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.     

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.     

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.     

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

Summary 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.     

Gray and Red Fragments of the Egg of the Ascidian Ciona savignyi: Preferential Development of Muscle Cells from Gray Fragments

The egg of the ascidian Ciona savignyi is pinkish red with brownish myoplasm that contains the putative determinants responsible for differentiation of muscle cells. When dechorionated unfertilized eggs were centrifuged at moderate speed, eggs were divided into centripetal, small gray fragments and centrifugal, large red fragments. The former contained the female pronucleus and clear cytoplasm, while most of the latter was filled with yolk granules. An antibody raised against the myoplasm of C. intestinalis eggs extensively stained the cortical region of gray fragments, while the antibody stained only small regions of the red fragments. After insemination, both fragments cleaved and gave rise to partial embryos. When development of muscle and epidermal cells in the partial embryos was examined with specific antibodies, muscle development was conspicuous in gray partial embryos, while epidermal differentiation was extensive in red partial embryos. Furthermore, when expression of markers of differentiation was examined in cleavage-arrested gray and red fragments, the number of arrested gray fragments exhibiting the muscle marker was about three-fold greater than in controls. These results suggest that putative muscle determinants are concentrated into gray fragments.     

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.     

Mapping the distribution of differentiation potential for intestine, muscle, and hypodermis during early development in Caenorhabditis elegans

We have analyzed the differentiation potential of cells in early embryos of Caenorhabditis elegans by assessing the production of markers for intestinal, muscle, and hypodermal cell differentiation in cleavage-arrested blastomeres. Our results show that differentiation potential does not always segregate during cleavage in a linear fashion, i.e., a blastomere can express a differentiation potential that is absent in its parent blastomere and vice versa. Furthermore, the expression of a particular differentiation program by certain cleavage-arrested blastomeres is an exclusive event in that each cell will express only one program of differentiation, even though it may have the potential to express several.     

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.     

Studies on the Cytoplasmic Determinant for Muscle Cell Differentiation in Ascidian Embryos: An Attempt at Transplantation of the Myoplasm

The 8-cell stage embryos of the ascidian Halocynthia roretzi which had been prevented from undergoing further divisions by continuous treatment with cytochalasin B could develop histospecific muscle acetylcholinesterase in two blastomeres (B4.1 and B4.1 cells). If the cytoplasm of a B4.1 or B4.1 cell was transplanted by microinjection into either an A4.1 or A4.1 cell of recipient embryos and the transplanted embryos were permanently cleavage-arrested with cytochalasin B, a few eventually developed AChE in three blastomeres instead of in just the two blastomeres found in cleavage-arrested control embryos. Judging from the relative positions of the blastomeres, the third AChE-producing cells appeared to be the A4.1 or A4.1 cells injected with the cytoplasm of B4.1 or B4.1. Although the success rate was considerably low, this result might indicate the presence in the cytoplasm of a determinant for the muscle-specific enzyme development.     

Quantitative control of end products in the melanocyte lineage of the ascidian embryo.

Terminal amounts of tyrosinase (EC 1.10.3.1) activity and melanin pigment in the giant melanocytes of cleavage-arrestedCiona intestinalis (L.) embryos are regulated independently of cell size and number of nuclei in the cells. Embryos were cleavage-arrested in cytochalasin B at a time before the last two divisions of the melanocyte lineage took place. The resulting two giant melanocytes, one from each of the two bilateral melanocyte lineages, developed tyrosinase and melanin. The cells were about three times larger in volume than the normal larval melanocytes and each contained four nuclei instead of just one. Quantitative measurements of melanin synthesized and tyrosinase activity in embryos with the giant melanocytes revealed amounts identical to those found in normal embryos. This specification of exact quantities differs markedly from the situation in mammalian melanocytes where cell volume and gene dosage influence the extent of melanotic differentiation. Quantitative control of differentiation in ascidian melanocytes appears to be mediated by a cytoplasmic determinant segregated through the melanocyte lineage and inherited by one daughter at each division of the lineages.     

Tail morphogenesis in the ascidian, Ciona intestinalis, requires cooperation between notochord and muscle

We present evidence that notochord and muscle differentiation are crucial for morphogenesis of the ascidian tail. We developed a novel approach for embryological manipulation of the developing larval tissues using a simple method to introduce DNA into Ciona intestinalis and the several available tissue-specific promoters. With such promoters, we misexpressed the Xenopus homeobox gene bix in notochord or muscle of Ciona embryos as a means of interfering with development of these tissues. Ciona embryos expressing bix in the notochord from the 64-cell stage develop into larvae with very short tails, in which the notochord precursors fail to intercalate and differentiate. Larvae with mosaic expression of bix have intermediate phenotypes, in which a partial notochord is formed by the precursor cells that did not receive the transgene while the precursors that express the transgene cluster together and fail to undergo any of the cell-shape changes associated with notochord differentiation. Muscle cells adjacent to differentiated notochord cells are properly patterned, while those next to the notochord precursor cells transformed by bix exhibit various patterning defects. In these embryos, the neural tube extends in the tail to form a nerve cord, while the endodermal strand fails to enter the tail region. Similarly, expression of bix in muscle progenitors impairs differentiation of muscle cells, and as a result, notochord cells fail to undergo normal extension movements. Hence, these larvae have a shorter tail, due to a block in the elongation of the notochord. Taken together, these observations suggest that tail formation in ascidian larvae requires not only signaling from notochord to muscle cells, but also a "retrograde" signal from muscle cells to notochord.     

Isolation of cDNA Clones for Epidermis-Specific Genes of the Ascidian Embryo

The present investigation was conducted to isolate cDNA clones that correspond to epidermis-specific genes of the ascidian embryo. When cleavage of fertilized eggs of Halocynthia roretzi is blocked by treatment with cytochalasin B and the arrested eggs are reared as one-celled embryos for about 30 hr, they develop features of differentiation of the epidermis only. Translation in vitro of poly(A)⁺ RNA from cleavage-arrested embryos and analysis of the products by two-dimensional gel electrophoresis revealed several predominant polypeptides that were not detected in a similar analysis of fertilized eggs, suggesting the appearance of epidermis-specific mRNAs in cleavage-arrested embryos. A cDNA library was constructed from arrested one-celled embryos. Differential screening of the library with a total cDNA probe from cleavage-arrested embryos and with a similar probe from fertilized eggs yielded eight different cDNA clones specific for the cleavage-arrested embryos. Northern blot analysis revealed that the mRNAs that corresponded to these cDNAs were present in normal tailbud embryos. In addition, in situ hybridization of whole-mount specimens showed that the mRNAs were restricted to the epidermal cells of tailbud embryos.     

Segregation during cleavage of a factor determining endodermal alkaline phosphatase development in ascidian embryos.

Localized alkaline phosphatase activity (EC 3.1.3.1) develops progressively in endodermal tissues of the presumptive digestive system in Ciona intestinalis embryos. It was first detected histochemically at late gastrulation, and a puromycin sensitivity period coincident with this time suggests that new alkaline phosphatase is synthesized. Embryos in which cell division was blocked with cytochalasin B at early cleavage stages up to the 64-cell stage, eventually differentiated strong alkaline phosphatase activity in certain cells at each cleavage-arrested stage. The maximum cell numbers and their positions were identical to those of the previously known endodermal cell lineage. Actinomycin D did not prevent development of endodermal alkaline phosphatase when administered from fertilization onwards, nor did other inhibitors of RNA synthesis (chromomycin A3, cordycepin, and daunomycin). There is probably a preformed maternal mRNA for endodermal alkaline phosphatase present in the unfertilizec Ciona egg. Either this RNA itself, or some related translation factor, is localized in the egg cytoplasm and segregated during early cleavages into the endodermal cell lineage of the embryo.     

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.     

Synthesis and secretion of the mouse whey acidic protein in transgenic sheep

The synthesis of foreign proteins can be targeted to the mammary gland of transgenic animals, thus permitting commercial purification of otherwise unavailable proteins from milk. Genetic regulatory elements from the mouse whey acidic protein (WAP) gene have been used successfully to direct expression of transgenes to the mammary gland of mice, goats and pigs. To extend the practical usefulness of WAP promoter-driven fusion genes and further characterize WAP expression in heterologous species, we introduced a 6.8 kb DNA fragment containing the genomic form of the mouse WAP gene into sheep zygotes. Two lines of transgenic sheep were produced. The transgene was expressed in mammary tissue of both lines and intact WAP was secreted into milk at concentrations estimated to range from 100 to 500 mg/litre. Ectopic WAP gene expression was found in salivary gland, spleen, liver, lung, heart muscle, kidney and bone marrow of one founder ewe. WAP RNA was not detected in skeletal muscle and intestine. These data suggest that unlike pigs, sheep may possess nuclear factors in a variety of tissues that interact with WAP regulatory sequences. Though the data presented are based on only two lines, these findings suggest WAP regulatory sequences may not be suitable as control elements for transgenes in sheep bioreactors.     

Cell contact induces multiple types of electrical excitability from ascidian two-cell embryos that are cleavage arrested and contain all cell fate determinants

Ascidian early embryonic cells undergo cell differentiation without cell cleavage, thus enabling mixture of cell fate determinants in single cells, which will not be possible in mammalian systems. Either cell in a two-cell embryo (2C cell) has multiple fates and develops into any cell types in a tadpole. To find the condition for controlled induction of a specific cell type, cleavage-arrested cell triplets were prepared in various combinations. They were 2C cells in contact with a pair of anterior neuroectoderm cells from eight-cell embryos (2C-aa triplet), with a pair of presumptive notochordal neural cells (2C-AA triplet), with a pair of presumptive posterior epidermal cells (2C-bb triplet), and with a pair of presumptive muscle cells (2C-BB triplet). The fate of the 2C cell was electrophysiologically identified. When two-cell embryos had been fertilized 3 h later than eight-cell embryos and triplets were formed, the 2C cells became either anterior-neuronal, posterior-neuronal or muscle cells, depending on the cell type of the contacting cell pair. When two-cell embryos had been fertilized earlier than eight-cell embryos, most 2C cells became epidermal. When two- and eight-cell embryos had been simultaneously fertilized, the 2C cells became any one of three cell types described above or the epidermal cell type. Differentiation of the ascidian 2C cell into major cell types was reproducibly induced by selecting the type of contacting cell pair and the developmental time difference between the contacting cell pair and 2C cell. We discuss similarities between cleavage-arrested 2C cells and vertebrate embryonic stem cells and propose the ascidian 2C cell as a simple model for toti-potent stem cells.