Determinative mechanisms in secondary muscle lineages of ascidian embryos: development of muscle-specific features in isolated muscle progenitor cells

The process of ascidian embryogenesis is regarded as an example of typical mosaic development. A significant body of information has accumulated about cytoplasmic determinants involved in differentiation of muscle cells (reviewed by Whittaker, 1979; Uzman and Jeffery, 1986; Satoh, 1987). Initially, it was thought that all muscle cells of the larval tail originated from a single pair of blastomeres of the 8-cell embryo (the posteriorvegetal blastomeres, B4.1) (Conklin, 1905; Ortolani, 1955; Mancuso, 1969), and the results of experiments with isolated blastomeres at the 8-cell stage were interpreted according to this hypothesis. Partial embryos derived from isolated B4.1 blastomeres of the 8cell embryo were shown, subsequently, to express autonomously certain muscle-specific features which could be detected histologically, histochemically and ultrastructurally (Reververi and Minganti, 1946; Whittaker et al. 197T, Crowther and Whittaker, 1983).

However, recent studies of cell lineage, involving intracellular injection of lineage tracer molecules have revealed that not only the B-line blastomeres (designated as the primary muscle lineage according to Meedel et al. (1987)) but also A-line (the anteriorvegetal) and b-line (the posterior-animal) blastomeres (designated as the secondary muscle lineages) contribute to formation of tail muscle (Nishida and Satoh, 1983, 1985; Nishida, 1987). Fig. 1 is a schematic representation of the muscle lineage of Halocynthia roretzi embryos.

Deno, Nishida and Satoh (1985) reinvestigated the autonomy of muscle differentiation in partial embryos derived from each pair of blastomeres of the 8-cell

Halocynthia embryo. Results clearly showed that most of the B-line partial embryos autonomously developed acetylcholinesterase, the putative marker of muscle differentiation, whereas few of the A-line and b-line partial embryos expressed the enzyme. The proportion of the A-line and b-line quarter embryos that expressed the enzyme was only about 3%. Essentially similar results were obtained with Ciona by Deno et al. (1985) and Meedel et al. (1987). From these observations, Meedel el al. (1987) suggested that interactions between cells are involved in the process of determination of the secondary muscle progenitor cells.

Isolation of blastomeres from cleavage-stage embryos and examination of the developmental autonomy of the cells is a powerful method for the investigation of the determinative state of embryonic cells, which is not evident during normal development. However, several factors make it difficult to interpret the results of differentiation of muscle in partial embryos derived from secondary-lineage blastomeres isolated from the 8-cell embryo: (a) the low proportion of positive specimens; (b) only a small number of muscle cells is derived from secondary lineages during normal development, as compared with the number of cells derived from primary lineage; and (c) the developmental fate is only partially segregated at the 8-cell stage, and each muscle-lineage blastomere gives rise to various kinds of larval tissue in addition to muscle.

In this study, muscle-lineage cells of three different lines were isolated from embryos at later cleavage stages, when restriction of their developmental fate to muscle was already advanced in A-line and b-line muscle-progenitor cells. If the failure of muscle development in A-line and b-line quarter embryos were due to a weaker restriction of their developmental fate, then A-line and b-line muscle-progenitor cells isolated from the 64-cell or 110-cell embryo might possibly develop features characteristic of differentiation to muscle. However, if such failure were due to the interruption of the intercellular interactions with other blastomeres that occur late in development, the secondary musclelineage cells isolated even at the late-cleavage stage might not develop muscle-specific features.

Furthermore, since mesodermal induction is involved in the formation of mesodermal tissues, including muscle, during amphibian embryogenesis, the possibility that mesodermal induction may play some role in ascidian embryogenesis was also investigated.

Adults of the ascidian, Halocynthia roretzi (Drasche), were collected in the vicinity of Asamushi Marine Biological Station, Aomori, Japan. They spawned yellowish, translucent eggs, about 280μm in diameter exclusive of the chorion. The translucency and large size of the eggs facilitated identification and isolation of each blastomere. Naturally spawned eggs were fertilized artificially with a dilute suspension of sperm from other individuals. Embryos were reared in Millipore-filtered seawater (MFSW) at 13°C, at which temperature embryos hatched about 35 h after fertilization.

Fertilized eggs were allowed to develop until the 16-cell stage. The 16-cell embryos were manually dechorionated with sharpened tungsten needles and reared in 0.9 % agar-coated plastic dishes filled with MFSW that contained 50μgml^-1 streptomycin sulphate. At desired stages between the 64-cell and the 110-cell stage (8-9h after fertilization), identified blastomeres were isolated from embryos with a fine glass needle under a stereomicroscope (OLYMPUS SZH-121). Isolated blastomeres were cultured separately in agar-coated plastic dishes filled with MFSW that contained streptomycin. Isolates were cultured until control larvae hatched, and they were then prepared for histochemical and immunohistochemical analysis.

Vital staining for mitochondria 3,3’-diethyloxacarbocyanin [DiOC₂ (3)] is a fluorescent probe for vital staining of mitochondria and its validity in ascidian embryos has been demonstrated (Zaloker and Sardet, 1984). Dechorionated embryos were incubated in MFSW that contained 0.5 μg ml^-1 DiOC₂ (3) (Molecular Probes Inc. Eugene) for 1 h at 13°C during the 16-cell and 32-cell stages, and then they were transferred to MFSW. The drug was not washed out in MFSW. At the 64-cell and 110-cell stages, blastomeres of various lineages were isolated from the embryos and then their mitochondria were examined with a Nikon Labophoto equipped with an epifluorescence optic unit EFD and a B2 excitation filter cassette (excitation wavelength 460-485 nm). In this system, mitochondria were visualized by green fluorescence emitted by the DiOC₂ stain.

Histochemical detection of AchE was carried out by the method described by Karnovsky and Roots (1964). Partial embryos were fixed for 10 min in 5% formalin in seawater and, after several washings with distilled water, they were incubated in the reaction mixture for 1-2 h at 30°C. Specimens were then washed and observed with Nomarsky differential interference optics (Nikon Optiphoto with NT equipment). Histochemical localization of AchE activity in larvae is shown in 4,Fig. 2A. Most of the activity is restricted to the tail muscle. At the larval stage, all of the muscle cells, including secondary muscle cells, show the same intensity of activity. The primordial pharynx and several cells in the trunk region also show evidence of the enzymatic activity, as described by Deno et al. (1985).

7-nitrobenz-2-oxa-l,3-diazole(NBD)-phallacidin is a drug that binds specifically to filamentous actin and a emits yellow fluorescence. Partial embryos were placed on glass coverslips and fixed for 7 min at 20°C in 4% formalin dissolved in Ca²⁺free artificial seawater buffered with 10 ran Tris-HCl (pH 7.0). Embryos were then extracted with acetone for 1 min at -20°C, and rinsed with buffered seawater. Extracted embryos were stained with a solution of 330 ng ml^-1 NBD-phallacidin (Wako Chemicals Ltd.) in buffered seawater for 30 min at 20°C, rinsed in buffered seawater, mounted in 50% glycerol and observed with a fluorescence microscope equipped with a B2 excitation filter cassette. When the larvae were stained with this drug, all tail muscle cells emitted an intense fluorescence (Fig. 2B). At high magnification, each myofibril was visible. Myofibrils were located only in the peripheral cytoplasm of muscle cells, and they ran at a slightly oblique angle to the anterior-posterior axis. The boundaries of non-muscle cells also emitted a weak fluorescence, most likely indicative of the presence of cytoskeletal actin. Fluorescence from cytoskeletal actin was faint and no filamentous structure was visible, so that such actin was easily distinguishable from myofibrils.

In order to examine the expression of antigens recognized by monoclonal antibodies in partial embryos, specimens were placed on glass coverslips, fixed for 10 min in methanol at -20°C, washed with phosphate-buffered saline (PBS) and incubated with hybridoma culture fluid for 1 h at room temperature. After washing with PBS, specimens were stained for 30 min with FITC-conjugated rabbit serum raised against mouse IgG (Miles-Yeda Ltd) diluted 1:60 in PBS. Specimens were washed again, mounted in 50% glycerol and observed with a fluorescence microscope equipped with a B2 excitation filter cassette.

The monoclonal antibody Mu-2 specifically recognizes differentiated muscle cells of Halocynthia larvae (Nishikata et al. 1987a) and it binds with the myosin heavy chain (Makabe and Satoh, 1989). In the larvae stained indirectly with the Mu2 antibody, fluorescence from FITC was completely restricted to muscle cells and no other cells emitted comparable fluorescence (4,Fig. 2C). With the exception of the nucleus, the entire cytoplasm of muscle cells was stained with this antibody. Even at high magnification, no filamentous structures of myofibrils were visible, possibly because of the method used for fixation. In all, three kinds of histochemical and immunohistochemical technique were used to evaluate the differentiation of muscle cells in partial embryos.

The monoclonal antibody Epi-2 recognizes cells of the larval epidermis and the larval tunic material which is secreted by the epidermal cells (Nishikata et al. 1987b). Figure 2D shows a larva stained with Epi-2 antibody.

The large size of Halocynthia eggs and use of a highresolution stereomicroscope permitted the identification and isolation of all blastomeres up to the 110-cell stage. Each muscle-progenitor blastomere was carefully isolated from embryos with a fine glass needle. Since embryos are bilaterally symmetrical (Fig. 1), the left and/or right blastomere(s) was isolated from each embryo.

The reliability of the identification and isolation of muscle lineage cells was examined in two ways. First, numbers of mitochondria in isolated muscle-lineage blastomeres were examined. Mitochondria in ascidian egg cytoplasm segregate preferentially to the musclelineage blasomeres, and ultimately they segregate to larval muscle cells (Reververi, 1956). DiOC₂ is a vital stain for mitochondria and is a useful marker for muscle-lineage cells of ascidian embryos (Zalokar and Sardet, 1984). In Halocynthia embryos, staining with DiOC₂ occurs mainly in muscle-lineage cells (Fig. 3A). Cells of the spinal cord and brain lineage contain some mitochondria, but fewer than muscle-lineage cells. The cells of other lineages contain few mitochondria. Embryos were stained with DiOC₂ at the 16-cell and 32-cell stages, and each muscle-lineage cell was then isolated at the 64-cell and 110-cell stage. The number of mitochondria in each isolated cell was immediately determined. Every isolated muscle-lineage cell (B7.4, B7.8, A7.8, b7.9 and b7.10 of the 64-cell embryo, and A8.16, b8.17 and b8.19 of the 110-cell embryo) was found to be rich in mitochondria when compared with isolated cells of other lineages (Fig. 3B and C).

After the 64-cell stage, the divisions of blastomeres become asynchronous. However, the timing of cell division is invariant in any individual, and the length of the cell cycle of each blastomere is unique and specific (Nishida, 1986). In a second examination, the timing of the divisions of isolated blastomeres was compared with that of blastomeres in normal whole embryos. In every case, at least one subsequent division of isolated cell occurred simultaneously with the division of their counterparts in normal embryos. Together, the results of these two kinds of test indicated the reliability of the identification and isolation of each muscle-lineage cell at the 64-cell and 110-cell stages.

Muscle differentiation in partial embryos was evaluated by histochemical staining for AchE (Fig. 4) and filamentous actin (Fig. 5A-C), and immunohistochemical staining for myosin heavy chains (4,Fig. 5D-F). Musclelineage blastomeres were separately isolated from 64cell embryos and 110-cell embryos and cultured until control larvae hatched. Each partial embryo was then examined for expression of features of muscle-specific differentiation. The results are summarized in Table 1. In the 64-cell embryos, almost all of the isolates of the B-line (B7.4, B7.8, and B7.5) developed three kinds of muscle-specific feature, but these features were not evident in isolates of the A and b-line (A7.8, b7.9, and b7.10). At the 110-cell stage, some isolated A8.16 cells developed the muscle-specific features, but none of the isolates of the b-line did so. Consistent results were obtained using the three kinds of marker for differentiation of muscle.

In normal embryogenesis, the B7.4 cell gives rise to eight muscle cells (Nishida, 1987). The partial embryos derived from the B7.4 cells consisted of eight AchEpositive cells (Fig. 4A) in most cases, which coincided with the developmental fate of this blastomere. When stained with NBD-phallacidin, filamentous actin was visible in each specimen (Fig. 5A). Myosin heavy chain (Mu-2 antigen) was also expressed in these partial embryos (4,Fig. 5D).

The developmental fate of the B7.8 cell in normal embryogenesis is four muscle cells. The partial embryos derived from this blastomere consisted of four cells, each of which expressed AchE (Fig. 4B), filamentous actin (4,Fig. 5B) and Mu-2 antigen (Fig. 5E). The development of B7.8 partial embryos coincided with the developmental fate of this cell in normal embryogenesis.

In present series of experiments, the B7.5 isolates were examined only for the expression of Mu-2 antigen (Table 1). All of the isolates developed the antigen. The developmental fate of this blastomere is two muscle cells and two endodermal cells.

The fate of the A7.8 cell of the 64-cell embryo includes two muscle cells and several spinal cord cells. None of the partial embryos derived from this blastomere developed muscle-specific features (Fig. 4C).

The developmental fate of the A8.16 cell of the 110cell embryo is two muscle cells and several spinal cord cells. In a previous study, incorporating the intracellular injection of horseradish peroxidase, the fate of the A8.16 cell in Halocynthia was shown to be only two muscle cells (Nishida, 1987). However, recent analysis using FITC-dextran as a tracer molecule indicates that the fate of each A8.16 cell is not just two muscle cells but also a small number of spinal cord cells (Nishida, unpublished observation). Moreover, tracing of the neural cell lineage in Clona intestinalis in serial optical sections had led to the same conclusion (Nicol and Meinertzhagen, 1988). These two lines of evidence strongly suggest that the fate of the A8.16 should be considered as two muscle cells plus a few spinal cord cells.

When the A8.16 cells were isolated at the 110-cell stage, 48%, 38% and 28% of the isolates developed AchE (Fig. 4D), filamentous actin (4,Fig. 5C), and Mu-2 antigen (Fig. 5F), respectively. Although the proportion of positive specimens with each kind of staining varied among batches of eggs, a significant fraction always stained positively in each case. Typical A8.16 embryos that were positive for staining (68% of the total number of positive specimens) had two larger positive cells and a few smaller negative cells, supporting the recent above-mentioned assertion of the fate of A8.16.

The fate of the b7.9 cell of the 64-cell embryo is three muscle cells, one cell of the endodermal strand or spinal cord, and a lot of epidermis cells. The fate of the b7.10 cell is two muscle cells, spinal cord cells and epidermis cells. In accordance with their developmental fates, the b7.9 and b7.10 partial embryos generated larval tunic materials, which are normally secreted by epidermal cells (Fig. 4E and F, arrowheads). However, no expression of any of the three kinds of marker of muscle differentiation was ever observed.

The fate of the b8.17 cell of the 110-cell embryo is mostly restricted to muscle, i.e., its fate is only a single cell of the endodermal strand or spinal cord in addition to three muscle cells. Nonetheless, the b8.17 partial embryos never showed any muscle-specific features (Fig. 4G). Likewise, although the fate of the b8.19 cell is two muscle cells and a few spinal cord cells, the b8.19 partial embryos also failed to express any musclespecific features (Fig. 4H). The b8.17 and b8.19 partial embryos did not secrete larval tunic materials, in accordance with the fact that the epidermal fate areas have segregated to their sibling cells.

The A-line progenitors of muscle cells isolated from the 64-cell embryos never developed muscle-specific features, while some of those isolated from the 110-cell embryos did so autonomously. The time at which isolates of A-line cells acquire the autonomous ability to develop muscle-specific features, i.e., the timing of determination, was therefore investigated in greater detail. My main aim was to assess whether the seventh cell division, which occurs between the 64-cell and the 110-cell stages, is the critical point in the determination.

The precise times of isolation adopted in this experiment are shown in 4,Fig. 6. The relative time of zero minutes indicates the beginning of division of the A7.8 cell, which yields the A8.15 cell (spinal cord progenitor) and A8.16 cell (muscle and spinal cord progenitor). The compaction of the A8.16 cell is accomplished at approximately 30min. The next division of the A8.16 cell occurs at 95 min, but it is not shown in Fig. 6. In the above-mentioned experiments, the isolation at the 64cell stage was performed between about -20 and -5 min relative to zero time, and the isolation at the 110-cell stage was performed between about 45 and 55 min. In this experiment, the A8.16 cells were isolated just after they were formed and at around the 110-cell stage. The isolation between relative times 5 and 15 min is referred to as early isolation, and that between 40 and 55 min, as late isolation. The A8.16 cell does not divide in the period between early and late isolation. The nuclei of the A8.16 cells were not yet visible at the time of early isolation, but they were clearly visible at the time of late isolation. The partial embryos that were derived from the isolates were examined for the expression of muscle-specific features.

Four independent experiments, with different batches of eggs, were carried out (Table 2). In two of these experiments, the embryos were examined for expression of AchE, and, in the other two experiments, for expression of the Mu-2 antigen. After early isolation, no expression of the muscle-specific features was detected. The results after late isolation were consistent with my previous results, with some (24%) of the partial embryos expressing muscle-specific features. The difference in results between times of isolation was shown to be statistically significant (Table 2). These results indicate that A8.16 cells isolated soon after they have been formed are not yet determined to give rise to muscle, while some portion of the cells isolated later in the cell cycle are determined to give rise to muscle.

During amphibian embryogenesis, mesodermal induction is involved in the formation of muscle (Nieuwkoop, 1973). In order to compare the properties of amphibian mesodermal induction and those of the process required for determination of muscle in the secondary lineage, differentiation of epidermis in partial embryos derived from secondary muscle-lineage cells (mainly bline) was examined (see also Discussion).

As mentioned earlier, most of the b7.8 and b7.9 cells, which are fated to give rise to both muscle and epidermis, generated a larval tunic, a feature of the epidermis, in isolation. Conversely, none of the b8.17 and b8.19 muscle-lineage cells examined, which have separated from their sibling cells with respect to their fate (i.e., from b8.18 and b8.20, which generate epidermis), developed larval tunic in isolation. Flowever, even when present, the larval tunic of partial embryos sometimes slipped off during the transfer of embryos between dishes, and it was also difficult to observe tunic material because of its transparency. Therefore, differentiation of epidermis was examined by immunohistochemical detection of the Epi-2 antigen (4,Fig. 2D).

As a positive control, epidermis precursor cells were isolated from the 110-cell embryos. Each of these isolates developed into a small permanent blastula, and in all cases the Epi-2 antigen was expressed (Fig. 7A). Another positive control was provided by the partial embryos that originated from isolated b7.9 and b7.10 cells, which are fated to become both epidermis and muscle. All partial embryos showed expression of Epi-2 (Fig. 7B). Flowever, the b8.17 cells (43 specimens) and b8.19 cells (22 specimens) which are b-line cells of the secondary muscle lineage, never developed the Epi-2 antigen. The expression of Epi-2 was also examined in the A8.16 partial embryos (14 specimens). No embryo expressed the antigen. In summary, differentiation to epidermis does not occur in partial embryos derived from the secondary muscle-lineage cells isolated before they acquire the capacity for autonomous development into muscle.

A detailed analysis of cell lineages during ascidian embryogenesis has revealed that tail muscle cells of larvae are derived from progenitor cells that belong to three different lines: the B-line (primary); and Aand b-lines (secondary) (Nishida and Satoh, 1983). When B4.1 blastomeres were isolated from 8-cell embryos and cultured until control larvae hatched, most of them autonomously developed muscle-specific features (AchE, Mu-2 antigen and myofilament). In contrast, A4.1 and b4.2 quarter embryos seldom expressed such features (Deno et al. 1985; Nishikata et al. 1987a; Meedel et al. 1987). Results such as these led Meedel et al. (1987) to suggest for the first time that different mechanisms may control the development of muscle cells in the primary and secondary muscle lineages.

As clearly shown in the present study, B-line musclelineage cells, isolated from 64-cell embryos, autonomously developed muscle-specific features (AchE, filamentous actin, Mu-2 antigen). In addition, some Aline isolates acquired the capacity for autonomous development around the 110-cell stage. If the isolation were performed at even later stages, we would expect that the proportion of the A-line partial embryos that showed muscle-specific features would increase. However, the isolation at stages later than the 110-cell stage is difficult because inaccuracies arise in identification of blastomeres under the stereomicroscope. Conversely, in the b-line isolates, none of the partial embryos showed any muscle-specific development even when cells were isolated as late as the 110-cell stage, at which time their developmental fates are predominantly restricted to muscle. For example, the fate of the b8.17 cell is three muscle cells and one small cell of the spinal cord or endodermal strand (see Fig. 6i in Nishida (1987)). Nevertheless, isolated b8.17 cells failed to show any muscle-specific development. Present results from isolation of blastomeres reconfirm that the consequences of isolation differ remarkably between primary and secondary lineages, and they provide strong evidence in support of the hypothesis that different mechanisms control the development of muscle in the two kinds of lineage, as advocated initially by Meedel et al. (1987).

Failure of muscle development in isolated cell of the secondary lineage is unlikely to be due to nonspecific damage caused by isolation procedures. The b7.9 and b7.10 cells, which are fated to generate both muscle and epidermis, did not express any muscle-specific features when isolated, but they did show autonomous development of epidermis-specific features. Thus, the failure to differentiate is specific to features of muscle cells in these cases. Moreover, the requirement for association between cells is not a nonspecific cell-mass effect because the B7.4 and B7.8 partial embryos expressed muscle-specific features, even though they consisted of only 8 and 4 cells. In contrast, A-line and b-line muscleprogenitor blastomeres that were isolated from 8-cell embryos developed into partial embryos which consisted of a lot of cells. Nevertheless, these embryos rarely developed muscle-specific features. The hypothesis that different mechanisms control the development of muscle in the primary and secondary lineages is also supported indirectly by the following two lines of evidence. 1. Expression of muscle-specific features in cleavage-arrested embryos: If the cleavage of cleavage-stage embryos is permanently arrested by treatment with cytochalasin B, an inhibitor of cytokinesis, the arrested embryos produce AchE and Mu-2 antigen only in blastomeres of the primary (B-line) muscle lineage. Blastomeres of the secondary (A-line and b-line) muscle lineage do not express the enzyme or the antigen (Whittaker, 1973; Satoh, 1979; Nishikata et al. 1987a). However, in the case of Ascidia ceratodes, Meedel et al. (1987) reported that cleavage-arrested 8cell embryos express AchE in the A-line blastomeres as well as in the B-line blastomeres. They also observed that A-line muscle-lineage blastomeres isolated from the 8-cell and 16-cell embryos develop AchE at high frequency, but they do not develop myofilaments. There are differences between species with respect to responses to isolation and cleavage-arrest of A-line cells. 2. Time of expression of differentiation-specific phenotypes: The expression of AchE in primary postmitotic precursors of muscle cells precedes that in secondary precursors of muscle cells (Nishida and Satoh, 1983; Deno et al. 1985; Meedel et al. 1987). In Halocynthia embryos, AchE activity is histochemically detectable first in B-line cells by the 14th hour of development (neurula), then in A-line cells by the 20th hour (early tailbud), and finally in b-line cells by the 25th hour (middle tailbud). It has also been reported that expression of mRNA for muscle actin in primary lineage cells precedes that in secondary lineage cells (Tomlinson et al. 1987).

It is noteworthy that, in Ciona, the three-quarter embryo that results from ablation of the pair of B4.1 blastomeres develops muscle cells in its caudal tip (Deno et al. 1984; Meedel et al. 1987). In Halocynthia, the three-quarter embryo also develops muscle-specific antigen in the caudal tip (our unpublished observation). These results indicate that cells of the primary muscle lineage are not required for the development of secondary muscle cells.

The precise timing of determination in the A-line muscle lineage appears to occur part of the way through the eighth generation. It should be noted that, irrespective of the stage of isolation (early isolation and late isolation during the eighth generation), the A8.16 cells have the same fate and contain the same cytoplasmic components, because no division occurs between the times of early and late isolations. Nevertheless, a clear difference is evident between the results of early and late isolations. Our experiment confirms that the low degree of restriction of the cell to a muscle-specific fate and/or the low degree of segregation of myoplasm are unlikely to be the only reasons for the observation that A-line muscle-lineage blastomeres do not show musclespecific development if isolated at early stages.

This result together with the results of isolation of the b-line muscle-lineage cells indicate that expression of muscle-specific features in the secondary lineages requires an association with cells from other lineages. Determination of muscle in the secondary lineages may be a process that is dependent on intercellular interactions. To test directly the role of such interactions in the determination of muscle in secondary lineages, experiments should be performed in which presumptive muscle cells are combined with other types of cell and the resultant effects on cell fates are followed.

During amphibian embryogenesis, the induction of mesoderm is involved in the formation of muscle (Nieuwkoop, 1973). It is known that the animal cap isolated from the blastula develops into epidermis when it is cultured as an explant. In contrast, when the animal cap is combined with vegetal cells, the cells of the animal cap give rise to muscle in addition to other mesodermal tissues and, of course, ectodermal tissues (Nieuwkoop, 1969). In ascidian embryos, b-line muscleprogenitor cells are cells of the animal hemisphere (Fig. 1). Moreover, all of the b-line cells other than four muscle-lineage cells of the 110-cell embryos are epidermis progenitors. By analogy with amphibian development, we might suppose that differentiation of the epidermis might have occurred in those b-line musclelineage partial embryos that did not show any evidence of muscle-specific differentiation. Epidermis fate is segregated and separated from muscle fate at the eighth generation (the 110-cell stage) in the b-line, facilitating the isolation of muscle progenitor cells that are not associated with epidermis fate, and allowing the possibility of differentiation to epidermis by b-line presumptive muscle cells to be tested.

Unlike the other cells of the b-line, isolated b8.17 and b8.19 cells do not develop into epidermis and, thus, bline muscle-lineage cells may already differ substantially from the other b-line epidermis-lineage cells even before the stage at which b-line muscle-lineage cells develop the capacity for autonomous development into muscle. It seems that, in the secondary muscle lineages, intercellular interactions may be required for differentiation of muscle phenotype, rather than the choice of a muscle pathway among the repertoire of developmental pathways. In this regard, the interactions between cells that are responsible for muscle development in secondary lineages may be considered to represent permissive induction (Gurdon, 1987). These results suggest that, although intercellular interactions are required for the process of muscle differentiation in the secondary lineage, the properties of the process differ from those of mesodermal induction in amphibian embryos.

However, we must not ignore another possible explanation for our results. All of the secondary musclelineage cells, such as the b8.17, b8.19, and A8.16 cells, are fated to give rise to spinal cord cells in addition to muscle cells. Therefore, the presumptive muscle cells that fail to develop into muscle cells in isolation may develop into spinal cord cells. Unfortunately, there is no way to examine this possibility, because we have no marker specific for the differentiation of spinal cord cells at present.

Evidence has accumulated to indicate the important roles of intercellular interactions in the development of certain kinds of tissue, even in animals with determinate and invariant cell lineages. In ascidian embryogenesis, the development of brain and pigment cells depends on such interactions (Rose, 1939; Reverberi et al. 1960; Nishida and Satoh, 1989). Recent results have demonstrated that such interactions are important in embryogenesis of C. elegans, even at the early cleavage stage (Schierenberg, 1987; reviewed by Emmons, 1987). Muscle development from the AB blastomere (minor muscle lineage) of C. elegans embryos depends on the presence of cells of other lineages (Priess and Thomson, 1987), as has also been shown to be the case in ascidian embryos, in this study.

I thank Dr N. Satoh for his valuable advice and constant encouragement throughout the course of this study, and for his critical reading of the manuscript. Thanks are also due to Dr T. Numakunai at the Asamushi Marine Biological Station for supplying the living materials and to Dr T. Nishikata, Kyoto Univ., for providing the monoclonal antibodies. I am also grateful to Dr A. Rossiter for his kind help in the preparation of the manuscript. This work was supported by a Grant-in-Aid from the Ministry of Education, Science and Culture, Japan (01740428).