Quantitative regulation of acetylcholinesterase development in the muscle lineage cells of cleavage-arrested ascidian embryos

There appears to be an egg cytoplasmic determinant that regulates larval muscle differentiation in ascidian embryos (subphylum Urochordata, class Ascidiacea). This determinant, or perhaps group of determinants, is localized in the egg and becomes segregated during early cleavages of the zygote into the muscle lineage cells where it eventually plays some role in initiating differentiation. Such an interpretation is implicit in Conklin’s (1905) classic observation that a specific yellow crescent region of cytoplasm in the fertilized egg (of some species) becomes segregated into the muscle lineage cells. A determinant hypothesis is supported also by cell lineage fate maps and the exceptional developmental autonomy expressed by isolated lineage cells in conforming to the predictions of these fate maps (reviewed by Whittaker, 1979a).

Experimental techniques combined with histochemical and ultrastructural observations provide results that suggest even more strongly that such an ascidian larval muscle determinant exists; it is associated with development of a histospecific larval muscle acetylcholinesterase and other features of muscle differentiation. Surgically isolated blastomeres from the muscle cell lineage of 4-cell- and 8-cell-stage embryos eventually develop acetylcholinesterase in division products of the isolated blastomeres (Durante, 1957; Whittaker, Ortolani & Farinella-Feruzza, 1977). In addition, embryos which are cleavage-arrested with cytochalasin B at various stages up to the 64-cell stage develop enzyme only in the muscle lineage blastomeres (Whittaker, 1973,1979b; Satoh, 1979). Recently, we have found that partial embryos resulting from isolated muscle lineage blastomeres as well as whole embryos cleavage arrested with cytochalasin B both develop myofilaments and partially organized myofibrils in the appropriate cells (Crowther & Whittaker, 1983). Cleavage-arrested ascidian embryos also acquire electrical excitability in membranes of their muscle lineage blastomeres (Takahashi & Yoshii, 1981).

Perhaps the most convincing evidence in favour of a determinant hypothesis is the result of those experiments which move presumptive myoplasm to new locations in the embryo. When yellow crescent myoplasm is moved mechanically into extra blastomeres at the 8-cell stage by altering the plane of third cleavage, it causes these extra cells to produce acetylcholinesterase (Whittaker, 1980). Presumptive myoplasm that is shifted into ectodermal lineage blastomeres by a direct microsurgical technique also initiates acetylcholinesterase development in some of the epidermal cell progeny (Whittaker, 1982).

Because cleavage-arrested embryos develop tyrosinase and melanin pigment only in the two melanocyte lineage cells of the larval brain, this observation suggests a segregation at each division in the two lineages of an egg cytoplasmic determinant that regulates melanocyte differentiation (Whittaker, 1973). Since cleavage-arrested embryos produce the same actual amounts of differentiation end products in the larger multinucleate cells as in the terminally differentiated normal melanocytes (Whittaker, 1979c, 1981), the determinant may possibly be exercising quantitative as well as qualitative control over melanocyte development. The present paper reports that muscle lineage cells of some cleavage-arrested embryos developed the same quantities of acetylcholinesterase activity as normal embryos. This finding also raises the prospect that the egg cytoplasmic agent involved in muscle expression might be exerting a quantitative control over enzyme development.

Clona intestinalis (L.) was collected in the vicinity of Woods Hole, Massachusetts, June through October, and maintained in tanks with running sea water under conditions of constant light. Eggs and sperm were obtained surgically from the gonoducts of animals. Embryos were cultured in filtered sea water at 18 ± 0·1 °C using a refrigerated constant temperature water bath (WilkensAnderson Lo-Temp). Some embryos were reared for periods of time in 2 μg/ml cytochalasin B (Sigma Chemical Company). Cytochalasin B was dissolved in a stock solution at 1 mg/ml in dimethyl sulfoxide (DMSO). The resulting sea water solutions contained 0·2 % DMSO, a concentration that had no detectable effect on normal embryonic development.

Acetylcholinesterase (E.C. 3.1.1.7) was localized in embryos by the Karnovsky & Roots (1964) procedure after 2-3 min fixation in cold (5 °C) 80 % ethanol. Incubation was for 60 or 120 min at 18 ± 0·1 °C; older embryos were incubated only 60 min to avoid over reaction. Various substrate and inhibitor controls for the identity of the Ciona enzyme are described elsewhere (Whittaker et al. 1977 ; Meedel & Whittaker, 1979).

Cytochrome oxidase (E.C. 1.9.3.1) was localized by the 3,3’-diaminobenzidine (DAB) reaction of Seligman, Karnovsky, Wasserkrug & Hanker (1968) containing cytochrome c substrate (0·5 mg/ml). Peroxidase activity was prevented by including 20 μg/ml Sigma C-40 catalase in the reaction medium. Fixation was for 10 min at 5 °C in Karnovsky’s (1965) fixative, but with formaldehyde and glutaraldehyde each reduced to 1·5%. Embryos were rinsed afterwards for 10 min (5 °C) in the cacodylate-buffered washing solution recommended by Karnovsky (1965), and incubated for 90 min at 18±0·l °C in the incubation medium.

After the respective histochemical reactions, embryos were dehydrated in ethanol, cleared in xylene, and mounted in damar resin. The two histochemical methods produced essentially permanent colour reactions. Embryos were handled throughout the histochemical and mounting procedures in 11mm diameter open glass tubes covered on one end with No. 21 Standard silk bolting cloth. All histochemical reagents were obtained from the Sigma Chemical Company.

Acetylcholinesterase activity can be measured quantitatively in similarly treated histochemical preparations by colorimetric assay of the relative rates of reaction product accumulation. Concentrations of the insoluble reaction product of the Karnovsky & Roots (1964) procedure, cupric ferrocyanide, obey the Beer-Lambert law. The amount of this product can, therefore, be measured in histochemically stained specimens by techniques of microdensitometry (Storm-Mathison, 1970; Wenk, Krug & Fletcher, 1973). Small spherical embryos which have not hatched from their chorions provide an almost ideal geometry for microdensitometry measurements provided, as in the case of Ciona, that measurements can be done adequately with lower-power objectives where there is a reasonable depth of focus, and where the size of the embryo conforms to a significant portion of the densitometer scanning area in the optical field (Whittaker, 1981).

Background staining from the Karnovsky & Roots (1964) direct colouring reaction for cholinesterase activity is not a serious difficulty for microdensitometry studies. Staining of enzyme-containing Ciona embryos with a reaction medium lacking the acetylthiocholine substrate or one in which butyrylthiocholine was substituted for acetylthiocholine revealed no localized enzyme activity. The background colouration in these embryos was no different from that found in non-muscle regions of normally stained embryos, nor from the background in normally stained embryos fixed at times before localized staining develops. Consequently, early embryos (taken at 2-6h of development before the occurrence of enzyme activity) were stained and used as ‘reagent’ subtraction blanks in the spectrophotometric measurements, on the assumption that optical density readings after such subtractions accurately express the real enzyme activity. Such reagent blank embryos were always removed from the same group being studied, placed in a refrigerator at 5 °C, and mixed with normal and experimental embryos at the appropriate later time of development so that the reagent blanks underwent identical fixation and reaction conditions.

A Vickers M85 scanning and integrating microdensitometer (Smith, Moore & Goldstein, 1975) was used to measure the reaction product in damar-mounted ascidian embryos. Measurements were made at 485 nm by the subtractive method. A 20x objective and a scanning spot of 2μm diameter were used in conjunction with a mask of 212 μm diameter (C-2) and a 400 x 400μm (3 x 3) scanning frame size. The fixed and stained embryos are 120-170 μm in diameter; shrinkage is variable according to embryonic stage used and the dehydration time sequence. Each slide contained embryos fixed at a time before normal acetylcholinesterase production (embryos 2-6h of age); the mean optical density measurement of 25 such embryos was used as a subtractive blank value for each measurement of the experimental and control embryos on the same slide. Final results were based on the mean value of 25 embryos selected as having the appropriate orientation for accurate measurements to be made. This selection was essentially random. Machine optical density readings of the enzyme activity were converted to absolute integrated optical density units by calibrating the instrument against a No. 96 Kodak 1·00 Neutral Density filter (10·0% transmission).

Identical settings of the Vickers instrument (as above) were used to measure optical density of the oxidative polymerization product of DAB in a quantitative cytochrome oxidase assay, except that a different wavelength (460 nm) was selected (Marinos, 1978). Subtractive blanks for these measurements consisted of embryos from the stages being studied which were incubated for 90 min in parallel reaction mixtures containing 10mM-KCN, a potent inhibitor of cytochrome oxidase activity. Such embryos were then mixed with normally stained embryos and mounted on the same slide. Suitability of the DAB reaction for quantitative histochemical measurements of cytochrome oxidase activity has been confirmed by Frasch, Itoiz & Cabrini (1978).

Two kinds of test indicated that the microdensitometry method of measuring acetylcholinesterase activity was capable of detecting significant differences in enzyme activity. Quantity of reaction product formed (absolute optical density units) in whole embryos was proportional to length of incubation time for the enzymatic reaction (Fig. 1). Also, enzyme activity in the whole embryos as measured by microdensitometry increased linearly with development time (Fig. 2).

At early development stages acetylcholinesterase occurs in either a posterior crescent of cells or in two thick muscle bands in the gradually developing tailbud. Orientations of the early embryo were selected for measurement such that the densitometer could be focused directly on either the crescent or the muscle bands (e.g., Fig. 3). At later ‘larval’ stages, when the tail had grown out and become thinner, measurements were made after focusing the microdensitometer on the central notochordal level of the embryo tail in lateral orientation (Figs 4,5). All normal embryos of appropriate age produced a localized histochemical reaction for acetylcholinesterase; only orientation of the embryo on the slide (Figs 3-5) served as a basis for selecting the embryos to be measured.

The relationship between product formed and enzyme incubation time was tested at three developmental stages: 10 h, 12 h, and 16 h. In 10 h embryos, product accumulation was proportional to time for 120 min (Fig. 1A). At later stages accumulation was proportional to time for about 60min (Figs 1A, IB). Extensions of the time intervals (not shown) all indicated that enzyme activity declined in relation to time after 60-120min. Activity measurements were based, therefore, on a 60-120min incubation period, depending on the age of embryo used.

Results of enzyme activity measurements in a series of developmental stages were consistent with the likelihood that activity is proportional to amount of enzyme present. There was a linearly increasing enzyme activity during 10-18 h of development, and activity was proportional during this time to age of the embryos (Fig. 2A). This activity closely paralleled specific activity measurements made on Ciona embryo homogenates by a radiometric assay of enzyme activity (Meedel & Whittaker, 1979), as shown in Fig. 2B. These curves are directly comparable because there is no change in total protein content per embryo during embryonic development (Meedel & Whittaker, 1979). A given weight of protein (1 mg) is equal to a fixed number of embryos at all developmental stages up to hatching. Ciona larvae begin hatching at 18 h, with the first few larvae breaking free of the chorionic membrane at that time.

Many cleavage-arrested early embryos eventually develop acetylcholinesterase activity in blastomeres of the muscle lineage, but the total number of embryos doing this is usually small and variable (Whittaker, 1973). Some cytochalasin-treated 1-cell-stage embryos developed enzyme in the whole egg (Fig. 6); cleavage-arrested 4-cell(Fig. 7) and 8-cell-stage (Fig. 8) embryos frequently developed acetylcholinesterase in each of their two muscle lineage blastomeres. Often the relative intensity of staining appeared to be the same in both blastomeres. Microdensitometry measurements were made on the experimental embryos by focusing the microdensitometer at a median level of the stained blastomeres and by selecting only embryos with two equal-staining blastomeres where the blastomeres occurred in approximately the same plane of focus. Except for these restrictions of orientation and blastomere pairs having approximately equal-staining intensity (in the 4-cell and 8-cell stages) embryos were measured as encountered during a progressive horizontal and vertical examination of the slides. Selection was assumed to be random but no statistical test of randomness was applied to the data.

Enzyme activity in cleavage-arrested embryos was examined at two time periods: 12 h and 16 h embryos. Since the ontogeny curve for acetylcholinesterase activity was linear with time during the later part of embryonic development (Figs 2A, 2B), it was probably not important which later time was selected for comparison between normal and cleavage-arrested embryos. One correction factor was incorporated directly into the experimental design. Cleavage-arrested embryos were about 30 min slower than the time at which enzyme was first observed histochemically in control embryos. Consequently, experimental embryos were arranged so that they were 30 min older than the normal control embryos to which they would be compared.

Embryos from the same collection of eggs (three to four animals) were taken for experimental, control, and subtraction blank measurements. Experimental embryos were fertilized first (with a mixed sperm suspension) and placed in 2 ig/ml cytochalasin B immediately after fertilization for 1-cell stages and shortly after 90 and 120 min for 4-cell and 8-cell stages. Control embryos were fertilized 30 min later; subtraction blank embryos were removed from control group dishes 2-3 h after fertilization and refrigerated at 5 °C. When the control group reached the appropriate age, all three groups were recombined in the same fixation tube, fixed and incubated together, and mounted on the same microscope slide. The possibility of inadvertently creating non-specific differences in enzyme content between experimental and control embryos was greatly reduced by using the same blanks and by keeping as many other variables as constant as possible.

The first comparisons were made at 12 h of normal development. Two sets of data are shown for each of the cleavage-arrested stages (Table 1). There was no large difference in enzyme activity between means of the cleavage-arrested and normal embryos; activity in normal and experimental embryos appeared to be the same.

Since it is possible that an accelerated enzyme synthesis might occur in experimental embryos for only a relatively short time, activities were also compared at a later development stage (16 h). Such measurements showed the higher activity levels of 16 h embryos and also revealed no difference in acetylcholinesterase activity between normal and cleavage-arrested embryos (Table 2). Since enzyme activity was still proportional to developmental time up to 18 h (Fig. 2A), the 16 h stage activity does not represent a saturation level for measurements by this technique.

Another enzyme, cytochrome oxidase, did not follow the same pattern of increase in cleavage-arrested embryos as it does in normal embryos. Cytochrome oxidase activity was measured in normal and cleavage-arrested embryos by a microdensitometry method. The DAB reaction for cytochrome oxidase resembles the acetylcholinesterase assay in being a simple, easily controlled reaction that yields very little background staining (Figs 9, 10). Enzyme activity in 16 h embryos was double that of normal 4-cell and 8-cell embryos (Table 3). This result agrees closely with that found by D’Anna (1966) where he measured cytochrome oxidase activity in homogenates of Ciona embryos by a manometric technique and found a doubling of activity during the same period of development. Comparison of the two results shows the microdensitometry method to be quite adequate for measuring changes of cytochrome oxidase activity during a 2-16 h time.

Cytochrome oxidase activity in cleavage-arrested embryos increased only slightly during a 16 h development period (Table 3). Other series (not shown) had activity changes within the same 2-19 % range. Obviously, no quantitative regulation of cytochrome oxidase development occurred. All embryos, cleavage-arrested as well as control, produced a localized cytochrome oxidase reaction. The only basis used in selecting embryos to measure quantitatively was their orientation on the slide (Figs 9, 10), such that strongly staining regions would fall within a median plane of focus of the microdensitometer.

Cleavage-arrested and normal 4-cell- and 8-cell-stage embryos could not be combined on the same slides because they were indistinguishable in their staining characteristics. Cleavage-arrested and cleavage-stage control embryos were incubated separately with 16 h control embryos from the same group. The measurement of each cytochalasin-arrested embryo was normalized to the 16 h value of the cleavage-stage control set by applying the ratio of the two 16 h mean (N = 25) values. A secondary problem is a variability in staining introduced by slight solubility of the enzyme reaction product in alcohols during the dehydration of embryos. This accounts for the differences in the 16 h measurements seen in Table 3, and necessitated the inclusion of 16 h control embryos in each group of embryos processed so that results could be calculated relative to the 16 h control value (% increase).

Microdensitometry measurements show clearly that some cleavage-arrested early Ciona embryos produced an amount of acetylcholinesterase activity similar in range to that of normal embryos developing for the same length of time. A serious bias, however, has been introduced into selection of the cleavage-arrested embryos to be measured. Many cleavage-arrested embryos make no enzyme, and others do not produce (by visual estimation) equivalent amounts of enzyme reaction product in the two lineage blastomeres at 4-cell and 8-cell stages; non-reactive embryos and these partially reactive embryos were disregarded in making the measurements reported in Tables 1 & 2. The acetylcholinesterase results presented in this paper are offered with the caveat that one can ignore ‘non-developing’ cleavage-arrested embryos and those in which development does not appear to be symmetrical or otherwise ‘normal’. Findings of future studies may conceivably invalidate this assumption.

The reasons why some cleavage-arrested embryos fail to produce enzyme in some or all of the muscle lineage cells are not known, but the most probable explanation is that a certain number of nuclear divisions (or DNA replications) must occur before the cell is competent to undergo differentiation (Satoh, 1982). Preliminary experimental results of my own are consistent with this explanation. In Ciona embryos dechorionated at fertilization, one can observe easily which of them become multinucleate during development in cytochalasin B. Those which are not obviously multinucleate in appearance do not later give reactions for acetylcholinesterase, whereas multinucleate embryos do. Continuity of nuclear divisions seems to fail early in some cytochalasin-treated embryos; such embryos are ‘non-developing’.

Regulation of acetylcholinesterase expression in cleavage-arrested embryos occurs independently of the normal cytoplasmic volume associated with larval muscle cells. The terminal number of larval muscle cells is 36 (Berrill, 1935), but most of the muscle lineage volume is already established at the 64-cell stage (Reverberi, 1971; Whittaker, 1979a). The volume of the lineage cells at the 64-cell stage represents approximately the relative cytoplasmic volume of larval muscle: l/8th (8 cells out of 64) of the original egg cytoplasmic volume (Fig. IIA). Cleavage-arrested 1-cell stages have, therefore, about eight times the volume of normal muscle lineage cell cytoplasm. One can calculate that the two muscle lineage cells of cleavage-arrested 4-cell stages have approximately four times the cytoplasmic volume of the normal larval muscle mass. Inspection of the muscle lineage territory at the 8-cell stage (Fig. 11B), as determined by the Ortolani (1954) marking experiments, shows that half of the two muscle lineage cells is future myoplasm: the two cells in cleavage-arrested 8-cell stages have about twice the volume of normal myoplasm.

Quantitative acetylcholinesterase expression is not so obviously exclusive of a fixed nuclear number. Cleavage-arrested embryos are very clearly multinucleate (Crowther & Whittaker, 1983), but without counting the nuclei histologically at the time of first enzyme expression one cannot know whether expression in cleavage-arrested embryos occurs in the presence of many more than the maximum number (36) usually associated with myoplasm. Perhaps there are never more than an average of 36 nuclei associated with myoplasm at the time of first expression in cleavage-arrested embryos. This seems an unlikely coincidence. Given a normal progression of nuclear divisions, 1-cell, 4-cell, and 8-cell cleavage-arrested embryos should have significantly different numbers of nuclei per myoplasm-containing cells. Certainly during early stages of treatment with cytochalasin B, nuclei are observed to divide regularly on approximate schedule (Brachet & Tencer, 1973; Whittaker, 1973).

Another example of end-product regulation is found in the melanocyte lineages of cleavage-arrested ascidian embryos: regulation of tyrosinase activity levels and amounts of melanin pigment occurs independently of cytoplasmic volume and also of nuclear number (Whittaker, 1979c, 1981). A cytoplasmic determinant seems to be segregated in the melanocyte lineages (Whittaker, 1973). Perhaps quantitative regulation in cleavage-arrested embryos occurs only in relation to differentiation pathways governed by egg cytoplasmic determinants.

Quantitative regulation of end products is not a general property of expression in cleavage-arrested ascidian embryos. Mitochondrial cytochrome oxidase, a universal rather than histospecific protein, changes only slightly in cleavage-arrested Ciona embryos. Large numbers of mitochondria become segregated into the muscle lineage cells of ascidian embryos, but mitochondrial enzymes are not known to be regulated by a differentially segregated determinant (Whittaker, 1979b). Since biosynthesis of cytochrome c oxidase requires participation of both mitochondrial and cytoplasmic translation systems (Poynton, 1980), its regulation in cleavage-arrested embryos may fail to occur for reasons unrelated to lack of involvement with a determinant factor.

The nature of the cytoplasmic determinant for muscle differentiation is unknown, but the hypothesis that determinants are the differential segregation of preformed maternal messenger RNA (mRNA) for histospecific proteins (Reverberi, 1971; Brachet, 1974) could explain most features of muscle determinant behaviour, including quantitative regulation of end product. A fixed amount of maternal mRNA for muscle acetylcholinesterase might, for example, give rise to a very definite quantity of enzyme irrespective of cytoplasmic volume or nuclear number. Substantial contrary evidence, however, denies that the acetylcholinesterase determinant is enzyme mRNA.

Results of experiments with actinomycin D, an inhibitor of RNA synthesis, identify a period of new RNA synthesis beginning at mid-gastrulation in various ascidian species as essential for later expression of muscle acetylcholinesterase (Whittaker, 1973, 1979b; Meedel & Whittaker, 1979; Satoh, 1979). Meedel & Whittaker (1983) have now found that RNA extracted from Ciona embryos at mid-gastrula and later stages elicits synthesis of immunospecific Ciona acetylcholinesterase in Xenopus laevis oocytes; RNA extracted from stages earlier than mid-gastrula does not. The mRNA for muscle acetylcholinesterase is apparently first synthesized at mid-gastrulation as implied by the results of actinomycin D experiments. Such findings also preclude occurrence of a maternally preformed but inactive proenzyme.

Similar amounts of acetylcholinesterase activity in normal and cleavage-arrested embryos indicate that some processes of genetic transcription and translation proceed normally in cleavage-arrested embryos. Possibly this quantitative control of differentiation end product is evidence of simple and as yet unappreciated properties of the transcriptional and translational control systems operating during embryonic development. More likely, however, quantitative control is a feature of development in those ‘mosaic’ embryos which have maternally preformed elements directing later pathways of development. Such control may be linked directly to the manner in which these egg cytoplasmic determinants function.

This work was supported by Grant HD-16547 from the National Institute of Child Health and Human Development, DHHS, and March of Dimes Birth Defects Foundation Grant 1-780. I thank Dr Vincent J. Cristofalo for permission to use the Vickers M85 microdensitometer at the Wistar Institute of Anatomy and Biology in Philadelphia.