Coral embryos and planula larvae

Early embryos and several stages of the planula larvae of A. tenuis (class Anthozoa; sub-class Hexacorallia, order Scleractinia, suborder Refertina, family Acroporidae) were kindly provided by Mr. Shuichi Mekaru at Onna Fisheries Cooperative, Okinawa, Japan. Larvae were allowed to develop at room temperature (∼25°C) in the Marine Genomics Unit, Okinawa Institute of Science and Technology Graduate University (OIST) until they reached the desired stages. Several batches of embryos and larvae were transferred to Kochi University.

Histology

Embryos at 12, 17, 20, 28, 40, 60, and 85 hours post-fertilization (shown as, for example, 20 hpf embryos) and larvae at 6, 8, 10, 14, and 18 days post-fertilization (shown as, for example, 10 dpf larvae) were fixed in Zamboni's fixative at room temperature for 30 min. They were serially dehydrated in a graded ethanol series and embedded in plastic resin, Technovit 8100 (Heraeus Kulzer, Wertheim, Germany). Samples were sectioned into 2-µm-thick slices with glass knives. Regarding light microscopic observations, sections were stained with 0.5% toluidine blue in 0.1 M phosphate buffer (PB) (pH 7.4) for 10 min and then observed under an ECLIPSE 80i (Nikon, Japan).

Production of antibodies

Antibodies against β-galactosidase (Atβ-Gal), lipoxygenase homology domain-containing protein 1 (AtLoxhd1), MAM-LDL receptor class A domain-containing protein 2 (AtMlrp2), AtSnail, and AtMyocyte-specific enhancer factor 2 (AtMef2) were used in the present study. The production of these antibodies was reported in part by Kawamura et al. (2021). Briefly, synthetic oligopeptides were produced by reference to deduced polypeptide sequence of respective proteins. They were

Before immunization, five oligopeptides each were conjugated to the carrier protein, keyhole limpet hemocyanin (KLH). Rabbit antibodies were raised by Eurofins Genomics, Tokyo. They were diluted 400-fold with phosphate-buffered saline (PBS) immediately before use. A goat anti-rabbit secondary antibody labeled with fluorescein isothiocyanate (FITC) (FI-1000) was purchased from Vector Laboratory (Burlingame, CA) and diluted 200-fold with PBS before use.

Immunohistochemistry

Sections were preincubated for 30 min with blocking solution containing 0.25% blocking reagent (Roche, Mannheim, Germany) and 2.5% skim milk in PBS. They were then incubated with primary antibodies containing 0.1 mg/ml KLH at room temperature for 1 h. Samples were stained with the FITC-labeled secondary antibody for 0.5 h and finally counterstained with DAPI (5 µg/mL) and 0.1 µM rhodamine phalloidin (Cytoskeleton Inc., Denver, CO, USA) for 5–10 min. Negative controls were employed using specific antibodies absorbed in advance by the corresponding oligopeptides at a concentration of 25 µg/mL. After antibody staining, sections were washed twice for 10 min with PBS containing 0.1% Tween 20. Specimens were observed under a confocal microscope (ECLIPSE C1si system; Nikon) by the single acquisition of images. Three types of Exc/Em were used as follows: 408/450, 488/515, and 561/605.

Western blot analysis

To examine the specificity of each antibody, we performed SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analyses using anti-AtMef2, anti-AtSnail, anti-AtLoxhd1, and anti-Atβ-Gal antibodies. Crude extracts of 8 dpf larvae (approximately 20 specimens) were prepared in 100 µL RIPA lysis buffer. They were briefly centrifuged and separated by SDS on a gel containing 12% polyacrylamide. After electrophoresis, proteins were blotted onto nitrocellulose membranes for 90 min at 300 mA. The membranes were then treated with the blocking solution and stained with the antibodies described above.

Cytological features of the gastrodermis and mesentery in A. tenuis polyps

The cytology of metamorphosing A. tenuis polyps is shown in Fig. 1. Polyps consisted of an ectoderm, gastrodermis, stomodeum, mesentery, and yolk cells (Fig. 1A1, B1). The stomodeum is derived from the oral concave and forms a clump in the middle region along the larval long axis (Kawamura et al., unpublished data). The gastrodermis invaginated to form the mesentery (Fig. 1A2, A3), which extended and connected to the stomodeum clump in order to separate the gastric cavity into several chambers (Fig. 1B1–B3).

Unlike the ectoderm showing non-specific fluorescence (Fig. 1A2, A3), the gastrodermis and mesentery were both strongly stained with rhodamine-phalloidin (Fig. 1A2, A3, B3). Yolk cells contained reflective yolk granules that were not observable by dark field microscopy (Fig. 1A1, A2). The gastrodermis and mesentery expressed endoderm-specific markers, such as β-galactosidase (Atβ-Gal) and lipoxygenase (At Loxhd1) (Fig. 1C1). Their nuclei were stained with anti-AtSnail and anti-myocyte-specific enhancer factor 2 (anti-AtMef2) antibodies (Fig. 1C2, C3). These features of the gastrodermis were utilized to chase the larval and embryonic origins of endodermal cells, as will be described below.

Phalloidin-stained cells in larvae and embryos

In 18 dpf larvae, the presumptive gastric cavity was located at the center of larvae (Fig. 2A1). The gastrodermis precursor beneath the mesoglea and stomodeum elongated along the oral-aboral axis and was stained with rhodamine phalloidin (Fig. 2A2, A3). In the gastrodermis, a few phalloidin-positive cells crowded with one another and were randomly located (Fig. 2A3), suggesting that epithelial integrity was still incomplete. On the other hand, in 6 to 8 dpf larvae, the inner endodermal layer was solid, filled with reflective yolk cells (Fig. 2B1, C1), and was strongly stained with rhodamine phalloidin (Fig. 2B2, C2). Phalloidin-positive cells were distributed in the interspace between yolk cells (Fig. 2B3, C3). In 60 to 85 hpf embryos, phalloidin-positive cells were also found in the whole area of the inner endodermal layer (Fig. 2D1–D3). In contrast, phalloidin-positive cells were not observable in 40 hpf or earlier stage embryos (Fig. 2E1–E3).

Fig. 1.

Characterization of the gastrodermis in metamorphosing polyps of A. tenuis. (A1–A3) Ectoderm (e), gastrodermis (g), and yolk cells (y) in the body wall. (A1) Toluidine blue staining. Bar, 20 µm. (A2) Rhodamine-phalloidin and DAPI staining. Bar, 20 µm. (A3) Mesenteries (m) developing from the gastrodermis. Bar, 40 µm. (B1–B3) The mesentery extending toward the stomodeum (s) and dividing the gastric cavity into chambers (gc). (B1) Toluidine blue staining. Bar, 100 µm. (B2) Rhodamine-phalloidin and DAPI staining. Bar, 100 µm. (B3) Connection of the mesentery to the clump of the stomodeum. Bar, 40 µm. (C1–C3) Immunostaining of polyp tissues. (C1) Anti-AtbGal staining. Bar, 40 µm. (C2) Rhodamine-phalloidin and DAPI staining. Arrowheads show nuclei. Bar, 20 µm. (C3) Anti-AtMef2 image of the same section as in (C2). Bar, 20 µm.

We then investigated when and how the endoderm dissociates into single cells and undergoes epithelial transformation to the gastrodermis.

Endodermal cell migration in the yolk cell layer

Nuclear localization was examined by means of DAPI staining. In sections, a 12 hpf prawnchip blastula took a U-shaped configuration (Fig. 3A1). The wall of the blastula consisted of outer and inner simple epithelia, both of which contained yolk granules in the cytoplasm (Fig. 3A2). At a higher magnification, the narrow lumen was observed between these epithelia (Fig. 3A3, white asterisk), indicating that the blastocoel collapsed in prawnchip embryos. The nuclei of the outer and inner epithelia were located near the apical surface of blastomeres (Fig. 3A2). In 17 hpf embryos, the blastocoel began to expand (Fig. 3B) and yolk cells appeared in the whole circumference beneath blastomeres (Fig. 4A).

During 20 to 28 hpf embryonic development, nuclei began to leave the inner layer of embryos to the blastocoel that was filled with yolk cells (Figs. 3C, D; 4B). At 28 hpf, embryos took a gastrula-like form (Fig. 3D1). The blastopore-like opening closed and the gastrocoel was completely included within the embryo (Fig. 3D2). Accordingly, the gastrocoel came from the lumen of the U-shaped prawnchip embryo (Fig. 3, red asterisks).

Fig. 2.

Tracing back of rhodamine-phalloidin staining from larvae to embryos. (A) 18 dpf larvae. (B) 8 dpf larvae. (C) 6 dpf larvae. (D) 85 hpf embryos. (E) 40 hpf embryos. (A1, B1, C1, D1, E1) Toluidine blue staining. The ectoderm (e), oral aperture (oa), presumptive gastric cavity (pgc), and mesoglea (m) are shown. Bars, 100 µm. (A2–E2, A3–E3) Rhodamine-phalloidin and DAPI staining. The stomodeum (s) is visible. Arrowheads show phalloidin-positive cells. (A2–E2) Bars, 50 µm. (A3–E3) Bars, 20 µm.

In 40 hpf embryos, the gastrocoel shrank and finally disappeared (Fig. 3E1, E2). At this stage, the mesoglea initially appeared in the yolk cell layer (Figs. 3E2; 4C, D) around the boundary between the outer yolk cell layer that developed under the outer epithelium (ectoderm) and the inner yolk cell layer that developed under the inner epithelium (endoderm precursor) (Fig. 4D). The nuclei of dissociated cells were dispersed in the whole area inside the mesoglea (Fig. 3E2). In 60 hpf embryos, dissociated cells were stained with rhodamine phalloidin (Fig. 3F1, F2), providing cytochemical evidence for the gastrodermis lineage of cells. The outer yolk cell layer gradually shrank, accompanying the preferential decrease in yolk contents, whereas the inner yolk cell layer appeared to remain intact (Fig. 4E). In 82 hpf embryos, yolk cells were almost lost from the outer yolk cell layer and, consequently, the mesoglea made direct contact with the ectoderm (Fig. 4F).

Fig. 3.

Nuclear localization shown by DAPI staining. White asterisks show the blastocoel. Red asterisks show a gastrocoel-like cavity. (A1, A2, A3) 12 hpf embryos. (A1) Longitudinal section. Bar, 300 µm. (A2) Outer and inner layers of the blastomere containing yolk granules (y). Bar, 20 µm. (A3) Higher magnification of blastomeres. Bar, 10 µm. (B) 17 hpf embryo. Bar, 200 µm. (C1, C2) 20 hpf embryos. Bars, 100 µm. (C1) Gastrula-like embryo. (C2) An embryo in which the blastocoel widely expanded. (D1, D2) 28 hpf embryos. Bars, 100 µm. (D1) The blastopore-like opening (arrowhead) now closed in the embryo. (D2) The gastrocoel included inside the embryo. (E1, E2) 40 hpf embryos. Bars, 100 µm. (E1) The gastrocoel collapsed in the embryo. The mesoglea (m) appears. (E2) Endodermal cells (e) scattering within the mesoglea. (F1, F2) 60 hpf embryo. Bars, 100 µm. (F1) Scattering endodermal cells; (F2) DAPI and rhodamine-phalloidin image of the same section.

Fig. 4.

Fate of yolk cells and formation of the mesoglea during embryogenesis. Toluidine blue staining. (A) 17 hpf embryo, transverse section. The blastocoel (bc) expands, and the yolk cell layer (ycl) appears beneath the blastoderm (bd) that encloses the blastocoel. Bar, 100 µm. (B) 20 hpf embryo. The outer, presumptive ectoderm (ect), the inner, presumptive endoderm (end), the yolk cell layer, and the gastrocoel-like cavity are visible. Bar, 50 µm. (C) 28 hpf embryo. Bar, 20 µm. (D) 40 hpf embryo. The mesoglea (m) appears between the outer and inner yolk cell layers. Bar, 20 µm. (E) 60 hpf embryo. Outer yolk cells decrease in number and reflective yolk granules are preferentially consumed (arrowheads). Bar, 20 µm. (F) 85 hpf embryo. Unlike the inner yolk cell layer, the outer yolk cell layer becomes narrower and most yolk granules are exhausted (arrowheads). Consequently, the mesoglea is located just beneath the ectoderm. Bar, 20 µm.

Immunohistochemistry for the nuclear factor Mef2

The gastrodermis-specific nuclear factors, Mef2 (molecular weight, 51,765) and Snail (mw, 29,813) were traced back to embryos. Anti-AtMef2 and anti-AtSnail antibodies recognized approximately 50- and 30-kDa bands, respectively (see  Supplementary Figure S1 (zs240032_FigS1.pdf), lanes 1, 2). Since Mef2 and Snail showed similar expression patterns, the results obtained with the anti-Mef2 antibody are mainly described here. In 12–20 hpf embryos, outer (presumptive ectoderm) and inner (presumptive endoderm) cells both emitted nuclear signals of AtMef2 (Fig. 5A1, A2, B1–B3). These nuclear signals were not as noticeable because of cytoplasmic autofluorescence, but disappeared in the negative control, in which the anti-AtMef2 antibody was absorbed in advance by a Mef2 peptide used for raising the antibody (Fig. 5A3). Mef2 immunostaining disappeared from 28 hpf embryos (Fig. 5C1–C3). However, it reappeared in 85 hpf embryos and, notably, only endodermal cells emitted the signal (Fig. 5D).

The stomodeum is ectoderm-derived endodermal tissue. In 18 dpf larvae, unlike the ectoderm, the oral aperture and stomodeum extending from it were stained with rhodamine phalloidin (Fig. 6A). Staining began in the oral concave of 8 dpf larvae (Fig. 6B1). The nucleus was stained by anti-Mef2 and anti-AtSnail antibodies (Fig. 6B2, B3). The nucleus of ectodermal cells surrounding the oral concave already expressed AtMef2 (Fig. 6C1, C2).

Immunohistochemistry for lipoxygenase

The gastrodermis preferentially expresses the cytoplasmic factors, β-GAL (mw, 75,470) and Loxhd1 (mw, 206,188). These factors were traced back to embryos and larvae. Anti-Atβ-Gal and anti-AtLoxhd1 antibodies recognized approximately 75- and 250-kDa bands, respectively (see  Supplementary Figure S1 (zs240032_FigS1.pdf), lanes 3, 4). The anti-AtLoxhd1 antibody stained 12, 17, 20, and 28 hpf embryos (Fig. 7A1–C1, A2–C2). However, the signals were not specific because similar staining was observed in the negative control, in which the antibody was absorbed in advance by the AtLoxhd1 peptide antigen (Fig. 7A3–C3). On the other hand, the antibody recognized the endodermal cells of 40 hpf embryos around the shrinking gastrocoel (Fig. 7D1–D3). Similar results were obtained using the anti-Atβ-Gal antibody (data not shown).

In 11 dpf larvae, the anti-Atβ-Gal and anti-AtLoxhd1 antibodies both stained endodermal cells that elongated and aggregated in the vicinity of the mesoglea (Fig. 8A1–A3, B1, B2). In the negative control, immunostaining was markedly weakened by absorption with a Loxhd1 peptide (Fig. 8B3). In 18 dpf larvae, both antibodies exclusively stained the gastrodermis de novo established in the larvae (Fig. 8A4, B4).

As described, the stomodeum is an ectoderm-derived endoderm tissue. The anti-AtMlrp2 antibody recognized the stomodeum (our unpublished data). In 11 dpf larvae, the antibody stained the stomodeum and elongated endodermal cells (Fig. 8C1–C3). In 18 dpf larvae, the stomodeum was mainly stained, whereas the larval gastrodermis was weakly stained (Fig. 8C4).

Fig. 5.

Immunohistochemistry for AtMef2 in embryos. (A1–A3) 12 hpf embryos. (A1) DAPI staining. Arrowheads show nuclei. Bar, 40 µm. (A2) FITC image of anti-AtMef2 immunostaining. Arrowheads show AtMef2-positive nuclei. Bar, 40 µm. (A3) Negative control. The antiAtMef2 antibody was preabsorbed with the Mef2 peptide. Bar, 20 µm. (B1–B3) 20 hpf embryos. (B1) Double staining for DAPI and the antibody. Arrowheads show the nuclei of outer blastomeres, and a broken circle shows the nuclei of the inner blastomere. Bar, 40 µm. (B2) FITC image of the antibody in outer blastomeres. Arrowheads show positive nuclei. Bar, 20 µm. (B3) FITC image of the antibody in inner blastomeres. Arrowheads show AtMef2-positive nuclei. Bar, 20 µm. (C1–C3) 28 hpf embryos. (C1, C2) Double staining for DAPI and the antibody. (C3) FITC image of antibody staining. (C1) Bar, 100 µm. (C2, C3) Bars, 40 µm. (D1–D3) 85 hpf embryos. (D1, D2) Double staining for DAPI and the antibody. (D3) FITC image of antibody staining. (D1) Bar, 100 µm. (D2) Arrowheads show nuclei. Bar, 20 µm. (D3) Arrowheads show positive nuclei. Bar, 20 µm.

Blastula and gastrula formation in Acropora tenuis

Stony corals (Cnidaria; Hexacorallia; Scleractinia) are one of the most well studied marine animals as a major component of coral-reef ecosystems with extremely high biodiversity. The order Scleractinia comprises of two suborders Refertina (sometimes called “Complex”) and Vacatina (sometimes called “Robust”) (Romano and Palumbi, 1996; Kitahara et al., 2010; Okubo, 2016; Quek et al., 2023). The former includes Acropora and Galaxia and the latter includes Fungia and Dipsastraea. Recent studies of coral embryogenesis have demonstrated that the two suborders exhibit different modes of early embryogenesis (Okubo, 2016; Okubo et al., 2013): especially, in contrast that corals of robust clade form a discrete blastocoel, those of complex clade develop no blastocoel (Okubo et al., 2013). In addition, the formation of two germ-layers is found during gastrulation in Vacatina (robust) corals but not seen in Refertina (complex) corals (Okubo, 2016; Okubo and Motokawa, 2007; Okubo et al., 2013, 2016). Therefore, the formation of gastrodermis and mesentery looks more complex in Refertina than in Vacatina, and the cellular and molecular mechanisms thereof remain to be elucidated.

Fig. 6.

Acquisition of endoderm-specific signals in the oral concave (oc), oral aperture (oa), and stomodeum (s) derived from the ectoderm (ect). (A) Oral region of 18 dpf larva. Rhodamine-phalloidin image merged with DAPI staining. Bar, 20 µm. (B) Oral concave of the ectoderm, 8 dpf larva. (B1) Rhodamine-phalloidin image merged with DAPI staining. Bar, 20 µm. (B2) Anti-AtSnail immunostaining merged with DAPI staining. Bar, 10 µm. (B3) Anti-AtSnail immunostaining. Bar, 10 µm. (C) Anti-AtMef2 immunostaining, 8 dpf larva. (C1) Merged rhodamine-phalloidin and DAPI images. Bar, 40 µm. (C2) Immunostaining of ectodermal nuclei in the presumptive oral concave. Bar, 20 µm.

The present study demonstrated for the first time the total pathway from the formation, development, and transformation of the endoderm to the gastrodermis in A. tenuis (Fig. 9). The blastula in A. tenuis commenced without a hollow blastocoel, since the blastocoel is compressed in ‘cushion-shaped’ embryos and then gradually swells, which is consistent with the results shown by Okubo and Motokawa (2007). Between U-shaped outer and inner epithelia, a narrow lumen expanded and became a blastocoel (Fig. 9). Gastrulation only accompanied the closure of the open extremity of the U-shaped embryo without apparent invagination or involution (Fig. 9).

The transcription factor Mef2 is preferentially expressed in muscle and mesodermal tissues and is essential for muscle development in triploblastic animals (Lilly et al., 1994). In diploblastic sea anemones, Mef2 is expressed in the columnar cells of the ectoderm in the gastrula and planula larva, but not in the gastrodermis of polyps (Martindale et al., 2004), whereas a splice variant of Mef2 appears in the endoderm (Genikhovich and Technau, 2011). Jellyfish Mef2 may be expressed in the endoderm or ectoderm after gastrulation (Boero et al., 1998). In A. tenuis, the gastrodermis in polyps was preferentially stained with the anti-Mef2 antibody. In 12–20 hpf embryos, Mef2 was temporarily expressed by both the outer and inner epithelia, and then reappeared in endodermal free cells from 40 hpf embryos onwards. Serial observations of embryonic nuclei showed that endodermal free cells came from the inner wall of early embryos. Therefore, the endoderm expressed Mef2 twice at embryonic stages at 12–20 hpf, possibly in endoderm of maternal origin, and then at 40 hpf in endoderm of zygotic origin (Fig. 9).

Fig. 7.

Anti-AtLoxhd1 immunostaining in embryos labeled with FITC. Specimens, except in (D2) and (D3), were also stained with DAPI. (A1–D1, A2–D2) Results of immunostaining. (A3–D3) Negative control by the absorption test. (A) 12 hpf embryos. (B) 17 hpf embryos. Arrowheads in (B2, B3) show fluorescent signals, which still remained after the absorption test. (C) 28 hpf embryos. The ectoderm (ect), presumptive endoderm (end), and gastrocoel (gc) are shown. (D) 40 hpf embryos. Endodermal cells around the regressing gastrocoel are shown. Bars in (A1, B1, C1) show 100 µm. Bars in (A2, B2, C2) and (A3, B3, C3) show 50 µm. Bars in (D1–D3) show 20 µm.

Cell dissociation of endodermal cells by ingression in A. tenuis

The endodermal cells of 28 hpf ‘donut’-stage embryos began to leave the basal area of the blastomere (Fig. 9). From 40 hpf to 60 hpf ‘pear’-stage embryos, endodermal cells were scattered in the blastocoel, which was filled with yolk cells by 40 hpf of embryonic development (Fig. 9). This cell behavior may be regarded as a large-scale ingression. In triploblastic animals, ingression is a mode of gastrulation that gives rise to the mesoderm. In the diploblastic hydrozoan Clytia hemisphaerica, the endoderm develops from individual cells detaching from the blastoderm and migrating inwards to fill the blastocoel (Byrum, 2001; van der Sande et al., 2020), which is similar to that in Acropora embryos.

Fig. 8.

Endoderm-specific immunostaining of 11 dpf larvae (A1–A3), (B1–B3), (C1–C3) and 18 dpf larvae (A4), (B4), (C4). (A1)–(A4) AntiAtβGal antibody staining. (B1)–(B4) Anti-AtLoxhd1 antibody staining. Both antibodies recognized elongated endodermal cells (arrowheads) beneath the mesoglea (m) and larval gastrodermis (g). (C1)–(C4) Anti-AtMlrp2 antibody staining. This antibody recognized the stomodeum (s) and elongated endodermal cells (arrowheads), but weakly stained the larval gastrodermis (g). (A1, B1, C1) FITC images merged with rhodamine-phalloidin and DAPI staining. (A2, A4, B2, B4, C2) FITC images merged with DAPI staining. (A3, C3, C4) FITC images. (B3) Negative control by the absorption test. Bars in (A1, A4, B4), and (C1–C3) indicate 40 µm. Bars in (A2), (A3), and (B1–B3) indicate 20 µm. The bar in (C4) indicates 100 µm.

In sea anemone embryos, Snail is involved in ingression through epithelial-mesenchymal transition (Fritzenwanker et al., 2004). In sea urchin embryos, Snail is required for the ingression of primary mesenchymal cells (Wu and McClay, 2007). It appears to suppress the function of the cell adhesion molecule cadherin and stimulate the endocytosis of cadherin. In A. tenuis, Snail localizes in vivo in the nucleus of the polyp gastrodermis. However, Snail expression was ambiguous around the ingression stage. Accordingly, it remains unclear whether Snail is involved in the ingression movement of endodermal cells in A. tenuis.

Fig. 9.

Summary of the developmental pathway of endodermal cells toward the gastrodermis of polyps in A. tenuis. Early embryos have a U-shaped configuration. The blastocoel soon expands and is embedded by yolk cells (yellow in color). During gastrulation, the endoderm forms without invagination and is dissociated into single cells via ingression. Single endodermal cells and then the larval oral concave and stomodeum become positive for rhodamine phalloidin (red in color). Endodermal cells become elongated and gather with one another to form the gastrodermis in association with the mesoglea (light blue in color).

Yolk cell behavior and formation of the mesoglea in A. tenuis

The present study demonstrated the novel and unique behavior of yolk cells. Prawnchip embryos contained a large number of reflective yolk granules in every blastomere, which is consistent with the findings reported by Okubo and Motokawa (2007). Yolk cells initially appeared in 17 hpf embryos and fell into the increasing volume of the blastocoel (Fig. 9). At 17 hpf, yolk granules remained in the cytoplasm of blastomeres, and they conspicuously decreased in number in 22 hpf embryos. This result suggests that yolk cells are segregated from blastomeres by unequal cell division.

Since yolk cells appeared in the whole circumference of the blastocoel, the outer yolk cell layer underlay the future ectoderm while the inner yolk cell layer underlay the future endoderm after approximately 20 h of embryonic development when the blastopore closed. The outer yolk cell layer has not yet been examined in detail because outer yolk cells were preferentially consumed during late embryonic stages and completely disappeared from swimming larvae (Fig. 9).

The mesoglea plays a leading role in the regeneration of the hydra body. According to early studies on reconstituting hydras, both ectoderm and endoderm synthesize the mesoglea (Epp et al., 1986). In the jellyfish Aurelia, amoeboid, mesogleal cells synthesize and secrete the extracellular component of mesoglea (Shaposhnikova et al., 2005). In A. tenuis, the mesoglea appeared in 40 hpf embryos at the boundary or in the vicinity of the boundary between outer and inner yolk cell layers. This result suggests that yolk cells synthesize and secrete the extracellular matrix components of the mesoglea.

Cell aggregation and formation of the gastrodermis in A. tenuis

One of the main questions examined in the present study is how endodermal cells form the gastrodermis associated with the mesoglea. As described, phalloidin-positive endodermal cells were scattered in the blastocoel of embryos and larvae. A similar finding was previously reported (Okubo and Motokawa, 2007). Immunohistochemical studies using anti-AtLoxhd1 and anti-Atβ-Gal antibodies showed that endodermal cells in 11 dpf larvae took an elongated configuration and aggregated to one another in the vicinity of mesoglea (Fig. 9). To the best of our knowledge, this is the first study to explicitly show the process of gastrodermis formation. The process can be regarded as mesenchymal-epithelial transition (Fig. 9).

In A. tenuis, the stomodeum acquired endodermal characteristics (rhodamine-phalloidin and Snail staining) when the oral concave invaginated and some ectodermal cells were dissociated into single cells. The stomodeum contains fibroblast-like elongated cells with pseudopodia (Kawamura et al., unpublished data). In vitro flattened amorphous cell lines (FAmC) are considered to be derived from the in vivo stomodeum (Kawamura et al., 2021; Kawamura et al., in preparation). AtMlrp2 was selected from the FAmC gene expression library for antibody production. The anti-AtMlrp2 antibody recognized the larval stomodeum. In the sea anemone N. vectensis, gastrodermal tissues other than the stomodeum and mesenterial filaments came from the embryonic endoderm (Steinmetz et al., 2017). Consistent with this notion, the elongated endodermal cells of A. tenuis consistently shared rhodamine-phalloidin stainability and Loxhd1 and β-Gal localization with the gastrodermis. Therefore, a relationship exists among dissociated endodermal cells, elongated cells, and gastrodermis. In contrast, the anti-AtMlrp2 antibody did not stain the gastrodermis, but stained elongated cells. These results suggest that the stomodeum participates in the formation of the gastrodermis or related tissues.

Perspective: Molecular and cell biology of endomesoderm and gastrodermis in coral embryos and planulae

The evolutionary origin of bilaterian mesoderm is one of the most intriguing issues of controversy. Mesodermal genes such as Mef2 and Snail in triploblastic Bilateria are expressed in cnidarian endoderm (Fritzenwanker et al., 2004; Genikhovich and Technau, 2011; Röttinger et al., 2012). The hypothesis proposed is that the mesoderm and endoderm of Bilateria could be derived from the endomesoderm of a diploblastic ancestor, otherwise the endomesoderm of cnidarians might be a secondary simplification, derived from an ancestral condition of triploblasty (Martindale et al., 2004). The present study suggests that zygotic AtMef2 appears in late stages of embryogenesis. The mechanism of de novo AtMef2 expression waits for analysis, which may afford insight into the evolution of the molecular network regulating AtMef2 gene expression.

Cell dissociation and reaggregation are another significant feature of the coral endomesoderm (Fig. 9). The present study confirmed that the former occurs by ingression which is a necessary prerequisite for the endomesoderm (gastrodermis precursor) to spread in the blastocoel. The latter is essential for the epithelial transformation of the gastrodermis. The mechanism of mesenchymal-epithelial transition in corals is unknown, and therefore, studies on the nature and dynamics of cell adhesion molecules in Cnidaria are desired.