Ectodermal Cell Differentiation by Unequal Cell Division in the Stony Coral Acropora tenuis

Ectodermal Cell Differentiation by Unequal Cell Division in the Stony Coral Acropora tenuis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Ectodermal Cell Differentiation by Unequal Cell Division in the Stony Coral Acropora tenuis Kaz Kawamura, Satoko Sekida, Koki Nishitsuji, Noriyuki Satoh This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8654320/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background In Anthozoa, the ectoderm contains the epidermis, progenitor/undifferentiated cells, and nerve cells; however, their embryonic origin and development remain unclear. In the stony coral Acropora tenuis , both blastula formation and gastrulation are quite unique. Endodermal cells form by ingression from the presumptive pseudo-archenteron, while ectodermal cell differentiation is poorly understood. Results Ultrastructural studies on A. tenuis showed that the ectoderm consisted of four cell layers stratified apicobasally from the glandular epithelium to yolk cells through undifferentiated-like cells and nerve lineage cells. A 12-hour ‘prawnchip’ embryo had a U-shaped outline devoid of blastocoel. Blastomeres of the embryo were monolayered and the outer blastomeres began to elongate in 17-hour embryos. The elongating blastomeres segregated Snail/Etv6-expressing yolk cells from their growing tips into the narrow blastocoel. In 28-hour ‘donut’ embryos, the nuclei of elongated ectodermal blastomeres translocated to the growing extremities of cells that became the glandular epithelium. Secondary segregation occurred herein in 40-hour embryos to produce ‘bottom’ cells labeled with SoxB2/SoxC near the yolk cells at the bottom of the ectoderm. In 60-hour embryos, the third segregation occurred to produce ‘middle’ cells in the middle region of the ectoderm. The segregated cells expressed lysine-specific demethylase 5 (KDM5) at embryonic stages, and they were tagged by trimethylated histone H3 at lysine 4 at larval stages. Conclusions The present study shows that in A . tenuis embryonic outer blastomeres and the blastomere-derived glandular epithelium expresses Snail, Etv6, C-Jun, SoxB2, SoxC, and KDM5. The glandular epithelium contains founder cells that give rise to yolk cells and progenitor/undifferentiated cells via three steps of unequal cell division. Every segregation accompanies the expression of C-Jun. The initial segregation event accompanies Snail/Etv6 expression, resulting in the yolk cell formation. The secondary and third segregation events accompany SoxB2/SoxC and KDM5, respectively. The former gave rise to nerve progenitors through ‘bottom’ cells, and the latter formed undifferentiated-like cells through ‘middle’ cells. Our results suggest that the ectodermal cell differentiation in Acropora depends on spatiotemporal condition of unequal segregation. Glandular epithelium Nerve progenitor Undifferentiated cell Yolk cell C-Jun KDM5 Snail Sox Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 INTRODUCTION Embryonic development in Cnidaria has attracted the interest of many researchers with special reference to the molecular and anatomical evolution of Bilateria. Blastula formation and gastrulation are two such examples. In the stony coral Acropora tenuis , the blastula forms without a blastocoel ( 1 , 2 ). The blastocoel subsequently appears, spreads widely, and plays an important role in the formation of the gastric cavity of polyps instead of the completely collapsing gastrocoel ( 2 ). Gastrulation occurs by invagination in jellyfishes ( 3 ), by invagination in the sea anemone Nematostella ( 4 ) and the combined activity of invagination and ingression in other Anthozoa ( 5 ), and by delamination in some hydrozoans ( 6 ). Endodermal cells in A . tenuis formed by ingression and behaved as if they were mesoderm-like cells until the gastrodermis formed ( 2 ). In contrast with the relative simplicity of the endodermal repertoire of differentiation, the anthozoan ectoderm contains complex cell types: an epidermis, gland/secretory cells, progenitor/undifferentiated cells, nerve cells, and cnidocytes ( 7 ). In A . tenuis , the outer and inner blastomeres (the presumptive ectoderm and endoderm) give off outer and inner yolk cells, respectively ( 2 ). Accordingly, outer yolk cells also belong to the ectoderm group. Recent studies on Nematostella polyps provided detailed information on the developmental pathways of neurons, gland cells, cnidocytes, and germ cells from putative stem cells through to progenitor cells ( 8 , 9 ). However, the embryonic origin of these differentiated cells remains unclear. Single-cell RNA sequencing technology has contributed to innovative advances in our understanding of cnidarian cells ( 7 , 10 , 11 ). In Nematostella , the epidermis expresses Claudin, a tight junction component ( 12 ), and other cell adhesion molecules ( 7 ). Unlike Hydra , the anthozoan epidermis does not have the epitheliomuscular type of cells other than the tentacle and oral disc ( 13 ). Some types of gland/secretory cells are scattered among the larval aboral ectoderm and express secretory protein genes ( 11 ). Progenitor/undifferentiated cells include a common progenitor for neurons, cnidocytes, and gland cells ( 14 ). They express nuclear factors such as Myc and SoxB ( 7 ) as well as Nanos and Piwi ( 8 , 9 ). Nerve and gland cells share the gene expression of FoxQ ( 11 ). Cnidocytes are characterized by multiple venom and capsule proteins ( 15 ). Although the gene catalog of each cell type has been extensively examined, limited information is currently available on the spatiotemporal origin and pathway of ectodermal cell differentiation in Cnidaria. We recently established many in vitro cell lines of A . tenuis that may be classified into three cell types ( 16 ). The gene expression profiles of cultured cells were found to partially reflect in vivo gene expression ( 17 ). Neuroblast differentiation-associated protein (AHNAK) is one such example and is highly expressed by brilliant brown cells (BBrC) belonging to the flattened amorphous cell type (FAmC). The in vivo nerve lineage from neuroblasts to neurons expresses AHNAK ( 17 ). Small smooth cells (SSmC) are another ectodermal type of cultured cells in A . tenuis . They express secretory proteins, such as endoglucanase and skeletal organic matrix proteins, which are specific to ectodermal secretory cells ( 17 ). These in vitro molecular probes of information may be utilized in the in vivo study of ectodermal cells. The primary purpose of the present study was to examine when and how ectodermal cell differentiation occurs in A . tenuis embryos. Larval ectodermal cells were classified ultrastructurally. We identified four types of stratified cell layers. We then investigated how embryonic blastomeres contribute to the spatiotemporal establishment of these four cell types. Antibodies against Snail, C-Jun, SoxB2, and SoxC were used to examine the formation of yolk cells and progenitor cells. We also investigated the developmental pathway from neuroblast precursors to neuroblasts and nerve cells using antibodies against nerve-specific tubulin beta-III (TUBB3), and the transcription factor, Sox. The results obtained herein provide evidence for ectoderm-derived cells originating from the developing glandular epithelium by unequal cell segregation. RESULTS Ectodermal Cell Morphology in A. tenuis Larvae The epidermis of 14 dpf larvae is shown ultrastructurally in Fig. 1 . It was divided into three regions (Fig. 1 A). The apical region mainly comprised elongated columnar cells (Fig. 1 B1). A tight junction was present between neighboring cells at the apical surface (Fig. 1 B2). Various types of granules and membrane-bound vacuoles were located in the cytoplasm (Fig. 1 B1), and some vacuoles were secreted from the apical cell surface (Fig. 1 B2 arrowhead). Accordingly, columnar cells were regarded as a new type of glandular/secretory epithelium. Vacuolated hyaline cells (VHCs) appeared among glandular epithelia (Fig. 1 C) and attained to the ectodermal basal region (not shown). Cnidocytes and their progenitors were also embedded in the apical region (Fig. 1 C, Suppl. Figure 1A). The middle region of the ectodermal layer was filled with small and round cells (Fig. 1 D). They appeared to be undifferentiated (Fig. 1 E). In the ectodermal basal region, large and oval cells were located in the vicinity of yolk cells (Fig. 1 D, F1, F2). They contained large granules in the cytoplasm (Fig. 1F1, F2), and the endoplasmic reticulum was located just beneath the plasma membrane (Fig. 1F1 inset). The nucleus showed an indented morphology (Fig. 1F1, F2 arrows). In addition to these features, some cells contained the Golgi apparatus and had a cytoplasmic protrusion (Fig. 1F2). This type of cell is a nerve progenitor (neuroblast) (see also Fig. 7 F). The nerve cell body was located at this basal area and enriched with synaptic vesicles (Fig. 1F3). It extended the cytoplasmic process towards the apical surface (Fig. 1F3 arrow, see also Fig. 7 G). VHC-like cells and cnidocytes sometimes appeared spontaneously in A. tenuis cell culture plates of FAmC and SSmC, respectively (Suppl. Figure 1B, C) ( 17 ). However, they have yet to be established as stable cell lines. Cell Elongation and Yolk Cell Segregation from Snail-expressing Blastomeres ‘Prawnchip’ embryos at 12 hpf had a U-shaped outline (Fig. 2A1) ( 2 ). The outer blastomeres (presumptive ectoderm) and inner blastomeres (presumptive endoderm) were both monolayered and had a round or cuboidal configuration (Fig. 2A2). Yolk granules were scattered within the cytoplasm of blastomeres (Fig. 2A3). In 17 hpf embryos, the blastocoel gradually expanded (Fig. 2B1-B3). Some outer and inner blastomeres began to elongate (Fig. 2B3, B4). Yolk granules were inclined to accumulate at the growing tip of blastomeres and segregated into de novo emerging yolk cells (Fig. 2B5). They fell into the developing blastocoel of ‘donut’ embryos (Fig. 2B5 bidirectional arrows). In 12 hpf embryos, AtSnail signals were observed in the nuclei of both outer and inner blastomeres and also in the whole cytoplasm (Fig. 3 A), similar to AtMef2 signals ( 2 ). In 17 hpf embryos, AtSnail signals were concentrated at the growing tip of elongated epithelial cells (Fig. 3 B white asterisks) or around yolk granules (Fig. 3 B red asterisks). In 22 hpf embryos, the blastocoel expanded throughout the whole embryo (Fig. 3C1, C2). Cytoplasmic AtSnail signals gradually attenuated, while nuclear signals were prominent in the outer blastomeres (Fig. 3C3). The gastrocoel-like lumen was observable, but came from the U-shaped concavity of the prawnchip embryo (Figs. 2A1, B1, 3C1, D1). Accordingly, the lumen was referred to as a pseudo-archenteron. The term pseudo-blastopore was also used in the present study. In 28 hpf embryos, the pseudo-blastopore closed and the blastocoel was filled with yolk cells (Figs. 3D1, D2, 4A). The yolk cell showed a moderate AtSnail signal, whereas the ectodermal gland epithelium scarcely emitted this signal (Fig. 3D3). Endodermal cells were released from the wall of the pseudo-archenteron and scattered in the blastocoel by ingression (Figs. 3D4 arrowheads). They were discernible histochemically from yolk cells because endodermal cell nuclei (Fig. 4 B) were stained with DAPI (Fig. 3D4, E3), whereas yolk cell nuclei (Fig. 4 C) were not ( 2 ). In the negative control, AtSnail signals disappeared from yolk cells after the pre-treatment of the anti-AtSnail antibody with the Snail peptide antigen (Fig. 3D5). Immunohistochemical results were consistent with those of RNA sequencing, which showed that AtSnail expression was the highest in early embryos (Table 1 ). In 40 hpf embryos, the pseudo-archenteron collapsed and the mesoglea appeared between the outer and inner yolk cell layers (Fig. 3E1, E2). The outer yolk cell layer was strongly stained with the anti-AtSnail antibody, whereas inner yolk cells were moderately stained (Fig. 3E2). Endodermal cells did not have AtSnail signals (Fig. 3E3, E3’). In the negative control, strong signals disappeared from outer yolk cells (Fig. 3E4). The pseudo-archenteron disappeared completely from 60–85 hpf embryos (Fig. 3F1, F2). Outer yolk cells still emitted strong Snail signals, but gradually decreased in number (Fig. 3F2, F3). In contrast, the inner yolk cell layer kept the cell number throughout larval stages, consistent with previous findings from histological studies using toluidine blue ( 2 ). AtETV6 was expressed in a similar pattern to AtSnail. In 17 hpf ‘prawnchip’ embryos, the growth tip of elongated blastomeres and the periphery of yolk granules were strongly stained (Suppl. Figure 2A1, A2). Nuclear signals were detected in both the presumptive ectoderm and endoderm of 22 hpf ‘donut’ embryos (Suppl. Figure 2B1, B2). Unlike AtSnail, 40 hpf ‘donut’ embryos did not emit Etv signals (not shown), whereas the outer yolk cell layer in 60 hpf ‘pear’ embryos was stained with the anti-AtEtv6 antibody (Suppl. Figure 2C1, C2), similar to AtSnail, but at a later stage. The number of Etv-positive outer yolk cells markedly decreased in 85 hpf embryos (Suppl. Figure 2D1, D2). Segregation of ‘Bottom’ and ‘Middle’ Cells in Embryos As already shown, blastomeres began to elongate in 17 hpf embryos (Fig. 2 B). Epithelial cells became slenderer from 22 to 40 hpf and aligned side by side (Fig. 5A1-A4). In 22 hpf embryos, they were 40–45 µm long in the major axis (Fig. 5B1 yellow broken line). The nucleus was located in the middle region of the elongated cell (Fig. 5B1). Non-elongated blastomeres remained near the apical surface of the embryo (Fig. 5B1 white asterisks). In 28 hpf embryos, ectodermal cells reached a maximum length of approximately 60 µm (Fig. 5B2 yellow broken line), and the nucleus translocated to the elongating tip of the cell. Vacuoles became prominent in the cytoplasm of columnar cells (Suppl. Figure 3), which was characteristic of the ectodermal gland epithelium (cf., Fig. 1B1, B2). Table 1 Gene expression of developing embryos, larvae, and polyps revealed by transcriptomic RNA sequencing. Gene symbol Gene ID TPM Embryos Larvae Polyps 3.5 hpf 15 hpf 25 hpf 36 hpf 60 hpf 85 hpf 5.5 dpf 8.5 dpf 14 dpf AtSnail2 s0006. g224.t1 231.39 173.77 36.97 40.87 66.45 49.95 47.7 64.14 19.5 AtC-Jun s0207. g13.t1 8.17 26.21 12.54 2.98 3.03 4.41 1.17 2.07 762.13 AtSoxB2 s0027. g3.t1 6.03 38.15 50.81 25.63 34.46 12.21 8.54 3.77 8.05 AtSoxC s0330. g5.t1 5.33 34.89 16.69 24.25 20.1 21.6 19.99 17.84 14.28 AtHES1 s0076. g32.t1 0 0 29.83 17.31 30.87 46.82 32.37 8.11 47.84 AtHES4 s0076. g33.t1 0 0 49.44 100.01 99.3 95.96 87.42 7.51 175.55 AtRFamide s0012. g36.t1 0 0 3.97 0 9.68 22.86 27.32 21.5 20.74 AtKDM5A s0006.g60.t1 5.45 21.09 123.2 92.4 85.06 87.62 106.1 27.73 26.42 Paired nuclei were often observed in 40 hpf embryos, suggesting that the gland epithelium gave rise to daughter cells by unequal cell division (Fig. 5B3). In 60 hpf embryos, the elongated gland epithelium shortened to approximately 40 µm, leaving daughter cells at the bottom of the ectodermal layer (Fig. 5B4 black arrowheads). ‘Bottom’ cells were thus formed (cf., Fig. 3E1 green circles). On the other hand, the gland cell continued to divide in situ (Fig. 5B4 broken circles), producing de novo paired cells in the middle of the ectodermal layer. Consequently, ‘middle’ cells were formed (cf., Fig. 3F1 blue circles). C-Jun Expression in Relation to Cell Segregation RNA sequencing data indicated that C-Jun, an AP-1 subunit, was expressed during the early embryonic stage and polyp stage (Table 1 ). The former was coincident with the segregation time period of ‘bottom’ cells and ‘middle’ cells. AtC-Jun expression was not immunohistochemically observable in 12 hpf embryos (Fig. 6 A). It initially appeared in segregating blastomeres and yolk cells at 17 hpf of embryonic development (Fig. 6 B). In 28 hpf ‘donut’ embryos, the majority of ectodermal nuclei were stained (Fig. 6 C). Mitotic figures were not detected, whereas amorphous, rectangular nuclei were often observed (Fig. 6 D arrowheads). AtC-J-Jun expression persisted in the paired nuclei of 40 hpf embryos (Fig. 6 E) and in ‘bottom’ and ‘middle’ cell nuclei until 60 hpf ‘pear’ embryos (not shown). Cytological Features of ‘Bottom’ and ‘Middle’ Cells According to RNA sequencing, Sox gene expression began at the ‘prawnchip’ stage and was maintained during the ‘donut’ stage when ‘bottom’ and ‘middle’ cells appeared (Table 1 ). Immunohistochemical studies showed that AtSoxB2 and AtSoxC were both expressed in the whole cytoplasm of elongated blastomeres in 17 hpf embryos (Fig. 7 A, B1). In the negative control, Sox signals markedly decreased after the absorption of antibodies with specific peptides (Fig. 7B2). In 28 hpf embryos, cytoplasmic signals were maintained, and nuclear signals were reinforced in the glandular epithelium (Fig. 7C1, C2), which was sensitive to the absorption test (Fig. 7C3). In 40 hpf embryos, not only single nuclei but also paired nuclei emitted strong Sox signals (Fig. 7D1, D2 arrowheads and broken circles), while cytoplasmic signals became attenuated. Sox signals were not apparent in 60–85 hpf embryos (Fig. 7 E); however, a weak expression signal reappeared at the bottom of the ectoderm in 6.5 dpf larvae (Fig. 7F1-F3). In 8 dpf larvae, Sox-expressing ‘bottom’ cells developed into neuroblasts that emitted nerve-specific TUBB3 signals (Fig. 7G2, H). They extended cytoplasmic processes towards the apical surface of the ectoderm. On the other hand, anti-Sox antibodies did not recognize ‘middle’ cells (Fig. 7 F-H). Lysine-specific demethylase 5 (AtKDM5) is one of epigenetic factors. It antagonizes trimethylation of histone H3K4 (H3K4me3). AtKDM5 appeared in ‘donut’ embryos (Table 1 ), and became prominent in ectodermal ‘middle’ cells of ‘pear’ embryos (Fig. 8 A). AtKDM5 disappeared from larvae (Fig. 8 B). In the meanwhile, H3K4me3 appeared in ectodermal ‘middle’ cells in 8 dpf larvae (Fig. 8 C), indicating that epigenetic markers would be available for lineage tracing of undifferentiated-like cells in A . tenuis . Histone methylation in Acropora progenitor cells as well as undifferentiated cells will be reported in detail elsewhere. DISCUSSION A Snail-expressing yolk cell is the primary segregation product from elongating Snail + blastomeres In A. millepora , a related species of A. tenuis , ectodermal Snail-expressing cells become elongated in 18–24 hpf embryos ( 18 ). Consistent with this finding, the present study showed that in A . tenuis , the elongating outer blastomeres (presumptive ectoderm) of ‘prawnchip’ embryos emitted Snail signals from the whole cytoplasm. As shown in this study, they also expressed Etv6, C-Jun, SoxB2, and SoxC (Fig. 9 ). A previous study reported that developing blastomeres expressed Mef2 ( 2 ). An increasing number of elongated blastomeres contained vacuoles in the cytoplasm and became the ectodermal glandular epithelium during the ‘donut’ stage (Fig. 9 ). It remains unclear whether Snail and/or other transcription factors are involved in the differentiation of blastomeres to the ectodermal epithelium. The ectodermal glandular epithelium in A. tenuis expressed a secreted protein, endoglucanase (Fig. 9 ) ( 17 ), similar to non-epithelial gland/secretory cells in the sea anemone Nematostella ( 7 , 11 ). Under electron microscopy, we did not observe non-epithelial gland cells in the Acropora larval ectoderm. Instead, the ectoderm contained VHCs, in which the vacuole was not solid but translucent. VHCs originate from the larval neuroblast (Kawamura et al., in preparation). In Nematostella , gland/secretory cells are derived from nerve progenitors ( 7 – 9 , 11 , 14 ). Therefore, VHCs in stony corals may substitute for Nematostella gland/secretory cells. The present study showed that elongating outer and inner blastomeres gave rise to yolk cells by unequal segregation, confirming our recent findings ( 2 ). Outer yolk cells arising from outer blastomeres preferentially emitted ring-like Snail signals from the gastrula stage (40 hpf ‘donut’ embryos). Inner yolk cells fulfilling the blastocoel emitted weak signals, while endoderm cells scattered among inner yolk cells had no apparent signals. The present results contrasted with previous findings on A . millepora showing that the endoderm expressed Snail during gastrulation ( 18 ). In Nematostella , Snail is also expressed in immigrating endodermal cells during gastrulation ( 19 , 20 ). It is important to note that in A . tenuis , gastrulation occurs by ingression and a very small number of presumptive endodermal cells migrate from the inner-most layer of the embryo into the blastocoel at the ‘donut’ stage when the pseudo-blastopore and pseudo-archenteron close ( 2 ). In situ hybridization studies on Acropora embryos showed that Snail signals were densely localized around the blastocoel ( 18 , 21 ). As discussed above, endodermal cells in A . tenuis were scattered at a low cell density in the blastocoel ( 2 ), and they did not emit Snail signals. Therefore, at least in A . tenuis , only yolk cells appear to express Snail in ‘donut’ embryos. ‘Bottom’ cells are secondary segregation products destined to nerve progenitors In A. millepora , both SoxB and SoxC is initially expressed in the presumptive ectoderm and later SoxC is found in putative sensory neuron ( 22 ). The present study confirmed and extended these findings: In A . tenuis , after the first segregation of yolk cells, the outer blastomeres became the glandular epithelium devoid of yolk granules; the ectodermal epithelium expressed AtSoxB2 and AtSoxC initially in the whole cytoplasm of elongating cells; then, AtSoxB2 and AtSoxC entered the nucleus of the epithelium and were dispensed into the secondary segregation cells at the bottom of the ectoderm (Fig. 9 ). The longest ectodermal cells in 28 hpf embryos are expected to be responsible for the deposition of ‘bottom’ cells near the outer yolk cell. To the best of our knowledge, the present study is the first to describe ‘bottom’ cells. ‘Bottom’ cells expressed C-Jun in addition to Sox (Fig. 9 ). SoxB2 and SoxC are both markers of neuroglandular precursors in Nematostella ( 8 , 14 , 23 ). In the present study, ‘bottom’ cells, nerve progenitors (neuroblasts), and nerve cells expressed AtSoxB2 and AtSoxC, and had cell bodies (nuclei) in common at the bottom of the ectoderm, indicating that AtSoxB2- and AtSoxC-expressing ‘bottom’ cells would develop into nerve progenitors and nerve cells in A . tenuis . The aboral region of anthozoan larvae is enriched with nerve cells ( 11 , 17 , 24 – 26 ). It is important to note that in A . tenuis , ‘bottom’ cells arise not only from the aboral area, but also from non-aboral positions. We recently demonstrated that non-aboral ‘bottom’ cells expressed similar nerve markers, but were destined for another cell fate (Kawamura et al., in preparation). In Nematostella , pluripotent stem cells appeared to arise from epithelial cells ( 23 , 27 – 29 ). They have been identified as the founder cells of neuroglandular progenitors ( 14 ), subsequently giving rise to nerve and gland cells when expressing Nanos ( 8 ). Consistent with these findings in Nematostella , it will be shown that neuroblasts in A . tenuis are developmentally multipotent in an oral-aboral position-dependent manner. Neuroblasts exhibit peculiar cytological features The neuroblast was morphologically characteristic of a large nucleus with an indented contour and the endoplasmic reticulum closely associated with the plasma membrane. An amorphous nucleus has been reported in amphibian and zebrafish primordial germ cells ( 30 , 31 ). A notched nucleus has also been observed in germline progenitor cells and smooth muscle precursors in colonial tunicates ( 32 – 34 ). Neuroblasts were also characterized by neuron-specific TUBB3. One of the in vitro Acropora cell lines, BBrC expressed TUBB3 and a neuroblast differentiation-associated protein (AtAHNAK) ( 17 ). BBrC appears to be a proliferative form of the in vitro FAmC line and is interchangeable with fibroblast-like cells ( 16 , 17 ). Fibroblast-like cells expressed hairy and enhancer of split ( Hes ) instead of AtAHNAK (17 and our unpublished data). In Nematostella , one group of progenitor/undifferentiated cells expresses Hes ( 7 ). Mammalian HES encodes a transcriptional repressor that suppresses neurogenesis in a Notch-dependent or -independent manner ( 35 ). In A . tenuis , AtHes1 and AtHes4 gene expression was maintained at high levels throughout the mid- and late embryonic stages (Table 1 ), which may explain why the expression of nerve-specific markers in bottom cells was suppressed to embryos until 6 dpf larvae. ‘Middle’ cells are the third segregation product at the ‘pear’ stage The present study showed that the gland epithelium shortened around 60 hpf, leaving segregated ‘bottom’ cells in situ , and unequal cell division occurred de novo in the middle region of the ectodermal layer. This was the third segregation from the gland epithelium to form ‘middle’ cells (Fig. 9 ). In the planula of Aurelia (class Scyphozoa), the ectoderm is pseudostratified with a superficial multilayer of densely packed nuclei ( 36 ). The ectodermal architecture of Acropora is similar to that of Aurelia , and embryonic ‘middle’ cells and larval undifferentiated-like cells may be homologous to ‘densely packed nuclei’ in Aurelia . More detailed information on densely packed cells is currently not available. In Nematostella , one group of progenitor/undifferentiated cells was shown to express SoxB2a , two Myc paralogs, and Nanos2 at the post-gastrula stages, while the other group expressed Hes and SoxB2 , enriched for gastrula-stage cells ( 7 ). In A. millepora , SoxC was initially expressed in the ectoderm as early as the ‘prawnchip’ stage and was maintained in putative sensory neurons throughout the larval stage ( 22 ). We expected undifferentiated-like cells in A . tenuis to express SoxB2 or SoxC; however, the present results indicated that both genes were restricted to nerve lineage cells. In vivo undifferentiated-like cells in A. tenuis showed a very similar morphology to the in vitro cell type, SSmC ( 17 ). Cell lines belonging to SSmC were highly proliferative and expressed high levels of polyubiquitin, ubiquitin ligase, ubiquitin-conjugating enzyme, and reverse transcriptase (our unpublished RNA sequencing data), of which reverse transcriptase was expressed by in vivo ‘middle’ cells and undifferentiated-like cells (Kawamura et al., in preparation). H3K4me3 is associated with active chromatin states ( 37 ). The dual modification of H3K4 and H3K27 appears to be essential for sustaining the pluripotency of mammalian embryonic stem cells ( 38 ). The present study has preliminarily shown that histone H3 of undifferentiated-like cells in A. tenuis was trimethylated at lysine 4. Undifferentiated-like cells also have H3K27me3 (a detailed account will be reported elsewhere). They were not endowed with histone trimethylation at embryonic stages, and ‘middle’ cells instead expressed AtKDM5, an antagonist of H3K4me3. The present results strongly suggest that ‘middle’ cells gradually acquire the undifferentiated state during the embryonic and larval stages. Conclusion In A . tenuis , four ectodermal cell layers arise through the unequal cell division of elongating outer blastomeres and the blastomere-derived glandular epithelium. These cell layers are stratified apicobasally, with yolk cells that segregated the earliest being located the most basally, indicating that the developing glandular epithelium is the source of yolk cells and progenitor/undifferentiated cells. The glandular epithelium expresses Snail, Etv6, C-Jun, SoxB2, SoxC in addition to Mef2. Yolk cell segregation is associated with Snail expression. Nerve lineage cells from neuroblast precursors (bottom cells) to nerves consistently express SoxB2 and SoxC throughout embryos and larvae. Undifferentiated-like cells emit signals for epigenetic histone methylation. The developmental fates of nerve progenitors and undifferentiated-like cells will be dealt with in the near future. METHODS Animals : The early embryos and planula larvae of Acropora tenuis (class Anthozoa; subclass Hexacorallia, order Scleractinia, 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. Embryos were fixed at adequate developmental stages. Fixed embryos and living larvae at 6–8 days post-fertilization (dpf) were transferred to Kochi University. Histology: Embryos at 12, 17, 22, 28, 40, 60, and 85 hours post-fertilization (hpf) and larvae at 6, 8, 10, 14, and 18 dpf were fixed in Zamboni’s fixative at room temperature for 30 min. They were serially dehydrated and embedded in plastic resin, Technovit 8100 (Heraeus Kulzer, Wertheim, Germany). Samples were sectioned into 2-µm-thick slices with glass knives. Primary antibodies: Rabbit antibody against Snail has already been described elsewhere ( 2 , 17 ). Additional antibodies were prepared as follows: VDSNEHSKQNSRLD to aa 47–60 of ETS translocation variant 6 (AtETV6), RPRRKPKSLLKKVDRYPFT to aa 78–96 of AtSoxB2, GGAQDSKTSSSNSDGHSHS to aa 118–136 of AtSoxC, and VNVQRQKEEDSKRKLGESL to aa 1193–1211 of lysine-specific demethylase 5 (AtKDM5). Before immunization, these oligopeptides were each conjugated to the carrier protein, keyhole limpet hemocyanin (KLH). Rabbit antibodies were raised by Eurofins Genomics Tokyo, Japan. They were diluted 400-fold with phosphate-buffered saline (PBS) immediately before use. A mouse anti-TUBB3 monoclonal antibody was purchased from Proteintech Group, Inc. (Tokyo, Japan). Rabbit anti-H3K4me3 (07-473, Millipore Temecula, CA, USA) and anti-H3K27me3 (07-449, Millipore) antibodies were purchased from commercial sources. They were used as previously described ( 17 , 39 ). Immunohistochemistry: The blocking of sections was performed in a mixture of 0.25% blocking reagent (Roche, Mannheim, Germany) and 2.5% skim milk in PBS for 30 min. Sections were then incubated with primary antibodies diluted 400-fold containing 0.1 mg/ml KLH at room temperature for 1 h. The goat anti-rabbit antibody labeled with fluorescein isothiocyanate (FITC) (FI-1000, Vector Laboratory, Burlingame, CA, USA) and the goat anti-mouse antibody labeled with rhodamine B (AM10706, Tago Immunologicals, CA, USA) were diluted 200-fold with PBS. Sections were stained with the secondary antibody for 0.5 h followed by washing twice for 10 min with PBS containing 0.1% Tween 20. Samples were finally counterstained with 4′6-diamidino-2-phenylindole (DAPI, 5 µg/mL) for 10 min. Some samples were stained with 0.1 µM rhodamine-phalloidin (Cytoskeleton Inc., Denver, CO, USA). In the negative control, primary antibodies were absorbed in advance by the corresponding oligopeptides at a concentration of 25 µg/mL. After antibody staining and washing with PBS containing 0.1% Tween 20, sections were observed under a confocal microscope (ECLIPSE C1si system; Nikon). Electron microscopy: Larvae were pre-fixed in Karnovsky’s fixative containing 2.5% glutaraldehyde, 2% paraformaldehyde, and 1.5% NaCl in 0.1 M cacodylate buffer (pH 7.4) on ice for 2 h, and then post-fixed with 1% osmium tetroxide in PB on ice for 14 h. After post-fixation, specimens were rinsed several times with PB containing 1.5% NaCl, dehydrated with a graded acetone series, and embedded in Spurr’s resin. The resulting resin blocks were sectioned with a diamond knife on an Ultracut UCT ultramicrotome (Leica, Vienna, Austria). Sections were mounted on formvar-coated grids and stained with 1% aqueous uranyl acetate for 1 h and with lead citrate for 15 min in sequence at room temperature. Samples were observed with a Jeol JEM-1400 transmission electron microscope (JEOL, Tokyo, Japan) and photographed using a charge-coupled device camera. RNA Sequencing : Total RNA was extracted from 3.5, 15, 25, 36, 60, and 85 hpf embryos, from 5.5 and 8.5 dpf larvae, and from 14 dpf metamorphosing polyps with TRIzol solution. cDNA libraries were produced using a TruSeq Stranded mRNA Library Prep Kit (Illumina) with IDT for Illumina-TruSeq DNA UD Indexes v2. RNA-seq libraries were quantified with a Real-Time PCR (StepOnePlus; Applied Biosystems) and quality control utilized capillary electrophoresis on a Bioanalyzer. Library sequencing was carried out on a MiSeq using MiSeq V2 reagent 300 cycles (Illumina). Transcriptomic analyses of raw data were carried out, as described previously ( 16 ). The abundance of annotated genes was estimated as transcripts per million (TPM). Declarations Acknowledgments We thank Mr. Shunichi Mekaru for proving Acropora tenuis larvae and all members of the Marine Genomics Unit, OIST, especially Kanako Hisata for help in preparation of manuscript. Prof. Shigeki Fujiwara is acknowledged for his support. Authors and Affiliation Department of Applied Science, Kochi University, Kochi 780-8520, Japan Dr. Kaz Kawamura, Emeritus Professor Kuroshio Science Unit, Multidisciplinary Science Cluster, Kochi University, Kochi 780-8520, Japan Dr. Satoko Sekida Department of Marine Science and Technology, Fukui Prefectural University, Obama, Fukui 917-0003, Japan Dr. Koki Nishitsuji Marine Genomics Unit, Okinawa Institute of Science and Technology Graduate University, Onna, Okinawa 904-0495, Japan. Dr. Nori Satoh Contributions KK and NS conceived and designed the study, and prepared the manuscript. KK, SS, and KN carried out immunohistochemical studies, electron microscopy, and RNA-seq analyses, respectively. All authors commented on it. Funding This work was supported by an OIST fund for collaboration with Kochi University, and in part by JSPS KAKENHI Grant number 22H03794 to KN, KK, and NS. Ethics declarations: No ethics approval was required for the work presented in this manuscript. Consent to Participate and Consent to Publish declarations: Not applicable. Competing interests: The authors declare they have no competing interests. Data Availability declaration: All relevant data are included within the manuscript. References Okubo N, Motokawa T. Embryogenesis in the reef-building coral Acropora spp. Zoolog Sci . 2007; 24(12): 1169–1177. 10.2108/zsj.24.1169 . PMID: 18271633. Kawamura K, Satoh N. Embryonic Development of the Gastrodermis in the Coral Acropora tenuis . Zoolog Sci. 2024;41:496–508. 10.2108/zs240032 . Kraus Y, Osadchenko B, Kosevich I. Embryonic development of the moon jellyfish Aurelia aurita (Cnidaria, Scyphozoa): another variant on the theme of invagination. PeerJ. 2022;10:e13361. 10.7717/peerj.13361 . Magie CR, Daly M, Martindale MQ. Gastrulation in the cnidarian Nematostella vectensis occurs via invagination not ingression. Dev Biol. 2007;305:483–97. 10.1016/j.ydbio.2007.02.044 . Marlow HQ, Martindale MQ. 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Dev Genes Evol. 2008;218:525–39. 10.1007/s00427-008-0254-8 . Grimaud C, Nègre N, Cavalli G. From genetics to epigenetics: the tale of Polycomb group and Trithorax group genes. Chromosome Res. 2006;14(4):363–75. 10.1007/s10577-006-1069-y . Grandy RA, Whitfield TW, Wu H, et al. Genome-wide studies reveal that H3K4me3 modification in bivalent genes is dynamically regulated during the pluripotent cell cycle and stabilized upon differentiation. Mol Cell Biol. 2015;36(4):615–27. 10.1128/MCB.00877-15 . Kimura-Nagano Y, Kishimoto K, Sekida S, Kawamura K. Stat stimulates histone H3K4 methylation via KDM5 inhibition in adult stem cells of budding tunicates. Dev Dyn. 2024. 10.1002/dvdy.754 . Additional Declarations No competing interests reported. Supplementary Files floatimage10.jpeg Suppl. Fig. 1 In vivo and in vitro VHC-like cells (vhc) and cnidocytes (c). (A) Electron microscopy of the larval ectoderm. Bar, 5 mm. (B,C) In vitro cells spontaneously appearing in the A. tenuis cell culture. Bars, 40 mm. (B) VHC-like cells. (C) Cnidocytes. floatimage11.jpeg Suppl. Fig. 2 Anti-AtEtv6 immunostaining of developing embryos. (A) A 17 hpf embryo. Bars, 20 mm. (B) A 22 hpf embryo. Bar, 20 mm. (C) A 60 hpf embryo. Bars, 40 mm. (D) An 85 hpf embryo. Bars, 40 mm. (A1-D1) FITC images of Etv6 expression. (A2-D2) FITC images merged with DAPI images. (A1, A2) White and red asterisks show an elongating blastomere and cytoplasmic Etv6 signals around yolk granules, respectively. (B1, B2) Arrowheads show Etv6-expressing nuclei. m, mesoglea; oy, outer yolk cell. floatimage12.jpeg Suppl. Fig. 3 Glandular epithelium formation. (A) A 17 hpf embryo. (B) A 20 hpf embryo. (C) A 28 hpf embryo. (D) A 40 hpf embryo. Bars, 10 mm. v, vacuole. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8654320","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":582045176,"identity":"b9ab132b-68b5-4be7-81b7-ad5f4bd61dbf","order_by":0,"name":"Kaz Kawamura","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA0UlEQVRIiWNgGAWjYBACCSBmbKhIALIYG6BiB4jRcoZkLY1tCRAWUUCy/fgzyZnz0uT4Zzc3MPMw2MkzMJ7Fb400T0Ka5MZtOcYSdw6CtCQbNjCcS8CrRY4h4Zjkw20ViRskEtt/8zAwA5WfMcCvhf9hm+TDOWAtIFvqCWuRlkhmk9zYkAPTcpiwFskZz5gtZxxLM5a4kdjAOMfguGEbIb9InE9/eLOnJlmOf0b6A4Y3FdXy/BIEQgwNAJ3EJnGGFB1gwN9DspZRMApGwSgY3gAADvtB2COCxxwAAAAASUVORK5CYII=","orcid":"","institution":"Kochi Univerity","correspondingAuthor":true,"prefix":"","firstName":"Kaz","middleName":"","lastName":"Kawamura","suffix":""},{"id":582045204,"identity":"7def4c08-8a6d-48d1-aae1-52451d0d0c69","order_by":1,"name":"Satoko Sekida","email":"","orcid":"","institution":"Kochi Univerity","correspondingAuthor":false,"prefix":"","firstName":"Satoko","middleName":"","lastName":"Sekida","suffix":""},{"id":582045221,"identity":"ac3df6e8-4648-4b80-a47d-457575d51482","order_by":2,"name":"Koki Nishitsuji","email":"","orcid":"","institution":"Fukui Prefectural University","correspondingAuthor":false,"prefix":"","firstName":"Koki","middleName":"","lastName":"Nishitsuji","suffix":""},{"id":582045234,"identity":"1c4bc856-0bf5-41a6-819b-e6eb0c660884","order_by":3,"name":"Noriyuki Satoh","email":"","orcid":"","institution":"Okinawa Institute of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Noriyuki","middleName":"","lastName":"Satoh","suffix":""}],"badges":[],"createdAt":"2026-01-21 02:39:09","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8654320/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8654320/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":101445201,"identity":"791943d9-f937-476c-9e13-8e3c5dc97836","added_by":"auto","created_at":"2026-01-29 18:14:15","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":645561,"visible":true,"origin":"","legend":"\u003cp\u003eUltrastructure of the ectoderm in 14 dpf larvae. (A) Ectodermal cell layers stratified apicobasally. Bar, 10 mm. (B) The glandular epithelium in the apical region. (B1) Many vacuoles and granules in columnar cells. Bar, 2 mm. (B2) The apical surface of the glandular epithelium. The arrowhead shows the secretion of vacuolar contents. Bar, 1 mm. (C) Vacuolated hyaline cells intervening between glandular cells. Bar, 10 mm. (D) The boundary between the middle and basal regions. Nerve progenitors were located at the bottom in the vicinity of yolk cells. Bar, 4 mm. (E) Undifferentiated-like cells. Bar, 2 mm. (F) Nerve lineage cells. (F1) A nerve progenitor cell (neuroblast). The arrow shows an indented contour of the nucleus. Bar, 2 mm. (Inset) Higher magnification of the cell periphery. Bar, 0.5 mm. (F2) A neuroblast with a cytoplasmic process (asterisk). Bar, 2 mm. (F3) Nerve cell. The arrow shows an extending axon. Bar, 1 mm. cp, cnidocyte precursor; e, endoplasmic reticulum; g, granule; ga, Golgi apparatus; l, lysosome; n, nucleus; np, nerve progenitor; p, plasma membrane; sv, synaptic vesicle; tj, tight junction; u, undifferentiated-like cell; v, vacuole; vhc, vacuolated hyaline cell; y, yolk cell.\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8654320/v1/35ef3a1956c421a1a5f76cef.jpeg"},{"id":101751320,"identity":"931979d0-0318-46ff-a2ca-fdc6f4e5f5c0","added_by":"auto","created_at":"2026-02-03 10:19:16","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":666151,"visible":true,"origin":"","legend":"\u003cp\u003eYolk cell segregation from outer and inner blastomeres in early embryos. (A) Twelve hpf embryos. (A1) Illustration of an embryo. Blastomeres colored yellow and red show presumptive ectoderm and endoderm cells, respectively. (A2) Confocal microscopy of monolayered blastomeres. Bar, 20 mm. (A3) Higher magnification of blastomeres that contain yolk granules in the cytoplasm. Bar, 10 mm. (B) Seventeen hpf embryos. (B1) Illustration of an embryo. Dark gray and light gray circles show outer and inner yolk cells, respectively. (B2) Confocal microscopy. Bar, 100 mm. (B3) A blastocoel expanding between outer and inner blastomeres. White and red arrowheads show outer and inner elongating blastomeres, respectively. Bar, 40 mm. (B4) Yolk cell formation (broken circles). Bar, 20 mm. (B5) Yolk cell segregation from blastomeres (bidirectional arrows). Bar, 10 mm. b, blastocoel; eb, elongating blastomere; ib inner blastomere; n, nucleus; ob, outer blastomere; yc, yolk cell; yg, yolk granule.\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8654320/v1/36dfa0629e969149b294bf03.jpeg"},{"id":101445206,"identity":"ea51d83d-e296-4f53-9455-48893710afb7","added_by":"auto","created_at":"2026-01-29 18:14:15","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":953160,"visible":true,"origin":"","legend":"\u003cp\u003eAnti-AtSnail immunohistochemistry of blastomeres and yolk cells in early embryos. (A) Outer and inner blastomeres of a 12 hpf embryo. Arrowheads show positive nuclei. Bars, 20 mm. (B) Outer blastomeres of 17 hpf embryos. White asterisks show elongating blastomeres. Red asterisks show the Snail-expressing cytoplasm around yolk granules. Bars, 10 mm. (C) Twenty-two hpf embryos. (C1) Illustration of an embryo. (C2) A sectioned whole embryo. Bar, 100 mm. (C3) Outer blastomeres. Bar, 10 mm. (D) Twenty-eight hpf embryos. (D1) Illustration of an embryo. The ingression of endodermal cells commenced and the pseudo-blastopore closed. (D2) A sectioned whole embryo. Bar, 100 mm. (D3) Glandular epithelium and outer yolk cells. Bar, 20 mm. (D4) Endodermal epithelium and inner yolk cells. Ingression of endoderm cell was in progress (arrowheads). Bar, 20 mm. (D5) Negative control. The antibody is pre-absorbed by the Snail peptide. Bar, 20 mm. (E) Forty hpf embryos. (E1) Illustration of an embryo. ‘Bottom’ cells (green circles) appeared. The pseudo-archenteron collapsed. (E2) The ectodermal gland epithelium and yolk cells. Bar, 40 mm. (E3) Endodermal cells (broken circles) among inner yolk cells. Bar, 10 mm. (E3’) The same picture as (E3) without DAPI image. (E4) Negative control. Bar, 40 mm. (F) Eighty-five hpf embryos. (F1) Illustration of an embryo. ‘Middle’ cells (blue circles) appeared. (F2) A sectioned whole embryo. Bar, 100 mm. (F3) The gland epithelium, ‘middle’ cells, and yolk cells. Bar, 40 mm. ge, glandular epithelium; ib, inner blastomere; iy, inner yolk cell; m, mesoglea; ob, outer blastomere; oy, outer yolk cell; pa, pseudo-archenteron; pb, pseudo-blastopore.\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8654320/v1/e53687055d98ba986f0cf7a1.jpeg"},{"id":101751384,"identity":"89315741-4f1b-4062-93f9-d7ef369b364e","added_by":"auto","created_at":"2026-02-03 10:19:53","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":315078,"visible":true,"origin":"","legend":"\u003cp\u003eUltrastructure of yolk cells and endoderm cells. (A) The blastocoel filled with yolk cells. Bar, 10 mm. (B) Endoderm cells (arrows) embedded among yolk cells. Bar, 5 mm. (C) The nucleus and cytoplasm of a yolk cell. Bar, 5 mm. n, nucleus; y, yolk cell.\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8654320/v1/2e5d471d7a837b8ebc0f4a89.jpeg"},{"id":101752374,"identity":"b72e6b0d-d050-423a-9b16-66421b6c9779","added_by":"auto","created_at":"2026-02-03 10:27:07","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":573982,"visible":true,"origin":"","legend":"\u003cp\u003eFormation of ectodermal ‘bottom’ and ‘middle’ cells by unequal cell division. (A) Dark field microscopy of the glandular epithelium. Bars, 20 mm. (A1) A 17 hpf embryo. (A2) A 22 hpf embryo. (A3) A 28 hpf embryo. (A4) A 40 hpf embryo. (B) Toluidine blue staining. Asterisks show nuclei left in the apical surface. All scale bars show 10 mm. (B1) Glandular cells of a 20 hpf embryo. Nuclei are located in the middle of elongating epithelial cells (yellow broken outline). (B2) A 28 hpf embryo. The glandular epithelium is the longest (broken lines), and the nucleus translocated to the growing extremity of the cell. (B3) A 40 hpf embryo. Pairs of nuclei are located at the elongated tip of epithelial cells (broken circles). (B4) A 60 hpf embryo. ‘Bottom’ cells segregated earlier (black arrowheads) and ‘middle’ cells with \u003cem\u003ede novo \u003c/em\u003epaired nuclei (broken circles) are shown.\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8654320/v1/8f4af4d157eb3946e8bdee30.jpeg"},{"id":101445211,"identity":"a65504fb-1ae3-4749-97e7-d8a1b6320d3d","added_by":"auto","created_at":"2026-01-29 18:14:15","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":877419,"visible":true,"origin":"","legend":"\u003cp\u003eAtC-Jun expression in elongated blastomeres, segregated yolk cells, and segregated cell nuclei. (A) A 12 hpf embryo. Bar, 20 mm. (B) A 17 hpf embryo. Arrowheads show segregating yolk cells. Bars, 20 mm. (C) A 28 hpf embryo. Arrowheads show positive nuclei. Bar, 10 mm. (D) A 28 hpf embryo. Arrowheads show amorphous, rectangular nuclei. Bar, 10 mm. (E) A 40 hpf embryo. Broken circles show paired nuclei. Bar, 10 mm. (A1-E1) FITC images. (A2-E2) FITC images merged with DAPI images.\u003c/p\u003e","description":"","filename":"floatimage6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8654320/v1/50e5c8fceb2181d267b03a60.jpeg"},{"id":101752269,"identity":"577535cb-015b-43d0-8986-e14f513751d7","added_by":"auto","created_at":"2026-02-03 10:26:27","extension":"jpeg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":835132,"visible":true,"origin":"","legend":"\u003cp\u003eExpression of AtSoxB2 (A, C, D, F1, F3, G1), AtSoxC (B, E, H), and TUBB3 (F2, F3, G2, H) in segregated ‘bottom’ cells and neuroblasts. (B2, C3) Negative controls. Some samples are counterstained with DAPI (B1, C2, D2, F, G, H). (A, B) Seventeen hpf embryos. (C) Twenty-eight hpf embryos. (D) A 40 hpf embryo. (E) A 60 hpf embryo. (F) A 6.5 dpf larva. (G, H) Eight dpf larvae. Arrowheads show immunopositive nuclei. Broken circles show paired nuclei. Bars except (E) show 10 mm. Bar in (E), 20 mm. m, mesoglea; oy, outer yolk cell.\u003c/p\u003e","description":"","filename":"floatimage7.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8654320/v1/70dac823235a5dc437eef915.jpeg"},{"id":101445212,"identity":"d810adda-57cf-489d-870f-f63c3f11ba32","added_by":"auto","created_at":"2026-01-29 18:14:15","extension":"jpeg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":743801,"visible":true,"origin":"","legend":"\u003cp\u003eAtKDM5 expression and epigenetic histone modifications of undifferentiated-like cells in embryos (A) and larvae (B, C). All figure scales show 10 mm. (A, B) Anti-AtKDM5 immunostaining. Arrowheads show KDM5-expressing nuclei. (C) Anti-H3K4me3 immunostaining. Broken circles show immunopositive nuclei. (A) An 85 hpf embryo. (B, C) Eight dpf larvae. (A1-C1) FITC images. (A2-C2) DAPI images. m, mesoglea.\u003c/p\u003e","description":"","filename":"floatimage8.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8654320/v1/c9bfca4c86f75eeca6b5a1ff.jpeg"},{"id":101445209,"identity":"836d1794-e41f-48e7-b501-3e6e5741dd8c","added_by":"auto","created_at":"2026-01-29 18:14:15","extension":"jpeg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":279810,"visible":true,"origin":"","legend":"\u003cp\u003eEctodermal cell differentiation from embryonic outer blastomeres. Snail-expressing blastomeres begin to elongate as early as the ‘prawnchip’ stage and segregate yolk granules and yolk cells from the growing tip of blastomeres (white broken circle). This is the first segregation that occurs in the ectodermal cell layer. Elongating cells become the glandular epithelium with the maximum long axis at the ‘donut’ stage when the nucleus moves to the extremity of elongation. The second segregation occurs herein to form ‘bottom’ cells (yellow broken circle), the nucleus of which is marked by Sox. At the ‘pear’ stage around 60 hpf of embryonic development, glandular cells shorten to some extent and shed off ‘middle’ cells in the middle region of the ectoderm layer (red broken circle), which is the third cell segregation. Every cell segregation is accompanied by C-Jun expression. In 8 dpf larvae, the ‘bottom’ cells become neuroblasts that express TUBB3, neuroblast-related differentiation markers (AHNAK), and the transcription factors, SoxB2/SoxC. Undifferentiated-like cells do not express these marker proteins, but are modified by histone trimethylation at H3lys 4 and H3lys 27. Four stratified layers of the ectoderm are formed.\u003c/p\u003e","description":"","filename":"floatimage9.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8654320/v1/aa30efb3e4bdde9df338dca7.jpeg"},{"id":103782870,"identity":"b8a73cfc-2eb7-442b-b2a9-742c77cfa314","added_by":"auto","created_at":"2026-03-02 21:24:37","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6909343,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8654320/v1/e2f9186d-aa3d-4971-a4c6-ddb458005fdc.pdf"},{"id":101445202,"identity":"064f9d4b-5fa7-40d9-9db9-3426cc005c9c","added_by":"auto","created_at":"2026-01-29 18:14:15","extension":"jpeg","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":432441,"visible":true,"origin":"","legend":"\u003cp\u003eSuppl. Fig. 1 \u003cem\u003eIn vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e VHC-like cells (vhc) and cnidocytes (c). (A) Electron microscopy of the larval ectoderm. Bar, 5 mm. (B,C) \u003cem\u003eIn vitro\u003c/em\u003e cells spontaneously appearing in the \u003cem\u003eA. tenuis\u003c/em\u003e cell culture. Bars, 40 mm. (B) VHC-like cells. (C) Cnidocytes.\u003c/p\u003e","description":"","filename":"floatimage10.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8654320/v1/a80eb116d2e3e2d03a5a3abc.jpeg"},{"id":101752347,"identity":"f93afbe1-e500-498a-9671-b49feb0ce6a1","added_by":"auto","created_at":"2026-02-03 10:26:58","extension":"jpeg","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":876376,"visible":true,"origin":"","legend":"\u003cp\u003eSuppl. Fig. 2 Anti-AtEtv6 immunostaining of developing embryos. (A) A 17 hpf embryo. Bars, 20 mm. (B) A 22 hpf embryo. Bar, 20 mm. (C) A 60 hpf embryo. Bars, 40 mm. (D) An 85 hpf embryo. Bars, 40 mm. (A1-D1) FITC images of Etv6 expression. (A2-D2) FITC images merged with DAPI images. (A1, A2) White and red asterisks show an elongating blastomere and cytoplasmic Etv6 signals around yolk granules, respectively. (B1, B2) Arrowheads show Etv6-expressing nuclei. m, mesoglea; oy, outer yolk cell.\u003c/p\u003e","description":"","filename":"floatimage11.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8654320/v1/5b709cf77a110778ea3bf7e0.jpeg"},{"id":101751594,"identity":"91dc20cc-7812-4758-a4f8-c0e8187f2a3e","added_by":"auto","created_at":"2026-02-03 10:21:35","extension":"jpeg","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":394125,"visible":true,"origin":"","legend":"\u003cp\u003eSuppl. Fig. 3 Glandular epithelium formation. (A) A 17 hpf embryo. (B) A 20 hpf embryo. (C) A 28 hpf embryo. (D) A 40 hpf embryo. Bars, 10 mm. v, vacuole.\u003c/p\u003e","description":"","filename":"floatimage12.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8654320/v1/a59df23a19cbd97c0674153b.jpeg"}],"financialInterests":"No competing interests reported.","formattedTitle":"Ectodermal Cell Differentiation by Unequal Cell Division in the Stony Coral Acropora tenuis","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eEmbryonic development in Cnidaria has attracted the interest of many researchers with special reference to the molecular and anatomical evolution of Bilateria. Blastula formation and gastrulation are two such examples. In the stony coral \u003cem\u003eAcropora tenuis\u003c/em\u003e, the blastula forms without a blastocoel (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). The blastocoel subsequently appears, spreads widely, and plays an important role in the formation of the gastric cavity of polyps instead of the completely collapsing gastrocoel (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). Gastrulation occurs by invagination in jellyfishes (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e), by invagination in the sea anemone \u003cem\u003eNematostella\u003c/em\u003e (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e) and the combined activity of invagination and ingression in other Anthozoa (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e), and by delamination in some hydrozoans (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). Endodermal cells in \u003cem\u003eA\u003c/em\u003e. \u003cem\u003etenuis\u003c/em\u003e formed by ingression and behaved as if they were mesoderm-like cells until the gastrodermis formed (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn contrast with the relative simplicity of the endodermal repertoire of differentiation, the anthozoan ectoderm contains complex cell types: an epidermis, gland/secretory cells, progenitor/undifferentiated cells, nerve cells, and cnidocytes (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). In \u003cem\u003eA\u003c/em\u003e. \u003cem\u003etenuis\u003c/em\u003e, the outer and inner blastomeres (the presumptive ectoderm and endoderm) give off outer and inner yolk cells, respectively (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). Accordingly, outer yolk cells also belong to the ectoderm group. Recent studies on \u003cem\u003eNematostella\u003c/em\u003e polyps provided detailed information on the developmental pathways of neurons, gland cells, cnidocytes, and germ cells from putative stem cells through to progenitor cells (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). However, the embryonic origin of these differentiated cells remains unclear.\u003c/p\u003e \u003cp\u003eSingle-cell RNA sequencing technology has contributed to innovative advances in our understanding of cnidarian cells (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). In \u003cem\u003eNematostella\u003c/em\u003e, the epidermis expresses Claudin, a tight junction component (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e), and other cell adhesion molecules (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). Unlike \u003cem\u003eHydra\u003c/em\u003e, the anthozoan epidermis does not have the epitheliomuscular type of cells other than the tentacle and oral disc (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). Some types of gland/secretory cells are scattered among the larval aboral ectoderm and express secretory protein genes (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). Progenitor/undifferentiated cells include a common progenitor for neurons, cnidocytes, and gland cells (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). They express nuclear factors such as Myc and SoxB (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e) as well as Nanos and Piwi (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). Nerve and gland cells share the gene expression of FoxQ (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). Cnidocytes are characterized by multiple venom and capsule proteins (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). Although the gene catalog of each cell type has been extensively examined, limited information is currently available on the spatiotemporal origin and pathway of ectodermal cell differentiation in Cnidaria.\u003c/p\u003e \u003cp\u003eWe recently established many \u003cem\u003ein vitro\u003c/em\u003e cell lines of \u003cem\u003eA\u003c/em\u003e. \u003cem\u003etenuis\u003c/em\u003e that may be classified into three cell types (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). The gene expression profiles of cultured cells were found to partially reflect \u003cem\u003ein vivo\u003c/em\u003e gene expression (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). Neuroblast differentiation-associated protein (AHNAK) is one such example and is highly expressed by brilliant brown cells (BBrC) belonging to the flattened amorphous cell type (FAmC). The \u003cem\u003ein vivo\u003c/em\u003e nerve lineage from neuroblasts to neurons expresses AHNAK (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). Small smooth cells (SSmC) are another ectodermal type of cultured cells in \u003cem\u003eA\u003c/em\u003e. \u003cem\u003etenuis\u003c/em\u003e. They express secretory proteins, such as endoglucanase and skeletal organic matrix proteins, which are specific to ectodermal secretory cells (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). These \u003cem\u003ein vitro\u003c/em\u003e molecular probes of information may be utilized in the \u003cem\u003ein vivo\u003c/em\u003e study of ectodermal cells.\u003c/p\u003e \u003cp\u003eThe primary purpose of the present study was to examine when and how ectodermal cell differentiation occurs in \u003cem\u003eA\u003c/em\u003e. \u003cem\u003etenuis\u003c/em\u003e embryos. Larval ectodermal cells were classified ultrastructurally. We identified four types of stratified cell layers. We then investigated how embryonic blastomeres contribute to the spatiotemporal establishment of these four cell types. Antibodies against Snail, C-Jun, SoxB2, and SoxC were used to examine the formation of yolk cells and progenitor cells. We also investigated the developmental pathway from neuroblast precursors to neuroblasts and nerve cells using antibodies against nerve-specific tubulin beta-III (TUBB3), and the transcription factor, Sox. The results obtained herein provide evidence for ectoderm-derived cells originating from the developing glandular epithelium by unequal cell segregation.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cp\u003e \u003cb\u003eEctodermal Cell Morphology in\u003c/b\u003e \u003cb\u003eA. tenuis\u003c/b\u003e \u003cb\u003eLarvae\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe epidermis of 14 dpf larvae is shown ultrastructurally in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. It was divided into three regions (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). The apical region mainly comprised elongated columnar cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB1). A tight junction was present between neighboring cells at the apical surface (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB2). Various types of granules and membrane-bound vacuoles were located in the cytoplasm (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB1), and some vacuoles were secreted from the apical cell surface (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB2 arrowhead). Accordingly, columnar cells were regarded as a new type of glandular/secretory epithelium.\u003c/p\u003e \u003cp\u003eVacuolated hyaline cells (VHCs) appeared among glandular epithelia (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC) and attained to the ectodermal basal region (not shown). Cnidocytes and their progenitors were also embedded in the apical region (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC, Suppl. Figure\u0026nbsp;1A).\u003c/p\u003e \u003cp\u003eThe middle region of the ectodermal layer was filled with small and round cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). They appeared to be undifferentiated (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). In the ectodermal basal region, large and oval cells were located in the vicinity of yolk cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD, F1, F2).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThey contained large granules in the cytoplasm (Fig.\u0026nbsp;1F1, F2), and the endoplasmic reticulum was located just beneath the plasma membrane (Fig.\u0026nbsp;1F1 inset). The nucleus showed an indented morphology (Fig.\u0026nbsp;1F1, F2 arrows). In addition to these features, some cells contained the Golgi apparatus and had a cytoplasmic protrusion (Fig.\u0026nbsp;1F2). This type of cell is a nerve progenitor (neuroblast) (see also Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eF). The nerve cell body was located at this basal area and enriched with synaptic vesicles (Fig.\u0026nbsp;1F3). It extended the cytoplasmic process towards the apical surface (Fig.\u0026nbsp;1F3 arrow, see also Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eG).\u003c/p\u003e \u003cp\u003eVHC-like cells and cnidocytes sometimes appeared spontaneously in \u003cem\u003eA. tenuis\u003c/em\u003e cell culture plates of FAmC and SSmC, respectively (Suppl. Figure\u0026nbsp;1B, C) (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). However, they have yet to be established as stable cell lines.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCell Elongation and Yolk Cell Segregation from Snail-expressing Blastomeres\u003c/h2\u003e \u003cp\u003e\u0026lsquo;Prawnchip\u0026rsquo; embryos at 12 hpf had a U-shaped outline (Fig.\u0026nbsp;2A1) (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). The outer blastomeres (presumptive ectoderm) and inner blastomeres (presumptive endoderm) were both monolayered and had a round or cuboidal configuration (Fig.\u0026nbsp;2A2). Yolk granules were scattered within the cytoplasm of blastomeres (Fig.\u0026nbsp;2A3). In 17 hpf embryos, the blastocoel gradually expanded (Fig.\u0026nbsp;2B1-B3). Some outer and inner blastomeres began to elongate (Fig.\u0026nbsp;2B3, B4). Yolk granules were inclined to accumulate at the growing tip of blastomeres and segregated into \u003cem\u003ede novo\u003c/em\u003e emerging yolk cells (Fig.\u0026nbsp;2B5). They fell into the developing blastocoel of \u0026lsquo;donut\u0026rsquo; embryos (Fig.\u0026nbsp;2B5 bidirectional arrows).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn 12 hpf embryos, AtSnail signals were observed in the nuclei of both outer and inner blastomeres and also in the whole cytoplasm (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA), similar to AtMef2 signals (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). In 17 hpf embryos, AtSnail signals were concentrated at the growing tip of elongated epithelial cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB white asterisks) or around yolk granules (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB red asterisks). In 22 hpf embryos, the blastocoel expanded throughout the whole embryo (Fig.\u0026nbsp;3C1, C2). Cytoplasmic AtSnail signals gradually attenuated, while nuclear signals were prominent in the outer blastomeres (Fig.\u0026nbsp;3C3). The gastrocoel-like lumen was observable, but came from the U-shaped concavity of the prawnchip embryo (Figs.\u0026nbsp;2A1, B1, 3C1, D1). Accordingly, the lumen was referred to as a pseudo-archenteron. The term pseudo-blastopore was also used in the present study. In 28 hpf embryos, the pseudo-blastopore closed and the blastocoel was filled with yolk cells (Figs.\u0026nbsp;3D1, D2, 4A). The yolk cell showed a moderate AtSnail signal, whereas the ectodermal gland epithelium scarcely emitted this signal (Fig.\u0026nbsp;3D3).\u003c/p\u003e \u003cp\u003eEndodermal cells were released from the wall of the pseudo-archenteron and scattered in the blastocoel by ingression (Figs.\u0026nbsp;3D4 arrowheads). They were discernible histochemically from yolk cells because endodermal cell nuclei (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB) were stained with DAPI (Fig.\u0026nbsp;3D4, E3), whereas yolk cell nuclei (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC) were not (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). In the negative control, AtSnail signals disappeared from yolk cells after the pre-treatment of the anti-AtSnail antibody with the Snail peptide antigen (Fig.\u0026nbsp;3D5). Immunohistochemical results were consistent with those of RNA sequencing, which showed that AtSnail expression was the highest in early embryos (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn 40 hpf embryos, the pseudo-archenteron collapsed and the mesoglea appeared between the outer and inner yolk cell layers (Fig.\u0026nbsp;3E1, E2). The outer yolk cell layer was strongly stained with the anti-AtSnail antibody, whereas inner yolk cells were moderately stained (Fig.\u0026nbsp;3E2). Endodermal cells did not have AtSnail signals (Fig.\u0026nbsp;3E3, E3\u0026rsquo;). In the negative control, strong signals disappeared from outer yolk cells (Fig.\u0026nbsp;3E4). The pseudo-archenteron disappeared completely from 60\u0026ndash;85 hpf embryos (Fig.\u0026nbsp;3F1, F2). Outer yolk cells still emitted strong Snail signals, but gradually decreased in number (Fig.\u0026nbsp;3F2, F3). In contrast, the inner yolk cell layer kept the cell number throughout larval stages, consistent with previous findings from histological studies using toluidine blue (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAtETV6 was expressed in a similar pattern to AtSnail. In 17 hpf \u0026lsquo;prawnchip\u0026rsquo; embryos, the growth tip of elongated blastomeres and the periphery of yolk granules were strongly stained (Suppl. Figure\u0026nbsp;2A1, A2). Nuclear signals were detected in both the presumptive ectoderm and endoderm of 22 hpf \u0026lsquo;donut\u0026rsquo; embryos (Suppl. Figure\u0026nbsp;2B1, B2). Unlike AtSnail, 40 hpf \u0026lsquo;donut\u0026rsquo; embryos did not emit Etv signals (not shown), whereas the outer yolk cell layer in 60 hpf \u0026lsquo;pear\u0026rsquo; embryos was stained with the anti-AtEtv6 antibody\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e(Suppl. Figure\u0026nbsp;2C1, C2), similar to AtSnail, but at a later stage. The number of Etv-positive outer yolk cells markedly decreased in 85 hpf embryos (Suppl. Figure\u0026nbsp;2D1, D2).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eSegregation of ‘Bottom’ and ‘Middle’ Cells in Embryos\u003c/h3\u003e\n\u003cp\u003eAs already shown, blastomeres began to elongate in 17 hpf embryos (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Epithelial cells became slenderer from 22 to 40 hpf and aligned side by side (Fig.\u0026nbsp;5A1-A4). In 22 hpf embryos, they were 40\u0026ndash;45 \u0026micro;m long in the major axis (Fig.\u0026nbsp;5B1 yellow broken line). The nucleus was located in the middle region of the elongated cell (Fig.\u0026nbsp;5B1). Non-elongated blastomeres remained near the apical surface of the embryo (Fig.\u0026nbsp;5B1 white asterisks). In 28 hpf embryos, ectodermal cells reached a maximum length of approximately 60 \u0026micro;m (Fig.\u0026nbsp;5B2 yellow broken line), and the nucleus translocated to the elongating tip of the cell. Vacuoles became prominent in the cytoplasm of columnar cells (Suppl. Figure\u0026nbsp;3), which was characteristic of the ectodermal gland epithelium (cf., Fig.\u0026nbsp;1B1, B2).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eGene expression of developing embryos, larvae, and polyps revealed by transcriptomic RNA sequencing.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"11\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eGene\u003c/p\u003e \u003cp\u003esymbol\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eGene ID\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"9\" nameend=\"c11\" namest=\"c3\"\u003e \u003cp\u003eTPM\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"6\" nameend=\"c8\" namest=\"c3\"\u003e \u003cp\u003eEmbryos\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c10\" namest=\"c9\"\u003e \u003cp\u003eLarvae\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c11\"\u003e \u003cp\u003ePolyps\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.5\u003c/p\u003e \u003cp\u003ehpf\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e15\u003c/p\u003e \u003cp\u003ehpf\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e25 hpf\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e36\u003c/p\u003e \u003cp\u003ehpf\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003e60\u003c/p\u003e \u003cp\u003ehpf\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003e85\u003c/p\u003e \u003cp\u003ehpf\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003e5.5\u003c/p\u003e \u003cp\u003edpf\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c10\"\u003e \u003cp\u003e8.5\u003c/p\u003e \u003cp\u003edpf\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c11\"\u003e \u003cp\u003e14 dpf\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAtSnail2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003es0006.\u003c/p\u003e \u003cp\u003eg224.t1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e231.39\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e173.77\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e36.97\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e40.87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e66.45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e49.95\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e47.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e64.14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e19.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAtC-Jun\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003es0207.\u003c/p\u003e \u003cp\u003eg13.t1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e8.17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e26.21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e12.54\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2.98\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e3.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e4.41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e1.17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e2.07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e762.13\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAtSoxB2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003es0027.\u003c/p\u003e \u003cp\u003eg3.t1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e38.15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e50.81\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e25.63\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e34.46\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e12.21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e8.54\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e3.77\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e8.05\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAtSoxC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003es0330.\u003c/p\u003e \u003cp\u003eg5.t1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5.33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e34.89\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e16.69\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e24.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e20.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e21.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e19.99\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e17.84\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e14.28\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAtHES1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003es0076.\u003c/p\u003e \u003cp\u003eg32.t1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e29.83\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e17.31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e30.87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e46.82\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e32.37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e8.11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e47.84\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAtHES4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003es0076.\u003c/p\u003e \u003cp\u003eg33.t1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e49.44\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e100.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e99.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e95.96\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e87.42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e7.51\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e175.55\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAtRFamide\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003es0012.\u003c/p\u003e \u003cp\u003eg36.t1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e3.97\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e9.68\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e22.86\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e27.32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e21.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e20.74\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAtKDM5A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003es0006.g60.t1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5.45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e21.09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e123.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e92.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e85.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e87.62\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e106.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e27.73\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e26.42\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003ePaired nuclei were often observed in 40 hpf embryos, suggesting that the gland epithelium gave rise to daughter cells by unequal cell division (Fig.\u0026nbsp;5B3). In 60 hpf embryos, the elongated gland epithelium shortened to approximately 40 \u0026micro;m, leaving daughter cells at the bottom of the ectodermal layer (Fig.\u0026nbsp;5B4 black arrowheads). \u0026lsquo;Bottom\u0026rsquo; cells were thus formed (cf., Fig.\u0026nbsp;3E1 green circles). On the other hand, the gland cell continued to divide \u003cem\u003ein situ\u003c/em\u003e (Fig.\u0026nbsp;5B4 broken circles), producing \u003cem\u003ede novo\u003c/em\u003e paired cells in the middle of the ectodermal layer. Consequently, \u0026lsquo;middle\u0026rsquo; cells were formed (cf., Fig.\u0026nbsp;3F1 blue circles).\u003c/p\u003e\n\u003ch3\u003eC-Jun Expression in Relation to Cell Segregation\u003c/h3\u003e\n\u003cp\u003e \u003c/p\u003e \u003cp\u003eRNA sequencing data indicated that C-Jun, an AP-1 subunit, was expressed during the early embryonic stage and polyp stage (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The former was coincident with the segregation time period of \u0026lsquo;bottom\u0026rsquo; cells and \u0026lsquo;middle\u0026rsquo; cells.\u003c/p\u003e \u003cp\u003eAtC-Jun expression was not immunohistochemically observable in 12 hpf embryos (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). It initially appeared in segregating blastomeres and yolk cells at 17 hpf of embryonic development (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). In 28 hpf \u0026lsquo;donut\u0026rsquo; embryos, the majority of ectodermal nuclei were stained (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). Mitotic figures were not detected, whereas amorphous, rectangular nuclei were often observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD arrowheads). AtC-J-Jun expression persisted in the paired nuclei of 40 hpf embryos (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE) and in \u0026lsquo;bottom\u0026rsquo; and \u0026lsquo;middle\u0026rsquo; cell nuclei until 60 hpf \u0026lsquo;pear\u0026rsquo; embryos (not shown).\u003c/p\u003e\n\u003ch3\u003eCytological Features of ‘Bottom’ and ‘Middle’ Cells\u003c/h3\u003e\n\u003cp\u003eAccording to RNA sequencing, Sox gene expression began at the \u0026lsquo;prawnchip\u0026rsquo; stage and was maintained during the \u0026lsquo;donut\u0026rsquo; stage when \u0026lsquo;bottom\u0026rsquo; and \u0026lsquo;middle\u0026rsquo; cells appeared (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Immunohistochemical studies showed that AtSoxB2 and AtSoxC were both expressed in the whole cytoplasm of elongated blastomeres in 17 hpf embryos (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA, B1). In the negative control, Sox signals markedly decreased after the absorption of antibodies with specific peptides (Fig.\u0026nbsp;7B2). In 28 hpf embryos, cytoplasmic signals were maintained, and nuclear signals were reinforced in the glandular epithelium (Fig.\u0026nbsp;7C1, C2), which was sensitive to the absorption test (Fig.\u0026nbsp;7C3). In 40 hpf embryos, not only single nuclei but also paired nuclei emitted strong Sox signals (Fig.\u0026nbsp;7D1, D2 arrowheads and broken circles), while cytoplasmic signals became attenuated. Sox signals were not apparent in\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e60\u0026ndash;85 hpf embryos (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE); however, a weak expression signal reappeared at the bottom of the ectoderm in 6.5 dpf larvae (Fig.\u0026nbsp;7F1-F3). In 8 dpf larvae, Sox-expressing \u0026lsquo;bottom\u0026rsquo; cells developed into neuroblasts that emitted nerve-specific TUBB3 signals (Fig.\u0026nbsp;7G2, H). They extended cytoplasmic processes towards the apical surface of the ectoderm. On the other hand, anti-Sox antibodies did not recognize \u0026lsquo;middle\u0026rsquo; cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eF-H).\u003c/p\u003e \u003cp\u003eLysine-specific demethylase 5 (AtKDM5) is one of epigenetic factors. It antagonizes trimethylation of histone H3K4 (H3K4me3). AtKDM5 appeared in \u0026lsquo;donut\u0026rsquo; embryos (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), and became prominent in ectodermal \u0026lsquo;middle\u0026rsquo; cells of \u0026lsquo;pear\u0026rsquo; embryos (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA). AtKDM5 disappeared from larvae (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB). In the meanwhile, H3K4me3 appeared in ectodermal \u0026lsquo;middle\u0026rsquo; cells in 8 dpf larvae (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eC), indicating that epigenetic markers would be available for lineage tracing of undifferentiated-like cells in \u003cem\u003eA\u003c/em\u003e. \u003cem\u003etenuis\u003c/em\u003e. Histone methylation in \u003cem\u003eAcropora\u003c/em\u003e progenitor cells as well as undifferentiated cells will be reported in detail elsewhere.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eA Snail-expressing yolk cell is the primary segregation product from elongating Snail\u003csup\u003e+\u003c/sup\u003e blastomeres\u003c/h2\u003e \u003cp\u003eIn \u003cem\u003eA. millepora\u003c/em\u003e, a related species of \u003cem\u003eA. tenuis\u003c/em\u003e, ectodermal Snail-expressing cells become elongated in 18\u0026ndash;24 hpf embryos (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e). Consistent with this finding, the present study showed that in \u003cem\u003eA\u003c/em\u003e. \u003cem\u003etenuis\u003c/em\u003e, the elongating outer blastomeres (presumptive ectoderm) of \u0026lsquo;prawnchip\u0026rsquo; embryos emitted Snail signals from the whole cytoplasm. As shown in this study, they also expressed Etv6, C-Jun, SoxB2, and SoxC (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e). A previous study reported that developing blastomeres expressed Mef2 (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). An increasing number of elongated blastomeres contained vacuoles in the cytoplasm and became the ectodermal glandular epithelium during the \u0026lsquo;donut\u0026rsquo; stage (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e). It remains unclear whether Snail and/or other transcription factors are involved in the differentiation of blastomeres to the ectodermal epithelium.\u003c/p\u003e \u003cp\u003eThe ectodermal glandular epithelium in \u003cem\u003eA. tenuis\u003c/em\u003e expressed a secreted protein, endoglucanase (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e) (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e), similar to non-epithelial gland/secretory cells in the sea anemone \u003cem\u003eNematostella\u003c/em\u003e (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). Under electron microscopy, we did not observe non-epithelial gland cells in the \u003cem\u003eAcropora\u003c/em\u003e larval ectoderm. Instead, the ectoderm contained VHCs, in which the vacuole was not solid but translucent. VHCs originate from the larval neuroblast (Kawamura et al., in preparation). In \u003cem\u003eNematostella\u003c/em\u003e, gland/secretory cells are derived from nerve progenitors (\u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). Therefore, VHCs in stony corals may substitute for \u003cem\u003eNematostella\u003c/em\u003e gland/secretory cells.\u003c/p\u003e \u003cp\u003eThe present study showed that elongating outer and inner blastomeres gave rise to yolk cells by unequal segregation, confirming our recent findings (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). Outer yolk cells arising from outer blastomeres preferentially emitted ring-like Snail signals from\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003ethe gastrula stage (40 hpf \u0026lsquo;donut\u0026rsquo; embryos). Inner yolk cells fulfilling the blastocoel emitted weak signals, while endoderm cells scattered among inner yolk cells had no apparent signals. The present results contrasted with previous findings on \u003cem\u003eA\u003c/em\u003e. \u003cem\u003emillepora\u003c/em\u003e showing that the endoderm expressed Snail during gastrulation (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e). In \u003cem\u003eNematostella\u003c/em\u003e, Snail is also expressed in immigrating endodermal cells during gastrulation (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). It is important to note that in \u003cem\u003eA\u003c/em\u003e. \u003cem\u003etenuis\u003c/em\u003e, gastrulation occurs by ingression and a very small number of presumptive endodermal cells migrate from the inner-most layer of the embryo into the blastocoel at the \u0026lsquo;donut\u0026rsquo; stage when the pseudo-blastopore and pseudo-archenteron close (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). \u003cem\u003eIn situ\u003c/em\u003e hybridization studies on \u003cem\u003eAcropora\u003c/em\u003e embryos showed that Snail signals were densely localized around the blastocoel (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). As discussed above, endodermal cells in \u003cem\u003eA\u003c/em\u003e. \u003cem\u003etenuis\u003c/em\u003e were scattered at a low cell density in the blastocoel (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e), and they did not emit Snail signals. Therefore, at least in \u003cem\u003eA\u003c/em\u003e. \u003cem\u003etenuis\u003c/em\u003e, only yolk cells appear to express Snail in \u0026lsquo;donut\u0026rsquo; embryos.\u003c/p\u003e \u003cp\u003e \u003cb\u003e\u0026lsquo;Bottom\u0026rsquo; cells are secondary segregation products destined to nerve progenitors\u003c/b\u003e \u003c/p\u003e \u003cp\u003eIn \u003cem\u003eA. millepora\u003c/em\u003e, both SoxB and SoxC is initially expressed in the presumptive ectoderm and later SoxC is found in putative sensory neuron (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e). The present study confirmed and extended these findings: In \u003cem\u003eA\u003c/em\u003e. \u003cem\u003etenuis\u003c/em\u003e, after the first segregation of yolk cells, the outer blastomeres became the glandular epithelium devoid of yolk granules; the ectodermal epithelium expressed AtSoxB2 and AtSoxC initially in the whole cytoplasm of elongating cells; then, AtSoxB2 and AtSoxC entered the nucleus of the epithelium and were dispensed into the secondary segregation cells at the bottom of the ectoderm (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e). The longest ectodermal cells in 28 hpf embryos are expected to be responsible for the deposition of \u0026lsquo;bottom\u0026rsquo; cells near the outer yolk cell. To the best of our knowledge, the present study is the first to describe \u0026lsquo;bottom\u0026rsquo; cells. \u0026lsquo;Bottom\u0026rsquo; cells expressed C-Jun in addition to Sox (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSoxB2 and SoxC are both markers of neuroglandular precursors in \u003cem\u003eNematostella\u003c/em\u003e (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). In the present study, \u0026lsquo;bottom\u0026rsquo; cells, nerve progenitors (neuroblasts), and nerve cells expressed AtSoxB2 and AtSoxC, and had cell bodies (nuclei) in common at the bottom of the ectoderm, indicating that AtSoxB2- and AtSoxC-expressing \u0026lsquo;bottom\u0026rsquo; cells would develop into nerve progenitors and nerve cells in \u003cem\u003eA\u003c/em\u003e. \u003cem\u003etenuis\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eThe aboral region of anthozoan larvae is enriched with nerve cells (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan additionalcitationids=\"CR25\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). It is important to note that in \u003cem\u003eA\u003c/em\u003e. \u003cem\u003etenuis\u003c/em\u003e, \u0026lsquo;bottom\u0026rsquo; cells arise not only from the aboral area, but also from non-aboral positions. We recently demonstrated that non-aboral \u0026lsquo;bottom\u0026rsquo; cells expressed similar nerve markers, but were destined for another cell fate (Kawamura et al., in preparation). In \u003cem\u003eNematostella\u003c/em\u003e, pluripotent stem cells appeared to arise from epithelial cells (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan additionalcitationids=\"CR28\" citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). They have been identified as the founder cells of neuroglandular progenitors (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e), subsequently giving rise to nerve and gland cells when expressing Nanos (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). Consistent with these findings in \u003cem\u003eNematostella\u003c/em\u003e, it will be shown that neuroblasts in \u003cem\u003eA\u003c/em\u003e. \u003cem\u003etenuis\u003c/em\u003e are developmentally multipotent in an oral-aboral position-dependent manner.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eNeuroblasts exhibit peculiar cytological features\u003c/h3\u003e\n\u003cp\u003eThe neuroblast was morphologically characteristic of a large nucleus with an indented contour and the endoplasmic reticulum closely associated with the plasma membrane. An amorphous nucleus has been reported in amphibian and zebrafish primordial germ cells (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e). A notched nucleus has also been observed in germline progenitor cells and smooth muscle precursors in colonial tunicates (\u003cspan additionalcitationids=\"CR33\" citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eNeuroblasts were also characterized by neuron-specific TUBB3. One of the \u003cem\u003ein vitro Acropora\u003c/em\u003e cell lines, BBrC expressed TUBB3 and a neuroblast differentiation-associated protein (AtAHNAK) (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). BBrC appears to be a proliferative form of the \u003cem\u003ein vitro\u003c/em\u003e FAmC line and is interchangeable with fibroblast-like cells (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). Fibroblast-like cells expressed hairy and enhancer of split (\u003cem\u003eHes\u003c/em\u003e) instead of AtAHNAK (17 and our unpublished data).\u003c/p\u003e \u003cp\u003eIn \u003cem\u003eNematostella\u003c/em\u003e, one group of progenitor/undifferentiated cells expresses \u003cem\u003eHes\u003c/em\u003e (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). Mammalian \u003cem\u003eHES\u003c/em\u003e encodes a transcriptional repressor that suppresses neurogenesis in a Notch-dependent or -independent manner (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e). In \u003cem\u003eA\u003c/em\u003e. \u003cem\u003etenuis\u003c/em\u003e, \u003cem\u003eAtHes1\u003c/em\u003e and \u003cem\u003eAtHes4\u003c/em\u003e gene expression was maintained at high levels throughout the mid- and late embryonic stages (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), which may explain why the expression of nerve-specific markers in bottom cells was suppressed to embryos until 6 dpf larvae.\u003c/p\u003e \u003cp\u003e \u003cb\u003e\u0026lsquo;Middle\u0026rsquo; cells are the third segregation product at the \u0026lsquo;pear\u0026rsquo; stage\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe present study showed that the gland epithelium shortened around 60 hpf, leaving segregated \u0026lsquo;bottom\u0026rsquo; cells \u003cem\u003ein situ\u003c/em\u003e, and unequal cell division occurred \u003cem\u003ede novo\u003c/em\u003e in the middle region of the ectodermal layer. This was the third segregation from the gland epithelium to form \u0026lsquo;middle\u0026rsquo; cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e). In the planula of \u003cem\u003eAurelia\u003c/em\u003e (class Scyphozoa), the ectoderm is pseudostratified with a superficial multilayer of densely packed nuclei (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e). The ectodermal architecture of \u003cem\u003eAcropora\u003c/em\u003e is similar to that of \u003cem\u003eAurelia\u003c/em\u003e, and embryonic \u0026lsquo;middle\u0026rsquo; cells and larval undifferentiated-like cells may be homologous to \u0026lsquo;densely packed nuclei\u0026rsquo; in \u003cem\u003eAurelia\u003c/em\u003e. More detailed information on densely packed cells is currently not available.\u003c/p\u003e \u003cp\u003eIn \u003cem\u003eNematostella\u003c/em\u003e, one group of progenitor/undifferentiated cells was shown to express \u003cem\u003eSoxB2a\u003c/em\u003e, two \u003cem\u003eMyc\u003c/em\u003e paralogs, and \u003cem\u003eNanos2\u003c/em\u003e at the post-gastrula stages, while the other group expressed \u003cem\u003eHes\u003c/em\u003e and \u003cem\u003eSoxB2\u003c/em\u003e, enriched for gastrula-stage cells (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). In \u003cem\u003eA. millepora\u003c/em\u003e, \u003cem\u003eSoxC\u003c/em\u003e was initially expressed in the ectoderm as early as the \u0026lsquo;prawnchip\u0026rsquo; stage and was maintained in putative sensory neurons throughout the larval stage (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e). We expected undifferentiated-like cells in \u003cem\u003eA\u003c/em\u003e. \u003cem\u003etenuis\u003c/em\u003e to express SoxB2 or SoxC; however, the present results indicated that both genes were restricted to nerve lineage cells.\u003c/p\u003e \u003cp\u003e \u003cem\u003eIn vivo\u003c/em\u003e undifferentiated-like cells in \u003cem\u003eA. tenuis\u003c/em\u003e showed a very similar morphology to the \u003cem\u003ein vitro\u003c/em\u003e cell type, SSmC (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). Cell lines belonging to SSmC were highly proliferative and expressed high levels of polyubiquitin, ubiquitin ligase, ubiquitin-conjugating enzyme, and reverse transcriptase (our unpublished RNA sequencing data), of which reverse transcriptase was expressed by \u003cem\u003ein vivo\u003c/em\u003e \u0026lsquo;middle\u0026rsquo; cells and undifferentiated-like cells (Kawamura et al., in preparation).\u003c/p\u003e \u003cp\u003eH3K4me3 is associated with active chromatin states (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e). The dual modification of H3K4 and H3K27 appears to be essential for sustaining the pluripotency of mammalian embryonic stem cells (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e). The present study has preliminarily shown that histone H3 of undifferentiated-like cells in \u003cem\u003eA. tenuis\u003c/em\u003e was trimethylated at lysine 4. Undifferentiated-like cells also have H3K27me3 (a detailed account will be reported elsewhere). They were not endowed with histone trimethylation at embryonic stages, and \u0026lsquo;middle\u0026rsquo; cells instead expressed AtKDM5, an antagonist of H3K4me3. The present results strongly suggest that \u0026lsquo;middle\u0026rsquo; cells gradually acquire the undifferentiated state during the embryonic and larval stages.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn \u003cem\u003eA\u003c/em\u003e. \u003cem\u003etenuis\u003c/em\u003e, four ectodermal cell layers arise through the unequal cell division of elongating outer blastomeres and the blastomere-derived glandular epithelium. These cell layers are stratified apicobasally, with yolk cells that segregated the earliest being located the most basally, indicating that the developing glandular epithelium is the source of yolk cells and progenitor/undifferentiated cells. The glandular epithelium expresses Snail, Etv6, C-Jun, SoxB2, SoxC in addition to Mef2. Yolk cell segregation is associated with Snail expression. Nerve lineage cells from neuroblast precursors (bottom cells) to nerves consistently express SoxB2 and SoxC throughout embryos and larvae. Undifferentiated-like cells emit signals for epigenetic histone methylation. The developmental fates of nerve progenitors and undifferentiated-like cells will be dealt with in the near future.\u003c/p\u003e "},{"header":"METHODS","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003cdiv id=\"Sec12\" class=\"Section3\"\u003e \u003ch2\u003e\u003cem\u003eAnimals\u003c/em\u003e:\u003c/h2\u003e \u003cp\u003eThe early embryos and planula larvae of \u003cem\u003eAcropora tenuis\u003c/em\u003e (class Anthozoa; subclass Hexacorallia, order Scleractinia, family Acroporidae) were kindly provided by Mr. Shuichi Mekaru at Onna Fisheries Cooperative, Okinawa, Japan. Larvae were allowed to develop at room temperature (~\u0026thinsp;25\u0026deg;C) in the Marine Genomics Unit, Okinawa Institute of Science and Technology Graduate University. Embryos were fixed at adequate developmental stages. Fixed embryos and living larvae at 6\u0026ndash;8 days post-fertilization (dpf) were transferred to Kochi University.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eHistology:\u003c/h2\u003e \u003cp\u003eEmbryos at 12, 17, 22, 28, 40, 60, and 85 hours post-fertilization (hpf) and larvae at 6, 8, 10, 14, and 18 dpf were fixed in Zamboni\u0026rsquo;s fixative at room temperature for 30 min. They were serially dehydrated and embedded in plastic resin, Technovit 8100 (Heraeus Kulzer, Wertheim, Germany). Samples were sectioned into 2-\u0026micro;m-thick slices with glass knives.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003ePrimary antibodies:\u003c/h2\u003e \u003cp\u003eRabbit antibody against Snail has already been described elsewhere (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). Additional antibodies were prepared as follows:\u003c/p\u003e \u003cp\u003eVDSNEHSKQNSRLD to aa 47\u0026ndash;60 of ETS translocation variant 6 (AtETV6), RPRRKPKSLLKKVDRYPFT to aa 78\u0026ndash;96 of AtSoxB2, GGAQDSKTSSSNSDGHSHS to aa 118\u0026ndash;136 of AtSoxC, and VNVQRQKEEDSKRKLGESL to aa 1193\u0026ndash;1211 of lysine-specific demethylase 5 (AtKDM5). Before immunization, these oligopeptides were each conjugated to the carrier protein, keyhole limpet hemocyanin (KLH). Rabbit antibodies were raised by Eurofins Genomics Tokyo, Japan. They were diluted 400-fold with phosphate-buffered saline (PBS) immediately before use.\u003c/p\u003e \u003cp\u003eA mouse anti-TUBB3 monoclonal antibody was purchased from Proteintech Group, Inc. (Tokyo, Japan). Rabbit anti-H3K4me3 (07-473, Millipore Temecula, CA, USA) and anti-H3K27me3 (07-449, Millipore) antibodies were purchased from commercial sources. They were used as previously described (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eImmunohistochemistry:\u003c/h2\u003e \u003cp\u003eThe blocking of sections was performed in a mixture of 0.25% blocking reagent (Roche, Mannheim, Germany) and 2.5% skim milk in PBS for 30 min. Sections were then incubated with primary antibodies diluted 400-fold containing 0.1 mg/ml KLH at room temperature for 1 h. The goat anti-rabbit antibody labeled with fluorescein isothiocyanate (FITC) (FI-1000, Vector Laboratory, Burlingame, CA, USA) and the goat anti-mouse antibody labeled with rhodamine B (AM10706, Tago Immunologicals, CA, USA) were diluted 200-fold with PBS. Sections were stained with the secondary antibody for 0.5 h followed by washing twice for 10 min with PBS containing 0.1% Tween 20. Samples were finally counterstained with 4\u0026prime;6-diamidino-2-phenylindole (DAPI, 5 \u0026micro;g/mL) for 10 min. Some samples were stained with 0.1 \u0026micro;M rhodamine-phalloidin (Cytoskeleton Inc., Denver, CO, USA). In the negative control, primary antibodies were absorbed in advance by the corresponding oligopeptides at a concentration of 25 \u0026micro;g/mL. After antibody staining and washing with PBS containing 0.1% Tween 20, sections were observed under a confocal microscope (ECLIPSE C1si system; Nikon).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eElectron microscopy:\u003c/h2\u003e \u003cp\u003eLarvae were pre-fixed in Karnovsky\u0026rsquo;s fixative containing 2.5% glutaraldehyde, 2% paraformaldehyde, and 1.5% NaCl in 0.1 M cacodylate buffer (pH 7.4) on ice for 2 h, and then post-fixed with 1% osmium tetroxide in PB on ice for 14 h. After post-fixation, specimens were rinsed several times with PB containing 1.5% NaCl, dehydrated with a graded acetone series, and embedded in Spurr\u0026rsquo;s resin. The resulting resin blocks were sectioned with a diamond knife on an Ultracut UCT ultramicrotome (Leica, Vienna, Austria). Sections were mounted on formvar-coated grids and stained with 1% aqueous uranyl acetate for 1 h and with lead citrate for 15 min in sequence at room temperature. Samples were observed with a Jeol JEM-1400 transmission electron microscope (JEOL, Tokyo, Japan) and photographed using a charge-coupled device camera.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e\u003cem\u003eRNA Sequencing\u003c/em\u003e:\u003c/h2\u003e \u003cp\u003eTotal RNA was extracted from 3.5, 15, 25, 36, 60, and 85 hpf embryos, from 5.5 and 8.5 dpf larvae, and from 14 dpf metamorphosing polyps with TRIzol solution. cDNA libraries were produced using a TruSeq Stranded mRNA Library Prep Kit (Illumina) with IDT for Illumina-TruSeq DNA UD Indexes v2. RNA-seq libraries were quantified with a Real-Time PCR (StepOnePlus; Applied Biosystems) and quality control utilized capillary electrophoresis on a Bioanalyzer. Library sequencing was carried out on a MiSeq using MiSeq V2 reagent 300 cycles (Illumina). Transcriptomic analyses of raw data were carried out, as described previously (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). The abundance of annotated genes was estimated as transcripts per million (TPM).\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Mr. Shunichi Mekaru for proving\u0026nbsp;\u003cem\u003eAcropora tenuis\u0026nbsp;\u003c/em\u003elarvae and\u0026nbsp;all members of the Marine Genomics Unit, OIST, especially Kanako Hisata for help in preparation of manuscript. Prof. Shigeki Fujiwara is acknowledged for his support.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors and Affiliation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDepartment of Applied Science, Kochi University, Kochi 780-8520, Japan\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eDr. Kaz Kawamura,\u0026nbsp;Emeritus Professor\u003c/p\u003e\n\u003cp\u003eKuroshio Science Unit, Multidisciplinary Science Cluster, Kochi University, Kochi 780-8520, Japan\u003c/p\u003e\n\u003cp\u003eDr. Satoko Sekida\u003c/p\u003e\n\u003cp\u003eDepartment of Marine Science and Technology, Fukui Prefectural University, Obama, Fukui 917-0003, Japan\u003c/p\u003e\n\u003cp\u003eDr. Koki Nishitsuji\u003c/p\u003e\n\u003cp\u003eMarine Genomics Unit, Okinawa Institute of Science and Technology Graduate University, Onna, Okinawa 904-0495, Japan.\u003c/p\u003e\n\u003cp\u003eDr. Nori Satoh\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eContributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eKK and NS conceived and designed the study, and prepared the manuscript. KK, SS, and KN carried out immunohistochemical studies, electron microscopy, and RNA-seq analyses, respectively. All authors commented on it.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by an OIST fund for collaboration with Kochi University,\u0026nbsp;and in part by JSPS KAKENHI Grant number 22H03794 to KN, KK, and NS.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics declarations:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo ethics approval was required for the work presented in this manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to Participate and Consent to Publish declarations:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability declaration:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll relevant data are included within the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eOkubo N, Motokawa T. 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Dev Dyn. 2024. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1002/dvdy.754\u003c/span\u003e\u003cspan address=\"10.1002/dvdy.754\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Glandular epithelium, Nerve progenitor, Undifferentiated cell, Yolk cell, C-Jun, KDM5, Snail, Sox","lastPublishedDoi":"10.21203/rs.3.rs-8654320/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8654320/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eIn Anthozoa, the ectoderm contains the epidermis, progenitor/undifferentiated cells, and nerve cells; however, their embryonic origin and development remain unclear. In the stony coral \u003cem\u003eAcropora tenuis\u003c/em\u003e, both blastula formation and gastrulation are quite unique. Endodermal cells form by ingression from the presumptive pseudo-archenteron, while ectodermal cell differentiation is poorly understood.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eUltrastructural studies on \u003cem\u003eA. tenuis\u003c/em\u003e showed that the ectoderm consisted of four cell layers stratified apicobasally from the glandular epithelium to yolk cells through undifferentiated-like cells and nerve lineage cells. A 12-hour \u0026lsquo;prawnchip\u0026rsquo; embryo had a U-shaped outline devoid of blastocoel. Blastomeres of the embryo were monolayered and the outer blastomeres began to elongate in 17-hour embryos. The elongating blastomeres segregated Snail/Etv6-expressing yolk cells from their growing tips into the narrow blastocoel. In 28-hour \u0026lsquo;donut\u0026rsquo; embryos, the nuclei of elongated ectodermal blastomeres translocated to the growing extremities of cells that became the glandular epithelium. Secondary segregation occurred herein in 40-hour embryos to produce \u0026lsquo;bottom\u0026rsquo; cells labeled with SoxB2/SoxC near the yolk cells at the bottom of the ectoderm. In 60-hour embryos, the third segregation occurred to produce \u0026lsquo;middle\u0026rsquo; cells in the middle region of the ectoderm. The segregated cells expressed lysine-specific demethylase 5 (KDM5) at embryonic stages, and they were tagged by trimethylated histone H3 at lysine 4 at larval stages.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eThe present study shows that in \u003cem\u003eA\u003c/em\u003e. \u003cem\u003etenuis\u003c/em\u003e embryonic outer blastomeres and the blastomere-derived glandular epithelium expresses Snail, Etv6, C-Jun, SoxB2, SoxC, and KDM5. The glandular epithelium contains founder cells that give rise to yolk cells and progenitor/undifferentiated cells via three steps of unequal cell division. Every segregation accompanies the expression of C-Jun. The initial segregation event accompanies Snail/Etv6 expression, resulting in the yolk cell formation. The secondary and third segregation events accompany SoxB2/SoxC and KDM5, respectively. The former gave rise to nerve progenitors through \u0026lsquo;bottom\u0026rsquo; cells, and the latter formed undifferentiated-like cells through \u0026lsquo;middle\u0026rsquo; cells. Our results suggest that the ectodermal cell differentiation in \u003cem\u003eAcropora\u003c/em\u003e depends on spatiotemporal condition of unequal segregation.\u003c/p\u003e","manuscriptTitle":"Ectodermal Cell Differentiation by Unequal Cell Division in the Stony Coral Acropora tenuis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-29 18:14:05","doi":"10.21203/rs.3.rs-8654320/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"10371169-e44d-4c74-8e81-099f46e205f1","owner":[],"postedDate":"January 29th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-03-02T21:24:18+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-29 18:14:05","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8654320","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8654320","identity":"rs-8654320","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}