Blastoderm formation in the silkworm egg (Bombyx mori L.)

By SACHIKO 2 4 KAZUO 2 4 0 Author's address: College of Medical Technology, Nagoya University , Nagoya 461 > Japan 1 Author's address: Institute for Developmental Research, Aichi Prefectural Colony , Kasugai, Aichi 480-03 , Japan 2 Institute for Developmental Research , Aichi Prefectural Colony 3 Author's address (for reprints): Biological Institute, Faculty of Science , Nagoya Uni- versity, Nagoya 464 , Japan 4 From the Biological Institute, Faculty of Science, Nagoya University and the SUMMARY Embryogenesis in the egg of the silkworm, Bombyx mori, up to 24 h after oviposition was studied, by light microscopy with special reference to nuclear migration and blastoderm formation. In Bombyx eggs blastoderm cells seem to form in a mechanism different from that usually seen in many other insect species; that is, in Bombyx eggs no typical cleavage furrows were seen. Cleavage nuclei which had migrated, accompanied by the associated cytoplasm, to the egg surface pushed up the plasma membrane and protruded beyond the initial level of the periplasm. The periplasm fused with their associated cytoplasm was partitioned among and pulled around the nuclei. Then each nucleus was separated by a laterally-invading limiting membrane from the yolk-granules-occupied region to yield a blastoderm cell. - There have been few morphological studies on early developmental stages up to 24 h after oviposition, i.e. until the stage of germ-band formation, in the egg of the silkworm, Bombyx mori. In a previous paper (Takesue, Keino & Endo, 1976), we have reported that in Bombyx eggs blastoderm and yolk cells begin to form about 6 and 24 h, respectively, after oviposition at 27 C. Anderson (1962) has stated that the mechanism of blastoderm formation is essentially similar among eggs of different insect species. A typical and wellstudied example can be found in Drosophila eggs, where the blastoderm is formed by cleavage furrows growing inward from the egg surface between nuclei located in a row in the periplasm (Huettner, 1923; Mahowald, 1963; Fullilove & Jacobson, 1971; Sanders, 1975). Iwasaki (1931) reported that the blastoderm is formed in Bombyx in the same manner as in Drosophila. In studying the early developmental stages of Bombyx eggs, however, we have made observations which suggest that blastoderm cells in Bombyx eggs might be formed in a mechanism different from that proposed by Iwasaki (1931). In the present work we have paid special attention to the movement of cleavage nuclei leading to blastoderm formation. A preliminary note of these results has been published previously (Takesue, Onitake & Keino, 1977). MATERIALS AND METHODS Non-diapause eggs from the cross between Daizo and Japanese 106 bivoltine races of the silkworm, Bombyx mori, were used as the material. The eggs were obtained from female moths which had hatched from eggs incubated at 15 C in the dark. Moths were allowed to lay eggs for 30 min at 25 C and the eggs were incubated at 25 C, not at 27 C used in the previous work (Takesue et al. 1976), in order to slow down the rate of development of the eggs. As described previously (Takesue et al. 1976), the eggs were deprived of the chorion and vitelline membrane with fine forceps in 2-5 % glutaraldehyde0-1 M potassium phosphate buffer (pH 7-5) and fixed in the same buffer for 2 h, followed by post-fixation with 2 % osmium tetroxide for 1-5 h. After block staining with uranyl acetate and dehydration in an ascending ethanol series and propylene oxide, they were embedded in Epon 812. Sections about 0-5 jum thick were cut with a glass knife on an ultramicrotome and stained with 1 % toluidine blue in 1 % borax for examination in the light microscope. In preliminary experiments, the eggs were punctured with a sharp needle at one site on each of the dorsal and ventral sides and at the anterior and posterior poles (four sites in all) and fixed by immersion in 2-5 % glutaraldehyde-0-1 M potassium phosphate buffer (pH 7-5) for 20 h, followed by post-fixation with 0-5 % osmium tetroxide for 18 h. After fixation, the chorion was removed and the eggs were block-stained with uranyl acetate, followed by the procedures as described above. Essentially the same morphological features were observed in the eggs fixed by the two procedures. Since the latter consumed much more time than the former, we have adopted the former fixation procedure in this work. In the egg less than 30 min after oviposition (Fig. 1 A), the whole surface of the egg was covered with microprojections and two kinds of yolk granules were seen: some (ygx) were intensely stained and located in the inner region of the egg, and the others (yg2), located in the peripheral region beneath the periplasm, were weakly stained, but had small densely-stained dots inside them (Takesue et al. 1976). In the periplasm there were a lot of small dots similar in density and size to those in yg2. In the egg 8 h after oviposition (8 h egg) (Fig. 1B), the microprojections became abundant and thick, implying intensive Fig. 1. Light micrographs of the eggs Oh (A) and 8 h (B) after oviposition. The whole surface of the eggs was covered with microprojections increasing in number and height with time. Cleavage nuclei accompanied by the associated cytoplasm had already appeared in the 8 h egg (B). ygu yolk granule in the inner region of the egg; ygz, yo'k granule in the peripheral region; Ip, lipid particle; ppl, periplasm; mp, microprojection; en, cleavage nucleus; ac, associated cytoplasm. synthesis of the plasma membrane during this period. Many cleavage nuclei, surrounded by the associated cytoplasm, were seen among ygl5 not yg2, near the anterior pole. Up to 9 h after oviposition, these cleavage nuclei with the associated cytoplasm had migrated into the peripheral region of the egg, though this nuclear invasion of the egg surface was not simultaneous over the entire periphery (Toyama, 1909). The blastoderm formed 9-10 h after oviposition at 25 C, though development of cleavage nuclei into blastoderm cells was not simultaneous over the entire periphery, consistent with the non-simultaneous nuclear invasion of the egg periphery described above. Figure 2 shows the process of blastoderm formation occurring in the eggs 9-10 h after oviposition. When a cleavage nucleus arrived near the periphery of the egg, the egg surface was raised into a hillock, still covered with abundant microprojections, over the nucleus and the periplasm fused with its associated cytoplasm (Fig. 2A and B). As cleavage nuclei continued to further migrate toward the surface and protruded beyond the initial level of the egg surface, the periplasm which had been previously Fig. 2. Light micrographs showing the process of blastoderm formation in the eggs 9-10 h after oviposition. See the text for the details. ygx andy#2> yolk granules; ygd and arrows, small densely-stained dot; en, cleavage nucleus; ac, associated cytoplasm; ppl, periplasm; mp, microprojection; blc, blastode (...truncated)