JEZ 845 THE JOURNAL OF EXPERIMENTAL ZOOLOGY 279:309–312 (1997) RAPID COMMUNICATIONS Evagination of the Amniotic Cavity in Larvae Derived from Lithium-Treated Embryos of a Direct Developing Echinoid, Peronella japonica CHISATO KITAZAWA AND SHONAN AMEMIYA* Department of Biological Sciences, Graduate School of Science, University of Tokyo, Tokyo 113, Japan ABSTRACT The effect of LiCl on the development of the sand dollar, Peronella japonica, a direct developer, was examined. Embryos treated with LiCl were vegetalized and exogastrulated, as reported for species showing indirect development. In addition, lithium treatment of Peronella embryos resulted in evagination of the amniotic cavity. Larvae whose amniotic cavity was evaginated did not complete the adult rudiment, but formed the adult skeletal system. The ratio of larvae with an evaginated amniotic cavity was not correlated with the concentration of LiCl enployed. The present results suggest that treatment of echinoid embryos with LiCl causes evagination of invaginating tissues as well as vegetalization. J. Exp. Zool. 279:309–312, 1997. © 1997 Wiley-Liss, Inc. Typical echinoids develop through a feeding and swimming larval stage called the pluteus, and metamorphose into juveniles several weeks after fertilization (Emlet et al., ’87). This mode of development is referred to as indirect development. Another mode of development, direct development, is a process in which part or most of the early stages of development are omitted, and the larva metamorphoses without feeding. This mode has evolved independently in several phylogenetic lineages of echinoids (Strathmann, ’78; Raff, ’87). Generally, the direct developers metamorphose without completing the typical pluteus (Raff, ’87). The development of the sand dollar, Peronella japonica, is unique (Wray and Raff, ’91). A larva of this species does not form the larval mouth, and metamorphoses within 3 days without feeding after going through an abbreviated pluteuslike stage (Mortensen, ’21; Okazaki, ’75). As the larvae metamorphoses within such a short time after fertilization, this species is considered to be well suited for studies of larval development. One of the specific features in the development of this species is that the amniotic cavity composing part of the adult rudiment is formed at an early stage (late gastrula) and invaginates to a markedly deeper extent than that in the typical larva (Mortensen, ’21; Okazaki and Dan, ’54). The amniotic cavity in sea urchins that are typical indirect developers is formed on the left lateral side of the larval body. In P. japonica, however, it is formed in the center of the ventral side, where the larval stomodeum is formed in the embryos © 1997 WILEY-LISS, INC. of typical sea urchins (Okazaki and Dan, ’54; Okazaki, ’75). The amniotic cavity of the Peronella embryo, after initial formation in the center of the ventral side, shifts toward the left dorsal side (Kajihara et al., ’93). In spite of the many interesting features in the development of this species, the process and mechanism of its development and metamorphosis have never been studied in detail, except for the reports of Mortensen (’21), Okazaki and Dan (’54) and Amemiya and Arakawa (’96). Several substances which cause vegetalization of sea urchin embryos have been reported so far. Among them, LiCl is known to be the most effective vegetalizing substance (Herbst, 1893; von Ubisch, ’29). von Ubisch (’29) examined the effect of LiCl on the animal cap consisting of presumptive ectoderm (eight mesomeres) isolated from the 16-cell-stage embryo. The LiCl-treated animal cap was vegetalized to form endo-mesodermal structures, which were expressed exclusively by the vegetal-half cells in undisturbed embryos. Undissected whole embryos treated with LiCl were vegetalized and developed into exogastrulae in which the area of the endodermal archenteron increased, resulting in evagination toward the outside (Lallier, ’75; Mitsunaga et al., ’83). The vegetalized embryos became less dissociable than normal embryos, because adhesiveness among cells in the endoderm *Correspondence to: S. Amemiya, Dept. of Biol. Sciences, Graduate School of Science, University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo 113, Japan. E-mail: email@example.com Received 13 May 1997; revision accepted 20 June 1997. 310 C. KITAZAWA AND S. AMEMIYA was stronger than that in the ectoderm (Fujisawa and Amemiya, ’82). The effects of LiCl on echinoid embryos have so far been studied using indirect developers; the effect of LiCl on the development of direct developers has never been reported. Moreover, the effects of LiCl on echinoid embryos have mainly been examined at early stages of development. Thus, the delayed effects of LiCl expressed at later stages in larvae treated at an early stage of development remain to be studied. In the present investigation, embryos of a direct developer, P. japonica, were treated with LiCl from cleavage to the early blastula stage, and examined at later stages. The results indicated that lithium treatment caused evagination of the amniotic cavity as well as exogastrulation. MATERIALS AND METHODS Adults of Peronella japonica were obtained in the vicinity of the Noto Marine Laboratory on the Japan Sea coast. Eggs and sperm were collected by artificial spawning induced by intracoelomic injection of a small amount of 100 mM acetylcholine chloride. The eggs were fertilized in artificial seawater (Jamarin U, JSW, Jamarin Laboratory) and the embryos were cultured at 25°C in JSW in plastic petri dishes through to the 16-cell stage. At this stage, aliquots of the embryos were transferred to 24-well plastic plates containing various concentrations of LiCl dissolved in JSW, and cultured for 2 h when the embryos were early blastulae. The embryos were then rinsed with JSW and removed into plastic petri dishes containing JSW for continued culture. The embryos were examined using a stereoscopic microscope (Leica MZ8, Deerfield, IL) at various intervals to count the numbers of embryos that had exogastrulated and/ or those with an evaginated amniotic cavity. The embryos were photographed using a light microscope (Nikon Biophot). RESULTS AND DISCUSSION The archenteron in normal embryos of Peronella japonica invaginated from the vegetal pole, as in embryos of other sea urchins. The amniotic cavity in Peronella embryos was formed on the area corresponding to the presumptive stomodeum of typical sea urchin embryos (Fig. 1A), confirming the previous report (Mortensen, ’21; Okazaki and Dan, ’54; Okazaki, ’75). Three types of morphological abnormalities were found in the Peronella embryos treated with LiCl. Some embryos exogastrulated (Fig. 1B), others evaginated the amniotic cavity (Fig. 1C), and a few embryos showed both phenomena (Fig. 1D). The endodermal area in the exogastrulae was increased (Fig. 1B) in comparison with that in normal embryos, indicating that lithium treatment caused vegetalization of the Peronella embryos. Embryos whose amniotic cavity became evaginated after treatment with LiCl developed into pluteus-like larvae and formed adult skeletons (Fig. 1E). The evaginated amniotic cavity was easily detached from the embryo because it tended to stick to the plastic dish during culture (Fig. 1E). Embryos with an evaginated amniotic cavity failed to differentiate an adult rudiment, and to metamorphose into juveniles. This appears to be reasonable because the adult rudiment is formed from a hydrocoel derived from a coelomic pouch through cooperation with the epidermis of the amniotic cavity. These results indicate that the adult skeleton is formed independently of the adult rudiment. It has been reported that the ratio of formation of exogastrulae in indirect developers is correlated with LiCl concentration (Lallier, ’75; Mitsunaga et al., ’83). Therefore, the effects of various concentrations of LiCl on evagination of the amniotic cavity were examined for Peronella embryos. The sensitivity to LiCl differed markedly among embryos derived from different mothers. Therefore, a typical experimental example is shown in Table 1. The experiments were carried out using concentrations of LiCl ranging from 10 to 60 mM. The highest ratio (53%) of the embryos developing an evaginated amniotic cavity was obtained with 20 mM LiCl. A higher concentration did not always produce a stronger effect, and in fact the weakest effect (7%) was obtained with 30 mM LiCl. The effect of 10 mM LiCl (30%) was weaker than that of 20 mM, but higher than that of 30 mM. Thus, a quantitative relationship between LiCl concentration and amniotic cavity evagination was not found in Peronella embryos. A total of six experiments was carried out using embryos obtained from six different batches, and the results were essentially similar to that shown in Table 1. It is unclear why a quantitative relationship was not found. However, one possibility is that the variation in lithium sensitivity among the embryos derived from the same batch was too great to allow detection of the effects resulting form use of the different concentrations. The effect of LiCl on echinoid embryos showing typical indirect development has been well studied, and exogastrulation based on vegetalization of the embryos has been reported to occur (von Ubisch, ’29; Mitsunaga et al., ’83; Lallier, ’75). In the present EVAGINATION OF AMNIOTIC CAVITY 311 Fig. 1. Normal and LiCl-treated embryos of Peronella japonica (scale bars 50 µm). A: A normal embryo at 18 h after fertilization. An arrow indicates the amniotic opening. B: An embryo at 18 h after fertilization, treated with 60 mM LiCl. The archenteron (arrow) evaginates to form an exogastrula. Arrowheads indicate the boundary between the ectoderm and endoderm. C: An embryo at 24 h after fertilization, treated with 20 mM LiCl. The amniotic cavity (arrowhead) evaginates toward the outside of the embryo. D: An embryo at 20 h after fertilization, treated with 30 mM LiCl. The amniotic cavity (arrowhead) as well as the archenteron (arrow) evaginates from the embryo. E: A pluteus larva at 4 days after fertilization derived from an embryo treated with 30 mM LiCl. The amniotic cavity has evaginated, but subsequently lost because it tended to stick to the plastic dish during culture. An asterisk indicates the place where the evaginated amniotic cavity was formed. Some spines (arrowheads) are formed in the larval body. study, it was found that echinoid embryos showing direct development also underwent exogastrulation after treatment with LiCl. The increased volume of tissue present in the exogastrulated archenterons implies that the embryos were vegetalized. In addition to exogastrulation, it was demonstrated that lithium treatment of Peronella embryos resulted in evagination of the amniotic cavity. So far, it has been considered that an increase in the endodermal territory by treatment of embryos with LiCl causes exogastrulation (Giudice, ’73). However, the amniotic cavity is formed in the animal half of the embryo, suggesting that evagination of the amniotic cavity does not depend on the endodermalizing effect of LiCl. We consider that LiCl has two different effects 312 C. KITAZAWA AND S. AMEMIYA TABLE 1. Relationship between concentration of LiCl and formation of embryos with an evaginated amniotic cavity1 Concentration of LiCl n Number of individuals with an evaginated amniotic cavity 10 mM 20 mM 30 mM 60 mM 30 30 30 30 9 (30%) 16 (53%) 2 (7%) 11 (37%) 1 All embryos were examined at 18 h after fertilization when control larvae were at the pluteus stage. on echinoid embryos: vegetalization and evagination of invaginating structures. ACKNOWLEDGMENTS We thank the members of the Noto Marine Biological Station for supplying materials. We also thank the stuff of the Misaki Marine Biological Station where most of the present work has been carried out. 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