Hiromi Takata

Gastrulation in the sea urchin embryo: A model system for analyzing the morphogenesis of a monolayered epithelium

Processes of gastrulation in the sea urchin embryo have been intensively studied to reveal the mechanisms involved in the invagination of a monolayered epithelium. It is widely accepted that the invagination proceeds in two steps (primary and secondary invagination) until the archenteron reaches the apical plate, and that the constituent cells of the resulting archenteron are exclusively derived from the veg2 tier of blastomeres formed at the 60‐cell stage. However, recent studies have shown that the recruitment of the archenteron cells lasts as late as the late prism stage, and some descendants of veg1 blastomeres are also recruited into the archenteron. In this review, we first illustrate the current outline of sea urchin gastrulation. Second, several factors, such as cytoskeletons, cell contact and extracellular matrix, will be discussed in relation to the cellular and mechanical basis of gastrulation. Third, differences in the manner of gastrulation among sea urchin species will be described; in some species, the archenteron does not elongate stepwise but continuously. In those embryos, bottle cells are scarcely observed, and the archenteron cells are not rearranged during invagination unlike in typical sea urchins. Attention will be also paid to some other factors, such as the turgor pressure of blastocoele and the force generated by blastocoele wall. These factors, in spite of their significance, have been neglected in the analysis of sea urchin gastrulation. Lastly, we will discuss how behavior of pigment cells defines the manner of gastrulation, because pigment cells recently turned out to be the bottle cells that trigger the initial inward bending of the vegetal plate.

Behavior of pigment cells in gastrula‐stage embryos of Hemicentrotus pulcherrimus and Scaphechinus mirabilis

The behavior of pigment cells in sea urchin embryos, especially at the gastrula stage, is not well understood, due to the lack of an appropriate method to detect pigment cells. We found that pigment cells emanated autofluorescence when they were fixed with formalin and irradiated with ultraviolet or green light. In Hemicentrotus pulcherrimus, fluorescent pigment cells became visible at the archenteron tip at the mid‐gastrula stage. The cells detached from the archenteron slightly before the initiation of secondary invagination and migrated toward the apical plate. Most pigment cells entered the apical plate. This entry site seemed to be restricted, because pigment cells could not enter the ectoderm and remained in the blastocoele at the vegetal pole side when elongation of archenteron was blocked. Pigment cells that had entered the apical plate soon began to migrate in the aboral ectoderm toward the vegetal pole. In contrast, pigment cells of Scaphechinus mirabilis embryos were first detected in the vegetal plate before the onset of gastrulation. Without entering the blastocoele, these cells began to migrate preferentially in the aboral ectoderm toward the animal pole. When the archenteron tip reached the apical plate, pigment cells had already distributed throughout the aboral ectoderm. Thus, the behavior of pigment cells was quite different between H. pulcherrimus and S. mirabilis..

Cellular Basis of Gastrulation in the Sand Dollar Scaphechinus mirabilis

The processes of gastrulation in the sand dollar Scaphechinus mirabilis are quite different from those in regular echinoids. In this study, we explored the cellular basis of gastrulation in this species with several methods. Cell-tracing experiments revealed that the prospective endodermal cells were convoluted throughout the invagination processes. Histological observation showed that the ectodermal layer remained thickened, and the vegetal cells retained an elongated shape until the last step of invagination. Further, most of the vegetal ectodermal cells were skewed or distorted. Wedge-shaped cells were common in the vegetal ectoderm, especially at the subequatorial region. In these embryos, unlike the embryos of regular echinoids, secondary mesenchyme cells did not seem to exert the force to pull up the archenteron toward the inner surface of the apical plate. In fact, the archenteron cells were not stretched along the axis of elongation and were in close contact with each other. Here we found that gastrulation was completely blocked when the embryos were attached to a glass dish coated with poly-L-lysine, in which the movement of the ectodermal layer was inhibited. These results suggest that a force generated by the thickened ectoderm, rather than rearrangement of the archenteron cells, may play a key role in the archenteron elongation in S. mirabilis embryos.

Behavior of Pigment Cells Closely Correlates the Manner of Gastrulation in Sea Urchin Embryos

Abstract To know whether behavior of pigment cells correlates the process of gastrulation or not, gastrulating embryos of several species of regular echinoids (Anthocidaris crassispina, Mespilia globulus and Toxopneustes pileolus) and irregular echinoids (Clypeaster japonicus and Astriclypeus manni) were examined. In M. globulus and A. crassispina, the archenteron elongated stepwise like in well-known sea urchins. In the embryos of both species, fluorescent pigment cells left the archenteron tip and migrated into the blastocoel during gastrulation. In T. pileolus, C. japonicus and A. manni, on the other hand, the archenteron elongated at a constant rate throughout gastrulation. In these species, no pigment cell was observed at the archenteron tip during invagination processes; pigment cells began to migrate in the ectoderm from the vegetal pole side toward the apical plate without entering the blastocoel. These results clearly indicate that the behavior of pigment cells closely correlated the manner of gastrulation. Further, it was examined whether the archenteron cells are rearranged during invagination, by comparing the number of cells observed on cross sections of the archenteron at the early and late gastrula stages. The rearrangement was not conspicuous in A. crassispina and M. globulus, in which archenteron elongated stepwise. In contrast, the archenteron cells were remarkably rearranged in C. japonicus, alothough the archenteron elon-gated continuously. Thus, neither the behavior of pigment cells nor the manner of gastrulation matches the current taxonomic classification of echinoids.

A monogamous pipefish has the same type of ovary as observed in monogamous seahorses

Syngnathid fish (pipefish and seahorses) are unique among teleost fish in that their ovary consists of a rolled sheet with germinal ridge(s) on the dorsal side running along the entire length of the sheet. A distinct difference is seen in the ovarian structure between polygamous Syngnathus pipefish and monogamous seahorses (Hippocampus spp.), the former having one germinal ridge and the latter with two ridges. This study examined the ovarian structure and the mode of egg production in a monogamous pipefish Corythoichthys haematopterus. The ovary of C. haematopterus had two germinal ridges like that observed in monogamous seahorses. There were two distinct groups of follicles in the ovary, one being a cohort of extremely small follicles and the other a cohort of follicles developing and increasing in size with the passage of time. We suggest that the ovarian structure and the mode of egg production in this pipefish are adaptations to monogamy.

Timing of Early Developmental Events in Embryos of a Tropical Sea Urchin Echinometra mathaei

Abstract Egg volume of a tropical sea urchin Echinometra mathaei is about one half that of other well-known species. We asked whether such a small size of eggs affected the timings of early developmental events or not. Cleavages became asynchronous from the 7th cleavage onward, and embryos hatched out before completion of the 9th cleavage. These timings were one cell cycle earlier than those in well-known sea urchins, raising the possibility that much earlier events, such as the increase in adhesiveness of blastomeres or the specification of dorso-ventral axis (DV-axis), would also occur earlier by one cell cycle. By examining the pseudopodia formation in dissociated blastomeres, it was elucidated that blastomeres in meso- and macromere lineages became adhesive after the 4th and 5th cleavages, respectively. From cell trace experiments, it was found that the first or second cleavage plane was preferentially employed as the median plane of embryo; the DV-axis was specified mainly at the 16-cell stage. Timings of these events were also one cell cycle earlier than those in Hemicentrotus pulcherrimus. The obtained results suggest that most of the early developmental events in sea urchin embryos do not depend on cleavage cycles, but on other factors, such as the nucleo-cytoplasmic ratio.

Involvement of Delta and Nodal signals in the specification process of five types of secondary mesenchyme cells in embryo of the sea urchin, Hemicentrotus pulcherrimus

Secondary mesenchyme cells (SMCs) of the sea urchin embryo are composed of pigment cells, blastocoelar cells, spicule tip cells, coelomic pouch cells and muscle cells. To learn how and when these five types of SMCs are specified in the veg2 descendants, Notch or Nodal signaling was blocked with γ‐secretase inhibitor or Nodal receptor inhibitor, respectively. All types of SMCs were decreased with DAPT, while sensitivity to this inhibitor varied among them. Pulse‐treatment revealed that five types of SMCs are divided into “early” (pigment cells and blastocoelar cells) and “late” (spicule tip cells, coelomic pouch cells and muscle cells) groups; the “early” group was sensitive to DAPT up to the hatching, and the “late” group was sensitive until the mesenchyme blastula stage. Judging from timing of the shift of Delta‐expressing regions, it was suggested that the “early” group and “late” groups are derived from the lower and the middle tier of veg2 descendants, respectively. Interestingly, numbers of SMCs were also altered with SB431542; blastocoelar cells, coelomic pouch cells and circum‐esophageal muscles decreased, whereas pigment cells and spicule tip cells increased in number. Pulse‐treatment showed that the “early” group was sensitive up to the mesenchyme blastula stage, while the “late” group up to the onset of gastrulation. Thus, it became clear that precursor cells of the “early” and “late” groups, which are located in different regions in the vegetal plate, receive Delta and Nodal signals at different timings, resulting in the diversification of SMCs. Based on the obtained results, the specification processes of five types of SMCs are diagrammatically presented.

Ectoderm exerts the driving force for gastrulation in the sand dollar Scaphechinus mirabilis.

How the ectodermal layer relates to the invagination processes was examined in the sand dollar Scaphechinus mirabilis. When the turgor pressure of blastocoele was increased, invagination was completely blocked. In contrast, an increase in turgor pressure did not affect elongation of the gut rudiment in the regular echinoid Hemicentrotus pulcherrimus. Rhodamine–phalloidin staining showed that the distribution of actin filaments was different between two species of embryos. In S. mirabilis gastrulating embryos, abundant actin filaments were seen at the basal cortex of ectoderm in addition to archenteron cells, while the intense signal was restricted to the archenteron in H. pulcherrimus. To investigate whether actin filaments contained in the ectodermal layer exert the force of invagination, a small part of the ectodermal layer was aspirated with a micropipette. If S. mirabilis embryos were aspirated from the onset of gastrulation, invagination did not occur at all, irrespective of the suction site. Even after the archenteron had invaginated to one‐half of its full length, further elongation of the archenteron was severely blocked by suction of the lateral ectoderm. In contrast, suction of the ectodermal layer did not affect the elongation processes in H. pulcherrimus. These results strongly suggest that the ectodermal layer, especially in the vegetal half, exerts the driving force of invagination in S. mirabilis.

Cell adhesion and the negative cell surface charges in embryonic cells of the starfish Asterina pectinifera

Spherical blastomeres of starfish embryos begin to adhere to neighboring blastomeres and to become columnar in shape from the 7th or 8th cleavage onward. Studying development of embryos in the presence of LiCl, we found that developmental changes in cell‐cell contacts were accelerated by LiCl. In order to learn why LiCl increased the adhesiveness between blastomeres, the negative surface charge density was estimated by the method of cell electrophoresis. It turned out that the electrophoretic mobility (EPM) of all blastomeres isolated from LiCl‐treated embryos before the 512‐cell stage was remarkably decreased. At the mid‐gastrula stage, however, when constituent cells were connected with each other more tightly, the EPM was significantly retarded irrespectively whether the cells had been isolated from control or from LiCl‐treated embryos. From these results of cell electrophoresis we conclude that reduction of the negative surface charge density may be one of the important factors that enhance the adhesion of starfish embryonic cells.

Process of pigment cell specification in the sand dollar, Scaphechinus mirabilis

The process of pigment cell specification in the sand dollar Scaphechinus mirabilis was examined by manipulative methods. In half embryos, which were formed by dissociating embryos at the 2‐cell stage, the number of pigment cells was significantly greater than half the number of pigment cells observed in control embryos. This relative increase might have been brought about by the change in the arrangement of blastomeres surrounding the micromere progeny. To examine whether such an increase could be induced at a later stage, embryos were bisected with a glass needle. When embryos were bisected before 7 h postfertilization, the sum of pigment cells observed in a pair of embryo fragments was greater than that in control embryos. This relative increase was not seen when embryos were bisected after 7 h postfertilization. From the size of blastomeres, it became clear that the 9th cleavage was completed by 7 h postfertilization. Aphidicolin treatment revealed that 10–15 pigment founder cells were formed. The results obtained suggest that the pigment founder cells were specified through direct cell contact with micromere progeny after the 9th cleavage, and that most of the founder cells had divided three times before they differentiated into pigment cells.