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On the 'clock' mechanism determining the time of tissue-specific enzyme development during ascidian embryogenesis. I. Acetylcholinesterase development in cleavage-arrested embryos.

During ascidian embryogenesis a tissue-specific enzyme, muscle acetylcholinesterase (AChE) may first be detected histochemically in the presumptive muscle cells of the neurula. In order to investigate the 'clock' or counting mechanism that is determining the time when AChE first appears, Whittaker's experiment (1973) has been repeated using eggs of the ascidian, Halocynthia roretzi. Embryos that had been permanently cleavage-arrested with cytochalasin B were able to differentiate AChE in their muscle lineage blastomeres. The time of first AChE occurrence in embryos that had been cleavage-arrested in the 32-cell stage with cytochalasin B was about the same as in normal embryos. This result indicates that the clock is not apparently regulated by the events of cytokinesis. The early gastrulae which had been arrested with colchicine or with colcemid could develop AChE activity, although no histochemically detectable AChE activity was observed in the cleavage-stage embryos that had been arrested with either drug. Therefore the clock does not seem to be controlled by the mitotic cycle of the nucleus. It is suggested that the cycle of DNA replication may be related to the regulation of the clock that is determining the time of development of histospecific protein.

Acetylcholinesterase

Cell differentiation in ascidian studied by nuclear transplantation.

The egg of the ascidian Ciona Intestinalis was divided 20 min after fertilization into two fragments, one nucleated and the other non-nucleated. Nucleus of ectodermal, mesodermal, or endodermal cell, taken from gastrula or tail-bud embryo was transplanted into the non-nucleated fragment. The fragment developed into abnormal or partial embryo, of which the cells or tissues were differentiated mainly according to the regional distribution of presumptive organ-forming substances of the egg cytoplasm that the fragment contained. The result indicated that differentiation of cells in ascidians was principally determined by the components of the mature egg cytoplasm, not by the transplanted nucleus, and the activity of the nucleus was, to some degree, controlled by the surrounding cytoplasm.

Animals

Development of acetylchilinesterase during embryogenesis of the ascidian Ciona intestinalis.

We have characterized the embryonic muscle cell cholinesterase of the solitary ascidian, Ciona intestinalis (L.). The effects of selective enzyme inhibitors and the inhibition of enzyme activity at high concentrations of substrate suggest that the muscle cell enzyme is an acetylcholinesterase (E.C. 3.1.1.7). After gastrulation and before hatching, acetylcholinesterase activity increased 35- to 40-fold; after hatching (18 hours postfertilization) this activity continued to increase, leveling off at about 36 hours of development. Histochemical observations showed that before hatching acetylcholinesterase was located principally in the muscle cells of the tail and, after hatching, it began to develop in cells of the adult musculature and brain. Inhibition of protein syntnesis by puromycin and of RNA synthesis by actinomycin D, suggest that both protein and RNA synthesis were required for the increase in acetylcholinesterase activity observed in unhatched embryos. Although the continued increase in enzyme activity duirng embryonic development was sensitive to puromycin at all times tested, the actinomycin D sensitivity of this increase was restricted to a discrete time that was completed by about 11 hours of development.

Acetylcholinesterase

Comparative studies on the structure of reproductive organs of four botryllid ascidians.

Reproductive organs of four botryllid ascidians, Botryllus primigenus, Botryllus schlosseri, Botrylloides violaceus and Botrylloides leachi, were studied histologically. In every species, the egg follicle consisting of an egg and its inner and outer follicles, is attached to the follicle stalk, the vesicle being composed of a flat epithelium, which in its turn is connected to the atrial epithelium or to the brood pouch specialized from it. In B. schlosseri, the egg is ovulated into the atrial cavity and remains there held by the brood cup, of which the inner epithelium is derived from the follicle stalk and the outer one from the atrial epithelium. In B. primigenus, the brood pouch develops as a diverticulum of the atrial cavity, around the entrance of which a fold differentiates from the atrial epithelium and closes the pouch during embryogenesis. In both species of Botrylloides, the brood pouch is formed by the outgrowth of the thickened atrial epithelium into the blood space, the entrance of which is closed during embryogenesis. The discarded outer follicle completely disintegrates soon after ovulation in B. schlosseri, but part of it remains throughout embryogenesis in the blood space in B. primigenus or projecting into the interior of the brood pouch in Botrylloides. In primigenus, the testis, when it accompanies the egg follicle, is placed at the bottom of the brood pouch and the sperm is shed through the pouch prior to ovulation. In B. schlosseri and the Botrylloides species, the testis is located independently from the egg follicle and the sperm matures after ovulation.

Animals

Endocrine cells in the oesophagus of the ascidian Styela clava, a cytochemical and immunofluorescence study.

Immunocytochemical studies have demonstrated the occurrence of an insulin-immunoreactive cell type in the oesophageal epithelium of the Ascidian Styela clava. Staining with aldehyde fuchsin has demonstrated a number of similar small, triangular, cells located on the basement membrane, which may have an endocrine function. Argyrophilic cells have also been found, suggesting the presence of a second endocrine cell type. The absence of argentaffin cells has led us to believe that the cells so far observed do not produce biogenic amines such as 5-HT (5-Hydroxytryptamine). The nature of these cells is discussed with reference to endocrine-like cells found in the digestive tracts of other protochordates.

Animals

Larval adhesive organs and metamorphosis in ascidians. II. The mechanism of eversion of the papillae of Distaplia occidentalis.

The cup-shaped adhesive papillae of Distaplia occidentalis evert at the onset of metamorphosis and each transforms into a hyperboloidal configuration. The rate of transformation is a function of temperature. At 14 degrees C complete eversion takes about 30 seconds. Myoepithelial cells that extend from the rim to the base on the cup contract. Simultaneously the central part of the papilla advances 60--70 micrometers. During the last phases of eversion, collocytes (cells that secrete adhesives) on the inner wall of the cup and on the sides of the axial protrusion flow outward and form a collar-like structure. The myoepithelial cells contain arrays of thick and thin filaments. These become compacted during contraction. The surfaces of these cells become extensively folded as they shorten to about 1/3 of rest length. According to the proposed model the myoepithelial cells are the driving force in papillary eversion. Immediately after eversion is completed the papillae begin to retract. Eversion of the papillae is not inhibited by cytochalasin B, but the process of retraction is reversibly inhibited. Some histological characteristics of five types of everting papillae in four families of ascidians are compared.

Animals

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.

Acetylcholinesterase

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.

Acetylcholinesterase

Calcitonin-like cells in the pharynx of the ascidian Styela clava.

A small group of granulated endocrine cells have been described in the endostylar region of the pharynx in Styela. These cells are argyrophilic and exhibit calcitonin-like immunofluorescence. Tests with antisera to other peptides all proved negative. Cells from animals exposed to elevated calcium levels showed a degree od degranulation. The possibility that these cells may represent ancestral ultimobranchial "C" cells is discussed.

Animals

A cytochemical and immunofluorescence study of endocrine cells in the gut of the ascidian Styela clava.

Strong secretin-like immunofluorescence has been demonstrated in endocrine-like cells from the gastric epithelium of Styela. These cells also stain with lead haematoxylin and exhibit a brilliant formaldehyde-induced fluorescence, but do not show any other cytochemical features characteristics of the mammalian APUD series. Tests with antisera to glucagon, gastrin and somatostatin all proved negative. In the oesophagus tests with all four antisera proved negative. The significance of these results is discussed in relation to the phylogeny of vertebrate gastro-intestinal hormones.

Animals