Nuclear division and migration during early embryogenesis of Bradysia tritici coquillet (syn. Sciara ocellaris) (diptera : Sciaridae)

Int. J. lnsectMorphol. &Embryol., Vol. 15, No. 3, pp. 155to 163, 1986.

0020/7322/86 $3.00 + .00 PergamonJournalsLtd.

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N U C L E A R DIVISION A N D M I G R A T I O N D U R I N G E A R L Y E M B R Y O G E N E S I S OF B R A D Y S I A TRITICI C O Q U I L L E T (SYN. S C I A R A OCELLARIS) ( D I P T E R A : S C I A R I D A E ) A. L. P. PERONDINI,* H. O. GUTZEIT~and L. MORI~ *Departamento de Biologia, Instituto de Bioci~ncias, Universidade de Sffo Paulo, CP 11461, S~o Paulo, Brazil; tlnstitut fur Biologie I, AlbertstraBe 21a, D-78 Freiburg i.Br., Federal Republic of Germany, SFundagffo " C e n t r o de Pesquisa de Oncologia", Rua Oscar Freire, 2396, Sffo Paulo, Brazil (Accepted 23 August 1985)

Abstract--Nuclear division and migration of cleavage nuclei in the embryos of Bradysia tritici (Diptera : Sciaridae) have been studied by light microscopy and nuclear staining. There are 8 cleavage cycles up to the syncytial blastoderm stage (4.5 hr), and during the 1 lth cycle cellularization begins (6.5 hr). The first 3 divisions take about 30 min each. During the 5th and 6th cycles, the maximum rate of division is reached (12 min/cycle at 22°C1. After pole cell formation, the duration of the following mitotic cycles increases progressively. During nuclear migration, the presumptive germ line nuclei reach the egg cortex first, followed by anterior somatic nuclei and finally, posterior somatic nuclei reach the egg cortex. Possibly as a result of this region-specific nuclear migration, nuclear divisions become parasynchronous after 3 hr of embryogenesis (4th cycle). Several mitotic cycle,; later, between the 8th and 10th cycle in different embryos, X-chromosome elimination in somatic nuclei begins at the anterior egg pole and progresses in anteroposterior direction. Our observations suggest that the observed region-specific differences may be due to the activity of localized factors in the egg that control migration and nuclear cycle of the somatic nuclei. Index descriptors (in addition to those in the title): Parasynchronous divisions, chromosome elimination.

INTRODUCTION THE EXISTENCE of localized developmental information in insect eggs has been inferred from a body of experimental evidence (reviewed by Sander, 1984). Unfortunately, little is known about the way in which these developmental signals "instruct" blastoderm cells or germ cells to differentiate appropriately. In the Bradysia egg, at least 3 different developmental signals may be distinguished: (1) factors involved in the determination of anterior body structures (Perondini et al., 1982); (2) factors that are thought to control the elimination of X-chromosomes (DuBois, 1932, 1933; Rieffel and Crouse, 1966; Mori and Perondini, 1980, 1984); and (3) localized factors in the germ plasm which are believed to be involved in germ cell determination (DuBois, 1932, 1933; Berry, 1941) and in the differential elimination of chromosomes in germ cells and in somatic cells (Rieffel and Crouse, 1966). The determination of the embryonic axis as well as chromosome elimination in somatic cells can be disturbed by experimental interference. By U.V.-irradiation of the anterior egg pole, monsters with an abdomen instead of thorax and head ("double-abdomen") may be produced (Perondini et al., 1982; Bischof and Gutzeit, unpublished). Errors in chromosome', elimination can be increased by "temperature shocks" (Perondini and Mori, in preparation) or genetically by making use of the mutant sepia (Mori et al., 1979; Mori and Perondini, 1980, 1984). 155

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PERONDINI, H. O. GUTZEIT and L. MORI

For further studies on the control of these developmental processes, an accurate description of the early cytological events in the embryo is required. Some data relevant to this topic were published by DuBois (1932, 1933), Butt (1934) and Berry (1941). We have extended these studies with particular emphasis on nuclear multiplication and movement of nuclei to the egg cortex where chromosome elimination occurs. MATERIAL

AND

METHODS

A bisexual laboratory strain of Bradysia tritici (syn. Sciara ocellaris) was used (Pavan and Perondini, 1967). The larvae were reared in vials on heat-sterilized hay dust as described elsewhere (Gutzeit, 1985). A large number of eggs (average about 100 per female) was obtained by inducing females to lay eggs in a short period of time according to the procedure of Carson (1946) or by decapitation. For light microscopic examination the eggs were kept in boiled and filtered tap water at 22°C. The chorion is transparent under water and need not be removed for staging and scoring of the embryos. Embryos were fixed with a solution of ethanol/acetic acid in heptane (phase partition fixation) followed by staining in toto with basic fuchsin according to Zalokar and Erk (1976). During the stages of intravitelline cleavage the nuclei were counted directly under the microscope. At later stages, when all nuclei were in the periplasm, only m i n i m u m estimates of the number of somatic nuclei could be given owing to occasional superposition of the nuclei in the optical pathway. For these estimates, the total surface area of each embryo was calculated by measuring both semi-axes and by considering the embryo a rotation ellipsoid. The surface area of each embryo was calculated using the formula E S = 2 b ( b + -a arc sin E); a and b stand for the lengths of the large and small semi-axis, respect.ively, and E

"J(a~

a

b2)

Since the nuclei are not evenly spaced, we calculated the average distance between 2 nuclei for each embryo (10 determinations) thus defining the average surface area surrounding each nucleus. The surface area (S) of a particular embryo divided by the average surface area of each nucleus gives an approximation of the total number of nuclei. The distance of the nuclei from the egg surface was measured in embryos stained at different stages of development. These data were corrected for fixation-induced shrinkage as determined from the change in egg diameter before and after fixation (shrinkage factor 0.63). Eliminated chromosomes in somatic anaphase nuclei can easily be identified after staining embryos of the appropriate stages with fuchsin. In embryos fixed after the elimination has occurred, the eliminated chromosomes can be seen as dense chromatin granules in the cytoplasm near the peripheral nuclei. RESULTS

1. Light microscopy Single eggs were taken from batches of eggs laid by different females and the morphological changes during early embryogenesis (up to syncytial blastoderm formation) studied under the microscope. At the time of egg deposition the pole plasm is visible as a clear area at the posterior pole (Fig. la) and there is little change during the first 3.5 hr of development. After this time 2 pole cells form at this area (Fig. lc) which later undergo several rounds of mitosis (Fig. ld). When there are 1 6 - 2 0 pole cells, the mitosis ceases and the pole cells enter a long resting stage (Berry, 1941). At the anterior pole of newly deposited eggs, only a very small clear area is visible (Fig. la), which is free of ooplasmic inclusions like yolk granules or lipid spheres (Gutzeit et al., 1985) and in this property resembles the "cytoplasmic cone" described previously in Smittia eggs (Zissler and Sander, 1973). Later, the clear area increases in thickness up to 2.5 hr of age, but later it becomes thinner, presumably owing to lateral spreading of the cortex material (Fig. lb). Nuclei that arrive in the periplasm during the nuclear migration stages show up as round clear areas, first at the anterior pole and progressively in the periplasm towards the posterior pole (Fig. lc). At about 4.5 hr a syncytial blastoderm is formed (Fig. ld).

Nuclear Division and Migration During Early Embryogenesis of Bradysia tritici

a

157

b

FIG. 1. Morphological changes in living embryo of Bradysia (anterior pole to left), a. embryo of age. b. 2.5 hr. c. pole cell stage (about 3.5 hr). d. syncytial blastoderm (4.5 hr). Note clear posterior pole (oosome) up to pole cell formation and first thickening and later regression of area at ~mterior pole; arrow = somatic nucleus entering the periplasm; open arrow = pole

20 min area at a clear cells.

2. Kinetics of nuclear division The number of nuclei was scored in embryos fixed at half-hour intervals. During the first 2 hr of embryogenesis the number of nuclei follows strictly a geometrical progression (1, 2, 4 or 8 nuclei). However, later in development the number of nuclei in some embryos deviated considerably from 2 n (only for n > 3; see Fig. 2). Some nuclei may fail to undergo mitosis along with the other nuclei, or may even degenerate (Zalokar and Erk, 1976). Despite the observed variation, it is clear from the data shown in Fig. 2 that most embryos had entered the 1 lth mitotic cycle at 6.5 hr of embryogenesis when cell formation (blastoderm stage) had just begun. The duration of the mitotic cycles was estimated by calculating the time of development when on the average 2 n nuclei were present. From these data, the average time intervals between the mitotic cycles were determined (Fig. 3), Meiosis is completed after about 1 hr 15 min of development. The following 3 mitotic cycles take about 30 min each. During the 5th and 6th cleavage divisions, the maximal rate is reached when it takes only about 12 min to complete one mitotic cycle. Later, the duration of the mitotic cycles increases progressively from 23 min (7th cycle) to about 60 rain (10th cycle).

3. Nuclear migration Nuclear syngamy occurs in the anterior-third of the egg at about 1 - 1.5 hr after egg

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PERONDINI, H. O. GUTZE1T and L. MORI

100 I. 5hr ! • 211 1.5 "t I I00 2hr n:|4 3.3~1 50 2 . 5hr 11=21 5_+3 50 3hr nt26 13±7 U') 0 >m =E W

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FIG. 2. Frequency distribution of embryos according to the number of nuclei during early embryogenesis. Note the change in scale on the abscissa after 8 nuclei. Due to the variation in the number of nuclei at later developmental stages the logarithmic scale was extended and the intervals between 2 n nuclei subdivided into 3 classes. The dark columns in the histogram represent the fraction of embryos that had undergone X-chromosome elimination. For each stage of development (in hr), the n u m b e r of analyzed embryos and the average number of nuclei (_+ S.D.) is given.

deposition. The zygote nucleus is located in the endoplasm at a minimum distance of about 65 ~tm from the egg membrane (Fig. 4). After the 1st mitotic division, the nuclei start their migration to the egg periphery. Frorh 2.5 hr (3rd and 4th cycle), the distribution of nuclei in the egg becomes increasingly asymmetric. The presumptive germ line nuclei complete their migration to the cell periphery first, followed by the anterior somatic nuclei and, finally, by the posterior somatic nuclei (Figs. 1 and 4). For this reason, the distance of the nuclei to the egg membrane in 3 hr-old embryos ranges from only 14 ~tm

Nuclear Division and Migration During Early Embryogenesis of Bradysia tritici

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FIG. 3. Duration of mitotic cycles in rain. On top of graph duration of developmental phases is indicated. N M = nuclear migration; P C = pole cell formation; SB = syncytial blastoderm.

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FIG. 4. Shortest distance of nuclei from egg cell m e m b r a n e in different regions of egg up to 5 hr of development. Average time when each nuclear cycle is completed is shown on top. Between 3.5 and 4 hr pole cells divide repeatedly (number of pole cells shown on abscissa). Timing of other events (see text) is indicated. AS = anterior somatic nuclei (from anterior 1/3); CE = X - c h r o m o s o m e elimination; GL = presumptive germ line nuclei; M R = mitotic rate; PC = pole cell; PRS = parasynchrony; PS = posterior somatic nuclei (posterior 1/3).

159

160

A.L.P. PERONDINI,H. O. GUTZEITand L. MORI

(presumptive germ line nuclei) to 20 ~tm (anterior somatic nuclei) and 28 ~tm (posterior somatic nuclei). Pole ceils are formed at a time (3.5 hr, 6th cycle) when the anterior somatic nuclei are still 15 lam away from the egg membrane. By 4.5 hr of development (8th cycle), all somatic nuclei are located in the egg cortex very close to the egg membrane (3 ~tm distance), and at this stage the formation of the syncytial blastoderm is completed.

4. Parasynchronous mitotic divisions During the first 3 hr of development, when the nuclei are in the endoplasm, nuclear divisions are generally synchronous. However, when the somatic nuclei are located at a distance of about 20 ~tm from the egg membrane (3 hr; 3rd or 4th cycle), parasynchronous divisions were observed in a small number of the analyzed embryos (4.4%, Table 1). The number of embryos with parasynchronous divisions increases rapidly up to the syncytial blastoderm stage (4.5 hr; 8th cycle), and remains high (average 88%) up to 6.5 hr of embryogenesis (Table 1). Parasynchronous nuclear divisions have been observed in embryos of m a n y insect species (Agrell, 1964; Lundquist 1981; van der Meer et al., 1982). During nuclear migration, the somatic nuclei reach the anterior periplasm first (Fig. 1), and it is from this end that parasynchrony is initiated. A wave of mitosis appears to pass along the egg in anteroposterior direction as illustrated in Fig. 5. The first few parasynchronous divisions of somatic nuclei usually show only 2 different phases of the mitotic process, for example, prophase and metaphase (Fig. 5a) or metaphase and anaphase (Fig. 5b). However, during later development there are often more than 2 phases of mitosis visible in a single embryo. Particularly during later stages of development the pattern of parasynchrony was found to be more complex, for example spreading of mitosis from the posterior pole or from the middle towards the egg poles. In several embryos at an early pole cell stage, the mitotic phase of the dividing pole cell nuclei appeared to be part of the parasynchronous mitotic wave. However, since pole cells divide 3 times between 3.5 and 4 hr, while the somatic nuclei divide at most twice (cycle 6 and 7, see Fig. 4), the mitotic cycle of the pole cell nuclei cannot be part of the parasynchronous wave that effects the somatic nuclei at that stage. 5. Chromosome elimination In sciarid flies, differential elimination of X-chromosomes occurs at the syncytial blastoderm stage (DuBois, 1932, 1933). In Sciara coprophila, which possesses germ line limited chromosomes (L-chromosomes), elimination of these chromosomes from the somatic nuclei occurs during the 5th division, and X - c h r o m o s o m e elimination in somatic nuclei follows one or 2 divisions later (DuBois, 1932, 1933). Since Bradysia tritici does not possess L-chromosomes (Metz and Lawrence, 1938), only elimination of X-chromosomes is expected to occur. We were able to score this phenomenon in our preparations. As shown in Fig. 2, elimination of X-chromosomes begins at 4.5 hr in about 10% of the embryos and ends at about 6.5 hr of development when more than 96% of the embryos have completed the process of chromosome elimination. This result shows that in this species, X - c h r o m o s o m e elimination is an asynchronous process that may occur at any time between the 8th and the 10th nuclear division. At this time ( 4 . 5 - 6 . 5 hr) parasynchronous mitotic divisions were typically observed (Table 1). In 96.5% of the analyzed embryos chromosome elimination was found to p~oceed in an anteroposterior direction in the embryo.

Nuclear Division and Migration During Early Embryogenesis of Bradysia tritici

161

TABLF 1. PARASYNCHRONOUS NUCLEAR DIVISIONS DURING EARLY FMBRYOGENESZS OF Bradysia tritici

Number of scored

Distance of somatic nuclei to egg membrane

Age (hr)

embryos

(lam)

Percentage of embryos with parasynchronous nuclear divisions

2 2.5 3 3.5 4 4.5 5 5.5 6 6.5

16 40 23 39 60 41 34 27 31 29

ca 60 ca 40 18 28 16-21 5 - 10 < 5 < 5 < 5 < 5 < 5

0.0 0.0 4.4 33.5 70.8 70.7 94.2 85.2 87.1 86.2

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FIG. 5. Camera lucida drawings of in toto stained embryos showing examples of parasynchronous nuclear divi:dons ( a - d ) . Nuclei were drawn only from median optical section upwards. Number of pole cells varied from 4 - 8. For clarity, only those pole cells visible in median optical section were drawn. A = anaphase; I = interphase; M = metaphase; P = prophase; PC = pole cells; T = telophase.

DISCUSSION

Nuclear division and migration has been studied in several insects (reviewed by Krause and Sander, 1962; Counce, 1973; see also Wolf, 1980; Foe and Alberts, 1983; Lundquist and LOwkvist, 1983). We have analyzed these processes in Bradysia tritici, since in this species developmental signals for early embryogenesis can be altered experimentally (Perondini et al., 1982; Bischof and Gutzeit, unpublished) and the egg cytoplasm shows many local morphological specializations (Perondini et al., 1982; Gutzeit et al., 1985; Zissler and Kyrieleis, pers. comm.); for this reason an attempt to correlate developmental information with structural specializations in the Bradysia egg seems promising. The 1st few cleavage divisions proceed rapidly and at increasing rate up to the 6th nuclear division. Between 3 and 3.5 hr of development, when the maximal rate of division is reached (5th and 6th cycles), it takes only little over 10 min at 22°C to complete a

162

A . L . P . PERONDINI, H. O. GUTZEIT and L. MORI

mitotic cycle. After this phase, the 1st nuclei approach the egg cortex and at the same time the rate of division decreases significantly. A similar observation has been made in Drosophila by Zalokar and Erk (1976). The observation that nuclear migration of somatic nuclei, parasynchronous divisions, and chromosome elimination take place in anteroposterior sequence in the egg, suggests that either signals controlling these processes are distributed in the egg in a graded fashion or, more likely, that these factors are localized in the egg cortex. In the latter case, the anteroposterior spreading of parasynchronous divisions may be considered a consequence of the observed regional differences in the migration of somatic nuclei to the egg cortex. If cortical factors are responsible for the observed lengthening of the mitotic cycles, the anterior nuclei would be exposed to these factors first, so that a wave of parasynchronous nuclear divisions spreading from the anterior egg pole would result. Figure 4 illustrates the timing of the studied events in relation to the distance of the nuclei from the oolemma. Parasynchronous nuclear divisions are initiated well before chromosome elimination begins. If chromosome elimination is controlled by cortical factors, their action appears to be delayed, since chromosome elimination takes place after all nuclei have reached the egg cortex (Fig. 4). Chromosome elimination is not controlled by a "mitotic clock", since elimination does not occur after a specific number of nuclear divisions (Fig. 2). The nuclear commitment for the future developmental pathways does not depend on the time of exposure to cortical factors. Centrifugation of Bradysia eggs, which greatly disturbs the pattern of nuclear migration, was often of no consequence for embryonic differentiation (Velloso, 1981; Perondini, unpublished). Furthermore, the direction of parasynchronous divisions in the egg of the pea beetle, Callosobruchus did not correlate with the determination of the longitudinal embryonic axis (van der Meer et al., 1982). The migration of presumptive pole cell nuclei deserves special mention. These nuclei are the first to reach the egg periphery, long before the 1st somatic nuclei reach the anterior egg cortex. The presumptive pole cell nuclei enter the pole plasm and, together with oosome material, become enclosed in the pole cells. Being separated in this way from the remaining ooplasm, the pole cell nuclei do not undergo chromosome elimination and pursue their own characteristic mitotic cycle. The early migration of presumptive pole cell nuclei and subsequent pole cell formation leads to a compartmentalization in the egg at an early stage, so that somatic and germ line nuclei may be exposed to different developmental signals. The importance of the correct timing in the migration of presumptive pole cell nuclei was shown in Drosophila by experimentally delaying the migration. Such embryos were unable to form functional germ cells (Okada, 1982). Delayed migration of nuclei to the pole plasm in the egg of the gall midge, Mayetiola, causes chromosome elimination also in presumptive germ line nuclei, thus giving rise to chromosome-deficient germ line cells, which are unable to form functional gametes (Bantock, 1970).

Acknowledgements--The collaboration was made possible by travelling grants to A.P. (Humboldt foundation) and to H.G. (Deutsche Forschungsgemeinschaft and FAPESP 83/1758-5). We further acknowledge the financial support of CNPq-Brasil. We thank Professor K. Sander, for helpful suggestions and critical reading of the manuscript.

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163

REFERENCES AGRELL, I. 19~4. Natural division synchrony and mitotic gradients in metazoan tissues, pp. 3 9 - 6 7 . In E. ZEUTHEN (ed.) Synehrony in Cell Division and Growth. Interscience, London. BANTOCK, C. R. 1970. Experiments on c h r o m o s o m e elimination in the gall midge, Mayetiola destructor. Y. Embryol. Exp. Morphol. 24: 2 5 7 - 86. BERRY, R. O. 1941. C h r o m o s o m e behavior in the germ cells and development of the gonads in Sciara ocellaris. J. Molphol. 68: 5 4 7 - 83. BUTT, F. H. 1934. Embryology of Sciara. Ann. Entomok Soc. Amer. 2 7 : 5 6 5 - 7 9 . CARSON, H. t . 1946. The selective elimination of inversion dicentric chromatids during meiosis in the eggs of Sciara impatiens. Genetics 31: 9 5 - 133. COUNCE, S. J. 1973. The causal analysis of insect embryogenesis, pp. l - 156. In S. J. COUNCE and C. H. WADDINGTON (eds.) Developmental Systems, Vol. 2. Academic Press, New York. DuBoIS, A. M. 1932. A contribution to the embryology of Sciara (Diptera). J. Morphol. 54:161 - 9 2 . DUBOlS, A. M. 1983. C h r o m o s o m e behavior during cleavage in the eggs of Sciara coprophila (Diptera) in relation to the problem of sex determination. Z. Zellforsch. Mikrosk. 19: 5 9 6 - 6 1 5 . FOE, V. E. and B. M. ALBERTS. 1983. Studies of nuclear and cytoplasmic behaviour during the five mitotic cycles that precede gastrulation in Drosophila embryogenesis. J. Cell Sci. 61:31 - 70. GUTZEIT, H. O. 1985. O o s o m e formation during in vitro oogenesis in Bradysia tritici (syn. Sciara ocellaris) Wilhelm Roux's Arch. Dev. Biok 194: 4 0 4 - 10. GUTZEIT, H. O., D. ZISSLER and A. L. P. PERONDIN1. 1985. Intracellular translocation of symbiotic bacteroids during late oogenesis and early embryogenesis of Bradysia tritici (syn. Sciara ocellaris) (Diptera : Sciaridae). Differentiation 29: 2 2 3 - 29. KRAUSE, G. and K. SANDER. 1962. Ooplasmic reaction systems in insect embryogenesis. Adv. Morphol. 2: 259 - 303. LUNDQUIST, A. 1981. A quantitative analysis of mitotic gradients during early development in Calliphora erythrocephala Meig. (Diptera). J. Morphol. 168: 2 3 9 - 45. LUNDQUIST, A. :and B. LOWKVIST. 1983. Nuclear kinetics during cleavage in a dipteran egg. J. Exp. Zool. 228: 151-55. METZ, C. W. and E. G. LAWRENCE. 1938. Preliminary observations on Sciara hybrids. J. Hered. 29: 1 7 9 - 8 6 . MORI, L. and A. L. P. PERONmNJ. 1980. Errors in the elimination of X-chromosomes in Sciara ocellaris. Geneth,s 94:663 - 73. MORJ, L. and A. L. P. PERONDINI. 1984. An analysis of Sciara ocellaris g y n a n d r o m o r p h s and the morphogenetic fate m a p of presumptive adult cuticular structures. J. Exp. Zool. 230: 2 9 - 35. MoRh L., E. M DESSEN and A. L. P. PERONDINI. 1979. A gene that modifies the sex-ratio in a bisexual strain of Sciara ocellaris. J. Here& 42:353 - 57. OKADA, M. 1982. Loss of the ability to form pole cells in Drosophila embryos with artificially delayed nuclear arrival at the posterior pole, pp. 3 6 3 - 7 2 . In M. M. BURGER and R. WEBER (eds.) Embryonic Develooment, Part A, Genetic Aspects. Alan R. Liss, New York. PAVAN, C. and A. L. P. PERONDJNI. 1967. Heterozygous puffs and bands in Sciara ocellaris Comstock (1982). Exp. CeIL Res. 48: 2 0 2 - 06. PERONDINI, A. L. P., H. O. GUTZEIT, L. MORI and K. SANDER. 1982. lnduktion von " D o p p e l a b d o m e n " und die Differenzierung anterioren Polplasmas in Embryonen von Sciara. Verh. Dtsch. ZooL Ges. 244 (abstra:t). RIEFFEL, S. M. and H. V. CROUSE. 1966. The elimination and differentiation of chromosomes in the germ line of Sciara. Chromosoma 19:231 - 7 6 . SANDER, K. 1984. Embryonic pattern formation in insects: Basis concepts and their experimental foundations, pp. 2 4 5 - 6 8 . In G. M. MALACINSKI and S. V. BRYANT (eds.) Pattern Formation, a Primer in Developmental Biology. Macmillan, New York. VAN DER MEER, J. M., W. KEMMER and D. M. MIYAMOTO. 1982. Evidence against a relation between the direction of mitotic waves in early insect development and egg polarity as reveated by segment sequence. Wilhelm Roux's Arch. Dev. Biol. 191: 3 5 5 - 65. VELLOSO,m. 19~:1. Efeitos da centrifugaego de ovocitos na determina¢go sexual e aspectos do desenvolvimento ovariano em Sciara ocellaris (Diptera, Sciaridae). Master thesis, Universidade de Sgo Paulo. WOLF, R. 1980. Migration and division of cleavage nuclei in the gall midge. Wachtliella persicaria. Wilhelm Roux's Arch. Dev. BioL 188: 6 5 - 7 3 . ZALOKAR, M. and I. ERK. 1976. Division and migration of nuclei during early embryogenesis of Drosophila melanogaster. J, Microsc. Biol. Cell. 25:97 106. ZISSLER, D. and K. SANDER 1973. The cytoplasmic architecture of the egg cell of Smittia spec. (Diptera, Chironamidae). I. Anterior and posterior pole regions. Wilhelm Roux's Arch. Dev. BioL 172: 1 7 5 - 86.