Muscle development and lineage-specific expression of CiMDF, the MyoD-family gene of Ciona intestinalis.

The expression pattern of CiMDF, the MyoD-family gene of Ciona intestinalis, was analyzed in unmanipulated and microsurgically derived partial embryos. CiMDF encodes two transcripts during development (coding for distinct proteins), the smaller of which, CiMDFa, was detected in maternal RNA. Zygotic activity of CiMDF initiated in cleaving embryos of 32-64 cells. Both CiMDFa and CiMDFb transcripts were detected at this time; however, CiMDFa accumulated more rapidly before declining in abundance such that, by the early tail-formation stage, CiMDFb was more prevalent. Microsurgical isolations of various lineage blastomeres from the eight-cell stage were used to analyze CiMDF expression in the two embryonic lineages that give rise to larval tail muscle-autonomously specified primary cells and conditionally specified secondary cells. CiMDFa and CiMDFb transcripts were detected in both lineages, suggesting that neither functioned in a lineage-specific manner. The data also demonstrated that CiMDF expression was autonomous in the primary lineage (i.e., cells derived from the B4.1 blastomeres) and correlated with histospecific differentiation of muscle. In the secondary lineage (i.e., cells derived from the A4.1 and b4.2 blastomeres), CiMDF expression was conditional and, as in the primary lineage, correlated with muscle differentiation. These experiments reveal similar patterns of CiMDF activity in the primary and secondary muscle lineages and imply a requirement for the expression of this gene in both lineages during larval tail muscle development.

Resistance mechanisms determining the in vitro sensitivity to paclitaxel of tumour cells cultured from patients with ovarian cancer.

Paclitaxel, a drug which stabilises microtubules, demonstrates marked activity against ovarian cancer. We investigated the sensitivity to paclitaxel of tumour cells from disaggregated solid tumours or tumour-bearing ascites from 7 ovarian cancer patients, and 21 established tumour cell lines (ovarian, melanoma and lung). Response was quantitated by [3H]-thymidine incorporation in 96-well plates or by colony growth. Dose-response curves to paclitaxel were biphasic with a dose-dependent phase providing an IC50 value (50% reduction in incorporation) and dose-dependent "plateau" phase where the effect was independent of paclitaxel concentration. IC50 values ranged from 2.5 to 110 nM with evidence of multidrug resistance in the two most resistant cell lines. The "plateau" killing values varied from 0.1 log10 to > 3.4 log10 units reduction, and were found to be significantly correlated (r = 0.86; P < 0.0001) with logarithmic culture doubling times of the cell lines. Cellular glutathione levels were measured and found not to be significantly associated with response to paclitaxel. The results suggest that the ratio of paclitaxel exposure time to the culture doubling time is a major factor in paclitaxel cytotoxicity. The relationship between tumour cell cytokinetics and paclitaxel pharmacokinetics in vivo may therefore be crucial in determining clinical paclitaxel response.

Serial repetition of cilia pairs along the tail surface of an ascidian larva.

Regularly spaced cilia pairs were found in two rows immediately opposite to each other mid-dorsally and mid-ventrally along the larval tail surface of the ascidian protochordate Ciona intestinalis. There were approximately ten such equidistantly placed dorsal-ventral sets embedded in the matrix of the extracellular larval test which forms the flattened vertical tail fin. These immotile cilia originate from pairs of cell bodies in mid-dorsal and mid-ventral peripheral nerves running beneath the tail epidermis. The cilia and neural cell bodies were visualized by immunocytochemical staining with anti-tubulin antibodies; their nature was confirmed by ultrastructural examination. This pattern of cilia and neural cell body placement is conceivably related to the segmentation found in vertebrates.

Structure of the caudal neural tube in an ascidian larva: vestiges of its possible evolutionary origin from a ciliated band.

Ultrastructural analysis and differential immunocytochemical staining with two antitubulin monoclonal antibodies were used to reexamine the organization and development of the neural tube in the larva of an ascidian, Ciona intestinalis, in appraisal of a theory that the dorsal tubular nervous system of the chordates evolved from two halves of a ciliated band in an auricularia-like larva of the kind found in echinoderms and hemichordates. One of the antibodies stained cilia in the nervous system and elsewhere; the other reacted primarily with neuronal axons. The caudal neural tube consists of four rows of large ciliated ependymal-glial cells enclosing an axial neural canal into which their single cilia extend. Two ventrolateral nerve tracts, containing axons, arise in the posterior brain region and extend along the length of the caudal tube, partially surrounded by the ependymal cells. The nonnervous, ciliated, ependymal neural tube of the ascidian larva with its two associated nerve tracts survives as a primitive early condition that could result from a ciliated band transformation. Tissues in the distal-most part of the ascidian larval tail have cell lineage origins that indicate an evolutionary history different from those in the proximal majority of the tail. The ependymal cells in this presumed later addition to the tail are not ciliated, although all of the others in the caudal ependymal tube appear to be.

Differentiation of tropomyosin-containing myofibrils in cleavage-arrested ascidian zygotes expressing acetylcholinesterase.

Two muscle differentiation programs, acetylcholinesterase and tropomyosin-containing filaments and fibrils, occur together in the same cleavage-arrested zygotes (1-celled) of the ascidian Ciona intestinalis. Coexpression in such undivided but developing 'embryos' is consistent with the idea that separate elements of muscle differentiation are related at some regulatory level, perhaps through a single multi-gene regulatory factor. Fertilized Ciona eggs were exposed to cytochalasin B for 20 h and then briefly reacted histochemically for acetylcholinesterase activity. Strongly reacting specimens were selected and processed for transmission electron microscopy to reveal regions of muscle ultrastructure. Every acetylcholinesterase-reactive zygote tested contained muscle contractile elements; no example lacking acetylcholinesterase was found with myofilaments and myofibrils. As demonstrated by immunogold labelling, a polyclonal antibody to tropomyosin from Ciona adult body wall reacted differentially with the presumed ultrastructural muscle elements in cleavage-arrested zygotes. Site-specific reactions were also observed in larval tail muscle and the siphon muscles of postmetamorphic zooids.

Determination of Alkaline Phosphatase Expression in Endodermal Cell Lineages of an Ascidian Embryo.

Ciona intestinalis embryos develop a strong histochemical localization of alkaline phosphatase activity in their known endodermal tissues. Such tissues arise solely from the four vegetal blastomeres at the 8-cell stage and six vegetal blastomeres at the 16-cell stage; these vegetal cells inherit an endodermal lineage cytoplasm. Pairs of blastomeres from the bilaterally symmetrical 8- and 16-cell stages were isolated and reared as partial embroys. Only those partial embryos derived from endoderm-containing lineages developed a histochemically localized alkaline phosphatase activity. From the results of such restricted developmental autonomy (self-differentiation), one can deduce that this enzymic expression of endodermal fate could be specified by events of cytoplasmic segregation that occur during the early cleavages. This conclusion offers additional support to the theory that specification of cell fate in ascidian embryos involves an early differential segregation of histodetermining egg cytoplasmic materials.

Two histospecific enzyme expressions in the same cleavage-arrested one-celled ascidian embryos.

Fertilized eggs of the ascidian, Ciona intestinalis, were prevented from undergoing cytokinesis but not nuclear division by treatment with cytochalasin B. After appropriate times, such cleavage-arrested multinucleate zygotes developed acetylcholinesterase of larval tail muscle and an alkaline phosphatase ordinarily localized in the larval endoderm tissues. Separate histochemical reactions on one of a pair of samples taken from the eggs of single animals provided examples (6/34) in which the numbers of cytochalasin-treated embryos displaying the respective reaction product overlapped sufficiently (15-29%) to indicate that some of the zygotes had developed both enzymes in the same uncleaved single cell. With an actual dual-staining technique that can be applied to single cleavage-arrested zygotes, 62% of those developing a strong alkaline phosphatase reaction also had a strong acetylcholinesterase reaction. In other experiments, quantitative measurements of enzyme activity in homogenates of 114 single cleavage-arrested zygotes confirm directly that 18% of the zygotes produce both enzymes. There was no obligatory mutual exclusion of the potential for simultaneous expression of two tissue-specific characteristics that would ordinarily be segregated into different lineages during early cleavages. The cytoplasmic determinants believed responsible for these histotypic expressions can apparently function independently in the same cell.

Cytokeratin expression in cervical epithelium: an immunohistological study of normal, wart virus-infected and neoplastic tissue.

In this study using a panel of anticytokeratin antibodies and an indirect immunoperoxidase method, we examined cervical squamous epithelia including mature stratified epithelium, immature squamous metaplasia, CIN 1, 2 and 3, wart virus infection and squamous carcinoma. Changes from the normal patterns of staining were inconsistently seen in CIN 1 and 2, but in CIN 3 the changes were more marked, and consisted of a loss of stratification of the staining pattern and a patchy reduction in staining. Invasive carcinomas showed a similar staining pattern to CIN 3 lesions.

Cell differentiation features in embryos resulting from interphylum nuclear transplantation: echinoderm nucleus to ascidian zygote cytoplasm.

When an echinoderm nucleus was transplanted into an ascidian zygote cytoplast there was developmental cooperation at the cellular level between nucleus and cytoplasm of these normally nonhybridizable species. A blastula stage nucleus from the sand dollar Echinarachnius parma was injected into an activated but nonnucleate egg fragment of the ascidian Ciona intestinalis. During culture, some of the "hybrid" embryos displayed ultrastructural evidence of cellular differentiation. Two recognizable features were (1) extracellular matrix components, and (2) neural cell characteristics, including elaboration of associated cilia. Nonnucleate zygote fragments alone, and such fragments injected with seawater or punctured by glass needle, did not develop organized subcellular structures. Morphologic expressions resulting from nuclear transplantations between these two phyla (Echinodermata and Chordata) seemingly indicate functional interactions at a gene regulatory level. Creation of such nuclear-cytoplasmic hybrids suggests thereby a means of exploring the nature of the egg cytoplasmic agents in ascidian embryos that appear to determine gene expression related to histospecific differentiation products.

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.

Differentiation without cleavage: multiple cytospecific ultrastructural expressions in individual one-celled ascidian embryos.

Multiple states of differentiation developed within the same undivided egg cytoplasm of ascidian zygotes cleavage-arrested with cytochalasin B. Complex ultrastructural traits of up to four quite diverse cell lineage components were observed in regions of the common cytoplasm in such multinucleate homokaryons of Ciona intestinalis: epidermal, muscle, notochordal, and neural. Almost all specimens among those selected as showing differentiation contained two such features, half of them had at least three, and a few expressed all four. The histospecific morphological characteristics noted were the extracellular test material of epidermal cell origin, muscle myofilaments and myofibrils, sheath components (leaflets and filaments) associated with notochordal cells, and the particular localized combinations of microtubules, filamentous structures, and cilia indicative of neural tissues. Cleavage-arrested one-celled embryos of Ascidia ceratodes served to demonstrate that those which were found cytochemically to contain muscle acetylcholinesterase always had myofibrils and myofilaments. Other arrested zygotes of Ascidia (unstained specimens) also had quite fully formed test material as well as myofilaments and myofibrils. The occurrence within the same cell of so many specific markers of diverse pathways of development is consistent with a theory about a primary level of regulation based on autonomous gene activation factors already present in the fertilized egg. If further investigation substantiates a real cytoplasmic continuity within these cleavage-arrested embryos, other theories that invoke cell interactions, temporal sequences of metabolically distinct microenvironments, and gradients of substances as causes of determinative change seem inadequate to account for the coexisting expressions of differentiation described here.

Lineage segregation and developmental autonomy in expression of functional muscle acetylcholinesterase mRNA in the ascidian embryo.

Acetylcholinesterase is a histospecific marker of cell differentiation occurring only in the muscle and mesenchyme tissues of the ascidian embryo. The distribution of functional mRNA coding for this enzyme has been investigated and it is shown here that only cells of muscle and mesenchyme lineages possess such a template. Blastomeres of four cell lineage quadrants were separated microsurgically from eight-cell-stage embryos of Ciona intestinalis and raised in isolation until muscle development was well advanced. Measurement of enzyme activity in the resulting partial embryos revealed that acetylcholinesterase was limited to descendants of one blastomere pair, the B4.1 blastomeres containing muscle and mesenchyme lineages. To study the tissue distribution of acetylcholinesterase mRNA, RNA from partial embryos was translated in Xenopus laevis oocytes. When oocytes were injected with an appropriate template, they synthesized a biologically active acetylcholinesterase that could be selectively immunopurified with an antiserum to the ascidian enzyme. Under the conditions used the quantity of acetylcholinesterase mRNA was directly related to the enzyme activity in immunoprecipitates. Acetylcholinesterase mRNA was found only in B4.1 lineage partial embryos where it occurred in approximately the same amount as in whole embryos of the same age. Since there is a limited period from gastrulation until the middle tail-formation stage when functional acetylcholinesterase mRNA accumulates, the results of our mRNA distribution experiments strongly suggest that the gene for ascidian acetylcholinesterase is active only in muscle and mesenchyme tissues. The histospecific occurrence of this enzyme apparently does not involve selective, cell-specific control of translation.

Differentiation of histospecific ultrastructural features in cells of cleavage-arrested early ascidian embryos.

Ultrastructural features of histospecific differentiation were found in early cleavage stage ascidian embryos treated with cytochalasin B and held thereby in cleavagearrest until hatching time. Markers characteristic of tissue differentiation during normal embryonic and larval stages ofCiona intestinalis were expressed in muscle and two brain cell lineages of cleavage-arrested whole embryos and in epidermal and notochordal cell lineages of cleavage-arrested partial embryos. These features were muscle myofilaments and myofibrils, melanosomes of the brain pigment cells, cilium-derived structures present in a "proprioceptive" brain cell, extracellular test material of epidermal cell origin, and the sheath filaments, membrane leaflets, and vacuolar colloid associated with notochord cells. All of these ultrastructural markers of differentiation were blocked in their development by treatment of gastrula stage embryos with actinomycin D, an inhibitor of RNA synthesis, and presumably result from the expression of new gene activity. At the time of cleavage-arrest the five cell lineages studies still contained two or more unsegregated lineage pathways. Subsequent developmental autonomy within the lineages is consistent with the hypothesis of segregation during early development of functionally independent gene regulatory factors.

Development of translationally active mRNA for larval muscle acetylcholinesterase during ascidian embryogenesis.

Relative quantities of translationally active acetylcholinesterase (acetylcholine acetylhydrolase, EC 3.1.1.7) mRNA present at various developmental stages were compared in embryos of the ascidian Ciona intestinalis. Purified RNA was tested for its translational capacity by microinjection into Xenopus laevis oocytes; the acetylcholinesterase produced was immunoprecipitated with antibody to Ciona acetylcholinesterase and enzyme activity was assayed radiometrically. With this protocol, enzyme synthesis was found to be directly related to the amount of RNA injected and to the oocyte incubation time. A functional template for acetylcholinesterase was first detected at 6 hr of development (late gastrula) and is probably present as early as 5 hr. The level of this template activity increased until the middle tail formation stage (11-12 hr after fertilization) and then remained constant until 16 hr of development (the final stage examined), 2 hr before hatching. These findings, and the results of previous actinomycin D inhibition experiments, indicate that mRNA for ascidian larval muscle acetylcholinesterase is first synthesized during gastrulation.

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

Some embryos of Ciona intestinalis which were permanently cleavage-arrested with cytochalasin B at the 1-cell, 4-cell, or 8-cell stages produced, after 12 or 16 h of development time (18 degrees C), a level of muscle acetylcholinesterase activity equal to that found in normal early and later larval stage embryos of the same age. Enzyme activity was measured quantitatively in single whole embryos by a colorimetric procedure using microdensitometry. Quantitative regulation of a differentiation end product indicated that the usual transcriptional and translational control mechanisms for that histospecific protein continued to operate normally in the cleavage-arrested embryos. Acetylcholinesterase expression was apparently regulated independently of the usual cell cytoplasmic volume in the muscle lineage cells and possibly also independently of the normal nuclear number in the lineage. There is an egg cytoplasmic determinant that is segregated into the muscle lineage cells during cleavage and which appears to specify the pathway of larval muscle development. Quantitative control of muscle acetylcholinesterase is possibly one of the consequences of how the agent releases genetic expression in the presumptive muscle cells. Quantitative regulation was not, however, a general functional activity of cleavage-arrested embryos. Mitochondrial cytochrome oxidase, an enzyme whose development is believed to be unaffected by cytoplasmic determinants, was not regulated quantitatively in cleavage-arrested embryos. Cytochrome oxidase activity of cleavage-arrested embryos, measured in single whole embryos by a colorimetric microdensitometry assay, increased only slightly during 16 h of development time whereas the activity in normal control embryos doubled during that time.

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.

Quantitative measurement by microdensitometry of tyrosinase (dopa oxidase) development in whole small ascidian embryos.

Embryos of the ascidian, Ciona intestinalis, were fixed in either cold (5 degree C) 70% ethanol or cold absolute methanol during their tyrosinase development phase and incubated in buffered (pH 7.2) solutions of the enzyme substrate L-dihydroxyphenylalanine. Optical density of the reaction product (melanin) was measured in the whole small embryos at 450 nm with a Vickers M85 scanning and integrating microdensitometer. The frequency distribution of the reaction density in embryos of a population was Gaussian, and the mean optical density in embryos samples (N = 25) increased linearly with incubation time when a saturation level of substrate was used. Absolute optical density units of dopa oxidase activity in embryos increased linearly in proportion to the development time preceding melanin granulogenesis thereby suggesting that the enzyme activity measured by this procedure is proportional to the amount of tyrosinase present. Since this developmental increase in activity was blocked by treatment of the embryos with puromycin, an inhibitor of protein synthesis, the change is apparently caused by new enzyme synthesis. The microdensitometry assay also confirmed results obtained previously with a radiometric assay: embryos cleavage-inhibited at 7 h development time with cytochalasin B to produce giant melanocytes developed only the same amount of enzyme activity as control embryos.