EXPERIMENTAL
CELL
194,316321
RESEARCH
(1991)
SHORT NOTE Microfilament Distribution in Cold-Treated Drosophila Embryos GIULIANO
CALLAINI,’
ROMANO DALLAI
Department of Evolutionary
001474827/91
should
Inc. reserved.
METHODS
Collection and staging of embryos.
Embryos of Drosophila melarw(Oregon-R strain) were collected at 24°C on agar plates, dechorionated in a 50% commercial bleach solution, and washed with distilled water. The age of the embryos was determined according to Campos-Ortega and Hartenstein [18] by direct observation with interference contrast or by counting the somatic nuclei. Cold treatment. After removal of excess liquid by blotting on tissue paper, embryos at appropriate stages were collected on a plastic film (Parafilm) and allowed to develop on melting ice for 1 h. Fluorescence microscopy. The dechorionated eggs were transferred to a solution containing 2.5 cc of 4% paraformaldehyde and 5 cc of heptane and gently agitated for 30 min. The embryos were then rinsed in PBS and their vitelline membrane was removed with fine needles. For double labeling the embryos were incubated for 5 h at room temperature with Rb188 antiserum, which specifically recognizes an antigen associated with the centrosome of Drosophila embryos [19] at a dilution of 1:400. The samples were then washed with PBS and incubated at room temperature in a goat anti-rabbit fluorescein-conjugated IgG at a dilution of 1:lOOO (Cappel, Westchester, PA). The embryos were then washed in PBS and stained for 30 min with 1.5 pglml phalloidin labeled with rhodamin (Molecular Probes, Inc.) for F-actin identification. The eggs were again rinsed in PBS and then exposed for 3-5 min to 1 aglml of the DNA-specific dye Hoechst 33258. Finally, the embryos were washed in PBS and mounted in 90% glycerol containing 2.5% n-propyl-gallate to reduce photobleaching [20]. The embryos were observed in a Leitz Aristoplan microscope equipped with Auorescein, rhodamin, and uv filters. Photomicrographs were taken with Kodak Tri-X pan film and developed in Kodak HC 110 for 7 min at 20°C.
RESULTS
When Drosophila embryos were exposed to low temperature and allowed to develop up to the syncytial blastoderm stage, the actin filaments were organized in cortical caps of irregular shape and size. Simultaneous Rh-phalloidin and Hoechst stains often showed that the number of F-actin caps was higher than expected from counting the somatic nuclei. Figure la is a detail of an embryo during interphase of the 10th mitotic cycle. It can be observed that there are several irregular cortical 316
Press, form
AND
gaster
be addressed.
$3.00
Copyright 0 1991 by Academic All rights of reproduction in any
4, 53100 Sienn, Italy
MATERIALS
The distribution of the actin filaments was recently investigated in the early Drosophila embryo at the syncytial blastoderm stage and during the process of cellularization. These studies revealed that once the somatic nuclei reach the cortex, the actin filaments are localized in cortical cytoplasmic domains above each nucleus [l41. These cytoplasmic regions also contain microtubules [2, 4-61, spectrin [7], myosin [8, 91 and actin-binding proteins [lo]; they undergo rapid morphological changes during mitosis [ 11,121. By the time mitosis begins, the cytoskeletal elements are rearranged in characteristic patterns of distribution; in particular the actin network expands as the nuclear cycle progresses and cleaves in two during later stages of mitosis. At the onset of cellularization the actin filaments form a hexagonal network associated with the leading edges of the extending plasmalemma [ 13-151. Certain features suggest that the change in actin localization is influenced by microtubules both during the syncytial blastoderm stage [2,6] and at the beginning of cellularization [ 12,16,17]. To better understand the relationship between microtubules and microfilaments we examined the distribution of microfilaments in Drosophila embryos exposed to cold during the early stages of development. Because cold treatment causes partial depolymerization of centrosome-associated microtubules, we applied immunofluorescence with Rh-phalloidin and requests
RIPARBELLI
anti-centrosome antibodies to cold-treated Drosophila embryos at different stages of development in order to investigate the possibility of a relation between the spatial organization of actin filaments and centrosomes.
INTRODUCTION
reprint
GIOVANNA
Biology, University of Siena, via Mattioli
Cold treatment of Drosophila embryos is observed to result in general alteration of microfilament distribution leading to deformation of the surface caps and to perturbation of the process of cleavage furrow extension. After exposure to low temperature the cortical actin caps underwent several morphological changes, despite the arrested nuclear cycle. These observations are discussed in relation to centrosome behavior during the cell cycle. 0 1991 Academic Preess, Inc.
i To whom
AND MARIA
SHORT
NOTE
FIG. 1. Fluorescence microscopy of whole cold-treated Drosophila embryos stained with rhodamine-labeled phalloidin for F-actin (a, c), Rb188 antibody to reveal the centrosomes (d) and counterstained with Hoechst dye to show the nuclei (b, e). (a, b) Cortical actin caps are single or double; arrows and arrowheads mark nuclei and the position of the nuclei inside the actin caps, open arrow indicates a cap without nucleus. (c-e) Centrosome replication occurs despite the arrested chromosome cycle. Cortical actin caps are in telophase-like configurations, while chromosomes are aligned in metaphase. A centrosome pair is visible under each cap. Open arrows mark the position of the chromosomes among the caps; arrows and arrowheads indicate the newly separated caps and the relative centrosomes, respectively. Bars, 50 pm (a, b); 10 pm (c, d, 4.
318
SHORT
actin caps. Most of them are round and irregularly scattered with a single nucleus. In some cases the caps appear double with a nucleus associated in the middle or shifted to an extremity. Sometimes small actin caps are without nuclei (Fig. lb). The experimental system employing cold treatment allows the centrosome cycle to move with respect to the chromosome cycle. Because at this time the number and disposition of chromosomes are the same as in normal metaphase plates, it seems that the centrosome cycle becomes uncoupled from the chromosome cycle. The centrosomes pass through anaphase- and telophase-like configurations, whereas the chromosomes remain in a metaphase-like configuration [21]. Hence, we observed chromosomes still in metaphase and the surface actin caps in a telophase-like configuration (Figs. lc and le). Below each actin cap we observed a centrosome pair (Fig. Id). In embryos recovered to 24’C for 30 min the chromosomes condensed in irregular figures and the centrosomes were no longer visible in pairs but moved away. Thus the organizing centers showed various degrees of splitting from each other and some of them lost contact with the spindle region and moved in the surrounding cytoplasm. The spindles often had only one main pole, the other being free in the cytoplasm [21]. Unlike in normal development in which the centrosomes are the focal point of the aster and spindle microtubules, in the cold-treated embryos the nucleation process associated with the centrosomes was only seen to a limited extent and very short aster microtubules were associated with the periphery of the centrosomal material. Double fluorescence with Rh-phalloidin and Rb188 serum, which specifically recognizes a centrosomal protein of Drosophila embryos [ 191, allowed us to compare the position of centrosomes and cortical actin caps. In recovered embryos the chromosomes were condensed in irregular figures and the cortical F-actin caps varied in shape and size. We often observed cap deformation and small caps devoid of nuclear material (Fig. 2a). Simultaneous observations with Rb188 serum and Rh-phalloidin revealed one or two centrosomes coinciding with each cortical actin cap (Fig. 2b). The dimensions and shape of the caps apparently depended on the number of the centrosomes and on the relative distance between them. Single or close pairs of centrosomes resulted in spheroidal actin caps, but as the centrosomes moved away the actin caps lengthened (Fig. 2b). The Rb188 antibody gives a feeble staining of the nuclear region [19], allowing visualization of the position of the centrosomes to the nuclei. Cold treatment is also known to affect the F-actin aggregates which have been observed as a uniform layer underneath the somatic nuclei in normal developing embryos [14]. In embryos exposed to low temperature the F-actin aggregates grouped in larger masses at the
IYUl’E =I---
actin cap edges and deeper down below the somatic nuclei (Figs. 2c-2e). When the embryos underwent cellularization during exposure to low temperature we observed small areas in which normal cleavage furrows failed to form (Figs. 3a and 3~). In these regions F-actin was not organized in the conventional system of hexagonal meshes around each forming cell. The somatic nuclei, which are localized in each small syncytium, were morphologically indistinguishable from the blastodermic cell nuclei (Fig. 3b). When the cellularized embryos were allowed to continue development we observed the formation of irregular gastrulae with small areas which failed to cellularize (Fig. 3d). DISCUSSION
The actin-based contractile network has been implicated in surface cap modification and cleavage furrow formation during the early development of the embryo of Drosophila (see [3, 15, 221 for review). In cold-treated embryos we observed cap deformation and irregular actin meshwork during the process of cellularization. Low temperature caused the depolymerization of the centrosome-associated microtubules, leaving spindle microtubules intact, and induced pole replication independently of the nuclear cycle [21]. Several spindles were observed in metaphase-like configuration with a pair of organizing centers at each pole. These organizing centers no longer occurred in pairs, as some of them had lost contact with the spindle region and moved off into the surrounding cytoplasm. Simultaneous observations with Rh-phalloidin, Rb188 antibody, and the DNA-specific Hoechst dye showed that during the syncytial blastoderm stages the actin filaments were arranged in irregular surface caps always associated with single or pairs of centrosomes. Sometimes we observed single nuclei associated with double caps as they were about to split or caps lacking nuclei. These observations raise the question of the relative importance of the nucleus and the centrosomes in organizing the cortical cytoskeleton of the embryo of Drosophila and agrees with findings in aphidicolintreated embryos in which the centrosomes, in the absence of nuclei, appear to organize the surface caps [23 ] and may initiate pole cell formation [24]. In drugtreated embryos, tubulin staining has shown that the isolated centrosomes are still able to nucleate microtubules, whereas in cold-treated embryos antitubulin fails to reveal evident aster microtubules in the centrosomal region, but only small tubulin-positive rings [21]. If intact centrosome microtubules interact with the cortical actin network in aphidicolin-treated embryos, influencing network dynamics during the formation of the sur-
SHORT
NOTE
319
FIG. 2. Fluorescence micrographs of whole Drosophila embryos stained for centrosomes (a) and F-actin (b-e) after cold treatment. ( a, b) Often the centrosomes move away from the nuclei and the actin caps become irregular in shape. Arrows and arrowheads indicate caps and relative centrosomes, respectively. (c-e) Three different levels of focus of the same embryo showing cortical actin caps, F-a&n aggreg :ates along the sides of the caps (arrows) and actin clusters in the lower periplasm (arrowheads). Bar, 50 pm.
320
SHORT
NOTE
FIG. 3. Fluorescence microscopy of cold-treated Drosophila embryos stained with rhodamine-labeled phalloidin (a, d), Hoechst dye (b) and scanning electron microscopy (c). (a, b) Small areas devoid of cleavage furrows are observed in cellularizing embryos (arrows). At this stage blastoderm cell nuclei and syncytial nuclei (arrowheads) are indistinguishable. (c) Detail of an embryo at the same stage as that in (a) showing several uncellularized areas (open arrows). (d) Embryos during the first phase of germ band elongation showing small areas which failed to cellularize (arrows). Bars, 25 pm (a, b); 5 pm (c); 50 pm (d).
face caps, it seems unlikely that centrosomes lacking astral microtubules, like those observed in cold-treated embryos, could have the same effect. A possible explanation is that the isolated centrosomes of cold-treated embryos nucleate a small number of microtubules, not detected in fluorescence observations, but capable of interacting with the cortical actin filaments. Treatment with colchicine before the beginning of cellularization prevents cleavage furrow formation [12, 171 and microinjection of antitubulin antibodies during the precellular stages leads to the local inactivation of the microtubules, disrupting the microfilament network within the caps [6]. Moreover, association of astral microtubules with the cortex has been observed in the egg of the dipteran Wachtliella [25] during the syncytial
blastoderm stage and in the embryo of Drosophila at the beginning of cellularization [161. These observations suggest that the dynamics of the actin-based cytoskeleton are influenced by the microtubular system. All these findings together indicate that when the centrosomal microtubules are disrupted at the beginning of cellularization by drugs, antitubulin antibodies, or cold treatment, as in the present case, cleavage furrow formation is prevented. This agrees partially with the discovery of areas where the cellularization is inhibited. Since the whole embryo was exposed to cold, we expected large areas devoid of cleavage furrows, but we only found small areas which failed to cellularize. Large areas of irregular cellularization were found in embryos exposed to low temperature during the later
SHORT
syncytial biastoderm stages. In this case these large irregularities reflect the abnormal organization of the surface caps before cellularization and the disposition of the somatic nuclei. After cold treatment some of the somatic nuclei move deeper into the cytoplasm as observed in antitubulin [6]- and antimyosin [22]-injected embryos. The observation that cellularization and gastrulation continue despite the presence of small uncellularized areas supports the hypothesis that the F-actin structures may act individually rather than together during the process of cellularization [15]. The presence of a layer of F-actin aggregates just below the somatic nuclei has already been observed in syncytial [l, 21 and in cellularizing embryos [14]. However, in cold-treated embryos the distribution pattern of the F-actin aggregates was different than that observed in normal developing embryos. After cold treatment the diffuse actin aggregates condensed into larger masses. This observation suggests a possible relation between microtubules and F-actin aggregates. Alternatively, cold treatment may have direct effects on actin itself. However, the functionality of the microfilaments was unaffected by cold, as revealed by the observation that gastrulation continued at low temperature. A further question is whether cold may affect other cytoplasmic components, which may cause the irregular behavior of the F-actin aggregates. Centrosome separation also occurred under these conditions indicating that this process is independent of the microtubules. This observation agrees with previous reports suggesting the dependence of centrosome separation in sea-urchin eggs on actin filaments [26, 271. We are indebted to Dr. W. Whitfield of the Department of Biochemistry, Imperial College of Science, Technology and Medicine, London, for his generous gift of the Rb188 antibody. This research project was supported by Grant 40% MPI to R.D.
REFERENCES 1.
Warn, R. M., Magrath, 156-162.
R., and Webb,
Received November 23, 1990 Revised version received February
27, 199 1
S. (1984)
J. CellBiol.
98,
321
NOTE
2. Karr,
T. L., and Alberts,
B. M.
(1986)
102, 1494-
J. CeU Biol.
1509.
3. Warn, R. M. (1986) Erp. Cell Res. 5,361-364. 4. Kellogg, D. R., Mitch&on, T. M., and Alberts, B. M. (1988) Deuelopment 103,675-686. 5. Warn, R. M., and Warn, A. (1986) Exp. Cell Res. 163,201-210. 6. Warn, R. M., Flegg, L., and Warn, A. (1987) J. Cell Biol. 105, 1721-1730.
7. Pesacreta, Branton,
T. C., Byers, T. J., Dubreuil, D., Kiehart, D. (1989) J. Cell Biol. 108,1697-1709.
D. P., and
8. Warn, 9. 10.
R. M., Bullard, B., and Magrath, R. (1980) Exp. Morphol. 57, 167-176. Lutz, D. A., and Kiehart, D. P. (1987) J. Cell Biol. Miller,
K. G., Field,
C. M., and Alberts,
J. Embryol.
105, 127a.
B. M. (1989)
J. Cell Biol.
109,2963-2975. 11. 12.
Warn, R. M., and Magrath, R. (1982) Dev. Biol. 89,540-548. Foe, V. E., and Alberta B. M. (1983) J. Cell Sci. 61,31-70.
13.
Warn, 114.
R. M.,
and Magrath,
14.
Warn,
R. M.,
and Robert-Nicoud,
R. (1983)
Exp.
M. (1990)
Cell Res.
143,103-
J. Cell Sci. B&35-
42. 15. 16.
Warn, (1990) Katoh,
R. M., Warn, A., Planques, V., and Robert-Nicoud, Ann. N.Y. Acad. Sci. 582,222-232. K., and Ishikawa; H. (1989) Protoplasma 150,83-95.
17. 18.
Zalokar, M., andErk, I. (1976) J. Microsc. Biol. Cell. 25,97-106. Campos-Ortega, J. A., and Hartenstein, V. (1985) in The Embryonic Development of Drosophila melarwgaster, p. 227, SpringerVerlag, Berlin.
19.
Whitfield, W. G. F., Millar, S. E., Saumweber, Glover, D. M. (1988) J. Cell Sci. 89,467-480.
H., Frash,
M.
M., and
20. Giloh, I. I., and Sedat, J. W. (1982) Science 217, 1252-1255. 21. Callaini, G., and Marchini, D. (1989) Exp. Cell Res. 184, 367374. 22. Kiehart, D. P., Ketchurn, A., Young, P., Lutz, D., AIfenito, M. R., Chang, X.-j., Awobuluyi, C. T., and Chen, T.-L. 251.
23. Raff,
J. W., and Glover,
M., Pesacreta, T. C., Inoue, S., Stewart, (1990) Ann. N.Y. Acad. Sci. 582, 233D. M.
(1988)
J. Cell Biol.
107, 2009-
2019.
24. 25. 26. 27.
Raff,
J. W., and Glover,
D. M. (1989)
Wolf, R. (1978) Deu. Biol. 62,464-472. Euteneuer, U., and Schliwa, M. (1985)
Cell 57,611-619. J. Cell Biol.
Schatten, H., Walter, M., Biessmann, H., (1988) Cell Motil. Cytoskeleton 11, 248-259.
and
101,96103. Schatten,
G.
CELL
194,316321
RESEARCH
(1991)
SHORT NOTE Microfilament Distribution in Cold-Treated Drosophila Embryos GIULIANO
CALLAINI,’
ROMANO DALLAI
Department of Evolutionary
001474827/91
should
Inc. reserved.
METHODS
Collection and staging of embryos.
Embryos of Drosophila melarw(Oregon-R strain) were collected at 24°C on agar plates, dechorionated in a 50% commercial bleach solution, and washed with distilled water. The age of the embryos was determined according to Campos-Ortega and Hartenstein [18] by direct observation with interference contrast or by counting the somatic nuclei. Cold treatment. After removal of excess liquid by blotting on tissue paper, embryos at appropriate stages were collected on a plastic film (Parafilm) and allowed to develop on melting ice for 1 h. Fluorescence microscopy. The dechorionated eggs were transferred to a solution containing 2.5 cc of 4% paraformaldehyde and 5 cc of heptane and gently agitated for 30 min. The embryos were then rinsed in PBS and their vitelline membrane was removed with fine needles. For double labeling the embryos were incubated for 5 h at room temperature with Rb188 antiserum, which specifically recognizes an antigen associated with the centrosome of Drosophila embryos [19] at a dilution of 1:400. The samples were then washed with PBS and incubated at room temperature in a goat anti-rabbit fluorescein-conjugated IgG at a dilution of 1:lOOO (Cappel, Westchester, PA). The embryos were then washed in PBS and stained for 30 min with 1.5 pglml phalloidin labeled with rhodamin (Molecular Probes, Inc.) for F-actin identification. The eggs were again rinsed in PBS and then exposed for 3-5 min to 1 aglml of the DNA-specific dye Hoechst 33258. Finally, the embryos were washed in PBS and mounted in 90% glycerol containing 2.5% n-propyl-gallate to reduce photobleaching [20]. The embryos were observed in a Leitz Aristoplan microscope equipped with Auorescein, rhodamin, and uv filters. Photomicrographs were taken with Kodak Tri-X pan film and developed in Kodak HC 110 for 7 min at 20°C.
RESULTS
When Drosophila embryos were exposed to low temperature and allowed to develop up to the syncytial blastoderm stage, the actin filaments were organized in cortical caps of irregular shape and size. Simultaneous Rh-phalloidin and Hoechst stains often showed that the number of F-actin caps was higher than expected from counting the somatic nuclei. Figure la is a detail of an embryo during interphase of the 10th mitotic cycle. It can be observed that there are several irregular cortical 316
Press, form
AND
gaster
be addressed.
$3.00
Copyright 0 1991 by Academic All rights of reproduction in any
4, 53100 Sienn, Italy
MATERIALS
The distribution of the actin filaments was recently investigated in the early Drosophila embryo at the syncytial blastoderm stage and during the process of cellularization. These studies revealed that once the somatic nuclei reach the cortex, the actin filaments are localized in cortical cytoplasmic domains above each nucleus [l41. These cytoplasmic regions also contain microtubules [2, 4-61, spectrin [7], myosin [8, 91 and actin-binding proteins [lo]; they undergo rapid morphological changes during mitosis [ 11,121. By the time mitosis begins, the cytoskeletal elements are rearranged in characteristic patterns of distribution; in particular the actin network expands as the nuclear cycle progresses and cleaves in two during later stages of mitosis. At the onset of cellularization the actin filaments form a hexagonal network associated with the leading edges of the extending plasmalemma [ 13-151. Certain features suggest that the change in actin localization is influenced by microtubules both during the syncytial blastoderm stage [2,6] and at the beginning of cellularization [ 12,16,17]. To better understand the relationship between microtubules and microfilaments we examined the distribution of microfilaments in Drosophila embryos exposed to cold during the early stages of development. Because cold treatment causes partial depolymerization of centrosome-associated microtubules, we applied immunofluorescence with Rh-phalloidin and requests
RIPARBELLI
anti-centrosome antibodies to cold-treated Drosophila embryos at different stages of development in order to investigate the possibility of a relation between the spatial organization of actin filaments and centrosomes.
INTRODUCTION
reprint
GIOVANNA
Biology, University of Siena, via Mattioli
Cold treatment of Drosophila embryos is observed to result in general alteration of microfilament distribution leading to deformation of the surface caps and to perturbation of the process of cleavage furrow extension. After exposure to low temperature the cortical actin caps underwent several morphological changes, despite the arrested nuclear cycle. These observations are discussed in relation to centrosome behavior during the cell cycle. 0 1991 Academic Preess, Inc.
i To whom
AND MARIA
SHORT
NOTE
FIG. 1. Fluorescence microscopy of whole cold-treated Drosophila embryos stained with rhodamine-labeled phalloidin for F-actin (a, c), Rb188 antibody to reveal the centrosomes (d) and counterstained with Hoechst dye to show the nuclei (b, e). (a, b) Cortical actin caps are single or double; arrows and arrowheads mark nuclei and the position of the nuclei inside the actin caps, open arrow indicates a cap without nucleus. (c-e) Centrosome replication occurs despite the arrested chromosome cycle. Cortical actin caps are in telophase-like configurations, while chromosomes are aligned in metaphase. A centrosome pair is visible under each cap. Open arrows mark the position of the chromosomes among the caps; arrows and arrowheads indicate the newly separated caps and the relative centrosomes, respectively. Bars, 50 pm (a, b); 10 pm (c, d, 4.
318
SHORT
actin caps. Most of them are round and irregularly scattered with a single nucleus. In some cases the caps appear double with a nucleus associated in the middle or shifted to an extremity. Sometimes small actin caps are without nuclei (Fig. lb). The experimental system employing cold treatment allows the centrosome cycle to move with respect to the chromosome cycle. Because at this time the number and disposition of chromosomes are the same as in normal metaphase plates, it seems that the centrosome cycle becomes uncoupled from the chromosome cycle. The centrosomes pass through anaphase- and telophase-like configurations, whereas the chromosomes remain in a metaphase-like configuration [21]. Hence, we observed chromosomes still in metaphase and the surface actin caps in a telophase-like configuration (Figs. lc and le). Below each actin cap we observed a centrosome pair (Fig. Id). In embryos recovered to 24’C for 30 min the chromosomes condensed in irregular figures and the centrosomes were no longer visible in pairs but moved away. Thus the organizing centers showed various degrees of splitting from each other and some of them lost contact with the spindle region and moved in the surrounding cytoplasm. The spindles often had only one main pole, the other being free in the cytoplasm [21]. Unlike in normal development in which the centrosomes are the focal point of the aster and spindle microtubules, in the cold-treated embryos the nucleation process associated with the centrosomes was only seen to a limited extent and very short aster microtubules were associated with the periphery of the centrosomal material. Double fluorescence with Rh-phalloidin and Rb188 serum, which specifically recognizes a centrosomal protein of Drosophila embryos [ 191, allowed us to compare the position of centrosomes and cortical actin caps. In recovered embryos the chromosomes were condensed in irregular figures and the cortical F-actin caps varied in shape and size. We often observed cap deformation and small caps devoid of nuclear material (Fig. 2a). Simultaneous observations with Rb188 serum and Rh-phalloidin revealed one or two centrosomes coinciding with each cortical actin cap (Fig. 2b). The dimensions and shape of the caps apparently depended on the number of the centrosomes and on the relative distance between them. Single or close pairs of centrosomes resulted in spheroidal actin caps, but as the centrosomes moved away the actin caps lengthened (Fig. 2b). The Rb188 antibody gives a feeble staining of the nuclear region [19], allowing visualization of the position of the centrosomes to the nuclei. Cold treatment is also known to affect the F-actin aggregates which have been observed as a uniform layer underneath the somatic nuclei in normal developing embryos [14]. In embryos exposed to low temperature the F-actin aggregates grouped in larger masses at the
IYUl’E =I---
actin cap edges and deeper down below the somatic nuclei (Figs. 2c-2e). When the embryos underwent cellularization during exposure to low temperature we observed small areas in which normal cleavage furrows failed to form (Figs. 3a and 3~). In these regions F-actin was not organized in the conventional system of hexagonal meshes around each forming cell. The somatic nuclei, which are localized in each small syncytium, were morphologically indistinguishable from the blastodermic cell nuclei (Fig. 3b). When the cellularized embryos were allowed to continue development we observed the formation of irregular gastrulae with small areas which failed to cellularize (Fig. 3d). DISCUSSION
The actin-based contractile network has been implicated in surface cap modification and cleavage furrow formation during the early development of the embryo of Drosophila (see [3, 15, 221 for review). In cold-treated embryos we observed cap deformation and irregular actin meshwork during the process of cellularization. Low temperature caused the depolymerization of the centrosome-associated microtubules, leaving spindle microtubules intact, and induced pole replication independently of the nuclear cycle [21]. Several spindles were observed in metaphase-like configuration with a pair of organizing centers at each pole. These organizing centers no longer occurred in pairs, as some of them had lost contact with the spindle region and moved off into the surrounding cytoplasm. Simultaneous observations with Rh-phalloidin, Rb188 antibody, and the DNA-specific Hoechst dye showed that during the syncytial blastoderm stages the actin filaments were arranged in irregular surface caps always associated with single or pairs of centrosomes. Sometimes we observed single nuclei associated with double caps as they were about to split or caps lacking nuclei. These observations raise the question of the relative importance of the nucleus and the centrosomes in organizing the cortical cytoskeleton of the embryo of Drosophila and agrees with findings in aphidicolintreated embryos in which the centrosomes, in the absence of nuclei, appear to organize the surface caps [23 ] and may initiate pole cell formation [24]. In drugtreated embryos, tubulin staining has shown that the isolated centrosomes are still able to nucleate microtubules, whereas in cold-treated embryos antitubulin fails to reveal evident aster microtubules in the centrosomal region, but only small tubulin-positive rings [21]. If intact centrosome microtubules interact with the cortical actin network in aphidicolin-treated embryos, influencing network dynamics during the formation of the sur-
SHORT
NOTE
319
FIG. 2. Fluorescence micrographs of whole Drosophila embryos stained for centrosomes (a) and F-actin (b-e) after cold treatment. ( a, b) Often the centrosomes move away from the nuclei and the actin caps become irregular in shape. Arrows and arrowheads indicate caps and relative centrosomes, respectively. (c-e) Three different levels of focus of the same embryo showing cortical actin caps, F-a&n aggreg :ates along the sides of the caps (arrows) and actin clusters in the lower periplasm (arrowheads). Bar, 50 pm.
320
SHORT
NOTE
FIG. 3. Fluorescence microscopy of cold-treated Drosophila embryos stained with rhodamine-labeled phalloidin (a, d), Hoechst dye (b) and scanning electron microscopy (c). (a, b) Small areas devoid of cleavage furrows are observed in cellularizing embryos (arrows). At this stage blastoderm cell nuclei and syncytial nuclei (arrowheads) are indistinguishable. (c) Detail of an embryo at the same stage as that in (a) showing several uncellularized areas (open arrows). (d) Embryos during the first phase of germ band elongation showing small areas which failed to cellularize (arrows). Bars, 25 pm (a, b); 5 pm (c); 50 pm (d).
face caps, it seems unlikely that centrosomes lacking astral microtubules, like those observed in cold-treated embryos, could have the same effect. A possible explanation is that the isolated centrosomes of cold-treated embryos nucleate a small number of microtubules, not detected in fluorescence observations, but capable of interacting with the cortical actin filaments. Treatment with colchicine before the beginning of cellularization prevents cleavage furrow formation [12, 171 and microinjection of antitubulin antibodies during the precellular stages leads to the local inactivation of the microtubules, disrupting the microfilament network within the caps [6]. Moreover, association of astral microtubules with the cortex has been observed in the egg of the dipteran Wachtliella [25] during the syncytial
blastoderm stage and in the embryo of Drosophila at the beginning of cellularization [161. These observations suggest that the dynamics of the actin-based cytoskeleton are influenced by the microtubular system. All these findings together indicate that when the centrosomal microtubules are disrupted at the beginning of cellularization by drugs, antitubulin antibodies, or cold treatment, as in the present case, cleavage furrow formation is prevented. This agrees partially with the discovery of areas where the cellularization is inhibited. Since the whole embryo was exposed to cold, we expected large areas devoid of cleavage furrows, but we only found small areas which failed to cellularize. Large areas of irregular cellularization were found in embryos exposed to low temperature during the later
SHORT
syncytial biastoderm stages. In this case these large irregularities reflect the abnormal organization of the surface caps before cellularization and the disposition of the somatic nuclei. After cold treatment some of the somatic nuclei move deeper into the cytoplasm as observed in antitubulin [6]- and antimyosin [22]-injected embryos. The observation that cellularization and gastrulation continue despite the presence of small uncellularized areas supports the hypothesis that the F-actin structures may act individually rather than together during the process of cellularization [15]. The presence of a layer of F-actin aggregates just below the somatic nuclei has already been observed in syncytial [l, 21 and in cellularizing embryos [14]. However, in cold-treated embryos the distribution pattern of the F-actin aggregates was different than that observed in normal developing embryos. After cold treatment the diffuse actin aggregates condensed into larger masses. This observation suggests a possible relation between microtubules and F-actin aggregates. Alternatively, cold treatment may have direct effects on actin itself. However, the functionality of the microfilaments was unaffected by cold, as revealed by the observation that gastrulation continued at low temperature. A further question is whether cold may affect other cytoplasmic components, which may cause the irregular behavior of the F-actin aggregates. Centrosome separation also occurred under these conditions indicating that this process is independent of the microtubules. This observation agrees with previous reports suggesting the dependence of centrosome separation in sea-urchin eggs on actin filaments [26, 271. We are indebted to Dr. W. Whitfield of the Department of Biochemistry, Imperial College of Science, Technology and Medicine, London, for his generous gift of the Rb188 antibody. This research project was supported by Grant 40% MPI to R.D.
REFERENCES 1.
Warn, R. M., Magrath, 156-162.
R., and Webb,
Received November 23, 1990 Revised version received February
27, 199 1
S. (1984)
J. CellBiol.
98,
321
NOTE
2. Karr,
T. L., and Alberts,
B. M.
(1986)
102, 1494-
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