Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

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 veg(2) descendants, Notch or Nodal signaling was blocked with gamma-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 veg(2) 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.

DOI： 10.1111/j.1440-169X.2010.01233.x

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：MARINE BIOLOGICAL LABORATORY  

In echinoderms, the circumesophageal muscle is mesodermal in origin. Several studies of sea urchins have reported that the molecular events of myogenesis occur during the differentiation of the circumesophageal muscle in early embiyogenesis. In contrast, few detailed reports have examined the differentiation of the circumesophagus muscle in larval starfish. Here, we examined the temporal-numeric distribution and differentiation of esophagus circular muscle fibers in the starfish Patina pectinifera by using rhodamine phalloidin staining. Muscle fibers were not detected in mouth-forming larvae, but a mean of about 10 muscle fibers was observed in 48-h larvae, and about 26 bundles were observed after 60 h. During the next 12 h, the number of muscle fiber bundles increased slightly to about 31 bundles and was stable until 96 h.

DOI： 10.1086/BBLv219n1p12

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ROYAL SOC  

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.

DOI： 10.1098/rsbl.2008.0157

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Language：English   Publishing type：Part of collection (book)   Publisher：Humana Press  

Sea urchin embryos have long been used as ideal experimental materials. In this chapter, we describe research areas to which sea urchin embryos have contributed, and the potential fields for which they may serve as a model system. The most valuable feature of sea urchin embryos is the availability of a large amount of homogeneous material, which facilitates biochemical and molecular biological approaches. The ease of gamete handling enables detailed analysis of the mechanism of fertilization, and the transparency and synchrony of fertilized eggs facilitate investigations on cell division and the cell cycle. Sea urchin embryos are an ideal model system for signal transduction, because the number of constituent cells is small. The simple organization of the embryo simplifies the analysis of morphogenetic movements. Both primary and secondary mesenchyme cells are interesting populations for studying cell movement. Sea urchin embryos will continue to contribute to the analysis of various unsolved problems. © 2008 Humana Press Inc.

DOI： 10.1007/978-1-59745-285-4_11

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：BLACKWELL PUBLISHING  

To gain information on the process of ectoderm patterning, the animal halves of sea urchin embryos were isolated at various stages, and their morphology was examined when control embryos developed into pluteus larvae. The animal halves separated at the 8-cell stage developed into 'dauerblastula', without showing any conspicuous ectoderm differentiation. In contrast, some of the animal halves isolated at the 60-cell stage (after the sixth cleavage) formed a ciliated band and oral opening, suggesting that some patterning signal was transmitted from the vegetal to animal hemisphere during early cleavage. Further patterning of the animal hemisphere did not seem to occur until hatching, since both the animal halves isolated at the 60-cell stage and hatching stage showed the same degree of ectoderm patterning. After hatching, the later animal halves were isolated, the more patterned ectoderm they formed. The animal halves isolated just prior to gastrulation differentiated well-patterned ectoderm. It is of note, however, that the level of separation was a more crucial factor than the timing of separation; even the animal fragments of newly hatched embryos differentiated well-patterned ectoderm if they had been separated at a subequatorial level. This suggests that the signal for ectoderm patterning is transmitted over the equator after hatching, and once the cells in the supra-equatorial region receive the signal, they, in turn, can transmit the signal upwardly. Interestingly, if the third cleavage plane was shifted toward the vegetal pole, the isolated animal pole-side fragments developed into 'embryoids' with fully patterned ectoderm. These results indicate that not the micromere descendants but the subequatorial cytoplasm plays an important role in ectoderm patterning.

DOI： 10.1111/j.1440-169x.2006.00850.x

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ZOOLOGICAL SOC JAPAN  

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 elongated continuously. Thus, neither the behavior of pigment cells nor the manner of gastrulation matches the current taxonomic classification of echinoids.

DOI： 10.2108/zsj.21.1025

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Language：English   Publisher：BLACKWELL PUBLISHING ASIA  

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.

DOI： 10.1111/j.1440-169x.2004.00755.x

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：BLACKWELL PUBLISHING ASIA  

In the tropical sea urchin Echinometra mathaei, pigment cells are just detectable before the onset of gastrulation, owing to an early accumulation of red pigment granules. Taking advantage of this feature, behavior of pigment cells was studied in relation to the processes of gastrulation. Before the initiation of primary invagination, pigment cells were arranged in a hemi-circle in the dorsal half of the vegetal plate. Inward bending of the vegetal plate first occurred at the position occupied by pigment cells, while the bending was not conspicuous in the ventral half of the blastopore. Rhodamine-phalloidin staining showed that actin filaments were abundant at the apical corticies of pigment cells. It was also found that the onset of gastrulation was considerably delayed in the NiCl2-treated embryos, in which pigment cells were drastically reduced in number. It is notable that the NiCl2-treated embryos began to gastrulate on schedule if they contained a number of pigment cells in spite of treatment. This shows that pigment cells are the bottle cells that trigger the onset of gastrulation. In the embryos devoid of pigment cells, a short stub-like gut rudiment formed in a delayed fashion, and several secondary mesenchyme cells (SMC) appeared at the tip of the rudiment and elongated gradually until its tip reached the apical plate. This observation suggests that the SMC that pull the gut rudiment upward are not pigment cells but blastocoelar cells, because pigment cells change their fate to blastocoelar cells upon NiCl2-treatment.

DOI： 10.1111/j.1440-169X.2004.00726.x

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：BLACKWELL PUBLISHING ASIA  

The behavior and differentiation processes of pigment cells were studied in embryos of a tropical sea urchin Echinometra mathaei, whose egg volume was about half of those of well-known sea urchin species. Owing to earlier accumulation of pigments, pigment cells could be detected in the vegetal plate even before the onset of gastrulation, distributed dorsally in a hemi-circle near the center of the vegetal plate. Although some pigment cells left the archenteron during gastrulation, most of them remained at the archenteron tip. At the end of gastrulation, pigment cells left the archenteron and migrated into the blastocoele, Unlike pigment cells in typical sea urchins, however, they did not enter the ectoderm, and stayed in the blastocoele even at the pluteus stage. It is of interest that the majority of pigment cells were distributed in the vicinity of the larval skeleton. Aphidicolin treatment revealed that eight blastomeres were specific to pigment cell lineage after the eighth cleavage, one cell cycle earlier than that in well-known sea urchins. The pigment founder cells divided twice, and the number of pigment cells was around 32 at the pluteus stage. It was also found that the differentiation of pigment cells was blocked with Ni2+, whereas the treatment was effective only during the first division cycle of the founder cells.

DOI： 10.1111/j.1440-169X.2003.00714.x

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ZOOLOGICAL SOC JAPAN  

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.

DOI： 10.2108/zsj.20.617

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：BLACKWELL PUBLISHING ASIA  

To learn how the dorso-ventral (DV) axis of sea urchin embryos affects the specification processes of secondary mesenchyme cells (SMC), a fluorescent dye was injected into one of the macromeres of 16-cell stage embryos, and the number of each type of labeled SMC was examined at the prism stage. A large number of labeled pigment cells was observed in embryos in which the progeny of the labeled macromere were distributed in the dorsal part of the embryo. In contrast, labeled pigment cells were scarcely noticed when the descendants of the labeled macromere occupied the ventral part. In such embryos, free mesenchyme cells (probably blastocoelar cells) were predominantly labeled. CH3COONa treatment, which is known to increase the number of pigment cells, canceled such patterned specification of pigment cells and blastocoelar cells along the DV axis. Pigment cells were also derived from the ventral blastomere in the treated embryo. In contrast, a similar number of coelomic pouch cells was derived from the labeled macromere, irrespective of the position of its descendants along the DV axis. After examination of the arrangement of blastomeres in late cleavage stage embryos, it was determined that 17-20 veg(2)-derived cells encircled the cluster of micromere descendants after the 9th cleavage. From this number and the numbers of SMC-derived cells in later stage embryos, it was suggested that the most vegetally positioned veg (2) descendants at approximately the 9th cleavage were preferentially specified to pigment and blastocoelar cell lineages. The obtained results also suggested the existence of undescribed types of SMC scattered in the blastocoele.

DOI： 10.1034/j.1600-0854.2004.00682.x

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-V C H VERLAG GMBH  

Spherical blastomeres of starfish embryos begin to adhere to neighboring blastomeres and to become columnar in shape from the 7(th) or 8(th) 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.

DOI： 10.1002/1522-2683(200207)23:13<2087::AID-ELPS2087>3.0.CO;2-R

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：BLACKWELL PUBLISHING ASIA  

The process of pigment cell specification in the sand dollar Scaphechinus mirabills 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 In postfertilization, the sum of pigment calls 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 In postfertilization. Aphiclicolin 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.

DOI： 10.1046/j.1440-169x.2002.00627.x

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ZOOLOGICAL SOC JAPAN  

Effects of LiCl on the specification process of pigment founder cells were examined in the sea urchin Hemicentrotus pulcherrimus. If embryos were treated with 30 mM LiCl during 4-7 or 7-10 hours postfertilization, pigment cells increased significantly. Aphidicolin treatment indicated that this increase was due to the increase in the pigment founder cells. Interestingly, if the embryos were treated sequentially with LiCl and Ca2+-free seawater during 4-7 and 7-10 hr, respectively, they differentiated only about the same number of pigment cells as control embryos. Further, the increase was scarcely discerned when the embryos were treated with LiCl in the absence of Ca2+ during 7-10 hr. These results suggested that effect of LiCl would be ascribed to the increase in cell adhesiveness. In fact, LiCl-treated embryos were more difficult to be dissociated into single cells. Cell electrophoresis showed that the amount of the negative cell surface charges decreased considerably in LiCl-treated embryos. It was also found that the number of pigment cells seldom exceeded 100, even if embryos were exposed to a higher concentration of LiCl. This suggested that only a subpopulation of the descendants of veg2 blastomeres received the inductive signal emanated from the micromere progeny.

DOI： 10.2108/zsj.19.299

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：BLACKWELL PUBLISHING ASIA  

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.

DOI： 10.1046/j.1440-169X.2001.00605.x

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ZOOLOGICAL SOC JAPAN  

By immersing sea urchin embryos in seawater containing various concentrations of sucrose, we found that the level of osmotic pressure of blastocoel changed during gastrulation; the level was low around the onset of gastrulation and higher during secondary invagination. To learn how such changes in osmotic pressure related to invagination processes, embryos were shrunken or expanded by means of sucrose treatments, and the degree of invagination was monitored using Nile blue staining. The method elucidated that the cells invaginated during primary invagination occupied only top one third of archenteron at the end of secondary invagination, and that the cells constituting intestine and the posterior half of stomach were recruited into the archenteron after secondary invagination. When embryos were expanded, the degree of invagination was remarkably decreased, indicating that a higher level of expanding force hindered the fulfillment of invagination. On the other hand, shrinkage of blastocoel during secondary invagination increased the degree of invagination. Cell tracing experiments showed that the change in the degree of invagination estimated with Nile blue staining was accompanied with the change in the number of cells incorporated into the archenteron. When embryos were expanded, only the descendants of veg2 constituted the gut rudiment, while a considerable amount of the veg1 descendants were recruited into archenteron when embryos were shrunken. Obtained results suggest that change in the level of osmotic pressure of blastocoel would be necessary for the progress of gastrulation.

DOI： 10.2108/zsj.18.1097

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：BLACKWELL PUBLISHING ASIA  

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.

DOI： 10.1046/j.1440-169x.2001.00576.x

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：MARINE BIOLOGICAL LABORATORY  

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.

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Presentation type：Oral presentation (general)  

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Language：Japanese   Presentation type：Oral presentation (general)  

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