Developmental autonomy of muscle fine structure in muscle lineage cells of ascidian embryos

We have observed ultrastructural features of muscle differentiation in the muscle lineage cells of cleavage-arrested whole embryos and partial embryos of ascidians. Whole embryos of Ciona intestinalis and Ascidia ceratodes were cleavage-arrested with cytochalasin B at the 8-cell stage and reared to an age equivalent to several hours after hatching; these embryos formed extensive myofilaments which were often further organized into myofibrils of different sizes and densities in the peripheral cytoplasm of the two muscle lineage blastomeres (B4.1 pair). Developing myofibrils in cleavage-arrested embryos resembled the muscle elements observed in normal hatched larvae, but were less uniformly organized. A similar development of myofilaments and myofibrils occurred in the muscle lineage cells of multicellular partial embryos reared to "hatching" age. These partial embryos resulted from the isolated muscle lineage pair (B4.1) of blastomeres of the 8-cell stage (Ciona and Ascidia), and from a muscle lineage blastomere pair (B5.2) isolated at the 16-cell stage (Ascidia). Muscle lineage cells in the partial embryos were readily identified by the dense aggregates of mitochondria in their cytoplasm. Taken together, these results from the two kinds of partial embryo effectively eliminate inductive interactions with embryonic tissues other than mesodermal as a necessary factor in the onset of self-differentiation in muscle lineage cells. The relative complexity of muscle phenotype expressed in cleavage-arrested and partial embryos attests to an unusually strong developmental autonomy in the ascidian muscle lineages. This autonomy lends further support to the theory that a localized and segregated egg cytoplasmic determinant is responsible for larval muscle development in ascidian embryos.     

Muscle lineage cytoplasm can change the developmental expression in epidermal lineage cells of ascidian embryos

Cytoplasm from muscle lineage blastomeres of an ascidian embryo can cause cells of a nonmuscle lineage to produce larval tail muscle acetylcholinesterase. Muscle cytoplasm was partitioned microsurgically into epidermal lineage blastomeres at the eight-cell stage. Posterior half-embryos (the two B3 cells) of Ascidia nigra were obtained first by separating the anterior and posterior blastomere pairs at the four-cell stage. At third cleavage, the two B3 cells divide into an ectodermal cell pair that gives rise solely to epidermal tissues, and a mesodermal-endodermal blastomere pair from which the tail muscle cells are derived. When the ectodermal and mesendodermal blastomere pairs were isolated from one another by microsurgery and reared as partial embryos, only cells originating from the mesendodermal blastomeres produced a histochemical acetylcholinesterase reaction. Immediately after cleavage of the isolated B3 cells into ectodermal and mesendodermal cell pairs, the cleavage furrows could be made to disappear by pressing firmly on the mesendodermal cells with a microneedle. Repeated up and down pressure with the microneedle at a new position across the mesendodermal cells caused furrows to reestablish in the new position, thereby incorporating mesodermal cytoplasm and increasing the size of the ectodermal cells. The cytoplasmically altered ectodermal blastomere pairs, which became detached from the mesendodermal cells by this microsurgical procedure, continued to divide and were reared to “larval” stages. One-third of these epidermal partial larvae produced patches of cells containing acetylcholinesterase. These results lend further support to the theory that choice of particular differentiation pathways (embryonic determination) in ascidian embryos is mediated by segregation of specific egg cytoplasmic determinants.     

Development of vestigial tail muscle acetylcholinesterase in embryos of an anural ascidian species.

1. The ascidian Molgula arenata produces an anural larva lacking a tail and other structural features of typical urodele larvae in the family Molglidae, yet its embryos developed a histochemically detectable acetylcholinesterase in the tail muscle rudiment. Development of the myoblasts seemed to fail during the neurula stage. 2. Larval enzyme activity occurred at a mean of 5--6% of the level found in the urodele species Molgula occidentalis and Molgula manhattensis, as measured by scanning integrating microdensitometry of the histochemical reaction product. Some anural larvae had as much as 20% of the enzyme activity in urodele larvae. 3. This example of vestigial expression in the absence of other urodele larval features further illustrates the autonomy of a histospecific enzyme development thought to be controlled by an egg cytoplasmic determinant. Partial suppression of the determinant might be the cause of this diminished expression. 4. Two other anural molgulid species, Molgula occulta and Bostrichobranchus pilularis, did not have vestigial larval enzyme and possibly have lost the determinant completely.     

Requirement of cell division for muscle actin expression in the primary muscle cell lineage of ascidian embryos

The role of cell division in the expression of muscle actin and its relationship to acetylcholinesterase (AChE) development was examined in cleavage-arrested embryos of the ascidian Styela. Muscle actin expression was detected by two-dimensional gel electrophoresis of radioactively labelled proteins and by in situ hybridization with a cDNA probe, whereas AChE activity was assayed by enzyme histochemistry. In the majority of cases, muscle actin expression was first detected in embryos arrested after the 16-cell stage. Some embryos showed muscle actin expression after arrest at the 8-cell stage, however, muscle actin mRNA did not accumulate in embryos arrested at earlier cleavages. The cells that expressed muscle actin in 8- to 64-cell cleavage-arrested embryos belonged to the primary muscle lineage; secondary muscle cell precursors did not express muscle actin. Zygotic muscle actin mRNA appeared to accumulate with myoplasmic pigment granules in the perinuclear region of cleavage-arrested embryos, suggesting that the myoplasm may have a role in the organization of muscle cells. In contrast to muscle actin, AChE was detected in a small proportion of embryos treated with cytochalasin as early as the 1- or 2-cell stage, and most embryos treated with cytochalasin at later cleavages expressed this enzyme in some of their cells. Most primary muscle lineage cells expressed both muscle actin mRNA and AChE, however, some cells expressed only muscle actin mRNA or AChE. The results suggest that at least three cleavages are required for muscle actin expression and that muscle actin and AChE expression can be uncoupled in cleavage-arrested embryos.     

Cell lineage and determination of cell fate in ascidian embryos

A detailed cell lineage of ascidian embryos has been available since the turn of the century. This cell lineage was deduced from the segregation of pigmented egg cytoplasmic regions into particular blastomeres during embryogenesis. The invariant nature of the cell lineage, the segregation of specific egg cytoplasmic regions into particular blastomeres, and the autonomous development of most embryonic cells suggests that cell fate is determined primarily by cytoplasmic determinants. Modern studies have provided strong evidence for the existence of cytoplasmic determinants, especially in the primary muscle cells, yet the molecular identity, localization, and mode of action of these factors are still a mystery. Recent revisions of the classic cell lineage and demonstrations of the lack of developmental autonomy in certain embryonic cells suggest that induction may also be an important mechanism for the determination of cell fate in ascidians. There is strong evidence for the induction of neural tissue and indirect evidence for inductive interactions in the development of the secondary muscle cells. In contrast to the long-accepted dogma, specification of cell fate in ascidians appears to be established by a combination of cytoplasmic determinants and inductive cell interactions.     

Determinative properties of muscle lineages in ascidian embryos

Blastomeres removed from early cleavage stage ascidian embryos and reared to 'maturity' as partial embryos often elaborate tissue-specific features typical of their constituent cell lineages. We used this property to study recent corrections of the ascidian larval muscle lineage and to compare the ways in which different lineages give rise to muscle. Our evaluation of muscle differentiation was based on histochemical localization and quantitative radiometric measurement of a muscle-specific acetylcholinesterase activity, and the development of myofilaments and myofibrils as observed by electron microscopy. Although the posterior-vegetal blastomeres (B4.1 pair) of the 8-cell embryo have long been believed to be the sole precursors of larval muscle, recent studies using horseradish peroxidase to mark cell lineages have shown that small numbers of muscle cells originate from the anterior-vegetal (A4.1) and posterior-animal (b4.2) blastomeres of this stage. Fully differentiated muscle expression in isolated partial embryos of A4.1-derived cells requires an association with cells from other lineages whereas muscle from B4.1 blastomeres develops autonomously. Clear differences also occurred in the time acetylcholinesterase activity was first detected in partial embryos from these two sources. Isolated b4.2 cells failed to show any muscle development even in combination with anterior-animal cells (a4.2) and are presumably even more dependent on normal cell interactions and associations. Others have noted an additional distinction between the different sources of muscle: muscle cells from non-B4.1 lineages occur exclusively in the distal part of the tail, while the B4.1 descendants contribute those cells in the proximal and middle regions. During the course of ascidian larval evolution tail muscle probably had two origins: the primary lineage (B4.1) whose fate was set rigidly at early cleavage stages and secondarily evolved lineages which arose later by recruitment of cells from other tissues resulting in increased tail length. In contrast to the B4.1 lineage, muscle development in the secondary lineages is controlled less rigidly by processes that depend on cell interactions.     

Ultrastructure and differentiation of ascidian muscle

Cell and Tissue Research - The larval muscle cells of Diplosoma macdonaldi contain subcortical and medullary myofibrils which are invested by fenestrated sheets of the sarcoplasmic reticulum....     

Ultrastructure and differentiation of ascidian muscle

Summary The larval caudal musculature of the compound ascidian Diplosoma macdonaldi consists of two longitudinal bands of somatic striated muscle. Approximately 800 mononucleate cells, lying in rows between the epidermis and the notochord, constitute each muscle band. Unlike the caudal muscle cells of most other ascidian larvae, the myofibrils and apposed sarcoplasmic reticulum occupy both the cortical and the medullary sarcoplasm.The cross-striated myofibrils converge near the tapered ends of the caudal muscle cell and integrate into a field of myofilaments. The field originates and terminates at intermediate junctions at the transverse cellular boundaries. Close junctions and longitudinal and transverse segments of nonjunctional sarcolemmata flank the intermediate junctions, creating a transverse myomuscular (TMM) complex which superficially resembles the intercalated disk of the vertebrate heart.A perforated sheet of sarcoplasmic reticulum (SR) invests each myofibril. The sheet of SR spans between sarcomeres and is locally undifferentiated in relation to the cross-striations. Two to four saccular cisternae of SR near each sarcomeric Z-line establish interior (dyadic) couplings with an axial analogue of the vertebrate transverse tubular system. The axial tubules are invaginations of the sarcolemma within and adjacent to the intermediate junctions of the TMM complex.The caudal muscle cells of larval ascidians and the somatic striated muscle fibers of lower vertebrates bear similar relationships to the skeletal organs and share similar locomotor functions. At the cellular level, however, the larval ascidian caudal musculature more closely resembles the vertebrate myocardium.This investigation was supported by Developmental Biology Training Grant No. 5-T01-HD00266 from the National Institute of Child Health and Human Development, National Institutes of Health, by National Research Service Award No. 1-F32-GM05259 (M.J.C.) from the National Institute of General Medical Sciences, National Institutes of Health, and by Research Grant No. BMS 7507689 (R.A.C.) from the National Science Foundation. A portion of this study was carried out at the Friday Harbor Laboratories of the University of Washington, and the authors gratefully acknowledge the cooperation and advice extended by the former Director, Dr. Robert L. FernaldResearch facilities were provided in part by Douglas E. Kelly, Professor and Chairman, Department of Anatomy, University of Southern California School of Medicine, Los Angeles, California 90033, USA. The provisions and counsel are warmly acknowledged     

Autonomous muscle cell differentiation in partial ascidian embryos according to the newly verified cell lineages

Recent analysis of cell lineages in ascidian embryos by the intracellular injection of a tracer enzyme has clearly demonstrated that muscle cells are derived not only from the B4.1-cell pair of the eight-cell stage embryo, as has hitherto been believed, but also from both the b4.2- and A4.1-cell pairs (H. Nishida and N. Satoh, 1983, Dev. Biol.99, 382–394). In order to reexamine the developmental autonomy in muscle lineage cells, the B4.1 pair was isolated from the eight-cell stage embryo. The progeny cells of the B4.1 pair, as well as those of the six other blastomeres, were then allowed to develop in isolation into partial embryos. Autonomous muscle cell differentiation not only in partial embryos originating from the B4.1 cells but also in those from the six other blastomeres was substantiated by (a) occurrence of localized histospecific muscle acetylcholinesterase and (b) development of myofibrils. These results support the validity of the recent cell lineage study and confirmed the self-differentiation potency of muscle lineage cells in ascidian embryos according to the newly verified cell lineages.     

Monitoring early neuronal differentiation by ion channels in ascidian embryos

According to the evolutionary tree proposed by Garstang, the tunicate larva has a central role in directing the ancestral sessile animal derived from primitive echinoderms into the stem for vertebrates by evolution through neoteny. The close similarity of the tunicate larval body plan to those of vertebrates and the extraordinary simplicity indicated by an extremely small cell population make the ascidian embryo and larva an excellent model system for analysis of vertebrate embryonic development. Furthermore, isolated anterior animal blastomeres from the Halocynthia eight-cell cleavage-arrested embryo, which are known to include presumptive brain vesicle region, autonomously develop long-lasting Ca-dependent action potentials which are characteristic of epidermal differentiation. However, when blastometeres are cultured in contact with the anterior vegetal blastomere, which are known to include presumptive notochordal region, and raised in contacted two cell systems, the same anterior animal blastomeres now develop neuronal Na⁺ spikes characterized by expression of Na⁺ channels and triethylammonium sensitive delayed rectifier K⁺ channels. This unique two-cell system enables us to examine roles of cell contact in various aspects of inductive differentiation at the cellular level. In this review, we focus on this simple cellular preparation and in particular, attempt to show how to make the preparation. © 1998 John Wiley & Sons, Inc. J Neurobiol 37: 3–22, 1998     

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

Muscle cells of the ascidian larva originate from three different lines of progenitor cells, the B-line, A-line and b-line. Experiments with 8-cell embryos have indicated that isolated blastomeres of the B-line (primary) muscle lineage show autonomous development of a muscle-specific enzyme, whereas blastomeres of the A-line and b-line (secondary) muscle lineage rarely develop the enzyme in isolation. In order to study the mechanisms by which different lines of progenitors are determined to give rise to muscle, blastomeres were isolated from embryos of Halocynthia roretzi at the later cleavage stages when conspicuous restriction of the developmental fate of blastomeres had already occurred. Partial embryos derived from B-line muscle-lineage cells of the 64-cell embryo (B7.4, B7.5 and B7.8) showed autonomous expression of specific features of muscle cells (acetylcholinesterase, filamentous actin and muscle-specific antigen). In contrast, b-line muscle-lineage cells, even those isolated from the 110-cell embryo (b8.17 and b8.19), did not express any muscle-specific features, even though their developmental fate was mainly restricted to generation of muscle. Isolated A-line cells from the 64-cell embryos (A7.8) did not show any features of muscle differentiation, whereas some isolated A-line cells from the 110-cell embryos (A8.16) developed all three above-mentioned features of muscle cells. This transition was shown to occur during the eighth cell cycle. These results suggest that the mechanism involved in the process of determination of the secondary-lineage muscle cells differs from that of the primary-lineage muscle cells. Interaction with cells of other lineages may be required for the determination of secondary precursors to muscle cells. The presumptive b-line and A-line muscle cells that failed to express muscle-specific features in isolation did not develop into epidermal cells. Thus, although interactions between cells may be required for muscle determination in secondary lineages, the process may represent a permissive type of induction and may differ from the processes of induction of mesoderm in amphibian embryos.     

Cell lineage and timing of fate restriction, determination and gene expression in ascidian embryos

Tadpole larvae of ascidians show the basic body plan of chordates. An ascidian larva consists of only few types of cells and has a relatively small number of cells. Cell lineages are simple and invariant among individuals and have been described in detail. The clonal restriction of developmental fate takes place considerably early in development. I review here the temporal relationship between fate restriction, determination and initiation of lineage-specific gene expression during ascidian embryogenesis. In several cases, determination and initiation of gene expression precede fate restriction and occur during the last cell cycle before fate restriction. Such a phenomenon contradicts the traditional view of fate specification and has several important implications for the understanding of the way in which cells execute the developmental pathway.     

Muscle cell differentiation in ascidian embryos analysed with a tissue-specific monoclonal antibody

Utilizing a muscle-specific monoclonal antibody (Mu-2) as a probe, we analysed developmental mechanisms involved in muscle cell differentiation in ascidian embryos. The antigen recognized by Mu-2 was a single polypeptide with a relative molecular mass of about 220 X 10(3). It first appeared at the early tailbud stage and continued to be expressed until the swimming larva stage. There were distinct and separate puromycin and actinomycin D sensitivity periods during the occurrence of the antigen, suggesting the new synthesis of the polypeptide by developing muscle cells. Embryos that had been permanently arrested with aphidicolin in the early cleavage stages up to the 32-cell stage did not express the antigen. DNA replications may be required for the antigen expression. Embryos that had been arrested with cytochalasin B in the 8-cell and later stages developed the antigen, and the number and position of the arrested blastomeres exhibiting the differentiation marker almost corresponded to those of the B4.1-line muscle lineage. Furthermore, in quarter embryos developed from each blastomere pair isolated from the 8-cell embryo, all the B4.1 as well as a part of b4.2 partial embryos expressed the antigen, while the a4.2 and A4.1 partial embryos did not show the antigen expression. These results may provide further support for the existence of cytoplasmic determinants for muscle cell differentiation in this mosaic egg.     

Fine structure and differentiation of ascidian muscle, 2. Morphometrics and differentiation of the caudal muscle cells of Distaplia occidentalis tadpoles

The locomotor function of the caudal muscle cells of ascidian larvae is identical with that of lower vertebrate somatic striated (skeletal) muscle fibers, but other features, including the presence of transverse myomuscular junctions, an active Golgi apparatus, a single nucleus, and partial innervation, are characteristic of vertebrate myocardial cells. Seven stages in the development of the compound ascidian Distaplia occidentalis were selected for an ultrastructural study of caudal myogenesis. A timetable of development and differentiation was obtained from cultures of isolated embryos in vitro. The myoblasts of the neurulating embryo are yolky, undifferentiated cells. They are arranged in two bands between the epidermis and the notochord in the caudal rudiment and are actively engaged in mitosis. Myoblasts of the caudate embryo continue to divide and rearrange themselves into longitudinal rows so that each cell simultaneously adjoins the epidermis and the notochord. The formation of secretory granules by the Golgi apparatus coincides with the onset of proteid-yolk degradation and the accumulation of glycogen in the ground cytoplasm. Randomly oriented networks of thick and thin myofilaments appear in the peripheral sarcoplasm of the muscle cells of the comma embryo. Bridges interconnect the thick and thin myofilaments (actomyosin bridges) and the thick myofilaments (H-bridges), but no banding patterns are evident. The sarcoplasmic reticulum (SR), derived from evaginations of the nuclear envelope, forms intimate associations (peripheral couplings) with the sarcolemma. Precursory Z-lines are interposed between the networks of myofilaments in the vesiculate embryo, and the nascent myofibrils become predominantly oriented parallel to the long axis of the muscle cell. Muscle cells of the papillate embryo contain a single row of cortical myofibrils. Myofibrils, already spanning the length of the cell, grow only in diameter by the apposition of myofilaments. The formation of transverse myomuscular junctions begins at this stage, but the differentiating junctions are frequently oriented obliquely rather than orthogonally to the primary axes of the myofibrils. With the appearance of H-bands and M-lines, a single perforated sheet of sarcoplasmic reticulum is found centered on the Z-line and embracing the I-band. The sheet of SR establishes peripheral couplings with the sarcolemma. In the prehatching tadpole, a second collar of SR, centered on the M-line and extending laterally to the boundaries with the A-bands, is formed. A single perforated sheet surrounds the myofibril but is discontinuous at the side of the myofibril most distant from the sarcolemma. To produce the intricate architecture of the fully differentiated collar in the swimming tadpole (J. Morph., 138: 349, 1972). the free ends of the sheet must elevate from the surface of the myofibril, recurve, and extend peripherally toward the sarcolemma to establish peripheral couplings. Morphological changes in the nucleus, nucleolus, mitochondria, and Golgi bodies are described, as well as changes in the ground cytoplasmic content of yolk, glycogen, and ribosomes. The volume of the differentiating cells, calculated from the mean cellular dimensions, and analyses of cellular shape are presented, along with schematic diagrams of cells in each stage of caudal myogenesis. In an attempt to quantify the differences observed ultrastructurally, calculations of the cytoplasmic volume occupied by the mqjor classes of organelles are included. Comparison is made with published accounts on differentiating vertebrate somatic striated and cardiac muscles.     

The genetic program to specify ectodermal cells in ascidian embryos

The ascidian belongs to the sister group of vertebrates and shares many features with them. The gene regulatory network (GRN) controlling gene expression in ascidian embryonic development leading to the tadpole larva has revealed evolutionarily conserved gene circuits between ascidians and vertebrates. These conserved mechanisms are indeed useful to infer the original developmental programs of the ancestral chordates. Simultaneously, these studies have revealed which gene circuits are missing in the ascidian GRN; these gene circuits may have been acquired in the vertebrate lineage. In particular, the GRN responsible for gene expression in ectodermal cells of ascidian embryos has revealed the genetic programs that regulate the regionalization of the brain, formation of palps derived from placode-like cells, and differentiation of sensory neurons derived from neural crest-like cells. We here discuss how these studies have given insights into the evolution of these traits.     

An evolutionary change in the muscle lineage of an anural ascidian embryo is restored by interspecific hybridization with a urodele ascidian

Anural ascidians do not develop into a conventional tailed larva with differentiated muscle cells, however, embryos of some anural ascidian species retain the ability to express acetylcholinesterase (AChE) in a vestigial muscle cell lineage. This study examines the number of AChE-positive cells that develop in the anural ascidian Molgula occulta relative to that in the closely related urodele (tailed) species, Molgula oculata. Histochemical assays showed that M. oculata embryos develop 36 to 38 AChE-positive cells, consistent with the number of tail muscle cells expressed in other urodele ascidians. In contrast, M. occulta embryos develop a mean of only 20 AChE-positive cells in their vestigial muscle lineage. Cleavage-arrested embryos of the anural species express AChE only in B-line blastomeres, showing that the vestigial muscle lineage cells are derived from the primary muscle lineage. Less than the expected number of AChE-positive B-line cells develop in cleavage-arrested anural embryos, however, implying that the allocation of primary muscle lineage cells is decreased. Eggs of the anural species can be fertilized with sperm of the urodele species resulting in the development of some larvae that contain a short tail and/or a brain melanocyte, specific features of urodele larvae. The typical urodele number of AChE-positive cells is restored in some of these hybrid embryos. Both primary and secondary muscle lineages are restored because cleavage-arrested hybrid embryos develop more AChE-positive cells in the B-line blastomeres and supernumerary AChE-positive cells in the A-line blastomeres. Hybrid embryos that develop the urodele complement of AChE-positive cells also form a tail and/or a brain melanocyte showing that restoration of muscle lineage cells is coupled to the development of other urodele features. AChE expression occurred in anural embryos with disorganized or dissociated blastomeres, indicating that AChE expression is determined autonomously. It is concluded that an evolutionary change in the allocation of larval muscle lineage cells occurs during development of the anural ascidian M. occulta which can be restored by interspecific hybridization with the urodele ascidian M. oculata.