DEVELOPMENTAL
BIOLOGY
46, 216-221 (1975)
Switch in Pattern Formation after Puncturing the Anterior Pole of Smittia
Eggs (Chironomidae,
OTTOSCHMIDT, DIETER ZISSLER, KLAUS
Diptera)’
SANDER, AND KLAUS KALTHOFF~
Biologisches Znstitut Z (Zoologie) der Universitiit, Katharinenstrasse
20, D 7800 Freiburg,
Federal Republic of
Germany Accepted
April 15, 1975
The aberrant pattern, “double abdomen,” previously induced in the egg of Smittia by uv irradiation of anterior pole regions was also produced by puncturing of the egg at the anterior pole. Double abdomens and embryos with anterior defects developed in eggs in which puncturing had locally prevented the regular arrangement of cleavage nuclei in the periplasm. The resulting gap in the blastoderm at the anterior pole was subsequently closed under exclusion of a small amount of egg material. Double abdomens did not develop in eggs where exclusion of anterior egg material was not observed. Thus a basic switch in the developmental program of the egg appears to depend upon the functional elimination of some crucial components in the anterior egg region. INTRODUCTION
METHODS
Spatial pattern formation during embryogenesis is a basic problem in developmental biology. In chironomid midges, the body segment pattern to be formed by the egg can be drastically altered by uv irradiation of the anterior pole region (8, 15). Instead of a normal embryo, the aberrant pattern, “double abdomen,” will then be formed. In this pattern, the head, thorax, and anterior abdominal segments are replaced by an additional set of posterior abdominal segments joined in mirrorimage symmetry to the corresponding parts of the original abdomen (see Fig. 5). This experimental system has been exploited to characterize the position, origin, and presumed nature of those egg components that on irradiation cause the formation of double abdomens (6, 7). Double abdomen induction by other methods should provide further cues to both nature and mode of action of these egg components. Such a method is described in this communication.
RESULTS
The developmental results after puncturing were classified as follows: Germ bands without defects visible upon external inspection under the stereo microscope were scored as normal. Embryos with dwarfed, deformed, or absent head lobes or mouth parts were subsumed under embryos with anterior defects. Germ bands with anal papillae (see Fig. 5) and a proctodaeum on each end were listed as double abdomens; their morphology was always symmetrical to the equatorial plane. All eggs that did not form identifia216
1975 by
Academic Press. Inc.
reproduction
in any form
reserved.
MATERIALS
Eggs obtained and prepared as described previously (5) were punctured during early stages of intravitelline cleavage, when the oolemma at the pole regions is separated from the egg shell by a cleft filled with perivitelline fluid (8). Puncturing was accomplished under water with glass needles mounted in a micromanipulator (12). Subsequent incubation was at 20°C in small dishes with water. For fixation, the egg shell was disrupted in the posterior region. Fixation and embedding techniques have been described previously (16).
1Supported by the Deutsche Forschungsgemeinschaft, SFB 46. *Author to whom correspondence and reprint requests should be addressed. Copyright 0 All rights of
AND
BRIJSF NOTES
ble germ band parts were pooled as undifferentiated; most of these had ceased development before blastoderm formation. Double abdomens were observed only if, after puncturing the anterior pole, a small dark blob of egg material had been excluded from the embryo. If material was extruded from the egg cell immediately after puncturing, the egg died soon. Eggs that later produced double abdomens invariably displayed a gap at the anterior pole in the superficial layer of cleavage energids (nuclei plus cytoplasm) which colonize the periplasm after intravitelline cleavage, The egg material located in this gap did not become incorporated into blastoderm cells and remained separated from the inner egg cytoplasm by a membrane that apparently derived from the oolemma (Fig. 1). The excluded material, referred to as extraovate, amounted to roughly 1% of the egg volume; it contained mainly lipid and proteid yolk, membrane bound vesicles, and swollen mitochondria (Figs. l-3). The extraovate became incorporated in or excluded from the embryo when the blastoderm gap was closed a few hours before germ anlage formation. In most cases, the bulk of the extraovate was pinched off from the embryo by blastoderm cells moving into the gap. Complementary to the resulting outer extraovate, a usually smaller inner extraovate remained underneath the blastoderm cells which closed the gap (Figs. 2 and 3). Double abdomens were observed only after formation of major outer extraovates (Figs. 4 and 5). The contents of outer and inner extraovates were very similar. In particular, swollen mitochondria were found in both fractions. Mainly during later blastoderm stages, abnormal mitochondria also occured in the endoplasm adjacent to the extraovate. A few abnormal mitochondria could be observed in the blastoderm cells closing the gap, too. However, large amounts of mitochondria in the anterior egg region retained the normal structures
217
(Figs. 1 and 2). The proportions and ultrastructural characteristics of extraovates and neighboring egg areas, shown in Figs. l-3, are representative of 12 punctured eggs investigated with the EM. No indications for extraovates were found in four unpunctured control eggs. In a series of experiments, 257 eggs were punctured at the anterior pole. Of these, 132 eggs remained undifferentiated while identifiable embryos developed in 125 eggs as listed in Table 1. Extraovate formation was observed in 44 eggs. In this group, 18 embryos were of the double abdomen type while most of the others carried anterior defects. No correlation was observed between size of the extraovate and type of resulting abnormality. In those eggs where no extraovate had been observed during blastoderm stages, no double abdomens and only a few anterior defects developed, while the majority of this group developed into normal embryos. As control experiments, 159 eggs were punctured at the posterior pole. Subsequent extraovate formation then interfered with pole cell and/or blastoderm development in various ways. The resulting defects did not include major pattern aberrations (12). In particular, “double heads,” as found by Yajima (15) after uv irradiation of posterior pole regions of Chironomus dorsalis eggs, were not observed after puncturing the posterior pole of Smittia eggs; double heads also failed to occur in Smittia after uv irradiation of the posterior egg pole (6) but can be induced by centrifugation (Kalthoff, unpublished results). The development of double abdomens after puncturing the anterior pole of Smittia eggs was considerably delayed as compared to the developmental rate of normal germ bands or embryos with anterior defects. Also, the egg shells readily burst after germ anlage formation, especially when the eggs were handled. Figures 4 and 5 represent double abdomens at the germ anlage and advanced germ band
218
DEVELOPMENTAL BIOLOGY
FIGS. 1 and 2
VOLUME46, 1975
219
BRIEF NOTES
5011 -5 FIGS. 1-3.
Electron micrographs of paramedian longitudinal sections through anterior pole region of Smittia at the anterior pole. Fixation after Franke et al. (3). B, Outpocketings of blastoderm cells; C, inner egg cytoplasm; L, lipid; M, abnormal mitochondrion; nM, normal mitochondrion; N, nucleus of blastoderm cell; 0, oolemma-derived membrane; V, membrane-bound vesicle; Y, proteid yolk. Fig. 1, Late preblastoderm stage; most blastoderm cells still connected to inner egg cytoplasm by cytoplasmic bridges; extraovate in blastoderm gap; x 3,900. Fig. 2, Blastoderm stage; extraovate becomes divided into “inner” and “outer” extraovate by cells closing the blastoderm gap; x 5,300. Fig. 3, Inner extraovate surrounded by blastoderm cells and inner egg cytoplasm, x 7,650. FIGS. 4 and 5. Light micrographs of double abdomens at germ anlage and germ band stage, respectively. Dorsal side up, anterior pole to the left, x 220. A, abdominal end; E, extraovate; P, anal papilla. eggs punctured
stages, respectively. Photographs and visual inspection of eggs after puncturing did not reveal any deviations from the course of development of uv-induced double abdomens as described previously (8).
DISCUSSION
Aberrant patterns of the double abdomen type occur spontaneously and have been generated by different ways of ex-
220
DEVELOPMENTAL BIOLOGY TABLE
1
DEVELOPMENTAL RESULTSIN SURVIVINGSmittia EGGS AVER PUNCTURING THE ANTERIORPOLE Pattern
Normal germ band Anterior defect Double abdomen Total
Extraovate at blastoderm stage Visible
Not visible
9 17 18 44
73 8 0 81
perimental interference in various orders of insects (2, 8, 10, 11, 13-15). However, double abdomens in Smith seem to be the first instance of longitudinal mirrorimage duplication produced by puncturing the egg. In other insects, this procedure has resulted in more or less defective embryos depending upon the components removed and on the developmental stage of the eggs at puncturing (1, 4, 13, 14). The predisposition of chironomid eggs for longitudinal pattern mirroring (8, 14, 15) is again demonstrated by the results reported here. The extraovate observed after puncturing the anterior pole comes from the egg region that reacted most effectively in the induction of double abdomens by uv irradiation; irradiation of neighboring areas also caused double abdomen formation but with less efficiency (6). It was also found that double abdomens can be uv induced most effectively by orienting the long axis of the egg parallel to the uv beam, with the anterior pole facing the uv source (Kalthoff, unpublished results). These results suggest that double abdomens result from uv inactivation or physical exclusion of the same egg material. However, it cannot be excluded so far that double abdomens after puncturing might result not from extraovate exclusion itself but from other changes in the egg, e.g., osmotic effects or disturbed communication between blastoderm cells, causing both extraovate and double abdomen formation. Actually, puncturing affects the egg structure beyond
VOLUME 46,197s
the limits of the extraovate as indicated by the occurrence of abnormal mitochondria in the blastoderm cells and the yolk endoplasm next to the extraovate. On a formal level of interpretation, two conclusions may be drawn: First, our results are incompatible with models of pattern formation based upon a detailed mosaic of determinants prelocalized in the ooplasm. If such models would apply to our system, puncturing as well as uv irradiation should cause only anterior defects with an extent corresponding to the amount of material removed or damaged (see also Ref. 6). Second, it seems important to note that in dipteran eggs double abdomens can be generated by several unrelated types of interference such as uv irradiation, puncturing, centrifugation (14), and mutagenesis (2). It seems hard to conceive that all these procedures could de novo generate or mimic putative posterior factors determining the formation of an abdominal end. Much more likely, the different methods have in common the removal or inactivation of crucial anterior egg components. This view is also corroborated by the photoreversibility of uv induction of double abdomens, since photoreversal is generally ascribed to the lightdependent repair mechanism (see Refs. 5, 7). The formation of double abdomens instead of normal embryos in dipteran eggs thus appears as the effect of a basic switch from one developmental pathway to another, triggered by the functional elimination of some crucial components at the anterior pole region. A formally identical bistable system is obviously underlying the alternative realization of one sex or the other in bisexual organisms. Bistable control circuits may also be involved in determination and transdetermination of imaginal discs (9). We thank Mrs. Heidrun Zimmermarm experienced technical assistance.
for her
BRIEF NOTES REFERENCES 1. BOWNES,M., and SANG,J. H. (1974). J. Embryol. Exp. Morphol. 32, 273-285. 2. BULL, A. L. (1966). J. Exp. 2001. 161, 221-242. 3. FRANKE,W. W., KRIEN, S., and BROWN,R. M., JR. (1969). Histochemie 19, 162-164. 4. ILLMENSEE,K. (1972). Wilhelm Roux Arch. Entwickhingsmech. Organismen 170.267-298. 5. KALTHOFF,K. (1971). Develop. Biol. 25, 119-132. 6. KALTHOFF, K. (1971). Wilhelm Roux Arch. Entwicklringsmech. Organismen 168,63-84. 7. KALTHOFF,K. (1973). Photochem. Photobiol. 18, 355-364. 8. KALTHOFF, K., and SANDER,K. (1968). Wilhelm Roux Arch. Entwickshingsmech. Organismen 161, 129-146.
221
9. KAUFFMAN,S. (1972). Science 181, 310-318. 10. PRICE, R. D. (1958).Ann. Entomol. Soc.Amer. 51, 600-604. 11. SANDER,K. (1960). Wilhelm Roux Arch. Entwicklu’ngsmech. Organismen 151,660-707. (1974). Staatsexamensarbeit, 12. SCHMIDT, 0. Fakultiit fur Biologie der Universitiit Freiburg. 13. SCHNETTER,W. (1965). Wilhelm Roux Arch. Entwicktingsmech. Organismen 155,637-692. 14. YAJIMA, H. (1960). J. Embryol. Exp. Morphol. 8, 198-215. 15. YAJIMA,H. (1964). J. Embryol. Exp. Morphol. 12, 89-100. 16. ZISSLER, D., and SANDER, K. (1973). Wilhelm Roux Arch. Entwickltingsmech. Organismen 172, 175-186.
BIOLOGY
46, 216-221 (1975)
Switch in Pattern Formation after Puncturing the Anterior Pole of Smittia
Eggs (Chironomidae,
OTTOSCHMIDT, DIETER ZISSLER, KLAUS
Diptera)’
SANDER, AND KLAUS KALTHOFF~
Biologisches Znstitut Z (Zoologie) der Universitiit, Katharinenstrasse
20, D 7800 Freiburg,
Federal Republic of
Germany Accepted
April 15, 1975
The aberrant pattern, “double abdomen,” previously induced in the egg of Smittia by uv irradiation of anterior pole regions was also produced by puncturing of the egg at the anterior pole. Double abdomens and embryos with anterior defects developed in eggs in which puncturing had locally prevented the regular arrangement of cleavage nuclei in the periplasm. The resulting gap in the blastoderm at the anterior pole was subsequently closed under exclusion of a small amount of egg material. Double abdomens did not develop in eggs where exclusion of anterior egg material was not observed. Thus a basic switch in the developmental program of the egg appears to depend upon the functional elimination of some crucial components in the anterior egg region. INTRODUCTION
METHODS
Spatial pattern formation during embryogenesis is a basic problem in developmental biology. In chironomid midges, the body segment pattern to be formed by the egg can be drastically altered by uv irradiation of the anterior pole region (8, 15). Instead of a normal embryo, the aberrant pattern, “double abdomen,” will then be formed. In this pattern, the head, thorax, and anterior abdominal segments are replaced by an additional set of posterior abdominal segments joined in mirrorimage symmetry to the corresponding parts of the original abdomen (see Fig. 5). This experimental system has been exploited to characterize the position, origin, and presumed nature of those egg components that on irradiation cause the formation of double abdomens (6, 7). Double abdomen induction by other methods should provide further cues to both nature and mode of action of these egg components. Such a method is described in this communication.
RESULTS
The developmental results after puncturing were classified as follows: Germ bands without defects visible upon external inspection under the stereo microscope were scored as normal. Embryos with dwarfed, deformed, or absent head lobes or mouth parts were subsumed under embryos with anterior defects. Germ bands with anal papillae (see Fig. 5) and a proctodaeum on each end were listed as double abdomens; their morphology was always symmetrical to the equatorial plane. All eggs that did not form identifia216
1975 by
Academic Press. Inc.
reproduction
in any form
reserved.
MATERIALS
Eggs obtained and prepared as described previously (5) were punctured during early stages of intravitelline cleavage, when the oolemma at the pole regions is separated from the egg shell by a cleft filled with perivitelline fluid (8). Puncturing was accomplished under water with glass needles mounted in a micromanipulator (12). Subsequent incubation was at 20°C in small dishes with water. For fixation, the egg shell was disrupted in the posterior region. Fixation and embedding techniques have been described previously (16).
1Supported by the Deutsche Forschungsgemeinschaft, SFB 46. *Author to whom correspondence and reprint requests should be addressed. Copyright 0 All rights of
AND
BRIJSF NOTES
ble germ band parts were pooled as undifferentiated; most of these had ceased development before blastoderm formation. Double abdomens were observed only if, after puncturing the anterior pole, a small dark blob of egg material had been excluded from the embryo. If material was extruded from the egg cell immediately after puncturing, the egg died soon. Eggs that later produced double abdomens invariably displayed a gap at the anterior pole in the superficial layer of cleavage energids (nuclei plus cytoplasm) which colonize the periplasm after intravitelline cleavage, The egg material located in this gap did not become incorporated into blastoderm cells and remained separated from the inner egg cytoplasm by a membrane that apparently derived from the oolemma (Fig. 1). The excluded material, referred to as extraovate, amounted to roughly 1% of the egg volume; it contained mainly lipid and proteid yolk, membrane bound vesicles, and swollen mitochondria (Figs. l-3). The extraovate became incorporated in or excluded from the embryo when the blastoderm gap was closed a few hours before germ anlage formation. In most cases, the bulk of the extraovate was pinched off from the embryo by blastoderm cells moving into the gap. Complementary to the resulting outer extraovate, a usually smaller inner extraovate remained underneath the blastoderm cells which closed the gap (Figs. 2 and 3). Double abdomens were observed only after formation of major outer extraovates (Figs. 4 and 5). The contents of outer and inner extraovates were very similar. In particular, swollen mitochondria were found in both fractions. Mainly during later blastoderm stages, abnormal mitochondria also occured in the endoplasm adjacent to the extraovate. A few abnormal mitochondria could be observed in the blastoderm cells closing the gap, too. However, large amounts of mitochondria in the anterior egg region retained the normal structures
217
(Figs. 1 and 2). The proportions and ultrastructural characteristics of extraovates and neighboring egg areas, shown in Figs. l-3, are representative of 12 punctured eggs investigated with the EM. No indications for extraovates were found in four unpunctured control eggs. In a series of experiments, 257 eggs were punctured at the anterior pole. Of these, 132 eggs remained undifferentiated while identifiable embryos developed in 125 eggs as listed in Table 1. Extraovate formation was observed in 44 eggs. In this group, 18 embryos were of the double abdomen type while most of the others carried anterior defects. No correlation was observed between size of the extraovate and type of resulting abnormality. In those eggs where no extraovate had been observed during blastoderm stages, no double abdomens and only a few anterior defects developed, while the majority of this group developed into normal embryos. As control experiments, 159 eggs were punctured at the posterior pole. Subsequent extraovate formation then interfered with pole cell and/or blastoderm development in various ways. The resulting defects did not include major pattern aberrations (12). In particular, “double heads,” as found by Yajima (15) after uv irradiation of posterior pole regions of Chironomus dorsalis eggs, were not observed after puncturing the posterior pole of Smittia eggs; double heads also failed to occur in Smittia after uv irradiation of the posterior egg pole (6) but can be induced by centrifugation (Kalthoff, unpublished results). The development of double abdomens after puncturing the anterior pole of Smittia eggs was considerably delayed as compared to the developmental rate of normal germ bands or embryos with anterior defects. Also, the egg shells readily burst after germ anlage formation, especially when the eggs were handled. Figures 4 and 5 represent double abdomens at the germ anlage and advanced germ band
218
DEVELOPMENTAL BIOLOGY
FIGS. 1 and 2
VOLUME46, 1975
219
BRIEF NOTES
5011 -5 FIGS. 1-3.
Electron micrographs of paramedian longitudinal sections through anterior pole region of Smittia at the anterior pole. Fixation after Franke et al. (3). B, Outpocketings of blastoderm cells; C, inner egg cytoplasm; L, lipid; M, abnormal mitochondrion; nM, normal mitochondrion; N, nucleus of blastoderm cell; 0, oolemma-derived membrane; V, membrane-bound vesicle; Y, proteid yolk. Fig. 1, Late preblastoderm stage; most blastoderm cells still connected to inner egg cytoplasm by cytoplasmic bridges; extraovate in blastoderm gap; x 3,900. Fig. 2, Blastoderm stage; extraovate becomes divided into “inner” and “outer” extraovate by cells closing the blastoderm gap; x 5,300. Fig. 3, Inner extraovate surrounded by blastoderm cells and inner egg cytoplasm, x 7,650. FIGS. 4 and 5. Light micrographs of double abdomens at germ anlage and germ band stage, respectively. Dorsal side up, anterior pole to the left, x 220. A, abdominal end; E, extraovate; P, anal papilla. eggs punctured
stages, respectively. Photographs and visual inspection of eggs after puncturing did not reveal any deviations from the course of development of uv-induced double abdomens as described previously (8).
DISCUSSION
Aberrant patterns of the double abdomen type occur spontaneously and have been generated by different ways of ex-
220
DEVELOPMENTAL BIOLOGY TABLE
1
DEVELOPMENTAL RESULTSIN SURVIVINGSmittia EGGS AVER PUNCTURING THE ANTERIORPOLE Pattern
Normal germ band Anterior defect Double abdomen Total
Extraovate at blastoderm stage Visible
Not visible
9 17 18 44
73 8 0 81
perimental interference in various orders of insects (2, 8, 10, 11, 13-15). However, double abdomens in Smith seem to be the first instance of longitudinal mirrorimage duplication produced by puncturing the egg. In other insects, this procedure has resulted in more or less defective embryos depending upon the components removed and on the developmental stage of the eggs at puncturing (1, 4, 13, 14). The predisposition of chironomid eggs for longitudinal pattern mirroring (8, 14, 15) is again demonstrated by the results reported here. The extraovate observed after puncturing the anterior pole comes from the egg region that reacted most effectively in the induction of double abdomens by uv irradiation; irradiation of neighboring areas also caused double abdomen formation but with less efficiency (6). It was also found that double abdomens can be uv induced most effectively by orienting the long axis of the egg parallel to the uv beam, with the anterior pole facing the uv source (Kalthoff, unpublished results). These results suggest that double abdomens result from uv inactivation or physical exclusion of the same egg material. However, it cannot be excluded so far that double abdomens after puncturing might result not from extraovate exclusion itself but from other changes in the egg, e.g., osmotic effects or disturbed communication between blastoderm cells, causing both extraovate and double abdomen formation. Actually, puncturing affects the egg structure beyond
VOLUME 46,197s
the limits of the extraovate as indicated by the occurrence of abnormal mitochondria in the blastoderm cells and the yolk endoplasm next to the extraovate. On a formal level of interpretation, two conclusions may be drawn: First, our results are incompatible with models of pattern formation based upon a detailed mosaic of determinants prelocalized in the ooplasm. If such models would apply to our system, puncturing as well as uv irradiation should cause only anterior defects with an extent corresponding to the amount of material removed or damaged (see also Ref. 6). Second, it seems important to note that in dipteran eggs double abdomens can be generated by several unrelated types of interference such as uv irradiation, puncturing, centrifugation (14), and mutagenesis (2). It seems hard to conceive that all these procedures could de novo generate or mimic putative posterior factors determining the formation of an abdominal end. Much more likely, the different methods have in common the removal or inactivation of crucial anterior egg components. This view is also corroborated by the photoreversibility of uv induction of double abdomens, since photoreversal is generally ascribed to the lightdependent repair mechanism (see Refs. 5, 7). The formation of double abdomens instead of normal embryos in dipteran eggs thus appears as the effect of a basic switch from one developmental pathway to another, triggered by the functional elimination of some crucial components at the anterior pole region. A formally identical bistable system is obviously underlying the alternative realization of one sex or the other in bisexual organisms. Bistable control circuits may also be involved in determination and transdetermination of imaginal discs (9). We thank Mrs. Heidrun Zimmermarm experienced technical assistance.
for her
BRIEF NOTES REFERENCES 1. BOWNES,M., and SANG,J. H. (1974). J. Embryol. Exp. Morphol. 32, 273-285. 2. BULL, A. L. (1966). J. Exp. 2001. 161, 221-242. 3. FRANKE,W. W., KRIEN, S., and BROWN,R. M., JR. (1969). Histochemie 19, 162-164. 4. ILLMENSEE,K. (1972). Wilhelm Roux Arch. Entwickhingsmech. Organismen 170.267-298. 5. KALTHOFF,K. (1971). Develop. Biol. 25, 119-132. 6. KALTHOFF, K. (1971). Wilhelm Roux Arch. Entwicklringsmech. Organismen 168,63-84. 7. KALTHOFF,K. (1973). Photochem. Photobiol. 18, 355-364. 8. KALTHOFF, K., and SANDER,K. (1968). Wilhelm Roux Arch. Entwickshingsmech. Organismen 161, 129-146.
221
9. KAUFFMAN,S. (1972). Science 181, 310-318. 10. PRICE, R. D. (1958).Ann. Entomol. Soc.Amer. 51, 600-604. 11. SANDER,K. (1960). Wilhelm Roux Arch. Entwicklu’ngsmech. Organismen 151,660-707. (1974). Staatsexamensarbeit, 12. SCHMIDT, 0. Fakultiit fur Biologie der Universitiit Freiburg. 13. SCHNETTER,W. (1965). Wilhelm Roux Arch. Entwicktingsmech. Organismen 155,637-692. 14. YAJIMA, H. (1960). J. Embryol. Exp. Morphol. 8, 198-215. 15. YAJIMA,H. (1964). J. Embryol. Exp. Morphol. 12, 89-100. 16. ZISSLER, D., and SANDER, K. (1973). Wilhelm Roux Arch. Entwickltingsmech. Organismen 172, 175-186.
