Fate mapping of the silkworm, Bombyx mori, using localized UV irradiation of the egg at fertilization

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1 Development 120, (1994) Printed in Great Britain The Company of Biologists Limited Fate mapping of the silkworm, Bombyx mori, using localized UV irradiation of the egg at fertilization Maroko Myohara Department of Insect Genetics and Breeding, National Institute of Sericultural and Entomological Science, Tsukuba, Ibaraki 305, Japan SUMMARY Bombyx eggs at the fertilization stage (0-2 hours after oviposition) were irradiated with a scanning UV-laser microbeam (355 nm) over an area of about 1% of the total egg surface. In spite of absence of nuclei or cells at the irradiated sites, larvae from treated eggs showed localized cuticle defects in the integument. The location and frequency of the defects within the cuticular pattern correlated closely to the site of irradiation both in the anteroposterior and the dorsoventral direction. Based on the correlation, presumptive regions for each larval segment were located and a fate map of the Bombyx egg was established. Key words: UV-laser irradiation, insect egg, fate map, pattern formation, Bombyx mori INTRODUCTION The silkworm, Bombyx mori, is a good subject for embryological research, owing to its well-documented genetics and its widespread use in research (Tazima, 1978). Accurate fate maps are an essential precondition for interpreting correctly the results of various types of developmental experiments. The establishment of Bombyx fate maps has so far been attempted by two methods, microcautery (Kuwana and Takami, 1957) and genetic mosaics (Katsuki et al., 1980). These methods, however, have yielded results of only limited value. The local cautery map by Kuwana and Takami (1957) is not as detailed as the segment pattern but roughly shows presumptive regions for the procephalon, gnathal segments, thorax and abdomen; and the stage of eggs at the cautery is unclear. The fate map by Katsuki et al. (1980) with regard to the topography totally depends on the local cautery map, because mosaic methods yield only the relative, not the absolute, positions of the primordia in the egg. Also, the indication of larval segments in the map is unclear and incomplete; there are only 11 segments illustrated on the map while a Bombyx larva has 13 segments in the thorax and abdomen alone. The most direct and most widely used method for fate mapping nowadays is marking individual blastomeres by intracellular injection of a tracer (for example, Technau and Campos-Ortega, 1985, for Drosophila; for review see Technau, 1987). Unfortunately, however, the method is not applicable to Bombyx, because the egg is enclosed by a thick, tough chorion, which cannot be removed from the unfixed early embryos. Recently, a UV-laser irradiation system which was designed for fluorescence excitation, cell surgery and cell ablation became commercially available. Equipped with a computercontrolled motorized microscope stage, the system (Workstation ACAS 470, Meridian) enables one to scan the laser microbeam on a strictly defined area in a highly reproducible fashion. In this study, this system was used to irradiate defined regions of the Bombyx egg and a detailed fate map was established based on the correlation between the site of irradiation and the site of the resultant defects in larval cuticle. MATERIALS AND METHODS Egg collection and rearing conditions Bombyx mori larvae of a non-diapause strain, pnd re ch (pigmented and non-diapausing egg; red egg; chocolate) were reared at 25 C and fed an artificial diet (Yakult). Egg collections were made on cellophane paper for 20 minutes. The average size of an egg was about 1150 µm in length anteroposteriorly, 950 µm in width dorsoventrally, and 650 µm in thickness. Stage of eggs for irradiation In Drosophila melanogaster, UV-laser irradiation of preblastoderm eggs rarely induces defects in the larval cuticle, thus fate mapping has been done by irradiating eggs at the cellular blastoderm stage (Lohs- Schardin et al., 1979b). However, in Bombyx in contrast to Drosophila, there is no clear cellular blastoderm stage and neither the penetration of cleavage nuclei into the egg periplasm nor the subsequent blastoderm cell formation occur simultaneously but begin first in the anterior part of the egg and then proceed toward the posterior (Takesue et al., 1980; Miya, 1984; Kobayashi and Miya, 1987). When cellularization has been completed at the posterior pole, cell density on the egg surface is much higher in the anterior half than in the posterior, since cells in the anterior region have already divided once or twice by then. Immediately after the completion of the cellular blastoderm, cells of the embryonic region begin to aggregate and the germ band anlage contracts. Thus in Bombyx the cellular blastoderm stage is not so suitable for fate mapping by UV irradiation as in Drosophila. To find an appropriate stage for fate mapping, a 200 µm square irra-

2 2870 M. Myohara diations were performed with Bombyx eggs at various stages between oviposition and the completion of cellular blastoderm, i.e. from 0 to 15 hours after egg laying (AEL). About 90% of larvae from the treated eggs showed cuticle defects irrespective of the developmental stage at irradiation, while the same treatment of Drosophila eggs at the early cleavage stage rarely induced cuticle defects (Myohara, unpublished data). It is very interesting that defective larvae could be induced by localized UV irradiation of Bombyx eggs as early as 0-2 hours AEL when even the union of female and male pronuclei has not been completed (Kawamura, 1978). Thus I chose this fertilization stage for UV irradiation for fate mapping. Irradiation procedure UV-laser irradiations were carried out on the ACAS 470 work station (Meridian) equipped with a 5 W argon ion laser tuned to the 355 nm line, a 16-bit microcomputer and an inverted microscope (IMT-2, Olympus) equipped with a motorized X-Y microscope stage. The laser beam is directed to the microscope system by a set of adjustable mirrors; the objective lens system delivers a focused laser beam to the sample plane and also collects fluorescent radiation from the specimen (Fig. 1). Irradiations were carried out using the area raster program provided by the ACAS microcomputer system. With this program, the size and shape of the irradiation areas were controlled by changing parameters for stage movement such as the width of the raster scan, the distance between scan lines (step size) and the number of lines in the raster (scans). For fate mapping in this study, irradiations of an area of µm were accomplished by setting the parameters as follows: width, 145 µm; step size, 0.25 µm; scans, 690. The speed of stage movement was 0.5 mm/second, thus an irradiation of the µm area took approximately 200 seconds. UV energy incident at the egg surface, which was measured directly for each experiment by an ultraviolet meter (DM-365N, Spectronics) on the microscope stage, was mw/cm 2. Position of irradiation Eggs were stuck on a piece of Scotch tape and placed into a hole made in the bottom of a 3.5 cm plastic dish; the dish was then set on the microscope stage of the ACAS 470 (Fig. 1). Eggs were oriented with either the lateral, ventral or dorsal side facing the laser beam and irradiated at a single site. By rotating the plastic dish on the microscope stage, the anteroposterior axis was set parallel to the Y direction of the stage movement. Anteroposterior polarity was recognized by the presence of a micropylar apparatus at the anterior pole and dorsoventral polarity was recognized by the differential convexity of the egg surface (the ventral side, where the germ anlage first appears, is more convex than the dorsal side). However, since the differential convexity is not always easy to distinguish, the dorsoventral polarity was checked again by examining the position of embryo in the egg at a later embryonic stage under the dissection microscope. With a 4 objective lens (Splan 4PL, Olympus), the laser beam is focused on the plane of the sample to a spot diameter of approximately 10 µm. The position of this spot was set by positioning the microscope stage and photographed at the beginning of each irradiation. On each photograph, the anteroposterior and dorsoventral position of the spot was measured and evaluated respectively in percentage egg length (% EL) and percentage ventrodorsal diameter (% VD), with 0% EL at the posterior egg pole and 0% VD at the ventral midline. The irradiation position of each experiment (the central position of the irradiation area) was expressed with Xc (% VD) and Yc (% EL) for dorsoventral and anteroposterior directions, respectively. Thus, when the irradiation area size is µm (15% of the total egg width 15% of the total egg length), irradiation at the position of Xc=20% VD and Yc=50% EL, for instance, refers to irradiation of an area between 13% and 28% VD and between 43% and 58% EL. Analysis of larvae After irradiation, eggs were kept in a dark, moist chamber at 25 C for Fig. 1. Diagrammatic drawing of the UV-laser irradiation system for Bombyx eggs. See text for detail days. Hatched larvae (first instar) were fixed in glycerol-acetic acid (1:4) for 3-4 hours at 60 C and then at room temperature for 24 hours or longer. Eggs with unhatched larvae were treated with 10% sodium hypochlorite solution (Wako Pure Chemical) at 70 C for several minutes to dissolve the chorion partially. The chorion was removed from larvae by pipetting of the eggs in the solution. Dechorionated larvae were washed with water and then fixed in the same way as hatched larvae. Fixed larvae were examined in the fixative solution under the stereomicroscope (SMZ-10, Nikon) for cuticle patterns and were mounted in Hoyer s mixture (Wieschaus and Nüsslein-Volhard, 1986) and photographed under the compound microscope (Optiphot, Nikon). The terminology for the cuticle structures of Bombyx first instar larvae was as described by Tanaka (1927) (Fig. 2). Calculations for fate mapping Irradiated eggs that did not develop analyzable cuticles were disregarded in calculations of defect frequencies or target centers. They usually did not exceed 10% of the irradiated individuals within a series (average 5%). Although half the irradiations were done on the right side of the egg, they were translated to the left side assuming bilateral symmetry and all the fate maps were presented for the left side of the egg. The data from lateral irradiations were divided into four dorsoventral sections: section a, 10-30% VD; section b, 30-50% VD; section c, 50-70% VD; and section d, 70-90% VD. In each section, defect frequency for each segment or structure at a certain anteroposterior position, n% EL, was calculated from data pooled for n± 2% EL. In order to provide a parameter representative of all irradiations causing defects in a particular segment or structure, a target center (Lohs-Schardin et al., 1979a) for that segment or structure was constructed. The anteroposterior location of each center was calculated as weighted mean value of the positions of defect-inducing irradiation sites. The following formula was used: x = 1 a n n a n, 100 n=1 100 n=1 where x = anteroposterior location of the target center (% EL), n = anteroposterior position of irradiation site (% EL), and a n = defect

3 Fate mapping of silkworm eggs 2871 Fig. 2. Cuticle pattern of a first instar larva of Bombyx mori. Subdorsal (a), supraspiracle (b), subspiracle (c), and baseline tubercles (d) are indicated in the third thoracic segment (T3) and the fourth abdominal segment (A4). A, antenna; Al-10, abdominal segments 1-10; AL, abdominal leg; CL, caudal leg; I, interparietal plate; L, labrum; LA, labium; M, mandible; MX, maxilla; O, ocellus; P, parietal plate; Tl-3, thoracic segments 1-3; TL, thoracic leg. Bar represents 0.5 mm. frequency for a certain segment or structure following irradiation at n±2% EL. RESULTS Size of irradiation area For accurate fate mapping, a small size irradiation is desirable; however, too small an irradiation area will yield defects that are likely to escape detection, or will not cause a defect at all. To find an appropriate size of irradiation area for fate mapping, irradiations of various area sizes were carried out with eggs at the fertilization stage (Table 1). The defect frequency was higher than 90% when the irradiated area was larger than µm, while it was less than 25% when the irradiated area was smaller than µm. The intermediate area sizes of irradiation yielded intermediate frequencies of defect. Thus, I fixed the irradiation area size for fate mapping at Table 1. Frequency of defective larvae following UV irradiation of different area sizes Size of Larvae with irradiation area* No. of eggs No. of eggs defective µm µm (%TEW %TEL) irradiated developed cuticles (2 2) (0%) (5 5) (0%) (10 10) (23%) (12 12) (55%) (14 14) (64%) (15 15) (92%) (21 17) (93%) Irradiations were carried out with eggs at the fertilization stage (0-2 hours AEL). The irradiation sites were laterally positioned on the egg surface within 15-60% VD (Xc) and 30-60% EL (Yc). Embryonic mortalities after irradiation were 0-5%, similar to those of the non-irradiated controls. *Sizes of irradiation area are shown by width length of the irradiated area. The width and the length are also given in parentheses as percentages of the total egg width (% TEW) and the total egg length (% TEL), respectively. µm (15% of the total egg width 15% of the total egg length), which amounts to approximately 1% of the reported total surface area of the Bombyx egg (Kawaguchi and Takizawa, 1942). A preliminary histological analysis of irradiated embryos showed that penetration of nuclei into the egg periplasm and the subsequent cellularization during the blastoderm formation were strictly inhibited at the irradiated region; the extent of the damaged area was roughly the same as the size of irradiated area (data not shown). General characteristics of the cuticular defects induced by irradiation The defective larvae always showed defects only on one side of their body corresponding to the irradiation site, with no injury other than a local defect in the cuticle, indicating that the effect of the laser was very specific to the irradiated area while it had no effect on development of the non-irradiated side of the egg. The cuticular defects induced by the irradiation generally consisted of missing structures (Fig. 3A-F). With low frequency, however, duplications were observed in the defective sites; 11 out of 99 appendage defects and 7 out of 43 ocellar defects were duplications (Fig. 3G-I). The hypoderm surrounding the site of defect was always fused together, thus producing a continuous cuticle. Cuticle defects were observed on all parts of the body, from head to telson. Despite the large size of irradiation, about 80% of the resulting cuticular defects were restricted to three segments or less. Of 389 defective larvae induced, 33% were affected in one segment only, 28% showed defects in two segments, 17 and 12% showed defects in three and four segments, respectively. The rest of the defective larvae (about 10%) showed defects spread over more than five segments. The average number of defective segments was 1.25±1.61 (s.d.) per irradiated individual that developed analyzable cuticle. Embryonic mortality For fate mapping, 789 Bombyx eggs at the fertilization stage (0-2 hours AEL) were irradiated either ventrally, dorsally or

4 2872 M. Myohara laterally at various positions (Fig. 4A). Among them, 38 (5%) died at the embryonic stage, 362 (46%) developed into apparently normal larvae and 389 (49%) resulted in larvae with defective cuticle (Fig. 4B). The percentages of embryonic mortality observed after irradiations were always low and similar to those of the non-irradiated controls (0-7%), except when irradiations were performed at very anterior egg regions (Yc=83-88% EL). In this case, of 35 eggs irradiated, nine (26%) died at the embryonic stage. This high mortality may be due to damaging egg nuclei (or pronuclei) by the UV radiation, since in Bombyx the male and female pronuclei are reported to unite at a position around 88% EL (Takami, 1969;

5 Fate mapping of silkworm eggs 2873 Fig. 3. Cuticle defects induced by UV-laser irradiation of Bombyx eggs at the fertilization stage. (A) Defects in the parietal plate (arrow), following irradiation at the position of Xc=83% VD and Yc=70% EL. No ocellus is formed on the parietal plate. (B) Defects in the right first and second thoracic legs, following irradiation at Xc=35% VD and Yc=48% EL. Only the claws (arrows) are formed. (C) Defects in the first and second abdominal segments, following irradiation at Xc=51% VD and Yc=31% EL. Subdorsal tubercle are deformed (left arrow) or missing (right arrow). (D) Defects in the fourth to sixth abdominal segments, following irradiation at Xc=29% VD and Yc=34% EL. Left leg of the fifth abdominal segment is missing, and legs of the fourth and sixth abdominal segments are fused (arrow). (E) Defects in the ocelli, following irradiation at Xc=83% VD and Yc=64% EL. Two ocelli are missing, only four are formed. (F) Ocelli of a non-irradiated normal larva. The sixth ocellus (arrowhead) is out of focus. (G) Duplication in the ocellus (arrow heads), following irradiation at Xc=55% VD and Yc=69% EL. (H) Duplication in the second thoracic leg, following irradiation at Xc=49% VD and Yc=37% EL. The second and third thoracic legs are fused and the claw of the second leg is duplicated (arrow). (I) Duplication in the second abdominal leg, following irradiation at Xc=31% VD and Yc=35% EL. The first and second abdominal legs are almost fused and the second leg is duplicated (arrow). Each bar represents 0.2 mm. Miya, 1984) at about 2 hours after oviposition (Kawamura, 1978). Defect frequency and location of the presumptive region for the entire larval integument Irradiations on either the ventral or the dorsal egg surface rarely resulted in defective cuticle (open circles and crosses in Fig. 4); the defect frequency was 8% and 7%, respectively. Low frequencies were also observed when eggs were irradiated near the anterior, posterior or dorsal periphery. The rest of the egg surface, where irradiations caused cuticular defects with high frequency, is considered to be the presumptive region for the entire larval integument. Taking the point of 50% defect frequency as the rim of the presumptive epidermal region, its position was estimated as lying between 10% and 68% VD and from 16% EL to 78% EL. However, in the region fated to make the head capsule (57-78% EL, see below), the presumptive region extended dorsally to 88% VD (Fig. 4B). Within the presumptive region for the entire larval integument, the defect frequency was 72% on the average. Detailed analysis showed that, even within the presumptive integument area, the defect frequency depended significantly on the site of irradiation, both in the dorsoventral (Fig. 5A,B) and in the anteroposterior direction (Fig. 5C,D). The highest frequency was obtained at the middle part of the presumptive integument, 36-57% EL (Fig. 5C), where 132 out of 145 larvae from irradiated eggs (91%) showed cuticle defects in the integument. The decline of defect frequency observed at around 45% VD (Fig. 5A) and 65% EL (Fig. 5C) is probably due to this region containing the anlagen for the stomodaeum and the intercalary segment that do not form integumental cuticle structures (Kuwana and Takami, 1957). Correlation between site of irradiation and position of defect The position of the defects within the larval cuticle closely correlated to the site of irradiation, both in the dorsoventral (Fig. 6) and in the anteroposterior direction (Fig. 7). As shown in Fig. 6, the highest frequency for the ventral, lateral and dorsal defects was respectively obtained by the irradiations at 15-35% VD, 35-50% VD, and 50-65% VD. Fig. 7 shows the defectinducing irradiation sites for each segment or structure. Although there is considerable overlap of defect-inducing areas for adjacent segments, it is evident that they are generally arranged anteroposteriorly in the order of the larval segments; head defects were induced only by irradiations at the anterior part of the egg while defects in thoracic and abdominal segments were induced by irradiations at the middle and the posterior part of the egg, respectively. Fate mapping The presumptive region for a particular larval segment or structure has been attributed to the area of the egg surface where irradiation causes cuticle defects in that segment with high frequency. Fig. 7 provides a rough indication of the location and extent of each presumptive segment; however, the data must be interpreted with consideration of complications caused by (1) about 20% of the resultant defects spread over four segments or more and (2) the distribution of irradiated sites was unequal (see Fig. 4A). Thus for fate mapping, a target center (a weighted mean value of the positions of defectinducing irradiation sites calculated using the defect frequency, see Materials and methods for the formula) was constructed for each segment or structure and used as a parameter representing the assumed center point of the presumptive region. Fig. 4. Sites and consequences of irradiation. Positions of the center point of each irradiation area are indicated. The size of irradiation area, µm (15% of the total egg width 15% of the total egg length), is shown in the center. All the lateral irradiation sites are shown as if they were carried out on the left side of the egg, although in fact half were on the right side. In ventral (open circles) and dorsal irradiations (crosses), eggs were oriented with the ventral or the dorsal side facing to the laser beam. (A) Sites where irradiation for fate mapping was performed and cuticles developed. (B) Sites where irradiation resulted in defective cuticles.

6 2874 M. Myohara Fig. 5. Frequency of defective cuticles following irradiation at various sites along the dorsoventral (A,B) and the anteroposterior axis (C,D). Extent of regions in which center points of irradiations fell (A, Yc=57-78% EL; B, Yc=16-56% EL; C, Xc=10-68% VD; D, Xc=69-88% VD; hatched area) and the size of irradiation area (rectangle) are diagramed at the right of each figure. The bars and the numbers at each spot indicate standard deviation of irradiation positions and number of analyzed larvae per site, respectively. Since the data shown in Fig. 7 suggested that the anteroposterior position of a presumptive region might be different along the dorsoventral axis, the data were divided into four dorsoventral sections (a=10-30% VD, b=30-50% VD, c=50-70% VD, and d=70-90% VD), and for each section the defect frequency and the target center were calculated for each segment and structure (Fig. 8). As clearly shown in Fig. 8, for the segments posterior to the maxilla, irradiations in the dorsalmost section (section d) rarely induce cuticle defects. It is also evident that the target centers for these segments locate slightly more anteriorly in the ventralmost section (section a) than in the middle sections (sections b and c); the difference in position of the target centers between sections a and b was Fig. 6. Dorsoventral correlation between the position of irradiation and the location of defects. Eggs were irradiated at various sites along the dorsoventral axis, and the defect frequency of ventral, lateral and dorsal cuticle was determined using the following structures as the markers; appendages for ventral cuticle (A), supraspiracle and subspiracle tubercles for lateral cuticle (B), and subdorsal tubercles for dorsal cuticle (C). Extent of region in which center points of irradiations fell (Yc=31-53% EL; hatched area) and the size of irradiation area (rectangle) are diagramed at the left of the figure. The transverse bars indicate the standard deviation of irradiation positions. The numbers at the top of the figure indicate number of analyzed larvae per site. For the terminology for the marker structures, see Fig % EL on average. In contrast, the difference was less than 1% EL on average between sections b and c, thus for these segments target centers were recalculated from the combined data of the two sections. Using these data, a fate map was constructed on the assumption that the anteroposterior position of the target center for these segments is constant in sections b and c, but is 3.5% EL more anterior in section a (Fig. 9). For the head parts anterior to the mandible, where structures do not appear as repeating similar units as in thorax and abdomen but rather as a three-dimensional complex of individual elements, fate mapping was achieved simply by plotting the target centers in each section (Fig. 9). For the segments posterior to the maxilla, the target centers were significantly different between two adjacent segments, except between maxilla and labium, abdominal segment 6 and 7, 7 and 8, and 9 and 10, if one regards two centers significantly different only when the difference is larger than the sum of the standard errors. Standard errors were % EL (0.9% EL on average) for the gnathal, thoracic and abdominal segments.

7 Fate mapping of silkworm eggs 2875 Because the presumptive region for the entire larval epidermis (the area enclosed with broken lines in Fig. 9, which was located by taking the point of 50% defect frequency as the rim of the presumptive epidermal region) includes the presumptive regions for all the larval segments, from the labrum to the tenth abdominal segment, and for all the cuticle structures from dorsal to ventral, the positions outside of this area presumably give rise in >50% of cases to visceral and extraembryonic tissues. From histological studies of Bombyx embryonic development (for review see Sakaguchi, 1978, or Miya, 1985) and the local cautery experiments (Takami, 1946; Miya, 1953; Kuwana and Takami, 1957), it is inferred that the ventral region of the extraepidermal area is the presumptive mesoderm and that the rest of this area gives rise to the extraembryonic envelopes, amnion and serosa. treatment yielded very consistent data for the localization of defects with respect to irradiation site (Fig. 7). Thus I conclude that the data is usable for constructing a fate map. In Drosophila, UV-laser irradiation of preblastoderm eggs has been reported to induce only a few defects in the larval cuticle (Lohs-Schardin et al., 1979b), while in this study localized cuticle defects were induced in Bombyx by irradiating eggs at the fertilization stage. The difference may be due to differential responses of the two insects to UV irradiation rather than the differences in the methods employed, because Drosophila eggs irradiated at the early cleavage stage rarely yielded cuticle defects even when irradiated under the same condition as for Bombyx (Myohara, unpublished data). DISCUSSION Comparisons to the Drosophila fate mapping A fate map for the Drosophila larval integument has been established by UV-laser irradiation of the eggs at the cellular blastoderm stage (Lohs-Schardin et al., 1979b). The map agrees well with that obtained by tracer injection method (Hartenstein et al., 1985); for instance, both maps locate the anterior edge of the presumptive thorax at 60-61% EL, the border between the presumptive thorax and abdomen at 48-49% EL, and the posterior border of the presumptive eighth abdominal segment at 20-21% EL. This good agreement indicates accuracy and usefulness of the UV-laser irradiation method for fate mapping. In the UV-laser fate mapping of Drosophila (Lohs-Schardin et al., 1979b), the irradiation spot was 20 µm diameter (approximately 12% of the total egg width 4% of the total egg length), and the average number of defective segments per larva from irradiated egg was 0.9 for the thorax and 0.7 for the abdomen. In comparison to this, the size of irradiation area employed here for Bombyx fate mapping (15% of the total egg width 15% of the total egg length) might be considered too large for fate mapping. However, the average number of defective segments per larva from irradiated egg, 1.25±1.61 (s.d.), was small enough to allow one to attempt to construct a fate map with the data. Also, defect frequency of resultant larvae from eggs irradiated in the presumptive epidermal area (72%) was sufficiently high for fate mapping, but was lower than that obtained in the Drosophila study (83%) in spite of the fact that the size of irradiation area was larger in the Bombyx study than in the Drosophila study. More importantly, this Fig. 7. Correlation between position of irradiation and location of defects. Eggs were irradiated at various sites as shown in Fig. 4 and defect-inducing irradiation sites were put together for each larval segment or structure. Each dot indicates center point of irradiation area.

8 2876 M. Myohara Fig. 8. Defect frequencies and target centers for each segment or structure. The data shown in Fig. 7 were divided into four dorsoventral sections (a=10-30% VD, b=30-50% VD, c=50-70% VD, d=70-90% VD) and, for each section, defect frequencies and target centers (arrow heads) were calculated for each segment or structure as described under Materials and methods. For the abbreviations for position of defects, see the legend of Fig. 2. There are two important differences between the Drosophila fate map (Lohs-Schardin et al., 1979b) and the Bombyx fate map established here; (1) presumptive regions for head structures and gnathal segments are mapped in Bombyx but not in Drosophila, (2) in Bombyx the presumptive regions for the abdominal segments (especially for the posterior several segments) are more narrowly spaced than those for the thoracic segments while they are equally spaced in Drosophila. However, in most features, the Bombyx egg belongs in the same category as the Drosophila egg, the long germ type (Sander, 1976): it has meroistic oogenesis; the presumptive germ anlage occupies most of the length of the egg and the duration of embryogenesis is relatively short. The present study also suggests that the Bombyx egg is a long germ type, as the ability to fate map all the larval segments at the fertilization stage suggests that all the abdominal segments are determined during the same time period as the rest of the germ band. Fig. 9. A fate map of the Bombyx mori egg at the fertilization stage. Lateral view of an egg with the anterior on the top and the ventral on the left. The broken lines outline the extent of presumptive region for the entire larval integument as defined by cuticular defect frequencies above 50%. The solid lines indicate center position of presumptive regions for each larval segment or structure. Presumptive mesodermal region (MS) is located as in the fate map by Kuwana and Takami (1957). A, antenna; Al-10, abdominal segment 1-10; EX, extraembryonic region; L, labrum; LA, labium; M, mandible; MS, mesodermal region; MX, maxilla; O, ocellus; P, parietal plate; Tl-3, thoracic segment 1-3. Comparisons to the previous Bombyx map by local cautery In addition to the increased resolution, the new Bombyx fate map established here by UV-laser irradiation is different from the previous map established by local cautery (Kuwana and Takami, 1957); in the laser map, anteroposterior extents of head and abdominal regions were each about 40% of the entire presumptive integument, whereas in the local cautery map, the head occupied only 15% while the abdomen occupied 70% of the entire presumptive integument. It is hard to determine whether the difference is due to the difference in the methods or difference in the egg stage at treatment, since the stage used for local cautery was referred to by the authors just as the blastoderm stage without any detailed description. However, the most probable explanation may be that the local cautery was

9 Fate mapping of silkworm eggs 2877 carried out soon after the completion of the cellular blastoderm, when the germ-band anlage had already begun to contract in the anterior region of the egg. If this is the case, the UV-laser fate map established here may possibly be the same as a presumptive fate map of the blastoderm stage before the beginning of the germ-band contraction. In fact, it has been reported in a moth, Tineola biselliella, that fate maps established by large-area UV irradiation of the egg at various stages begun to get distorted essentially only after the cellular blastoderm stage (Lüscher, 1944). Primary targets of the UV radiation Because defective larvae were induced by UV irradiation of eggs at the fertilization stage, it is obvious that the primary targets of the UV laser were not nuclei. In fact, no photorecovery was observed in the defect frequency of irradiated eggs incubated under visible light (data not shown). Moreover, the wavelength used (355 nm) is much more likely to affect proteins than nucleic acids. It has been reported that partial embryos can also be induced by local cautery of Bombyx eggs at the fertilization stage (Takami, 1942, 1943). However, these results do not necessarily imply that the Bombyx egg cortex consists of a segmental prepattern already fixed in position at egg laying. A preliminary histological analysis of embryos irradiated at the fertilization stage showed that penetration of nuclei into the egg periplasm and the subsequent cellularization during the blastoderm formation were strictly inhibited at the irradiated region (data not shown), suggesting that the cytoplasmic instructions to form a structure could arrive after the time of irradiation in the respective region where no nucleus or cell responsive to these instructions existed as yet. Thus the most probable explanation for the larval defect induced in the present study is absence of cells or nuclei in the irradiated region at the time when segment specification occurs, which could be much later than the time of irradiation. A more detailed study of the morphological events between irradiation and cuticle formation is required to understand the mechanism underlying the defect induction in larval cuticle by UV irradiation of the egg at the fertilization stage. Moreover, to elucidate how and when determination of segment-specific fate occurs in the Bombyx egg, further research including transplantation experiments of cytoplasm or cells must be carried out. I am particularly grateful to Dr Fukumi Sakai of National Institute of Agrobiological Resources, Tsukuba, Japan, for allowing me to use the ACAS 470 work station in his laboratory. I wish to thank Dr Kenji Kiguchi and other members of my laboratory for technical support. Thanks are also due to Drs Lisa Nagy, Judy Willis and Masukichi Okada for helpful comments on an earlier version of the manuscript. I am also grateful to Dr Klaus Sander for critical reading of the manuscript and valuable comments. This work was supported in part by a Grant-in-Aid (Bio Media Program) from the Ministry of Agriculture, Forestry and Fisheries (BMP 93-I-2-5). REFERENCES Hartenstein, V., Technau, G. M. and Campos-Ortega, J. A. (1985). Fatemapping in wild-type Drosophila melanogaster. III. A fate map of the blastoderm. Roux s Arch. Dev. Biol. 194, Katsuki, M., Murakami, A. and Watanabe, I. (1980). Fate mapping of some tissues in the genetic mosaics of the silkworm, Bombyx mori. Zool. Mag. 89, (Japanese with an English summary). Kawaguchi, E. and Takizawa, Y. (1942). Genetics of egg color. II. Heredity of the number and size of serosa cells. J. Sericult. Sci. Japan 13, (Japanese). Kawamura, N. (1978). 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