Marina Dan-Sohkawa

Cell dynamics of the blastulation process in the starfish, Asterina pectinifera

Abstract Embryonic cells of the starfish, Asterina pectinifera, both enclosed in and deprived of the fertilization membrane, were observed through the process of blastula formation by light and electron microscopy. The first few divisions of the denuded egg produce blastomeres which are virtually unconnected. However, these cells start to pack closely together after the eighth cleavage (28-cell stage) and soon arrange themselves into a tightly packed sheet. This cell sheet, then, turns its free edges up at the late 29- or very early 210-cell stage and forms itself into an irregularly shaped, hollow blastula, usually before another cell division. Cleavage of these cells takes place in perfect synchrony with the cells of the normal embryo. Ultrastructural studies of the contact area between these cells and cells of a normal embryo reveal a good correlation between the dynamic closing movement of blastulating cells and the developmental appearance of septate desmosomes. Basement membrane was laid down only after the cell sheet has closed itself into a hollow blastula, at the early 211-cell stage. Cellular activities, mentioned above, are discussed in connection with a process in which “epithelium” is organized from relatively independent morular cells.

A “NORMAL” DEVELOPMENT OF DENUDED EGGS OF THE STARFISH, ASTERINA PECTINIFERA*

Experimental conditions that allow “normal” development of starfish eggs stripped of the fertilization membrane are reported in this paper. Four kinds of intercellular relation are distinguished during the pre‐hatching stages of these eggs.

Defense system by mesenchyme cells in bipinnaria larvae of the starfish, Asterina pectinifera

Here we characterize starfish larval mesenchyme cells, in terms of not only their phagocytic behavior, but also their structural and functional properties as a defense system. Our study reveals the following: (1) most mesenchyme cells construct a dynamic network structure beneath the body wall; (2) mesenchyme cells phagocytically respond to almost all foreign materials and form syncytial aggregates to conceal relatively large amounts and large sizes of foreign material; (3) the morphologies of the syncytial aggregates differ from one another depending on the species and the surface configuration of the cellular foreign material; (4) no mesenchyme cells respond to live mesenchyme cells even though they phagocytose chemically fixed cells; (5) mesenchyme cells phagocytose both cellular constituents effluxed from the ectodermal cells and foreign materials taken into the blastocoel through the body wall. Together, these results suggest that mesenchyme cells are equipped with a spectrum of abilities to engage in a defense system in starfish larva.

Characterization of Coelomocytes of the Ascidian Halocynthia roretzi Based on Phase-contrast, Time-lapse Video and Scanning Electron Microscopic Observations

Abstract Coelomocytes (blood cells) of the solitary ascidian Halocynthia roretzi were observed in culture by phase-contrast, time-lapse video and scanning electron microscopy. Seven types and one sub-type of cells were distinguished with respect to their morphology and behavior. These cell types are discussed in relation to those appearing in the literature. A table of correlation between cells reported under various names in the literature is presented in the Appendix.

MESENCHYME CELLS IN STARFISH DEVELOPMENT: EFFECT OF TUNICAMYCIN ON THEIR DIFFERENTIATION, MIGRATION AND FUNCTION

The effect of tunicamycin, an inhibitor of protein glycosylation, on starfish development was investigated. Specific developmental events such as 1) bulging of the archenteron tip, 2) migration of mesenchyme cells, 3) formation of coelomic pouches and 4) mouth formation, are inhibited in the presence of this drug. These events are discussed in connection with differentiation, migration and function of mesenchyme cells. The possibility is discussed that tunicamycin exerts its effect by interfering with de novo synthesis of a cell surface factor(s) supporting dynamic cell surface activities.

Primary Culture of Mesodermal and Endodermal Cells of the Starfish Embryo

Abstract A method is described for culturing embryonic cells of the starfish Asterias amurensis. A network composed purely of mesenchyme cells can be obtained by treating dissociated cells of embryos at the mesenchyme migration stage with 0.6 M glycine in half strength Ca2+, Mg2+ free Jamarin (artificial sea water) for 24 hr and subsequently culturing them for 24 hr. When the treatment period is shortened to 12 hr, aggregates of epithelial cells come to coexist with the network. After 3 days of culture, these aggregates form monolayer sheets constituted of four types of cells which can be identified morphologically and by 8-anilino-1-naphthalenesulfonic acid (1, 8-ANS) stainability. These cell types correspond to cells composing the esophagus, the stomach, the intestine and the coelomic pouch. No ectodermal cells were found in the sheet. These results suggest that there is an order of resistibility to 0.6 M glycine treatment among cells of the three germ layers, i. e. mesoderm, endoderm and ectoderm in descending order.

Study on the Nature of Starfish Larval Muscle Cells In Vitro

Abstract We describe a culture method in which larval muscle cells of the starfish Asterias amurensis develop from epithelial cells, probably deriving from the coelomic pouches. The nature of the muscle which appears in the culture is described morphologically, physiologically and ultrastructurally. Cells were dissociated from the late gastrula stage, treated with 0.6 M of glycine in half-strength sea water free of Ca2+ and Mg2+ for 12 hr, and cultured for various periods. Elongated cells appeared after about a week from small aggregates of epithelial cells which were found among the mesenchymal network on the 1st day of culture. The characteristics of the elongated cells are as follows: (1) they possess two or more arms; (2) they adhere to the cultural substratum, mesenchyme cells, and themselves at all parts of the cell body; (3) they contract in response to acetylcholine; (4) they contain an abundance of fibrous actin and myosin throughout their cytoplasm; (5) the cytoplasm contains bundles of thick (12–19 nm) and thin (5–8 nm) filaments without any dense material; and (6) no proliferative activity was observed while the cells were kept in culture for up to 14 days. These features were compared with those of the larval muscle cells in vivo.

FORMATION OF JOINED LARVAE IN THE STARFISH, ASTERINA PECTINIFERA

A method for joining larvae in the starfish, Asterina pectinifera, is presented. Any number of embryos are stably united by simple contact of the cell clusters (descendents of individual denuded eggs) before reaching the early blastula stage (ca. 6 1/2 hr after insemination at 21°C). Embryos do not combine with each other after closing into hollow blastulae. This is considered to indicate the transformation of the cell cluster from a mere collection of cells to an individual, multicellular system. Biological meanings of the fertilization membrane are discussed.

Fibrous component of the blastocoelic extracellular matrix shapes epithelia in concert with mesenchyme cells in starfish embryos

By using a monoclonal antibody (4H11 Mab), we have investigated morphogenetic functions of a fibrous component of the blastocoelic extracellular matrix in relation to cellular activities during early development of the starfish Asterina pectinifera. The 4H11 fibers fill the blastocoele from the late‐cleavage to late‐gastrula stage and contain the 370‐kDa proteinaceous molecule secreted only by the epithelial cells. When 4H11 Mab is introduced into the blastocoele of blastulae, the embryos reveal three distinct morphological abnormalities after the mid‐gastrula stage: (1) Distribution of mesenchyme cells confined near the tip of the archenteron, (2) swelling of the posterior ectoderm, and (3) suppressed growth of the mouth, esophagus, and coelomic pouches. These abnormalities occur together with alterations in the distribution of the 4H11 fibers. In embryos recovering from the effect of 4H11 Mab, the mesenchyme cells rearrange the 4H11 fibers. We propose that 4H11 fibers play direct roles in the morphogenesis of starfish embryos by providing a dynamic scaffold not only for the mesenchyme cells but also for the epithelial cells. Moreover, 4H11 fibers have a resist force from within, in concert with the mesenchyme cells, to counter the bulging force intrinsic to the epithelia and hold the epithelia in specific positions, once the positions have been decided. Developmental Dynamics 232:915–927, 2005.

Selective inhibition of gastrulation in the starfish embryo by albuside B, an inosine analogue

External application of 0.2–100 μg/ml albuside B inhibits gastrulation of the starfish (Asterina pectinifera) embryo. Treated embryos retain the late blastula morphology with the vegetal plate. However, the vegetal plate is unreactive to soybean agglutinin, a probe for observing the progenitor cells of the archenteron (mesendoderm) in a normal embryo. The effective period of the treatment is limited from 4 to 6 h after fertilization, a period immediately before the onset of blastulation. RNA synthesis is unaffected during the period of sensitivity. The selectivity of the inhibition shows that albuside B may be a useful tool for studying the mechanisms of mesendoderm differentiation.