Cytochalasin B inhibits morphogenetic movement and muscle differentiation of activin-treated ectoderm in Xenopus

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1 Develop. Growth Differ. (1999) 41, Cytochalasin B inhibits morphogenetic movement and muscle differentiation of activin-treated ectoderm in Xenopus Keiko Tamai, 1 Chika Yokota, 2 Takashi Ariizumi 2 and Makoto Asashima 1,2 * 1 Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Hongo, Bunkyo-ku, Tokyo and 2 Department of Life Sciences, CREST Project, Graduate School of Arts and Sciences, The University of Tokyo, Komaba, Meguro-ku, Tokyo , Japan. Xenopus ectodermal explants (animal caps) begin to elongate after treatment with the mesoderm inducing factor activin A. This phenomenon mimics the convergent extension of dorsal mesoderm during gastrulation. To analyze the relationship between elongation movement and muscle differentiation, animal caps were treated with colchicine, taxol, cytochalasin B and hydroxyurea (HUA)/aphidicolin following activin treatment. Cytochalasin B disrupted the organization of actin filaments and inhibited the elongation of the activin-treated explants. Muscle differentiation was also inhibited in these explants at the histologic and molecular levels. Colchicine and taxol, which are known to affect microtubule organization, had little effect on elongation of the activin-treated explants. Co-treatment with HUA and aphidicolin caused serious damage on the explants and they did not undergo elongation. These results suggest that actin filaments play an important role in the elongation movement that leads to muscle differentiation of activin-treated explants. Key words: activin A, cytochalasin B, morphogenetic movement, muscle differentiation, Xenopus. Introduction Gastrulation is the most dynamic cell movement during early amphibian development, and includes the processes of epiboly, involution, convergent extension and directed cell migration (Keller et al. 1992). When Xenopus animal caps are treated with dorsal mesodermal inducers, they begin to show elongation movement that mimics the convergent extension of dorsal mesoderm during gastrulation (Symes & Smith 1987). This morphological change in animal cap explants has been used to test certain factors for dorsal mesoderm inducing activity (Graff et al. 1996; Ladher et al. 1996; Horb & Thomsen 1997; Philpott et al. 1997). Activin A, a dorsal mesoderm inducer, is known to induce ventral to dorsal mesoderm in animal cap explants in a dose-dependent manner (Asashima et al. 1990; Smith et al. 1990; Ariizumi et al. 1991; Asashima 1994). When animal caps are treated with 5 10 ng/ml activin A, which mainly induces muscle in the explants, they begin to elongate within several hours after treatment (Ariizumi et al. 1991). *Author to whom all correspondence should be addressed. cmasa@komaba.ecc.u-tokyo.ac.jp Received 3 June 1998; revised 7 August 1998; accepted 7 August A number of factors including fibronectin, integrin, and C-cadherin have been implicated in the process of gastrulation in Xenopus. Gastrulation is inhibited by the injection of cytochalasin B (CCB) into the blastocoele at the blastula stage (Nakatsuji 1979). The CCB also inhibits the migration of dissociated animal cap cells treated with activin A on fibronectin-coated plates (Howard & Smith 1993; Symes et al. 1994). Therefore, CCB may be useful in determining whether there is any direct relationship between morphogenetic movement and cell differentiation. In the present paper we tested four drugs, colchicine (an inhibitor of tubulin polymerization), taxol (a stabilizer of microtubules), CCB (an inhibitor of actin polymerization) and hydroxyurea (HUA)/aphidicolin, (inhibitors of DNA synthesis) to see if they would inhibit the elongation of activin-treated animal caps. We found that CCB specifically inhibited the elongation movement of activin-treated animal caps and subsequent differentiation into muscle. Materials and Methods Embryos Eggs were obtained after injecting females and males with 600 IU of human chorionic gonadotropin (Gestron;

2 42 K. Tamai et al. Denka Seiyaku, Tokyo, Japan). Embryos were dejellied with 3% cystein hydrochloride dissolved in Steinberg s solution (SS; 58 mmol/l NaCl, 0.67 mmol/l KCl, 0.34 mmol/l Ca(NO 3) 2, 0.85 mmol/l MgSO 4, 3 mmol/l Hepes, 0.1 g/l kanamycin sulfate; ph 7.4), then washed with SS and incubated at 20 C until they reached the late blastula stage (stage 9; Nieuwkoop & Faber 1956). Animal caps and test solutions Human recombinant activin A was a generous gift from Dr Y. Eto (The Central Research Laboratory of Ajinomoto Co., Kawasaki, Japan). Animal caps ( mm) were dissected from stage 9 embryos using tungsten needles, and treated with 0 10 ng/ml activin A dissolved in SS containing 0.1% bovine serum albumin (BSA; Sigma Chemical Co., St Louis, MO, USA) for 3 h at 20 C. They were then rinsed with SS and treated with CCB ( µmol/l), colchicine (1 100 µmol/l), taxol (2 200 µg/ml) or HUA (2 40 mmol/l)/aphidicolin (5 100 µg/ml; Sigma) in SS for 3 h. The animal caps were subsequently transferred to fresh SS and cultured for 3 days at 20 C for histologic and reverse transcription-polymerase chain reaction (RT-PCR) analyses. The concentrations of drugs used were chosen based on the reports of Schaeffer et al. (1993), Perry (1975), Nakatsuji (1979), Harris and Hartenstein (1991) and Lane and Keller (1997). Confocal laser scanning microscopy Explants were fixed with 3.7% formaldehyde in F buffer (50 mmol/l Hepes, 100 mmol/l KCl, 0.2 mmol/l CaCl 2, 5 mmol/l MgCl 2; ph 7.5) for 3 h, transferred to F buffer containing 0.1% Triton X for 20 min, and postfixed with 3.7% formaldehyde in F buffer for more than 1 h. They were then stained with rhodamine-phalloidin (Molecular Probes, Eugene, OR, USA), which specifically binds to F-actin, for 1 h and examined under a confocal laser scanning microscope (LSM510, Zeiss Co., Oberkochen, Germany). Histologic preparations Animal cap explants were fixed with Bouin s fluid for 3 h, dehydrated in an ethanol series, and embedded in paraffin. Sections were made at 8 µm and stained with hematoxylin/eosin. RT-PCR analysis Extraction of total RNA, RT-PCR reactions, subcloning and sequencing of RT-PCR fragments were performed as previously described (Yokota et al. 1998) with one modification: The RT-PCR reaction was carried out in a 50 µl volume. N-CAM (Kintner & Melton 1987), XAG1 (Sive et al. 1989), Xotx2 (Pannese et al. 1995), Xbra (Smith et al. 1991), XmyoD (Hopwood et al. 1989), Chordin (Sasai et al. 1994), Xwnt-8 (Christian et al. 1991), alpha skeletal muscle actin (muscle specific actin: ms-actin; Stutz & Spoh 1986), XlHbox6 (Wright et al. 1990), Endodermin (Sasai et al. 1996) and EF1- (Krieg et al. 1989) were analyzed as previously described (Hemmati-Brivanlou & Melton 1994; LaBonne & Whitman 1994; Wilson & Melton 1994; Hatada et al. 1995; Lai et al. 1995; Sasai et al. 1995; Bouwmeester et al. 1996; Sasai et al. 1996; Takahashi et al. 1998). Fig. 1. Time-course of elongation of the animal caps in response to different concentrations of activin A. Elongation is not observed in the 0 and 0.5 ng/ml activin-treated explants (A). Explants treated with activin A at 5 and 10 ng/ml begin to elongate at about 6 h after dissection (A,B). The 10 ng/ml activintreated explants elongated more consistently than the explants treated with 5 ng/ml activin A, and when they reached their maximum length they began to contract. Means ± standard error (SE) are given; n 4 in all cases. ( ), Control; ( ), activin 0.5 ng/ml; ( ), activin 5 ng/ml.

3 Cytochalasin effects on Xenopus ectoderm 43 Results Time-course of the elongation of activin-treated animal caps The lengths of the animal caps after treatment with different concentrations of activin A were measured at 20 C using a micrometer. At 0 and 0.5 ng/ml of activin A, the explants remained spherical throughout the culture period (Fig. 1A). Explants treated with 5 and 10 ng/ml of activin A began to elongate about 6 h after the dissection (Fig. 1A,B). Explants treated with 5 ng/ml of activin A elongated rapidly, maintained their maximum length for 2 or 3 h, and began to contract about 20 h after the dissection. Explants treated with 10 ng/ml Fig. 2. External view of the animal cap explants treated with activin A and drugs. Explants were photographed at 15 h after dissection. The square area in each photograph shows the explants without activin treatment. (A) 10 ng/ml activin A treatment, (B) activin A and 1 µmol/l colchicine treatment, (C) activin A and 10 µmol/l colchicine treatment, (D) activin A and 2 µg/ml taxol treatment, (E) activin A and 20 µg/ml taxol treatment, (F) activin A and 2 mmol/l HUA/5 µg/ml aphidicolin treatment, (G) activin A and 20 mmol/l HUA/50 µg/ml aphidicolin treatment, (H) activin A and 1 µmol/l CCB treatment, (I) activin A and 10 µmol/l CCB treatment. Colchicine and taxol did not inhibit elongation of the activintreated explants, but CCB significantly inhibited elongation. Explants treated with HUA/aphidicolin did not elongate, and they became fragile. Bar, 0.5 mm.

4 44 K. Tamai et al. Fig. 3. Effects of drugs on elongation of activin-treated animal caps. Activin-treated explants were incubated with colchicine (A), taxol (B), HUA/aphidicolin (C), and CCB (D). The length of the explants was measured at 3, 9 and 15 h after dissection from the embryo. Means SE are given; n 38 in all cases.

5 Cytochalasin effects on Xenopus ectoderm 45 of activin A steadily elongated until they reached their maximum length, then began to contract immediately. At 15 h after the dissection, both 5 and 10 ng/ml activin-treated explants had elongated sufficiently for the effects of activin A to be recognized. However, the explants treated with 10 ng/ml activin A showed a more stable time-course curve than those treated with 5 ng/ml activin A (Fig. 1B). We therefore used the 10 ng/ml concentration of activin A to induce elongation of the animal caps and to examine the effect of drugs on this process. Cytochalasin B specifically inhibits elongation of activin-treated animal caps Explants were treated with 10 ng/ml of activin A for 3 h and then incubated with colchicine, taxol, HUA/aphidicolin, or CCB for the next 3 h. The length of the explants was then measured to determine the effects of the drugs on the elongation movement. At 1 and 10 µmol/l of colchicine, or 2 and 20 µg/ml of taxol, there was almost no effect on elongation of the activin-treated animal caps 15 h after dissection (Fig. 2B E). They showed good elongation when compared with the control activin-treated explants (Fig. 2A). After 3 days of culture, few explants survived when treated with a 10 µmol/l or more concentration of colchicine (Fig. 3). Co-treatment with HUA (2 mmol/l) and aphidicolin (5 µg/ml) caused serious damage to the explants at 9 h. Explants appeared fragile at 15 h and no elongation was observed (Fig. 2F,G). On the other hand, the 1 and 10 µmol/l concentrations of CCB inhibited elongation of the activin-treated explants without causing serious damage (Figs 2H,I,3). The explants curled up during 3 h of activin treatment, but the spherical explants reopened their inner blastocoelic surfaces within the first 30 min of CCB treatment. After rinsing with SS, the explants became spherical again within 1 h and survived after 3 days of culture. None of the other drugs Table 1. Tissues induced in animal cap explants by activin and cytochalasin B Activin 10 ng/ml Cytochalasin B (µmol) 0 0* * No. of specimens Atypical epidermis Epidermis Cement gland Neural tissue Notochord Muscle Mesenchyme Endothermal cells Values indicate the frequencies of inductions as a percentage of the number of specimens. *Containing 1% of dimethyl sulfoxide. Fig. 4. Histologic sections of animal cap explants. No mesodermal or neural derivatives were found in untreated animal caps (A). A cement gland was found in the 2.5 µmol/l CCB-treated animal caps (B). Both (A) and (B) formed irregularshaped epidermis (atypical epidermis). Activin A at 10 ng/ml induced mesenchyme, muscle and neural tissues in animal caps (C). Muscle formation in these activin-treated explants was decreased by 2.5 µmol/l CCB treatment (D). ae, atypical epidermis; cg, cement gland; epi, epidermis; mes, mesenchyme; mus, muscle; neu, neural tissue. Bar, 100 µm.

6 46 K. Tamai et al. produced such dramatic changes in the explants. Elongation was completely suppressed in about onethird of explants by 1 and 10 µmol/l CCB treatment (Figs 2H,I,3). Histologic analysis and specific gene expression of the activin/cytochalasin B-treated animal caps To examine the effects of CCB on activin-treated explants at the histologic level, sections were made after 3 days of culture. The results of the histologic analysis are summarized in Table 1. A 1% solution of dimethyl sulfoxide (DMSO), the CCB solvent, had no effect on elongation of the activin-treated explants (data not shown), and as shown in Table 1, there was little difference in mesodermal tissue differentiation in activin-treated animal caps irrespective of DMSO treatment. Cytochalasin B at 0.1 µmol/l did not inhibit the elongation of the activin-treated explants (data not shown), which differentiated into various mesodermal tissues, as seen in the control activin-treated explants (Fig. 4C; Table 1). However, the frequency and size of the dorsal mesoderm tissues, such as muscle and notochord, decreased in the 1 µmol/l CCB-treated explants that did not exhibit elongation (Fig. 2H; Table 1). The cement gland was seen in the CCBtreated explants irrespective of activin treatment (Fig. 4B,D; Table 1). The results of RT-PCR analysis using various marker genes are shown in Fig. 5. Explants were harvested immediately after the end of CCB treatment (6 h after the dissection of animal caps), cultured for 3 h more (9 h after the dissection; Fig. 5A), or cultured for 3 days more (Fig. 5B). No expression of Xbra, a panmesodermal marker, was detected in the activin/ccbtreated explants at 6 h and expression was low in the explants at 9 h (Fig. 5A). The expression of XmyoD, a muscle specific marker, was completely inhibited by CCB treatment at 9 h. Chordin, a dorsal marker, and Xwnt-8, a lateral-ventral marker, on the other hand, were consistently expressed irrespective of CCB treatment. After 3 days of culture, expression of the muscle specific marker ms-actin was reduced by activin/ccb treatment. Expression levels of the pan-neural marker N-CAM and the endodermal marker Endodermin were unchanged irrespective of CCB treatment (Fig. 5B). However, expression of Xotx2, an anterior neural marker, was increased by CCB treatment and expression of XlHbox6, a posterior neural marker, was decreased. Expression of XAG1, a cement gland marker, was detected in all CCB-treated explants, a finding consistent with the results of histologic analysis (Table 1). Effect of cytochalasin B on the organization of actin filaments in activin-treated animal caps Cortical actin filament organization was seen in the untreated explants at each time examined (4,9,15 h; Fig. 6A,D,G). Actin filaments were observed more Fig. 5. Changes in specific gene expression in animal cap explants. Animal caps were treated with or without 10 ng/ml activin for 3 h, and then cultured in 2.5 µmol/l CCB for the next 3 h. The CCB ( ) explants were cultured in SS containing DMSO diluted 1:1600 instead of CCB. Expression of marker genes was tested by RT-PCR. Explants were harvested immediately after CCB treatment (6 h), at 3 h after CCB treatment (9 h; A) or after 3 days of culture (B). (A) Expression of Xbra and XmyoD was decreased in the CCB-treated explants, but expression of Chordin (Chd) and Xwnt-8 was unchanged. (B) Expression of the muscle marker, ms-actin, and the posterior neural marker, XlHbox6, were suppressed in activin/ccb-treated explants as compared with activin-treated explants. On the other hand, expression of the cement gland marker, XAG1, and the anterior neural marker, Xotx2, was enhanced. WE, whole embryo.

7 Cytochalasin effects on Xenopus ectoderm 47 clearly in the activin-treated explants than in the untreated explants (Fig. 6B,E,H). In the activin/ CCB-treated explants, the actin filaments were observed to be disrupted at 4 h (1 h after the CCB treatment was started; Fig. 6C). Actin filament organization recovered by 9 h (3 h after the end of CCB treatment; Fig. 6F). Discussion In the present study, we showed that CCB, which causes reversible disaggregation of microfilaments, inhibited elongation of activin-treated animal caps, while both colchicine and taxol, which affect microtubule structure, had no marked effect. Cytochalasin B inhibits the migration of dissociated activin-treated animal cap cells on fibronectin-coated plates (Howard & Smith 1993; Symes et al. 1994). Microfilaments are seen in protrusive filopodia during migration of dissociated gastrula head mesoderm cells on fibronectin-coated plates (Selchow & Winklbauer 1997; Wacker et al. 1998). Thus, accumulation of motilities driven by actin filaments in each cell may be the source of the elongation movement of activin-treated animal caps. Muscle differentiation was significantly decreased in the activin/ccb-treated explants in which morphogenetic movement was inhibited. The volume of muscle tissue in these explants was observed to be clearly decreased when examined histologically. These phenomena were also confirmed by RT-PCR analysis, in which the expression of Xbra, XmyoD and ms-actin was remarkably suppressed by CCB treatment. No expression of Xbra was detected in activin/ccb-treated explants immediately after the end of CCB treatment, and it was reduced at 3 h after CCB removal as compared with activin-treated explants. No expression of XmyoD was detected in the activin/ccb-treated explants at 3 h after CCB removal although the expression was induced in the explants treated with activin alone. The suppression of Xbra expression by CCB may be responsible for the failure to express XmyoD in these explants, which finally leads to the inhibitory effects of muscle formation in the activin-treated explants. By contrast, expression of Chordin, which is upstream of Endodermin (Sasai et al. 1996), was unchanged by CCB treatment, a finding consistent with the absence of any effect on endoderm formation. Cytochalasin B also has no effect on the lateral-ventral Fig. 6. Organization of actin filaments in animal cap explants. (A,D,G) untreated control animal caps; (B,E,H) activin-treated animal caps; (C,F,I) activin/ccb-treated animal caps; h indicates the time after dissection of the animal caps from the embryo. Organization of the actin filaments was disrupted in the activin/ccb-treated animal caps at 4 h (C). At 9 h (3 h after CCB removal), cortical actin filaments had begun to reorganize (arrow heads in F). Bar, 20 µm.

8 48 K. Tamai et al. differentiation of the mesoderm induced by activin, because expression of Xwnt-8 was also unchanged by CCB treatment. Using CCB treatment, cement gland was seen in the explants irrespective of activin treatment. As adhesion of the animal cap cells became weak during CCB treatment, it is possible to assume that a decreased number of BMP4 (antineuralizing factor) molecules in the extracellular matrix directly caused neuralization and cement gland formation. However, the neural tissues seen in the activin-treated animal caps can also be explained as a secondary interaction between noninduced cells and mesodermalized cells (muscle cells), because animal cap cells are fated to differentiate into mesoderm during activin treatment and Xbra (mesodermal marker) has expressed in the activin/ CCB-treated explants 3 h after CCB removal. As shown by RT-PCR analysis, expression of XlHbox6 (posterior neural marker) of the activin-treated animal caps was decreased by CCB treatment, while expression of Xotx2 (anterior neural marker) was increased. Decreased expression of Xbra, which has caudalizing ability, may anteriorize the pattern of neural tissue induced in the activin/ccb-treated explants. What is the mechanism of inhibition of muscle differentiation by CCB? Fibronectin (Winklbauer 1990; Howard et al. 1992; Winklbauer & Keller 1996), cadherin (Brieher & Gumbiner 1994; Lee & Gumbiner 1995), and heparin (Mitani 1989; Itoh & Sokol 1994) are known to play important roles in morphogenetic movement. Gastrulation is inhibited by injection of Arg- Gly-Asp (RGD) peptide (Winklbauer 1990) and antifibronectin antibody into the blastocoele (Winklbauer & Keller 1996). Ectopic expression of dominant-negative type C-cadherin in the animal cap inhibits the elongation induced by activin (Lee & Gumbiner 1995). 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