesis, establish gradients that lead to segment determination in the early embryo. Further analysis of these processes by embryological means is difficult, partly because many components may be inactivated or displaced by an operation, whereas a mutation will inactivate or alter only one component (although, of

6 V. French course, this may have arange of confusing secondary effects!). Rapid progress is now being made in analysing segmentation in Drosophila by using early mutant phenotypes to identify genes specifically involved in generating and interpreting spatial information, and then cloning those genes and studying the distribution and function of the RNA and protein products. Genetic and molecular evidence for gradients Mutations in the genes involved in establishing determinants and gradients during oogenesis should be maternal (i.e. phenotype determined by the genotype of the female producing the egg), while those involved in the interpretation of this spatial information are expected to be zygotic, involving genes expressed in nuclei of the embryo. Several systematic searches have now identified most of the Drosophila genes of both types which are specifically involved in forming the embryonic segment pattern (see Ntisslein- Volhard et al. L987; Ntisslein-Volhard & Wieschaus, 1gg0). The zygotic 'segmentation genes' have been classified (Ntisslein-Volhard & Wieschaus, 1980) into gap genes (whose inactivation primarily results in absence of one large region of the pattern), pair-rule genes (loss of alternate segment-wide strips of the pattern) and segment-polarity genes (deletion of part of each segment). The results of intensive genetic and molecular analysis of these genes (see many chapters in this volume) indicate that these classes correspond to successive stages in interpretation of the information in the egg: into broad bands of gap gene expression, leading to repeating stripes of pair-rule gene expression and hence to expression of segment-polarity genes in part of each segment (see ngham, 1"988). The spatial information present in the oocyte and very early egg results from the activity of the maternal 'segmentation genes', most of which fall into three clear categories, with severe mutations primarily causing deletion of almost non-overlapping regions of the pattern (reviewed by Ntisslein-Volhard et al. 1987). Mutations in the anterior group (e.g. bicoid, exuperantia) remove the head and thorax, of the posterior group (e.g. oskar, pumilio, nanos) remove the abdometr, and of the terminal group (e.g. torso) remove the extreme anterior (acron) and posterior (telson) structures (see Fig. 3). The effects are approximately additive (e.9. the bicoid oskar double mutant lacks all but terminal structures), leading to the surprising conclusion that the three parts of the pattern depend on different spatial cues and that mutations in one gene group will inactivate (itt different ways) one cue and leave the others intact. (i) The anterior pattern Several maternal genes are required for the formation of the anterior part of the embryonic pattern, and there is now very strong evidence that the product of one of these, bicoid, actually provides information in the form of a gradient. Strong mutations of bicoid result in embryos that lack the head and thorax, and also have variable deletions and fusions in the anterior abdominal segments. n addition, the anterior tip of the embryo forms, not the acron, but a second telson. n weaker alleles, there are deletions of parts of the acron, head and thorax, but the ectopic telson is not formed, and, in the weakest alleles, deletions are confined to the acron and head (Frohnhofer & Ni.isslein-Volhard, 1986). The segmental abnormalities resulting from the lack of maternal gene activity can be seen much earlier, tt blastoderm stage, in the patterns of expression of the zygotic pair-rule genes such as ftz and eve (see Lehmarr, this volume). n bicoid embryos (strictly, in embryos from bicoid mothers), all the segmental primordia are displaced anteriorly and, in strong mutant alleles, head and thorax are missing and replaced by the telson primordium, while the abdomen is greatly expanded and its anterior segmental primordi a are indistinct and fused (Frohnhofer & Ni.isslein-Volhard, 1987; see Fig. 5A). bicoid eggs can be rescued phenotypically to form a normal complete pattern if injected anteriorly with cytoplasm taken from the anterior tip of a wild-type egg (Fig. 4Ai). For a high frequency of rescue, host and donor must be no older than late cleavage stage (Frohnhofer & Ntisslein-Volhard, L986). Rescue is often not complete (injected eggs forming thoracic but no head structures and retaining part of the telson), and the degree of rescue increases with the volume of cytoplasm injected and with the number of copies of the bicoid * gene in the female producing the donor egg (Frohnhdfer & Ni.isslein-Volhard, 1986). njection into other positions along the host egg can also result in anterior structures and changes in polarity around the injection site, but the frequency and degree of response falls with distance from the anterior pole (Fig.4Aii). These injection experiments show that bicoid eggs lack the anterior determinant (see previous section) which has a longrange quantitative effect and which is inhibited by proximity to the posterior determinant. The gene, bicoid, has now been cloned and its expression pattern determined (Berleth et al. 1988). bicoid is indeed transcribed in oogenesis, in the nuclei of the nurse cells, and the RNA accumulates in the adjacent tip of the developing oocyte, remains strictly localized in the peripheral cytoplasm of the anterior 20 % of the egg throughout cleavage stages and disappears before cellularization of the blastoderm

7 Gradients and insect segmentation Ant Post A AA TT 11,te B pp Fig. 3. The blastoderm fate map (A) and larval segment pattern (B) of Drosophila. (A) Positions of segment primordia in the gnathocephalon (G1-G3), thorax (71-rc) and anterior abdomen (A1-A8) are derived from cell marking (Hartenstein et al. 1985). The segments of the procephalon (Prl-Pr3) plus the acron (ac), and of the posterior abdomen (A9-Al/) plus the telson (te) are identified by deletion mapping and homeotic mutant phenotypes (see Ji.irgens et al. 1986; Jiirgens, 1987). Segment borders are only drawn on the ectoderm - other blastoderm regions form the anterior (o*) and posterior Qtm) midgut, the mesoderm (m), the procephalic ganglia (pil and extraembryonic membrane ("). (B) Structure of the larva. Most of the head structures are invaginated (dashed lines), and the anterior end of the larva is formed by the pseudocephalon (pc), consisting of parts of the2nd procephalic and gnathal segments. Prominent cuticular structures shown are the denticle belts (d.), the thoracic Keilin's organs (K), the posterior spiracle (sp) and the anterior mouth-hooks (mh). The anterior stippled region, termed the'acron'(niisslein-volhard et al. 1987), is derived from the non-segmental acron, the labrum (segment Pr) and parts of the other head segments. The posterior stippled area, termed theotelson'(ni.isslein-volhard et al. 1987), derives from the non-segmental telson, segments A11-A9 and dorsal A8. The arrows denote parts of the pattern missing in mutants of maternal genes of anterior (AA), posterior (PP) and terminal QT) groups - for illustrations, see Lehmann (this volume). (Berleth et al. 1988). No biciod protein is detectable during oogenesis but it appears shortly after the egg is laid and accumulates (mainly within the nuclei) to form a concentration gradient over the anterior 70% of the egg (Driev er & Ni.isslein-Volhard, 988a - see Fig. 5ts). Gradient levels rise slightly until blastoderm stage and then the protein disappears during gastrulation. The gradient is approximately exponential and, considering plausible values for the half-life of the protein and its diffusion constant in the syncytial early egg, Driever & Ntisslein-Volhard (1988a) concluded that the gradient could be established simply by diffusion from the anterior source. These observations suggest that bicoid is not just required for development of the anterior determinant, but that the RNA (trapped at its point of entry into the oocyte) is the determinant, and that translation and diffusion results in a protein gradient, giving spatial information for the anterior part of the segment pattern. The maternal mutant, dicephalic, produces some egg clusters in which the 15 nurse cells are separated into groups at both ends of the oocyte, and some embryos (presumably developing from those oocytes) form double-anterior segment patterns (Lohs-Schardin, 1982). Perhaps in these oocytes the bicoid RNA is trapped at both ends and gives rise

8 t a 10 V. French A (i) 7o rescue Ant Torescue 100 (ii) Position in donor O%EL Position in host 20 O% EL B (i) 7" rescue 100 Post 7o rescue 100 (ii) Position in donor O%EL /" EL Position in host Fig. 4. Phenotypic rescue of eggs from maternal segmentation mutants by injection at early cleavage stage of cytoplasm from wild-type ( +) eggs. (A) Rescue of the anterior group mutant bicoid (bcd) - redrawn from Frohnhdfer & Niisslein- Volhard (1986). Rescue occurs when the host forms head andf or thoracic structures (dashed line), and graphs also show frequency of head formation (strong response - continuous line). When cytoplasm is injected into the anterior tip of the host, rescue depends strongly on its position of origin within the wild-type donor (i); and when cytoplasm is taken from the anterior tip rescue, declines as it is injected into more posterior positions in the host (ii). Schematic diagram shows the most effective injection. (B) Rescue of the posterior group mutant oskar (osk) - redrawn from Lehmann & Ntisslein-Volhard (1986). Rescue occurs when the host forms abdominal segments and is most frequent when cytoplasm is (i) taken from the posterior tip of the donor egg, and (ii) placed in the presumptive abdominal region (20-50 %F,L) ot the host - the latter effect is even more pronounced when injections are done at later cleavage stage (not shown). to a symmetrical protein gradient, resulting in the formation of two anterior ends. A patterning function for the bicoid protein gradient is directly indicated by cases in which a change in pattern is preceded by a similar change in the gradient (Driever & Ntisslein-Volhard, L988b). Females with only one copy or with duplications of the bicoid * gene all produce eggs that eventually form normal larvae, but which have (respectively) decreased or increased protein levels and have their segment primordia displaced on the blastoderm f.ate map (Fig. sbi,ii). Two other genes of the anterior group, exuperantia and swallow, have mutant phenotypes with deletions of extreme anterior structures and enlargement of remaining head and thoracic segments (Schiipbach & Wieschaus, 1986). Genetic and injection studies indicate that these genes are required for the normal localization of. bicoid activity (Frohnhofer & Ntisslein-Volhard, 1987), and there is now direct evidence for this,?s the mutants form eggs with shallow gradients of bicoid RNA (Berleth et al. 1988) and protein (Driever & Ni.isslein-Volhard, 198Sb) and have a correspondingly distorted blastoderm fate map (Fig. sbiii). Although the bicoid protein gradient provides spatial information, it is clear that the different segment primordia are not directly specified by small changes in concentration (us, for example, in the gradient model in Fig.2). Changes in the gradient result in similar, but smaller, changes in segment location and, conversely, in mutants of the posterior and terminal groups, the bicoid protein gradient is normal (Driever & Ni.isslein-Volhard, 1988b) but the head and thoracic primordia are shifted on the blastoderm (see below). t seems likely that broad concentration ranges of the protein, in conjunction with other cues, specify a few bands of. zygotic gap gene expression, &S a first step in the formation of the anterior region of the segment pattern (Driever & Ni.isslein-Volhard, 7988b; Meinhardt, this volume). The gap genes, hunchback and Kriippel, are expressed in successive belts corresponding to regions of high and low bicoid protein level. n bicoid mutants, the hunchback region is abolished (Tautz, 1988) and the Kriippel region is moved anteriorly (Gaul et al. 1987), suggesting that high protein levels activate the former and inhibit the latter (see Fig. 6,4.). bicoid levels may also have a role in

9 Gradients and insect segmentation 11 A (i) bcd+ f bcd* bcd bcd G bicoid protein G3 (i) bcd+ luca (ii) 2. bcd+ f bcd+ (iii) exuf exu % ELLC/0^ 80 O%EL r00 80 O% EL Fig. 5. Blastoderm fate maps and levels of bicoid protein. Fate maps are schematic drawings based on photographs (Frohnhofer & Ntisslein-Volhard, 1987; Driever & Ntisslein-Volhard, 1988b) of in situ hybridizations to RNA from the zygotic pair-rule segmentation genes, ftz (fine stippling) and eve (coarse stippling). ftz is expressed in the evennumbered and eve in the odd-numbered parasegments (see references cited above). The parasegment is a metameric unit corresponding to the posterior compartment of one segment and the anterior compartment of the next (see Lawrence, this volume) - e.g. parasegment 4 rs posterior prothorax Q) plus anterior mesothorax (72). The graphs (redrawn from Driever & Ntisslein-Volhard, 1988b) show intensity (in arbitrary units) of staining along the anterior-posterior axis (100 %-}%F.L) of late cleavage stage embryos exposed to antibody raised against bicoid fusion proteins. (A) Fate maps of (i) wild-type (bcd+lbcd+) and (ii) strongbicoid mttant (bcdlucal blastoderm stage embryos. n bicoid mutants, parasegments /-5 are missing and replaced by an ectopic 14 (identity of parasegments is established by study of a phenotypic series of alleles - see Frohnhdfer & Niisslein-Volhard, 1987). (B) Correlation between bicoid protein levels and fate map distortions. (i) n the bicoidheterozygote (bcd+lbcd) anterior protein levels are lower than in wild type (dashed line), and segment primordia are displaced anteriorly. (ii)'n eggs from females with a duplication of the bicoid* region (2.bcd+lbcd+) protein levels are higher than normal and segments are displaced posteriorly. (iii) n exuperantia mutants (exuf exu) bicoid protein levels are much lower, and the anterior part of the fate map is greatly expanded. Changes in the gradient are more extreme than distortions in the fate map (because segments are not specified directly by protein level - see text), and these are usually more extreme than changes in the cuticular patterns eventually formed (because of subsequent size regulation within segments). controlling expression of other gap genes expressed at the anterior tip of the embryo (Driev er & Ni.isslein- Volhard, 1988b). (ii) The posterior pattern Maternal genes of the posterior group are required for the formation of the abdominal segments. They all have similar mutant phenotypes and, zt present, there is only indirect evidence about which of them provide spatial information in the early egg, and in what form. Strong mutations of the posterior group genes delete all 8 abdominal segments from the larval pattern and, dt least in the case of. oskar, part of the posterior metathorax as well. Weaker mutant alleles show partial deletions in which the middle abdominal segments (A4-A6) are readily lost while the end ones (A1 and A8) are retained (Lehmann & Ntisslein- Volhard, 1986; Lehmann, this volume). Anterior structures and posterior telson are normal but, in all of these genes except nanos and pumilio, mutation disrupts the pole plasm at the posterior tip of the eee, and no germ cells are formed (Niisslein-Volhard et al.

10 T2 V. French A conc conc An,a + \ 2.-if- 3 4 j <- v Fig. 6. llustration of a possible relationship between maternally synthesized determinants, gradients, zygotic gap gene expression and segment patterns. (A) The normal embryo. The upper diagram shows the anterior-posterior (Ant-Post) axis of the egg, with spatial information consisting of the demonstrated anterior concentration (conc) gradient (A) from the anterior determinant (A'), a postulated posterior gradient (P) from the posterior determinant (P') and 'terminal activity' (T) labelling both ends. The lower diagram shows a simple scheme of gap gene expression on the blastoderm, where / is expressed in the presence of both T and high A levels; 2 at high A levels but in the absence of T; 3 in the absence of T and at low (below r) levels of both A and P; 4 at high P levels in the absence of T;5 in the presence of Z and the absence of high A levels. (The rules for genes 2-4 define the main regions of hunchback, Krilppel and knirps expression, but all these genes are also expressed in other regions, and gap genes expressed at the ends have not been fully characterized.) Subsequent elaboration of pattern and polarity occurs by interactions between adjacently expressed gap gene products (see text) and is indicated by arrows. (B) n the bicoid mutant (bcd) A' rs absent and no A gradient is formed, so the anterior end expressed 5 instead of /, and 2 is not expressed. P gradient levels rise to Pi so the expression of 3 is shifted anteriorly. nteractions between 5, 4 and 3 result in an enlarged posterior pattern with normal polarity. 5 and 3 arc not normally adjacently expressed, so they do not interact and the region expressing 5 forms an isolated posterior tip, the telson. The embryo forms the bicoid phenotype (i). f the P levels rise to Pii,3 is eliminated and at each end of the embryo 5 and 4 interact, giving posterior ends with opposite polarity (ii). This is the bicaudal phenotype and occurs occasionally in bicoid mutants (Niisslein-Volhard et al. L987; Lehmann & Sander, 1988). (C) After injection of posterior cytoplasm (containing P') into the middle of a host containing only terminal activity (bcd osk), a low symmetrical P gradient causes the middle to express 4,while both ends express 5. The bicaudal phenotype results. (D) After injection into a host lacking all activity (bcd ask tor), low P gradient levels and the absence of T cause 3 to be expressed at the ends of the egg. nteractions between 4 and 3 result in a symmetrical posterior pattern with its end (lacking the telson) in the middle of the egg.

11 Gradients and insect segmentqtion ). Early effects on the segment pattern can be seen in pair-rule gene expression on the blastoderm and, in strong mutants, primordia of the head and telson are normal, while the thoracic segments ate displaced posteriorly (Lehmann, this volume). The region between the expanded thorax and the telson shows no clear segmentation and later undergoes extensive cell death. Eggs from females mutant for posterior group genes can be phenotypically rescued if they are injected before blastoderm stages with cytoplasm from the posterior tip (the pole plasm) of a wild-type embryo (Lehmann & Ni.isslein-Volhard, 1986 see Fig. 4Bi). Rescue can also be achieved using cytoplasm taken from immature oocytes or from nurse cells (Lehmann & Sander, 19BB), even those from posterior group mutants except nanos (Lehmann cited in North, 1988). These results indicate that the posterior determinant is synthesized by the nurse cells, probably consists of nanos RNA and gradually becomes localized at the posterior tip of the mature oocyte, perhaps through being degraded except within the pole plasm (Lehmann & Sander, 1988). Several maternal genes such as bicaudal mutate to give phenotypes ranging from anterior deletions (similar to those resulting from bicoid mutations) to a double-posterior pattern with a plane of symmetry in the mid to posterior abdomen (Ntisslein-Volhard, 1977; Mohler & Wieschaus, 1986). There is good evidence that this results from abnormal localization of the posterior determinant, which now occurs at both poles (Lehmann & Ntisslein-Volhard, 1986) and suppresses the anterior determinant. Hence the double-mutant bicaudal oskar (which lacks the posterior determinant) has the oskar phenotype, forming a normal anterior part of the pattern (Lehmann & Ntisslein-Volhard, 1986). Unlike the injections of anterior cytoplasm to rescue bicoid mutant eggs, rescue of posterior group mutants is most effective when the wild-type posterior cytoplasm ts not placed at the normal location of the determinant, but more anteriofly, in the normal position of the abdominal primordia (Fig. 4Bii). Rescue is often incomplete (segments 4L and A8 forming most readily) and, regardless of the site of injection, abdominal segments only appear in a normal position, without disturbing overall embryonic polarity (Lehmann & Ntisslein-Volhard, 1986). These results may indicate that the signal from the posterior determinant is specifically translocated to a more anterior position. Alternatively, the signal may form a concentration gradient from the posterior source, but not specify abdomen in the posten or 20 "h of the egg because this region forms telson in response to other cues. Mutants of pumilio produce eggs that possess the determinant but, nonetheless, form a maximum of two abdominal segments, unless they are also mutant for a terminal group gene such as torso, in which case they lack the telson (see below) but form more abdominal segments (Lehmann & Niisslein-Volhard, 1986). Presumably the signal is attentuated in pumilio but can reach a receptive atea of the blastoderm if the telson is not formed. Present evidence is compatible with the existence of a strictly \ocahzed posterior determinant (perhaps nanos RNA) which, by analogy with bicoid, results in a protein gradient that specifies patterns of gap gene expression in posterior regions not occupied by the telson. n posterior group mutants (e.g. oskar), the expression of. Krilppel is extended posteriorly (Gaul et al. 9S7), suggesting that it is normally inhibited by high posterior gradient levels. The gap gene knirps rs expressed posterior to Kriippel (Jackle et al. cited in ngham, 1988) and is likely to depend on high posterior gradient levels (Lehmann & Ni.isslein- Volhard, 1986; Lehmann, this volume). (iii) The terminal structures Maternal genes of the terminal group are required for the normal development of both ends of the embryonic pattern, but there is no direct evidence concerning the distribution or function of their products. Females mutant for terminal group genes such as torso produce embryos that lack acron, telson and, in extreme genotypes, parts of abdominal segments A8 and A7 (Schiipbach & Wieschaus, 1986; Dengleman et al. 1986). The eggs contain normal anterior and posterior determinants, 3S assessed by cytoplasmic injections (Ni.isslein-Volhard et al. 1987), and a normal bicoid gradient (Driever & Niisslein-Volhard, 1988b), but the patterns of gap and pair-rule gene expression (Tautz, 1988; Dengleman et al. 1986; Klingl er et al. 1988) indicate that the terminal primordia are missing, while the rest of the pattern is spread to the ends of the blastoderm. 'Terminal activity' is required for the formation of the acron (at high bicoid gradient levels) and the telson (regardless of posterior 'gradient' level). t seems likely that this activity (and hence the product of at least one of the genes) is ocalized at the ends of the egg, since the bicoid oskar double mutant, which presumably lacks all other spatial information, forms a telson at each end (Ntisslein-Volhard et al. 9B7). Mutant torso embryos can be phenotypically rescued by injection of cytoplasm from the ends or middle of the wild-type egg, indicating that the torso gene product is not localized (Klingler et al. 1988), but the other terminal group genes have not yet been tested in this way. (iv) Anterior and posterior gradients The pattern of gap gene expression develops around

12 L4 V. French the blastoderm stage, apparently in response to terminal cues and levels of gradients established from the anterior (A') and posterior (P') determinants (Fig. 6,4.). There seem to be major differences between these gradients, suggesting that they may be formed by different mechanisms. Abnormal proximity to P' seems to suppress A' (itt bicaudal mutants or in injection experiments) but the anterior gradient is normally independent of posterior or terminal activity, &S bicoid protein levels are normal in oskar and torso mutants (Driever & Niisslein-Volhard, 1988b). However, the posterior gradient may not be independent of anterior activity since Kriippel expression in bicoid mutants is shifted (not merely extended) anteriorly, so that the middle of the egg no longer expresses Krilppel (Gaul et al. 9B7), presumably because of elevated posterior gradient levels. This suggests that bicoid protein suppresses posterior activity and contributes to the normal posterior gradient profile (Fig. 68). The anterior gradient may form by diffusion and decay of. bicoid protein, but the posterior signal seems to be capable of reorganization, and may not be a simple diffusion gradient (Ni,isslein-Volhard et al. 1987). When injected into the middle of a bicoid oskar egg, anterior cytoplasm provokes the formation of head or thorax at the site of injection, whereas posterior cytoplasm frequently causes a posterior end to form at both poles of the egg (the bicaud a pattern) or at one pole, but not at the site of injection (Ni.isslein-Volhard et al. 1987). This may indicate a diffusionf reaction system that can reorganize to form a gradient with terminal peak(s) (see Meinhardt, this volume). However, when the posterior cytoplasm is injected into the completely neutral bicoid oskar torso egg, the posterior end of the pattern forms at the injection site (Lehmann, cited in North, L988). This is not readily explained by the diffusi onf reaction model and suggests that the sequence of segments eventually formed may not always correspond to the gradient profile. t seems that gap genes are initially expressed in partially overlapping regions, and that interactions occur between them to restrict their zones of expression (Jiickle et al. 1986) and to establish the subsequent patterns of pair-rule and homeotic gene expression that define the segment pattern (reviewed by ngham, 19BB). Fig. 6 illustrates how these interactions may result in different segment patterns after injection of posterior cytoplasm into eggs of different genotypes. Direct evidence that posterior activity is in the form of a gradient awaits the cloning of posterior group genes and the study of their products. f a posterior gradient exists it will then be possible to study directly the mechanism by which it is established, its interactions with the anterior gradient and the relationship between maternal gradient levels and patterns of gap gene expression. Gradients in the eggs of insects other than Drosophila The double-gradient model of insect segment specification was first suggested (Sander, 1960) in the context of ligature experiments (Fig. 2). These give very similar results when performed on a range of intermediate and long germ eggs which develop in association with nurse cells in meroistic ovaries. There are some differences in the time of segment determination and the extent to which the fate of anterior fragments are altered by isolation (see Sander, this volume), but it seems a reasonable expectation that the maternally provided anterior and posterior gradients demonstrated in Drosophila underlie segmentation in all these embryos (although with differences in the details of their formation, interaction and interpretation). The analysis of the formation of bicoid and bicaudal patterns in Drosophila (Fig. 6) suggests an obvious explanation of the symmetrical segment patterns rn Smittia. f the ends of the Smittia egg are also labelled independently of the gradients, the P gradient is also inhibited by A and, unlike the situation in Drosophila, A is inhibited by P, then the destruction of the A' determinant by u.v. will result in a rise in the P gradient, giving the double abdomen (ar in Fig. 6Bii), and posterior u.v. will similarly generate the double cephalon. f the determinants are mixed by centrifugation, one will dominate and either the double abdomen (see Fig. 6C) or the double cephalon will form. Oocytes formed in panoistic ovaries have no nurse cells and hence no external anterior source of RNA, so the spatial information in these eggs may well differ from that described above. Ligature and similar experiments were performed on the intermediate germ eggs of the damselfly, Platycnemis (Seidel, 1929) and the cricket, Acheta (Yollmar - see Sander, 1976). Much of the anterior of the egg is not required in forming the segment pattern and,?s discussed by Sander (1976), there is evidence of a signal (which could be a gradient) from the posterior end of the embryonic region, but not of a similar signal from the anterior end. Thus the early stages of segment specification in these eggs may depend on a single posterior gradient. Almost nothing is known of the spatial information in early short germ eggs. The tiny embryo normally forms in a specific region although, in some species, it may be induced elsewhere (see Sander, 1976), but most studies (e.g. Krause & Krause,1957; Mee, 1986)

13 Gradients and insect segmentation 15 have involved damaging or dividing the embryo once it has started to form visible segments. t is now clear from the genetic and molecular analysis of segmentation rn Drosophila that the determinants and gradients inferred from earlier embryological studies are involved in insect segmentation, but that they provide a coarse positional framework within which the segment pattern is gradually elaborated. Because gradient profiles cannot be deduced directly from the segment pattern, investigation of gradients in other eggs may not progress much further until the molecular techniques can be transferred from Drosophila. t is obvious how much the field owes to the elegant and innovative work of Klaus Sander and of Christiane Ni.isslein-Volhard and her colleagues. This review has grown largely out of discussions with Phil ngham (who is, of course, innocent of the errors and idiocies that it may contain). References ANoEnsoN, D. (1973). Embryology and Phylogeny in Annelids and Arthropods. Oxford, New York, Sydney: Pergamon. BpnrErH, T., BunR, M., TuotvtA, G., Boru, D., RrcusrEN, S., FrucERo, G., Nott, M. & NUssLEN- VorHnno, C. (1938). The role of localisation of bicoid RNA in organising the anterior pattern of the Drosophila embryo. EMBO J. 7, Cnrcr, F. (1970). Diffusion in embryogenesis. '{ature, Lond. 225, DnNcTEMANN, A., HRnoy, A., PEnnMoN, N. & M*rowALD, A. (1936). Developmental analysis of the torso-like phenotype in Drosophila produced by a maternal-effect locus. Devl Biol. 1.15, DnrBvBR, W. & NUssrprN-VoLHARD, C. (1988a). A gradient of bicoid protein in Drosophila embryos. Cell 54, 83-93, DnrBvER, W. & NussrstN-VoLHARD, C. (1988b). The bicoid protein determines position in the Drosophila embryo in a concentration-dependent manner. Cell 54, ErsErHrEHA, A. & KnrrHoFF, K. (1988). Anterior determinants in embryos of Chironomus samoensis: characterisation by rescue bioassay. Development 104, FnouNH6nnn, H.-G., LnuuANN, R. & NUssLEN- VorueRo, C. (1986). Manipulating the anteroposterior pattern of the Drosophila embryo. J. Embryol. exp. Morph. 97 Supplement, FnosNHorEn, H.-G. & NUssrem-VorHARD, C. (1986). Organizatton of anterior pattern in the Drosophila embryo by the maternal gene bicoid. Nature, Lond. 324, r20-r25. FnouNuonBn, H.-G. & NussrBm-VorHARD, C. (1987). Maternal genes required for the anterior localtzation of bicoid activity in the embryo of Drosophila. Genes & Development 1, GRur, U. & JAcrrs, H. (1987). Pole region-dependent repression of the Drosophila gap gene Krilppel by maternal gene products. Cell 51, Grpnnn, A. & MBrNH^Rnot, H. (1972). A theory of biological pattern formation. Kybernetik 12,, Gurznrr, H. & SnNonn, K. (1985). Establishment of polarity in the insect egg. n The biology of fertilization (ed. C. Metz & A. Monroy), vol. 1, pp New York: Academic Press. HnnrENSrErN, V., TncuNRu, G. & Cnupos-OntEcR, J. (1985). Fate-mapping in wild type Drosophila melanogaster.. A fate map of the blastoderm. Wilhelm Roux Arch. devl Biol. 194,213-21,6. TTuENSEE, K. & MnHowALD, A. (1974). Transplantation of posterior polar plasm in Drosophila. nduction of germ cells at the anterior pole of the egg. Proc. natn. Acad. Sci. U.S.A. 71, NcuRrvr, P. (1988). The molecular genetics of embryonic pattern formation in Drosophila. Nature, Lond. 335, JAcrrB, H.,Tnvrz, D., ScnuH, R., Semnnr, E. & LButunNN, R. (1986). Cross-regulatory interactions among the gap genes of Drosophila. Nature, Lond. 324, JUncsNS, G. (1987). Segmental organisation of the tail region in the embryo of Drosophila melanogaster. Wilhelm Roux Arch. devl Biol. 196, 41,-L57. JunceNS, G., LnHvrANN, R., ScHRnDN, M. & NUssLEN- VorHnno, C. (1986) Segmental organisation of the head in the embryo of Drosophila melanogaster. Wilhelm Roux Arch. devl Biol. 195, KnrruorE, K. (1978). Pattern formation in early insect embryogenesis - data calling for modification of a recent model. ". Cell Sci. 29, -5. Knrruorr, K. (1983). Cytoplasmic determinants in dipteran eggs. n Time, Space and Pattern in Embryonic Development (ed. W. Jeffery & R. Raff), pp New York: Alan R. Liss. KnrrHonr, K., RA,u, K.-G. & EpvtoND, J. (1982). Modifying effects of ultraviolet irradiation on the development of abnormal body patterns in centrifuged insect embryos (Smittia sp., Chironomidae, Diptera). Devl Biol. 91, Kerrnorr, K. & SaNpnn, K. (1968). Der Entwicklungsgang der Missbildung Doppelabdomen im partiell UV-bestrahlten Ei von Smittia parthenogenetica (Dipt., Chironomidae). Wilhelm Roux Arch. EntwMech. Org. 16l, L KrrNcpR, M., EnonLy, M., Szasnp, J. & NussrBN- Vorunno, C. (1988). Function of torso in determining the terminal anlagen of the Drosophila embryo. Nature, Lond. 335, Knnusp, G. (1939). Die Eitypen der nsekten. Biol. Zbl 59, Knnusn, G. & Knnusn, J. (1957). Die Regulation der Embryonalanlage von Tachycines (Saltatoria) im Schnittversuch. Zool. Jahrb. 75, LpHunNN, R. & NUssrprN-VorHARD, C. (1986). Abdominal segmentation, pole cell formation, and embryonic polarity require the loc alized activity of oskar, a maternal gene tn Drosophila. Cell 47,l4-52.

14 16 V. French LpuunNN, R. & NUssrBrN-VorHARD, C. (1987). nvolvement of the pumilio gene in the transport of an abdominal signal in the Drosophila embryo. {ature, Lond. 329, LnuuaNN, R. & SnNoBn, K. (1988). Drosophila nurse cells produce a posterior signal required for embryonic segmentation and polarity. Nature, Lond. 335, Lons-ScHARDN, M. (1982). Dicephalic - a Drosophila mutant affecting polarity in follicle organrzation and embryonic patterning. Wilhelm Roux Arch. devl Biol. 191, MnuowALD, A. (1972). Oogenesis. n Developmental Systems: nsecrs (ed. S. Counce & C. Waddington), vol. 1, pp. 1,-47. New York: Academic Press. MEn, J. (1986). Pattern formation in fragmented eggs of the short germ insect Schistocerce gregaria. Wilhelm Roux Arch. devl Biol. 195, MErNn.e.not, H. (1977). A model of pattern formation in insect embryogenesis. ". Cell Sci 23, MBrNHanot, H. (1982). Models of Biological Pattern Formation. New York: Academic Press. MoHrBR, J. & WTEscHAUS, E. (1986). Dominant maternal effect mutations of Drosophila melanogaster causing the production of double-abdomen embryos. Genetics 112, MoncnN, T. (1901). Regeneration New York: Macmillan. NpwunN, S. & ScnuBGER, G. (1980). A morphological and developmental study of Drosophila embryos ligated during nuclear multiplication. Devl Biol. 79, Nonrs, G. (1938). n my beginning is my end. Nature, Lond. 332, NUssrpm-VorHARD, C. (1977). Genetic analysis of pattern formation in the embryo of Drosophila melanogaster. Wilhelm Roux Arch. devl Biol. 183, Nussrsw-VorHARD, C., FRottNHonEn, H.-G. & LEuunNN, R. (1987). Determination of anteroposterior polarity in Drosophila. Science 238, Nussrnn-VorHARD, C. & WrpscHAUS, E. (1980). Mutations affecting segment number and polarity in Drosophila. Nature, Lond. 287, '. Reu, K.-G. & KarrHonr, K. (1980). Complete reversal of antero-posterior polarity in a centrifuged insect egg. Nature, Lond. 287, SeNonn, K. (1959). Analyse des ooplasmatischen Reakionssytems von Euscelis plebejzs Fall. (Circadina) durch solieren und Kombinieren von Keimteilen.. Die Differenzierungsleistungen vorderer und hinderer Eiteile. Wilhelm Roux Arch. EntwMech. Org. 15t, SnNonn, K. (1960). Analyse des ooplasmatischen Reakionssystems von Euscelis plebeirzs Fall. (Circadina) durch solieren und Kombinieren von Keimteilen.. Die Differenzierungsleistungen nach Verlagern von Hinterpolmaterial. Wilhelm Roux Arch. EntwMech. Org. 151, SnNoBn, K. (1976). Specification of the basic body pattern in insect embryogenesis. Adv. nsect Physiol. 12,, SaNopn, K. (1984). Embryonic pattern formation in insects: basic concepts and their experimental foundations. n Pattern Formation (ed. G. Malacinski & S. Bryant). New York, London: Macmillan. SnNpsn, K., GvrzErr, H. & JAcrrB, H. (1984). nsect embryogenesis: morphology, physiology, genetical and molecular aspects. n Comprehensive nsect Physiology, Biochemistry and Pharmacology (ed. G. Kerkut & L. Gilbert), vol. 1, pp Oxford, New York, Sydney: Pergamon. ScHusrcER, G., MosEty, R. & Wooo, W. (1977). nteraction of different egg parts in determination of various body regions in Drosophila melanogster. Proc. natn. Acad. Sci. U.S.A. 74, ScHUpsACH, T. & WrnscHAUs, E. (1986). Maternal-effect mutations altering the anterior-posterior pattern of the Drosophila embryo. Wilhelm Roux Arch. devl Biol. 195, Snronr, F. (1929). Untersuchungen uber das Bildungsprinzip der Keimanlage im Ei der Libelle Platycnemis pennipes -V. Wilhelm Roux Arch. EntwMech. Org. ll9, Trurz, D. (1988). Regulation of the Drosophila segmentation gene hunchback by two maternal morphogenetic centres. Nature, Lond. 332, 281,-284. VoEcsL, O. (1977). Regionalisation of segment-forming capacities during early embryogenesis in Drosophila melanogaster. Wilhelm Roux Arch. devl Biol. 182, WorppRr, L. (1969). Positional information and the spatial pattern of cellular differentiation. ". theor. Biol. 25,, -47, Yl.rrun, H. (1960). Studies on embryonic determination of the harlequin-fly, Chironomus dorsalis.. Effects of centrifugation and of the combination with constriction and puncturing. /. Embryol. exp. Morph. 8, Ynruua, H. (1964). Studies on embryonic determination of the harlequin-fly, Chironomus dorsalis.. Effects of partial irradiation of the egg by UV light. J. Embryol. exp. Morph. 12, Yarrnnq, H. (1983). nduction of longitudinal double malformations by centrifugation or by partial UVirradiation of eggs in the Chironomid species, Chironomus samoensis (Diptera: Chironomidae). Entomol. Gen. 8,, 7L-9.